Method and system for producing nutritional supplements from microbial cells

Abstract

A method for producing a nutritional supplement from microbial cells is disclosed. The method includes culturing microbial cells to obtain a biomass, incubating the biomass by heat treatment at a temperature between 55° C. and 80° C. for an incubation time between 10 and 60 minutes, and concentrating the biomass by separating and removing the liquid phase from the solid phase to obtain a dry matter content between 2% and 40% of the total weight of the nutritional supplement. A system for producing a nutritional supplement from microbial cells using the aforementioned method is also disclosed. The system includes a bioreactor, a heat exchanger, and a separator for carrying out each step of the aforementioned method.

Description

[Technical Field]

[0001] The present disclosure relates generally to bioprocessing of grown microbial mass, and more particularly to methods and systems for producing nutritional supplements from microbial cells. In this regard, the present disclosure relates to removing endotoxins from food ingredients, particularly single-cell nutritional supplements, during production, particularly during downstream processing. [Background technology]

[0002] With the current explosion of innovation in food science and technology, alternative and novel food concepts, such as alternative foods and especially alternative proteins, are being introduced. In this regard, growing microorganisms in bioreactors with minimal use of natural resources and minimal greenhouse gas emissions, and then converting the biomass into food or feed, is a promising and sustainable protein production method. Few commercial examples already exist, such as mycoprotein (fungal biomass) in the form of meat substitute (Quorn®) or various bacterial biomasses used as feed additives (e.g., Pekilo®, Uniprotein®). In these cases, the microbial biomass constitutes the majority of the food or feed, rather than being a contaminant found in small amounts. When preparing these food and feed concepts using gram-negative bacteria (e.g., E. coli and Bacteroidetes) as a sole source or as part of a larger bacterial flora, controlling endotoxin content becomes crucial.

[0003] In particular, Gram-negative bacteria are part of our environment and contribute to the human intestinal microbiota as a result of ingestion or inhalation. Gram-negative bacteria have the ability to migrate from the intestine into the bloodstream and stimulate the human immune system. Specifically, microbial endotoxins, a type of lipopolysaccharide (LPS), are responsible for the aforementioned stimulation that causes toxic effects. More specifically, when released into the blood in sufficiently large quantities, endotoxins can cause toxic effects including fever and potentially fatal septic shock, as well as low-grade inflammation, liver damage, diabetes, obesity, and cognitive impairment associated with low levels of endotoxin in the blood.

[0004] The largest source of endotoxin content in the daily diet of humans (or livestock) is food contamination with Gram-negative bacteria. Other common sources of ingested endotoxins are probiotic capsules, supplements, or other food ingredients. The majority of Gram-negative probiotics are strains of Escherichia coli (E. coli) or belong to the Enterobacteriaceae family. Normally, when food is ingested, intestinal epithelial cells act as a physical barrier by producing a mucus layer that prevents bacteria and LPS from translocating into the bloodstream. However, in cases of endotoxemia or leaky gut syndrome, bacterial translocation can occur due to mucosal degradation. Furthermore, since ingested endotoxins pose no health risk, there are no endotoxin limits set for ingested cases, which is also related to the low cell density resulting from such uptake compared to the Gram-negative bacterial levels present in the intestine (discussed in Wassenaar and Zimmerman, 2018).

[0005] Conventional techniques for preventing endotoxins from entering the body include pre-testing any food or pharmaceutical product that may enter the body for the presence of endotoxins. In this regard, pharmaceutical products, including parenteral drugs and injection devices, are tested before being released to the market. In this regard, advanced assays and sterilization techniques are employed to estimate the amount of endotoxins and destroy microbial endotoxins, respectively. However, microbial endotoxins are highly heat-stable and are not destroyed under typical sterilization conditions. Traditionally, on a laboratory scale, microbial endotoxins can be inactivated by exposure to temperatures of 250°C for more than 30 minutes, 180°C for more than 3 hours, or an acid or alkali with a strength of at least 0.1 M. Other methods for removing endotoxins from protein solutions are typically based on chromatography or detergent phase partitioning, including ion exchange chromatography, affinity adsorbents such as immobilized L-histidine, poly-L-lysine, poly(methyl L-glutamate), and polymyxin B, gel filtration chromatography, ultrafiltration, sucrose gradient centrifugation, and Triton X114 phase separation, filtration, affinity adsorbents, activated carbon, and nanoparticle-based methods. In particular, multiple cycles are required to completely remove endotoxins and render the product nonpyrogenic. These processes are utilized in the pharmaceutical industry, for example, to eliminate the risk of contamination from gram-negative bacteria in the production of antibiotics or intravenous drugs and devices.

[0006] However, food is considered different from intravenous pharmaceuticals in terms of endotoxinology (presence of LPS in the blood). Therefore, the aforementioned techniques are not applicable to food manufacturing due to the associated high costs, potential toxicity introduced by the reagents, and different purity level requirements in food and pharmaceutical applications. Furthermore, there are no known processes specifically used in the food industry that eliminate the risk of food contamination, which means keeping good manufacturing and handling practices in place.

[0007] Therefore, in light of the foregoing discussion, a need exists to overcome the drawbacks associated with conventional techniques for producing nutritional supplements from microbial cells while reducing endotoxins. Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure seeks to provide a method for producing a nutritional supplement from microbial cells. The present disclosure also seeks to provide a system for producing a nutritional supplement from microbial cells. The present disclosure seeks to provide a solution to the existing problem of efficiently reducing endotoxins in foods, functional foods, and the like. An object of the present disclosure is to provide a solution that at least partially overcomes the problems encountered in the prior art, and to provide an efficient and robust technology for producing a nutritional supplement from microbial cells that results in reduced endotoxin levels in the nutritional supplement, and thereby using the nutritional supplement for food or feed production in the form of a slurry or powder, essentially described as a heat-inactivated dry nutritional supplement. [Means for solving the problem]

[0009] In one aspect, an embodiment of the present disclosure is a method for producing a nutritional supplement from microbial cells, the method comprising: - culturing said microbial cells to obtain a biomass comprising Gram-negative bacteria; - incubating the biomass by heat treatment at a temperature of from 55°C to 80°C for an incubation time of from 10 to 60 minutes in order to at least partially decompose the walls of the microbial cells; - concentrating the incubated biomass by separating and removing the liquid phase from the solid phase to obtain a dry matter content of between 2% and 40% of the total weight of the nutritional supplement; and - homogenizing said nutritional supplement in at least one high pressure homogenization step to at least partially degrade said walls of said microbial cells.

[0010] In another aspect, an embodiment of the present disclosure is a system for producing a nutritional supplement from microbial cells, the system comprising: a bioreactor for culturing said microbial cells to obtain a biomass comprising Gram-negative bacteria; a heat exchanger for partially decomposing the walls of the microbial cells, with an incubation temperature of between 55°C and 80°C and an incubation time of between 10 and 60 minutes; a separator for separating the solid and liquid phases to obtain a dry matter content of between 2% and 40% of the total weight of the nutritional supplement; and a high-pressure homogenizer for at least partially disrupting the walls of the microbial cells of the nutritional supplement.

[0011]

[0009] Embodiments of the present disclosure substantially eliminate or at least partially address the aforementioned problems in the prior art and provide a bioprocess comprising culturing, incubating, and separating bacteria for easy use in food processing. Furthermore, the selected conditions of the aforementioned bioprocess result in a final nutritional supplement comprising Gram-negative bacteria produced from microbial cells and having reduced endotoxin levels, without adding significant operational costs to the production.

[0012] Further aspects, advantages, features and objects of the present disclosure will become apparent from the drawings and detailed description of illustrative embodiments taken in conjunction with the appended claims.

[0013] It will be appreciated that features of the present disclosure can be combined in various combinations without departing from the scope of the present disclosure as defined by the appended claims.

[0014] (Drawing summary) The foregoing summary, as well as the following detailed description of exemplary embodiments, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the disclosure, there are shown in the drawings exemplary configurations of the disclosure. However, the disclosure is not limited to the particular methods and instrumentalities disclosed herein. Moreover, those skilled in the art will appreciate that the drawings are not to scale. Wherever possible, like elements will be designated by like numerals.

[0015] Embodiments of the present disclosure will now be described, by way of example only, with reference to the following figures:

[0016] It should be noted that in the accompanying drawings, underlined numbers are used to represent the item the underlined number is located above or adjacent to. Non-underlined numbers refer to items identified by a line connecting the non-underlined number to the item. When a number is not underlined and has an associated arrow, the non-underlined number is used to identify the general item to which the arrow is pointing. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a flow chart illustrating steps in a method for producing a nutritional supplement from microbial cells, according to one embodiment of the present disclosure. [Figure 2] FIG. 2 is a flow chart illustrating steps in a method for producing a nutritional supplement from microbial cells, according to one embodiment of the present disclosure. [Figure 3] FIG. 3 is an illustration of a low-endotoxin bacterial biomass production process according to one embodiment of the present disclosure. [Figure 4] FIG. 4 is a block diagram of a system for producing a nutritional supplement from microbial cells according to one embodiment of the present disclosure. [Figure 5] FIG. 5 is a block diagram of a system for producing high moisture meat substitutes according to one embodiment of the present disclosure. [Figure 6] FIG. 6 is a schematic diagram of a process for producing a high moisture meat substitute according to one embodiment of the present disclosure. [Figure 7] 7A and 7B are LAL assay results of different samples showing intermediate endotoxin activity, according to one embodiment of the present disclosure. [Figure 8] FIG. 8 shows LAL assay results of different samples showing intermediate endotoxin activity, according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0018] The following detailed description sets forth embodiments of the present disclosure and how they can be made. While several ways of making the present disclosure are disclosed, those skilled in the art will recognize that other embodiments are possible for making or carrying out the present disclosure.

[0019] In one aspect, embodiments of the present disclosure provide a method for producing a nutritional supplement from microbial cells, the method comprising: - culturing said microbial cells to obtain a biomass comprising Gram-negative bacteria; - incubating the biomass by heat treatment at a temperature of from 55°C to 80°C for an incubation time of from 10 to 60 minutes in order to at least partially decompose the walls of the microbial cells; - concentrating the incubated biomass by separating and removing the liquid phase from the solid phase to obtain a dry matter content of between 2% and 40% of the total weight of the nutritional supplement; and - homogenizing the nutritional supplement with at least one high pressure homogenization to at least partially degrade the walls of the microbial cells.

[0020] In another aspect, embodiments of the present disclosure provide a system for producing a nutritional supplement from microbial cells, the system comprising: a bioreactor for culturing said microbial cells to obtain a biomass comprising Gram-negative bacteria; a heat exchanger for partially decomposing the walls of the microbial cells, with an incubation temperature of between 55°C and 80°C and an incubation time of between 10 and 60 minutes; a separator for separating the solid and liquid phases to obtain a dry matter content of between 2% and 40% of the total weight of the nutritional supplement; and a high-pressure homogenizer for at least partially breaking down the walls of the microbial cells of the nutritional supplement.

[0021] The present disclosure provides a method for producing a nutritional supplement from microbial cells. The disclosed method includes growing microbial cells to produce biomass and processing the biomass through downstream processing to produce a nutritional supplement for use in food or feed. The processing process for biomass containing Gram-negative bacteria includes culturing, incubation, and separation steps to effectively disrupt the microbial cell membranes and release microbial endotoxins (lipopolysaccharides) from the membranes. Advantageously, the processing process provides a concentrated nutritional supplement with significantly reduced endotoxin levels for the production of feed, food, or food ingredients. Furthermore, the disclosed method enables the growth and processing of a wide variety of cells, including, but not limited to, bacteria, fungi, protozoa, plant cells, animal cells, etc., for the production of nutritional supplements, thereby resolving issues related to the undeniable need for alternative foods, particularly alternative proteins, in the human (and livestock) diet due to the fact that the current food system is unsustainable and a major source of environmental damage. Furthermore, the selected conditions of the aforementioned method result in a final nutritional supplement produced from microbial cells with reduced endotoxin levels without adding significant operational costs to production.

[0022] As used herein throughout this disclosure, the term "nutritional supplement" refers to a nutritional supplement extracted from microbial cells. The nutritional supplement provides a concentrated source of protein that contains no or negligible amounts of carbohydrates, fat, or any other compounds. Alternatively, nutritional supplements produced from microbial cells contain protein and are fortified with compounds such as vitamins and minerals, e.g., iron. In particular, protein is a building block of the body and is typically essential for muscle building and repair. It will be understood that protein consumption should be monitored to avoid reported effects on the kidneys, liver, and bone and calcium balance of the body from excessive protein consumption over a long period of time. Typically, a healthy body should consume approximately 0.5 to 2.5 grams of protein per kilogram of body weight per day. Optionally, the nutritional supplement can be mixed with water, milk, fruit or vegetable juice, smoothies, etc. for consumption by humans or animals (including birds, fish, etc.).

[0023] A method for producing a nutritional supplement from microbial cells involves culturing the microbial cells to obtain biomass. As used herein, the term "microbial cells" refers to microorganisms that are a rich source of protein. Microorganisms typically include bacteria, archaea, fungi, protists, etc. Microbial cells are cultured under controlled conditions to build a microbial mass. As used herein, the term "biomass" refers to a measure of the amount of biological components (i.e., microorganisms) in a sample, such as a culture medium, that grow and continue to reproduce from an initial amount of microbial cells (inoculum). It can be understood that a single type of microorganism or a combination of microorganisms can be grown to obtain biomass. It can be understood that microorganisms have a shorter propagation time and can therefore be grown rapidly to produce a microbial mass with a high cell density. Furthermore, microbial cells are a rich source of protein and can therefore be utilized to produce single-cell proteins, i.e., potential alternatives to animal- or plant-derived proteins, for example, for human consumption. However, due to the small size and low density of microbial cells, large amounts of microbial mass must be produced to meet consumer protein demands. Furthermore, harvesting larger amounts of microbial mass is easier and more cost-effective than recovering single microbial cells and proteins therefrom, as this requires highly efficient microscale laboratory equipment. In other words, obtaining sufficient and viable cell densities for producing food ingredient nutritional supplements from microbial cells is important and is achieved by culturing the microbial cells under optimal growth conditions.

[0024] In particular, microorganisms have the ability to grow in different types of growth conditions, ranging from aerobic, anaerobic, and facultative conditions. Furthermore, microorganisms are grown in appropriate natural environments or artificial systems. Artificial systems are configured to mimic the natural environment suitable for a given microorganism. Typically, an inoculum (i.e., a small amount) of a microorganism, acting as a starting material, is used to grow more microorganisms under optimal growth conditions in the artificial system. Furthermore, microorganisms can be grown in a controlled environment for a defined period of time to achieve optimal growth (i.e., biomass). Optimal growth of a microorganism is associated with its biomass, or by-products of microbial growth, being subsequently recovered for subsequent use, such as in human nutrition, including proteins, lipids, carbohydrates, vitamins, minerals, fiber, etc., and in this case, for producing nutritional supplements from microbial cells.

[0025] Optionally, the artificial system is implemented as, for example, a bioreactor. The term "bioreactor" refers to a vessel intended for culturing an inoculum of microbial cells (e.g., from a stock solution), growing the microorganisms, and carrying out the biological and / or biochemical reactions necessary for the production of biomolecules that help meet the nutritional, pharmaceutical, or energy needs of consumers under defined and controlled physical and chemical conditions. The controlled conditions necessary to obtain biomass include optimal gas (carbon dioxide, hydrogen, oxygen, etc.), mineral, and liquid phase conditions. Optionally, in a bioreactor, microbial cell growth is continuous, and once a target cell density of biomass is achieved, the biomass is extracted from the bioreactor continuously or batchwise, for example, to produce a nutritional supplement from the microbial cells. In an exemplary embodiment, a biomass cultivation process is carried out in a bioreactor with microbial cells loaded into the bioreactor from a stock solution. The biomass is cultivated in a growth medium containing a carbon source, a nitrogen source, an energy source, minerals, and other specific nutrients under controlled conditions, depending on the microorganism. In a bioreactor, cell growth is continuous and the biomass exits the bioreactor once a target cell density is reached. The harvested biomass is subjected to a treatment step to reduce or remove microbial endotoxins from the biomass or the final product obtained from the treated biomass. It can be appreciated that in some applications, the final product may be only partially dewatered or not dewatered at all.

[0026] The biomass contains gram-negative bacteria. Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method. Gram-negative bacteria are part of our environment and are ingested or inhaled daily. Examples of gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Chlamydia trachomatis, and Yersinia pestis. Gram-negative bacteria contain an outer membrane that protects them from antibiotics, detergents, lysozyme, and other physical or chemical degradation methods. Additionally, the outer membrane contains complex lipopolysaccharide (LPS).

[0027] LPS is a complex molecule with a lipid region covalently linked to a polysaccharide region. It forms a dense, slightly negatively charged network on the outer membrane of the cell wall, shielding the cell from compounds and other cells that could damage the inner cell, but the layer is loose enough to allow nutrients in (reviewed in Wassenaar and Zimmerman, 2018). From a structural perspective, three distinct regions of LPS can be distinguished. The first is the lipid A, which is the only lipid portion of the molecule, and its fatty acids, like an anchor, allow the molecule to insert into the outer membrane of microbial cells. The second is the core, which is an oligosaccharide linked to the lipid portion and contains rare and specific carbohydrates. The third is the O-chain, which consists of repeating oligosaccharide units that form a long glycan chain that can be considered an O-antigenic polysaccharide. Notably, the toxicity of LPS is primarily due to lipid A, while the polysaccharide is less toxic. Although some LPS in microbial cells is endotoxin, not all LPS possesses toxic activity and therefore cannot be considered endotoxin. In particular, some fatty acids of lipid A are β-hydroxylated, which has not been described in other biomolecules and is therefore considered a specific marker of endotoxin.

[0028] Endotoxins are LPS (composed of lipid and polysaccharide regions) in the outer membrane of Gram-negative bacteria. Human pathogens (e.g., Salmonella, Neisseria, Yersinia, Bordetella), commensal bacteria (e.g., Escherichia, Bacteroides, Veillonella), and even environmental bacteria (e.g., Rhizobia, Xanthomonas, and some Pseudomonas) contain endotoxins. When released into the blood in sufficiently large quantities, endotoxins are potent stimulators of the immune system. Specifically, the lipid portion of endotoxins (i.e., lipid A) contributes to their toxic activity. It can be understood that while all endotoxins are LPS, not all LPS is endotoxin. Therefore, it is important to prevent microbial endotoxins from entering the human or animal body. In this regard, biomass is treated to reduce endotoxins therefrom in order to provide consumers with a safer and more suitable end product (i.e., a nutritional supplement from microbial cells). It will be understood that gram-positive bacteria generally do not require endotoxin removal. However, typically, when culturing gram-positive bacteria, gram-negative bacteria may also be present if a pure culture is not required. In such cases, the gram-positive bacterial culture needs to be treated to reduce microbial endotoxins originating from the gram-negative bacteria.

[0029] Optionally, the microbial cell comprises the isolated bacterial strain deposited under VTT-E-193585 or a derivative thereof. The isolated bacterial strain or its derivative is a Gram-negative bacterium that is essentially a source of microbial endotoxin. It can be understood that the isolated bacterial strain or its derivative is genetically stable and can be grown over time under a wide range of processing conditions, from optimal to stressful. As used herein, the term "genetically stable" refers to the characteristic of a species or strain / isolate that resists change and maintains its genotype over multiple generations or cell divisions (ideally hundreds to thousands of cell divisions). Optionally, the isolated bacterial strain or its derivative utilizes hydrogen gas as an energy source and carbon dioxide as a carbon source. Advantageously, the strain or its derivative contains iron and vitamin B12. Furthermore, the end product obtained from the strain or its derivative does not have a bean-off flavor and is therefore easy to flavor. Potentially, the end product also has an umami (i.e., savory or "meaty") flavor. Additionally, the final product upon further downstream processing yields a high protein nutritional supplement.

[0030] As used herein, the term "downstream processing" refers to processes following microbial cell culture in a bioreactor to obtain biomass. The downstream processing of biomass allows for the reduction or removal of microbial endotoxins by subjecting the biomass to physiological and mechanical conditions, providing a reduced-endotoxin or endotoxin-free final product that is safe for consumer, human, and animal use. Typically, downstream processing includes steps such as heat treatment (hereinafter referred to as "incubating"), hydrolysis, concentration (hereinafter referred to as "separating"), and optionally homogenization and dehydration (hereinafter referred to as "drying"). The downstream process may be understood to be a step in the method for producing a nutritional supplement from microbial cells disclosed in the present disclosure.

[0031] In this regard, the method includes obtaining biomass and then incubating the biomass by heat treatment at a temperature ranging from 55°C to 80°C for an incubation time ranging from 10 to 60 minutes. As used herein, the terms "incubating" or "incubation" refer to heat treatment carried out at a selected temperature for a selected incubation time. In other words, by incubating, the biomass is subjected to appropriate conditions, such as temperature, and optionally other complementary conditions, including, for example, humidity and atmospheric composition, to allow microbial growth and further generation of biomass. In particular, by incubating the biomass, the microorganisms are allowed to grow through different stages of their life cycle, such as the lag phase, log phase, stationary phase, and decline phase. It can be appreciated that late-logarithmic or early stationary phase biomass is then typically used for its nutritional benefits, such as, for example, producing nutritional supplements from microbial cells as disclosed herein. Advantageously, incubation at an appropriate temperature allows the microorganisms to undergo specific chemical and structural changes to enable further downstream processing. Incubation of biomass by heat treatment at temperatures ranging from 55°C to 80°C for incubation times ranging from 10 to 60 minutes results in hydrolysis of the outer membrane of the microbial cell wall structure, resulting in partial removal of LPS-containing components (e.g., endotoxins) during further processing of the biomass, e.g., during its separation stage. Incubation temperatures can be, for example, from 55, 56, 57, 58, 59, 60, 65, 70, or 75°C to 56, 57, 58, 59, 60, 65, 70, 75, or 80°C. Incubation periods can be, for example, from 10, 15, 20, 25, 30, 35, or 40 minutes to 20, 25, 30, 35, 40, 55, or 60 minutes. Furthermore, partial degradation of the outer membrane cell wall results in a final product with at least a 10- to 1000-fold lower endotoxin response. Furthermore, this temperature range stops the metabolic activity of the microorganisms, for example, this temperature range inactivates the biomass.

[0032] Optionally, the incubation temperature is from 55° C. to 70° C., or preferably from 60° C. to 68° C. The incubation temperature can be, for example, from 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67° C. to 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70° C. The incubation temperature can be, for example, from 60, 61, 62, 63, 64, 65, 66, or 67° C. to 61, 62, 63, 64, 65, 66, 67, or 68° C.

[0033] Optionally, the incubation time is from 15 to 40 minutes, or preferably from 20 to 30 minutes. The incubation period can be, for example, from 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, or 35 minutes to 20, 22, 24, 26, 28, 30, 35, or 40 minutes. The incubation period can be, for example, from 20, 22, 24, 26, or 28 minutes to 22, 24, 26, 28, or 30 minutes.

[0034] Advantageously, the aforementioned incubation of biomass at a selected temperature range and incubation time results in hydrolysis of the outer membrane structure of the cell wall, resulting in partial removal of LPS from the cell wall. Furthermore, incubation of biomass at a temperature of 60°C to 68°C and an incubation time of 20 to 30 minutes resulted in the most efficient removal of LPS from the cell wall. Optionally, the incubation of the biomass is selected to be at least one of batch incubation and continuous incubation. In particular, batch incubation involves incubating biomass in batches for a longer, predetermined incubation time, removing the total amount of incubated biomass together, i.e., all-in all-out, and then starting anew for the next incubation cycle. Continuous incubation involves shorter incubation times and routinely removing the biomass for further processing to obtain the final product, i.e., nutritional supplement, therefrom.

[0035] The method further includes concentrating the biomass by separating and removing the liquid phase from the solid phase to obtain a dry matter content of 2% to 40% of the total weight of the nutritional supplement. The dry matter content of the total weight of the nutritional supplement can be, for example, from 2, 4, 6, 8, 10, 15, 20, 25, 30, or 35% to 6, 8, 10, 15, 20, 25, 30, 35% to 40%. The higher the dry matter content, the higher the protein level in the nutritional supplement. However, if the dry matter content exceeds 40% of the total weight of the nutritional supplement, the nutritional supplement becomes less flowable and is difficult to use in food manufacturing processes. Concentrating the biomass containing Gram-negative bacteria allows the removal of excess liquid phase from the solid phase of the biomass. The liquid phase of the biomass contains hydrolyzed components of cell wall structures, including LPS-containing endotoxins. Separating and removing the liquid phase from the solid phase leaves the concentrated biomass with reduced endotoxins. During incubation, some of the LPS is released and leaks out of the cells. During separation, most of the liquid phase is removed, and the LPS present in the liquid phase is removed.

[0036] Optionally, the dry matter content is from 5% to 30%, or preferably from 8% to 25% of the total weight of the nutritional supplement. The dry matter content can be, for example, from 5, 6, 7, 8, 9, 10, 15, or 20% to 8, 10, 15, 20, or 25% of the total weight of the nutritional supplement.

[0037] Optionally, the separation is performed using a separation method selected from at least one of centrifugation and filtration. Centrifugation is a technique for separating particles from a solution, typically including solid and liquid phases, according to size, shape, density, viscosity, or the speed of the rotor used for separation. In this regard, the solution is placed in a centrifuge tube, which is then placed in a rotor and spun at a constant speed. Optionally, the centrifugation is performed at a centrifugal force ranging from 10,000 x g to 20,000 x g. Centrifugation separates approximately 90-95% of the liquid phase from the solid phase of the culture broth. Centrifugation is the most efficient and simplest method for separating the liquid and solid phases, thereby removing endotoxins present primarily in the liquid phase from the concentrated biomass. Filtration techniques typically separate the liquid and solid phases through a semipermeable membrane, allowing the liquid phase to pass through while retaining the solid phase on the semipermeable membrane. Optionally, filtration can be performed using a ceramic filter. Filtration provides the most energy efficient method of separating the liquid and solid phases, thereby removing endotoxins, which are primarily present in the liquid phase, from the concentrated biomass.

[0038] The method includes homogenizing the nutritional supplement with at least one high-pressure homogenization to at least partially degrade the walls of the microbial cells. As used herein, the term "homogenizing" refers to a means of physical disruption of microbial cell walls. It can be understood that incubating microbial cells partially disrupts the outermost membrane of the microbial cell walls, and homogenizing the biomass further disrupts the microbial cell walls. High-pressure homogenization (HPH) is the most effective technique for disrupting microbial cell walls by physical (mechanical) disruption. It can be understood that the cell wall disruption resulting from homogenizing the biomass partially degrades remaining endotoxins from the microbial cells. The high-pressure homogenization process results in partial lysis of the cells and an increase in the soluble protein content of the biomass, thereby improving the functional properties of the biomass as a food ingredient. Furthermore, homogenizing the biomass further removes endotoxins remaining after the separation process, thereby further reducing them from the biomass. Therefore, homogenization reduces the endotoxin levels in nutritional supplements made from microbial cells and containing Gram-negative bacteria, making the dietary supplement safe for human consumption.

[0039] Typically, homogenization techniques utilize fluid flow, particle-particle interactions, and pressure drop to promote cell disruption. As used herein, the term "high-pressure homogenization" refers to a physical or mechanical process in which a stream of sample, such as a biomass containing a solid phase (remaining after a separation process) and a liquid phase, is forced through a system implemented as a homogenization device (described in more detail below) that subjects the sample to multiple forces, such as any combination of high pressure or shear forces, intended to homogenize the sample and / or reduce the particle size of any components within the sample.

[0040] Optionally, the homogenization is carried out as at least one high-pressure homogenization run at a pressure of 800 bar to 2000 bar. Optionally, the homogenization pressure can be typically from 800, 1000, 1200, 1400, 1600, or 1800 bar to 1000, 1200, 1400, 1600, 1800, or 2000 bar. As used herein, the term "at least one run" refers to the number of cycles or passes the concentrated biomass undergoes to increase cell disruption efficiency. Optionally, the concentrated biomass is subjected to the above-mentioned homogenization pressure of 800 to 2000 bar once, twice, or three times. It can be understood that during the homogenization process, proteins are partially released from the microbial cells into the remaining liquid phase of the biomass (i.e., after the separation process). Advantageously, the homogenization process results in partial lysis of the cells and an increase in the soluble protein content of the biomass, thereby improving the functional properties of the biomass as a food ingredient. Additionally, homogenizing the biomass further removes and thereby further depletes the endotoxins remaining after the separation process.

[0041] Optionally, high pressure homogenization is carried out at a pressure of from 900 bar to 1000 bar. Optionally, the homogenization pressure can be typically from 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 bar to 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 bar. This range of homogenization pressure provides the best results with increased soluble protein content and reduced endotoxin levels in the homogenized biomass, with the ratio of soluble protein content compared to reduced endotoxin levels being optimal.

[0042] Optionally, the method includes drying the nutritional supplement to obtain a dry matter content of 94% to 99% of the total weight of the nutritional supplement. As used herein, the term "drying" refers to the process of drying a liquid from a source material, such as biomass. Optionally, drying the biomass is accomplished by subjecting the biomass to relatively low temperatures while rotating it in a closed system, such as a drying drum, or by rapidly drying it using hot gas. It can be appreciated that drying the biomass allows for easy and effective storage of the nutritional supplement or powder derived therefrom. Furthermore, drying the biomass protects the nutritional supplement or powder from potential infestation and thereby rendering it unsuitable for human or animal consumption. Furthermore, drying the biomass increases the dry matter content of the nutritional supplement. The dry matter content of the nutritional supplement after the drying process is typically 94, 94.5, 95, 96, 97, or 98% to 94.5, 95, 96, 97, 98, or 99%. Optionally, after the drying process, the final product is homogenized to obtain a powder form of the final product, i.e., the nutritional supplement.

[0043] Optionally, the drying is selected as at least one of drum drying or spray drying, and the drying temperature is from 120°C to 180°C. Drum drying is a process in which the nutritional supplement is rotated in a relatively low-temperature, rotating, high-volume drying drum to produce a drum-dried product sheet. The drum-dried product sheet is then pulverized into the final product, i.e., the nutritional supplement. Advantageously, drum drying is suitable for highly viscous samples that cannot be dried using other drying techniques. Spray drying utilizes a spray of hot gas (such as nitrogen or oxygen) to rapidly dry biomass. Spray drying is suitable for drying heat-sensitive samples such as food and pharmaceutical products. After the incubation, separation, and homogenization steps, the dried nutritional supplement has reduced endotoxin levels compared to samples that have not been incubated and / or homogenized. Furthermore, the nutritional supplement has endotoxin levels reduced to food-acceptable levels. Drying is typically performed at drum temperatures ranging from 120, 125, 130, 135, 140, 150, 160, or 170°C to 125, 130, 135, 140, 150, 160, 170, or 180°C. Drying at the aforementioned temperature ranges can be understood to dry out the liquid (or water) in the nutritional supplement to obtain its powder form, which is easier to store and has a longer shelf life. The aforementioned temperature ranges also kill undesirable microorganisms or pathogens that may grow with the nutritional supplement during any of the aforementioned culturing and / or downstream processing steps of the biomass. Furthermore, drying the nutritional supplement facilitates efficient comminution of the nutritional supplement to obtain a final product with a desired particle size.

[0044] Optionally, the method further includes extruding the nutritional supplement via high-moisture extrusion. As used herein, the term "extrusion" refers to a process for forming a material, such as a food product, into a product of a fixed cross-section (desired form), such as a slice, block, piece, or cube. In this regard, the material is forced through a die of the desired cross-section and subjected to compressive and shear stresses. As used herein, the term "high-moisture extrusion" refers to a thermomechanical cooking process often used to produce high-moisture meat substitutes (HMMA). Typically, HMMA is made from plant-based ingredients to mimic a meat-like texture and mouthfeel. HMMA products may exhibit, for example, a microfibrous structure resembling meat fibers. Related products are textured vegetable protein (TVP) used in vegan nuggets and burger patties, or gluten-based meat-type products. HMMA products may also be blended with other ingredients, such as spices, nutrients, and pharmaceuticals, to enhance the nutritional and flavor profiles of the HMMA product.

[0045] Furthermore, HMMA products produced from dry nutritional supplements using high moisture extrusion exhibited dramatically reduced endotoxin levels compared to the levels in the nutritional supplement itself. High moisture extrusion can reduce endotoxins in HMMA products from >4000 EU / g to <0.5 EU / g. Furthermore, high moisture extrusion of nutritional supplements resulted in HMMA products that did not exhibit an endotoxin response.

[0046] In some cases, after extrusion of the biomass, the nutritional supplement may be stored in a container such as a can or pouch packet, which is evacuated and sealed at its ends. Advantageously, proper storage prevents post-processing contamination of the product. Unhygienic processing or post-processing contamination is commonly responsible for increased endotoxin levels in fresh produce and other foods.

[0047] The present disclosure also relates to such a system, and the various embodiments and variants disclosed above apply to the system mutatis mutandis.

[0048] Optionally, the bioreactor may have a shape such as a cylinder, a cone, a rectangular parallelepiped, or a cube. Optionally, the volume of the bioreactor is, for example, 10 liters, 100 liters, 200 liters, 1000 liters, etc. Optionally, the bioreactor is fabricated from a material that is inert to the contents being processed within the bioreactor. In one example, the material of fabrication is stainless steel (e.g., type 304 ,also The bioreactor may be made of a large capacity (316 liters), other suitable metals or alloys, glass materials, fibers, ceramics, plastic materials, and / or combinations thereof. Additionally, the materials of construction are typically waterproof and strong enough to withstand the abrasive effects of various biological, biochemical, and / or mechanical processes, such as microbial concentrations, biomass production, agitation forces, aeration forces, operating pressures, temperatures, acids, alkalis, etc. Typically, the bioreactor is thick enough to support the weight of its contents and to carry out various biological, biochemical, and / or mechanical processes. Additionally, the bioreactor should preferably be such that it can withstand sterilization conditions.

[0049] Biomass incubation is typically carried out in a heat exchanger. A heat exchanger is a system user for transferring heat between two or more fluids. In this regard, the heat exchanger may have a flow configuration selected from either parallel flow or counter-current, so that the heat exchange fluids move parallel to each other or in opposite directions to each other. Heat exchangers are generally widely used on an industrial scale and are well known to those skilled in the art.

[0050] Optionally, the heat exchanger is selected to be at least one of a tank heat exchanger, a tubular heat exchanger, or a plate heat exchanger. Optionally, the tank heat exchanger is, for example, a jacketed-tank heat exchanger. A jacketed-tank heat exchanger is a heating vessel designed to heat (or cool) its contents by using a heating (or cooling) "jacket" around the heating vessel through which a heating (or cooling) fluid circulates. A tubular heat exchanger has a set of tubes containing a fluid to be heated (or cooled), and a second fluid (for heat exchange) flows over the set of tubes to provide (or absorb) heat. Tubular heat exchangers are suitable for high-pressure applications. A plate heat exchanger includes multiple heat transfer plates bundled together with a gasket arrangement, with each pair of plates providing two separate channel systems for fluid flow.

[0051] The separation of the liquid and solid phases of the biomass can be carried out in a centrifuge or a filtration unit, such as a membrane filter or a ceramic filter, which are widely used on an industrial scale. Centrifuges typically use centrifugal separation technology to separate components in a solution based on their particle size. Membrane filtration units operate on the basis of filtration technology, which utilizes semipermeable membranes to separate the solid and liquid phases of a solution. It can be seen that centrifuges are preferred for concentrating bacterial cells compared to filtration.

[0052] Homogenization devices use physical or mechanical methods to disrupt or homogenize materials. Typically, homogenization devices used include mortars and pestles, blenders, bead mills, sonicators, rotor-stators, etc. High-pressure homogenization (HPH) involves at least one pass, e.g., 1, 2, or 3 passes, through the homogenization device to increase cell disruption efficiency.

[0053] Optionally, the homogenization device is a high-pressure homogenizer, and the pressure in the high-pressure homogenizer is from 800 bar to 2000 bar, or preferably from 900 bar to 1000 bar. High-pressure homogenizers typically use very high pressure to disrupt cellular structure. Optionally, the homogenization device is a liquid mill. Liquid mills typically use shear forces to disrupt cellular structure.

[0054] The biomass is dried in a dryer. Typically, the dryer uses a direct or indirect heat supply to dry the liquid phase of the sample, here the nutritional supplement. In this regard, the dryer uses a gas stream that has been heated (or cooled, such as by freeze-drying) using various techniques known to those skilled in the art. Optionally, the dryer is selected to be at least one of a drum dryer or a spray dryer. A drum dryer is a rotary, large-capacity vessel configured to accommodate a slurry material, such as biomass, and rotate the material therein at a relatively low temperature to produce a sheet of drum-dried product. The drum-dried product may be further ground or polished into flake or powder form. Optionally, the drum dryer is a double drum dryer providing a steam pressure in the range of 2 to 7 bar. The pressure of the double drum dryer may be, for example, from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 bar to 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 bar. Spray dryers allow for rapid drying of slurry materials by using hot gases, and are typically suited to heat-sensitive materials.

[0055] Optionally, the system further comprises an extruder for extruding the nutritional supplement in a high moisture extrusion. An extruder is typically a system with a defined (or fixed) cross-section that is used to pass material through to give the material a shape or desired cross-section. Extruders usually use heat resulting from friction (between the passing material and the extruder) and the resulting pressure from friction to shape the final product that is extruded from the extruder.

[0056] Optionally, the microbial cell comprises the isolated bacterial strain deposited under VTT-E-193585 or a derivative thereof. [Example]

[0057] (Experimental results) Limulus Amebocyte Extract (LAL) Assay: The LAL assay is a calorimetric method for identifying and quantifying microbial endotoxins in samples. The LAL assay is highly sensitive and is required by Pharmacopoeias for release into the market. The LAL assay works by an enzyme cascade from horseshoe crab amebocytes, which can clot lymph in the presence of endotoxins. Based on a universal reference standard endotoxin (RSE) prepared from an Escherichia coli strain, the following value has been established: 0.2 ng LPS = 1 Endotoxin Unit [EU]. It should be understood that the LAL assay is intended only to measure activity, and its conversion to quantity is based solely on the E. coli LPS response. Furthermore, structurally distinct LPS react differently to the LAL assay. Furthermore, because the LAL assay is a colorimetric method, it cannot be used unless the material is soluble. To assay insoluble medical devices, it is necessary to follow the protocol defined by the European Pharmacopoeia. Based on the parameters defined in the regulation, the powder samples were subjected to extraction prior to LAL assay, such as by dispersion in water, stirring for a defined time, centrifugation, and filtering the supernatant. The filtrate was subjected to LAL assay to quantify endotoxin therein.

[0058] The LAL assay was used by the present applicant to analyze microbial biomass samples obtained from microbial cell cultures and nutritional supplement samples obtained through different downstream processing routes of the biomass. Regardless of the downstream processing route, all nutritional supplements exhibited toxic activity (measured as endotoxin units per gram of sample (EU / g)) higher than, for example, powdered milk, but comparable to lactobacillus tablets or spirulina powder analyzed within the same set. Table 1 below provides the results of LAL assays performed on several microbial biomass samples, showing high endotoxin activity (100,000-3,000,000 EU / g) and its reduction with downstream processing. LAL assay values ​​represent replicates of the same sample using either the kinetic colorimetric or chromogenic LAL assay. Day 1 and day 2 nutritional supplement samples were separated by 12 days, and day 2 and day 3 nutritional supplement samples were separated by 30 days. As shown, nutritional supplement samples that did not undergo high-pressure homogenization (HPH) before incubation and drying (i.e., sample V0) exhibited moderate endotoxin activity (i.e., 6000 EU / g to 15000 EU / g). However, nutritional supplement samples that were subjected to the HPH process before incubation and drying (i.e., sample V1) exhibited a clear decrease in endotoxin activity (i.e., values ​​below 50 EU / g to 200 EU / g).

[0059] [Table 1] Table 1: LAL assays performed on several microbial biomass samples

[0060] LAL assays were also performed on the separator supernatant (referred to as the separated liquid phase above). We observed that the separated liquid phase of the incubated biomass (V1) repeatedly exhibited higher EU / mL values ​​compared to the non-incubated batch (V0). This indicates that LPS detaches from the outer cell membrane during incubation and is removed along with the separated liquid phase during the separation process.

[0061] Table 2 below provides the LAL assay results for two nutritional supplement samples and their respective extrudates (obtained after high moisture extrusion).

[0062] [Table 2] Table 2: LAL assay results for nutritional supplement samples and their respective extrudates

[0063] Detailed Description of the Drawings Referring to FIG. 1 , a flowchart 100 illustrating steps of a method for producing a nutritional supplement from microbial cells according to one embodiment of the present disclosure is shown. In step 102, microbial cells are cultured to obtain a biomass. In step 104, the biomass is incubated by heat treatment at a temperature of 55° C. to 80° C. for an incubation time of 10 to 60 minutes. In step 106, the biomass is concentrated by separating and removing the liquid phase from the solid phase to obtain a dry matter content of 2% to 40% of the total weight of the nutritional supplement. In step 108, the nutritional supplement is homogenized in at least one high-pressure homogenization to at least partially decompose the walls of the microbial cells.

[0064] Steps 102, 104, 106, and 108 are exemplary only, and other alternatives may be provided in which one or more steps are added, one or more steps are deleted, or one or more steps are provided in a different order without departing from the scope of the claims.

[0065] Those skilled in the art will appreciate that Figure 1 is merely an example for clarity and should not unduly limit the scope of the claims. Those skilled in the art will recognize many variations, alternatives, and modifications of the disclosed embodiments. In one example, the process described in steps 102, 104, and 106 may follow a different sequence to arrive at the final product.

[0066] Referring to FIG. 2, a flowchart 100 illustrating steps of a method for producing a protein powder according to any embodiment of the present disclosure is shown. In step 102, microbial cells are cultured to obtain a biomass. In step 104, the biomass is incubated by heat treatment at a temperature of 55° C. to 80° C. for an incubation time of 10 to 60 minutes. In step 106, the biomass is concentrated by separating and removing the liquid phase from the solid phase to obtain a dry matter content of 2% to 40% of the total weight of the microbial cells. In step 108, the biomass is homogenized to at least partially decompose the walls of the microbial cells. In step 110, the biomass is dried to obtain a dry matter content of 94% to 99% of the total weight of the nutrient supplement.

[0067] Steps 102, 104, 106, 108, and 110 are exemplary only, and other alternatives may be provided in which one or more steps are added, one or more steps are deleted, or one or more steps are provided in a different order without departing from the scope of the claims.

[0068] Referring to FIG. 3, a diagram of a low-endotoxin bacterial biomass production process according to one embodiment of the present disclosure is shown. As shown, the microbial cell walls contain lipopolysaccharide-containing endotoxins, represented as lines protruding from the microbial cell walls. The microbial cells are cultured, for example, in a bioreactor (not shown). The microbial cells (represented as single microbial cells) are incubated with heat treatment to partially hydrolyze the outer membrane of their cell walls. The partially hydrolyzed microbial cells are then passed through a separator to separate a liquid phase of the microbial cells containing endotoxins removed from the cell walls during the partial hydrolysis. The solid phase of the microbial cells is concentrated with reduced endotoxin content due to the separation of the liquid phase containing the partially removed endotoxins. The concentrated microbial cells (or biomass) are then homogenized and then dried. Upon drying, the biomass is obtained in a powder form with endotoxin levels typically 10-1000 times lower, as measured using an assay such as the LAL assay described above.

[0069] 4, a block diagram of a system 300 for producing a nutritional supplement from microbial cells 320 is shown, according to one embodiment of the present disclosure. The system 300 includes a bioreactor 302 for culturing microbial cells 304 to obtain biomass, the bioreactor comprising a growth medium 306 and microbial cells 304 for their growth, a heat exchanger 308 for incubation at a temperature of 55° C. to 75° C. for an incubation time of 15 to 40 minutes, a separator 310 for separating a liquid phase 312 from a solid phase 314 of the biomass, a homogenization device 316 for at least partially degrading the walls of the microbial cells 304 of the biomass, and a dryer 318 for drying the biomass to obtain a powder 320 form of biomass having a dry matter content of 94% to 99%.

[0070] 5, a block diagram of a system 400 for producing a high moisture meat substitute 422 is shown according to one embodiment of the present disclosure. The system 400 comprises a bioreactor 402 for culturing microbial cells 404 to obtain biomass, the bioreactor comprising a growth medium 406 and the microbial cells 404 for their growth, a heat exchanger 408 for incubation at a temperature of from 55° C. to 75° C. for an incubation time of from 15 to 40 minutes, a separator 410 for separating a liquid phase 412 from a solid phase 414 of the biomass, a homogenization device 416 for at least partially breaking down walls of the microbial cells 404 of the biomass, a dryer 418 for drying the biomass, and an extruder 420 for extruding the biomass in a high moisture extrusion process after drying to obtain a high moisture meat substitute 422.

[0071] Referring to FIG. 6, a schematic diagram of a process for producing a high-moisture meat substitute 510 is shown, according to one embodiment of the present disclosure. As shown, a biomass sample 502 is dried in a dryer 504 and passed through an extruder 506. The extruder 506 is supplied with water from a water feed 508 to provide moisture to the dried biomass sample. The extruder 506 is configured to extrude the biomass sample through multiple barrel sections, such as sections 506A, 506B, and 506C, within the extruder 506. The extruder 506 includes a cooling die 510 at its end, configured to operate at a predetermined temperature, such as 50° C., and to cool the high-moisture meat substitute 512 that exits the extruder 506 and subsequently the cooling die 510. The high-moisture meat substitute 512 is endotoxin-free. Furthermore, the extruder 506 is operated by a motor M.

[0072] 7A, 7B, and 8, LAL assay results for different samples showing intermediate endotoxin activity are shown, according to one embodiment of the present disclosure. FIG. 6A shows the results of an LAL assay performed on a powder sample. FIG. 6B shows the results of an LAL assay performed on the separator supernatant or liquid phase containing endotoxin. The LAL assay involves a powder sample being dispersed in water, vortexed, centrifuged, the supernatant portion filtered, and mixed with an LAL reagent using a kinetic colorimetric method.

[0073] As shown in Figure 7A, endotoxin activity is reduced in the nutritional supplement samples processed by incubation at a temperature of 68°C for a 30 minute incubation time. As shown, the nutritional supplement sample that did not undergo high-pressure homogenization (HPH) before incubation and drying (i.e., sample V0) exhibited moderate endotoxin activity (i.e., 6000 EU / g to 15000 EU / g). However, the nutritional supplement sample that was subjected to the incubation and HPH process (i.e., sample V1) exhibited a clear decrease in endotoxin activity, i.e., values ​​of less than 50 EU / g to 200 EU / g.

[0074] As shown in Figure 7B, endotoxin activity is higher in separator supernatant samples incubated at temperatures ranging from 68°C to 75°C for incubation times of 20 to 35 minutes (i.e., sample V1) compared to separator supernatant samples that were not subjected to incubation (i.e., sample V0).

[0075] As shown in Figure 8, endotoxin activity, measured by the amount of endotoxin units per gram of sample (EU / g), is highest for the whole biomass, i.e., biomass that was not incubated or homogenized (i.e., V0). Furthermore, the EU / g values ​​for the non-incubated, homogenized samples (i.e., V0.5) are lower compared to the non-incubated, homogenized samples (i.e., V0). Furthermore, the samples that were subjected to incubation and homogenization (i.e., V1) have the lowest EU / g values. A clear decrease in toxic activity was repeatedly observed for the incubated and homogenized samples (V1) compared to the samples that were not incubated and homogenized (V0) or the samples that were only homogenized (V0.5).

[0076] Modifications can be made to the embodiments of the present disclosure described above without departing from the scope of the disclosure, which is defined by the claims. The terms "including," "comprising," "incorporating," "have," "is," and the like, used to describe and claim the present disclosure, are intended to be construed in a non-exclusive manner, i.e., allowing for the presence of items, components, or elements not expressly recited. References to the singular should also be construed to relate to the plural.

Claims

1. 1. A method for producing a nutritional supplement from microbial cells, the method comprising: - culturing said microbial cells to obtain a biomass comprising Gram-negative bacteria; - incubating the biomass by heat treatment at a temperature of between 55°C and 80°C for an incubation time of between 10 and 60 minutes in order to partially decompose the walls of the microbial cells; - concentrating the incubated biomass by separating and removing the liquid phase from the solid phase to obtain a dry matter content of between 2% and 40%; and - homogenizing in at least one high pressure homogenization at a pressure of between 800 and 2000 bar in order to at least partially decompose the walls of the microbial cells, The method wherein the incubating, concentrating, and homogenizing reduces endotoxin levels in the nutritional supplement.

2. The dry matter content of the nutritional supplement is: -5% to 30%, or -8% to 25%, The method of claim 1, wherein

3. The method described in claim 1 or 2, further comprising drying the nutritional supplement to obtain a dry matter content of 94% to 99% of the total weight of the microbial product.

4. The incubation temperature is -55°C to 70°C, or -60°C to 68°C, The incubation time is: -15 to 40 minutes, or 3. The method according to claim 1, wherein the heating time is from 20 minutes to 30 minutes.

5. 4. The method of claim 3, further comprising extruding the nutritional supplement by high moisture extrusion.

6. 3. The method of claim 1 or 2, wherein the homogenizing is carried out under a pressure of from 900 bar to 1000 bar.

7. 3. The method of claim 1 or 2, wherein the separating is performed using a separation method selected from at least one of centrifugation, filtration.

8. 4. The method of claim 3, wherein the drying is selected as at least one of drum drying or spray drying, and the drying temperature is from 120°C to 180°C.

9. 3. The method of claim 1 or 2, wherein the microbial cell comprises the isolated bacterial strain deposited as VTT-E-193585.

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