Chlorella pyrenoidosa composite nutritional plant meat rich in organic selenium and omega-3 fatty acid and preparation method thereof

Abstract

The invention discloses chlorella pyrenoidosa compound nutritional plant meat rich in organic selenium and omega-3 fatty acid and a preparation method thereof. Comprising the following steps: (1) inoculating chlorella pyrenoidosa into a culture medium containing an inorganic selenium source and an omega-3 fatty acid precursor substance, carrying out fermentation culture, collecting algae, and carrying out wall breaking treatment and drying to obtain wall-broken chlorella pyrenoidosa powder rich in organic selenium and omega-3 fatty acid; 2) uniformly mixing the wall-broken chlorella pyrenoidosa powder with a vegetable protein raw material to obtain a mixture; the mass of the wall-broken chlorella pyrenoidosa powder accounts for 5-15% of the total mass of the mixture; and (3) continuously feeding the mixture into a double-screw extruder, synchronously injecting water to carry out high-moisture extrusion treatment, extruding through a cooling mold after extrusion, and curing and forming, thereby obtaining the chlorella pyrenoidosa compound nutrient plant meat rich in organic selenium and omega-3 fatty acid. By coupling microalgae targeted nutrition enrichment, high-pressure homogenization wall breaking and double-screw high-moisture extrusion technologies, the comprehensive improvement of the texture, nutrition and digestibility of the plant meat is realized.

Description

Technical Field

[0001] This invention relates to the field of plant-based food processing technology, and in particular to a protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids, and its preparation method. Background Technology

[0002] In recent years, with global economic development, rapid population growth, and increasing public concern for health, environmental protection, and animal welfare, plant-based meat (plant-based meat) has experienced rapid development as a sustainable alternative to traditional animal meat. Currently, high-moisture extrusion (HME) is the most core and cutting-edge technology for producing plant-based meat. This technology, under the influence of high temperature, high pressure, and high shear, causes the intermolecular disulfide bonds and hydrogen bonds of plant proteins such as soybeans and peas to depolymerize and rearrange, subsequently undergoing directional cross-linking in a cooling mold to form a fibrous structure with anisotropy similar to animal muscle tissue.

[0003] However, existing high-moisture extruded plant-based meat products still have significant limitations in terms of nutrition and texture. First, from a nutritional perspective, while single plant protein sources such as soybeans are rich in protein, they generally lack certain key micronutrients found in animals (especially deep-sea fish), such as highly bioavailable organic selenium and omega-3 polyunsaturated fatty acids (EPA, DHA, ALA, etc.) beneficial to cardiovascular health. Second, the conventional addition of inorganic nutritional supplements not only has low absorption rates, but excessive inorganic selenium (such as sodium selenite) can also easily cause toxicity.

[0004] Microalgae (such as *Chlorella proteoglycans*) are considered highly promising future food substrates due to their high protein content, balanced amino acid ratios, and ease of enriching specific nutrients through directed fermentation. Studies have shown that *Chlorella* can efficiently convert inorganic selenium in its culture medium into safe, highly absorbable organic selenium (such as selenomethionine); simultaneously, by adjusting the carbon-nitrogen ratio and adding precursor substances, the accumulation of ω-3 fatty acids in its cells can be significantly increased. However, directly applying microalgae to high-moisture extruded plant-based meat still faces three major technical bottlenecks:

[0005] First, Chlorella has a tough cellulose cell wall, and if it is not effectively broken down, the human body will have a very low rate of digestion and absorption of its rich nutrients.

[0006] Second, the high temperature and high shear environment during the high moisture extrusion process can easily cause the oxidation, degradation and loss of heat-sensitive nutrients such as organic selenium and ω-3 fatty acids.

[0007] Third, microalgae cells and their rich polysaccharide and lipid components can severely interfere with the fiber rearrangement of plant proteins in the high-moisture extrusion cold zone mold. Academic research shows that when the amount of untreated algae powder added exceeds a certain proportion, it will hinder protein cross-linking, resulting in extruded products that are loose in texture, resembling "dough" or "lumps," completely losing the tearing and chewiness of meat.

[0008] Therefore, how to develop a plant-based meat that has both high content of organic selenium and ω-3 fatty acids, excellent fiber texture and high digestibility by using reasonable algae enrichment culture and cell wall breaking process, and accurately matching it with high moisture extrusion technology parameters, is a technical problem that urgently needs to be solved in the current plant-based food field. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a protein-nucleated Chlorella complex nutritional plant-based meat rich in organic selenium and ω-3 fatty acids, and its preparation method. By coupling microalgae targeted nutrient enrichment, high-pressure homogenization and cell wall disruption, and twin-screw high-moisture extrusion technology, the plant-based meat achieves a comprehensive improvement in texture, nutrition, and digestibility.

[0010] The objective of this invention is achieved as follows: a method for preparing a protein-nucleated Chlorella complex nutritional plant-based meat rich in organic selenium and ω-3 fatty acids, comprising the following steps:

[0011] (1) Preparation of algal powder: Chlorella pyrenoidosa was inoculated into a culture medium containing inorganic selenium source and ω-3 fatty acid precursor substances for fermentation culture. After collecting the algae, the cell wall was broken and dried to obtain Chlorella pyrenoidosa powder rich in organic selenium and ω-3 fatty acids.

[0012] (2) Mixing: The cell wall broken Chlorella protein powder obtained in step (1) is mixed evenly with the plant protein raw material to obtain a mixture; wherein, the mass of the cell wall broken Chlorella protein powder accounts for 5% to 15% of the total mass of the mixture;

[0013] (3) Extrusion molding: The mixture obtained in step (2) is continuously fed into a twin-screw extruder and water is injected simultaneously for high-moisture extrusion treatment. After extrusion, it is extruded through a cooling mold and solidified to obtain an extruded material with a moisture content of 50%~65%, namely, protein-core Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids.

[0014] Furthermore, the inorganic selenium source mentioned in step (1) is sodium selenite, and the concentration of the inorganic selenium source added to the culture medium is 10~25 mg / L; the ω-3 fatty acid precursor is flaxseed oil or α-linolenic acid, and the mass concentration of the ω-3 fatty acid precursor in the culture medium is 1%~3%.

[0015] Furthermore, the fermentation culture conditions described in step (1) are: cultured at 25~28℃ for 4~7 days; the cell wall breaking treatment specifically involves: mixing the collected algae with water to prepare a suspension, and using a high-pressure homogenizer to perform 3~5 homogenization cycles at a pressure of 600~850 bar, so that the average particle size of the broken Chlorella pyrenoidosa is reduced to 1.0~2.0 μm.

[0016] Furthermore, the fermentation culture conditions in step (1) are: cultured at 25~28℃ for 4~7 days; the cell wall breaking treatment specifically involves: mixing the collected algae with water to prepare a suspension, and using a high-pressure homogenizer to perform 3~5 homogenization cycles at a pressure of 600~850 bar, so that the average particle size of the broken Chlorella pyrenoidosa is reduced to 1.0~2.0 μm.

[0017] Furthermore, the plant protein raw material mentioned in step (2) is one or more of soy protein isolate, pea protein isolate, wheat gluten and peanut textured protein; the total protein content of the plant protein raw material is ≥85%.

[0018] Furthermore, the specific parameters of the high moisture extrusion treatment in step (3) are as follows: the fixed rotation speed of the twin screw is 150~200 rpm; the heating zone temperature of the twin screw extruder from the feeding section to the die section is set to 35~60℃, 80~100℃, 120~140℃ and 150~170℃ respectively; and the temperature of the cooling die is controlled at 40~50℃.

[0019] Furthermore, the mixture in step (2) also contains edible polysaccharides and edible vegetable oil; the mass of the edible polysaccharides accounts for 0.5% to 3% of the total mass of the mixture, and the mass of the edible vegetable oil accounts for 2% to 10% of the total mass of the mixture.

[0020] Furthermore, the edible polysaccharide is one or more of xanthan gum, carrageenan, sodium alginate, and locust bean gum; the edible vegetable oil is one or more of soybean oil, sunflower seed oil, corn oil, flaxseed oil, and coconut oil.

[0021] Furthermore, the specific operation of mixing in step (2) is as follows: place each raw material in a powder mixer, mix and stir at a speed of 150~250 rpm for 20~40 minutes, and adjust the pH value of the mixture to 6.5~7.5.

[0022] This invention also provides a protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids, prepared by the above method.

[0023] Compared with existing technologies, the beneficial effects of this invention are as follows: 1) Targeted nutrient enrichment and high bioavailability: This invention introduces 10-25 mg / L sodium selenite and ω-3 precursor substances during the cultivation stage of Chlorella proteoglycans. Utilizing the efficient biotransformation of microalgae, potentially toxic inorganic selenium is converted into highly safe and easily absorbed organic selenium (such as selenomethionine), and a large amount of ω-3 polyunsaturated fatty acids are accumulated in vivo. Simultaneously, a high-pressure homogenization cell wall disruption process at 600-850 bar completely breaks down the hard microalgal cellulose cell walls, reducing the average particle size of the algal powder to 1.0-2.0 μm, greatly releasing intracellular nutrients and significantly improving the in vitro digestibility and bioavailability of the plant-based meat final product in the human gastrointestinal environment.

[0024] 2) Overcoming the bottleneck of texture degradation to achieve a superior biomimetic muscle fiber structure: Adding algae powder to conventional high-moisture extrusion processes easily interferes with the rearrangement of disulfide and hydrogen bonds in plant proteins, resulting in a loose, "dough-like" extrudate. This invention not only precisely controls the amount of cell-wall-broken algae powder added within the golden range of 5% to 15%, but also adjusts the pH of the mixture to a neutral range (6.5 to 7.5) far from the isoelectric point of proteins such as soybeans. Furthermore, it cleverly utilizes 0.5% to 3% of edible macromolecular polysaccharides (such as xanthan gum) and 2% to 10% of edible vegetable oil to exert synergistic plasticizing and viscoelasticity-regulating effects, successfully guiding the depolymerized protein molecules to undergo highly directional cross-linking and fibrous rearrangement in a cooling mold at 40 to 50°C. The resulting plant-based meat possesses a highly anisotropic three-dimensional network fiber structure, giving the product excellent tearing texture, chewiness, and elasticity comparable to real animal muscle.

[0025] 3) Precise extrusion process effectively protects heat-sensitive nutrients: This invention achieves precise coupled control of the temperature (gradually increasing from 35℃ to 170℃) and screw speed (150~200 rpm) in each section of the parallel twin-screw extruder. This ensures that the plant protein fully melts and depolymerizes in the high-temperature, high-shear cooking zone while shortening the residence time of the material in the extreme high-temperature zone. Combined with rapid cooling and solidification by the long cooling die, this maximally inhibits the thermal oxidative degradation of heat-sensitive nutrients such as organic selenium and ω-3 fatty acids during extrusion, ensuring excellent nutrient preservation in the final product. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1Comparison of the appearance of protein-core Chlorella complex nutrient plant-based meat patties prepared by different processing techniques after cooking: (a) Unbroken algae powder + no polysaccharide / oil group (UAN); (b) High-pressure homogenization + no polysaccharide / oil group (HAN); (c) Algae powder with excessive addition + no polysaccharide / oil group (EAN); (d) Unbroken algae powder + polysaccharide / oil synergistic group (UAP); (e) High-pressure homogenization + polysaccharide / oil synergistic group (HAP); (f) Algae powder with excessive addition + polysaccharide / oil synergistic group (EAP).

[0028] Figure 2 Low-field nuclear magnetic resonance (LF-NMR) spectra of water distribution in protein-nucleated Chlorella composite nutrient plant meat prepared by different processing techniques; T2b: bound water; T21: immobilized water; T22: free water; T23: structural water.

[0029] Figure 3 Protein secondary structure analysis of protein-nucleated Chlorella complex nutritional plant meat prepared by different processing techniques; Figure 3 a is the Fourier transform infrared (FTIR) spectrum; Figure 3 b is a bar chart showing the relative content of protein secondary structures. 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, all raw materials involved in the embodiments and comparative examples of this invention can be obtained commercially, and all equipment and analytical instruments used are conventional equipment and instruments in the art.

[0032] Performance testing and evaluation methods:

[0033] Determination of organic selenium content: Referencing GB 5009.93-2017 National Food Safety Standard for the Determination of Selenium in Food, and using high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for qualitative and quantitative analysis of total selenium and organic selenium (selenomethionine, selenocysteine, etc.), the mass percentage of organic selenium in total selenium was calculated.

[0034] ω-3 fatty acid content determination: Gas chromatography (GC) was used, and extraction, methylation and determination were performed in accordance with the "GB 5009.168-2016 National Food Safety Standard - Determination of Fatty Acids in Food".

[0035] In vitro digestibility (bioaccessibility) determination: The internationally recognized INFOKES standardized in vitro simulated gastrointestinal digestion model was used for testing. After digestion with simulated gastric juice (containing pepsin, pH 2.0, reaction time 2 h) and simulated intestinal juice (containing pancreatic enzymes and bile salts, pH 7.0, reaction time 2 h), the samples were centrifuged, and the supernatant was collected. The soluble nitrogen content was determined by the Kjeldahl method, and the in vitro digestibility of the protein was calculated.

[0036] Texture properties (TPA) and fiber drawability testing: A dual compression test was conducted using a texture analyzer (equipped with a P / 36R cylindrical probe) to determine the hardness, elasticity, cohesiveness, and chewiness of the samples. The extrudate was cut into strips along the extrusion direction, and the anisotropic fiber structure was evaluated using a combination of sensory evaluation and the instrument's transverse / longitudinal shear force ratio (fiber drawability index). The closer the fiber drawability index is to or greater than 1.5, the closer the fiber structure is to real animal muscle.

[0037] Example 1

[0038] A protein-rich Chlorella complex nutrient plant meat, rich in organic selenium and ω-3 fatty acids, is prepared by the following steps:

[0039] (1) Preparation of algal powder: Chlorella proteoglycans were inoculated into a culture medium containing 15 mg / L sodium selenite and 2% (w / w) linseed oil and fermented at 25°C for 5 days. The algae were collected, water was added to prepare a suspension, and a high-pressure homogenizer was used to perform four homogenization cycles at 750 bar. The suspension was then spray-dried to obtain algal powder with an average particle size of 1.5 μm.

[0040] (2) Mixing: Place 10% of broken cell wall protein Chlorella powder, 83.5% of soy protein isolate (total protein content 90%), 1.5% of xanthan gum and 5% of soybean oil in a powder mixer and mix at 200 rpm for 30 minutes. Adjust the pH to 7.0 to obtain the mixture (the percentages are all mass fractions of the total mass of the mixture).

[0041] (3) Extrusion molding: The mixture is continuously fed into a parallel twin-screw extruder, and the amount of water added is controlled to ensure that the total moisture content of the material reaches 58%. The twin-screw speed is set to 180 rpm, and the heating zone temperatures from the feed section to the die section are set to 45℃, 90℃, 130℃, and 160℃, respectively. After the material melts, it enters a cooling die with the temperature controlled at 45℃ for extrusion and solidification to obtain plant-based meat.

[0042] Example 2

[0043] A protein-rich Chlorella complex nutrient plant meat, rich in organic selenium and ω-3 fatty acids, is prepared by the following steps:

[0044] (1) Preparation of algal powder: The concentration of sodium selenite in the culture medium was 10 mg / L, the concentration of α-linolenic acid was 1%, and the fermentation culture was carried out at 28℃ for 4 days. The homogenization pressure was 600 bar, and the cycle was repeated 3 times to obtain algal powder with an average particle size of 2.0 μm.

[0045] (2) Mixing: The mixture formula is: 5% cell wall broken algae powder, 92.5% pea protein isolate, 0.5% carrageenan, and 2% sunflower seed oil. Mix at 150 rpm for 20 minutes and adjust the pH to 6.5.

[0046] (3) Extrusion molding: The amount of water added is controlled to 50% of the total water content, the screw speed is 150 rpm, the heating zone temperature is 35℃, 80℃, 120℃, and 150℃; the cooling mold temperature is 40℃, and the plant meat is obtained by extrusion and solidification.

[0047] Example 3

[0048] A protein-rich Chlorella complex nutrient plant meat, rich in organic selenium and ω-3 fatty acids, is prepared by the following steps:

[0049] (1) Preparation of algal powder: The concentration of sodium selenite in the culture medium was 25 mg / L, the concentration of flaxseed oil was 3%, and the fermentation culture was carried out at 26℃ for 7 days. The homogenization pressure was 850 bar, and the cycle was 5 times to obtain algal powder with an average particle size of 1.0 μm.

[0050] (2) Mixing: The mixture formula is: 15% cell wall broken algae powder, 72% wheat gluten powder and soy protein isolate compound, 3% sodium alginate and locust bean gum compound, and 10% flaxseed oil. Mix at 250 rpm for 40 minutes and adjust the pH to 7.5.

[0051] (3) Extrusion molding: The amount of water added is controlled to 65% of the total water content, the screw speed is 200 rpm, the heating zone temperature is 60℃, 100℃, 140℃, and 170℃; the cooling mold temperature is 50℃, and the plant meat is obtained by extrusion and solidification.

[0052] Comparative Example 1 (without cell wall breaking treatment)

[0053] The only difference from Example 1 is that after collecting the algae in step (1), they are not subjected to high-pressure homogenization and cell wall disruption treatment, but are directly dried to obtain unbroken Chlorella powder with an average particle size of about 5.8 μm. The remaining steps are exactly the same as in Example 1.

[0054] Comparative Example 2 (without added polysaccharides and vegetable oil synergy)

[0055] The only difference from Example 1 is that xanthan gum and soybean oil are not added to the mixture formulation in step (2), and the mass fraction is made up with soy protein isolate (i.e., 10% cell wall broken algae powder and 90% soy protein isolate). The remaining steps are exactly the same as in Example 1.

[0056] Comparative Example 3 (Excessive Algae Powder Addition)

[0057] The only difference from Example 1 is that in the mixture formulation of step (2), the amount of broken cell wall algae powder added is increased to 25% (i.e., 25% broken cell wall algae powder, 68.5% soy protein isolate, 1.5% xanthan gum, and 5% soybean oil). The remaining steps are exactly the same as in Example 1.

[0058] The experimental test results and data analysis are shown in Table 1:

[0059] The plant-based meats prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to performance tests, and the results are shown in Table 1:

[0060]

[0061] Data Analysis and Discussion:

[0062] To further investigate the impact of different processing techniques on the quality of plant-based meat, this invention conducted macroscopic and microscopic characterization of representative formulation processes (see attached figures). Figure 1 To be continued Figure 3 In the figure, the HAP group (high-pressure homogenization and cell disruption + polysaccharide / oil synergy) corresponds to the optimal process embodiment 1 of the present invention; the UAP group (no cell disruption) corresponds to embodiment 1; the HAN group (no polysaccharide / oil) corresponds to embodiment 2; and the EAP group (algae powder exceeding limits) corresponds to embodiment 3. The UAN and EAN groups serve as auxiliary reference groups under the condition of no polysaccharide / oil. The specific correlation analysis is as follows:

[0063] 1) Nutritional content, digestibility, and cell wall breaking pressure range:

[0064] From Example 1 (corresponding) Figure 1 e's HAP group) and Comparative Example 1 (corresponding to Figure 1 As shown in the comparison with the UAP group (d), the in vitro digestibility of plant-based meat was significantly improved after high-pressure homogenization and cell wall disruption. Figure 1 As shown in the macroscopic morphology, the extrudate without cell wall disruption treatment ( Figure 1 d) The surface has a noticeable grainy texture and a weak tearing sensation, while Example 1, which has undergone moderate cell wall disruption treatment ( Figure 1e) This results in a fibrous structure with excellent tearability. This directly demonstrates that the cell wall disruption process not only releases nutrients by breaking down the dense cellulose cell walls of microalgae, but is also essential for maintaining texture. Further analysis of Table 2 (Examples 4-1 to 4-4) shows that the 600-850 bar range specified in this invention is the key pressure range for achieving dual optimization of nutrition and texture. When the pressure is below the lower limit (e.g., 300 bar in Example 4-1), the shear force is insufficient to completely disrupt the cell wall, resulting in ineffective dissolution of intracellular nutrients (digestibility of only 74.2%), and the large, rigid algal particles block the continuous protein network, resulting in a stringiness of only 1.15. Conversely, when the pressure is above the upper limit (e.g., 1000 bar in Example 4-4), excessive and intense cavitation effects and instantaneous high temperatures trigger severe thermo-oxidative degradation of ω-3 fatty acids (content drops to 435.2 mg / 100g), and the excessively released lipids and polysaccharides form an excessive "lubricating barrier," leading to a decrease in stringiness. Only within the 600~850 bar range can organic selenium and ω-3 fatty acids be fully released (in vitro digestibility >89.5%) while retaining a moderate amount of micron-sized shell residue as active microparticle filler to synergistically promote phase separation and fiber crosslinking.

[0065] 2) Texture, spinnability, and range of polysaccharide addition:

[0066] From Example 1 (corresponding) Figure 1 e's HAP group) and Comparative Example 2 (corresponding to Figure 1 A comparison with the HAN group (b) shows that, in the absence of the synergistic plasticizing effect of edible polysaccharides and oils, the extrudate macroscopically exhibits a loose "dough-like" appearance (e.g., ...). Figure 1 As shown in b), its spinnability index is only 0.95. Its internal rheological and cross-linking mechanisms can be confirmed by characterization results of water distribution and protein conformation: such as... Figure 2 The low-field nuclear magnetic resonance (LF-NMR) spectrum of the water distribution shows that, compared with the polysaccharide-oil-free group (HAN group), the peak area distribution of bound water (T2b) and immobilized water (T21) in the polysaccharide-oil synergistic group (HAP group) was significantly reshaped, demonstrating that the addition of polysaccharides significantly enhanced the hydration binding capacity of the system, providing a stable rheological basis for cross-linking between macromolecules; at the same time, the bound water... Figure 3 Protein secondary structure analysis (FTIR and relative content plots) revealed that the addition of appropriate amounts of polysaccharides and lipids effectively promoted the rearrangement of hydrogen bonds between protein molecules, resulting in a significant increase in the relative content of higher-order structures such as β-sheets, which contribute to the construction of continuous three-dimensional networks. This ultimately led to the formation of perfectly fibrous fibers on a macroscopic scale (e.g., ...). Figure 1(As shown in e). This fully demonstrates the necessity of polysaccharide at the microscopic molecular scale and in terms of the water phase. Further analysis of the data in Tables 3 (Examples 5-1 to 5-4) shows that the 0.5%~3% polysaccharide addition specified in this invention is the core range for inducing a reasonable rheological phase transition. When the polysaccharide addition is below 0.5% (e.g., 0.1% in Example 5-1), the hydrocolloid concentration in the system is insufficient to generate effective hydration binding, resulting in low melt viscosity. The protein chains cannot obtain sufficient hydrodynamic resistance in the cooling mold for high orientation (fiber stretching is only 1.05). When the polysaccharide addition exceeds 3% (e.g., 4.5% in Example 5-4), a "phase reversal" occurs in the system. Supersaturated polysaccharide molecules form an extremely thick steric hindrance layer around the protein chains, completely severing the recombination crosslinking between protein subunits, causing the fiber stretching to plummet to 0.88. Therefore, the specific ratio of 0.5%~3% precisely plays a role in viscoelastic regulation and interfacial lubrication, guiding the smooth crosslinking of protein fibers.

[0067] 3) Algae powder addition threshold and golden window:

[0068] From Example 1 (corresponding) Figure 1 e's HAP group) and Comparative Example 3 (corresponding to Figure 1 A comparison with the EAP group (f) shows that adding excessive amounts of broken-cell wall algae powder will completely destroy the muscle fiber structure, such as Figure 1 Macroscopic observation of f shows that the extrudate is loose and lumpy or pasty, completely losing the fibrous texture of animal muscle. Meanwhile, combined with... Figure 2 and Figure 3 The test results also show that the massive amount of non-protein components from microalgae (EAP group) caused severe "over-lubrication," disrupting the stable binding state of water and severing the cross-linking pathway of proteins to β-sheet rearrangement. Further analysis of the data in Table 4 (Examples 6-1 to 6-4) reveals that the 5%~15% range defined in this invention represents the optimal window for balancing targeted nutritional fortification and superior texture. When the amount of algae powder added is less than 5% (e.g., 1% in Example 6-1), although the stringiness is excellent (1.65), the absolute enrichment of organic selenium and ω-3 fatty acids in the final product is at a very low level (ω-3 only 65.4 mg / 100g), thus losing the specific nutritional advantages of compound plant-based meat. When the amount added exceeds 15% (e.g., 25% in Example 6-4), the massive amount of non-protein components of microalgae forms a dense insulating layer around the ununwound protein subunits, blocking the cross-linking pathway of disulfide bonds (-SS-). At the same time, a strong "over-lubrication effect" occurs in the extrusion chamber, causing a sharp drop in mechanical shear energy. Not only does the stringiness drop below 0.85, but the highly enriched ω-3 fatty acids also undergo drastic degradation due to the loss of thermodynamic protection from the dense protein network. Only in the range of 5% to 15% does the moderate interfacial lubrication provided by the microemulsion coating layer neither block protein cross-linking nor fail to achieve effective retention of high-content nutrients and a perfect stringiness structure.

[0069] Since the plant-based meat prepared in Example 1 has the best overall performance, it is used to compare and illustrate the role of each formulation and process in this application.

[0070] Example 4: The only difference between this example and Example 1 is that after collecting algae in step (1), only the pressure range during high-pressure homogenization and cell disruption is changed; the comparison results are shown in Table 2.

[0071] Table 2

[0072]

[0073] As can be seen from the experimental data in Table 2, the pressure of the high-pressure homogenization cell disruption process has a decisive influence on the release efficiency of intracellular nutrients in Chlorella pyrenoidosa and the final texture characteristics of plant meat, and exhibits a significant "threshold effect" and "overload degradation effect".

[0074] When the homogeneous pressure is at a low level (such as 300 bar in Example 4-1), the fluid shear force is insufficient to completely break the hard and dense thick-walled cellulose shell of Chlorella pyrenoidosa. At this time, the average particle size of the algal powder is still as high as 3.8 μm. The abundant organic selenium and ω-3 fatty acids are physically wrapped by intact or semi-intact cell walls and cannot be effectively dissolved, resulting in an in vitro digestibility of only 74.2% for the plant-based meat end product. At the same time, the large, rigid, free algal particles that are not fully broken down act as "macromolecular stress concentration points" in the high shear field of the twin-screw extruder, locally blocking the development of the continuous network of plant proteins, resulting in a rough extrudate surface and a fiber draw index of only 1.15.

[0075] As the pressure increases to the core range of 600-850 bar defined in this invention (750 bar being optimal in Example 1), the cell wall undergoes deep structural fragmentation, and the average particle size of the algal powder is precisely reduced to the ideal range of 1.0-2.0 μm. At this scale, intracellular nutrients are fully exposed, resulting in an in vitro digestibility of 89.5%-93.4%. Simultaneously, the moderately fragmented micron-sized algal powder acts as a high-quality microparticle filler, uniformly dispersed within the protein matrix, synergistically promoting phase separation and fiber cross-linking during high-moisture extrusion, giving the product excellent tearability and a high fiber draw index of 1.62.

[0076] However, excessive pursuit of high cell wall breakage rates can lead to unforeseen technological degradation. When the pressure reaches an extreme of 1000 bar (as in Examples 4-4), the intense cavitation and localized instantaneous high temperatures generated by the fluid in the extremely narrow homogeneous valve gap directly trigger severe oxidative degradation of highly unsaturated and heat-sensitive ω-3 fatty acids, resulting in a sharp decrease in their content. Strong free radical reactions also simultaneously induce the inactivation and loss of some organic selenium. More seriously, from the perspective of macromolecular interaction mechanisms, excessive pulverization (particle size <1.0 μm) caused by extremely high pressure results in the complete and excessive release of free lipids and intracellular polysaccharides within microalgal cells in a very short time. Within the high-temperature melting zone of the extruder, these overloaded lipids and polysaccharides form an overly coated "lubricating barrier" at the plant protein (soybean / pea protein) interface, severely hindering the dimerization of disulfide bonds and the rearrangement of hydrogen bonds after the protein molecular chains depolymerize. Ultimately, this results in the extrudate failing to form a tightly oriented anisotropic three-dimensional network, macroscopically exhibiting deterioration phenomena such as softening, gelatinization, and a significant decrease in stringiness.

[0077] Based on the above experimental and mechanistic verifications, the present invention strictly limits the high-pressure homogenization pressure to 600~850 bar. This is not an arbitrary parameter selection, but rather a clever avoidance of "low absorption caused by undertreatment" and "thermo-oxidative degradation and protein cross-linking blockage caused by overtreatment". It truly achieves a perfect unity of efficient and targeted release of microalgae nutrients and the excellent biomimetic muscle fiber texture of plant meat.

[0078] Example 5: The only difference between this example and Example 1 is that the mass of the edible polysaccharide added in step (2) is changed to the range of the total mass of the mixture; the comparison results are shown in Table 3.

[0079] Table 3

[0080]

[0081] As can be seen from the experimental data and macroscopic morphological observations in Table 3, the addition ratio of edible polysaccharides (such as xanthan gum and other hydrocolloids) plays a crucial role in rheological regulation and interfacial intervention in the twin-screw high-moisture extrusion system. Its concentration changes not only directly trigger the phase transformation of the material, but also profoundly affect the nutrient retention and multidimensional texture of the microalgae composite plant meat.

[0082] When the polysaccharide content is extremely low (e.g., 0.1% in Example 5-1), the hydrocolloid concentration in the system is insufficient to effectively hydrate and bind high-moisture (50%~65%) materials and increase viscosity. Inside the high-temperature, high-shear extruder barrel, the plant protein has a high free water content and low melt viscosity, resulting in insufficient hydrodynamic resistance and directional shear stress for the protein molecular chains in the cooling die. Consequently, protein chain rearrangement and cross-linking efficiency are low, and the extrudate cannot form a continuous anisotropic three-dimensional network, macroscopically appearing as loose, non-fibrous fragments (fiberability index only 1.05). Simultaneously, due to the lack of physical encapsulation and viscous flow protection by polysaccharide macromolecules, the heat-sensitive ω-3 fatty acids and organic selenium contained in microalgae are more easily exposed to strong shear heat and oxidation, leading to nutrient degradation and loss.

[0083] As the polysaccharide content entered the core range of 0.5%~3.0% defined in this invention (with 1.5% in Example 1 showing optimal performance), a moderate degree of thermodynamic incompatibility and microphase separation occurred between the soybean / pea protein macromolecules and the polysaccharide macromolecules. In this state, the polysaccharide formed a highly dispersed aqueous phase network within the protein matrix, acting as an excellent plasticizing lubricant, reducing the mechanical specific energy (SME) during extrusion, and cleverly guiding the highly directional cross-linking of protein disulfide bonds and hydrogen bonds after depolymerization in the cooling laminar flow zone. This scientific phase distribution not only endowed the plant-based meat with high chewiness (845.2 g) comparable to animal muscle and a perfect tearing texture (stretch index 1.62), but the dense hydrogel network constructed by the polysaccharide also provided excellent barrier protection for the released intracellular ω-3 fatty acids and organic selenium, greatly inhibiting thermo-oxidative degradation caused by the instantaneous high temperature in the extrusion chamber, maintaining an in vitro digestibility of up to 92.6% and a high content of microalgae-specific nutrients.

[0084] However, blindly increasing the polysaccharide content to 4.5% (as in Examples 5-4) will result in a catastrophic "phase inversion" in the rheological behavior within the extrusion chamber. Excessive high-molecular-weight polysaccharides cause the melt viscosity to spike exponentially. This excessive viscosity dissipates locally in the screw, generating extremely intense internal frictional heat. This uncontrollable extreme thermal stress directly leads to severe thermal decomposition of ω-3 fatty acids, with the content dropping below 400 mg / 100g, and significant deactivation of organoselenium. Even more fatally, the supersaturated polysaccharide molecules form an extremely thick steric hindrance layer around the plant protein chains, completely severing the possibility of recombination and cross-linking between protein subunits. This prevents the material from forming a continuous fibrous gel within the cooling mold, ultimately causing the extrudate to irreversibly deteriorate into a viscous, gel-like dough with no chewiness and completely lost its stringy structure (stringiness index plummets to 0.88).

[0085] The above mechanisms confirm that the precisely formulated polysaccharide concentration window of 0.5% to 3.0% in this invention is key to overcoming the industry's technical prejudice that "microalgae addition destroys the fleshy texture of plants." This specific ratio, through sophisticated phase separation and interfacial rheological control, successfully constructs a dual-network matrix structure with high nutrient retention and excellent biomimetic filament-drawing properties.

[0086] Example 6: The only difference between this example and Example 1 is that the amount of broken algae powder added in the mixture formula in step (2) is changed; the comparison results are shown in Table 4.

[0087] Table 4

[0088]

[0089] As shown in Table 4, the mass proportion of broken-cell wall protein Chlorella powder in the plant-based meat mixture is the core limiting parameter that determines the thermodynamic compatibility of the high-moisture extrusion system and the nutritional-texture balance of the final product. Changes in this parameter directly affect the mechanical specific energy (SME) input of the plant protein fluid and the topological structure of the macromolecular cross-linking network during extrusion.

[0090] When the amount of algae powder added is at an extremely low level (such as 1% in Example 6-1), the extruded material system still exhibits typical homogeneous rheological characteristics dominated by soybean / pea single protein. At this time, due to the lack of steric hindrance interference from exogenous non-protein macromolecules, the plant protein can fully melt and depolymerize in the high-temperature, high-shear zone, and successfully complete the dimerization of disulfide bonds and hydrogen bond rearrangement in the cooling mold, thus obtaining an extremely high fiber draw index (1.65). However, due to the extremely low microalgae biomass, the absolute enrichment of organic selenium and ω-3 fatty acids in the end product is at an extremely low level, which completely fails to meet the original intention of targeted nutritional fortification and loses the specific functional advantages of compound plant meat.

[0091] As the amount of algal powder added entered the strictly defined core window period of 5% to 15% in this invention (with 10% in Example 1 as a typical example), the system exhibited a remarkable "multiphase synergistic plasticizing effect" at the microscopic level. The micron-sized microalgal cellulose shell remnants released after appropriate cell disruption acted as rigid "active micro-fillers" in the continuous protein matrix droplets; while the natural lipids and polysaccharides exuded from the microalgal cells formed a uniformly dispersed microemulsion coating layer at the multiphase fluid interface. This sophisticated physicochemical coordination not only did not block the main cross-linking of plant proteins, but also guided protein molecules to undergo highly ordered stretching and orientation along the laminar flow direction in the long die section of the extruder cooling process through appropriate interfacial lubrication. Therefore, at an addition amount of 5% to 15%, the plant-based meat not only successfully accumulated a high concentration of highly bioavailable organic selenium and ω-3 fatty acids, but also maintained an excellent stringiness index as high as 1.51-1.62, achieving a realistic chewy texture reminiscent of animal muscle.

[0092] However, once the compatibility threshold is crossed, blindly pursuing ultimate nutrition and drastically increasing the amount of algae powder added to 25% (as in Examples 6-4) will lead to a catastrophic collapse in the system's polymeric phase behavior. The excessive introduction of microalgal contents (especially a high proportion of free lipids and large intracellular polysaccharides) generates an extremely strong "over-lubrication effect" within the extrusion chamber. This effect causes a precipitous drop in the coefficient of friction of the material within the twin-screw extruder, preventing the system from generating sufficient mechanical shear energy (SME) to disrupt the natural dense spherical structure of the plant protein. Simultaneously, the massive amount of non-protein components from the microalgae forms a dense steric barrier layer around the incompletely unwound protein subunits, completely blocking the cross-linking pathway of cysteine ​​residue thiol (-SH) oxidation to form disulfide bonds (-SS-). Macroscopically, the extrudate is completely unable to form a continuous three-dimensional network, resulting in severe structural collapse and gelatinization, with the stringiness dropping below 0.85 (appearing as a loose dough). Furthermore, due to the loss of the thermodynamic protective barrier of the dense protein network, the highly enriched ω-3 fatty acids undergo drastic thermo-oxidative degradation in the high-temperature zone, failing to achieve a linear increase in content and instead experiencing a significant decline in in vitro digestibility.

[0093] In summary, this invention successfully overcomes the technical limitations of traditional plant-based meats, such as nutritional deficiencies, texture degradation due to microalgae addition, and low digestibility, by combining scientific microbial fermentation for targeted enrichment, cell wall disruption, and precise high-moisture extrusion processes. It has extremely high industrial application value and broad market prospects.

[0094] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a protein-rich Chlorella complex nutritional plant-based meat rich in organic selenium and ω-3 fatty acids, characterized in that, Includes the following steps: (1) Preparation of algal powder: Chlorella pyrenoidosa was inoculated into a culture medium containing inorganic selenium source and ω-3 fatty acid precursor substances for fermentation culture. After collecting the algae, the cell wall was broken and dried to obtain Chlorella pyrenoidosa powder rich in organic selenium and ω-3 fatty acids. (2) Mixing: The cell wall broken Chlorella protein powder obtained in step (1) is mixed evenly with the plant protein raw material to obtain a mixture; wherein, the mass of the cell wall broken Chlorella protein powder accounts for 5% to 15% of the total mass of the mixture; (3) Extrusion molding: The mixture obtained in step (2) is continuously fed into a twin-screw extruder and water is injected simultaneously for high-moisture extrusion treatment. After extrusion, it is extruded through a cooling mold and solidified to obtain an extruded material with a moisture content of 50%~65%, namely, protein-core Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids.

2. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The inorganic selenium source mentioned in step (1) is sodium selenite, and the concentration of the inorganic selenium source added to the culture medium is 10~25 mg / L; the ω-3 fatty acid precursor is flaxseed oil or α-linolenic acid, and the mass concentration of the ω-3 fatty acid precursor in the culture medium is 1%~3%.

3. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The fermentation culture conditions described in step (1) are: cultured at 25~28℃ for 4~7 days; the cell wall breaking treatment is specifically: the collected algae are mixed with water to form a suspension, and a high-pressure homogenizer is used to perform 3~5 homogenization cycles at a pressure of 600~850 bar to reduce the average particle size of Chlorella proteoglycans after cell wall breaking to 1.0~2.0 μm.

4. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The fermentation conditions described in step (1) are: cultured at 25~28℃ for 4~7 days; the cell wall breaking treatment is specifically: the collected algae are mixed with water to form a suspension, and a high-pressure homogenizer is used to perform 3~5 homogenization cycles at a pressure of 600~850 bar, so that the average particle size of Chlorella proteoglycans after cell wall breaking is reduced to 1.0~2.0 μm.

5. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The plant protein raw material mentioned in step (2) is one or a mixture of soy protein isolate, pea protein isolate, wheat gluten and peanut textured protein; the total protein content of the plant protein raw material is ≥85%.

6. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The specific parameters for the high moisture extrusion treatment in step (3) are as follows: the fixed rotation speed of the twin screw is 150~200 rpm; the heating zone temperatures of the twin screw extruder from the feeding section to the die section are set to 35~60℃, 80~100℃, 120~140℃ and 150~170℃ respectively; and the temperature of the cooling die is controlled at 40~50℃.

7. The method for preparing the protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The mixture in step (2) also contains edible polysaccharides and edible vegetable oils; the mass of the edible polysaccharides accounts for 0.5% to 3% of the total mass of the mixture, and the mass of the edible vegetable oils accounts for 2% to 10% of the total mass of the mixture.

8. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 7, characterized in that, The edible polysaccharide is one or a mixture of xanthan gum, carrageenan, sodium alginate and locust bean gum; the edible vegetable oil is one or a mixture of soybean oil, sunflower seed oil, corn oil, flaxseed oil and coconut oil.

9. The method for preparing protein-nucleated Chlorella complex nutritional plant meat rich in organic selenium and ω-3 fatty acids according to claim 1, characterized in that, The specific operation of mixing in step (2) is as follows: place each raw material in a powder mixer, mix and stir at a speed of 150~250 rpm for 20~40 minutes, and adjust the pH value of the mixture to 6.5~7.

5.

10. A protein-rich Chlorella complex nutritional plant-based meat rich in organic selenium and ω-3 fatty acids, characterized in that, Prepared by the method according to any one of claims 1 to 9.

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