Fungus-based protein meat as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food, and particularly relates to fungus-based protein meat as well as a preparation method and application thereof. The invention provides fungus-based protein meat, which is prepared from the following raw materials in parts by mass: 1 to 6 parts of edible fungus powder and 4 to 6 parts of gluten protein, the edible mushroom powder comprises pleurotus eryngii and / or cordyceps militaris powder. According to the present invention, the edible fungus powder is adopted as the raw material, and is mixed with the gluten protein according to a certain ratio, such that the obtained fungus-based protein meat has similar lightness with the cooked chicken breast and the cooked beef, has characteristics of good texture structure, high tissue degree, rich protein content, no cholesterol, containing cordyceps polysaccharide and other bioactive substances, and rich natural delicate flavor; the method can be used for preparing food, especially meat products.

Description

Technical Field

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

[0002] With the continued growth of the world's population and the increasing disposable income, global meat consumption has surged. However, to achieve good flavor during meat processing, additional flavor enhancers are often added. As people become more health-conscious, the negative health effects of consuming these substances are gradually being exposed. Plant-based meat, with its advantages of high protein, low fat, and low cholesterol, is attracting increasing attention from researchers and consumers.

[0003] The main raw material for plant-based meat is soy protein. Through electrospinning, extrusion, and 3D printing technologies, soy protein is processed into a fibrous structure similar to meat, thus mimicking the structure, flavor, and texture of real meat. However, soy protein is prone to developing various flavor compounds during processing and storage, as well as some perceptible bitter and metallic tastes and perceptible beany odors. Currently, existing plant-based meat still lags significantly behind real meat in terms of texture, color, flavor, and mouthfeel, necessitating further improvements to enhance the characteristics of alternative plant-based meats.

[0004] Edible fungi are a type of large fungi that can be consumed by humans. They are diverse in form and rich in various natural active ingredients such as high-molecular-weight polysaccharides, proteins, β-glucan and RNA complexes, natural organic germanium, nucleic acid degradation products, cAMP, and triterpenoids. They have important utilization value for maintaining human health and also possess certain medicinal and health-preserving value with long-term use. However, current technologies for the development and utilization of edible fungi mainly focus on agricultural and industrial waste treatment and resource utilization, energy conservation and environmental protection, and health product development. There is no record of using edible fungi to develop protein-based meat substitutes. Summary of the Invention

[0005] The purpose of this invention is to provide a fungal-based protein meat, its preparation method and application, which enriches the types of natural plant edible resources. The obtained fungal-based protein meat has significant improvements in texture, color and degree of texturing.

[0006] This invention provides a fungal-based protein meat, comprising, by weight parts: 1-6 parts edible fungus powder and 4-6 parts gluten protein; wherein the edible fungus powder includes king oyster mushroom and / or cordyceps militaris powder.

[0007] Preferably, the gluten protein is wheat gluten protein.

[0008] Preferably, when the edible fungus powder includes king oyster mushroom and cordyceps militaris powder, the mass ratio of king oyster mushroom and cordyceps militaris powder is (1~5):1.

[0009] Preferably, the edible fungus powder can pass through a 60-mesh sieve.

[0010] This invention provides a method for preparing the fungal-based protein meat described in the above technical solution, comprising the following steps: Edible mushroom powder and gluten protein are mixed to obtain a premix; The premixed material is extruded, and the material after extrusion is cooled to obtain a high-moisture extrudate. The high-moisture extrudate is sheared to obtain fungal-based protein meat.

[0011] The extrusion process is performed using a twin-screw extruder. During the extrusion process, the feeding speed is 2~3 kg / h, the water feeding speed is 4~5 L / h, and the screw speed is 300~500 rpm.

[0012] Preferably, the twin-screw extruder includes an extrusion processing module and a cooling processing module; the extrusion processing module includes seven sections, the temperatures of which are 50~70℃, 70~90℃, 100~120℃, 120~140℃, 130~150℃, 130~150℃ and 130~150℃ respectively. The cooling module comprises two sections, with temperatures of 60~80℃ and 30~50℃ respectively.

[0013] Preferably, the moisture content of the high-moisture extrudate is 90-120%.

[0014] Preferably, after shearing, the process further includes packaging the sheared high-moisture extrudate.

[0015] This invention provides the application of the fungal-based protein meat described in the above-described technical solution or the fungal-based protein meat obtained by the preparation method described in the above-described technical solution in food.

[0016] Beneficial effects: This invention provides a fungal-based protein meat, comprising, by weight parts: 1-6 parts edible fungus powder and 4-6 parts gluten protein; the edible fungus powder includes king oyster mushroom and / or cordyceps militaris powder. This invention uses edible fungus powder as a raw material, mixing it with gluten protein in a certain proportion to obtain a fungal-based protein meat with a similar brightness to cooked chicken breast and beef, possessing good texture and structure, high density, rich protein content, no cholesterol, containing bioactive substances such as cordyceps polysaccharides, and rich in natural umami flavor. It can be used to prepare food products, especially meat products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The results are scanning electron microscopy images of the extruded meat protein obtained in Examples 1-2 and Comparative Examples 1-2. Figure 2 The results of surface hydrophobicity measurements for Examples 1-2 and Comparative Examples 1-2 before and after extrusion are shown. Figure 3 The results are the determination results of free thiol content before and after extrusion in Examples 1-2 and Comparative Examples 1-2; Figure 4 The results of disulfide bond content determination before and after extrusion in Examples 1-2 and Comparative Examples 1-2 are shown. Figure 5 The results of secondary structure measurements before and after extrusion for Examples 1-2 and Comparative Examples 1-2 are shown. Figure 6 The results of intrinsic fluorescence measurement of the premixes of Examples 1-2 and Comparative Examples 1-2 before and after extrusion are shown. Figure 7 The macroscopic observation results of the extruded meat protein obtained in Examples 3-11 are shown below; Figure 8 The results are scanning electron microscopy of the extruded meat protein obtained in Examples 3-11. Detailed Implementation

[0019] This invention provides a fungal-based protein meat, comprising, by weight parts: 1-6 parts edible fungus powder and 4-6 parts gluten protein; wherein the edible fungus powder includes king oyster mushroom and / or cordyceps militaris powder.

[0020] In one embodiment, the gluten protein described in this invention is wheat gluten protein. In another embodiment, the edible fungus powder described in this invention can pass through a 60-mesh sieve.

[0021] In one embodiment, when the edible fungus powder of the present invention includes king oyster mushroom and cordyceps militaris powder, the mass ratio of king oyster mushroom to cordyceps militaris powder is (1~5):1; in another embodiment, when the edible fungus powder of the present invention includes king oyster mushroom and cordyceps militaris powder, the mass ratio of king oyster mushroom to cordyceps militaris powder is 3:1. The present invention combines king oyster mushroom and cordyceps militaris powder in a specific ratio, which can further improve the texturization degree of the obtained fungal-based protein meat.

[0022] This invention provides a method for preparing the fungal-based protein meat described in the above technical solution, comprising the following steps: Edible mushroom powder and gluten protein are mixed to obtain a premix; The premixed material is extruded, and the material after extrusion is cooled to obtain a high-moisture extrudate. The high-moisture extrudate is sheared to obtain fungal-based protein meat.

[0023] This invention mixes edible mushroom powder and gluten protein to obtain a premix. The mixing method is not strictly required; conventional methods in the art can be used.

[0024] After obtaining the high-moisture extrudate, the present invention extrudes the premixed material, and cools the material after extrusion to obtain the high-moisture extrudate.

[0025] In one embodiment, the extrusion process of the present invention employs a twin-screw extruder. In one embodiment, the screw speed in the extrusion process is 300-500 rpm; in another embodiment, the screw speed is 400 rpm. In one embodiment, the feeding rate in the extrusion process is 2-3 kg / h; in another embodiment, the feeding rate is 2.5 kg / h. In one embodiment, the water feeding rate in the extrusion process is 4-5 L / h; in another embodiment, the water feeding rate is 4.5 L / h. The continuous water addition during the extrusion process of the present invention allows for precise control of the material texture, resulting in a more uniform texture.

[0026] In one embodiment, the twin-screw extruder of the present invention includes an extrusion processing module and a cooling processing module. In one embodiment, the extrusion processing module of the present invention includes seven sections, with temperatures in the seven sections sequentially set to 50~70℃, 70~90℃, 100~120℃, 120~140℃, 130~150℃, 130~150℃, and 130~150℃; in another embodiment, the temperatures in the seven sections of the extrusion processing module of the present invention are sequentially set to 60℃, 80℃, 110℃, 130℃, 140℃, 140℃, and 140℃. The present invention uses segmented extrusion, limiting different section temperatures, which, compared to one-step extrusion, offers advantages such as precise control of the fiberization process, improved product texture uniformity, and enhanced process stability.

[0027] In one embodiment, the cooling module of the present invention includes two sections, with temperatures of 60-80°C and 30-50°C respectively; in another embodiment, the temperatures of the two sections of the cooling module of the present invention are 60°C and 40°C respectively. The present invention uses different temperature conditions in stages for cooling, which, compared to one-step cooling, has the advantage of resulting in a more uniform fiber structure and optimal filament drawing strength.

[0028] In one embodiment, the moisture content of the high-moisture extrudate of the present invention is 90-120%; in another embodiment, the moisture content of the high-moisture extrudate of the present invention is 100-110%; in yet another embodiment, the moisture content of the high-moisture extrudate of the present invention is 105%.

[0029] After obtaining the high-moisture extrudate, the present invention cuts the high-moisture extrudate to obtain fungal-based protein meat.

[0030] In one embodiment, the present invention further includes packaging the high-moisture extrudate after shearing. In another embodiment, the packaging includes vacuum packaging. Packaging the high-moisture extrudate after shearing in this invention prevents moisture loss.

[0031] This invention provides the application of the fungal-based protein meat described in the above-described technical solution or the fungal-based protein meat obtained by the preparation method described in the above-described technical solution in food.

[0032] In one embodiment, the food products described in this invention include meat products.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a fungal-based protein meat, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Test Example 1 1. Analysis of main components of raw materials The protein content of shiitake mushroom powder (LE), cordyceps militaris powder (CM), button mushroom powder (AB), king oyster mushroom powder (PE), and wheat gluten protein (WG) was tested according to GB 5009.5-2016 (Method I). The results showed that WG had the highest protein content, at 70.16±0.37%. The protein content of the four edible fungi ranged from 20% to 30%, with AB having the highest protein content at 30.72±0.03%. The protein content of LE and PE was around 20%.

[0035] 2. Testing of raw material water absorption index and water solubility index Water absorption index (WAI) measures the ability of extruded raw materials to absorb and retain water under specific conditions. A high WAI means the raw material can absorb more water to form a viscous gel, which is the basis for constructing high-moisture extrudates. Water solubility index (WSI) measures the proportion of soluble components in water that an extruded raw material can dissolve under specific conditions. In the extruder cavity, the raw material transforms into a viscoelastic molten state under high temperature, high pressure, and high shear. WAI and WSI together determine its rheological properties.

[0036] Shiitake mushroom powder, Cordyceps militaris powder, button mushroom powder, and king oyster mushroom powder were mixed with wheat gluten protein at a mass ratio of 3:7 to obtain four premixes. Approximately 2 g (m0) of each premix was weighed into a constant-weight centrifuge tube (m1), and 25 mL of deionized water was added. The mixture was vortexed for 2 min to ensure thorough mixing. After equilibration at room temperature for 2 h, the mixture was centrifuged at 4000 r / min for 20 min. The supernatant was transferred to a dried, constant-weight glass dish (m2), and the dish was dried in a 105℃ oven until constant weight. The mass of the dried glass dish and supernatant residue (m3) and the mass of the centrifuge tube and precipitate (m4) were weighed. The water absorption index and water solubility index were calculated using the following formulas. Each sample group underwent three parallel experiments, and the results are shown in Table 1.

[0037] Water absorption index (g / g) = (m4-m1) / m0; Water solubility index (%) (m3-m2) / m0×100%.

[0038] Table 1 Results of water absorption index and water solubility index determination

[0039] Note: Different lowercase letters in the same column indicate significant differences. p <0.05), the same applies below.

[0040] Table 1 shows significant differences in the water-soluble protein (WAI) and water-soluble protein (WSI) values ​​of different edible fungi raw materials, revealing the essential differences in structure and composition among the four types of edible fungi. WAI primarily reflects the ability of hydrophilic colloids such as starch and dietary fiber to retain water through hydrogen bonds, while WSI characterizes the solubility of soluble sugars, proteins, and small-molecule polysaccharides. Shiitake mushrooms have the highest WAI value (2.99 g / g), indicating that their cell wall or dietary fiber structure has a stronger water-holding capacity compared to other edible fungi powders. This may be related to their dense fruiting bodies and high polysaccharide content. Simultaneously, shiitake mushrooms have the lowest WSI (13.83%), indicating a lower rate of solids dissolution in water and a more compact structure. Conversely, Cordyceps militaris exhibits the lowest WAI (2.35 g / g) and the highest WSI (19.96%), suggesting that its cell wall structure may be more porous or that it has a higher content of water-soluble components.

[0041] Example 1 1. Pass the Cordyceps militaris powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 3:7 to obtain a premix.

[0042] 2. The premix obtained in step 1 is subjected to high-moisture extrusion using a twin-screw extruder. Specifically, the premix is ​​fed into the twin-screw extruder at a rate of 10 kg / h, while moisture is introduced into the chamber at a rate of 17 L / h, so that the moisture content of the extruded plant protein meat is maintained at 65%~70%. The extrusion conditions are as follows: screw speed: 396 r / min, extrusion temperature ranges are 160℃, 170℃, 160℃, 130℃, 110℃, and 90℃, and cooling is performed at 60℃ and 40℃ in the cooling section.

[0043] 3. After the extruder output stabilizes, cut the extruded protein meat into long strips. To prevent moisture loss, use a vacuum packaging machine to vacuum seal the extruded protein meat (i.e., fungal-based protein meat), number them sequentially, and then freeze them in a -20℃ refrigerator.

[0044] Example 2 1. Pass the king oyster mushroom powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 3:7 to obtain a premix.

[0045] 2. Following the steps of Example 1, the pre-raw material obtained in step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for storage.

[0046] Comparative Example 1 1. Pass the shiitake mushroom powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 3:7 to obtain a premix.

[0047] 2. Following the steps of Example 1, the pre-raw material obtained in step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for storage.

[0048] Comparative Example 2 1. Pass the button mushroom powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 3:7 to obtain a premix.

[0049] 2. Following the steps of Example 1, the pre-raw material obtained in step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for storage.

[0050] Comparative Example 3 Place the chicken breast in a heat-resistant sealed bag and heat it in a hot water bath until the core temperature reaches 80°C. Let it cool at room temperature, carefully pat dry any excess moisture, and cut it into pieces.

[0051] Comparative Example 4 Place the beef tenderloin in a heat-resistant sealed bag and heat it in a hot water bath until the core temperature reaches 80°C. Let it cool at room temperature, carefully pat dry any excess moisture on the surface, and cut it into pieces.

[0052] Test Example 2 Product performance testing 1. Color difference analysis The extruded protein meat obtained in Examples 1-2 and Comparative Examples 1-2, and the meat blocks obtained in Comparative Examples 3 and 4, were dried, and then measured using a fully automated colorimeter in reflectance mode. This indicates the lightness of the sample; the higher the value, the whiter and brighter the sample. The red-green value represents the sample's color; a positive value indicates a reddish tint, while a negative value indicates a greenish tint. The value represents the yellow-blue tint, with a positive value indicating a yellowish tint and a negative value indicating a bluish tint. Measurements were taken at six different locations on the extruded protein, outliers were removed, and the average value was calculated. The results are shown in Table 2.

[0053] Table 2. Color difference analysis results of different samples

[0054] As shown in Table 2, after high-moisture extrusion, the L of the king oyster mushroom-gluten extruded protein and cooked beef in Example 2 were significantly different. The similar values ​​indicate that they have similar brightness. However, there is a significant difference in brightness between other edible fungi-gluten protein meat and chicken breast / beef. The redness values ​​(a) of chicken breast and beef are... Similarly, the red and yellow hue values ​​of all edible fungi-gluten protein meats differed significantly from those of real meats, with Cordyceps militaris-gluten protein meat a being the most notable exception. The highest value was 12.90 ± 0.26, b The highest value was 22.13±0.42. These unusual red and yellow values ​​may be due to the color of the Cordyceps militaris powder itself influencing the color of the extruded meat protein, resulting in its unique color. These results indicate that different edible fungi varieties significantly affect the color of extruded meat protein. The mechanism may primarily stem from the synergistic effect of natural pigments and the Maillard reaction during processing. This can provide a reference for the selection of edible fungi varieties when preparing fungal meat.

[0055] 2. Determination of texture and degree of organization (1) The texture of the extruded protein obtained in Examples 1-2 and Comparative Examples 1-2 was evaluated using a physical property testing instrument, including hardness, elasticity, and chewiness. The samples were cut into 20mm × 20mm × 7mm shapes and measured using a P / 36R probe in TPA mode. The operating parameters were: speed 2.0 mm / s before testing, speed 1.0 mm / s during testing, speed 2.0 mm / s after testing, pressure level 75%, and trigger force 5g. Each sample was measured at least six times, outliers were removed, and the final average value was taken. The results are shown in Table 3.

[0056] (2) The extruded protein obtained in Examples 1-2 and Comparative Examples 1-2 were cut into shapes of 20 mm × 20 mm × 7 mm. The shear force perpendicular to and parallel to the extrusion direction of the samples was measured using a physical property tester equipped with an HDP / BS probe. The degree of texturization was calculated according to the following formula, with the operating parameters being a speed of 2.0 mm / s before testing, a speed of 1.0 mm / s during testing, and a speed of 2.0 mm / s after testing, and a shear degree of 100%. Each sample was measured at least six times, outliers were removed, and the average value was taken. The results are shown in Table 3.

[0057] Degree of organization = Shear force perpendicular to the extrusion direction / Shear force parallel to the extrusion direction.

[0058] Table 3 Results of different quality profile measurements

[0059] Table 3 shows that the textures of the four edible fungi-gluten protein meats differed significantly after high-moisture extrusion. Example 2, *Pleurotus eryngii*-gluten protein meat, exhibited the highest hardness (2178.60) and chewiness (1399.61), with the strongest texture. This may be due to the dense fibrous structure of *Pleurotus eryngii* acting as a physical filler and reinforcement. The hardness of Example 2 *Pleurotus eryngii*-gluten protein meat was similar to that of Example 1 *Cordyceps militaris*-gluten protein meat, at 2178.60±227.04g and 1947.40±170.38g respectively, but the chewiness was slightly lower (1133.93). This indicates that the addition of *Pleurotus eryngii* and *Cordyceps militaris* effectively enhanced the deformation resistance and chewiness of the protein gel network. The elasticity of the four edible fungi-gluten protein meats was highly consistent, indicating that the addition of different edible fungi varieties did not significantly affect the resilience of the extruded protein gel network. This is likely because wheat gluten protein primarily provides elastic support to the mixture. Since the four raw materials have the same wheat gluten protein content, the elasticity of the extruded protein is similar after high-moisture extrusion. This result suggests that the texture of the extruded protein can be selectively controlled by choosing the type of edible fungus.

[0060] From the perspective of shear mechanics properties, both Example 2 (King Oyster Mushroom-Gluten Protein Meat) and Example 1 (Cordyceps Militaris-Gluten Protein Meat) exhibit high shear strength. Both showed significantly higher vertical shear forces (1.71 kg and 1.65 kg, respectively) and parallel shear forces (1.05 kg and 1.09 kg, respectively), indicating that the protein gel network they formed possesses stronger overall shear resistance and fiber toughness, which perfectly matches the aforementioned textural properties. Texture degree is a key indicator for evaluating the anisotropy of extruded fiber structure. Example 2 (King Oyster Mushroom-Gluten Protein Meat) showed the highest texture degree (1.63), indicating its most pronounced fibrous structure and significantly stronger resistance to shearing in the vertical direction than in the parallel direction, which is closer to the textural properties of real muscle tissue. Example 1 Cordyceps militaris-gluten protein meat (1.52) also showed good texture, followed by Comparative Example 1 Shiitake mushroom-gluten protein meat (1.45), while Comparative Example 2 Agaricus bisporus-gluten protein meat sample (1.17) had the lowest texture, indicating that its structural anisotropy was weak and its degree of fibrosis was the lowest.

[0061] Based on comprehensive textural properties and texture indicators, the *Pleurotus eryngii* sample exhibited outstanding performance in hardness, chewiness, and shear properties, suggesting that its components (such as specific dietary fibers and polysaccharides) may have a stronger interaction with gluten proteins, more effectively promoting the formation of oriented fiber networks during high-temperature shear extrusion. While *Cordyceps militaris* can form a highly textured fiber structure, its unique pigment components may affect the similarity of the fungal flesh to meat simulations, therefore its addition should not be excessive. *Agaricus bisporus* showed relatively weak performance in all textural indicators, and its components may not be conducive to the formation of a strong, ordered protein gel structure.

[0062] 3. Microstructure determination The microstructure of extruded protein meat (numbered a, b, c, and d sequentially) obtained by Comparative Example 1, Example 1, Comparative Example 2, and Example 2 was observed using scanning electron microscopy. Fresh samples were sliced ​​into thin sections (5×5×2 mm) to expose the internal fibrous network. The sample structure was first fixed with 2.5% glutaraldehyde at 4°C for 24 hours, then washed three times with 0.1 M phosphate-buffered saline (PBS). The samples were then eluted with fractionated ethanol at 30%, 50%, 70%, 80%, 90%, and 100% (v / v) for 5 min each. After dehydration, the samples were freeze-dried. Finally, the samples were fixed on an aluminum stage with double-sided conductive adhesive, sputter-coated with gold, and observed and photographed under a scanning electron microscope at a magnification of ×1500. The results are as follows: Figure 1As shown in the SEM images, all four groups of samples exhibited a fibrous structure along a single direction, with the fiber bundles arranged in parallel. This is due to the shearing and stretching action of different edible fungi raw materials during high-moisture extrusion, forming oriented fibers, which is consistent with the typical characteristics of high-moisture extrusion simulating meat myofibril tissue. Example 2: The extruded protein-meat fiber structure of king oyster mushroom was coarse and obvious, with the highest density, regular arrangement, clear interface, and no obvious breaks or wrinkles. This indicates that the melting, shearing, and orientation processes of the king oyster mushroom were more complete during the shearing process, and the molecular chain / protein network was stretched and formed more completely. However, a small number of flocculent protrusions were observed in the fiber structure. Figure 1 (d). Example 1: The fibrous structure of the extruded protein from Cordyceps militaris showed localized fiber aggregation, slight wrinkling, and localized curling. Its density was lower than that of Pleurotus eryngii, and some fiber gaps were slightly wider. Figure 1 (b) In Comparative Example 2, the extruded protein fibers from the button mushroom showed obvious aggregation, with poorer fiber orientation, disordered arrangement, and a single round amorphous particle in the matrix. Figure 1 (c) In Comparative Example 1, the pores and gaps of the extruded protein fibers from shiitake mushrooms were most pronounced, resulting in a fine fibrous structure. Figure 1 (a)

[0063] 4. Determination of the hydrophobicity of the surface of extruded raw materials and extruded powder The surface hydrophobicity index is a key indicator characterizing the interfacial properties of high-moisture extruded protein meat, reflecting the degree of exposure of hydrophobic groups on the surface of the material before and after extrusion. The surface hydrophobicity of proteins was determined using the ANS fluorescent probe method. The extrusion premixes or extrudates of Examples 1-2 and Comparative Examples 1-2 were lyophilized, ground, and passed through an 80-mesh sieve. 100 mg each of the lyophilized premix and extrudate powder were weighed and dissolved in 10 mmol / L PBS phosphate buffer (pH 7.0). After treatment at room temperature for 2 h, the solution was centrifuged at 4000 r / min for 25 min. The protein content in the supernatant was determined using the BCA method, repeated three times and the average value was taken. The protein solution was serially diluted 2, 4, 6, 8, and 10 times with PBS to a concentration between 0.005 mg / mL and 0.5 mg / mL. 8-aniline-1-naphthalenesulfonic acid (ANS) was dissolved in PBS in a light-protected environment to prepare an 8 mM solution. 12 mL of raw material and extruded protein dilution solution of different concentrations were mixed with 60 μL of ANS, shaken vigorously, and allowed to stand in the dark for 10 min. Fluorescence intensity was measured using a fluorescence spectrophotometer with excitation wavelength of 370 nm, emission wavelength of 470 nm, slit width of 5 nm, and scan frequency of 20 nm / s. A fluorescence intensity versus protein concentration curve was plotted, with the surface hydrophobicity index as the initial slope between fluorescence intensity and protein concentration. Each sample was measured in triplicate. Results are as follows: Figure 2As shown, *Agaricus bisporus* has the highest surface hydrophobicity index, followed by *Cordyceps militaris* and *Lentinula edodes*, while *Pleurotus eryngii* has the lowest. This may be because *Agaricus bisporus* has more exposed hydrophobic groups, such as hydrophobic amino acid residues, resulting in higher initial hydrophobicity, while *Pleurotus eryngii* has a higher proportion of hydrophilic components, such as polysaccharides, leading to lower initial hydrophobicity. After high-moisture extrusion, the hydrophobicity index of *Lentinula edodes* and *Cordyceps militaris* extrudates increased significantly, with *Cordyceps militaris* showing the largest increase. The hydrophobicity index of *Agaricus bisporus* and *Pleurotus eryngii* extrudates was lower than before extrusion, with *Pleurotus eryngii* showing the most significant decrease. The high temperature, high pressure, and shear force during the extrusion process alter the molecular structure and interfacial properties of the materials. Shear force disrupts the cell walls of edible fungi, exposing hidden hydrophobic groups. Simultaneously, protein molecules denature and aggregate, leading to increased hydrophobicity in *Lentinula edodes* and *Cordyceps militaris*. The cell wall structure of *Cordyceps militaris* may be more porous, resulting in a much higher degree of exposure of hydrophobic groups compared to other species. During the extrusion process, the high temperature and pressure cause a large amount of hydrophilic polysaccharides from button mushrooms and king oyster mushrooms to dissolve and adsorb onto the surface, resulting in a decrease in hydrophobicity.

[0064] 5. Changes in internal forces of extruded raw materials and extruded protein (1) Determination of free thiol content in extruded raw materials and extruded powder The extruded premixes or extrudates of Examples 1-2 and Comparative Examples 1-2 were lyophilized, ground into powder, and passed through an 80-mesh sieve. 80 mg of each was weighed and dissolved in 10 ml of TGE buffer solution, shaken every 10 minutes to ensure homogeneity, and reacted at room temperature for 30 min. Then, 50 μL of Ellman's reagent was added, and the mixture was incubated in the dark at room temperature for 1 h. Subsequently, the mixture was centrifuged at 3000 × g for 10 min, and the supernatant was collected. A solution containing only Ellman's reagent but no sample was used as a blank control. The absorbance of the sample and the blank group at 412 nm was measured using a spectrophotometer, with each group analyzed in triplicate and the average value taken. The results are as follows: Figure 3 As shown, the free sulfhydryl content of the four edible fungi after high-moisture extrusion was significantly higher than that of the extruded raw materials. Cordyceps militaris showed the most significant increase, followed by Agaricus bisporus, while the increases in Lentinus edodes and Pleurotus ostreatus were relatively gradual. This may be because the high temperature, high pressure, and high shear force during the extrusion process damage the protein structure in the samples, causing the disulfide bonds originally hidden inside the molecules to break and decompose into free sulfhydryl groups, thus increasing the free sulfhydryl content after extrusion.

[0065] (2) Determination of disulfide bond content in extruded raw materials and extruded powder The extruded premixes or extrudates from Examples 1-2 and Comparative Examples 1-2 were lyophilized, ground, and passed through an 80-mesh sieve. 30 mg of each was weighed and dissolved in a mixture of 10 mL TGE buffer and 100 μL β-mercaptoethanol, and incubated at room temperature for 1 h. 10 mL of 12% trichloroacetic acid (TCA) solution was added, and incubation continued for 1 h. The solution was centrifuged at 4000 rpm for 10 min, and the precipitate was washed twice with 5 mL TCA solution. The precipitate was redissolved in 5 mL TGE buffer. Then, 40 μL LDTNB reagent was added, and the absorbance was measured at 412 nm after incubation at room temperature for 30 min. Three replicates were taken for each group, and the average value was calculated. The results are as follows: Figure 4 As shown, the disulfide bond content was higher after high-moisture extrusion than before extrusion, possibly due to the "break-recombination" effect between disulfide bonds and free thiol groups. During extrusion, some free thiol groups undergo oxidation, reforming disulfide bonds and increasing the total disulfide bond content. Agaricus bisporus has a high proportion of hydrophobic amino acids, and its conformational changes lead to sufficient exposure of thiol groups, resulting in more oxidized and recombined disulfide bonds. While Cordyceps militaris has the highest release of free thiol groups, some thiol groups do not complete oxidative recombination, thus resulting in a lower increase in disulfide bonds.

[0066] 6. Characterization of protein structure in extruded raw materials and extrudates (1) Determination of the secondary structure of proteins in extruded raw materials and extruded powder The extruded premixes or extrudates of Examples 1-2 and Comparative Examples 1-2 were freeze-dried, ground into powder, and passed through an 80-mesh sieve. 5 mg each of the freeze-dried extruded raw material and extrudate powder were weighed and mixed separately with 200 mg of dry KBr. After grinding for 3 minutes, the mixture was compressed into tablets using a tablet press to form uniform and transparent thin sheets. The sample thin sheets were analyzed using a Fourier transform infrared spectroscopy (FTIR) instrument at 4000 cm⁻¹. -1 -400cm -1 The wavelength range was scanned 16 times across the entire wavelength band, with a scanning resolution of 4 cm. -1 Each sample group was tested in triplicate, and the average value was taken. The relative proportions of protein secondary structures were quantified using Omnic and Peakfit v4.12 software. Results are as follows: Figure 5As shown, Fourier transform infrared spectroscopy is an important method for characterizing protein secondary structure, which is the core representation of protein spatial conformation. The high temperature, high pressure, and high shear force environment of high-moisture extrusion disrupts intramolecular interactions in proteins, causing the β-sheet structure to transform into α-helices and random coils. High-moisture extrusion treatment significantly altered the proportion of secondary structure proteins in shiitake mushrooms, cordyceps militaris, button mushrooms, and king oyster mushrooms: except for king oyster mushrooms, the proportion of α-helices and random coils increased after extrusion, while the proportion of β-sheets and β-turns decreased. King oyster mushrooms showed the opposite trend: a decrease in the proportion of α-helices and random coils and an increase in the proportion of β-sheets, with only a slight decrease in β-turns. This may be because the amino acid arrangement in its β-sheet region is more stable, and the shear force did not unwind it but instead promoted the α-helices to refold into β-sheets. Cordyceps militaris showed the most significant change in secondary structure proportion, while its β-turn proportion was not affected by extrusion. This may be because the high proportion of sulfur-containing amino acids exacerbates the breaking and recombination of disulfide bonds, leading to the unfolding of the β-sheet region and the formation of random curls.

[0067] (2) Determination of intrinsic fluorescence of proteins in extruded raw materials and extruded powder The extruded premixes or extrudates from Examples 1-2 and Comparative Examples 1-2 were lyophilized, ground, and passed through an 80-mesh sieve. 30 mg each of the extruded raw material and the lyophilized extrudate powder were weighed and dissolved in PBS phosphate buffer. The mixture was vortexed until homogeneous and then centrifuged at 4000 r / min for 10 min. The supernatant was collected and analyzed using a fluorescence spectrophotometer. The test conditions were: excitation wavelength 280 nm, scanning wavelength 300-500 nm, slit width 5 nm, scanning rate 240 nm / min, and test voltage 700 V. The fluorescence intensity of the solution was recorded, and the spatial conformation of different edible fungi before and after extrusion was characterized using intrinsic fluorescence spectroscopy. The results are as follows: Figure 6As shown, except for Cordyceps militaris after extrusion, the fluorescence signals of all edible fungi were concentrated at 350 nm (the characteristic peak of tryptophan endogenous fluorescence). Before extrusion, there was a clear gradient in fluorescence intensity among different types of edible fungi, with Pleurotus ostreatus (PE) showing the highest fluorescence intensity, followed by Agaricus bisporus (AB), Lentinus edodes (LE), and Cordyceps militaris (CM) showing the lowest. After high-moisture extrusion, the fluorescence intensity of all fungi showed a significant decrease, except for Cordyceps militaris, where there was no obvious shift, only intensity decay, and the intensity difference narrowed, showing overall convergence. This may be because the high temperature and high shear force of high-moisture extrusion disrupted the intramolecular / intermolecular hydrogen bonds and hydrophobic interactions of protein molecules, exposing tryptophan / tyrosine residues from the hydrophobic core to the hydrophilic environment, reducing the fluorescence quantum yield and leading to a decrease in intensity. The characteristic peak of Cordyceps militaris shifted from 350 nm (tryptophan) before extrusion to 310 nm (tyrosine) after extrusion, mainly because tyrosine fluorescence is dominant and tryptophan is targeted and quenched by Cordyceps militaris polysaccharides. It is possible that the proportion of tyrosine residues in Cordyceps militaris protein is much higher than that of the other three edible fungi. The high temperature and high shear after extrusion caused a large number of tyrosine residues to be exposed from the hydrophobic core of the protein, and its characteristic fluorescence signal intensity of 310 nm surpassed that of tryptophan (350 nm), becoming the main peak.

[0068] Example 3 1. Pass the king oyster mushroom powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 1:9 to obtain a premix.

[0069] 2. The pre-raw material obtained in step 1 is subjected to high-moisture extrusion using a twin-screw extruder. Specifically, the raw material is fed into the twin-screw extruder at a rate of 2.5 kg / h, while moisture is introduced into the chamber at a rate of 4.5 L / h, so that the moisture content of the extruded edible fungus protein meat is maintained at 90%~120%. The extrusion conditions are as follows: screw speed: 400 rpm, extrusion temperature in each section is 60℃, 80℃, 110℃, 130℃, 140℃, 140℃, and 140℃, and cooling is performed at 60℃ and 40℃ in the cooling section.

[0070] 3. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20℃ freezer.

[0071] Example 4 1. Pass the king oyster mushroom powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 3:7 to obtain a premix.

[0072] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0073] Example 5 1. Pass the king oyster mushroom powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 5:5 to obtain a premix.

[0074] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0075] Example 6 1. Pass the Cordyceps militaris powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 1:9 to obtain a premix.

[0076] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0077] Example 7 1. Pass the Cordyceps militaris powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 3:7 to obtain a premix.

[0078] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0079] Example 8 1. Pass the Cordyceps militaris powder through a 60-mesh sieve, and mix the sieved material with wheat gluten protein at a mass ratio of 5:5 to obtain a premix.

[0080] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0081] Example 9 1. Pass the king oyster mushroom powder and cordyceps militaris powder through a 60-mesh sieve, collect the sieve-passing material, and mix them evenly according to the mass ratio of king oyster mushroom powder: cordyceps militaris powder: wheat gluten protein of 1:1:8 to obtain a premix.

[0082] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0083] Example 10 1. Pass the king oyster mushroom powder and cordyceps militaris powder through a 60-mesh sieve, collect the sieve-passing material, and mix them evenly according to the mass ratio of king oyster mushroom powder: cordyceps militaris powder: wheat gluten protein of 3:1:6 to obtain a premix.

[0084] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0085] Example 11 1. Pass the king oyster mushroom powder and cordyceps militaris powder through a 60-mesh sieve, collect the sieve-passing material, and mix them evenly according to the mass ratio of king oyster mushroom powder: cordyceps militaris powder: wheat gluten protein of 5:1:4 to obtain a premix.

[0086] 2. Following the steps of Example 3, the pre-raw material obtained in Step 1 is subjected to high-moisture extrusion. After the extruder output stabilizes, the extruded protein meat is cut into long strips. To prevent moisture loss, the extruded protein meat is vacuum-sealed using a vacuum packaging machine, numbered sequentially, and then frozen in a -20°C freezer for preservation.

[0087] Test Example 3 1. Cut the extruded protein meat obtained in Examples 3-11 (numbered 1-9 sequentially) into equal-sized pieces and photograph them. The results are as follows: Figure 7 As shown, the king oyster mushroom-gluten extruded protein meat fiber bundles obtained in Examples 2-5 are arranged in a loose network, with a large gap ratio, and the individual fibers are thick and easy to separate; the cordyceps militaris-gluten extruded protein meat fibers obtained in Examples 6-8 are tightly interwoven, with a very low gap ratio, and the fiber bundles are thin and arranged longitudinally in a regular manner, but the fiber bundles are small and easy to break; the king oyster mushroom compound cordyceps militaris-gluten extruded protein meat obtained in Examples 9-11 retains the framework structure formed by the coarse fibers of king oyster mushroom, while the fine fibers of cordyceps militaris fill the gaps in the framework, and the fiber bundles are uniform and tough.

[0088] 2. Following the steps of Test Example 2, the extruded protein meat obtained in Examples 3 to 11 was cut into shapes of 20 mm × 20 mm × 7 mm and subjected to texture and texturization tests. The results are shown in Tables 4 and 5.

[0089] Table 4 Results of different quality profile measurements

[0090] Table 5 Results of tissue degree determination for different samples

[0091] Table 4 shows that the extruded protein meat with different formulations exhibits significant differences in hardness, elasticity, and chewiness. The ratio of king oyster mushroom to gluten of 3:7 exhibits the firmest overall texture, with significantly higher hardness (21568.98g) and chewiness (15983.36g) compared to other groups, and its elasticity (0.93) remains at a high level. In contrast, the ratio of cordyceps to gluten of 3:7 generally shows the lowest hardness, elasticity, and chewiness. The trends in hardness and chewiness are highly consistent, and the wheat gluten protein content is the most critical factor determining the product's hardness and chewiness. When the gluten protein ratio reaches 70%, the product exhibits the highest hardness (21568.98g) and chewiness (15983.36g); conversely, a lower ratio leads to a significant decrease in these two indicators. The addition of mushrooms systematically softens the product texture, and the "softening" effect of cordyceps is stronger than that of king oyster mushroom. However, the changes in elasticity and hardness are not entirely synchronized. High elasticity (>0.94) depends more on a high proportion (≥80%) and continuous gluten network, while excessive mushroom addition (50%) will disrupt the network continuity, resulting in a significant reduction in elasticity.

[0092] As shown in Table 5, compared with extruded protein containing a single edible fungus, the overall vertical shear force and texture of Cordyceps militaris extruded protein were generally higher than those of King Oyster Mushroom extruded protein in the same proportion: when added at 50%, the vertical shear force (3.80 kg vs 2.71 kg) and texture (2.13 vs 1.81) of Cordyceps militaris extruded protein in Example 8 were significantly better than those of King Oyster Mushroom extruded protein in Example 5, indicating that Cordyceps militaris has greater potential in enhancing product elasticity and fiber structure. Compared with single additions, the compound group significantly improved the fiber structure of the product. The texture of Example 10 (King Oyster Mushroom: Cordyceps militaris: gluten = 3:1:6) reached the highest value among all 9 samples (2.29 ± 0.06). This is even much higher than any formulation with a single edible fungus (the highest being 2.13). This indicates that when King Oyster Mushroom and Cordyceps militaris are combined in a mass ratio of approximately 3:1, a synergistic effect of "1+1>2" is produced with gluten protein. The possible mechanism is that the polysaccharides and other functional components in Cordyceps militaris improve the water retention and viscoelasticity of the protein network, while the fibers of Pleurotus eryngii provide additional physical support. Together, they promote the formation of a more uniform, dense, and tearable fibrous structure.

[0093] 3. Following the steps in Test Example 2, the microstructure of the extruded protein meat (numbered 1-9 sequentially) obtained in Examples 3-11 was determined. Results Figure 8As shown, the layered structure of the king oyster mushroom-gluten extruded protein obtained in Examples 3-5 is obvious, but the fiber structure is obviously loose and the pores between fibers are large; the pore size of the cordyceps militaris-gluten extruded protein obtained in Examples 6-8 is smaller than that of the king oyster mushroom group, and the fiber structure is fine and easily broken; the king oyster mushroom compound cordyceps militaris-gluten extruded protein obtained in Examples 9-11 forms a typical oriented and rich fiber structure, with fibers arranged along the extrusion direction and smooth and dense pore walls, which is a characteristic structure of high-moisture extruded protein.

[0094] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fungal-based protein meat, characterized in that, The ingredients, by weight, include the following: 1-6 parts edible fungus powder and 4-6 parts gluten protein; the edible fungus powder includes king oyster mushroom and / or cordyceps militaris powder.

2. The fungal-based protein meat according to claim 1, characterized in that, The gluten protein is wheat gluten protein.

3. The fungal-based protein meat according to claim 1, characterized in that, When the edible fungus powder includes king oyster mushroom and cordyceps militaris powder, the mass ratio of king oyster mushroom and cordyceps militaris powder is (1~5):

1.

4. The fungal-based protein meat according to any one of claims 1 to 3, characterized in that, The edible mushroom powder can pass through a 60-mesh sieve.

5. The method for preparing fungal-based protein meat according to any one of claims 1 to 4, characterized in that, Includes the following steps: Edible mushroom powder and gluten protein are mixed to obtain a premix; The premixed material is subjected to extrusion and cooling processes in sequence to obtain a high-moisture extrudate. The high-moisture extrudate is sheared to obtain fungal-based protein meat.

6. The preparation method according to claim 5, characterized in that, The premixed material is extruded and cooled sequentially using a twin-screw extruder. During the extrusion and cooling processes, the feeding speed is 2-3 kg / h, the water feeding speed is 4-5 L / h, and the screw speed is 300-500 rpm.

7. The preparation method according to claim 6, characterized in that, The twin-screw extruder includes an extrusion processing module and a cooling processing module; the extrusion processing module includes seven sections, the temperatures of which are 50~70℃, 70~90℃, 100~120℃, 120~140℃, 130~150℃, 130~150℃ and 130~150℃ respectively. The cooling module comprises two sections, with temperatures of 60~80℃ and 30~50℃ respectively.

8. The preparation method according to any one of claims 5 to 7, characterized in that, The moisture content of the high-moisture extrudate is 90-120%.

9. The preparation method according to claim 5, characterized in that, The process after shearing also includes packaging the high-moisture extrudate.

10. The use of the fungal-based protein meat according to any one of claims 1 to 4 or the fungal-based protein meat obtained by the preparation method according to any one of claims 5 to 9 in food.

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