A method for improving color and texture of plant-based meat products using recombinant porcine myoglobin

Abstract

The application discloses a method for improving color and texture of plant-based meat products by using recombinant pig myoglobin, and belongs to the field of food biotechnology. In view of the problems of light color and poor texture of plant-based meat products, the recombinant pig myoglobin prepared by fermentation of Pichia pastoris is added into a mixture of soybean protein concentrate and gluten protein at a proportion of 0.5% or 1.0%, and then high-moisture extrusion molding is performed. The redness of the obtained product is significantly improved, and the color is close to that of real meat; the fiber structure and mechanical anisotropy are not affected under the addition amount of 0.5%. The method effectively improves the sensory appearance of plant-based meat products, and provides technical support for the development of high-quality alternative meat.

Description

Technical Field

[0001] This invention relates to a method for improving the color and texture of plant-based meat products using recombinant porcine myoglobin, belonging to the fields of food engineering and enzyme engineering technology. Background Technology

[0002] Plant-based meat products typically use plant proteins such as soy protein isolate and pea protein as the main raw materials, mimicking the fibrous texture of animal muscle through processing methods such as extrusion and heat-induced gelation. However, existing products still differ significantly from real meat in key quality attributes such as color, flavor, and texture, with color being particularly prominent—plant proteins are naturally light in color, often turning pale yellow or grayish-white after heat processing, lacking the bright red color characteristic of animal meat. Currently, the industry often improves the product's appearance by adding natural pigments such as betaine and red yeast rice, or nitrites for color development, but these solutions all have significant drawbacks: natural pigments have poor thermal stability, beginning to degrade at around 50°C, easily fading the red color to brown; while nitrites can fix the color, they pose potential safety hazards. In addition, existing products generally contain excessive additives, resulting in a significant difference in taste from real meat, severely restricting consumer acceptance and market promotion.

[0003] Myoglobin is a heme-binding protein found in animal muscle tissue. Its heme prosthetic group gives it a red color through iron-oxygen binding, making it a core carrier for meat color formation. Studies have shown that adding myoglobin to plant-based meat products can significantly deepen and redden the product color, giving it a color similar to real meat. In February 2025, the US FDA officially approved myoglobin as a color additive for plant-based minced meat and minced poultry-like products, providing a clear regulatory basis for their commercial application. Existing research has found that porcine myoglobin (P-Mb) derived from Pichia pastoris has good safety and no significant sensitization in animals. Therefore, providing a method for improving the color and texture of plant-based meat products using recombinant porcine myoglobin is of great significance for improving the quality and promoting the market of plant-based meat products. Summary of the Invention

[0004] This invention addresses the issues of poor color and lack of fibrous chewiness in plant-based meat products by adding recombinant porcine myoglobin, giving the products a color resembling real meat. This visually optimizes the sensory appearance of plant-based meat products and improves the texture of the product without damaging the original structure and mechanical anisotropy of the fibers.

[0005] The purpose of this invention is to provide a method for improving the color and / or texture of plant-based meat products by adding recombinant porcine myoglobin to plant protein raw materials and processing them through high-moisture extrusion to produce plant-based meat products.

[0006] This invention also provides a method for preparing plant-based meat products, comprising the following steps: (1) Add recombinant porcine myoglobin to the plant protein raw material and mix evenly to obtain a mixture; (2) The mixture is subjected to high-moisture extrusion processing to obtain plant-based meat products; The amount of recombinant porcine myoglobin added is 0.5% to 1.0% of the total mass of the plant protein raw materials.

[0007] In one embodiment, the plant protein raw material comprises soy protein concentrate and gluten protein.

[0008] In one embodiment, the mass ratio of soy protein concentrate to gluten protein is 7:3.

[0009] In one embodiment, the moisture content of the material in the high-moisture extrusion process is 50%.

[0010] In one embodiment, the extrusion temperature in the high-moisture extrusion process is set sequentially to 25°C, 40°C, 60°C, 90°C, 110°C, 130°C, and 140°C, and the end cooling temperature is 75°C.

[0011] In one embodiment, the screw speed in the high-moisture extrusion process is 180 rpm.

[0012] The present invention also provides plant-based meat products prepared by the above method.

[0013] In one embodiment, the plant-based meat product includes plant-based hamburger patties, plant-based minced meat, or plant-based meatballs.

[0014] The present invention also provides the application of the method in the food industry.

[0015] The beneficial effects of this invention are: This invention utilizes the color-producing effect of the heme cofactor in recombinant porcine myoglobin to add recombinant porcine myoglobin to plant-based meat products, followed by high-moisture extrusion processing. This allows the plant-based meat products to achieve a red color close to that of real meat. After adding 0.5% recombinant porcine myoglobin, the lightness value (L) of the prepared plant-based meat products decreased from 56.8 to 50.7, while the redness value (a) increased from 5.5 to 6.4. The color changed from dark yellow to a flesh-colored hue, effectively overcoming problems such as the pale or yellowish color after heat processing of plant proteins, the poor thermal stability of natural pigments, and the safety hazards associated with nitrites.

[0016] This invention also optimizes the amount of porcine myoglobin added. Adding it at a level of 0.5% does not damage the layered fiber structure and mechanical anisotropy formed by high-moisture extrusion processing. The anisotropy index of the product's fracture stress and fracture strain does not change significantly, indicating that it can maintain a fibrous chewiness and texture similar to muscle while obtaining good color. This solves the problems of poor color and lack of fibrous texture in existing plant-based meat products. Attached Figure Description

[0017] Figure 1 Macroscopic structures of SPC-WG extrudates from different groups.

[0018] Figure 2 Fracture stress, fracture strain, and anisotropy index of SPC-WG extrusions from different groups.

[0019] Figure 3 CLSM images of SPC-WG extrudates from different groups. Detailed Implementation

[0020] Soy protein concentrate (SPC) was purchased from Kerry Protein Industries, Qinhuangdao, Hebei Province (its composition is 67.07% protein, 24.96% carbohydrates, 6.59% moisture, 0.56% ash, and 0.82% fat). Wheat gluten protein (WG) was purchased from Shandong Binzhou Zhongyu Food Co., Ltd., and its composition was 6.0% moisture, 85.6% dry-basis protein, 12.7% polysaccharide, 0.9% ash, and 0.8% fat. B-Hb was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a total protein content of 95% and a purity of 98%. Example 1: Preparation of Recombinant Porcine Myoglobin (1) The highly efficient mitochondrial targeting signal peptide MTS181 and heme synthesis-related transport protein F2QQD0 were combined and applied to the inducible expression system of P-Mb to construct the engineered strain P181-D0.

[0021] (2) Feed-on-batch fermentation was carried out in a 10 L fermenter using BSM medium. By optimizing the feeding strategy during the methanol induction phase (using μ-STAT exponential feeding with an initial feeding coefficient F0=20), the induction pH (maintaining pH 5.5 during the glycerol growth phase and adjusting it to pH 6.0 during the methanol induction phase), and the medium salt concentration (2 / 3 BSM), efficient secretory expression was achieved. After 132 h of fermentation, the P-Mb yield in the fermentation broth reached 1.68 g / L, and the heme binding rate was 55.2 ± 3.2%.

[0022] (3) After fermentation, the cells were removed by centrifugation, and the supernatant was concentrated by ultrafiltration, purified by Ni-NTA affinity chromatography (elution with 200 mM imidazole), diluted with PBS buffer and desalted and concentrated to finally obtain recombinant P-Mb samples for evaluation of plant-based food applications.

[0023] Example 2 (1) Raw material pretreatment Soy protein concentrate and gluten protein were separately pulverized and sieved, then mixed evenly at a mass ratio of 7:3 and set aside. Recombinant porcine myoglobin freeze-dried powder was refrigerated at 4°C for later use.

[0024] (2) High moisture extrusion processing High-moisture extrusion was performed using a twin-screw co-rotating extruder with a screw diameter of 15.5 mm and a length-to-diameter ratio of 25:1. The extruder barrel was equipped with seven independently heated temperature control zones. Water was continuously added to the extruder through a water injection system to maintain the final moisture content of the material at 50%. P-Mb was pre-dissolved in water and added at concentrations of 0.5% and 1.0% (w / w), respectively, and the resulting samples were labeled as the 0.5% P-Mb group and the 1.0% P-Mb group. A control group was prepared by adding 0.5% (w / w) B-Hb, and the samples were labeled as the 0.5% B-Hb group; samples without added P-Mb or B-Hb served as a blank control group. Based on preliminary test results, the extrusion conditions were set as follows: feed rate 300 g / h, screw speed 180 rpm, and barrel temperatures successively set to 25 ℃, 40 ℃, 60 ℃, 90 ℃, 110 ℃, 130 ℃, and 140 ℃. A cooling die (5 mm × 20 mm × 240 mm, height × width × length) was connected to the end of the extruder, and the temperature was maintained at 75 ℃ through circulating water. After the extrusion process stabilized, the extrudate was collected and placed in a self-sealing bag for storage at -18 ℃ for later use.

[0025] (3) Evaluation of coloring effect The extrudate was stored at -18℃ for later use. Color parameters of the extrudate surface were measured using a colorimeter, the microstructure was observed using a laser confocal scanning microscope, and tensile strength was measured and anisotropy index calculated using a texture analyzer. The results showed that the lightness value (L) of the sample with 0.5% recombinant porcine myoglobin added decreased from 56.8 to 50.7, while the redness value (a) increased from 5.5 to 6.4, and the color changed from dark yellow to a flesh-colored hue. Table 1. Color parameters of plant-based meat products with different amounts of recombinant porcine myoglobin.

[0026] (4) Evaluation of texture properties By comparing the macroscopic structure of SPC-WG extrudates, the control group and the samples with added 0.5% P-Mb and 0.5% B-Hb all showed a relatively obvious layered structure arranged along the extrusion direction, while the 1% P-Mb group showed a uniform gel-like structure, and no clear layering phenomenon was observed inside the sample.

[0027] To further evaluate the degree of microstructure and mechanical properties of the samples, the fracture stress (σ) and fracture strain (ε) of the SPC-WG extrudate were measured in parallel and perpendicular to the extrusion direction, and the anisotropy index (AI) was calculated. (See attached...) Figure 2 As shown, the AI ​​value of σ in the control group extruder was 1.4, while the AI ​​value of ε was 1.0, indicating that the sample had a certain directionality in fracture stress, but its overall microstructure was limited. After adding 1% P-Mb, the AI ​​value of σ decreased to 1.1, while the AI ​​value of ε did not change significantly compared with the control group, indicating that the high amount of P-Mb reduced the mechanical differences of the sample in different directions and weakened its layered microstructure. In contrast, the AI ​​values ​​of σ and ε did not change significantly in the samples with added 0.5% P-Mb and 0.5% B-Hb.

[0028] Overall, the microstructure of SPC-WG extrudates exhibits a relatively clear V-shaped layered structure, consistent with the structural characteristics formed by the combined effects of material flow, orientation, and cooling during high-moisture extrusion. Adding 0.5% P-Mb or 0.5% B-Hb did not significantly alter the layered banded structure of the samples, indicating that at this level, the heme-binding protein had minimal interference with the microstructure of the system. However, when the P-Mb addition increased to 1%, the original banded fiber structure essentially disappeared, and the structure became more homogeneous, suggesting that higher concentrations of P-Mb significantly affect the phase distribution and structure formation in high-moisture extrusion systems.

[0029] Therefore, in summary, 0.5% is a more suitable addition amount.

[0030] Example 3: Preparation of plant-based hamburger pork patties using porcine myoglobin In this embodiment, the total amount of the plant-based hamburger pork patty recipe is calculated as 1 kg: Take 300g of soy protein concentrate, add 2-3 times its weight of water for rehydration, soak for 10-20 minutes, drain excess water, and set aside. Mix 70g of soy protein isolate with 80-150g of wheat gluten, add water, and stir in a high-speed shear mixer for 5-10 minutes to form a uniform protein gel system.

[0031] Dissolve 5g of recombinant porcine myoglobin in 30g of water to form a myoglobin solution. Add this solution to the protein gel system described above and continue stirring for 2-3 minutes until evenly dispersed. Add rehydrated textured soybean protein, 20g of methylcellulose, 20g of konjac gum, 120g of vegetable oil, 1g of salt, and 13g of spices to the mixture. Continue mixing in a high-shear mixer for 5-8 minutes until a dough-like material with viscoelasticity and resilience is formed.

[0032] Transfer the dough-like material to a patty forming machine and press it into hamburger patty embryos of 80-120g each. Freeze the formed hamburger patty embryos at -20°C to -30°C for 1-2 hours to set the shape. Vacuum seal the frozen and set plant-based hamburger pork patties and store them at -20°C.

[0033] Example 4: Preparation of plant-based meat paste (dumpling / steamed bun filling) using porcine myoglobin In this embodiment, the total amount of the plant-based minced meat formula is calculated as 1 kg: Take 200g of soy protein concentrate, add 2-3 times its weight of water for rehydration, soak for 10-20 minutes, drain excess water, and set aside. Take 150g of textured soy protein, add 2-3 times its weight of water for rehydration, soak for 10-20 minutes, drain excess water, and shred into thin strands, and set aside.

[0034] Mix 50g of soy protein isolate with 40g of wheat gluten, add water to bring the total volume to 1000g, and chop in a food processor for 5-10 minutes to form a homogeneous protein gel system. Dissolve 5g of recombinant porcine myoglobin in 30g of water to form a myoglobin solution, add it to the protein gel system above, and continue chopping for 2-3 minutes until evenly dispersed.

[0035] Add the rehydrated soy protein concentrate, textured soy protein, 5g methylcellulose, 3g carrageenan, 7g konjac gum, 70g vegetable oil, 12g salt, 10g sugar, 15g light soy sauce, 5g dark soy sauce, 10g oyster sauce, 3g thirteen-spice powder, 15g minced ginger, and 30g chopped green onions to the above mixture. Continue chopping in a food processor for 5-10 minutes until a filling-like material with viscoelasticity, cohesiveness, and toughness is formed.

[0036] Transfer the above filling materials to a container and let it stand at 0-4°C for 30-60 minutes to allow the filling to fully absorb the flavor and set. Use the set plant-based minced meat filling to wrap dumplings or buns. After shaping, freeze at -20°C to -30°C for 1-2 hours to set the product. Vacuum seal the frozen and set plant-based dumplings or buns and store them at -20°C.

[0037] Example 5: Preparation of plant-based meatballs using porcine myoglobin In this embodiment, the total amount of the plant-based meatball formula is calculated as 1 kg: Take 250g of soy protein concentrate, add 2-3 times its weight of water for rehydration, soak for 10-20 minutes, drain excess water, and set aside.

[0038] Mix 70g of soy protein concentrate and 75g of wheat gluten evenly, add water to bring the total volume to 1000g, and stir in a high-speed shear mixer for 5-10 minutes to form a homogeneous protein gel system. Dissolve 5g of recombinant porcine myoglobin in 20-50g of water to form a myoglobin solution, add it to the above protein gel system, and continue stirring for 2-3 minutes until evenly dispersed.

[0039] Add rehydrated soy protein concentrate, 60g potato starch, 20g methylcellulose, 70g vegetable oil, 12g salt, 10g white sugar, 20g spices, and 1g transglutaminase to the above mixture. Continue mixing in a high-shear mixer for 8-12 minutes until a viscoelastic and resilient meat-like material is formed.

[0040] Transfer the above-mentioned minced meat material to a meatball forming machine and extrude it into round meatball embryos of 15-25g each. Place the formed meatball embryos in 80-90°C hot water and cook for 8-12 minutes to set and cook the meatballs. Remove the cooked meatballs and quickly transfer them to ice water to cool for 5-10 minutes, then drain off the surface moisture. Freeze the cooled meatballs at -20°C to -30°C for 2-4 hours until they are completely frozen. Vacuum seal the frozen and set plant-based meatballs and store them at -20°C.

[0041] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for improving the color and / or texture of plant-based meat products, characterized in that, Recombinant porcine myoglobin is added to plant protein raw materials and processed through high-moisture extrusion to produce plant-based meat products.

2. A method for preparing plant-based meat products, characterized in that, Includes the following steps: (1) Add recombinant porcine myoglobin to the plant protein raw material and mix evenly to obtain a mixture; (2) The mixture is subjected to high-moisture extrusion processing to obtain plant-based meat products; The amount of recombinant porcine myoglobin added is 0.5% to 1.0% of the total mass of the plant protein raw materials.

3. The method according to claim 2, characterized in that, The plant protein raw materials include soy protein concentrate and gluten protein.

4. The method according to claim 3, characterized in that, The mass ratio of soy protein concentrate to gluten protein is 7:

3.

5. The method according to claim 2, characterized in that, The moisture content of the material in the high-moisture extrusion process is 50%.

6. The method according to claim 2, characterized in that, In the high-moisture extrusion process, the extrusion temperatures are set sequentially to 25℃, 40℃, 60℃, 90℃, 110℃, 130℃ and 140℃, and the final cooling temperature is 75℃.

7. The method according to claim 6, characterized in that, The screw speed in the high-moisture extrusion process is 180 rpm.

8. Plant-based meat products prepared by the method described in any one of claims 2 to 7.

9. The plant-based meat product according to claim 8, characterized in that, Includes plant-based pork patties, plant-based minced meat, and plant-based meatballs.

10. The application of the method according to any one of claims 2 to 7 in the food industry.

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