Method for preparing microbial protein meat based on TGase enzyme method of neurospora crassa hypha protein

Abstract

The invention relates to the technical field of food processing, in particular to a method for preparing microbial protein meat based on a TGase enzyme method of neurospora crassa hypha protein, which comprises the following steps: S101, crushing hypha protein biomass of neurospora crassa, sieving with a 100-mesh sieve to obtain hypha protein powder, mixing the hypha protein powder with auxiliary materials, and uniformly stirring to obtain a mixed material; and S102, adding transglutaminase into the mixed material, uniformly stirring, filling into a mold, and reacting at-80 DEG C for 24 hours to obtain the product. The preparation method of the hypha protein biomass comprises the following steps: inoculating a neurospora crassa spore suspension in a culture medium according to the inoculation amount of 1-10%, and fermenting for 3-5 days under the conditions of 150-180rpm and 28-30 DEG C to obtain a fermentation product; a fermentation product is washed with ultrapure water, two-stage heat treatment is carried out, and hyphae are completely washed with ultrapure water; and pre-freezing the obtained hyphae, and freeze-drying at-80 DEG C for 48 hours.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and more specifically, to a method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein. Background Technology

[0002] With the continued growth of the global population and the upgrading of dietary structures, the demand for animal protein is rising, putting traditional animal husbandry under multiple pressures, including high resource consumption, significant environmental pollution, and increased disease risks. At the same time, consumers' growing focus on healthy and sustainable foods is driving the development of novel protein resources such as plant-based and microbial-derived proteins. Against this backdrop, cultured meat and alternative protein technologies are rapidly developing and have become a research hotspot in the intersection of food science and agri-food fields.

[0003] Currently, there are various protein products on the market based on microbial fermentation, such as protein raw materials prepared using microorganisms like Fusarium venenatum, Aspergillus, Pleurotus eryngii, Yarrowia lipolytica, and Saccharomyces cerevisiae. These microorganisms generally have characteristics such as rapid growth, low nutritional requirements, and high protein content, and some have already been industrialized and used in the development of meat analogues. However, existing microbial protein meat products still have certain limitations in terms of texture simulation, flavor control, and nutritional balance, especially lacking products that can highly simulate the fiber structure and chewing characteristics of real meat. Summary of the Invention

[0004] To overcome the shortcomings mentioned above, this invention provides a method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein, producing a product that can highly simulate the fiber structure and chewing characteristics of real meat.

[0005] The objective of this invention is achieved through the following technical solution: A method for preparing microbial meat protein based on Neurospora crassa mycelial protein using the TGase enzymatic method includes the following steps: S101. The mycelial protein biomass of Neurospora crassa is pulverized and passed through a 100-mesh sieve to obtain mycelial protein powder. The mycelial protein powder is mixed with excipients and stirred evenly to obtain a mixture. S102. Add transglutaminase to the mixture, stir evenly, fill the mold, and react at -80°C for 24 h to obtain the product.

[0006] Furthermore, the *Neurospora crassa* is named *Neurospora crassa* HJDF, and its accession number is CGMCC NO:41518.

[0007] Furthermore, the method for preparing mycelial protein biomass in step S101 includes the following steps: S201. Inoculate the culture medium with a suspension of Neurospora crassa spores at an inoculation rate of 1% to 10%, and ferment at 150 to 180 rpm and 28 to 30°C for 3 to 5 days to obtain the fermentation product; S202. The fermentation product obtained in step S201 is washed with ultrapure water, and then subjected to two-stage heat treatment. After heat treatment, the mycelium is completely washed with ultrapure water. S203. The mycelium obtained in step S202 is pre-frozen and freeze-dried at -80°C for 48 h to obtain the desired mycelial protein biomass.

[0008] Furthermore, in step S201, the culture medium consists of: 30 g / L sucrose; 10 g / L yeast extract; 0.5 g / L potassium chloride; 4 g / L potassium dihydrogen phosphate; and 0.5 g / L magnesium sulfate heptahydrate.

[0009] Furthermore, in step S202, the heat treatment method is as follows: first, treat at 65~70°C for 20min~30min, then further heat to 90~95°C and hold for 30min~40min.

[0010] Furthermore, in step S101, the excipients are fish gelatin, sodium alginate, and water.

[0011] Furthermore, in step S101, the amount of mycelial protein powder added is 12%, the amount of fish gelatin added is 2% to 6%, the amount of sodium alginate added is 1% to 4%, and the remainder is water.

[0012] Furthermore, in step S102, the amount of transglutaminase added is 1% to 3%.

[0013] Furthermore, in step S101, the mycelial protein biomass of Neurospora crassa is pulverized by an ultra-micro pulverizer.

[0014] The present invention also provides a microbial protein meat of Neurospora crassa HJDF prepared according to the above preparation method.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This is the first time that mycelial protein from the strain Neurospora crassa HJDF has been developed and used as a core ingredient in artificial meat. This mycelial protein is naturally rich in high-quality protein and dietary fiber, and is low in fat and cholesterol-free. Its safety and nutritional value lay a solid foundation for the health attributes of the final product.

[0016] A three-dimensional gel network structure with excellent elasticity, chewiness, and adhesiveness was successfully constructed through the cross-linking reaction of TGase enzyme under specific low-temperature conditions, combined with the synergistic effect of excipients. This structure can highly simulate the texture of real meat, solving the technical problem of some existing substitutes having a soft texture or lacking chewiness.

[0017] The fermentation, heat treatment, and molding processes provided by this invention are stable and reliable. The resulting product has low cooking loss, only 3% to 5%, indicating good structural stability. It can effectively maintain its shape and juices during cooking and has good processing adaptability.

[0018] This invention provides a method for preparing microbial meat protein using the TGase enzymatic process based on Neurospora crassa mycelial protein. By adjusting the formulation to modify the gel texture properties, the texture of the protein mimics that of real meat, producing a healthier and more sustainable novel artificial meat product. This offers a potential solution to the growing demand for protein and health foods. The final product has a balanced amino acid composition, with the essential amino acid ratio (IAA / TAA) approaching 0.4, indicating it is a high-quality protein source. Simultaneously, the total fatty acid content is significantly lower than that of commercially available fish tofu and other similar products, better meeting the needs of a low-fat, healthy diet. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a diagram showing the growth of Neurospora crassa HJDF in a shake flask.

[0020] Figure 2 This image shows the heat treatment of mycelial protein from Neurospora crassa HJDF.

[0021] Figure 3 The images show the appearance of the microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5.

[0022] Figure 4 The bar chart shows the cooking loss of the microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5.

[0023] Figure 5 The bar chart shows the comparison of the post-steaming elasticity of the microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5 with that of the comparative examples.

[0024] Figure 6 The bar chart shows the post-steam recovery of the microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5, compared with the comparative example.

[0025] Figure 7 This is a bar chart comparing the chewiness of steamed microbial protein meat based on *Neurospora crassa* HJDF mycelium in Examples 3-5 with that of the comparative examples.

[0026] Figure 8 The bar chart shows the post-steam adhesiveness of the microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5 compared with the comparative examples.

[0027] Figure 9 The bar chart shows the post-steam coagulation properties of the microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5, compared with the comparative examples.

[0028] Figure 10 The bar chart shows the comparison between the hardness of the steamed microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5 and the comparative examples.

[0029] Figure 11 The image shows a comparison of the sensory evaluation of the steamed microbial protein meat based on the mycelial protein of Neurospora crassa HJDF in Examples 3-5 with that of the comparative examples. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] Example 1 – Fermentation culture of Neurospora crassa mycelial protein: Prepare the culture medium according to the following formula: sucrose 30 g / L; yeast extract 10 g / L; potassium chloride 0.5 g / L; potassium dihydrogen phosphate 4 g / L; magnesium sulfate heptahydrate 0.5 g / L.

[0033] Collect *Neurospora crassa* spores, dilute with sterile water, determine spore concentration using a hemocytometer, and then dilute to 2.0 × 10⁻⁶. 6 The concentration was determined by counting spores per mL, and the spore solution was inoculated into the culture medium at an inoculum rate of 1%. The medium was then incubated at 29°C and 150 rpm for 3 days in a shaker. The condition after 3 days of fermentation was as follows. Figure 1 As shown.

[0034] After fermentation, the culture was collected and washed three times with ultrapure water to obtain wet-based Neurospora crassa mycelium, which was then used for the preparation of mycelial protein raw materials.

[0035] Example 2 – Preparation of mycelial protein raw material: Add an appropriate amount of ultrapure water to the wet-based mycelium obtained in Example 1 until it becomes a paste. Place it in a water bath at 65-70°C for 20-30 minutes, then further heat it to 90-95°C and hold for 30-40 minutes. The state is shown in the figure. Figure 2 After heat treatment, the mycelial protein was collected using a filter screen, washed three times with ultrapure water and wrung out. The mycelial protein was spread out in a large glass dish, covered with plastic wrap and punctured, pre-frozen overnight at -80°C, and then freeze-dried for 48 hours. The freeze-dried material was collected, pulverized using an ultra-micro pulverizer, and passed through a 100-mesh sieve. The resulting powder was the desired mycelial protein raw material.

[0036] Example 3: The mycelial protein biomass of *Neurospora crassa* was pulverized using an ultrafine pulverizer and passed through a 100-mesh sieve to obtain mycelial protein powder. 0.3 g of fish gelatin was dissolved in 7 g of ultrapure water and stirred in a 40°C water bath until completely dissolved to obtain a fish gelatin solution. 1.2 g of mycelial protein powder and 0.3 g of sodium alginate were dissolved in the obtained fish gelatin solution and stirred evenly to obtain a mixture. 0.3 g of transglutaminase was dissolved in 1 g of water and added to the above mixture. The mixture was stirred evenly, filled into a 25 mm × 25 mm × 25 mm mold, and placed at -80°C for 24 h. Afterward, it was removed, thawed at room temperature, and steamed for 10 min. The finished product is shown below. Figure 3 As shown in TG3.

[0037] Example 4: The mycelial protein biomass of *Neurospora crassa* was pulverized using an ultrafine pulverizer and passed through a 100-mesh sieve to obtain mycelial protein powder. 0.3 g of fish gelatin was dissolved in 7 g of ultrapure water and stirred in a 40°C water bath until completely dissolved to obtain a fish gelatin solution. 1.2 g of mycelial protein powder and 0.3 g of sodium alginate were dissolved in the obtained fish gelatin solution and stirred evenly to obtain a mixture. 0.1 g of transglutaminase was dissolved in 1 g of water and added to the above mixture. The mixture was stirred evenly, filled into a 25 mm × 25 mm × 25 mm mold, and placed at -80°C for 24 h. Afterward, it was removed, thawed at room temperature, and steamed for 10 min. The finished product is shown below. Figure 3 As shown in SA3.

[0038] Example 5: The mycelial protein biomass of *Neurospora crassa* was pulverized using an ultrafine pulverizer and passed through a 100-mesh sieve to obtain mycelial protein powder. 0.4 g of fish gelatin was dissolved in 7 g of ultrapure water and stirred in a 40°C water bath until completely dissolved to obtain a fish gelatin solution. 1.2 g of mycelial protein powder and 0.3 g of sodium alginate were dissolved in the obtained fish gelatin solution and stirred evenly to obtain a mixture. 0.1 g of transglutaminase was dissolved in 1 g of water and added to the above mixture. The mixture was stirred evenly, filled into a 25 mm × 25 mm × 25 mm mold, and placed at -80°C for 24 h. Afterward, it was removed, thawed at room temperature, and steamed for 10 min. The finished product is shown below. Figure 3 As shown in G4.

[0039] Measurement of cooking loss: Let the initial mass of the sample be m1, and the mass after steaming be m2. Calculate the cooking loss using the following formula: Cooking loss (%) = (m1-m2)×100 / m1 Cooking loss results as follows Figure 4 As shown, the cooking loss of the three-component products prepared in Examples 3-5 was between 3% and 5%, indicating that the three-component products prepared in Examples 3-5 performed well under cooking treatment.

[0040] Texture determination: Commercially available fish fillets and fish tofu were purchased as comparative examples. The three product groups prepared in Examples 3-5, along with the commercially available fish fillets and fish tofu, were cut into 12.5 mm × 12.5 mm × 12.5 mm cubes. The texture of the samples was measured using a texture analyzer (TA.XT Plus, Stable Micro Systems E, UK) with a P / 50 probe. Compression was performed at 1 mm / s. -1 The speed was measured at a trigger force of 0.10 N, with a compression deformation of 40%. The measurements were performed at room temperature and repeated four times.

[0041] TPA measurement results are as follows Figure 5 As shown, the results indicate that the elasticity, resilience, chewiness, adhesiveness, cohesiveness, and hardness of the three-component products prepared in Examples 3-5 are close to those of the comparative example, indicating that the three-component products prepared in Examples 3-5 have good textural properties and can simulate real commercially available products.

[0042] Sensory evaluation: Nine participants conducted sensory evaluations of the three products prepared in Examples 3-5 and commercially available fish fillets and fish tofu products. A nine-point scale was used for hedonic testing (0-2: very dislike, 3-6: moderately like, 7-9: very like) and descriptive testing (0-2: not noticeable, 3-6: noticeable, 7-9: very noticeable) to assess color, flavor, texture, and overall acceptability.

[0043] Sensory evaluation results such as Figure 6 As shown, the selected sample lacks a fish-like flavor and has a mushroom-like flavor from fungi. The texture is also too soft. Overall, its acceptability is different from commercially available fish tofu products. When applying it to products, an appropriate amount of flavoring substances can be added to improve its acceptability.

[0044] Nutritional components: The amino acid and protein content of the three products prepared in Examples 3-5 and commercially available fish fillets and fish tofu products were determined. Amino acid content was determined according to GB 5009.124-2016, and protein content was determined according to GB 5009.5-2016. The results are shown in Table 1 below.

[0045] The amino acid content of the three-component products prepared in Examples 3-5 is close to that of the control fish tofu, indicating that the quality of the three-component products prepared in Examples 3-5 is close to that of commercial products in this respect. In addition, the IAA / TAA ratio of the three-component products prepared in Examples 3-5 is close to 0.4, indicating that its protein is of high quality and has actual nutritional value.

[0046] Table 1. Amino acid composition and protein content of the samples Testing items Result Unit <![CDATA[TG3]]> <![CDATA[G4]]> <![CDATA[SA3]]> Fish fillet Fish tofu Aspartic acid (Asp) g / 100g 0.72 0.74 0.66 1.37 0.74 Threonine (Thr) g / 100g 0.37 0.38 0.35 0.62 0.38 Serine (Ser) g / 100g 0.40 0.42 0.37 0.58 0.42 Glutamic acid (Glu) g / 100g 0.92 0.99 0.87 2.30 0.99 Glycine (Gly) g / 100g 0.96 1.13 0.91 0.58 1.13 Alanine (Ala) g / 100g 0.66 0.73 0.62 0.75 0.73 Valine g / 100g 0.29 0.30 0.27 0.54 0.30 Methionine (Met) g / 100g 0.088 0.10 0.081 0.27 0.10 Isoleucine (Ile) g / 100g 0.23 0.23 0.22 0.56 0.23 Leucine (Leu) g / 100g 0.47 0.48 0.44 1.06 0.48 Tyrosine (Tyr) g / 100g 0.19 0.19 0.18 0.45 0.19 Phenylalanine (Phe) g / 100g 0.29 0.29 0.27 0.52 0.29 Histidine (His) g / 100g 0.29 0.27 0.26 0.28 0.27 Lysine (Lys) g / 100g 0.64 0.66 0.60 1.59 0.66 Arginine (Arg) g / 100g 0.59 0.65 0.56 0.86 0.65 Proline (Pro) g / 100g 0.63 0.73 0.60 0.50 0.73 IAA / TAA % 0.42 0.41 0.42 0.49 0.41 Protein content g / 100g 8.85 9.86 8.96 13.4 11.2 The fatty acid content of the three-component products prepared in Examples 3-5 and commercially available fish fillets and fish tofu products was determined. The fatty acid content was measured according to GB 5009.168-2016, and the results are shown in Table 2 below. The total fatty acid content of the three-component products prepared in Examples 3-5 was close to that of the fish fillets, while the total fatty acid content of the fish tofu was very high. This may be related to the low-fat characteristics of mycelial protein, indicating that this product is healthier than commercially available fish tofu in this respect.

[0047] Table 2 Fatty acid composition of samples Testing items Result Unit <![CDATA[TG3]]> <![CDATA[G4]]> <![CDATA[SA3]]> Fish fillet Fish tofu C14:0 g / 100g ND ND ND 0.00823 0.0746 C16:0 g / 100g 0.115 0.128 0.0766 0.133 1.127 C16:1n7 g / 100g 0.00795 0.00930 0.00626 ND 0.104 C17:0 g / 100g ND ND ND ND 0.0131 C18:0 g / 100g 0.0384 0.0416 0.0191 0.0426 0.425 C18:1n9c g / 100g 0.147 0.176 0.125 0.14 1.427 C18:2n6c g / 100g 0.183 0.215 0.149 0.0574 0.92 C20:0 g / 100g ND ND ND ND 0.0128 C20:1 g / 100g ND ND ND 0.00473 0.0306 C18:3n3 g / 100g 0.00846 0.0109 0.00724 0.00377 0.107 C20:2 g / 100g ND ND ND ND 0.0163 C20:3n6 g / 100g ND ND ND 0.00816 ND C20:4n6 g / 100g ND ND ND 0.0236 0.0334 C24:0 g / 100g 0.00714 0.00928 ND ND ND C20:5n3 g / 100g ND ND ND ND 0.0547 C22:6n3 g / 100g ND ND ND 0.00629 0.0787 Total fatty acids g / 100g 0.506 0.590 0.383 0.427 4.42 Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing microbial meat protein based on TGase enzymatic method using Neurospora crassa mycelial protein, characterized in that, Includes the following steps: S101. The mycelial protein biomass of Neurospora crassa is pulverized and passed through a 100-mesh sieve to obtain mycelial protein powder. The mycelial protein powder is mixed with excipients and stirred evenly to obtain a mixture. S102. Add transglutaminase to the mixture, stir evenly, fill the mold, and react at -80°C for 24 h to obtain the product.

2. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 1, characterized in that, The Neurospora crassa was named Neurospora crassa HJDF, and its accession number is CGMCC NO: 41518.

3. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 1 or 2, characterized in that, The method for preparing mycelial protein biomass in step S101 includes the following steps: S201. Inoculate the culture medium with a suspension of Neurospora crassa spores at an inoculation rate of 1% to 10%, and ferment at 150 to 180 rpm and 28 to 30°C for 3 to 5 days to obtain the fermentation product; S202. The fermentation product obtained in step S201 is washed with ultrapure water, and then subjected to two-stage heat treatment. After heat treatment, the mycelium is completely washed with ultrapure water. S203. The mycelium obtained in step S202 is pre-frozen and freeze-dried at -80°C for 48 h to obtain the desired mycelial protein biomass.

4. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 3, characterized in that, In step S201, the culture medium consists of: 30 g / L sucrose; 10 g / L yeast extract; 0.5 g / L potassium chloride; 4 g / L potassium dihydrogen phosphate; and 0.5 g / L magnesium sulfate heptahydrate.

5. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 4, characterized in that, In step S202, the heat treatment method is as follows: first, treat at 65~70°C for 20min~30min, then further heat to 90~95°C and hold for 30min~40min.

6. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 3, characterized in that, In step S101, the excipients are fish gelatin, sodium alginate, and water.

7. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 6, characterized in that, In step S101, the amount of mycelial protein powder added is 12%, the amount of fish gelatin added is 2% to 6%, the amount of sodium alginate added is 1% to 4%, and the remainder is water.

8. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 7, characterized in that, In step S102, the amount of transglutaminase added is 1% to 3%.

9. The method for preparing microbial meat protein based on the TGase enzymatic method of Neurospora crassa mycelial protein according to claim 8, characterized in that, In step S101, the mycelial protein biomass of Neurospora crassa is pulverized by an ultra-micro pulverizer.

10. A type of Neurospora crassa HJDF microbial protein meat prepared by the preparation method according to any one of claims 1-9.

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