Pasteurization of microbial biomass for food applications

Abstract

The present invention relates to a method for pasteurizing biomass, such as fungal biomass, using specific time and temperature parameters to obtain pasteurized biomass, which is suitable for use as a source of single-cell protein in food products. The present invention also relates to pasteurized products and food products containing the same.

Description

[Technical Field]

[0001] [Field of the Invention] The present invention relates to a method for pasteurizing biomass, such as fungal biomass, using specific time and temperature parameters to obtain pasteurized biomass, which is suitable for use as a source of single-cell protein in food products. The present invention also relates to pasteurized products and food products containing the same.

[0002] [Background technology] Single-cell proteins (SCPs) are of interest as meat protein substitutes and as protein sources in many other food applications, such as breakfast cereals, bread, pasta, dairy products, ice cream, chocolate, and soups. When fungal SCPs are produced, they yield many more tons of protein per hectare than meat production, and because they have lower nitrogen emissions, they can be produced from sugar-rich crops in a more sustainable manner than meat protein production. One way to produce protein-rich foods or animal feed is to produce SCPs by fermentation (Suman et al., 2015, Int J. Curr. Microbiol. Appl. Sci., Vol. 4, No. 9, pp. 251-262). In this context, fermentation is understood as the microbial conversion of carbohydrate-rich raw materials into protein-rich products composed of microbial cells, such as bacteria, yeast, or fungi. The use of SCP as an animal feed and food ingredient offers the additional advantage that microbial cells have a high content of essential amino acids and, when applied, for example, to supplement a grain-based diet, they produce useful enzymes such as phytase, xylanase, pectinase, protease, cellulase, amylase, etc., all of which can positively affect the digestibility of formulated feeds that are high in, for example, the anti-nutritional compound phytate, indigestible fiber, etc. Furthermore, fungal cells in particular can be very rich in trace elements and vitamins that make fermented feeds very nutritious.

[0003] SCPs are already being used in foods for human consumption. For example, Quorn™ contains mycoprotein produced by Fusarium venenatum, which contains vitamin B1 (thiamin), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), and biotin. One issue in SCP production, especially in the case of submerged fermentation with bacteria or yeast, is the concentration of SCP biomass produced in the fermentation broth. Another problem is the need for expensive enzymes to convert inexpensive polymeric carbon sources into monomeric fermentable sugars. Furthermore, to avoid infection when using mesophilic microorganisms for SCP production, sterile fermentation conditions must be applied, which leads to prohibitive operating costs due to high capital investment and energy demands (WO 2018 / 029353). Some of these issues have been addressed by using solid-state fermentation with thermophilic fungi (WO 2018 / 029353). WO 2018 / 029353 describes a fungal-derived SCP that is easily collected and compressed in a sieving process.

[0004] U.S. Patent No. 8,481,295 discloses the production of thermophilic fungi as an animal feed ingredient using batch fermentation on thin stillage obtained from ethanol refineries, however the fungal strains used there do not perform well at pH below 4 and temperatures above 45°C, making the process sensitive to bacterial and yeast contamination.

[0005] Gregory KF et al. (1977, Anim. Feed Sci. Technol. 2:7-19) described attempts to use thermotolerant fungi in the conversion of cassava, isolating many of these fungi in the process. However, these attempts did not lead to commercialization because their organisms remained susceptible to contamination, they used undesirable human pathogens (e.g., Aspergillus fumigatus), and their Mucor strains proved indigestible in rat studies. Several sources have also reported studies involving the thermotolerant fungus Cepalosporium eichornia in the production of SCP, as described in WO 2018 / 029353.

[0006] To improve shelf stability, SCP can be cooled, frozen, or dried. However, when SCP is sold as an ingredient in the food industry, microbial growth may continue or resume in the final food product. Therefore, SCP for use in food products generally must have a bacterial count reduced to less than 10 fungi / gr, which is the standard for typical foods. Such a bacterial count reduction is conveniently achieved by pasteurization.

[0007] Pasteurization equipment is well known and commercially available in many shapes and forms. Pasteurization is a type of heat treatment and therefore consumes a large amount of energy. For example, when pasteurization is performed after submerged fermentation of unicellular organisms, the volumes to be processed are quite large, since fungal biomass generally does not reach a biomass dry matter content of more than 5% dry matter. This is because oxygen transfer is generally limited during such fermentation conditions as a result of the viscous behavior of the broth. For this reason, the fermentation broth is sometimes intentionally further diluted.

[0008] The goal of milk pasteurization is to achieve a 99.999% (5-log) reduction in viable microorganisms. Pasteurization is typically achieved using high-temperature, short-time equipment using continuous heat treatment. The temperature and time combinations typically used in milk pasteurization are selected to optimize microbial destruction while minimizing the impact on the nutritional quality of the milk. Pasteurized milk (heated to 60°C for approximately 20 minutes) must be refrigerated and has a relatively short shelf life. In contrast, ultra-pasteurized (UP) milk (heated to 125–138°C for 2–4 seconds) can be stored refrigerated for up to 3 months. Milk that has undergone ultra-high-temperature (UHT) processing (heated to 135–140°C for a few seconds) can be stored at room temperature for 3–6 months. However, consumer panel surveys have revealed that many consumers can distinguish pasteurized milk from UHT milk by taste. Consumers often prefer pasteurized milk for its flavor, describing UHT milk as having a "cooked" flavor (L. Meunier-Goddik, S. Sandra, Encyclopedia of Dairy Sciences, 2011, pp. 274-280). High temperature processing has also been associated with adversely affecting the taste of foods containing Mucorales fungi (WO 0167886).

[0009] An object of the present invention is to improve pasteurization of single-cell protein biomass. An object of the present invention is to reduce the energy requirements of pasteurization. An object of the present invention is to reduce the complexity of the equipment and fermentation facilities required for the production of pasteurized SCP. An object of the present invention is to produce (pasteurized) SCP with improved organoleptic qualities, such as improved taste, odor, and / or mouthfeel.

[0010] [Summary of the Invention] The present invention provides a method for pasteurizing biomass, comprising: i) providing biomass; ii) pasteurizing the biomass to obtain a pasteurized biomass, wherein the biomass is pasteurized at a temperature of at least 70°C for a maximum of 45 minutes; and iii) Optionally, recovering the pasteurized biomass. The present invention provides a method comprising:

[0011] Preferably, the biomass is derived from a fermentation broth, preferably from a submerged fermentation process. The fermentation broth preferably comprises a biomass dry matter content of at most 6%, preferably at most 5%, more preferably at most 4%. Preferably, to obtain the biomass, the fermentation broth is sieved, preferably using a sieve belt, a vibrating sieve, or a screen sieve. Preferably, the provided biomass comprises at least 7% dry matter, preferably at least 10% dry matter. Preferably, the biomass is biomass derived from a fungal strain, preferably the fungal strain is a strain of the genus Rasamsonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Myceliophthora, Thermoascus, Thiella, Preferably, the fungal strain is a strain of a fungal genus selected from the group consisting of Thielavia, Mucor, Stibella, Melanocarpus, Malbranchea, Dactylomyces, Canariomyces, Scytalidium, Myriococcum, Corynascus, and Coonemeria, preferably the genus is Rhizomucor, preferably the strain is Rhizomucor pusillus, more preferably Rhizomucor pusillus strain CBS143028, or a strain which is a single colony isolate or derivative thereof.

[0012] In a preferred embodiment, pasteurization is carried out in an in-line heating unit, preferably comprising a pipe heater, a heating block, or a steam injection element, more preferably comprising a steam injection element, and optionally comprising a mixing element such as a static mixer. Preferably, pasteurization is carried out for a maximum of 5 minutes, preferably from about 0.5 to about 3 minutes, more preferably from about 1 to 2 minutes. Preferably, pasteurization is carried out at a temperature of at least 74°C, preferably at least 80°C, more preferably at least 86°C. Preferably, the bacterial count of the pasteurized biomass is at least 7, preferably at least 11, more preferably at least 12 log lower than that of the provided biomass. 10 Has a decrease.

[0013] In a preferred embodiment, in step i) a biomass derived from submerged fermentation of a Rhizomucor pusillus strain is provided, and in step ii) the biomass flows through an in-line heating unit in which the biomass has a residence time of about 1 to 2 minutes at a temperature of about 86°C, and the pH of the biomass in step ii) is preferably at most 4.5, more preferably at most 3.5.

[0014] The present invention also provides pasteurized biomass obtained by the above-described method, preferably containing about 6% to 12% lipid and about 35% to 55% protein on a dry weight basis. A food or feed product comprising the pasteurized biomass is also provided. The present invention also provides the use of the pasteurized biomass in the production of a food product or as a source of at least one of fiber or protein for use in the production of a food product.

[0015] [Description of the embodiment] The inventors have surprisingly found that single cell proteins pasteurized at temperatures of at least 70°C for up to 45 minutes have a sufficiently low bacterial count and are more sensorially pleasant. The present invention encompasses methods and products obtained thereby, as well as uses of such products. Accordingly, the present invention provides a method for pasteurizing biomass, comprising: i) providing biomass; ii) pasteurizing the biomass to obtain a pasteurized biomass, wherein the biomass is pasteurized at a temperature of at least 70°C for a maximum of 45 minutes, preferably by flowing the biomass through an in-line heating unit to obtain the pasteurized biomass, wherein the biomass has a residence time in the heating unit of at most 45 minutes, and the heating unit has a temperature of at least 70°C; and iii) Optionally, recovering the pasteurized biomass. The present invention provides a method comprising:

[0016] Such a method is hereinafter referred to as a method or process according to the invention. The term "single cell protein", abbreviated as "SCP", is understood herein to refer to a biomass consisting essentially of a single cell or cells of an organism existing in a single-cell state, including single-cell bacteria, yeasts, fungi or algae, and further including microorganisms growing in a filamentous form (mycelia), which biomass is preferably in dry form and is suitable as a dietary source of protein or protein supplement in human food or animal feed.

[0017] Method Step i) Providing biomass In step i), a biomass is provided. This biomass is preferably the biomass of a unicellular or filamentous organism, among which bacteria, yeast, fungi, or algae are preferred. In some embodiments, the biomass is a unicellular biomass. In other embodiments, the biomass is from a filamentous organism. In a preferred embodiment, the provided biomass is derived from a fungal strain, preferably a filamentous fungal strain. It is preferably not pasteurized biomass and is derived from a non-heat-treated fermentation.

[0018] The fungi used in the methods of the present invention, i.e., the fungi grown in the process, are preferably thermophilic fungi. Thermophilic fungi for use in the present invention are preferably fungi that grow at temperatures of at least 45, 46, 47, 48, 50, 51, 52, or 55°C, and sometimes above 56°C. Thermophilic fungi for use in the present invention are also preferably fungi that grow at low, i.e., acidic, pH. Preferred thermophilic fungi grow at a pH of 3.8, 3.75, 3.74, 3.73, 3.72, 3.71, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, or less, more preferably 3, or less. Preferably, the fungi are grown at that pH. Thermophilic fungi for use in the present invention are preferably cellulolytic and / or hemicellulolytic fungi.

[0019] "Fungi" is defined herein as a eukaryotic microorganism, including all species of the subgenus Eumycotina (Alexopoulos et al., 1962, Introductory Mycology, John Wiley & Sons, Inc., New York). Thus, the term fungi includes both filamentous fungi and yeasts. "Filamentous fungi" is defined herein as a eukaryotic microorganism, including all filamentous forms of the subgenus Eumycotina and Oomycota (Hawksworth et al., 1983, in Ainsworth and Brisby's Dictionary of the Fungi, 7th ed., Commonwealth Mycological Institute, Kew, Surrey). Filamentous fungi are characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic.

[0020] The thermophilic fungi for use in the methods of the present invention are preferably filamentous fungi. Preferred thermophilic fungi for use in the present invention are strains of fungal genera selected from the group consisting of Razamthonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Myserioptra, Thermoascus, Thielavia, Mucor, Stibella, Melanocarpus, Malbranchia, Dactyromyces, Canariomyces, Scytharrhizolium, Myriococcum, Corynascus, and Kuhnemeria. Preferably, the genus is Rhizomucor, and preferably, the strain is Rhizomucor pusillus, and more preferably, Rhizomucor pusillus strain CBS143028 or a single colony isolate or derivative thereof. More preferably, the thermophilic fungus is a strain of a fungal species selected from the group consisting of Rasamsonia composticola, Talaromyces emersonii, Rasamsonia emersonii, Thermomucor indicae-seudaticae, Rhizomucor miehei, Rhizomucor pusillus, Thielavia terricola var. minor, Rhizopus sp. and Thermoascus thermophilus. Suitable strains of these thermophilic fungi can be isolated, for example, from Dutch compost and have been successfully used by the present inventors to demonstrate that these thermophilic fungi grow well on complex nutrients at high temperatures and low pH. Following this, many thermophilic strains, such as Thielavia tericola var. minor and Thermoascus thermophilus, are also suitable for use in the present invention and also grow well at high temperatures and low pH.The Rhizopus species can be any one of Rhizopus oryzae, Rhizopus chlamydosporus, Rhizopus microsporus, Rhizopus stolonifer, or Mucor indicus. Alternatively, the Rhizopus species can be an unidentified Rhizopus or Mucor species corresponding to Rhizopus species CBS143160. Preferably, the Rhizopus species is safe for use in food, and more preferably, the Rhizopus species is a tempeh fungus.

[0021] Preferred strains of the above-mentioned thermophilic fungi for use in the present invention are obtained from the Westerdijk Institute for Fungal Biodiversity (formerly Centraalbureau voor ) in Utrecht, The Netherlands, under the provisions of the Budapest Treaty. The following strains were deposited in the Schimmelcultures (formerly known as CBS) on the indicated dates and assigned the indicated accession numbers: Rasamsonia composticola CBS141695 (July 29, 2016), Thermomucor indicae seudataicae CBS143027 (July 21, 2017), Rhizomucor miehei CBS143029 (July 21, 2017), Rhizomucor pusillus CBS143028 (July 21, 2017), Rasamsonia emersonii strain CBS143030 (July 30, 2017), and Rhizopus species CBS143160 (August 11, 2017). Further preferred strains for use in the present invention include Thermomucor indicae saudaticae CBS104.75, Thermoascus thermophilus CBS528.71, Thielavia terrestris CBS546.86, Talaromyces emersonii CBS393.64 and Thermothelomyces thermophila CBS117.65. Particularly preferred for use in the present invention are Rasamsonia composticola strain CBS141695, Rasamsonia emersonii CBS143030, Thermomucor indicae seudataicae CBS143027, Rhizomucor miehei CBS143029, Rhizopus sp. CBS143160 and Rhizomucor pusillus CBS143028.

[0022] Thermophilic fungi for use in the present invention are more preferably those capable of obtaining biomass with a high protein content. Preferably, the protein content of the biomass is at least 30, 35, 40, 45, 50, or 55% (w / v) on a dry matter basis. High-protein strains are most likely to have a low content of carbon accumulation and / or storage compounds, such as trehalose, glycogen, and / or lipids.

[0023] The thermophilic fungus for use in the present invention is more preferably a fungus whose biomass contains proteins containing one or more essential amino acids. Preferably, the proteins are rich in such essential amino acids. In this specification, essential amino acids are understood to include at least one or more of lysine, phenylalanine, threonine, methionine, valine, arginine, histidine, tryptophan, isoleucine, and leucine, of which lysine, threonine, and methionine are most preferred.

[0024] Since the SCP product is intended for use in food for human consumption or animal feed, production of mycotoxins such as ochratoxin A and fumonisins by the applied thermophilic fungi is undesirable. Therefore, thermophilic fungi for use in the present invention are preferably selected to not produce mycotoxins. This screening is preferably carried out by genetic means, such as PCR or whole genome sequencing, to confirm the absence of genes in the mycotoxin pathway, and, if such genes are present, to confirm that they are not expressed and / or that these toxic compounds are not produced under the process conditions used.

[0025] Preferably, the microorganisms contained in the biomass produce their own extracellular hydrolytic enzymes, which allow the process to be carried out under non-sterile conditions, since at temperatures above 45°C and a pH below 3.8, other (micro)organisms cannot invade and / or compete with the target microorganism, preferably a fungus, more preferably a thermophilic fungus. Thus, preferably, thermophilic fungi are used that are capable of growing on energy-rich, carbon-based feedstocks containing both simple sugars such as sucrose, glucose, and fructose, and polymeric sugars such as starch, inulin, cellulose, hemicellulose, chitin, and pectin, as well as organic acids such as lactic acid, acetic acid, formic acid, ethanol, and methanol (these metabolic products are often formed during the silage process or derived from their separation from pectin and hemicellulose), and lipids present in the form of triglycerides or phospholipids. Conversion of other sugars, such as those present in hemicellulose; rhamnose, fucose, galactose, xylose, arabinose, mannose, galacturonic acid, and glucuronic acid, is also necessary. Raffinose, melibiose, and stachyose are preferred to enhance the protein content of feed ingredients and minimize the carbon load from filtrate that must go to wastewater treatment / biogas facilities. Fungal conversion of betaine, ferulic acid, and coumaric acid is also preferred to maximize yield. The advantage of many thermophilic fungi occurring in processes such as composting is that they can tolerate very harsh conditions and produce enzymes that degrade and convert polymeric substrates, such as carbohydrates, into simple sugars.

[0026] Alternatively, the thermophilic fungi used in the process of the invention have been genetically modified to produce increased amounts of hydrolytic enzymes, preferably invertases (e.g., in the case of Rathamsonia), cellulolytic enzymes and / or lignocellulolytic enzymes, such as those described in WO 2011 / 000949. Enhanced enzyme production can lead to shorter hydrolysis times and the use of smaller tanks, and the enzyme-containing filtrate / decant can be commercialized as a secondary product.

[0027] The biomass is preferably derived from a fermentation broth, more preferably from a submerged fermentation process. Providing the biomass preferably comprises the step of (i) growing a thermophilic fungus in a medium comprising a fermentable, carbon-rich feedstock. Preferably, in step (i), the fungus is grown in submerged culture. Preferably, in step (i), the fungus is grown under non-sterile conditions. Preferably, in step (i), the fungus is grown at a temperature of 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55°C or higher. Preferably, in step (i), the fungus 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, or 2.5 or lower. The process preferably further comprises a step ib) of recovering the microorganisms from the culture medium in the form of a (concentrated) biomass of the thermophilic fungus grown in step ia). The biomass to be pasteurized preferably has a high cell density.

[0028] As known from WO 2018 / 029353, one problem to be solved in the production of SCP at low pH is the toxicity of many carbon-rich fermentable feedstocks. Hydrolyzed biomass or silage products, in particular, can contain compounds that are toxic to most microorganisms, including organic acids such as acetic acid, lactic acid, ferulic acid, coumaric acid, and formic acid. These acids are particularly toxic at low pH, where they are in an undissociated form and can easily penetrate cell walls and acidify the interior of the cells. If such carbon-rich fermentable feedstocks are applied to fermentations at low pH and with feedstock dry matter concentrations (w / v) higher than 2, 5, or 10%, their toxicity will inhibit fungal growth.

[0029] Therefore, preferably, in the process of the present invention, the concentration of the carbon-rich feedstock in step ia) is at a level such that toxic compounds in the feedstock do not reduce the growth rate of the fungus. More preferably, the carbon-rich feedstock is fed to the medium at a rate such that toxic compounds in the feedstock do not reduce the growth rate of the fungus. For example, in the process according to the present invention, the carbon-rich feedstock in the medium is fed to the medium at a rate that maintains a concentration of, or less than 5, 4, 3, or 2% (w / v) dry matter. The dry matter in the feedstock is generally the substrate for fermentation and is different from the biomass dry matter, which is a measure of the amount of microorganisms in the broth or biomass. The feedstock dry matter is preferably present at 1-20% or 2-20%, more preferably 2-15%, even more preferably 2-10%, and even more preferably 2-6% dry matter (w / v). In a preferred embodiment, the fermentation broth contains a biomass dry matter content in the range of 0.5-7%, preferably up to 6%, preferably up to 5%, and more preferably up to 4%. It is even more preferably at most 3%, even more preferably at most 2%, even more preferably at most 1.5%, and most preferably at most 1%. The dry matter content is preferably at least 0.2%, more preferably at least 0.5%, even more preferably at least 1%, even more preferably at least 2%, even more preferably at least 3%, and most preferably at least 3.5%. A higher biomass dry matter content reduces the amount of pasteurization required, and a lower biomass dry matter content allows for improved fermentation conditions such as aeration and mixing.

[0030] The fermentability of a feedstock can be conveniently checked or monitored by measuring at least one of the CO2 and O2 contents of the fermenter exhaust gas. Therefore, the maximum concentration at which a feedstock can be used without adversely affecting the growth rate of the fungus can be determined by increasing the concentration of the feedstock in the medium until a concentration is reached at which the CO2 production rate and / or oxygen consumption rate decreases. Therefore, preferably, in the process of the present invention, the concentration at which toxic compounds in the feedstock do not decrease the growth rate of the fungus is determined and / or defined as the highest concentration of carbon-rich feedstock that does not cause a decrease in the CO2 production rate and / or O2 consumption rate by the fungus. A well-fermented feedstock will allow a rapid increase in the CO2 production rate or oxygen consumption rate, as determined by an increase in the CO2 concentration or a decrease in the oxygen concentration, respectively, in the off-gas from fermentation. If the CO2 release rate is low (and other factors such as nitrogen, carbon, minerals, and trace elements are not limiting), growth will be slow and can be enhanced by dilution with water until growth begins to take off and CO2 production accelerates.

[0031] WO 2018 / 029353 discloses a fed-batch process in which hydrolyzed biomass is diluted to less than 2% dry matter before inoculation, followed by a complete batch phase. Once organic acids and sugars have been consumed, the feeding of hydrolyzed biomass is initiated at a slow rate under glucose-limited conditions (e.g., glucose <2 g / L) to allow the fungus to consume all of the toxic organic acids fed to the fermenter. Thus, preferred biomass for use in the methods of the present invention is derived from a fed-batch process, a repeated fed-batch process (in which portions of the fermentation broth are repeatedly withdrawn), or a continuous process. Preferably, the dilution rate in such processes, i.e., the rate at which the feedstock is fed to the fermenter, should be as high as possible but not higher than the maximum specific growth rate of the fungus to prevent fungal washout. The dilution rate is preferably in the range of 0.05 to 0.5, preferably up to 0.4, more preferably 0.2 L / h, which corresponds to a residence time in the fermenter of 5 to 20 hours. Therefore, the dilution rate is preferably at least 0.05 or 0.1 l / h and preferably does not exceed 0.4 or optionally 0.2 l / h.

[0032] Further preferred biomass is derived from a process involving the use of two or more fermenters, in which at least a first fermenter is emptied for harvesting and optionally cleaning, while fungal growth continues in at least a second fermenter. Cleaning of the empty fermenter preferably includes sterilization, e.g., by rinsing with an acid (such as sulfuric acid or phosphoric acid), an alkali (such as NaOH or KOH), a disinfectant (such as hydrogen peroxide or peracetic acid), or heat (e.g., steam), e.g., to control infection of the fermentation by bacteria or yeast. Cleaning is preferably performed using CIP equipment. In one embodiment, the biomass is derived from a process carried out in at least one pair of fermenters that are alternately emptied for harvesting and optional cleaning once every 1, 2, 3, 4, or 5 days, or once a week, or once a month. This operation is a process improvement that allows the practice of non-sterile conditions without process instability or deviations in quality or process stability. In a further preferred embodiment of the process, after harvesting and optional cleaning, the empty first fermenter is filled with at least a portion of the contents of the second fermenter, and growth continues while the first fermenter is harvested and optionally cleaned. In the next round of the process to provide biomass, the second fermenter is harvested and optionally cleaned, and then filled with at least a portion of the contents of the first fermenter, and growth continues while the second fermenter is harvested and optionally cleaned, and so on. In yet another embodiment, the harvested fermentation batch is collected in further continuous fermentation stages, allowing for higher product yields and / or a steady feed to downstream processing areas.

[0033] In the process of the present invention, it is preferable to manage the dry matter concentration (of the feedstock) for feeding biomass so that the oxygen consumption rate does not exceed the oxygen transfer capacity of the fermentor (leading to insufficient aeration and incomplete substrate consumption).

[0034] Note that the biomass to be pasteurized does not need to be identical to the fermentation mixture, as it may be further processed prior to pasteurization. The dry matter concentration in the medium is preferably optimized to maximize cost-effective downstream processing. To minimize the amount of recovered fermentation medium pasteurized in step ii), the dry matter concentration of the provided biomass is preferably as high as possible. On the other hand, if the dry matter concentration of the feedstock in the medium is too high, the viscosity of the fungal broth will increase and oxygen transfer will become a problem. We have found that the optimal dry matter concentration of the feedstock in the medium in the fermenter is in the range of 1-20%, preferably 2-15%, dry matter (w / v). Variations are possible depending on the raw material, salt stress, toxic metabolites, and the type of fermenter used. The biomass dry matter content can also be at least 5%, preferably at least 6%. In a preferred embodiment, the biomass contains at least 7% dry matter, preferably at least 8%, more preferably at least 9%, even more preferably at least 10%, even more preferably at least 11%, and most preferably at least 12%. This biomass dry matter content preferably relates to the content during pasteurization. The dry matter content is preferably at most 15%, more preferably at most 13%, even more preferably at most 12%, even more preferably at most 11%, and even more preferably at most about 10%. A preferred range is 5-15%, more preferably about 7-12%, and even more preferably about 7-10%.

[0035] Furthermore, the rheology of the broth is determined in part by the growth morphology of the fungus. The preferred growth morphology of the fungus in the process of the present invention is a hyphal length that is short enough to reduce the viscosity of the broth and facilitate oxygen transfer and mixing, but long enough to facilitate filtration or decantation at low g values. Thus, the hyphal length is preferably in the range of 10 to 500 μm (micrometers), and preferably the hyphal length is not excessively branched. More preferably, the hyphal length is in the range of 30 to 300 μm. The mycelium is preferably easily recoverable by retention on a sieve or screen, preferably having pores with a diameter of 0.1, 0.5, 1, or 2 mm.

[0036] It is further preferred that nitrogen limitation be avoided when the biomass is fermented. Therefore, it is preferred that microorganisms such as fungi are grown under carbon limitation. This can maximize the protein content of the biomass produced and avoid the accumulation of carbon accumulation and / or storage compounds, such as trehalose, glycogen and / or lipids, that would result from a carbon excess.

[0037] The carbon-rich fermentable feedstock that can be used to provide the biomass to be pasteurized in the process of the present invention can be any feedstock that can serve as a carbon and energy source for thermophilic fungi. Such carbon-rich feedstocks can be crops freshly recovered from the primary production of edible sugars, such as corn, sugar beet, thin juice, thick juice, or sugarcane juice. However, particularly when SCP is intended for animal feed applications, it is more logical and preferable to use carbon-rich by-products or by-products or waste streams from agriculture and / or food production, such as sugar beet pulp, liquid C starch from grain processing, vegetable waste from peeled or cut vegetable production, or vegetable waste from non-conforming vegetables, such as potato peels, and off-cuts from French fries, pea cream, and rejected potatoes, as feedstocks. Palm oil mill effluent (POME), which mainly contains palm oil and palm oil fatty acids, and palm mill residues, which contain empty fruit bunches (EFB) or palm leaves, can also be used as feedstocks. Preferred feedstocks can be silaged, allowing for year-round processing into SCP, while still recovering the feedstock in one operation, as in the case of sugar beet pulp or potato or sugar beet leaves. Silage from whole feed beets, for example, in combination with corn or whole corn or its ensiled form, can also be used, although lignin-rich corn stover and sugarcane bagasse are not preferred. For example, pentoses from lignocellulosic hydrolysates can also be used. These syrups contain primarily glucose, xylose, arabinose, mannose, and galactose. Another suitable source of raw material as a fermentable, carbon-rich feedstock for the process of the present invention is the organic fraction of municipal solid waste (MSW).

[0038] The method according to the present invention is highly useful for producing pasteurized SCP that can be used in food or feed products. For the production of SCP for food production (edible use), any plant-derived product that is suitable for or acceptable for food applications can be applied to the present invention as a carbon-rich feedstock for use in providing biomass for pasteurization, including, for example, corn, potato, wheat, rice, cassava, sugarcane or sugarcane juice, sugarbeet or sugarbeet juice, molasses, sugarcane molasses, glucose syrup, fructose syrup, and other vegetable products suitable for food use. Because the selected organisms also consume triglycerides, including, for example, soybean oil, olive oil, corn oil, palm oil, coconut oil, rapeseed oil, or sunflower oil, lipid-rich fractions, such as vegetable oils or fractions thereof, can also be applied to the present invention as carbon-rich feedstocks.

[0039] The medium used to provide the biomass further preferably contains and / or is supplied with a nitrogen source. Preferably, the nitrogen source comprises one or more of ammonia, urea, and nitrate (sources). More preferably, the nitrogen source is a reductant such as urea or ammonium. NH3 or HNO3 can also be used to control the pH in the fermenter, or urea can be used as a pH-independent nitrogen source. Nitrogen sources from waste streams are also preferred. These include, for example, molasses, evaporation of sugar beet or sugar cane vinasse, vinasse from the wine industry, grape residue, potato protein liquor (PPL), corn steep liquor (CSL), ammonia from animal farm exhaust gas cleaning scrubbers, and one or more of the amines present in the burden condensate obtained from the thin fraction of manure processing.

[0040] In a preferred embodiment of the method according to the present invention, biomass is grown or cultivated in a chemically defined medium. The term "chemically defined" is understood to refer to a fermentation medium that is essentially composed of chemically defined components, i.e., the chemical composition of essentially all chemicals used in the medium is known. A fermentation medium that is essentially composed of chemically defined components includes a medium that does not contain complex carbon and / or nitrogen sources, i.e., does not contain complex raw materials with a chemically undefined composition. Chemically defined media preferably do not contain chemically undefined yeast, animal, or plant tissues; they do not contain peptones, extracts, or digests, or other components that may contribute to chemically poorly defined proteins and / or peptides and / or hydrolysates. Chemically undefined or poorly defined chemical components are those whose chemical composition and structure are not known, are poorly defined and exist in various compositions, or can only be defined through extensive experimental effort.

[0041] Nevertheless, a fermentation medium consisting essentially of chemically defined components may further comprise a medium containing essentially small amounts of complex nitrogen and / or carbon sources, the amounts being defined below, but the amounts are typically insufficient to sustain microbial growth and / or ensure the formation of sufficient amounts of biomass. In this respect, complex feedstocks have compositions that are not chemically defined, for example, due to the fact that these feedstocks contain many different compounds (among which complex heteropolymeric compounds) and have varying compositions due to seasonal variations and differences in geographic origin. Typical examples of complex feedstocks that serve as complex carbon and / or nitrogen sources in fermentation are soybean meal, cottonseed meal, corn steep liquor, yeast extract, casein hydrolysate, molasses, etc.

[0042] A small amount of complex carbon and / or nitrogen source may be present in the chemically defined medium of the present invention, for example, as carryover contamination from the inoculum for the main fermentation. The inoculum for the main fermentation is not necessarily obtained by fermentation in a chemically defined medium. In most cases, carryover contamination from the inoculum can be detected by the presence of a small amount of complex nitrogen source in the chemically defined medium for the main fermentation. The use of a complex carbon and / or nitrogen source in the fermentation process of the inoculum for the main fermentation can be advantageous, for example, in accelerating biomass formation, i.e., increasing the growth rate of the microorganisms, and / or facilitating internal pH control. For the same reason, it can be advantageous to add a small amount of complex carbon and / or nitrogen source, such as yeast extract, to the early stage of the main fermentation, particularly to speed up biomass formation in the early stage of the fermentation process. The essentially small amounts of complex carbon and / or nitrogen sources that may be present in the chemically defined medium of the present invention are defined herein as amounts of up to about 10% of the total amount of carbon and / or nitrogen (Kjeldahl N) present in the chemically defined medium, preferably up to 5, 2, 1, 0.5, 0.2 or 0.1% (w / v) of the total amount of carbon and / or nitrogen.

[0043] However, in a preferred embodiment, no complex carbon and / or nitrogen sources are present in the chemically defined media of the present invention, except for an optional antifoaming agent, so long as the antifoaming agent can be used as a carbon source by the biomass strain, such as a thermophilic fungus, cultivated in the process of the present invention. The fungal strain is preferably grown in a relatively pure carbohydrate (e.g., glucose) solution and ammonia combined with an inorganic salt solution (e.g., as described in U.S. Patent Application Publication No. 2014 / 0342396). The pure carbohydrate / sugar solution allows for production with low levels of heavy metals, low toxicity substances such as herbicides, insecticides, and fungicides, and low levels of mycotoxins from the feedstock that may form during growth in soil or during storage and processing.

[0044] It will be further understood that the term "chemically defined medium" as used herein includes a medium in which all necessary components are added to the medium before the start of the fermentation process, and also includes a medium in which at least some of the necessary components are added before the start and some are added or fed to the medium during the fermentation process.

[0045] The chemically defined medium used in the process of the present invention typically contains so-called structural elements and so-called catalytic elements. Structural elements are understood to be elements that are constituents of microbial macromolecules, namely, hydrogen, oxygen, carbon, nitrogen, phosphorus, and sulfur. The structural elements hydrogen, oxygen, carbon, and nitrogen are typically contained in carbon and nitrogen sources. Phosphorus and sulfur are typically added as phosphate ions and sulfate ions and / or thiosulfate ions. The type of carbon and nitrogen sources used in the chemically defined medium is not critical to the present invention, as long as the carbon and nitrogen sources have essentially chemically defined characteristics.

[0046] In a preferred embodiment, the carbon source in the chemically defined medium is or consists of a hydrophilic carbon source, such as a carbohydrate. The inventors have found that problems with fungal pellet morphology arise when fungi are cultured at low pH in a chemically defined medium lacking hydrophobic substances such as lipids (e.g., because the carbon source is hydrophilic), and that the desired dispersed morphology can be induced by including a small amount of a hydrophobic substance, i.e., an antifoaming agent, in the medium. Therefore, the present process preferably comprises growing a microorganism, such as a thermophilic fungal strain, at a pH below 5.0 in a chemically defined medium containing at least one hydrophobic compound or hydrophobic substance. Preferably, the hydrophobic compound or substance is an antifoaming agent. Antifoaming agents are generally well known in the art (e.g., en.wikipedia.org / wiki / Defoamer(See, for example, "Antifoaming Agents" in ...

[0047] More specifically, in one embodiment, the thermophilic fungal strain is cultured in a chemically defined medium consisting of a carbon source, a nitrogen source, and additional components necessary for fungal growth, wherein the carbon source in the chemically defined medium consists of at least one of a hydrophilic carbon source and an antifoaming agent. The hydrophilic carbon source preferably comprises or consists of at least one of a carbohydrate and an organic acid. Preferably, the carbohydrate comprises at least one source of glucose, fructose, galactose, xylose, arabinose, rhamnose, fucose, galactose, and mannose, of which glucose and fructose are preferred, and glucose is most preferred. Suitable carbohydrate carbon sources, including, for example, sources of glucose and / or fructose, include, for example, maltose, isomaltose, maltodextrin, starch, glucose syrup (e.g., corn syrup such as HCFS), invert (sugarcane or sugar beet) sucrose, crude starch, starch liquefaction (e.g., liquefied with alpha amylases such as Lycozyme (Novozymes) or Veretase (BASF)), inulin, raffinose, melibiose, and stachyose. Organic acids that can be included in the carbon source include lactic acid, acetic acid, galacturonic acid, and glucuronic acid. Antifoaming agents can be used as (at least a portion of) the carbon source, for example, when the antifoaming agent contains an oil that can be utilized as a carbon source by the fungal strain, such as the vegetable oils described above.

[0048] The nitrogen source in the chemically defined medium used in the process of the present invention preferably comprises or consists of at least one of urea, ammonia, nitrate, ammonium salts such as ammonium sulfate, ammonium phosphate, and ammonium nitrate, and amino acids such as glutamic acid and lysine. More preferably, the nitrogen source is selected from the group consisting of ammonia, ammonium sulfate, and ammonium phosphate. Most preferably, the nitrogen source is ammonia. The use of ammonia as a nitrogen source has the advantage that ammonia can also function as a pH regulator. Preferably, when ammonia is used to control pH, its concentration is controlled to be 10, 20, 50, 100, 200, 500, 750, or 1000 mg / L or less. When ammonium sulfate and / or ammonium phosphate are used as nitrogen sources, some or all of the sulfur and / or phosphorus requirements of the fungal strain can be met.

[0049] Catalytic elements are elements that are components of enzymes or enzyme cofactors. These elements include, for example, magnesium, iron, copper, calcium, manganese, zinc, cobalt, molybdenum, selenium, and boron. In addition to the aforementioned structural and catalytic elements, cations such as potassium and / or sodium are preferably present to function as counterions and control intracellular pH and osmolality. Mineral compositions suitable for the chemically defined media of the present invention are described in U.S. Patent Application Publication No. 2014 / 0342396.

[0050] Compounds that may optionally be included in chemically defined media are buffers such as mono- and dipotassium phosphate, calcium carbonate, etc. Buffers are preferably added only when operating a process without external pH control.

[0051] Vitamins refer to a group of structurally unrelated organic compounds necessary for the normal metabolism of thermophilic fungi. Fungi are known to vary widely in their ability or inability to synthesize the vitamins they require. Vitamins only need to be added to the fermentation medium of fungal strains that cannot synthesize the vitamin. Typically, chemically defined fermentation media for lower fungi, such as Mucorales, may require supplementation with one or more vitamin(s). Higher fungi often do not require vitamins. Vitamins are selected from the group consisting of thiamine, riboflavin, pyridoxal, nicotinic acid or nicotinamide, pantothenic acid, cyanocobalamin, folic acid, biotin, lipoic acid, purines, pyrimidines, inositol, choline, and hemin.

[0052] In one embodiment, the thermophilic fungus grown in the process of the present invention is a strain that does not require the presence of vitamins in a chemically defined medium. The inventors have found that Rhizomucor species, such as Rhizomucor pusillus CBS143028, Rhizomucor miehei CBS143029, and Rhizopus species CBS143160, do not require vitamins even when grown in a mineral medium. Therefore, in a preferred embodiment, in step i) of the process, the biomass is derived from a thermophilic fungal strain that does not require any vitamins and is grown in a chemically defined medium without added vitamins, preferably a chemically defined medium consisting of a carbon source as defined herein above, a nitrogen source as defined herein above, and minerals as described, for example, in U.S. Patent Application Publication No. 2014 / 0342396. Further definitions of chemically defined medium are found in EP 20153414.

[0053] Biomass can also be provided by other means, for example, suitable biomass can be procured from commercial suppliers.

[0054] Step i) preferably also includes step ib) which involves treating the fermentation broth of step ia) to facilitate the subsequent pasteurization of step ii). This can be by recovering or partially recovering biomass from the fermentation, for example by concentrating the fermentation broth. In a preferred embodiment, the fermentation broth, such as that of step ia), is concentrated to reduce the energy requirements of the pasteurization. This is preferably achieved via at least one of sieving, filtration, and decantation. Preferably, the biomass of step ia) is sieved to obtain the provided biomass, more preferably using filtration via a sieve belt, a vibrating sieve, or a screen sieve.

[0055] The biomass can be concentrated by filtration, such as by rotary drum filtration, filter press, belt filter, decanter centrifuge, and / or sieving. Preferably, the biomass is recovered by sieving on a sieve or screen having a pore size of 0.1, 0.5, 1, or 2 mm. More preferably, the biomass is recovered by at least two, three, or four successive rounds of sieving on a sieve or screen, where smaller diameter pores are applied in each subsequent round of sieving. For example, an initial round of sieving using a 2 mm pore size is followed by rounds of 1, 0.5, and / or 0.1 mm. Preferably, the biomass is concentrated into a pumpable slurry, such as a pumpable slurry with a dry matter content of >10%. This advantageously allows pasteurization to be initiated in a smaller process volume. Pasteurization of such slurries can be performed in a manner that minimizes the equipment required, allowing process operators to reduce capital and operational costs and minimize the energy required for pasteurizing the biomass. As noted above, preferably, the dry matter concentration of the sieved, filtered, or decanted biomass when subjected to pasteurization is at least 7%, more preferably at least 10%. In preferred embodiments, it is about 7% to about 20%, or up to about 15%. Most preferably, it is about 9-12%. Therefore, it is preferred that the microorganisms, such as thermophilic fungi, contained in the provided biomass have good filtration properties.

[0056] In one embodiment of the process of the present invention, the filtrate containing water and enzymes produced by the microorganisms, such as fungi, can be recycled and used in the next round of fermentation. Preferably, water utilization throughout the process is minimized. Thus, preferably, in the present process, the water fraction (filtrate) obtained after sieving, filtering, decanting, and / or further pressing the biomass (cake) is recycled back into fermentation and / or (re)used in further fermentation batches. This is particularly preferred when the fermentation is carried out with low dry matter (e.g., less than 10, 5, or 2% dry matter). Preferably, at least 10, 20, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99%, or more of the filtrate from the recovery process is recycled. Preferably, substantially all of the filtrate is recycled. If too high a concentration of salts and non-consumables accumulates as a result of recycling, a portion of the filtrate may be sent to wastewater treatment and / or used for fertilizer production. Therefore, the titrant in the present process is preferably selected so that the filtrate yields a suitable fertilizer composition, preferably one or more of N, P, K, S, Mg, and Ca. Recycling the water fraction will improve the overall economics of the process by reducing wastewater treatment capacity and / or freshwater usage. Optionally, the filtrate from the first fermentation can be used in a second fermentation, allowing a second organism to consume the carbon source that the first organism cannot consume. It may also be possible to apply more than one organism in a single fermentation. Applying two or more thermophilic fungi simultaneously or sequentially to perform two or more fermentations may allow for optimization of yield, amino acid profile, taste, physical behavior, and more. In a preferred embodiment, the filtrate from a first fermentation using one or more strains of thermophilic fungi is used in a second fermentation using one or more strains of thermophilic fungi, where at least one thermophilic fungal strain in the second fermentation is different from the strain used in the first fermentation. Preferably, the strains for the first and second fermentations are selected to be complementary with respect to their biomass amino acid profile and / or ability to consume fractions of the carbon-rich feedstock.Preferably, the thermophilic fungal strains used in the first and second fermentations, and optionally in further fermentations, are selected from the thermophilic fungi and strains described above. Examples of two complementary thermophilic fungi that may subsequently be used are, for example, a strain of Thermomucor and a strain of Rasamsonia, or a strain of Thermomucor and a strain of Rhizomucor, or a strain of Rhizomucor and a strain of Rasamsonia.

[0057] Alternatively, the enzymes can be recovered after filtration and sold as enzyme preparations for use in animal feed or detergent cleaning, industrial cleaning, etc.

[0058] Another advantage of using thermophilic fungi is that the fermenter can be operated without cooling or with minimal cooling depending on the growth rate and biomass concentration (Suman et al., 2015, supra), e.g., without (active) cooling devices requiring energy input. Therefore, no internal cooling coils in the fermenter, no cooling coils in the baffles of stirred fermenters, no cooling coils in the fermenter walls, no Riesel cooling, and no cooling towers are required. No external cooling loops using heat exchangers are required. This will reduce plant or equipment investment, since cooling relies solely on CO2-vented fermenter gas exhaust and / or evaporation of water leaving the fermenter via heat passively exchanged with the fermenter environment.

[0059] Preferably, fermentation is carried out in a fermenter having means for introducing sterile air (to prevent the introduction of adventitious fungal spores or yeasts) and preferably means for controlling pH, e.g., with NH3 and / or H2SO4, HNO3, or H3PO4. In some instances, the need for phosphate may be evident, and in such cases, the use of ammonium phosphate or, optionally, H3PO4 is preferred in the process of the present invention. As known to those skilled in the art, other minerals and trace elements may be preferred in some instances as well. The fermenter used to provide the biomass can, in principle, be any type of fermenter known in the art. Advantageously, the fermenter is a simple bubble column, e.g., >100 m 3 , >200m 3 , >500m 3, >1000m 3 , >2000m 3 , or >3000m 3 etc., which reduces the number of fermenters per plant and the total investment and operating costs.

[0060] Method step ii) Pasteurization of biomass In step ii), the biomass is pasteurized to obtain pasteurized biomass. Pasteurization is a well-known technique in the art and can be carried out using commercially available equipment. Pasteurization can be carried out in a batch process or in a continuous process. Continuous process pasteurization can be referred to as in-line pasteurization, which is preferred for the method according to the present invention as it reduces the complexity of the equipment. When pasteurization is carried out in a continuous process, the method is a continuous method. In such a case, the biomass provided in step i) can be described as a biomass stream, and the pasteurized biomass can be referred to as a pasteurized biomass stream.

[0061] Thus, in a preferred embodiment, in step ii), the biomass is passed through an in-line heating unit to obtain pasteurized biomass. In-line heating units are well known and various types are commercially available, some of which are actively marketed as in-line pasteurization units or flow pasteurization devices. Such heating units can generally be set to a predetermined temperature, and the residence time of the liquid flowing through the heating unit is generally controllable. In principle, any of these in-line heating units can be used to practice the present invention, as long as it can reach the required residence time and temperature. In a preferred embodiment, the duration of pasteurization is expressed as the duration of contact with the heating unit. In a preferred embodiment, the duration of pasteurization is expressed as the duration of residence in the in-line heating unit. Preferably, the residence time in the in-line heating unit is expressed as the amount of time the biomass is approximately equal to or above the designated pasteurization temperature. In a preferred embodiment, the duration of pasteurization is expressed as the time the biomass is approximately equal to or above the designated pasteurization temperature.

[0062] While any heating unit known in the art may be used, the heating unit is preferably an in-line heating unit such as a pipe heater or a heating block. In such an in-line heater, a liquid, in this case biomass, flows through a channel and is heated therein to the desired temperature. The biomass enters the in-line heater through a first opening, is heated inside the first channel, and exits the first channel through a second opening. Heating is preferably controlled by one or more temperature sensors located within the heating unit or immediately downstream thereof, preferably within the channel transporting the pasteurized biomass. Preferably, two temperature sensors are located within the element. In the most preferred configuration, one sensor is located at or near the inlet opening and one sensor is located at or near the outlet opening.

[0063] In a preferred embodiment, a method according to the present invention is provided, wherein pasteurization is carried out in an in-line heating unit, preferably comprising a pipe heater, a heating block, or a steam injection element, more preferably a steam injection element, and the in-line heating unit optionally comprises a mixing element such as a static mixer.

[0064] In the case of a pipe heater, the in-line heating unit is configured as a pipe, typically an aluminum or stainless steel pipe, wrapped with heating wire or fitted with a jacket heated by hot water or steam. In such cases, the interior of the pipe forms the channel through which the biomass is transported. The heating wire is in intimate contact with this channel. A preferred heating unit comprises a heating wire enclosed inside an aluminum tube filled with a non-conductive medium such as magnesium oxide powder. In the case of a heating block, the in-line heating unit is configured as a block, typically a thermally conductive block, which is traversed by a biomass channel and heated throughout. This may be via integrated heating wire, transverse heating wire, or other means known in the art. Suitable in-line heating units are preferably formed from a thermally conductive material. Suitable materials are ceramics and metallic materials such as stainless steel, cast iron, copper, aluminum, brass, zinc, and their alloys.

[0065] Steam injection elements are preferred. Steam injection is widely known (Encyclopedia of Agricultural, Food, and Biological Engineering, 2nd ed., pp. 1581-1586, doi:10.1081 / E-EAFE2-120045618). The advantages of direct steam heating are a simpler design, no condensate return, and reduced wall fouling. Typically, when heating through a wall, a temperature gradient would occur from the wall to the center of the pipe. This could result in the biomass burning to the wall. This is avoided by steam injection. Preferably, when steam injection is used, it is used in combination with biomass containing at least 8%, preferably at least 10%, dry matter. The steam preferably consists essentially of water and contains no further additives; for example, preferred steam is food-grade steam or culinary steam.

[0066] As is known in the art, in an in-line heating unit, the combination of flow rate, internal channel volume, and heating will determine the effective heating of the material being heated. Thus, when biomass is pasteurized in an in-line heating unit, the channel through which the biomass flows preferably has a diameter configured to allow a specific residence time within the heating unit. Preferably, the diameter is at most 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm. Preferably, the diameter is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm. The flow velocity of the biomass is preferably at least about 0.05 m / s, more preferably at least 0.1 m / s, and even more preferably at least 0.25 m / s. In some embodiments, the flow velocity is at most 2 m / s.

[0067] Heating is conveniently expressed as the amount of time the liquid remains at a given temperature. Preferably, the indicated time does not include the time required for the liquid to equilibrate, or nearly equilibrate, with the prevailing temperature in the heating unit. For convenience, the temperature of the biomass at pasteurization conditions is referred to as the temperature of the heating unit. For batch processes, the time is generally the same as the duration of the process. For continuous processes, for example, using an in-line heating unit, the duration is generally expressed as the residence time of the liquid in the heating unit. The same residence time can be achieved with a higher flow rate if the in-line heating unit is longer. This reduces baking of the biomass to the walls of the channel.

[0068] The in-line heating unit optionally includes mixing elements such as static mixers. Such means are well known in the art (Paul, Edward L. (2004). Handbook of Industrial Mixing - Science and Practice, Hoboken, NJ: John Wiley & Sons. Page 399, Section 7-3.1.4) and are beneficial when the pipe has a larger diameter, as they ensure a more uniform distribution of heat. For thinner pipes, such as those up to 5 cm in diameter, it is preferable that there be no static mixer. Preferred static mixers are plate-type static mixers, helical static mixers, and baffles.

[0069] In step ii), the biomass has a residence time in the heating unit at a temperature of at least 70°C and up to 45 minutes. Preferably, when a temperature is indicated to be at least a given temperature, the temperature at which pasteurization is carried out is close to the given temperature, preferably within 20°C, more preferably within 10°C, and even more preferably within 5°C above the indicated temperature. This helps reduce energy requirements. When heating is indicated for at most a given time, it is preferably carried out for the indicated amount of time. This contributes to the uniformity of the process. Pasteurization for more than the indicated amount of time leads to increased energy consumption and may contribute to the development of off-flavors in the biomass. Pasteurization for substantially less than the indicated amount of time may lead to an insufficient reduction in the bacterial count. The inventors have found that these parameters lead to an adequate reduction in the bacterial count in the biomass. In a preferred embodiment, pasteurization is carried out at 74°C for 37 minutes, which results in a 12 log reduction in the bacterial spore count. 10 It was found to lead to a decrease.

[0070] In the context of the present application, one of the main goals of pasteurization is the reduction of bacterial counts in the biomass. The bacterial counts may be based on mycelium counts. The bacterial counts may also be based on spore counts. Preferably, in the context of the present application, the reduction includes spore counts in addition to mycelium counts. The reduction in bacterial counts is measured by log 10 This is conveniently expressed as a decrease in 10 The reduction means that only about 1 in 100 microorganisms survives after treatment, a 7 log 10 The reduction means that only about 1 in 10,000,000 microorganisms survives. This is the common format for reporting such data. log 10Reduction values ​​can be assessed using any method known in the art, such as dye reduction tests using methylene blue or resazurin (see, for example, Bapat et al., J Microbiol Methods. 2006 Apr;65(1):107-16, DOI: 10.1016 / j.mimet.2005.06.010). More preferably, dilution series are plated and colony forming units are counted. Heat inactivation kinetics is preferably determined as described by Casolari (2018, Microbial death, Physiological Models in Microbiology: Vol. II). The bacterial count of the pasteurized biomass should be at least 7 log lower compared to the provided biomass. 10 A reduction is preferred, as this substantially extends the shelf life of the pasteurized biomass. Typical food standards for fungi are that the bacterial count should be less than 10 fungi per gram of food. Of course, this depends on the amount of single-cell protein present in the food and the bacterial count of other ingredients that may be present in the food.

[0071] In a preferred embodiment, the bacterial count of the pasteurized biomass is at least 7, preferably at least 8, more preferably at least 9, even more preferably at least 10, even more preferably at least 11, and most preferably at least 12 log higher than the provided biomass. 10 Thus, step ii) preferably reduces this log 10 It is carried out at such a temperature or for such a period of time to achieve the reduction. Based on the examples and teachings provided herein, one of ordinary skill in the art will be able to adjust these parameters to achieve this reduction.

[0072] Another goal of pasteurization is to reduce the enzymatic activity of biomass. Enzymes can contribute to the degradation of cellular components, and degradation products can contribute to the development of off-flavors in SCP. Preferably, the method according to the present invention reduces the activity of one or more enzymes that catalyze or promote the oxidation and / or degradation of at least one of (poly)peptides and lipids, more preferably at least one enzyme selected from lipases, lipoxidases, peptidases, proteases, and aminopeptidases. These enzymes are widely known. Examples of peptidases are endopeptidases and exopeptidases. More preferably, the activity of each two of these enzymes, and most preferably each enzyme, is reduced. The reduction in activity in pasteurized biomass should be compared to the activity in non-pasteurized biomass. The reduction in total activity of the assayed enzyme is preferably at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or 100%, which may mean that no residual activity is detectable. The reduction is more preferably at least 10%, and even more preferably at least 80%. Those skilled in the art know how to assess the reduction in enzyme activity and can select an appropriate assay for any of the types of enzymes described.

[0073] A shorter duration of pasteurization or heat exposure or residence time in the heating unit can lead to improved efficiency, as it allows for a greater amount of biomass to be processed in a shorter amount of time. In the case of a batch process, it will allow for more processes to be carried out within a given time frame. Shorter heating also generally reduces energy consumption. In preferred embodiments, pasteurization is carried out for a maximum of 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 minutes. More preferably, it is carried out for a maximum of 30 minutes. Even more preferably, it is carried out for a maximum of 25 minutes. Even more preferably, it is for up to 20 minutes. Even more preferably, it is for up to 15 minutes. Even more preferably, it is for up to 10 minutes. Even more preferably, it is for up to 5 minutes. Even more preferably, it is for up to 3 minutes. Even more preferably, it is for up to 2 minutes. Most preferably, it is for up to 1 minute. In a preferred embodiment, pasteurization is carried out for up to 5 minutes, preferably about 0.5 to about 3 minutes, more preferably about 1 to 2 minutes.

[0074] A minimum duration of pasteurization can help ensure adequate bacterial reduction. In preferred embodiments, pasteurization is carried out for at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 minutes. More preferably, it is carried out for at least 1 minute. In other preferred embodiments, it is carried out for at least 2 minutes. In other preferred embodiments, it is carried out for at least 3 minutes. In other preferred embodiments, it is carried out for at least 4 minutes. In other preferred embodiments, it is carried out for at least 5 minutes. In other preferred embodiments, it is carried out for at least 6 minutes.

[0075] Pasteurization at higher temperatures can help achieve the same bacterial count reduction with shorter pasteurization durations. In preferred embodiments, pasteurization is carried out at a temperature of at least 74°C, preferably at least 80°C, and more preferably at least 86°C. In other preferred embodiments, pasteurization is carried out at a temperature of at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, or 92°C. More preferably, it is carried out at a temperature of at least 75°C, even more preferably at least 77°C, even more preferably at least 79°C, even more preferably at least 81°C, even more preferably at least 82°C, even more preferably at least 83°C, and even more preferably at least 84°C, with 85°C being even more preferred, and 86°C being even more preferred. Pasteurization at 87°C can achieve a 7 log reduction in less than 30 seconds. 10 Pasteurization at 88°C allows for a 12 log reduction in approximately 30 seconds. 10 Even reduction is possible.

[0076] In some embodiments, the pasteurization in step ii) is carried out twice, while in preferred embodiments it is carried out once.

[0077] Pasteurization is preferably carried out at a pH of less than 5, more preferably less than 4.5, even more preferably less than 4, 4.9, 4.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, or 3, and most preferably at a pH of about 3.5. Pasteurization is preferably carried out at a pH of at least 1, more preferably at least 2, even more preferably at least 2.5, 2.6, 2.7, 2.8, 2.9, or 3, and most preferably at least about 3.5. The pH of the biomass to be pasteurized is generally the pH of the biomass as provided, and can be controlled as described for step i). Preferably, the pH is not adjusted between steps i) and ii). Preferably, the pH is not adjusted as part of step ii). The pH during pasteurization is preferably substantially similar to the pH at which the fermentation is carried out when it is carried out as part of step i), or the pH at which the fermentation from which the biomass in step i) was derived was carried out.

[0078] If pasteurization is carried out for up to 45 minutes, it is preferably carried out at at least 72°C. If pasteurization is carried out for up to 40 minutes, it is preferably carried out at at least 73°C. If pasteurization is carried out for up to 35 minutes, it is preferably carried out at at least 73°C. If pasteurization is carried out for up to 30 minutes, it is preferably carried out at at least 75°C. If pasteurization is carried out for up to 25 minutes, it is preferably carried out at at least 74°C. If pasteurization is carried out for up to 20 minutes, it is preferably carried out at at least 75°C. If pasteurization is carried out for up to 15 minutes, it is preferably carried out at at least 77°C. If pasteurization is carried out for up to 10 minutes, it is preferably carried out at 8 log 10For reduction, it is preferably carried out at at least 77°C or 78°C. If pasteurization is carried out for up to 9 minutes, it is preferably carried out at at least 77°C. If pasteurization is carried out for up to 8 minutes, it is preferably carried out at at least 78°C. If pasteurization is carried out for up to 7 minutes, it is preferably carried out at at least 79°C. If pasteurization is carried out for up to 6 minutes, it is preferably carried out at at least 80°C. If pasteurization is carried out for up to 5 minutes, it is preferably carried out at at least 79°C. If pasteurization is carried out for up to 4 minutes, it is preferably carried out at at least 81°C. If pasteurization is carried out for up to 3 minutes, it is preferably carried out at least 7 log 10 For reduction, at least 81°C, 12 log 10 For reduction, it is preferable to do it at 83°C. If pasteurization is done for a maximum of 2 minutes, it is 7 log 10 For reduction, at least 82°C, 12 log 10 For reduction, it is preferable to carry out the process at 84°C. If pasteurization is carried out for a maximum of 1 minute, the reduction will be 7 log. 10 For reduction, at least 84°C, 12 log 10 In the case of reduction, it is preferable to carry out the reduction at 86°C.

[0079] If pasteurization is carried out at at least 72°C, it is preferably carried out for a maximum of 45 minutes. If pasteurization is carried out at at least 74°C, it is preferably carried out for a maximum of 40 minutes. If pasteurization is carried out at at least 76°C, it is preferably carried out for a maximum of 12 minutes (7 log 10 reduction) or up to 20 minutes (12 log 10 If pasteurization is carried out at least at 78°C, it is recommended to carry out the pasteurization for a maximum of 7 minutes (7 log reduction). 10 reduction) or up to 11 minutes (12 log 10 If pasteurization is carried out at least at 79°C, it is recommended to do so for a maximum of 5 minutes (7 log reduction). 10 reduction), or up to 8 minutes (12 log 10If pasteurization is carried out at least at 80°C, it is recommended to carry out the pasteurization for a maximum of 4 minutes (7 log reduction). 10 reduction), or up to 6 minutes (12 log 10 If pasteurization is carried out at least at 81°C, it is recommended to carry out the pasteurization for a maximum of 2.5 minutes (7 log reduction). 10 reduction), or up to 4.5 minutes (12 log 10 If pasteurization is carried out at least at 82°C, it is recommended to do so for a maximum of 2 minutes (7 log reduction). 10 reduction), or up to 3.5 minutes (12 log 10 If pasteurization is carried out at least at 83°C, it is recommended to do so for a maximum of 1.5 minutes (7 log reduction). 10 reduction), or up to 2.5 minutes (12 log 10 If pasteurization is carried out at least at 84°C, a maximum of 1 minute (7 log reduction) is recommended. 10 reduction), or up to 2 minutes (12 log 10 If pasteurization is carried out at least at 85°C, it is preferable to carry out the pasteurization for a maximum of 1.5 minutes (12 log reduction). 10 If pasteurization is carried out at least at 86°C, it is preferably carried out for a maximum of 1 minute (12 log reduction). 10 decrease).

[0080] Pasteurization at a temperature of at least 78°C was found to be associated with improved sensory quality. In this context, a temperature of 80°C was preferred, with 84°C resulting in a 7 log reduction in just 1 minute. 10 Excellent results have been obtained at 86°C for up to 12 minutes (7 log 10 reduction) or up to 20 minutes (12 log 10 It is preferable to reduce the

[0081] Pasteurization for up to 12 minutes was found to be associated with improved sensory quality. In this context, a maximum duration of 6 minutes was preferred, while a maximum of 4.5 minutes at 81°C was associated with a 12 log 10Excellent results have been obtained with pasteurization for up to about 3 minutes. In this context, a maximum of 2 minutes or even 1 minute is preferred.

[0082] Method step iii) Recovery of pasteurized biomass Step iii) is an optional step for the recovery of pasteurized biomass. This generally results in isolated SCP that can be used in further applications, preferably in food or feed products. In a preferred embodiment of the method according to the invention, step iii) is not optional.

[0083] The method for recovering the pasteurized biomass can be the same as known methods for recovering biomass from a suspension. For example, the pasteurized biomass can be further concentrated as described in step ib), for example to form a pasteurized biomass cake. Preferably, the dry matter concentration of the sieved, filtered, or decanted biomass (cake) is at least 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 52%, 53%, 54%, or 55% (w / v). Optionally, the dry matter concentration of the sieved, filtered, or decanted biomass (cake) is further increased by further removal of water, i.e., drying.

[0084] If the microorganisms used have good filtration properties (see above), protein-rich animal feed or food cake can be obtained by simple filtration such as rotary drum filtration, screen straps, filter presses, belt filters, etc., or decanter centrifuges operated at low g-forces (Suman et al., 2015, supra), sieves, DSM screens, belt sieves, belt presses, screw presses, etc.

[0085] The recovered product may be moist. The recovered product may be stabilized by maintaining a pH of 4.5 or less, preferably 3.5 or less, by adding organic acids, such as formic acid, acetic acid, benzoic acid, phosphoric acid, or sulfuric acid, optionally in combination, to prevent microbial deterioration. If the pH of the biomass provided in step i) is sufficiently low, such as below 4.5, it may be maintained within that range, for example, by reducing the intensity of dilution or washing of the recovered biomass. While liquid feeds and foods generally have lower production costs, optional drying of the feed or food, such as animal feed cake, may also be considered if transportation, logistics, and / or storage stability require drying, for example, using fluidized bed drying, vacuum drying, drum drying, belt drying, or any other drying means. In a particularly preferred process, the biomass is concentrated in multiple steps and combinations, for example, by subsequent sieving through sieves with pore sizes selected from at least two of 2 mm, 1 mm, 0.1 mm, and 50 μm; and then concentrating by at least one of squeezing using a screen strap, a screw press, a hydraulic press, and a pneumatic press. The most preferred method for concentrating the biomass may simply be a combination of a DSM screen (with an optimized diameter screen), a screen strap, and a belt press. The filtrate containing water and enzymes produced by the fungus can be recycled and used in the next round of fermentation, as described for step ib).

[0086] The recovered pasteurized biomass can be further dried, for example, by squeezing out (more) of the remaining water, using compressed air, for example, with a pneumatic press, and / or mechanical pressing, for example, with a belt press or screw press. In warm climates, the biomass (cake) can simply be air-dried (in the sun). After pressing the biomass into a cake, the cake can optionally be crushed or extruded, for example, to allow drying, preferably air-drying. Preferably, the particle size of the pressed mycelial biomass cake is reduced by physical means to allow (more efficient) drying of the pressed cake. This can optionally be done by extruding the mycelial cake through holes with a diameter of 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2 mm, using extruders known in the art. However, if 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., the cake is too hard to extrude), the particle size of the cake can be reduced by combining grinding and sieving. For the grinding step, any type of grinder known in the art, such as a knife mill or hammer mill, can be used. To obtain a uniform particle size of the ground pressed cake, large particles still present after grinding can be removed by sieving the ground cake through a sieve with a pore size of 0.5, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, or 3 mm before drying. The resulting ground cake will preferably have a particle size of 1 to 3 mm before drying. Reducing the particle size allows for more efficient and rapid evaporation of water from the pressed cake.

[0087] Preferably, the cake is dried using waste heat, for example from plants where hot water is obtained after condensation of gases (e.g., ethanol distillation, potato cooking, potato steam peeling, etc.). A heat exchanger can be used to heat the dried air with hot water from a heat source.

[0088] Drying of the extruded or milled cake is preferably carried out at temperatures between 30 and 70°C. Hot air can then be used to dry the cake in a gentle and cost-effective manner in a belt dryer or fluidized bed dryer. Steam drying at high temperatures (e.g., >80°C) is preferably avoided, as this can adversely affect protein digestibility by denaturing and baking amino acids via the Maillard reaction, as well as chemical degradation. Flash drying under vacuum is another preferred method.

[0089] Preferably, step iii), if present, also includes heat recovery techniques. The heat generated by pasteurization and remaining in the pasteurized biomass can be conveniently used to preheat provided biomass that has not yet been pasteurized or to heat the biomass in a fermenter. For example, the outlet and inlet channels of an in-line heating unit can be configured to allow heat exchange between the two channels. This can be done by routing the channels through a heat-conducting medium such as an aluminum block. Alternatively, the channels can be intertwined or arranged in a spiral around one another. Those skilled in the art will know how to configure channels to allow heat exchange. Alternatively, the outlet channel can be configured to allow heat exchange between the outlet channel and a fermenter, preferably the fermenter in which the biomass is fermented in step i). This allows the heat remaining in the pasteurized biomass to contribute to maintaining the desired fermentation temperature. A useful side effect of recovering heat from pasteurized biomass is that it can help cool the pasteurized biomass.

[0090] Thus, in a preferred embodiment, the method of the present invention comprises step iii) comprising recovering the pasteurized biomass and exchanging heat between the pasteurized biomass and non-pasteurized biomass, for example between the pasteurized biomass stream and the biomass stream provided in step i). Alternatively, heat can be exchanged between the pasteurized biomass and the fermentor from which the biomass provided in step i) originated.

[0091] Further definition of the method In a preferred embodiment, in step i) a biomass derived from the fermentation, preferably submerged fermentation, of a fungus, preferably a thermophilic fungus, preferably having 2-5% dry matter is provided, which is then preferably in step ib) sieved on a belt or vibrating sieve or DSM screen to achieve a dry matter content of 7-15%, such as about 10% dry matter, and preferably in step ii) pasteurization is carried out using an in-line heating unit, preferably comprising a steam injector.

[0092] In a preferred embodiment, in step i) a biomass derived from the fermentation, preferably submerged fermentation, of a fungus, preferably a thermophilic fungus, and preferably having 2-5% dry matter is provided, which is then preferably in step ib) sieved on a belt or vibrating sieve or DSM screen to achieve a dry matter content of 7-15%, such as about 10% dry matter, and in step ii) the biomass flows through an in-line heating unit, preferably comprising a steam injector, where it has a residence time of up to 10 minutes at a temperature of at least 76°C, preferably at least 77°C.

[0093] In a preferred embodiment, in step i), a biomass derived from the fermentation, preferably submerged fermentation, of a Rhizomucor sp., preferably a Rhizomucor pusillus strain, preferably having 2-5% dry matter is provided, which is then, preferably in step ib), sieved on a belt or vibrating sieve or DSM screen to achieve a dry matter content of 7-15%, such as about 10% dry matter, and in step ii), the biomass flows through an in-line heating unit, preferably comprising a steam injector, where it has a residence time of up to 10 minutes, preferably about 1-2 minutes, at a temperature of at least 77°C, preferably at least 80°C, most preferably about 86°C.

[0094] In a preferred embodiment, in step i), a biomass of 2-5% dry matter derived from submerged fermentation of a Rhizomucor pusillus strain is provided, which is then preferably sieved in step ib) on a belt or vibrating sieve or DSM screen to achieve a dry matter content of 7-15%, such as about 10% dry matter, and in step ii), the biomass flows through an in-line heating unit, preferably including a steam injector, where it has a residence time of about 1-2 minutes at a temperature of about 86°C.

[0095] Products obtained by this method The present invention provides pasteurized biomass obtainable by the method according to the invention, which preferably contains, on a dry weight basis, about 6% to 12% lipids and about 35% to 55% protein.

[0096] The lipid content of the pasteurized biomass is preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%. It is more preferably at least 6%, even more preferably at least 8%, and even more preferably at least about 10%. The lipid content of the pasteurized biomass is preferably at most 30, 28, 26, 24, 22, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10%. It is more preferably at most 25%, even more preferably at most 20%, more preferably still at most 15%, and even more preferably at most 12%.

[0097] The protein content of the pasteurized biomass is preferably at least 20, 25, 30, 35, 40, or 45%. It is more preferably at least 30%, even more preferably at least 35%, and even more preferably at least about 40%. The protein content of the pasteurized biomass is preferably at most 70, 65, 60, 55, 50, 45, or 40%. It is more preferably at most 65%, even more preferably at most 60%, more preferably still at most 55%, and even more preferably at most 50%.

[0098] It is highly preferred that the pasteurized biomass is based on fungal biomass and contains about 35% to 55%, preferably 40 to 50 wt-% protein, for example 45% protein, plus 8% to 12% (w / w, for example about 10 wt-%) lipids.

[0099] Lipids and proteins in pasteurized biomass may be degraded by enzymatic means, such as by degradation using endogenous lipases and / or proteases, which may contribute to the generation of unpleasant sensory experiences upon consumption, for example, due to off-flavors. The integrity of the ingredients can be further improved, for example, by using antioxidants.

[0100] The inventors have surprisingly found that biomass pasteurized by the method of the present invention provides a superior sensory experience. For example, as shown in Example 2, it was found that pasteurized biomass provides the same olfactory experience as unpasteurized biomass produced by the same method except for the pasteurization step. In contrast, biomass pasteurized at temperatures below 75°C was described by taste panels as having a metallic, iron, or oxidized odor. Therefore, preferred biomass is pasteurized at a temperature of at least 75°C, preferably at least 80°C, more preferably at least 81°C, more preferably at least 82°C, even more preferably at least 83°C, even more preferably at least 84°C, more preferably still at least 85°C, and most preferably about 86°C.

[0101] In Example 2, biomass pasteurized for more than 35 minutes was described by a taste panel as having a metallic, iron, or oxidized odor. Thus, preferred biomass is pasteurized for a maximum of 35 minutes, preferably 30 minutes, more preferably 25 minutes, and even more preferably 20, 15, or 10 minutes. In a highly preferred embodiment, pasteurization is carried out for a maximum of 10 minutes, preferably 9 minutes, more preferably 8 minutes, more preferably 7 minutes, more preferably 6 minutes, more preferably 5 minutes, more preferably 4 minutes, even more preferably 3 minutes, even more preferably 2 minutes, and most preferably about 1 minute.

[0102] For excellent aroma, excellent results can be obtained by pasteurizing the biomass at at least 78°C for up to 11 minutes, at least 79°C for up to 8 minutes, at least 80°C for up to 5 minutes, at least 81°C for up to 4 minutes, at least 82°C for up to 3 minutes, at least 83°C or 84°C for up to 2 minutes, or at 85°C or above, such as 86°C for up to 1 minute.

[0103] In Example 3, freeze-dried biomass was pasteurized under different conditions and subjected to sensory testing. All pasteurized samples had a better odor than their non-pasteurized counterparts. In one embodiment, the freeze-dried biomass is pasteurized batchwise or using an in-line steam injector. Preferably, the freeze-dried biomass is pasteurized at a temperature of 86°C or higher, preferably for 45-75 seconds, more preferably about 1 minute.

[0104] In-line pasteurization of freeze-dried biomass with an in-line steam injector has achieved superior sensory results compared to batch pasteurization. Thus, in one embodiment, freeze-dried biomass is pasteurized using an in-line steam injector. Preferably, the freeze-dried biomass is pasteurized in the in-line steam injector at a temperature of at least 86, 90, 92, 94, 96, 98, 100, 102, or 105°C, preferably for a time of up to 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0.5 seconds.

[0105] In one embodiment, the freeze-dried biomass is pasteurized using an in-line steam injector at a temperature of about 105° C. for 0.2 to 1.0 seconds, more preferably about 0.3 seconds. In a preferred embodiment, the freeze-dried biomass is pasteurized using an in-line steam injector at a temperature of about 96° C. for 1 to 5 seconds, more preferably 2 to 4 seconds, and most preferably about 3 seconds.

[0106] The present invention also provides a food or feed product comprising the pasteurized biomass according to the present invention. The pasteurized biomass can be a product in itself, optionally after undergoing inspection or certification required by local regulations. The pasteurized biomass can also be used as an ingredient in a food or feed product. When used as an ingredient, the pasteurized biomass is preferably a source of at least one of fiber or protein. In a preferred embodiment, a food product, preferably a food product for human consumption, is provided. Examples of preferred food products are meat substitutes, breakfast cereals, bread, pasta, dairy products or dairy substitutes, ice cream, chocolate, and soup. More preferred are meat substitutes and dairy substitutes. In another preferred embodiment, a feed product is provided, preferably for feeding livestock or fish, more preferably livestock.

[0107] The pasteurized biomass obtained by the process of the present invention can be used to supplement the feed of various different types of livestock animals, including pigs, poultry, ruminants, and aquatic fish and crustacean species. For fish feed applications, the feed is preferably enriched with a source of omega-3 fatty acids, such as fish oil, or lipid-rich algae, such as Cryptocodinium cohnii or Traustochytrium aureum. A further advantage of the pasteurized biomass obtained by the process of the present invention is that the acidic pH at which the SCP is produced prevents contamination of the SCP with problematic bacteria, such as E. coli, Salmonella, Bacillus cereus, Enterobacteriaceae, and Listeria, which may be present, dead or alive, in pasteurized biomass produced in other processes.

[0108] For example, the lysine content of soybeans is approximately 6% of the total amino acids, whereas the lysine content of the total amino acids of existing fungal SCPs is 8.3% for Fusarium venenatum biomass (Quorn™) and 5.6% for Pekilo protein (Paecilomyces varioti). Preferably, the sum of all essential amino acids is 16 percentage points higher than that of soybean protein (e.g., when Rasamsonia composticola is used) or even 25 percentage points higher than that of soybean protein (e.g., when Thermomucor indicae saudaticae is used). SCP derived from Thermomucor indicae saudaticae (e.g., strain CBS143027) has a lysine content of 8.5% or more, or even 10% or more, of the total amino acids, and a phenylalanine content of at least 10% of the total amino acids. Therefore, SCP derived from Thermomucor strains not only has a high protein content, but also a high lysine and phenylalanine content. Thus, Thermomucor SCP has surprisingly high nutritional value. For food products, the thermophilic fungal strain is preferably selected from the group consisting of Rathamsonia composticola strain CBS141695, Rathamsonia emersonii CBS143030, Thermomucor indicae saudaticae CBS143027, Rhizomucor miehei CBS143029, Rhizopus species CBS143160, and Rhizomucor pusillus CBS143028.

[0109] In a further aspect, the present invention relates to a pasteurized SCP product containing protein from biomass obtainable or produced by the process described hereinabove. Preferably, the pasteurized SCP product comprises or consists of dry biomass having a dry matter concentration of at least 25%, 30%, 35%, 40%, 45%, 50%, 52%, 53%, 54%, or 55% (w / v), which is preferably ground or extruded to an average particle size in the range of 1-3 mm, allowing the product to be transported for packaging or to a subsequent processing step. The protein-rich product can then be dried.

[0110] Preferably, the pasteurized SCP product according to the present invention is selected from the group consisting of Lasamsonia composticola strain CBS141695, Lasamsonia emersonii CBS143030, Thermomucor indicae seudataicae CBS143027 and CBS104.75, Rhizomucor miehei CBS143029, Rhizomucor pusillus CBS143028, Thermoascus thermophilus CBS528.71, Thielavia . The pasteurized SCP product comprises protein from the biomass of at least one thermophilic fungal strain selected from the group consisting of Streptomyces terrestris CBS546.86, Talaromyces emersonii CBS393.64, Thermotheromyces thermophila CBS117.65, and Rhizopus sp. CBS143160, of which strains CBS141695, CBS143030, CBS143027, CBS143029, CBS143160, and CBS143028 are preferred. Thus, the pasteurized SCP product can be biomass or biomass cake that has been recovered, pressed, dried, milled, and / or extruded as described hereinabove. Preferably, the pasteurized SCP product (or protein in the biomass) has a sum of total essential amino acids that is at least 10% higher than the sum of total essential amino acids in soy protein. More preferably, the pasteurized SCP product (or protein in the biomass) has at least one of a lysine content of at least 8.5% of the total amino acids and a phenylalanine content of at least 10% of the total amino acids.

[0111] In a further aspect, the present invention provides Rasamsonia composticola strain CBS141695, Rasamsonia emersonii CBS143030, Thermomucor indicae seudataicae CBS143027 and CBS104.75, Rhizomucor miehei CBS143029, Rhizomucor pusillus CBS143028, Thermoascus thermophilus CBS528.71, Thielavia terrestris CBS546.86, Talaromycetes thaliana CBS528.71, Talaromycetes thaliana CBS528.72, Talaromycetes thaliana CBS528.73, Talaromycetes thaliana CBS528.74, Talaromycetes thaliana CBS528.75 ... The present invention relates to a pasteurized food or feed product comprising proteins from the biomass of at least one thermophilic fungal strain selected from the group consisting of Rhizopus emersonii CBS393.64, Rhizopus sp. CBS143160 and Thermotheromyces thermophila CBS117.65, of which strains CBS141695, CBS143030, CBS143027, CBS143029, CBS143160 and CBS143028 are preferred.

[0112] The present invention also encompasses the use of the pasteurized biomass described above in the production of food products or as a source of at least one of fiber or protein. The use is as a source suitable for use in the production of food products. Characteristics and definitions are as described elsewhere.

[0113] General definition In this document and its claims, the verb "comprise" and its conjugations are used in an open-ended sense, meaning that the items following the word are included, but not excluding items not specifically mentioned. In addition, referring to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be only one element. Thus, the indefinite article "a" or "an" typically means "at least one." The words "about" or "approximately," when used in connection with a numerical value (e.g., about 10), mean that the value may be within a given range, preferably within 5% of that value. As used herein, a "subject" refers to any animal, preferably a mammal, and most preferably a human. In a preferred embodiment, the subject is a non-human.

[0114] In the context of the present invention, a decrease or increase in an evaluated parameter means a change of at least 5% in the value corresponding to that parameter. More preferably, a decrease or increase in value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In the latter case, there may be no detectable value associated with that parameter.

[0115] Throughout this document, when percentages are used to express amounts of substances in mixtures, weight percentages are intended unless otherwise stated or clear from the context.

[0116] The present invention has been described above with reference to several exemplary embodiments. Modifications and alternative implementations of some parts or elements are possible, and where possible, features of the embodiments can be combined. All citations to literature and patent documents are incorporated herein by reference. [Brief explanation of the drawings]

[0117] [Figure 1A]7D mortality curves for pasteurization of fungal biomass containing 10% dry matter. [Figure 1B] 12D mortality curves for pasteurization of fungal biomass containing 10% dry matter.

[0118] [Example] Example 1 - Production of pasteurized single cell protein Rhizomucor pusillus strain CBS143028 was fermented as described in WO 2018 / 029353. Alternatively, Rhizomucor pusillus CBS143028 strain, KCl 0.5gr / L; KH2PO44, Na2HPO41.1, citric acid 1.5gr / L, MgSO4.7aq 2gr / L, FeSO4.7aq 0.1gr / L, CaCl2.2aq 0.1gr / L, ZnSO4.7aq 0.125gr / L, MnCl2.4aq 0.012, CuSO4.5aq 0.0016gr / L, CoCl2.6aq 0.0015gr / L, Na2B4O7.10aq 0.009gr / L, KI 0.0009gr / L, Na2MoO4.2aq The preculture medium is inoculated into 200 ml of a pH 5.5 preculture medium containing a defined mineral composition including 0.0015 gr / L; 11 g dextrose per liter as a carbon source; 4 g (NH4)2SO4 per liter as an nitrogen source; and 7.5 g tartaric acid per liter. The culture is incubated at 46°C for 24 hours in a 1 L Erlenmeyer flask equipped with a baffled, air-permeable stop on an orbital shaker at 200 rpm. The preculture is then used to inoculate a pH 3.5 medium containing the mineral medium defined above, containing 77 g dextrose per liter as a carbon source; 1.4 g (NH4)2SO4 per liter as an nitrogen source, and supplemented with NH3 as a titrant. Olive oil is continuously supplied. Further definitions are described in EP 20153414.

[0119] The fermentation broth, which reached a dry matter content ranging from 2 to 5 weight percent, was concentrated using a vibrating sieve to achieve at least 10% (by weight) dry matter. The biomass was then pumped through an in-line heating unit containing a pipe equipped with a steam injector. The pumping speed, pipe diameter, and temperature were configured to achieve different residence times at different temperatures. Systematic screening of the parameters resulted in various degrees of log 10 Decrease, especially 7 logs 10 reduction (see Figure 1A), and 12 logs 10 This allowed the generation of a kill curve for reduction (see Figure 1B). The data are shown in the table below. Pasteurized samples were briefly dried in a fluidized bed dryer (70°C for 10 minutes).

[0120] [Table 1]

[0121] Example 2 - Sensory quality of pasteurized single cell protein The pasteurized biomass obtained as described above was subjected to analysis by a test panel. Sample 1 was pasteurized at 74°C for 37 minutes. Sample 2 was pasteurized at 86°C for 1 minute. Sample 3 was not pasteurized.

[0122] A test panel of 12 people reported the following odor sensations for Sample 1: metallic, iron, oxidation, French fries, sunflower oil, raw egg. Samples 2 and 3 were generally described as odorless. The average score given for the odor was 51% for Sample 1 and 86% for Samples 2 and 3.

[0123] Example 3 - Sensory quality of pasteurized single cell protein Biomass was produced as described in Example 1, but the freeze-dried biomass was pasteurized either batchwise (Sample 1) or with an in-line steam injector (Samples 2, 3, and 4). Samples 5 and 6 were not pasteurized, the latter being the only samples without an antioxidant. Samples (1–5) contained 2000 ppm of NaturFORT™ 15L, an antioxidant from Kemin (www.kemin.com). The temperature / residence time during pasteurization was 86°C for 1 minute for Samples 1 and 2, 96°C for 3 seconds for Sample 3, and 105°C for 0.5 seconds for Sample 4. An eight-person test panel identified the non-pasteurized sample as the most unpleasant based on odor. The batch-pasteurized sample was ranked worst among the pasteurized samples. As shown in Table 2, the sample pasteurized with an in-line steam injector at 96°C was ranked the highest, followed by the 86°C sample, and the 105°C sample was ranked third.

[0124] [Table 2]

Claims

1. 1. A method for pasteurizing biomass, comprising: i) providing biomass; ii) pasteurizing the biomass to obtain a pasteurized biomass, wherein the biomass is pasteurized at a temperature of 70-105°C for a maximum of 45 minutes; and iii) Recovering the pasteurized biomass Including, The method, wherein the biomass is biomass derived from a fungal strain of the genus Rhizomucor.

2. The method of claim 1 , wherein the biomass is derived from a fermentation broth.

3. 3. The method of claim 2, wherein the fermentation broth comprises a biomass dry matter content of up to 6% or the fermentation broth is sieved to obtain the biomass.

4. 4. The method according to any one of claims 1 to 3, wherein the pH of the biomass in step ii) is at most 4.

5.

5. 5. The method of any one of claims 1 to 4, wherein the biomass comprises at least 7% dry matter.

6. 6. The method of any one of claims 1 to 5, wherein the fungal strain is Rhizomucor pusillus or a strain which is a single colony isolate or derivative thereof.

7. The method described in claim 6, wherein the fungal strain is Rhizomucor pusillus CBS143028 or a strain that is a single colony isolate or derivative thereof.

8. The method according to any one of claims 1 to 7, wherein the pasteurization is carried out in an in-line heating unit.

9. 9. The method according to any one of claims 1 to 8, wherein the pasteurization is carried out for a maximum of 5 minutes.

10. 10. The method according to any one of claims 1 to 9, wherein the pasteurization is carried out at a temperature of at least 74°C.

11. The bacterial count of the pasteurized biomass is at least 7 log lower than that of the provided biomass. １０ The method according to any one of claims 1 to 10, wherein the method comprises reducing

12. 12. The method according to any one of claims 1 to 11, wherein in step i) a biomass derived from submerged fermentation of a Rhizomucor pusillus strain is provided, and wherein in step ii) the biomass flows through an in-line heating unit, in which the biomass has a residence time of 1 to 2 minutes at a temperature of 86°C.

13. 13. Pasteurized biomass obtainable by the method according to any one of claims 1 to 12, comprising 6% to 12% lipids and 35% to 55% protein based on dry weight.

14. 14. A food or feed product comprising the pasteurized biomass of claim 13.

15. 14. Use of the pasteurized biomass of claim 13 in the production of food products or as a source of at least one of fiber or protein for use in the production of food products.

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