Method for producing meat-like food ingredients

Abstract

A method for producing a meat analog food ingredient is disclosed, which includes culturing bacterial cells to obtain a bacterial biomass, downstream processing including separation of the biomass into liquid and solid phases, concentrating the biomass by removing the liquid phase, and drying the biomass to obtain a first protein powder, mixing the first protein powder with a liquid and NaCl to obtain a powder mixture, extruding the powder mixture by high moisture extrusion, chopping the extruded mixture, and cooling the extruded mixture.

Description

[Technical Field]

[0001] The present disclosure relates generally to meat analogs and, more particularly, to methods of making meat analog food ingredients. [Background technology]

[0002] A balanced human diet should contain proteins, carbohydrates, fats, vitamins, and minerals in appropriate proportions. In the human diet, plants (e.g., soybeans) and animals (e.g., cattle, pigs, poultry, and fish) are potential sources of the above nutrients, especially high-quality protein. Currently, the demand for high-quality protein continues to grow, while the limited land available for growing plants and raising animals makes it difficult to meet the protein needs of an exponentially growing world population. Furthermore, animal-derived proteins are unattractive to a wide range of consumers who identify as vegetarian or vegan, as well as some non-vegetarians who are trying to reduce their meat consumption. The global food industry has therefore had to adapt to relatively sustainable and healthier alternatives to animal-derived meat products.

[0003] Typical alternatives to animal-derived products include meat analogs (i.e., artificial meat) produced from plants such as soybeans, corn, peanuts, etc. In this regard, plants may be grown naturally or by using three-dimensional (3D) printing technology to produce plant-based meat analogs. However, plant-based meat analogs cannot adequately mimic standard meat in terms of appearance, texture, flavor, chewiness, and juiciness. For example, it is difficult for plant-based meat analogs to achieve a fibril structure similar to meat fibers. Additionally, plant-based meat analogs typically have a bean-off flavor and are difficult to season to mimic a meat-like flavor. Additionally, production of plant-based meat analogs is highly labor-intensive. Plant-based meat analogs also lack other nutrients, such as iron and vitamins.

[0004] Recent advances in food technology have expanded the production of meat analogs using microorganisms such as yeast and algae. In this regard, techniques such as cell culture followed by extrusion processes and 3D printing technology have been used to produce microbial-based meat analogs. However, specific food-grade 3D printing equipment is not readily available and is quite expensive. Furthermore, microbial-based meat analogs, like plant-based meat analogs, lack meat-like texture and other characteristics and are not suitable for consumption by mammals, such as humans or animals, primarily due to their low digestibility. Furthermore, low digestibility may be related to low availability of nutrients from such microbial-based meat analogues. Furthermore, microbial-based meat analogs may represent the largest source of endotoxin content in the daily diet of humans (or livestock). Normally, when such meat analogs are ingested, intestinal epithelial cells produce a mucus layer that acts as a physical barrier and prevents endotoxins from passing into the bloodstream. However, in cases of endotoxemia and leaky gut syndrome, endotoxins can enter the bloodstream through the breakdown of mucous membranes, causing health risks ranging from allergic to fatal toxic reactions.

[0005] Therefore, in view of the foregoing discussion, there is a need to overcome the shortcomings associated with conventional techniques for producing meat analog food ingredients having a meat-like texture and improved digestibility. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure aims to provide a method for producing a meat analog food ingredient. The present disclosure aims to provide a solution to the existing problem of producing a meat analog food ingredient from a microorganism. It is an object of the present disclosure to provide a solution that at least partially overcomes the problems encountered in the prior art. [Means for solving the problem]

[0007] In one aspect, embodiments of the present disclosure provide a method of producing a meat analog food ingredient, the method comprising: - downstream processing, - culturing fungal cells to obtain bacterial biomass; - Separation of the biomass into liquid and solid phases; - Concentration of the biomass by removing the liquid phase, and - drying the biomass to obtain a first protein powder; and - mixing the first protein powder with a liquid and NaCl to obtain a powder mixture; - extrusion of the powder mixture by high moisture extrusion; - cutting the extruded mixture, and - cooling the extruded mixture.

[0008] Embodiments of the present disclosure substantially eliminate or at least partially address the aforementioned problems in the prior art and provide an efficient and reliable method for producing a meat analog food ingredient that mimics a meat-like texture and is digestible by mammals, e.g., humans and animals.

[0009] Additional 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.

[0010] It will be understood 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.

[0011] (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.

[0012] Embodiments of the present disclosure will now be described, by way of example only, with reference to the following figures:

[0013] 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]

[0014] [Figure 1] FIG. 1 is a flow chart illustrating steps in a method for producing a meat analog food ingredient according to one embodiment of the present disclosure. [Figure 2] FIG. 2 is a flow chart illustrating upstream and downstream processing of a meat analog food ingredient according to various embodiments of the present disclosure. [Figure 3] FIG. 3 is a flow chart illustrating upstream and downstream processing of a meat analog food ingredient according to various embodiments of the present disclosure. [Figure 4] FIG. 4 is a flow chart illustrating upstream and downstream processing of a meat analog food ingredient according to various embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] In one aspect, embodiments of the present disclosure provide a method of producing a meat analog food ingredient, the method comprising: - downstream processing, - culturing fungal cells to obtain bacterial biomass; - Separation of the biomass into liquid and solid phases; - Concentration of the biomass by removing the liquid phase, and - drying the biomass to obtain a first protein powder; and - mixing the first protein powder with a liquid and NaCl to obtain a powder mixture; - extrusion of the powder mixture by high moisture extrusion; - cutting the extruded mixture, and - cooling the extruded mixture.

[0017] The present disclosure provides a method for producing the meat analog food ingredient described above. The method of the present disclosure includes utilizing a protein powder derived from microbial biomass mixed with a liquid, salt, and spices, and producing the meat analog food ingredient using an extrusion process. Advantageously, the method is efficient and less labor-intensive. Furthermore, this method provides a healthier alternative to standard meat in terms of appearance, texture, flavor, and nutrition. Furthermore, the meat analog food ingredients produced using the above-described method are animal-free and therefore suitable for vegetarian and vegan consumers. Advantageously, the meat analog food ingredients are easily digestible by humans and animals and provide high-quality protein, iron, and vitamins such as B12.

[0018] As used herein throughout this disclosure, the term "meat analogue food ingredient" refers to a meat-like product made from animal-free products. Typically, meat analogue food ingredients are derived from, for example, plants or microorganisms. In general, meat analogue food ingredients can be used as whole foods or food ingredients that resemble meat from a particular type of animal, typically due to certain favorable properties (e.g., texture, appearance, flavor, etc.) or chemical characteristics (e.g., protein content, etc.). Meat analog ingredients can be seen as a more sustainable, healthier, and cruelty-free alternative to standard animal-derived meat, which is obtained after animals are slaughtered. Furthermore, meat analog substitutes appeal to a wide range of consumers who identify as vegetarian or vegan, as well as some non-vegetarians who are trying to reduce their meat consumption. Furthermore, the production of meat analogue ingredients has a negligible impact on global warming compared to the production of animal-derived meat, which releases large amounts of carbon dioxide into the environment.

[0019] As used herein throughout this disclosure, the term "protein powder" refers to a dietary supplement extracted from plants and / or microorganisms, e.g., in dehydrated form. Generally, protein powders provide a concentrated source of protein with no or negligible concentrations of carbohydrates, fats, and other compounds. Alternatively, protein powders may contain protein and be fortified with vitamins and minerals, e.g., calcium, iron, and other compounds. It will be appreciated that protein is essential for muscle building and recovery. Therefore, protein consumption needs to be monitored to provide the necessary amount of protein in the diet while avoiding long-term excessive protein intake, which can affect the kidneys, liver, bone and calcium balance of the body. Optionally, the protein powder can be mixed with water, milk, fruit or vegetable juice, smoothies, etc. for human or animal (including bird, fish, etc.) consumption. Optionally, the protein powder can also be used to make meat analogue food ingredients, as described below.

[0020] The method involves downstream processing, beginning with culturing bacterial cells (i.e., an inoculum) to obtain biomass. As used herein, the term "biomass" refers to a measure of the amount of living components (i.e., bacteria) in a sample. Specifically, biomass includes a solid phase (i.e., bacterial cells) and a liquid phase (growth medium). Bacterial cells may be cultivated or cultured in vessels called bioreactors under controlled conditions (e.g., temperature, humidity, pH, either aerobic, anaerobic, or facultative conditions) by gas fermentation or sugar fermentation in a medium suspension (containing a carbon source, a nitrogen source, an energy source, minerals, and other specified nutrients). Optionally, the bacterial cells are cultured by gas fermentation, and the feed comprises at least one selected from CO2, CH4, H2, O2, and NH3, and at least one mineral. In some cases, the biomass can be produced by continuous or batch culture of the bacterial cells. It can be appreciated that microorganisms have short regeneration times and can grow rapidly to produce biomass at high cell densities. Advantageously, the high cell density of the biomass is sufficient for the production of protein powders, for example, for human consumption. Furthermore, advantageously, large-scale production and harvesting of biomass is easier and more cost-effective than harvesting proteins from single bacterial cells, since it requires highly efficient microscale laboratory equipment.

[0021] The culture biomass having a high cell density can then be harvested and subjected to further processing steps, such as incubation, separation, homogenization and drying, to obtain the desired end product.

[0022] The method may also include upstream processing prior to downstream processing. Typically, upstream processing involves creating an optimal environment for microbial cells, e.g., bacterial cells, to grow and produce the desired intracellular protein. Optionally, upstream processing includes genetically engineering the microbial cells to produce high yields of desired proteins and / or other nutritional components such as antioxidants, iron, vitamins, etc. It can be understood that one or more batches of bacterial cells that produce the desired intracellular protein are selected as starting material or inoculum for their further propagation. As used herein, the term "downstream processing" refers to processes that follow the selection of bacterial cells that produce high yields of protein. Typically, downstream processing is a unit operation that facilitates the production of an end product in a manner that is useful to a consumer (human or animal). In this context, downstream processing involves subjecting bacterial cells to physiological, chemical, and mechanical conditions to provide a safe end product suitable for consumer use.

[0023] Downstream processing includes separating the liquid and solid phases of the biomass and concentrating the biomass by removing the liquid phase. Optionally, the separating is performed using a separation method selected from at least one of centrifugation and filtration. Centrifugation is a technique for separating particles, typically according to their 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. It can be seen that centrifugation is the most efficient and simplest method for separating the liquid and solid phases. Filtration techniques typically separate liquid and solid phases through a semipermeable membrane, allowing the liquid phase to pass through while retaining the solid phase on the membrane. Filtration offers the most energy-efficient method of separating liquid and solid phases. It can be seen that along with the liquid phase, hydrolyzed components of cell wall structures containing endotoxins are removed from the concentrated biomass, thus leaving an endotoxin-reduced concentrated biomass.

[0024] Downstream processing includes drying the biomass to obtain a first protein powder. As used herein, the term "drying" refers to the process of drying liquid from a feedstock such as biomass. Optionally, drying of biomass is accomplished by subjecting the biomass to a relatively low temperature while rotating the biomass in a closed system, such as a drying drum, or by rapidly drying the biomass using hot gases. Drying is typically carried out at temperatures ranging from 120, 125, 130 or 135°C to 125, 130, 135 or 140°C and at pressures ranging from 2, 2.5, 3 or 3.5 bar to 2.5, 3, 3.5 or 4 bar. Drying of biomass may typically be understood to increase the dry matter content of the biomass, for example, to a range of from 96, 96.5, 97 or 97.5% to 96.5, 97, 97.5 or 98%. Optionally, the drying is selected as at least one of drum drying or spray drying. Optionally, the dryer is selected to be at least one of a drum dryer or a spray dryer. Optionally, after the drying process, the final product is milled to obtain a powder form of the final product, i.e., protein powder. Advantageously, drying at the aforementioned temperature ranges dries out the liquid (or water) in the biomass to obtain a powder form that is easy to store. Furthermore, drying the biomass prevents the potential for pathogenic bacteria to infest the biomass and extends the shelf life of the biomass. Furthermore, drying the biomass facilitates efficient comminution of the biomass to obtain a final product with a desired particle size.

[0025] The method includes mixing a first protein powder with a liquid and NaCl to obtain a powder mixture. As used herein, the term "first protein powder" refers to a dehydrated (or powdered) form of a protein derived from a microorganism, and is therefore commonly referred to as a single-cell protein (or SCP). The dry matter content of the first protein powder is 96% to 98%. For example, the dry matter content of the protein powder ranges from 96, 96.5, 97, or 97.5% to 96.5, 97, 97.5, or 98%. It can be understood that the first protein powder typically contains edible microbial cells. Advantageously, the first protein powder is rich in protein as well as iron and vitamins (such as B12). Furthermore, liquid and NaCl (or salt) are mixed with the first protein powder to form a flavored dough therefrom. Optionally, other salts, such as KCl and monosodium glutamate (MSG), may also be included. If desired, in addition to the liquid and NaCl, spices and preservatives may be mixed with the first protein to simulate a meat-like flavor. It may be understood that the first protein powder, liquid, NaCl, and other additives are all used in accordance with good manufacturing practices.

[0026] Optionally, downstream processing further comprises heat-treating and incubating the biomass at a temperature of from 55° C. to 75° C. for a time period of from 15 to 40 minutes. In particular, heat-treating and incubating the biomass promotes certain chemical and structural changes in the bacterial cells. Specifically, incubation promotes cell wall breakdown, releasing endotoxins that can be harmful to humans if they migrate from the intestine into the bloodstream. Optionally, an incubation is carried out before the separation step. The incubation temperature can be, for example, from 55, 56, 57, 58, 59, 60, 65, or 70° C. to 56, 57, 58, 59, 60, 65, 70, or 75° C., and the incubation period can be, for example, from 15, 20, 25, 30, or 35 minutes to 20, 25, 30, 35, or 40 minutes. 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. Advantageously, degradation of the outer cell wall results in a final product with at least a 10- to 1000-fold lower endotoxin response as a result of incubation of the biomass. Furthermore, incubation at the aforementioned temperature ranges prevents the growth of undesirable microorganisms, resulting in a pure culture of only the desired bacteria.

[0027] Optionally, downstream processing further comprises homogenizing the bacterial cells of the biomass prior to the drying step. In particular, homogenization at least partially breaks down the cell walls of the bacterial cells. As used herein, the term "homogenization" refers to a means of physically disrupting bacterial cell walls. It can be understood that incubating the bacterial cells partially disrupts their cell walls, and homogenizing the biomass further disrupts the cell walls. Typically, homogenization utilizes fluid flow, particle-particle interactions, and pressure drop to facilitate cell disruption. Advantageously, homogenization partially lyses the bacterial cells and increases the soluble protein content of the biomass, thereby improving the functional properties of the biomass as a food ingredient. Typically, homogenization devices used include mortars and pestles, blenders, bead mills, sonicators, rotor-stators, etc. Additionally, homogenizing the biomass further removes endotoxins remaining in the concentrated biomass, thereby further reducing endotoxins from the homogenized biomass.

[0028] Optionally, homogenization may be carried out using high pressure homogenization (HPH) or grinding techniques. As used herein, the term "high pressure homogenization" refers to a physical or mechanical process in which a stream of a sample, such as a concentrated biomass, is forced through a high pressure homogenizer to homogenize the sample and / or reduce the particle size of all components within the sample. Typically, the high-pressure homogenizer applies multiple forces to the sample, such as any combination of high pressure or shear forces. Optionally, the homogenization is carried out at a pressure of from 800 bar to 2000 bar in at least one run. The homogenization pressure may be, for example, 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 (e.g., one, two, or three) that the concentrated biomass undergoes to increase cell disruption efficiency. Preferably, homogenization is carried out at 700 bar to 1000 bar. The homogenization pressure may be, for example, from 700, 750, 800, 850, 900, or 950 bar to 750, 800, 850, 900, 950, or 1000 bar. Even more preferably, homogenization is carried out at 900 bar. Advantageously, the above range of homogenization pressure provides the best results of increasing soluble protein content and reducing endotoxin levels in the homogenized biomass.

[0029] Optionally, downstream processing further comprises filtering the homogenized bacterial cells of the biomass by at least one selected from nanofiltration or ultrafiltration. After homogenization, a biomass slurry is obtained. The biomass slurry is filtered by ultrafiltration to remove cellular debris and by nanofiltration to concentrate the protein content in the biomass slurry. Alternatively, precipitation of a homogenized biomass slurry can be used to increase the protein content in the biomass slurry. A filtration step can be performed after homogenization to disrupt the bacterial cells of the biomass. If the cells are not disrupted, there is nothing to filter from the biomass. Filtration by nanofiltration or ultrafiltration can increase the protein content in the biomass. Beneficially, the higher protein content of the biomass improves the texture of the meat analog food ingredient during the extrusion step, making the meat analog food ingredient fibrous and more meat-like in texture.

[0030] The term "milling" as used herein refers to a mechanical method for breaking down larger particle size components into smaller, e.g., nano-sized particles. Milling is performed by a grinding means that exerts shear forces that break down larger size particles into smaller size particles. Optionally, milling techniques include liquid milling (i.e., bead milling or ball milling) and ultrasonication. Bead mill homogenization techniques utilize beads in a mill homogenization device and rapid agitation to mill and homogenize the sample. It can be appreciated that the disruption of cell walls resulting from homogenization of biomass removes residual endotoxins from bacterial cells. Advantageously, the soluble protein content of the biomass is increased by milling and homogenization. Furthermore, milling also reduces endotoxin levels within the biomass.

[0031] Optionally, the method further comprises adjusting the pH of the biomass to 7.4 to 8.5 after at least one step selected from liquid phase separation or homogenization. The pH can be, for example, from 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9 to 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. It can be appreciated that an appropriate pH is an essential element for a growth medium to promote bacterial growth. In this regard, acids and bases can be added to the concentrated biomass to adjust its pH. Optionally, the pH adjuster can be selected from potassium hydroxide (KOH) or calcium hydroxide (Ca(OH)2). Furthermore, it can be appreciated that if the pH is lower than 7.0, the extruded product will not provide a meat-like texture upon extrusion.

[0032] Optionally, the liquid is selected to be at least one of water and a protein slurry. For example, mixing the first protein powder with water results in a dough. Optionally, the water is double-distilled water. As used herein, the term "protein slurry" refers to a fluid comprising a solid phase essentially consisting of protein and a liquid phase. Optionally, in addition to protein, the solid phase of the protein slurry includes carbohydrates, fat, dietary fiber, ash, etc. Optionally, the protein slurry includes 90% to 95% water, 1% to 10% of a third protein powder. The amount of water in the protein slurry may be, for example, from 90, 91, 92, 93, or 94% to 91, 92, 93, 94, or 95% of the total amount of protein slurry, and the third protein powder may be from 1, 2, 3, 4, 5, 6, 7, 8, or 9% to 2, 3, 4, 5, 6, 7, 8, 9, or 10%. In one example, the protein slurry has a 6% solid phase and a 94% liquid phase. In one example, the protein slurry contains 5% solid phase and 95% water, with the solid phase containing 65% protein, 10% fat, and 25% minerals and fiber. It can be seen that best results are obtained within the aforementioned ranges. Furthermore, protein slurries containing more water tend to be too watery and fail to achieve a meat-like texture in the final product. Also, if too much protein powder is present, the protein slurry may be too thick for processing.

[0033] Optionally, the third protein powder contains another protein in powder form. The third protein powder may differ from the first protein powder in terms of its structural and / or functional characteristics. For example, the third protein powder may be an enzyme required for the first protein powder to function properly.

[0034] Optionally, at least one selected from the first protein powder and the third protein powder comprises the isolated bacterial strain deposited under VTT-E-193585 or a derivative thereof. The isolated bacterial strain or a derivative thereof is typically a Gram-negative bacterium (i.e., does not retain crystal violet staining used in the Gram staining method). It can be understood that the isolated bacterial strain or a derivative thereof is genetically stable and can grow over a long period of time under a wide range of process conditions, from optimal to stressful conditions. As used herein, the term "genetically stable" refers to the characteristic of a species or strain / isolate to resist change and maintain its genotype over multiple generations or cell divisions, ideally over hundreds to thousands of cell divisions. Optionally, the isolated bacterial strain or a derivative thereof utilizes hydrogen gas as an energy source and carbon dioxide as a carbon source. Advantageously, the strain or a derivative thereof contains iron and vitamin B12. Furthermore, the final product obtained from said strain or its derivatives does not have the off-flavor of bean and is therefore easy to flavor, possibly including an umami (i.e., savory or "meaty") flavor in the final product.

[0035] Optionally, the powder mixture is mixed in a mixer selected from at least one of a preconditioner, a flour mixer, and a twin-screw extruder. It can be understood that the first protein powder, liquid, and NaCl are mixed to obtain a homogeneous mixture thereof. Furthermore, mixing the aforementioned ingredients ensures high moisture retention by the first protein powder, allowing for a softer and more consistent final product. A preconditioner is a mixer that heats, hydrates, and homogenously mixes dry ingredients to produce a pre-treated product for further processing, e.g., extrusion. Flour mixers can be used to mix large quantities of dough. Flour mixers are standard kitchen appliances used to knead dough, e.g., using flour. A twin-screw extruder is typically a system with a defined (or fixed) cross-section used to pass material through and impart a shape or desired cross-section to the final product that emerges from the extruder. In this regard, the extruder uses friction (between the material passing through and the extruder) and heat from the resulting pressure to shape the final product. Twin-screw extruders typically consist of two co-rotating screws positioned on shafts within a sealed, fixed barrel. Specifically, twin-screw extruders are well-suited for extruding highly viscous and rigid mixtures while mixing them.

[0036] The method further includes extruding the powder mixture by high moisture extrusion. As used herein, the term "extrusion" refers to a process in which a material, such as a food product, is formed into a product of a defined cross-section (desired shape), such as a slice, block, piece, cube, or the like. In this regard, the material is forced through a die of desired cross section (i.e., a perforated plate designed to produce the required shape) connected to a given extruder, where it is 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 products such as high moisture meat analogs (HMMA). Typically, high moisture extrusion processes facilitate continuous mixing, kneading, and shaping of the extruded material. In this regard, high moisture extrusion is carried out using a high moisture extruder that utilizes barrel heating and screw shear (such as the standard twin screw extruder described above) to produce HMMA. Alternatively, the powder mixture can be extruded using dry extrusion.

[0037] HMMA typically has a moisture content of about 40% to 70% and mimics the texture and mouthfeel of meat. Furthermore, HMMA may exhibit a fibril structure similar to, for example, meat fibers. Advantageously, HMMA provides a much improved fibrous and textured meat analog compared to conventional textured vegetable protein (TVP) produced using a low-moisture extrusion process. Additionally, HMMA can be blended with other ingredients, such as spices, nutrients, and pharmaceuticals, to enhance the nutritional content and flavor of the HMMA. Furthermore, HMMA produced from dried biomass using high-moisture extrusion has demonstrated dramatically lower endotoxin levels compared to the endotoxin levels in the powder itself. High-moisture extrusion typically reduces endotoxins in HMMA from >4000 EU / g to <0.5 EU / g. Furthermore, high-moisture extrusion of protein powders produced without downstream processing operations resulted in HMMA that exhibited no endotoxin response.

[0038] Optionally, high moisture extrusion is carried out using the following parameters: - Torque from 1.0Nm to 1.3Nm, - Die pressure from 15 bar up to 18 bar, - Die temperature of 140℃~160℃, and - Melting temperature of 135℃~155℃.

[0039] In this regard, the term "torque" as used herein typically refers to the rotational (i.e., twisting) force between the shafts (on which the screw is loaded) that cause the mating shafts to co-rotate during the extrusion process. Optionally, the torque may be, for example, from 1.0, 1.1, or 1.2 Nm (abbreviation for Newton meters) to 1.1, 1.2, or 1.3 Nm. As used herein, the term "die pressure" refers to the pressure typically generated at the front end of the extruder coupled to the die. The die pressure may be, for example, from 15, 16, or 17 bar to 16, 17, or 18 bar. As used herein, the term "die temperature" typically refers to the temperature at the first end of the die as a result of the die pressure. The die temperature may be, for example, from 140, 145, 150, or 155°C to 145, 150, 155, or 160°C. As used herein, the term "melting temperature" typically refers to the temperature at which a product begins to melt. The melting temperature usually increases with mold pressure. The melting temperature may be, for example, from 135, 140, 145, or 150°C to 140, 145, 150, or 155°C. In one example, high moisture extrusion is carried out for at least 5 minutes at a torque of 1.2 Nm, a die pressure of 14 bar, a die temperature of 160°C, and a melt temperature of 151°C. In particular, high die temperatures and high melt temperatures produce fibrous HMMA. It can be appreciated that the aforementioned conditions can be controlled based on the desired product to ensure uniformity of the final product.

[0040] Even more advantageously, high moisture extrusion takes an input powder mixture and extrudes a final product (i.e., HMMA) that has a meat-like texture, additional nutrients as a result of the addition of supplemental protein and / or nutrients, and / or higher digestibility as a result of the addition of soluble fiber. It will be appreciated that the steps of the methods for producing meat analogs disclosed hereinabove can be modified (by addition or omission) to produce final products of different qualities. For example, the incubation and homogenization steps can be omitted to produce HMMA that exhibits higher human digestibility. Furthermore, in the above example, the addition of soluble fiber further enhances the desirability of the meat analog.

[0041] It can be seen that extrusion disrupts the cell walls of microorganisms, improving digestibility. Optionally, extruding a powder mixture has been shown to improve the digestibility-indispensable amino acid score (DIAAS). The change in DIAAS was improved from 0.22 to 0.79, as shown in Table 1. As shown, the non-incubated, non-homogenized powder mix has a reduced digestibility as described by a DIAAS of 0.22 and a protein digestibility of 39, while the extrudate shows an improved digestibility as described by a DIAAS of 0.79 and a protein digestibility of 69. According to FAO recommendations, a DIAAS below 0.75 can be considered to be of poor protein quality.

[0042] [Table 1] Table 1. Protein digestibility scores

[0043] Optionally, the method further comprises adding at least one selected from at least one second protein powder and at least one soluble fiber to the powder mixture before mixing. As used herein, the term "second protein powder" refers to, for example, a dehydrated (or powdered) protein derived from a plant. It can be understood that the second protein powder typically comprises an edible plant isolate. The second protein powder is generally intended to provide a comprehensive protein column that is not derived solely from microbial proteins. Additionally, the addition of a second protein powder improves the binding properties of the powder mixture, ensuring uniformity of the final product.

[0044] Optionally, the at least one second protein powder is selected to be at least one of pea isolate powder, wheat gluten powder, vital wheat gluten powder, soy protein concentrate powder, and soy isolate powder. It can be appreciated that the structure of the final product is improved by using at least one of the aforementioned second protein powders. Furthermore, the at least one second protein powder can further supplement the final product with protein, minerals such as iron, elasticity, etc.

[0045] Optionally, the total weight of the powder mixture is - 20% to 40% of the first protein powder, - at least one second protein powder, up to 20% to 40%; - 20% to 40% water, - 0.5% to 1.5% NaCl, and - Contains at least one soluble fiber, up to 2% to 4%.

[0046] In this regard, optionally, each of the first protein powder and the second protein powder may be, for example, 20, 25, 30, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 23 5-pronged 35% to 2 5-pronged Optionally, the first protein powder and the second protein powder together comprise 64.0 to 64.5% of the powder mixture. Optionally, water may be added, for example, in an amount of 20, 2% of the powder mixture. 5-pronged 35% to 2 5-prongedOptionally, NaCl may be, for example, from 0.5, 0.7, 0.9, 1.1 or 1.3% to 0.7, 0.9, 1.1, 1.3 or 1.5%, preferably from 0.5, 0.6, 0.7, 0.8 or 0.9 to 0.6, 0.7, 0.8%, 0.9 or 1.0% of the powder mixture. Optionally, the at least one soluble fiber may be, for example, from 4.0, 4.5, 5.0, or 5.5% to 4.5, 5.0, 5.5, or 6.0% of the powder mixture, preferably 5%. Optionally, the at least one soluble fiber is pectin. More optionally, the pectin is obtained from apples, citrus fruits, vegetables, and the like. It may be appreciated that the use of pectin provides a better structure to the final product.

[0047] The method further includes cutting the extruded mixture. The extruded mixture is recovered by cutting it into smaller blocks or pieces, whereby each cut can be achieved by a single movement of a cutter along a direction perpendicular to a die located at the end of the extruder. It can be seen that a single cut clearly removes a block of extruded mixture for further use. Advantageously, making the cut along a cross section perpendicular to the die ensures that the quality and structure of the extruded mixture so produced are preserved. The block of extruded mixture can then be recovered and layered to produce the desired product, i.e., HMMA.

[0048] The method further includes cooling the extruded mixture. It will be appreciated that the extruded mixture is inherently at an elevated temperature, e.g., above room temperature. Therefore, to extend the shelf life of the extruded mixture, the extruded mixture is cooled by any suitable method known to those skilled in the art.

[0049] Optionally, the cooled extruded mixture is stored in a container such as a sealed vacuum-ended can or pouch packet. Advantageously, storing the extruded mixture in a cool place prevents oxidation and infiltration of the extruded mixture.

[0050] Optionally, the method further comprises freezing the extruded mixture. Optionally, the extruded mixture is frozen at below -9.5°C. Advantageously, freezing the extruded mixture further extends the shelf life of the product. Additionally, freezing the extruded mixture prevents the extrusion of the extruded mixture by various pathogens.

[0051] Detailed Description of the Drawings Referring to Figure 1, a flow chart 100 illustrating steps in a method for producing a meat analog food ingredient according to one embodiment of the present disclosure is shown. Downstream processing 102 includes producing a first protein powder. In step 104, bacterial cells are cultured to obtain a biomass. In step 106, the liquid and solid phases of the biomass are separated. In step 108, the biomass is concentrated by removing the liquid phase. In step 110, the biomass is dried to obtain a first protein powder.

[0052] In step 112, a first protein powder is mixed with a liquid and NaCl to obtain a powder mixture. In step 114, the powder mixture is extruded by high moisture extrusion. In step 116, the extruded mixture is cut. In step 118, the extruded mixture is cooled.

[0053] Steps 102, 104, 106, 108, 110, 112, 114, 116 and 118 are merely exemplary, 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.

[0054] Those skilled in the art will appreciate that FIG. 1 is merely an example for clarity, and should not unduly limit the scope of the claims. One of ordinary skill in the art would recognize many variations, alternatives, and modifications of the disclosed embodiments. In one example, the process described in steps 102, 104, 106, and 108 may be performed according to different orders to provide the final product, i.e., the meat analog food ingredient.

[0055] Referring to Figures 2, 3 and 4, there are shown flow charts 200, 300 and 400, respectively, illustrating upstream and downstream processing of a meat analog food ingredient (hereinafter referred to as high moisture meat analog (HMMA)) according to various embodiments of the present disclosure. 2 and 3, bacterial cells are subjected to bioreactor cultivation. In bioreactor cultivation, the bacterial cells must be supplied with carbon dioxide gas, oxygen gas, hydrogen gas, and a growth medium. In addition, oxygen gas and hydrogen gas can be obtained by electrolyzing water using electricity. This water is further used to prepare a growth medium further comprising ammonium hydroxide, macronutrients and micronutrients, and is then sterilized. It can be understood that bioreactor cultivation is carried out under predetermined conditions that promote bacterial cell growth and have a high cell density of biomass. It can be understood that during bioreactor cultivation, water gas and excess oxygen gas are released to the atmosphere or recycled as needed.

[0056] After achieving a desired high cell density of the biomass during bioreactor cultivation, the biomass is subjected to heat treatment (i.e., incubation), for example, in a heat exchanger. The incubated biomass is then concentrated by separating the liquid phase from the solid phase of the biomass. The separated liquid phase, or supernatant, is purified to produce water and reuse it to remove endotoxins therein. The solid phase of the biomass, or cell slurry, is subjected to homogenization. The homogenized biomass is subjected to drum drying, and the water vapor removed during the drying process is directed to its purification. The flour product obtained from drum drying is mixed with other ingredients, such as liquid, NaCl, at least one second protein powder, and soluble fiber, during extrusion premixing. In this case, the powder mixture to obtain a total of 300 g of protein powder contains 192 g (64%) of the first and second protein powders, 90 g (30%) of water, 3 g (1%) of NaCl, and 15 g (3%) of at least one soluble fiber derived from apple or citrus fruits. The flour mixture obtained from extrusion premixing is subjected to high-moisture extrusion (or wet extrusion) to obtain the extruded product, high-moisture meat analog (or HMMA). The HMMA is cut into blocks or small pieces. The HMMA can be optionally mixed with seasonings and cooled or frozen to extend its shelf life.

[0057] Also, as shown in Figure 3, the cell slurry is mixed with a pH adjuster such as potassium hydroxide (KOH) or calcium hydroxide (Ca(OH)2) to adjust the pH of the cell slurry to a range of 7.4 to 8.0. It can be seen that if the pH value is lower than 7.0, the extruded product cannot provide a meat-like texture upon extrusion. Furthermore, the extrusion premixing consists of mixing only the powdered product with NaCl.

[0058] Furthermore, after biomass is harvested from the bioreactor culture, the cell slurry is directly drum-dried without heat treatment and omitting homogenization, as shown in Figure 4. In this case, the powder mixture contains 120 g (i.e., 70%) of the first protein powder and the second protein powder, and 51 g (i.e., 30%) of water, resulting in a total of 171 g of protein powder. Furthermore, extrusion premixing consists of mixing only the powder product with water.

[0059] 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 meat analog food ingredient, the method comprising: - downstream processing, - culturing the bacterial cells to obtain the bacterial biomass; - incubating the biomass at a temperature between 55°C and 75°C for a time between 15 and 40 minutes; - Separation of the liquid and solid phases of the biomass; - adjusting the pH of the biomass to between 7.4 and 8.5; - Concentration of the biomass by removing the liquid phase; homogenizing the bacterial cells of the biomass, and - drying the biomass to obtain a first protein powder; - mixing the first protein powder with water and NaCl to obtain a powder mixture; - extrusion of the powder mixture carried out using the following parameters: - torque from 1.0 Nm to 1.3 Nm, - die pressure from 15 bar up to 18 bar, - a die temperature of 140°C to 160°C, and a melting temperature of 135°C to 155°C, - cutting the extruded mixture, and - cooling the extruded mixture, wherein the steps are carried out in the above order.

2. 10. The method of claim 1, further comprising freezing the extruded mixture.

3. 3. The method of claim 1 or 2, further comprising adding at least one selected from at least one second protein powder and at least one soluble fiber to the powder mixture prior to mixing.

4. The total weight of the powder mixture may include: - 20% to 40% of a first protein powder, - 20% to 40% of at least one second protein powder, - 20% to 40% water, - 0.5% to 1.5% NaCl, and 3. The method according to claim 1 or 2, comprising from 2% to 4% of at least one soluble fiber.

5. 4. The method of claim 3, wherein the at least one second protein powder is selected to be at least one of pea isolate powder, wheat gluten powder, vital wheat gluten powder, soy protein concentrate powder, soy isolate powder.

6. 3. The method of claim 1 or 2, wherein the powder mixture is mixed in a mixer selected from at least one of a preconditioner, a flour mixer, and a twin-screw extruder.

7. The method of claim 1 or 2, wherein the water is contained in a protein slurry.

8. 8. The method of claim 7, wherein the protein slurry comprises 90% to 95% water and 1% to 10% of a third protein powder.

9. 3. The method of claim 1 or 2, wherein at least one selected from the first protein powder and the third protein powder comprises the isolated bacterial strain deposited as VTT-E-193585 or a derivative thereof.

10. 10. The method of claim 1, wherein the homogenization is carried out at a pressure of from 800 bar to 2000 bar, or from 700 bar to 1000 bar in at least one run.

11. 10. The method of claim 1, further comprising filtering the homogenized bacterial cells of the biomass by at least one selected from nanofiltration or ultrafiltration.

12. 10. The method of claim 1, further comprising adjusting the pH of the biomass to between 7.4 and 8.5 after the liquid phase homogenization.

13. The bacterial cells are cultivated by gas fermentation and the feed is CO ２ , C.H. ４ , H ２ , O ２ , N.H. ３ 10. The method of claim 1, further comprising at least one mineral selected from the group consisting of:

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