Ethanol clostridium protein-based microbial protein meat, preparation method thereof and application of ethanol clostridium protein

Abstract

The invention provides ethanol clostridium protein-based microbial protein meat, a preparation method thereof and application of ethanol clostridium protein. The preparation method of the ethanol clostridium protein-based microbial protein meat comprises the following steps: S1, carrying out wall breaking treatment on ethanol clostridium protein by adopting enzyme to obtain wall-broken ethanol clostridium protein; s2, adding the wall-broken ethanol clostridium protein into a twin-screw extruder for extrusion processing, and controlling the moisture content in the extrusion processing process of the ethanol clostridium protein; the extrusion temperature of the twin-screw extruder is 150-200 DEG C; the conditioning water content is 40%-65%; the rotating speed of screws of the double-screw extruder is 120-200 rpm; the feeding speed is 20 to 60 g / min. According to the method, the tissue meat with high fibrosis degree is prepared from the clostridium ethanolicum protein, the taste of the product is more appropriate, and the tissue meat containing the clostridium ethanolicum protein is high in protein content, stable in quality and balanced in amino acid content.

Description

Technical Field

[0001] This invention belongs to the field of protein meat preparation technology, specifically relating to a Clostridium ethanol protein-based microbial protein meat, its preparation method, and the application of Clostridium ethanol protein. Background Technology

[0002] Tissue meat, also known as protein meat, is a food product made from plant proteins, microbial proteins, etc., through physical or chemical processing methods to resemble the fibrous structure and texture of real meat. Tissue meat processing is a complex and delicate process aimed at simulating the texture, taste, and nutritional components of animal meat using scientific and technological means. Currently, the main raw materials include agricultural crops rich in plant protein such as soybeans, peas, wheat, and peanuts. Tissue meat processing primarily utilizes high-moisture extrusion technology, which involves uniformly mixing pre-treated plant protein raw materials, auxiliary materials, and water, controlling the stirring speed and temperature to ensure thorough integration and the formation of a homogeneous material system, before feeding it into a twin-screw extruder. Inside the extruder, under the action of high temperature, high pressure, and mechanical shearing, the proteins denature and aggregate and cross-link, forming a fibrous network structure. While high-moisture extrusion technology, as the main processing technology for tissue meat, can create a fibrous structure similar to meat, its complexity and realism still need improvement compared to the fibrous structure of real animal meat. This may result in tissue meat not being able to completely simulate the chewiness and texture of real animal meat.

[0003] Currently, the plant protein raw materials used in high-moisture extrusion research and application are relatively limited, and single plant proteins suffer from problems such as limiting amino acids and incomplete nutritional components. Chinese patent document CN118285453A discloses a type of textured meat containing yeast protein and its preparation method. Using soybean protein and yeast protein as the main raw materials, it produces a meat analogue that can be processed into Chinese and Western dishes as a substitute for animal meat. The textured meat has a reasonable amino acid composition, elasticity, chewiness, and meat-like texture, and is easily digestible. This patent utilizes yeast protein. Clostridium ethanolica protein is an innovative and environmentally friendly new single-cell protein product. It uses Clostridium autoethanogenum as the fermentation strain and utilizes gases such as carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (H2) through liquid fermentation, centrifugal separation and concentration, and spray drying to artificially synthesize Clostridium ethanolica protein. The crude protein content of Clostridium ethanolica protein is as high as 80% or more, and the content of impurities such as crude ash, moisture, and ammonium salts is controlled at low levels. Its amino acid structure is balanced and rich in functional substances such as nucleotides, making it widely applicable. Currently, Clostridium ethanolaceum protein is mainly used in animal feed. Due to its high protein content and excellent nutritional value, it has become an important feed protein source. In terms of food innovation and diversity, the development of Clostridium ethanolaceum protein offers opportunities for such innovation. By designing the structure and function of the protein, foods with specific taste, texture, and nutritional characteristics can be created.

[0004] However, using Clostridium ethanolae protein alone to prepare textured meat presents challenges. As a single-cell protein, Clostridium ethanolae protein has low solubility and gelling properties due to the presence of its cell wall, resulting in weaker interactions between protein molecules and hindering the formation of fibrous structures during processing. Therefore, compared to plant proteins, it suffers from poor fibrosis and inferior texture in textured meat. Furthermore, compared to plant proteins, Clostridium ethanolae protein has lower viscosity due to the presence of its cell wall, which is detrimental to its application in extrusion processing. Protein viscosity significantly impacts the fibrous structure of high-moisture extruded products. In addition, the tough cell walls of bacterial proteins make them difficult for the human body to directly digest and absorb. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a Clostridium ethanol protein-based microbial protein meat, its preparation method and the application of Clostridium ethanol protein. Using Clostridium ethanol protein to prepare tissue meat with high fibrous degree, the product has a more suitable taste. The tissue meat of Clostridium ethanol protein has high protein content, stable quality and balanced amino acid content.

[0006] To address the above problems, the first aspect of this invention provides a method for preparing Clostridium ethanol-based microbial protein meat, comprising the following steps:

[0007] S1. The Clostridium ethanol protein was subjected to enzyme-mediated cell disruption to obtain the disrupted Clostridium ethanol protein.

[0008] S2. The cell-wall-broken Clostridium ethanolis protein is added to a twin-screw extruder for extrusion processing, and the moisture content of the Clostridium ethanolis protein is controlled during the extrusion process; the extrusion temperature of the twin-screw extruder is 150-200℃; the conditioning moisture content is 40%-65%; the screw speed of the twin-screw extruder is 120-200 rpm; and the feed rate is 20-60 g / min.

[0009] Preferably, in step S1, lysozyme and chitinase are used to break down the cell walls of Clostridium ethanol.

[0010] Preferably, step S1 specifically includes the following steps:

[0011] S101. Add ethanol Clostridium protein to the first alkaline solution, then add lysozyme and chitinase, mix, and obtain the first suspension;

[0012] S102. Freeze the first suspension into blocks to obtain frozen blocks;

[0013] S103. The frozen block is crushed to obtain small particles, which are then added to a heated second alkaline solution and mixed to obtain a second suspension;

[0014] S104. Homogenize the second suspension;

[0015] S105. The homogenized second suspension is subjected to alcohol removal treatment to obtain a concentrated suspension;

[0016] S106. The concentrated suspension is dried to obtain ethanol Clostridium protein with broken cell walls.

[0017] Preferably, the first alkaline solution is an aqueous solution of sodium bicarbonate; the second alkaline solution is an aqueous solution of sodium bicarbonate.

[0018] Preferably, the extrusion temperature of the twin-screw extruder is 170–190°C; the moisture content of the tempered material is 40%–50%; the screw speed of the twin-screw extruder is 120–200 rpm; and the feed rate is 30–50 g / min.

[0019] Preferably, the extrusion temperature of the twin-screw extruder is 179.11℃; the conditioned moisture content is 47.52%; the screw speed of the twin-screw extruder is 160 rpm; and the feed rate is 36.15 g / min.

[0020] Preferably, step S2 specifically includes the following steps:

[0021] S201. Mix Clostridium ethanolis protein with water to obtain the first mixed raw material;

[0022] S202. Knead and sieve the mixed raw materials;

[0023] S203. Further mix the sieved raw materials to obtain a second mixed raw material;

[0024] S204. Determine the moisture content of the second mixed raw material;

[0025] S205. The second mixed raw material is added to a twin-screw extruder for extrusion processing. The amount of water to be added is calculated based on the target conditioned moisture content and the measured moisture content in the second mixed raw material. Water is added at the front end of the twin-screw extruder according to the required amount of water to be added, thereby controlling the moisture content during the extrusion process.

[0026] Preferably, the temperature of the twin-screw extruder is 40-60℃ in zone one, 80-100℃ in zone two, 120-140℃ in zone three, 160-180℃ in zone four, 160-180℃ in zone five, and the cooling temperature is 40-60℃.

[0027] A second aspect of the present invention provides a Clostridium ethanol-based microbial protein meat obtained by the above-described preparation method.

[0028] A third aspect of the present invention provides the application of Clostridium ethanolis protein in the preparation of meat protein.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1. The method for preparing Clostridium ethanol-based microbial protein meat of the present invention uses Clostridium ethanol as the single raw material for the protein meat. The crude protein content of Clostridium ethanol is as high as 80% or more, and the contents of impurities such as crude ash, moisture, and ammonium salts are all controlled at low levels. Its amino acid structure is balanced, rich in functional substances such as nucleotides, and has high protein content and excellent nutritional value. In addition, compared with traditional animal husbandry, microbial protein production does not require a large amount of land, water resources, and feed, thereby reducing resource consumption and waste, and also reducing greenhouse gas emissions. It is both efficient and environmentally friendly, and has high sustainability.

[0031] 2. The method for preparing Clostridium ethanolis protein-based microbial protein meat of the present invention firstly involves breaking down the cell walls of Clostridium ethanolis protein. This cell wall breaking process improves the texture and mouthfeel of the meat by altering the water absorption, water solubility, protein dispersion index, modulus, and viscosity of the Clostridium ethanolis protein, making it softer and more delicate, thus enhancing the taste and flavor of the food. Furthermore, the nutrients in the cell wall-broken microbial protein are more easily absorbed by the human body, which helps to improve the nutritional value of the food.

[0032] 3. The method for preparing Clostridium ethanol-based microbial meat of the present invention further optimizes the extrusion temperature, conditioning moisture content, screw speed, and feed rate during extrusion processing. Temperature and pressure during extrusion are key factors affecting the denaturation of microbial proteins and the formation of fibrous structures. Moisture content has a significant impact on the denaturation of plant proteins and the formation of fibrous structures during extrusion processing. The screw configuration and speed have a significant impact on the material conveying and mixing effect. Selecting an appropriate screw speed can improve production efficiency and product quality. By precisely controlling the extrusion temperature, conditioning moisture content, screw speed, and feed rate, the present invention can control the texture and taste of plant-based meat products, and prepare high-fiber meat with more meat-like characteristics and a more suitable taste. Attached Figure Description

[0033] Figure 1 The figures represent the water absorption, water solubility, and protein dispersion index of Clostridium ethanolica protein before and after cell wall disruption treatment in Example 20 of this invention, where WAI is the water absorption index, WSI is the water solubility index, and PDI is the protein dispersion index.

[0034] Figure 2 The storage modulus (a), loss modulus (b), and loss angle (c) of Clostridium ethanolis protein before and after cell wall disruption treatment in Example 20 of this invention are:

[0035] Figure 3 In Embodiment 20 of the present invention, a) is the effect of shear rate on the viscosity characteristics of Clostridium ethanol before and after cell wall disruption treatment; b) is the effect of temperature on the viscosity characteristics of Clostridium ethanol before and after cell wall disruption treatment.

[0036] Figure 4 The images show the torn surface, appearance, and cross-section of the products obtained in Examples 1-6, where A, B, C, and D are the torn surfaces of the products, and E and F are the appearance and cross-section of the products.

[0037] Figure 5 The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 3 and 7-11, where A, B, C, and D are the torn surfaces of the products, and E and F are the appearance and cross-section of the products.

[0038] Figure 6 The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 3, 12-15, where A, B, C, and D are the torn surfaces of the products, and E and F are the appearance and cross-section of the products.

[0039] Figure 7 The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 3, 16-19, where A, B, C, and D are the torn surfaces of the products, and E and F are the appearance and cross-section of the products.

[0040] Figure 8 Figure a is a comparison chart of transverse shear force, longitudinal shear force, and degree of fiberization of the products obtained in Examples 1-6; Figure b is a comparison chart of transverse shear force, longitudinal shear force, and degree of fiberization of the products obtained in Examples 3 and 7-11; Figure c is a comparison chart of transverse shear force, longitudinal shear force, and degree of fiberization of the products obtained in Examples 3 and 12-15; Figure d is a comparison chart of transverse shear force, longitudinal shear force, and degree of fiberization of the products obtained in Examples 3 and 16-19.

[0041] Figure 9 In Figure a, the textural properties of the products obtained in Examples 1-6 are compared; in Figure b, the textural properties of the products obtained in Examples 3 and 7-11 are compared; in Figure c, the textural properties of the products obtained in Examples 3 and 12-15 are compared; and in Figure d, the textural properties of the products obtained in Examples 3 and 16-19 are compared.

[0042] Figure 10 This is the response surface processing parameter optimization diagram in Embodiment 27 of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] The first aspect of this invention provides a method for preparing Clostridium ethanol-based microbial protein meat, comprising the following steps:

[0045] S1. The Clostridium ethanol protein was subjected to enzyme-mediated cell disruption to obtain the disrupted Clostridium ethanol protein.

[0046] S2. The cell-wall-broken Clostridium ethanolis protein is added to a twin-screw extruder for extrusion processing, and the moisture content of the Clostridium ethanolis protein is controlled during the extrusion process; the extrusion temperature of the twin-screw extruder is 150-200℃; the conditioning moisture content is 40%-65%; the screw speed of the twin-screw extruder is 120-200 rpm; and the feed rate is 20-60 g / min.

[0047] The method for preparing Clostridium ethanol-based microbial protein meat according to this invention uses Clostridium ethanol as the single raw material for the protein meat. The crude protein content of Clostridium ethanol is as high as 80% or more, and the contents of impurities such as crude ash, moisture, and ammonium salts are all controlled at low levels. Its amino acid structure is balanced, rich in functional substances such as nucleotides, and it has high protein content and excellent nutritional value. Furthermore, compared with traditional animal husbandry, microbial protein production does not require large amounts of land, water resources, and feed, thereby reducing resource consumption and waste, and also reducing greenhouse gas emissions. It is both efficient and environmentally friendly, and has high sustainability.

[0048] Using only Clostridium ethanolis protein to prepare textured meat results in low fibrousness, poor texture, and the tough cell walls of the microbial protein make it difficult for the human body to directly digest and absorb. The method for preparing Clostridium ethanolis protein-based microbial protein meat according to this invention first involves cell wall disruption of the Clostridium ethanolis protein. Cell wall disruption alters the water absorption, water solubility, protein dispersion index, modulus, and viscosity of the Clostridium ethanolis protein, improving the texture and texture of the meat, making it softer and more delicate, and enhancing the taste and flavor of the food. Furthermore, the nutrients in the cell protein after cell wall disruption are more easily absorbed by the human body, contributing to improved nutritional value of the food.

[0049] The method for preparing Clostridium ethanol-based microbial meat according to this invention further optimizes the extrusion temperature, conditioning moisture content, screw speed, and feed rate during extrusion processing. Temperature and pressure during extrusion are key factors affecting the denaturation of microbial proteins and the formation of fibrous structures. Moisture content has a significant impact on the denaturation of plant proteins and the formation of fibrous structures during extrusion processing. The screw configuration and speed have a significant impact on the material conveying and mixing effect. Selecting an appropriate screw speed can improve production efficiency and product quality. By precisely controlling the extrusion temperature, conditioning moisture content, screw speed, and feed rate, this invention can control the texture and taste of plant-based meat products, preparing high-fiber meat with more meat-like characteristics and a more suitable taste.

[0050] Preferably, in step S1, lysozyme and chitinase are used to break down the cell walls of Clostridium ethanol.

[0051] Preferably, step S1 specifically includes the following steps:

[0052] S101. Add ethanol Clostridium protein to the first alkaline solution, then add lysozyme and chitinase, mix, and obtain the first suspension;

[0053] S102. Freeze the first suspension into blocks to obtain frozen blocks;

[0054] S103. The frozen block is crushed to obtain small particles, which are then added to a heated second alkaline solution and mixed to obtain a second suspension;

[0055] S104. Homogenize the second suspension;

[0056] S105. The homogenized second suspension is subjected to alcohol removal treatment to obtain a concentrated suspension;

[0057] S106. The concentrated suspension is dried to obtain ethanol Clostridium protein with broken cell walls.

[0058] This invention utilizes enzymatic hydrolysis to disrupt the cell wall of Clostridium ethanolis protein. However, during the inactivation process, the Clostridium ethanolis protein agglomerates, hindering sufficient contact with enzymes. Therefore, this invention first adds the Clostridium ethanolis protein to an alkaline solution. In alkaline solutions, especially at pH values ​​above the isoelectric point, the surface hydrophobicity index of the Clostridium ethanolis protein decreases, increasing its solubility. This is because, at pH conditions far from the isoelectric point, the net surface charge of the Clostridium ethanolis protein increases, leading to increased protein-water interactions and thus improved solubility, facilitating sufficient contact with enzymes. Furthermore, simple enzymatic hydrolysis is insufficient for cell wall disruption. Therefore, this invention further enhances the efficiency of cell wall disruption through mechanical pressure. During freezing, water in the Clostridium ethanolis protein crystallizes to form ice crystals. The formation and expansion of these ice crystals exert mechanical pressure on the protein molecules, damaging the protein wall structure and initiating cell wall disruption. The protein is then pulverized into small particles, which facilitates rapid contact with the hot alkaline solution in the subsequent reaction, resulting in a more complete cell wall disruption. In addition, the present invention also performs a final alcohol removal process. Since some alcohols may remain in the ethanol clostridium protein during production, which may affect its odor, the alcohol removal process is performed to remove the residual alcohols.

[0059] Preferably, the first alkaline solution is an aqueous solution of sodium bicarbonate; the second alkaline solution is an aqueous solution of sodium bicarbonate.

[0060] Preferably, step S101 specifically includes the following steps:

[0061] Prepare an aqueous solution of sodium bicarbonate with a molar concentration of 1.1–1.3 mol / L; mix Clostridium ethanolis protein with the aqueous solution of sodium bicarbonate to prepare a Clostridium ethanolis protein-sodium bicarbonate suspension with a water content of 75%–85%; add lysozyme and chitinase to the Clostridium ethanolis protein-sodium bicarbonate suspension so that the suspension contains 0.3–0.8 g / L of lysozyme and 0.3–0.8 g / L of chitinase, and mix uniformly in a horizontal shaker at 30–40°C for 1–3 h to obtain the first suspension.

[0062] More preferably, step S101 specifically includes the following steps:

[0063] Prepare an aqueous solution of sodium bicarbonate with a molar concentration of 1.2 mol / L; mix Clostridium ethanolis protein with the aqueous solution of sodium bicarbonate to prepare a Clostridium ethanolis protein-sodium bicarbonate suspension with a water content of 80%; add lysozyme and chitinase to the Clostridium ethanolis protein-sodium bicarbonate suspension so that the suspension contains 0.5 g / L of lysozyme and 0.5 g / L of chitinase, and mix at a constant speed in a horizontal shaker at 37°C for 2 h to obtain the first suspension.

[0064] Preferably, the lysozyme activity is 20,000 U / mg, and the chitinase activity is 100,000 U / mg.

[0065] Preferably, step S102 specifically includes the following steps:

[0066] The first suspension was transferred to a -80°C freezer and frozen into blocks to obtain frozen blocks.

[0067] Preferably, step S103 specifically includes the following steps:

[0068] The frozen block was crushed into small particles using a crusher and quickly mixed with an aqueous solution of sodium bicarbonate at pH=8 and heated to 100°C to obtain a second suspension.

[0069] Preferably, step S104 specifically includes the following steps:

[0070] The second suspension was homogenized in a high-speed homogenizer at 13,000-15,000 rpm for 30 min at room temperature.

[0071] Preferably, step S105 specifically includes the following steps:

[0072] The homogenized second suspension was subjected to alcohol removal treatment using a rotary evaporator at 90°C to obtain a concentrated suspension.

[0073] Preferably, step S106 specifically includes the following steps:

[0074] The concentrated suspension was spray-dried using a spray dryer to obtain ethanol Clostridium protein with disrupted cell walls.

[0075] Preferably, step S2 specifically includes the following steps:

[0076] S201. Mix Clostridium ethanolis protein with water to obtain the first mixed raw material;

[0077] S202. Knead and sieve the mixed raw materials;

[0078] S203. Further mix the sieved raw materials to obtain a second mixed raw material;

[0079] S204. Determine the moisture content of the second mixed raw material;

[0080] S205. The second mixed raw material is added to a twin-screw extruder for extrusion processing. The amount of water to be added is calculated based on the target conditioned moisture content and the measured moisture content in the second mixed raw material. Water is added at the front end of the twin-screw extruder according to the required amount of water to be added, thereby controlling the moisture content during the extrusion process.

[0081] Preferably, step S201 specifically includes the following steps:

[0082] Weigh the Clostridium ethanolis protein to be compounded and add water during the mixing process using a peristaltic pump to adjust the moisture content of the mixed raw materials to 20%-30%. Add some water in advance to ensure sufficient contact between the raw materials and water, but the moisture content should not be too high, and the moisture content required for extrusion processing should not be used directly.

[0083] Preferably, step S202 specifically includes the following steps:

[0084] Pour the mixed raw materials onto a 40-mesh sieve and knead them through the sieve. The ethanol-containing Clostridium protein in the raw material will clump together when it combines with water. Raw materials with poor water solubility cannot react well with water because they cannot come into sufficient contact with it. This will lead to uneven distribution of moisture in the raw materials. By kneading and sieving, the clumps in the raw materials can be broken up, further improving the uniformity of moisture distribution in the raw materials.

[0085] Preferably, step S203 specifically includes the following steps:

[0086] The sieved raw materials are further mixed to maintain a uniform texture, resulting in a second mixed raw material. In the previous step, a specific sieve was used to disperse the clumps of raw materials; in this step, by remixing, the raw materials are formed into a mixture with relatively uniform moisture content.

[0087] Preferably, step S204 specifically includes the following steps:

[0088] Take a portion of the second mixed raw material and quickly determine its moisture content. Seal the remaining second mixed raw material in a sealed container for at least 1 hour to allow the moisture content to equalize. In this step, the moisture determination is used to calculate the amount of water that needs to be added during the twin-screw extrusion process based on the target conditioning moisture content and the measured moisture content in the second mixed raw material.

[0089] Among them, the moisture content of conditioning is the moisture content after pressing.

[0090] Preferably, the extrusion temperature of the twin-screw extruder is 170–190°C; the moisture content of the tempered material is 40%–50%; the screw speed of the twin-screw extruder is 120–200 rpm; and the feed rate is 30–50 g / min.

[0091] Most preferably, the extrusion temperature of the twin-screw extruder is 179.11℃; the conditioned moisture content is 47.52%; the screw speed of the twin-screw extruder is 160rpm; and the feed rate is 36.15g / min.

[0092] Preferably, the temperature of the twin-screw extruder is 40-60℃ in zone one, 80-100℃ in zone two, 120-140℃ in zone three, 160-180℃ in zone four, 160-180℃ in zone five, and the cooling temperature is 40-60℃.

[0093] Preferably, it further includes:

[0094] S206. Cut the obtained Clostridium ethanol-based microbial protein meat into fixed-size pieces and immediately seal them using a vacuum sealing machine.

[0095] A second aspect of this invention provides Clostridium ethanolis protein-based microbial protein meat prepared using the above-described method. This Clostridium ethanolis protein-based microbial protein meat has high fiber content, good taste, and is rich in amino acids, meeting the needs of different age groups for various amino acids and satisfying public demand.

[0096] A third aspect of the present invention provides the application of Clostridium ethanolis protein in the preparation of meat protein.

[0097] Preferably, Clostridium ethanolae protein is used as the sole component of the protein meat.

[0098] Example 1

[0099] The method for preparing Clostridium ethanol-based microbial protein meat in this embodiment includes the following steps:

[0100] S101. Prepare an aqueous solution of sodium bicarbonate with a molar concentration of 1.2 mol / L; mix Clostridium ethanolis protein with the aqueous solution of sodium bicarbonate to prepare a Clostridium ethanolis protein-sodium bicarbonate suspension with a water content of 80%; add lysozyme and chitinase to the Clostridium ethanolis protein-sodium bicarbonate suspension until the concentration of lysozyme is 0.5 g / L, the concentration of chitinase is 0.5 g / L, the enzyme activity of lysozyme is 20000 U / mg, and the enzyme activity of chitinase is 100000 U / mg; mix the suspension at a constant speed in a horizontal shaker at 37℃ for 2 h to obtain the first suspension.

[0101] S102. Transfer the first suspension to a -80°C freezer and freeze it into blocks to obtain frozen blocks.

[0102] S103. Crush the frozen block into small particles using a crusher, and quickly mix it with an aqueous solution of sodium bicarbonate at pH=8 heated to 100°C to obtain a second suspension.

[0103] S104. Homogenize the second suspension at 13000rpm-15000rpm for 30min in a high-speed homogenizer at room temperature.

[0104] S105. The homogenized second suspension is subjected to alcohol removal treatment using a rotary evaporator at 90°C to obtain a concentrated suspension.

[0105] S106. The concentrated suspension is spray-dried using a spray dryer to obtain ethanol Clostridium protein with cell wall disruption.

[0106] S201. Weigh the Clostridium ethanolis protein to be compounded and add water during the mixing process using a peristaltic pump to condition the moisture content of the mixed raw materials to 25%, thus obtaining the first mixed raw material.

[0107] S202. Pour the mixed ingredients onto a 40-mesh sieve, knead and sieve.

[0108] S203. Further mix the sieved raw materials to keep the raw materials uniform in texture and obtain a second mixed raw material.

[0109] S204. Take a portion of the second mixed raw material and quickly determine its moisture content. Seal the remaining second mixed raw material in a sealed container for no less than 1 hour to allow the moisture content to equalize.

[0110] S205. The second mixed raw material is fed into a twin-screw extruder at a constant speed via a rotary feeder for extrusion processing. The required amount of water to be added is calculated based on the target tempered moisture content and the measured moisture content in the second mixed raw material. Water is then added at a constant speed using a peristaltic pump to the front end of the twin-screw extruder according to the required amount, controlling the moisture content during the extrusion process. Processing parameters: Extrusion temperature of the twin-screw extruder is 150℃; tempered moisture content is 50%; screw speed of the twin-screw extruder is 160 rpm; feed rate is 40 g / min. The temperature of the twin-screw extruder is as follows: Zone 1: 50℃; Zone 2: 90℃; Zone 3: 130℃; Zone 4: 170℃; Zone 5: 170℃; Cooling temperature: 50℃.

[0111] S206. Cut the obtained Clostridium ethanol-based microbial protein meat into fixed-size pieces and immediately seal them using a vacuum sealing machine.

[0112] Example 2

[0113] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 1, except that the extrusion temperature of the twin-screw extruder is 160°C.

[0114] Example 3

[0115] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 1, except that the extrusion temperature of the twin-screw extruder is 170°C.

[0116] Example 4

[0117] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 1, except that the extrusion temperature of the twin-screw extruder is 180°C.

[0118] Example 5

[0119] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 1, except that the extrusion temperature of the twin-screw extruder is 190°C.

[0120] Example 6

[0121] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 1, except that the extrusion temperature of the twin-screw extruder is 200°C.

[0122] Example 7

[0123] The preparation method of Clostridium ethanol-based microbial protein meat in this embodiment is the same as that in Example 3, except that the moisture content of the conditioning is 40%.

[0124] Example 8

[0125] The preparation method of Clostridium ethanolis protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the moisture content of the conditioning is 45%.

[0126] Example 9

[0127] The preparation method of Clostridium ethanolis protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the moisture content of the conditioning is 55%.

[0128] Example 10

[0129] The preparation method of Clostridium ethanol-based microbial protein meat in this embodiment is the same as that in Example 3, except that the moisture content of the conditioning is 60%.

[0130] Example 11

[0131] The preparation method of Clostridium ethanol-based microbial protein meat in this embodiment is the same as that in Example 3, except that the moisture content of the conditioning is 65%.

[0132] Example 12

[0133] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the screw speed of the twin-screw extruder is 120 rpm.

[0134] Example 13

[0135] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the screw speed of the twin-screw extruder is 140 rpm.

[0136] Example 14

[0137] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the screw speed of the twin-screw extruder is 180 rpm.

[0138] Example 15

[0139] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the screw speed of the twin-screw extruder is 200 rpm.

[0140] Example 16

[0141] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the feeding rate is 20g / min.

[0142] Example 17

[0143] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the feeding rate is 30g / min.

[0144] Example 18

[0145] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the feeding rate is 50g / min.

[0146] Example 19

[0147] The preparation method of Clostridium ethanol protein-based microbial protein meat in this embodiment is the same as that in Example 3, except that the feeding rate is 60g / min.

[0148] Example 20 Effect of cell wall disruption treatment on Clostridium ethanol protein

[0149] The water absorption, water solubility, and protein dispersion index of Clostridium ethanolica protein after cell wall disruption treatment and Clostridium ethanolica protein without cell wall disruption treatment in Example 3 were measured.

[0150] like Figure 1 As shown, the water absorption, water solubility, and protein dispersion index of the treated Clostridium ethanolis protein were significantly increased. The water absorption of protein directly affects the texture and taste of the product. Proteins with high water absorption can absorb more water during extrusion, forming a softer, moister texture and improving the product's taste. Conversely, proteins with low water absorption may result in a dry, hard texture and poor taste. Proteins with good water absorption help the product retain moisture during processing and storage, improving its water retention and stability. This is crucial for extending the product's shelf life and maintaining its quality. The water solubility of protein determines its solubility and dispersion in water. Proteins with good water solubility dissolve more easily in water, forming a uniform dispersion system. These proteins are also more easily digested and absorbed by the human body, thus improving nutrient release and utilization. This is significant for improving the nutritional value of the product and meeting consumers' health needs. The protein dispersion index reflects the uniformity of its dispersion in water. Proteins with a high dispersion index form a finer, more uniform particle distribution. This helps improve the product's microstructure and appearance, making it more delicate and smooth. Proteins with a high dispersibility index provide better texture and flavor. Smaller protein particles distribute more evenly throughout the food, resulting in a smoother, more uniform texture. At the same time, proteins with a high dispersibility index also help improve the product's flavor release and stability.

[0151] The elastic modulus and storage modulus of Clostridium ethanole protein after cell wall disruption treatment and Clostridium ethanole protein without cell wall disruption treatment in Example 3 were measured.

[0152] Increased elastic and storage moduli in proteins signify stronger intermolecular forces, thus enhancing the structural strength of the product. This helps the product maintain its shape stability during extrusion, reducing the likelihood of deformation and breakage. Proteins with high elastic and storage moduli are better able to resist external pressure or shear forces, making the product less prone to deformation during processing and storage. In meat substitutes or vegetarian products, highly elastic proteins can mimic the chewiness and texture of real meat. During extrusion processing, proteins with high elastic and storage moduli better maintain their structural integrity, reducing nutrient loss. Increased protein elastic and storage moduli have multifaceted implications for extrusion processing. They not only improve the physical stability and texture of the product but also optimize the extrusion process and enhance its nutritional value. Figure 2As shown, the protein modulus of Clostridium ethanol was significantly increased after treatment, which optimized its potential for use in high-moisture extrusion processing.

[0153] Compared to plant proteins, bacterial proteins have lower viscosity due to the presence of cell walls, which makes them less suitable for extrusion processing. Protein viscosity significantly impacts the fiber structure of high-moisture extruded products. For example... Figure 3 As shown in Figures a and b, the treated Clostridium ethanolis protein significantly increased its viscosity. Proteins with moderate viscosity can better form fibrous structures during extrusion, giving the product a fibrous appearance and texture similar to meat. High-viscosity proteins may form a denser microstructure during extrusion, which helps improve the product's water retention and stability. Protein viscosity directly affects the texture and mouthfeel of the product. Proteins with moderate viscosity can impart suitable firmness and chewiness to the product, making the texture more delicate and elastic. Protein viscosity may also affect the nutritional value of the product. Proteins with moderate viscosity can better retain their nutrients during extrusion, while also being easier for the human body to digest and absorb. Protein viscosity also has an important impact on the shelf life and stability of the product. Proteins with moderate viscosity can give the product better stability and water retention, helping to extend the product's shelf life.

[0154] As shown in Table 1, the endothermic peak temperature and enthalpy of the treated Clostridium ethanolis protein were significantly lower than those of the untreated Clostridium ethanolis protein. This indicates that the treated Clostridium ethanolis protein is more likely to undergo phase transition under heat. In high-moisture extrusion processing, a lower temperature can achieve better results, which is more conducive to the extrusion processing of the product.

[0155] Table 1

[0156] Parameters CAP (before processing) CAP (after processing) T0(℃) <![CDATA[96.12±1.52 b ]]> <![CDATA[79.87±0.94 a ]]> TP (°C) <![CDATA[117.35±2.14 b ]]> <![CDATA[109.47±1.25 a ]]> TE (°C) <![CDATA[138.3±1.15 b ]]> <![CDATA[116.76±1.64 a ]]> △H(J / g) <![CDATA[3.98±0.09 b ]]> <![CDATA[3.07±0.26 a ]]>

[0157] Clostridium ethanolica protein that has not undergone cell wall disruption and Clostridium ethanolica protein that has undergone cell wall disruption but not mechanical treatment in steps S102-S104, and only enzymatic hydrolysis, were respectively prepared into protein meat using the method of Example 3. The degree of fiber was measured, and the degree of fiber of the protein meat obtained in Example 3 was also measured. As shown in Table 2 below, it can be seen that the degree of fiber of the protein meat prepared from Clostridium ethanolica protein after cell wall disruption is significantly higher than that of the un-cell wall-disrupted Clostridium ethanolica protein and the Clostridium ethanolica protein that has only undergone enzymatic hydrolysis.

[0158] Table 2

[0159] Transverse shear (kg) Longitudinal shear (kg) Fiberization Unprocessed 1.33±0.02 <![CDATA[1.36±0.02 b ]]> <![CDATA[0.98±0.01 a ]]> Enzymatic hydrolysis only, without mechanical treatment 1.31±0.02 <![CDATA[1.13±0.02 b ]]> <![CDATA[1.13±0.02 b ]]> After cell wall breaking process 1.30±0.02 <![CDATA[0.98±0.02 a ]]> <![CDATA[1.31±0.03 b ]]>

[0160] Example 21: Apparent characteristics of Clostridium ethanol-based microbial protein meat

[0161] Under the condition of keeping other processing parameters constant, this experiment systematically investigated the effects of extrusion temperature, moisture content, screw speed, and feed rate on the texturized extrusion processing of Clostridium ethanolae protein. The apparent characteristics were determined using the following method: the sample was cut into a cuboid approximately 3 cm long, and the sample was cut at three cross-sections using a sharp blade. Images were taken using a Canon 052D camera against a white background with LED light.

[0162] like Figure 4 The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 1-6. Examples 1-6 differ only in extrusion temperature; other process parameters are the same. The temperature range is 150℃-200℃. Experimental results show that in Example 6, when the extrusion temperature reaches 200℃, the sample cannot maintain its intact shape, and is accompanied by machine instability and "explosion" phenomena, indicating that the raw material components are prone to excessive melting at high temperatures, reducing viscosity and leading to product appearance defects. Examples 1-5 represent suitable extrusion temperature ranges, i.e., the suitable processing temperature range is 150℃-190℃. Among them, the cross-sections of the samples in Examples 3 and 4 are the most uniform, indicating that an extrusion temperature of 170℃-180℃ is a more preferred range. In Example 5, irregular voids appear inside when the extrusion temperature is 190℃, reflecting that while high temperatures enhance fluidity and plasticity, they may damage the fiber structure. Figure 4 Tear analysis showed that in Examples 3-6, the fibrous structure was obvious when the extrusion temperature was above 170°C, while it was not obvious at the low temperature (150°C-160°C) in Examples 1 and 2, indicating the importance of moderate temperature for protein denaturation and fibrous structure formation.

[0163] like Figure 5 The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 3 and 7-11. Examples 3 and 7-11 differ only in the conditioned moisture content; other process parameters are the same. The conditioned moisture content ranges from 40% to 65%. Experiments revealed that in Examples 10 and 11, sample molding became difficult when the moisture content exceeded 60%. In Example 11, with a moisture content of 65%, only irregularly shaped products could be produced, and the machine operation was unstable. Excessive moisture reduces material viscosity and molding ability, while increasing fluidity but hindering stable structure formation. In Examples 3 and 7-9, with a conditioned moisture content in the range of 40%-55%, pores appeared on the sample cross-section as the moisture content increased, but... Figure 5 All of the above can form a good fibrous structure. High moisture content contributes to the formation of fibrous structures by reducing viscosity, promoting protein denaturation and intermolecular interactions.

[0164] like Figure 6The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 3 and 12-15. Examples 3 and 12-15 differ only in screw speed; other process parameters are the same. With the screw speed set within the range of 120 rpm to 180 rpm, it was observed that the speed had no significant effect on the appearance and cross-section of the samples. However, in Example 15, the cross-section was rough at a screw speed of 200 rpm, indicating that excessively high speeds may be detrimental. Screw speed primarily affects the material flow rate and mixing effect; a suitable speed facilitates mixing and plasticizing, but excessive speed increases mechanical wear and energy consumption.

[0165] like Figure 7 The images shown are photographs of the torn surface, appearance, and cross-section of the products obtained in Examples 3 and 16-19. Examples 3 and 16-19 differ only in the feed rate; other process parameters are the same. The feed rate was set between 20 g / min and 60 g / min. The results show that the feed rate has a limited effect on the cross-section of the samples, but the tear images show that the fibrous structure weakens when the feed rate is higher than 50 g / min in Examples 18 and 19. A low feed rate prolongs the residence time of the protein in the barrel, promoting unfolding and rearrangement, and improving the texture; while a high feed rate shortens the residence time, which may lead to incomplete cross-linking and reduced texture.

[0166] Example 22: Product shape, moisture content, and density of Clostridium ethanol-based microbial protein meat.

[0167] Moisture content was determined according to the method in GB / T 6435-2006, with modifications. The sample was cut into slices no more than 2 mm thick, perpendicular to the extrusion direction. Ten slices were weighed and laid flat on the bottom of a glass dish, then dried in an oven at 105℃ until constant weight. The moisture content calculation formula is as follows:

[0168]

[0169] In the formula, m1: weight of the petri dish after drying, m2: weight of the sample, and m3: weight of the sample and petri dish after drying.

[0170] To determine the density of a sample using the specific gravity method, first, a completely clean and dry specific gravity beaker needs to be prepared. Fill it with distilled water at a constant temperature and weigh it (m1). Then, gently immerse the object to be tested in the water, allowing some water to overflow. Weigh the beaker on a balance (m2). Remove the object and weigh the beaker and remaining water (m3). The density calculation formula is:

[0171]

[0172] Table 3 shows the shape, moisture content, and density of the samples obtained in Examples 1-5. The difference between Examples 1-6 is the extrusion temperature. Compared to other extrusion temperatures, when the extrusion temperature of Examples 5 and 6 reached or exceeded 190°C, the height and width of the samples significantly exceeded those of other groups, showing a significant increase in expansion at this temperature, but the density did not increase significantly. This may be due to the rapid evaporation of moisture during high-temperature demolding, showing a trend of gradually decreasing moisture content as the extrusion temperature increases.

[0173] Table 3

[0174] Temperature (°C) Sample height (mm) Sample width (mm) <![CDATA[Density (g / cm 3 )]]> Moisture (%) Example 1 150 <![CDATA[9.07±0.02 c ]]> <![CDATA[19.00±0.03 a ]]> <![CDATA[1.15±0.00 a ]]> <![CDATA[47.62±0.03 d ]]> Example 2 160 <![CDATA[8.86±0.01 a ]]> <![CDATA[19.06±0.02 a ]]> <![CDATA[1.17±0.00 b ]]> <![CDATA[46.37±0.04 c ]]> Example 3 170 <![CDATA[8.96±0.02 b ]]> <![CDATA[19.00±0.02 a ]]> <![CDATA[1.16±0.00 b ]]> <![CDATA[45.78±0.03 b ]]> Example 4 180 <![CDATA[8.95±0.01 b ]]> <![CDATA[19.02±0.02 a ]]> <![CDATA[1.16±0.00 b ]]> <![CDATA[45.50±0.12 ab ]]> Example 5 190 <![CDATA[10.19±0.03 d ]]> <![CDATA[19.25±0.01 b ]]> <![CDATA[1.16±0.00 b ]]> <![CDATA[45.12±0.05 a ]]>

[0175] Table 4 shows the shape, moisture content, and density of the samples obtained in Examples 3 and 7-9. Compared to Examples 3 and 7-11, the sample density gradually decreased as the processing moisture content gradually increased from 40%. This is because the increased volume percentage of water molecules reduced the proportion of solid matter. High moisture conditions lower the denaturation temperature of proteins, making it easier for protein polypeptide chains to extend and rearrange. This helps to form a more porous structure, which can reduce the sample density to some extent.

[0176] Table 4

[0177]

[0178] Table 5 shows the shape, moisture content, and density of the samples obtained in Examples 3 and 12-15. Compared with Examples 3 and 12-15, the screw speed in the range of 120 rpm to 200 rpm has no significant effect on the expansion, density, and moisture content of the product, indicating that the degree of plasticization of the material is relatively stable in this range.

[0179] Table 5

[0180]

[0181] Table 6 shows the shape, moisture content, and density of the samples obtained in Examples 3 and 16-19. Compared with Examples 3 and 16-19, the change in feed rate mainly affects the moisture content of the product. Low feed rate increases the moisture content because the long residence time of the material is conducive to moisture binding, while high feed rate has the opposite effect, revealing the regulatory effect of feed rate on moisture dispersion and moisture binding inside the product.

[0182] Table 6

[0183]

[0184] Example 23: Product color of Clostridium ethanol-based microbial protein meat

[0185] The color of the sample was measured using a colorimeter. Measurements were taken at six different locations of the sample after collection, and the average value was recorded. The color was represented by L (brightness index: + indicates whiteness, - indicates darkness), a (color index: + indicates redness, - indicates greenness), and b (color index: + indicates yellowness, - indicates blueness).

[0186] Extrusion processing parameters have a significant impact on the color of the sample.

[0187] Table 7 shows the color values ​​of the products obtained in Examples 1-5. The difference between Examples 1-6 is the extrusion temperature. The increase in extrusion temperature, especially in Examples 3-5 (170℃-190℃), significantly improved the L value (brightness) of the samples, indicating that higher temperatures enhance the brightness of the samples. The red-green value a and yellow-blue value b of the samples reached their highest values ​​at an extrusion temperature of 180℃ (Example 4). The total color difference E value, in contrast to the L value, was significantly higher than the low-temperature group at temperatures of 150℃ (Example 1) and 160℃ (Example 2), indicating that the E value is greatly affected by the L value.

[0188] Table 7

[0189] Temperature (°C) L a b △E Example 1 150 <![CDATA[49.95±0.26 a ]]> <![CDATA[6.60±0.10 a ]]> <![CDATA[12.90±0.12 a ]]> <![CDATA[46.80±0.22 b ]]> Example 2 160 <![CDATA[48.41±0.27 a ]]> <![CDATA[6.14±0.06 a ]]> <![CDATA[12.58±0.12 a ]]> <![CDATA[48.13±0.27 b ]]> Example 3 170 <![CDATA[53.20±0.32 b ]]> <![CDATA[7.72±0.06 bc ]]> <![CDATA[15.10±0.10 b ]]> <![CDATA[44.57±0.27 a ]]> Example 4 180 <![CDATA[53.06±0.52 b ]]> <![CDATA[8.26±0.20 c ]]> <![CDATA[15.69±0.12 c ]]> <![CDATA[44.99±0.51 a ]]> Example 5 190 <![CDATA[54.33±0.44 b ]]> <![CDATA[7.57±0.18 b ]]> <![CDATA[15.22±0.07 b ]]> <![CDATA[43.54±0.46 a ]]>

[0190] Table 8 shows the color values ​​of the products obtained in Examples 3 and 7-9. The difference between Examples 3 and 7-11 is the amount of moisture removed during extrusion. When the moisture content during extrusion is in the range of 50%-55% (Examples 3 and 9), the L value of the samples also increases significantly. During high-moisture extrusion, the presence of a large amount of free water reduces the viscosity and residence time of the material in the extruder, thereby reducing the mechanical action on the material in the extruder. This reduces the degree of protein subunit polymerization and cross-linking, and increases the synergistic effect between disulfide bonds and hydrogen bonds, and disulfide bonds and hydrophobic interactions, ultimately making the extruded protein product lighter in color.

[0191] Table 8

[0192] Conditioning moisture content (%) L a b △E Example 7 40 <![CDATA[47.65±0.24 a ]]> <![CDATA[7.55±0.05 ab ]]> <![CDATA[14.29±0.07 a ]]> <![CDATA[49.50±0.21 c ]]> Example 8 45 <![CDATA[49.66±0.26 b ]]> <![CDATA[8.03±0.06 c ]]> <![CDATA[15.06±0.07 b ]]> <![CDATA[47.91±0.22 b ]]> Example 3 50 <![CDATA[53.20±0.32 c ]]> <![CDATA[7.72±0.06 bc ]]> <![CDATA[15.10±0.10 b ]]> <![CDATA[44.57±0.27 a ]]> Example 9 55 <![CDATA[53.80±0.43 c ]]> <![CDATA[7.16±0.18 a ]]> <![CDATA[14.81±0.11 b ]]> <![CDATA[43.82±0.45 a ]]>

[0193] Table 9 shows the color values ​​of the products obtained in Examples 3 and 12-15. The difference between Examples 3 and 12-15 is the screw speed. The screw speed initially increases and then decreases the L and E values ​​(total color difference) of the samples. The optimal screw speed is 160 rpm (Example 3), at which point the brightness and color uniformity of the samples are optimal. This also indicates that a suitable screw speed is crucial for optimizing the macroscopic structure of the product. Appropriate screw speeds contribute to obtaining products with uniform appearance, good color, and regular shape.

[0194] Table 9

[0195]

[0196] Table 10 shows the color values ​​of the products obtained in Examples 3 and 16-19. The difference between Examples 3 and 16-19 is the feed rate. Changes in feed rate resulted in a trend of initial increase followed by a decrease in both product brightness and color uniformity. The product brightness was lowest at a feed rate of 60 g / min (Example 19), while the color uniformity was poor at 50 g / min (Example 18). This indicates that feed rate not only affects product brightness but may also be closely related to the color stability and formation mechanism of the extrudate. Lower speeds prolong heating time, potentially deepening the color; while excessively high speeds may shorten the critical heating process, affecting the uniform formation of color.

[0197] Table 10

[0198]

[0199] Example 24: Texture characteristics of Clostridium ethanol-based microbial protein meat

[0200] Five indicators—hardness (kg), chewiness (kg), elasticity, cohesiveness, and resilience—were used to characterize the product's textural properties. Six squares with sides of 1.5 cm were randomly cut from the sample along the extrusion direction. A texture analyzer (Boston TVT6700) and a probe (P-CY30S) were used. Operating parameters were set as follows: TPA mode, pre-test speed 2.0 mm / s, test speed 1.0 mm / s, post-test speed 2.0 mm / s, and pressure reduction 50%.

[0201] Analysis of the data in the table shows that temperature, moisture, screw speed, and feed rate during the extrusion process have a significant impact on the physical properties of the product, especially hardness, chewiness, resilience, and elasticity.

[0202] Table 11 shows the hardness, chewiness, elasticity, cohesiveness, and resilience of Examples 1-5. The difference between Examples 1-6 is the extrusion temperature. As the extrusion temperature increases, the hardness and chewiness of the samples initially increase and then decrease. Within the 150℃-160℃ range (Examples 1 and 2), the sample hardness and chewiness are significantly higher than other temperature groups, indicating that protein denaturation is incomplete in this temperature range, the gel network is loose, and there are insufficient cross-linking points, resulting in a relatively rigid physical structure. However, when the temperature rises to 180℃-190℃ (Examples 4 and 5), the hardness and chewiness significantly decrease. This is attributed to the high temperature promoting the full unfolding and rearrangement of protein molecules, enhancing the cross-linking of the gel network, and accelerating the evaporation and migration of water, making the internal structure of the sample more uniform and soft. Furthermore, the high temperature also promotes the formation of fibrous structures, significantly improving the resilience of the samples.

[0203] Table 11

[0204]

[0205] Table 12 shows the hardness, chewiness, elasticity, cohesiveness, and resilience of Examples 3 and 7-9. The difference between Examples 3 and 7-11 lies in the conditioning moisture content, i.e., the extrusion moisture content. Increased extrusion moisture significantly softens the hardness, chewiness, and resilience of the products. Compared to Examples 3 and 7-11, within the moisture content range of 40%-55% (Examples 3 and 7-9), the hardness and chewiness of the products gradually decrease with increasing moisture content. This is mainly because moisture, acting as a plasticizer, promotes the flow and deformation of the material, forming a softer structure. Simultaneously, increased moisture also promotes the interaction between protein molecules, which is beneficial for forming a more uniform gel network.

[0206] Table 12

[0207]

[0208] Table 13 shows the hardness, chewiness, elasticity, cohesiveness, and resilience of Examples 3 and 12-15. The difference between Examples 3 and 12-15 is the screw speed. At 140 rpm and 160 rpm (Examples 13 and 3), the hardness and chewiness of the products reached their lowest values. This may be because the appropriate speed promotes uniform mixing of the materials and moderate cross-linking of the proteins, thereby optimizing the physical structure of the product.

[0209] Table 13

[0210]

[0211] Table 14 shows the hardness, chewiness, elasticity, cohesiveness, and resilience of Examples 3 and 16-19. The difference between Examples 3 and 16-19 lies in the feed rate, which has a complex impact on the physical properties of the products. Compared to Examples 3 and 16-19, at low feed rates of 20 g / min and 30 g / min (Examples 16 and 17), the product hardness is significantly reduced; while the chewiness initially increases and then decreases with increasing feed rate, reaching a peak at 40 g / min (Example 3) and then decreasing to a minimum at 60 g / min (Example 19). This reflects the dual influence of feed rate on the degree of protein cross-linking and the residence time of the material in the barrel. Meanwhile, the feed rate has a relatively small impact on the product's elasticity, but exhibits a significant negative impact on cohesiveness and resilience at 60 g / min, which may be related to incomplete protein cross-linking and uneven internal structure at high speeds.

[0212] Table 14

[0213]

[0214] Example 25: Shear force and degree of fibrosis of Clostridium ethanol-based microbial protein meat

[0215] The degree of fibrillation was characterized by shear forces perpendicular and parallel to the extrusion direction, using a texture analyzer (Boston TVT6700) and a sharp cutter (P-CKB). Operating parameters were set as follows: pre-test speed 2.0 mm / s, test speed 1.0 mm / s, post-test speed 2.0 mm / s, and shear degree 75%.

[0216] Figure 8 Figure a is a comparison chart of the transverse shear force, longitudinal shear force, and degree of fibrillation of the products obtained in Examples 1-6. The difference between Examples 1-6 is the extrusion temperature. Figure 8 Analysis of Example a revealed that, compared to Examples 1-6, extrusion temperature had a significant impact on the longitudinal shear force of the product, exhibiting a trend of first decreasing and then increasing. It reached its peak at 150℃ (Example 1), significantly higher than the other temperature groups, while decreasing to its lowest point at 180℃ (Example 4), significantly lower than the other groups. This trend was highly correlated with the degree of fibrillation; the degree of fibrillation was highest at 180℃, indicating the formation of a significant fibrous structure at this temperature. The longitudinal shear force was lower than the transverse shear force, consistent with the observations in the sample appearance diagram.

[0217] Figure 8 Figure b is a comparison chart of the transverse shear force, longitudinal shear force, and degree of fibrillation of the products obtained in Examples 3 and 7-11. The difference between Examples 3 and 7-11 is the different moisture content during extrusion. Figure 8 As shown in Figure b, changes in moisture content also significantly affect the shear force characteristics of the product. Compared to Examples 3 and 7-11, with increasing moisture content, the transverse shear force gradually and significantly decreases, while the longitudinal shear force first increases and then decreases, reaching its lowest point at 50% moisture content (Example 3). The degree of fibrillation is relatively stable in the 40%-50% moisture range (Examples 7, 8, and 3), but decreases significantly at 55% (Example 9). This may be related to the significant decrease in transverse shear force and the relative increase in longitudinal shear force, suggesting the formation of a denser fiber structure.

[0218] Figure 8 Figure c is a comparison chart of the transverse shear force, longitudinal shear force, and degree of fibrillation of the products obtained in Examples 3 and 12-15. The difference between Examples 3 and 12-15 is the screw speed. Figure 8 As shown in Figure c, the screw speed has no significant effect on the transverse shear force, longitudinal shear force, and degree of fiberization of the product within the experimental range, indicating that the changes in the physical properties of the product are relatively stable within this speed range. The effect of the feed rate on the product shear force and degree of fiberization is more complex.

[0219] Figure 8 Figure d is a comparison chart of the transverse shear force, longitudinal shear force, and degree of fiberization of the products obtained in Examples 3 and 16-19. The difference between Examples 3 and 16-19 is the different feed rates. Figure 8 As shown in Figure d, compared to Examples 3 and 16-19, the transverse shear force was lower at feed rates of 20 g / min (Example 16) and 40 g / min (Example 3), while the longitudinal shear force was highest at 60 g / min (Example 19). The texture was higher in the range of 30 g / min-50 g / min (Examples 17, 18, and 3). However, it is worth noting that excessively low feed rates (such as 20 g / min) also led to a significant decrease in texture, possibly due to insufficient material filling, inadequate extrusion, and reduced outlet pressure under low load conditions.

[0220] Example 26 Mechanical response parameters of Clostridium ethanol-based microbial protein meat

[0221] Methods for determining mechanical response parameters: Pressure, torque, and load are recorded once per second, and averaged per minute. Mechanical response parameters are recorded for each sample during a 9-minute equilibrium sampling period. Sampling is performed once per minute, with 9 samples taken from each group, and the average yield is calculated.

[0222] The unit mechanical energy (SME) is calculated from the mass of extruded material in 1 minute, based on the screw speed and its corresponding average torque, using the following formula (Chang et al., 1999): the torque is recorded every 6 seconds, the SME is calculated based on 10 data points, and the average value is taken for each processing condition.

[0223]

[0224] Figure 9 Figure a is a comparison chart of the textural properties of the products obtained in Examples 1-6. The difference between Examples 1-6 is the extrusion temperature. Figure 9 As shown in Figure a, in this experiment, compared to Examples 1-6, the die pressure showed a trend of first increasing and then decreasing with the increase of extrusion temperature, reaching a peak at 170℃ (Example 3), significantly higher than other temperature groups. Then, when the temperature climbed to the 180℃-190℃ range (Examples 4 and 5), the die pressure decreased significantly. This phenomenon reflects the promoting effect of high temperature on material flowability, reducing friction and adhesion between material particles by accelerating moisture evaporation and vaporization, thereby alleviating resistance during extrusion. However, extreme high temperatures may also trigger chemical and physical changes in the material, such as protein denaturation, leading to an abnormal increase in material viscosity, thus requiring even higher pressure to pass through the die. Simultaneously, the increase in temperature was accompanied by a significant decrease in torque, load, and mechanical energy, indicating an improvement in processing efficiency and a reduction in energy consumption.

[0225] Figure 9 Figure b is a comparison diagram of the textural properties of the products obtained in Examples 3 and 7-11. The difference between Examples 3 and 7-11 is the difference in moisture content upon extrusion. Figure 9 As shown in Figure b, under high moisture extrusion conditions, moisture acts as a natural lubricant, effectively reducing friction between materials, further reducing die pressure and required torque, and promoting a significant increase in output.

[0226] Figure 9 Figure c is a comparison chart of the textural properties of the products obtained in Examples 3 and 12-15. The difference between Examples 3 and 12-15 is the screw speed. Figure 9 As shown in Figure c, adjusting the screw speed has a limited impact on output, but significantly affects torque, load, and unit mechanical energy. As the speed increases, torque and load gradually decrease, while unit mechanical energy increases, indicating that while higher speed reduces direct resistance, it also increases the mechanical energy consumed per unit time. Notably, the pressure reaches its lowest point at 160 rpm, reflecting the optimal material flow at this speed. Changes in feed rate have a complex impact on system pressure, load, torque, and output.

[0227] Figure 9 Figure d is a comparison chart of the textural properties of the products obtained in Examples 3 and 16-19. The difference between Examples 3 and 16-19 is the different feed rates. Figure 9 As shown in Figure d, compared to Examples 3 and 16-19, the pressure initially increases and then decreases with increasing feed rate, remaining relatively low at 20 g / min (Example 16) and 40 g / min (Example 3). Load and torque are significantly lower at 20 g / min (Example 16), while output continuously increases, and unit mechanical energy gradually decreases. This indicates that a suitable feed rate helps optimize the residence time of material in the barrel, promoting protein cross-linking and fiber formation, while reducing energy consumption and improving production efficiency. However, excessively high or low feed rates may be detrimental to the physical properties of the product and processing efficiency.

[0228] In summary, based on the degree of fiberization, the preferred process parameters are: extrusion temperature of the twin-screw extruder is 170–190℃; conditioned moisture content is 40%–50%; screw speed of the twin-screw extruder is 120–200 rpm; and feed rate is 30–50 g / min.

[0229] Example 27: Optimization of Processing Parameters Using Response Surface Methodology

[0230] During the high-moisture extrusion texturization of Clostridium ethanolica protein, extrusion temperature, moisture content, screw speed, and feed rate all have a certain impact on the corresponding system parameters and product quality. Compared with screw speed, feed rate, extrusion temperature, and moisture content have a more significant impact on the product's textural properties and appearance characteristics. Based on the above experimental results, this experiment preliminarily determined that a screw speed of 160 rpm, an extrusion temperature of 170-190℃, a moisture content of 40-50%, and a feed rate of 30-50 g / min are suitable ranges for this experiment. A three-factor response surface methodology was then designed. The specific experimental parameters are shown in Table 15.

[0231] Table 15

[0232] Extrusion temperature (°C) Moisture (%) Feed rate (g / min) 170 40 40 170 45 30 170 50 40 170 45 50 180 45 40 180 45 40 180 40 30 180 45 40 180 40 50 180 50 50 180 50 30 180 45 40 180 45 40 190 50 40 190 45 50 190 45 30 190 40 40

[0233] This experiment selected the degree of fiberization as the target value to optimize processing parameters. Table 16 shows that under the parameter settings of this experiment, extrusion temperature, moisture content, and feed rate all have a significant impact on the target value. The lack of fit test showed no significant difference, indicating that the parameter setting threshold range is reasonable. Table 17 shows that the actual R-squared = 0.9822, Adj-R-squared = 0.9592, and the difference between Adj-R-squared and Pred-R-squared is less than 0.2, indicating that the response surface equation is reasonable.

[0234] Table 16

[0235]

[0236] Table 17

[0237] R-Squared 0.9822 Adj R-Squared 0.9592 Pred R-Squared 0.7831 Adeq Precision 19.6133

[0238] Depend on Figure 10 According to the response surface methodology, under the processing conditions of this experiment, the optimal degree of fiberization of the product can be obtained when the extrusion temperature is 179.11℃, the moisture content is 47.52%, and the feed rate is 36.15g / min.

[0239] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing Clostridium ethanol-based microbial protein meat, characterized in that, Includes the following steps: S1. The Clostridium ethanol protein was subjected to enzyme-mediated cell disruption to obtain the disrupted Clostridium ethanol protein. S2. The cell-wall-broken Clostridium ethanolis protein is added to a twin-screw extruder for extrusion processing, and the moisture content of the Clostridium ethanolis protein is controlled during the extrusion process; the extrusion temperature of the twin-screw extruder is 150-200℃; the conditioning moisture content is 40%-65%; the screw speed of the twin-screw extruder is 120-200 rpm; and the feed rate is 20-60 g / min.

2. The preparation method according to claim 1, characterized in that: In step S1, lysozyme and chitinase are used to break down the cell walls of Clostridium ethanol.

3. The preparation method according to claim 2, characterized in that: Step S1 specifically includes the following steps: S101. Add ethanol Clostridium protein to the first alkaline solution, then add lysozyme and chitinase, mix, and obtain the first suspension; S102. Freeze the first suspension into blocks to obtain frozen blocks; S103. The frozen block is crushed to obtain small particles, which are then added to a heated second alkaline solution and mixed to obtain a second suspension; S104. Homogenize the second suspension; S105. The homogenized second suspension is subjected to alcohol removal treatment to obtain a concentrated suspension; S106. The concentrated suspension is dried to obtain ethanol Clostridium protein with broken cell walls.

4. The preparation method according to claim 3, characterized in that: The first alkaline solution is an aqueous solution of sodium bicarbonate; the second alkaline solution is an aqueous solution of sodium bicarbonate.

5. The preparation method according to claim 1, characterized in that: The extrusion temperature of the twin-screw extruder is 170–190℃; the moisture content of the tempered material is 40%–50%; the screw speed of the twin-screw extruder is 120–200 rpm; and the feed rate is 30–50 g / min.

6. The preparation method according to claim 5, characterized in that: The extrusion temperature of the twin-screw extruder is 179.11℃; the conditioned moisture content is 47.52%; the screw speed of the twin-screw extruder is 160rpm; and the feed rate is 36.15g / min.

7. The preparation method according to claim 1, characterized in that: Step S2 specifically includes the following steps: S201. Mix Clostridium ethanolis protein with water to obtain the first mixed raw material; S202. Knead and sieve the mixed raw materials; S203. Further mix the sieved raw materials to obtain a second mixed raw material; S204. Determine the moisture content of the second mixed raw material; S205. The second mixed raw material is added to a twin-screw extruder for extrusion processing. The amount of water to be added is calculated based on the target conditioned moisture content and the measured moisture content in the second mixed raw material. Water is added at the front end of the twin-screw extruder according to the required amount of water to be added, thereby controlling the moisture content during the extrusion process.

8. The preparation method according to claim 1, characterized in that: The temperature of the twin-screw extruder is 40-60℃ in zone one, 80-100℃ in zone two, 120-140℃ in zone three, 160-180℃ in zone four, 160-180℃ in zone five, and the cooling temperature is 40-60℃.

9. A Clostridium ethanol-based microbial protein meat prepared by any one of claims 1-8.

10. The application of an ethanol Clostridium protein in the preparation of meat protein.

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