Fermented composite vegetable protein and preparation method thereof

Abstract

The invention discloses a fermented composite vegetable protein and a preparation method thereof. According to the process, the high-purity composite vegetable protein is prepared through a multi-source vegetable protein synergistic treatment and biotransformation technology. Soybeans, peas, oats, chenopodium quinoa willd and chia seeds are selected for pretreatment, neutral protease, cellulase and pectinase are adopted for staged ultrasonic-assisted enzymolysis, and protein and functional components are fully released. Then flora metabolism is regulated and controlled through step-by-step fermentation of composite strains, and the synergistic interaction of bacteriostasis, corrosion prevention and probiotics is achieved in combination with additives. The method comprises the following steps of: removing macromolecular impurities from fermentation liquor through ceramic membrane separation, accurately separating target protein by utilizing ion-exchange chromatography gradient elution, and obtaining a low-moisture final product while protecting heat-sensitive components through a drying technology. The process integrates enzymolysis, fermentation, separation and purification and drying technologies, realizes efficient extraction of vegetable protein, functional activity retention and product stability improvement, and is suitable for development of nutrition-enhanced food and functional ingredients.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and particularly relates to a fermented composite plant protein and a preparation method thereof. Background Art

[0002] Soybeans are an important source of plant protein, and its fermentation technology mainly focuses on improving nutritional value and functionality. Fermented pea protein is usually applied to plant-based yogurt, meat substitutes, and functional beverages. Oat fermentation can provide essential amino acids. There is less research on chia seed fermentation, but its high protein and amino acid balance characteristics make it have potential. Existing research focuses on enzymolysis to prepare antioxidant peptides. For example, the antioxidant activity is significantly enhanced after hydrolysis by alkaline protease. Therefore, its functional characteristics can be improved through fermentation treatment.

[0003] Plant protein ferment is a product prepared by microbial fermentation technology and can be used in the production of functional foods. The industrial production of plant protein faces technical bottlenecks such as low raw material utilization rate, insufficient functional characteristics, and flavor defects. Traditional processes mostly rely on a single protein source, such as soy protein isolate, which has an unbalanced amino acid composition and poor processing adaptability. In recent years, composite plant protein has become a research hotspot because it can integrate the nutritional complementary advantages of multiple raw materials. However, problems such as insufficient raw material pretreatment, low enzymolysis efficiency, interference of miscellaneous bacteria in the fermentation process, and oxidative degradation of products in the existing technology seriously restrict the product quality and yield.

[0004] Plant protein ferment is a protein raw material that uses microorganisms such as lactic acid bacteria, Aspergillus oryzae, and Bacillus subtilis to grow and reproduce in plant raw materials, and accumulates bacteria, enzymes, and intermediate products through metabolic activities. The plant cell wall is composed of complex components such as polysaccharides and lignin. The fermentation process can lyse the cell wall and release intracellular nutrients, significantly improving the protein digestibility. The fermented product not only retains the characteristics of plant protein with low fat and no cholesterol, but also generates small peptides and amino acids through protein degradation, enhancing the nutrient absorption efficiency.

[0005] Therefore, how to provide a scientific plant ratio, enhance nutritional complementarity and functional enhancement is one of the key research directions of the present invention. During the whole fermentation process of plant protein, the compatibility between the strain and the plant matrix needs to be further studied. Temperature and pH value are commonly used to optimize the fermentation process, increase the fermentation efficiency and product yield. How to precisely ferment a composite plant formula efficiently is also one of the research focuses of the present invention. Summary of the Invention

[0006] The purpose of the present invention is to provide a fermented composite plant protein and a preparation method thereof.

[0007] To solve the above technical problems, the specific preparation method of the present invention is as follows:

[0008] Use 40 - 50 parts of soybeans, 25 - 30 parts of peas, 10 - 15 parts of oats, 10 - 15 parts of quinoa, and 5 - 8 parts of chia seeds for raw material pretreatment: After microwave puffing treatment at a power of 800 watts for 60 seconds, it is ultrafinely pulverized to a particle size of 50 microns, and soaked in 50°C warm water at a material - to - water ratio of 1:3 for 2 hours;

[0009] Subsequently, add 0.4% neutral protease, 0.2% cellulase, and 0.1% pectinase by mass fraction, and perform ultrasonic - assisted enzymatic hydrolysis at 50°C and a pH value of 6.5 for 2 hours. Then, raise the temperature to 55°C and a pH value of 7.0 and continue enzymatic hydrolysis for 1.5 hours, and inactivate at 85°C for 10 minutes;

[0010] Next, add a composite strain for three - stage fermentation treatment; Add citric acid to the solution obtained by filtering the above fermentation to adjust the pH to 3.5, load the sample at a flow rate of 1.5 CV / h, select strong cation - exchange resin SP SepHarose 6FF as the stationary phase, balance the column bed with a sodium citrate buffer solution with a pH of 4.0, and control the flow rate at 2 CV / h for gradient elution separation: Buffer A: Sodium citrate buffer solution with a pH of 4.0; Buffer B: 1M NaCl; From 0 - 10 minutes, the mobile phase A is 90%, and the mobile phase B is 10%; From 10 - 15 minutes, the mobile phase A decreases uniformly to 70%, and the mobile phase B increases uniformly to 30%; From 15 - 30 minutes, the mobile phase A is 50%, and the mobile phase B is 50%; From 30 - 40 minutes, the mobile phase A increases uniformly to 30%, and the mobile phase B decreases uniformly to 70%;

[0011] Pass the ion - exchange eluate through a 10 kDa ultrafiltration system, with a transmembrane pressure of 0.2 - 0.4 MPa, and recycle and concentrate to 1 / 10 of the original volume. Add 5% trehalose to the concentrated solution, adjust the pH to 6.8, sublime free water at - 45°C and 50 Pa vacuum for 24 hours, and then place it at 25°C and 10 Pa vacuum to remove bound water for 12 hours. The moisture content of the final product is ≤3%, and the composite plant protein is obtained.

[0012] Among them, the fermentation treatment is as follows: In the first stage, add 2.5% Lactobacillus plantarum at 37°C and perform static anaerobic fermentation for 12 hours. In the second stage, add 1.25% Candida utilis and 1.25% Bacillus natto at 42°C and 120 rpm, and perform oscillating micro - aerobic fermentation for 12 hours. In the third stage, stand at 30°C for after - ripening until the pH value naturally drops to 4.3; After the fermentation broth is autolyzed at 55°C for 2 hours, use a 50 kDa ceramic membrane separation system to remove macromolecular impurities.

[0013] Among them, the addition of additives is also included during the fermentation treatment process: Add 0.03 - 0.08% of ε - polylysine hydrochloride in the first stage.

[0014] Among them, the addition of additives is also included in the fermentation process: 0.3-0.5% fucoidan is added in the second stage.

[0015] Among them, the addition of additives is also included in the fermentation process: 0.05% ε-polylysine hydrochloride and 0.5% fucoidan are added in the third stage.

[0016] The beneficial effects of the present invention are as follows:

[0017] 1. The production process of this composite plant protein realizes efficient preparation through the combination of multi-source plants and the coordination of multiple technologies. Its core feature is that in the raw material pretreatment stage, microwave puffing combined with ultrafine grinding technology is used to fully destroy the plant cell wall structure, and the protein dissolution efficiency is significantly improved by combining gradient temperature and multi-enzyme system for staged enzymatic hydrolysis; through a three-stage fermentation process, the strain combination and fermentation process are precisely regulated to achieve the directional metabolism of functional strains, and targeted additives are used to stepwise control the dynamic balance of the microbial community, effectively inhibiting the contamination of miscellaneous bacteria while promoting the generation of flavor substances; subsequently, the combined technology of strong cation exchange resin gradient elution and ultrafiltration concentration is used to achieve high-precision separation and purification of the target protein, and a stable system is constructed by combining the vacuum gradient freeze-drying process, completely retaining the functional activity of the protein.

[0018] 2. This plant protein formula covers legumes, grains and functional seeds, with strong nutritional complementarity. Moreover, the high fiber and ω-3 fatty acid content of quinoa and chia seeds can enhance the functional characteristics of the product. The scientific ratio of multi-source plant matrices realizes nutritional complementarity and functional enhancement: the compound of soybean, pea, oat, quinoa and chia seeds breaks through the nutritional limitations of single plant protein; soybean and pea provide high-quality protein, oat and chia seeds add dietary fiber and ω-3 fatty acids, and quinoa supplements complete amino acids. Microwave puffing combined with ultrafine grinding pretreatment improves the enzymatic hydrolysis efficiency, and intermittent ultrasound can reduce the thermal inactivation of enzymes, improve the enzyme activity retention rate, increase the phytic acid degradation rate, and significantly reduce anti-nutritional factors; secondly, by integrating physical and chemical treatment and biological fermentation technology, the production cycle is greatly shortened and the energy consumption is reduced; finally, the fermentation system is precisely regulated to solve the problems of product inhibition and flavor loss, significantly improving the protein recovery rate and product purity. The overall process has the advantages of high efficiency, environmental protection and product functionalization.

[0019] 3. Through multi-enzyme synergistic staged enzymatic hydrolysis, active peptide segments are efficiently released: neutral protease, cellulase and pectinase act in stages: in the first stage, the cell wall is broken, and in the second stage, small molecule peptides are directionally generated. Intermittent ultrasound assistance shortens the enzymatic hydrolysis time. Among them, the content of ACE inhibitory peptides and antioxidant peptides obtained by enzymatic hydrolysis doubles, and at the same time, the generation of bitter peptides is reduced, which can improve the palatability of the product.

[0020] 4. Dynamic fermentation with three strains to produce high - value - added metabolites: Lactobacillus plantarum, Bacillus natto, and Candida utilis are co - fermented in stages. In the anaerobic stage, it rapidly produces acid to inhibit bacteria. In the micro - aerobic stage, it secretes antibacterial peptides and γ - polyglutamic acid. In the post - ripening stage, it synthesizes glutathione and flavor esters. Autolysis treatment releases nucleotides and B - group vitamins, enhancing the function and flavor.

[0021] 5. ε - Polylysine can target and bind to the teichoic acid on the surface of the cell membrane of Gram - positive bacteria through its cationic polypeptide chain, destroy the membrane integrity and form transmembrane ion channels. In the first stage, it destroys the phospholipid bilayer of the cell membrane of Gram - positive bacteria and inhibits their growth, but does not affect the proliferation of Lactobacillus plantarum, making up for the lag of the antibacterial effect of organic acids. At the initial stage of anaerobic fermentation, the risk of miscellaneous bacteria contamination is the highest. ε - Polylysine hydrochloride can reduce the complexity of the initial flora, enabling Lactobacillus plantarum to quickly dominate. Compared with other broad - spectrum preservatives, its lethal concentration threshold for Lactobacillus plantarum is higher, achieving selective antibacterial.

[0022] 6. In the second stage, the sulfate groups of fucoidan specifically bind to the heterologous binding proteins on the surface of Bacillus natto, activating the quorum - sensing system, up - regulating the gene expression of alkaline protease and plasmin, and significantly increasing the enzyme production. Under the condition of oscillating micro - aerobic, fucoidan cross - links with bacterial exopolysaccharides through hydrogen bonds, promoting the formation of a three - dimensional biofilm by Bacillus, restricting the oxygen diffusion rate, stabilizing the dissolved oxygen gradient, avoiding enzyme inactivation or by - product accumulation caused by over - oxidation. Moreover, fucoidan has significant antioxidant activity, can scavenge free radicals and reduce the damage of oxidative stress to microorganisms. In addition to providing certain nutritional supplements for Candida utilis, it can also increase enzyme activity and improve fermentation efficiency.

[0023] 7. In the third stage, ε - polylysine hydrochloride and fucoidan are compounded in a certain proportion. Among them, ε - polylysine hydrochloride can destroy yeast cells to a certain extent, damage the membrane integrity, accelerate the autolysis of Candida utilis, and release intracellular flavor substances. And fucoidan, as an osmoprotectant, combines with free water to form a vitreous matrix, preventing the oxidative degradation of active peptides when the cells rupture, forming a protection mechanism. Compared with traditional single - function additives, it shows significant advantages in terms of bioconversion efficiency, product stability, and functional characteristics.

[0024] 8. This process integrates microwave pretreatment, dynamic fermentation, and membrane - chromatography coupling technology, reducing the production cost compared with traditional biological extraction methods and greatly increasing the peptide recovery rate. The product has both full - nutrition coverage and functions such as blood - pressure lowering and antioxidant, adapting to the vegetarian, sports nutrition, and special medical food markets, providing a new process method for the high - value development of plant proteins. Detailed implementation mode

[0025] The present invention will be further described in detail below in conjunction with embodiments. The strain number of Lactobacillus plantarum used in the following embodiments is CICC 20279; the strain number of Bacillus natto is CGMCC 1.1086; the strain number of Candida utilis is ACCC 20060.

[0026] Example 1

[0027] Perform raw material pretreatment with 45 parts of soybeans, 28 parts of peas, 12 parts of oats, 13 parts of quinoa, and 7 parts of chia seeds: After microwave puffing treatment at a power of 800 watts for 60 seconds, it is ultrafinely pulverized to a particle size of 50 microns, and soaked in 50°C warm water at a material-to-water ratio of 1:3 for 2 hours;

[0028] Subsequently, add 0.4% neutral protease, 0.2% cellulase, and 0.1% pectinase by mass fraction, and perform ultrasonic-assisted enzymatic hydrolysis for 2 hours at 50°C and a pH value of 6.5. Then, raise the temperature to 55°C and continue enzymatic hydrolysis for 1.5 hours at a pH value of 7.0, and inactivate at 85°C for 10 minutes; then add a composite strain for three-stage fermentation treatment; in the first stage, add 2.5% Lactobacillus plantarum at 37°C, and add 0.05% ε-polylysine hydrochloride and let it stand for anaerobic fermentation for 12 hours; in the second stage, add 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan sulfate at 42°C and 120 rpm, and perform oscillating microaerobic fermentation for 12 hours; in the third stage, let it stand at 30°C for after-ripening until the pH value naturally drops to 4.3, add 0.05% ε-polylysine hydrochloride and 0.5% fucoidan sulfate. After the fermentation broth is autolyzed at 55°C for 2 hours, use a 50 kDa ceramic membrane separation system to remove macromolecular impurities.

[0029] Add citric acid to the solution obtained by the above fermentation and filtration to adjust the pH to 3.5, load the sample at a flow rate of 1.5 CV / h, select strong cation exchange resin SP SepHarose 6FF as the stationary phase, and use sodium citrate buffer with a pH of 4.0 to balance the column bed, and control the flow rate at 2 CV / h for gradient elution separation: Buffer A: Sodium citrate buffer with a pH of 4.0; Buffer B: 1M NaCl; from 0 to 10 minutes, the mobile phase A is 90%, and the mobile phase B is 10%; from 10 to 15 minutes, the mobile phase A decreases uniformly to 70%, and the mobile phase B increases uniformly to 30%; from 15 to 30 minutes, the mobile phase A is 50%, and the mobile phase B is 50%; from 30 to 40 minutes, the mobile phase A increases uniformly to 30%, and the mobile phase B decreases uniformly to 70%;

[0030] Pass the ion exchange eluate through a 10 kDa ultrafiltration system with a transmembrane pressure of 0.3 MPa, circulate and concentrate it to 1 / 10 of the original volume. Add 5% trehalose to the concentrated solution, adjust the pH to 6.8, and sublime free water at -45°C and 50 Pa vacuum for 24 hours. Then place it at 25°C and 10 Pa vacuum to remove bound water for 12 hours. The moisture content of the final product is ≤ 3%, and the composite plant protein is obtained.

[0031] Example 2

[0032] Use 40 parts of soybeans, 30 parts of peas, 10 parts of oats, 15 parts of quinoa, and 8 parts of chia seeds for raw material pretreatment: After microwave puffing treatment at 800 watts of power for 60 seconds, it is ultrafinely ground to a particle size of 50 microns, and soaked in 50°C warm water at a material-to-water ratio of 1:3 for 2 hours;

[0033] Subsequently, add 0.4% neutral protease, 0.2% cellulase, and 0.1% pectinase by mass fraction, and perform ultrasonic-assisted enzymatic hydrolysis at 50°C and a pH value of 6.5 for 2 hours. Then raise the temperature to 55°C and a pH value of 7.0 and continue enzymatic hydrolysis for 1.5 hours, and inactivate at 85°C for 10 minutes; then add a composite strain for three-stage fermentation treatment; in the first stage, add 2.5% Lactobacillus plantarum at 37°C, and add 0.08% ε-polylysine hydrochloride and let it stand for anaerobic fermentation for 12 hours; in the second stage, at 42°C and 120 rpm, add 1.25% Candida utilis, 1.25% Bacillus natto, and 0.3% fucoidan sulfate, and perform oscillating microaerobic fermentation for 12 hours; in the third stage, let it stand at 30°C for after-ripening until the pH value naturally drops to 4.3, add 0.05% ε-polylysine hydrochloride and 0.5% fucoidan sulfate, and after the fermentation broth is autolyzed at 55°C for 2 hours, use a 50 kDa ceramic membrane separation system to remove macromolecular impurities.

[0034] Add citric acid to the solution obtained by the above fermentation and filtration to adjust the pH to 3.5, load the sample at a flow rate of 1.5 CV / h, select strong cation exchange resin SP SepHarose 6FF as the stationary phase, use sodium citrate buffer solution with a pH of 4.0 to balance the column bed, and control the flow rate at 2 CV / h for gradient elution separation: Buffer A: Sodium citrate buffer solution with a pH of 4.0; Buffer B: 1 M NaCl; from 0 to 10 minutes, the mobile phase A is 90% and the mobile phase B is 10%; from 10 to 15 minutes, the mobile phase A decreases uniformly to 70% and the mobile phase B increases uniformly to 30%; from 15 to 30 minutes, the mobile phase A is 50% and the mobile phase B is 50%; from 30 to 40 minutes, the mobile phase A increases uniformly to 30% and the mobile phase B decreases uniformly to 70%;

[0035] Pass the ion exchange eluate through a 10 kDa ultrafiltration system with a transmembrane pressure of 0.4 MPa, circulate and concentrate it to 1 / 10 of the original volume. Add 5% trehalose to the concentrated solution, adjust the pH to 6.8, sublime free water at -45 °C and 50 Pa vacuum for 24 hours, and then place it at 25 °C and 10 Pa vacuum to remove bound water for 12 hours. The moisture content of the final product is ≤ 3%, and the composite plant protein is obtained.

[0036] Example 3

[0037] Use 50 parts of soybeans, 25 parts of peas, 15 parts of oats, 10 parts of quinoa and 5 parts of chia seeds for raw material pretreatment: After microwave puffing treatment at a power of 800 watts for 60 seconds, it is ultrafinely ground to a particle size of 50 microns, and soaked in 50 °C warm water at a material-to-water ratio of 1:3 for 2 h;

[0038] Subsequently, add 0.4% neutral protease, 0.2% cellulase and 0.1% pectinase by mass fraction, and perform ultrasonic-assisted enzymatic hydrolysis at 50 °C and a pH value of 6.5 for 2 h. Then, raise the temperature to 55 °C and a pH value of 7.0 and continue enzymatic hydrolysis for 1.5 h, and then inactivate at 85 °C for 10 min; Then add a composite strain for three-stage fermentation treatment; In the first stage, add 2.5% Lactobacillus plantarum at 37 °C, and add 0.03% ε-polylysine hydrochloride and let it stand for anaerobic fermentation for 12 h; In the second stage, at 42 °C and 120 rpm, add 1.25% Candida utilis, 1.25% Bacillus natto, and 0.5% fucoidan sulfate, and perform oscillating microaerobic fermentation for 12 h; In the third stage, let it stand and ripen at 30 °C until the pH value naturally drops to 4.3, add 0.05% ε-polylysine hydrochloride and 0.5% fucoidan sulfate. After the fermentation broth is autolyzed at 55 °C for 2 h, use a 50 kDa ceramic membrane separation system to remove macromolecular impurities.

[0039] Add citric acid to the solution obtained by the above fermentation and filtration to adjust the pH to 3.5, load the sample at a flow rate of 1.5 CV / h, select strong cation exchange resin SP SepHarose 6FF as the stationary phase, balance the column bed with a sodium citrate buffer solution with a pH of 4.0, and control the flow rate at 2 CV / h for gradient elution separation: Buffer A: Sodium citrate buffer solution with a pH of 4.0; Buffer B: 1 M NaCl; From 0 to 10 min, the mobile phase A is 90% and the mobile phase B is 10%; From 10 to 15 min, the mobile phase A decreases uniformly to 70% and the mobile phase B increases uniformly to 30%; From 15 to 30 min, the mobile phase A is 50% and the mobile phase B is 50%; From 30 to 40 min, the mobile phase A increases uniformly to 30% and the mobile phase B decreases uniformly to 70%;

[0040] Pass the ion exchange eluate through a 10 kDa ultrafiltration system with a transmembrane pressure of 0.2 MPa, circulate and concentrate it to 1 / 10 of the original volume. Add 5% trehalose to the concentrated solution, adjust the pH to 6.8, sublime the free water at -45°C and 50 Pa vacuum for 24 hours, and then place it at 25°C and 10 Pa vacuum to remove the bound water for 12 hours. The moisture content of the final product is ≤ 3%, and the composite plant protein is obtained.

[0041] Comparative Example 1

[0042] The difference between this comparative example and Example 1 is that the raw material pretreatment in this comparative example is simple crushing and sieving. Specifically: Use 45 parts of soybeans, 28 parts of peas, 12 parts of oats, 13 parts of quinoa, and 7 parts of chia seeds for raw material pretreatment: Crush them with a crusher and sieve through a 200-mesh sieve, soak them in 50°C warm water at a material-to-water ratio of 1:3 for 2 h; the rest is the same as in Example 1.

[0043] Comparative Example 2

[0044] The difference between this comparative example and Example 1 is that only neutral protease is added in this comparative example. Specifically: Add 0.7% by mass of neutral protease, perform ultrasonic-assisted enzymatic hydrolysis at 50°C and a pH value of 6.5 for 2 h, then raise the temperature to 55°C and continue enzymatic hydrolysis at a pH value of 7.0 for 1.5 h and inactivate it at 85°C for 10 min; then add a composite strain for three-stage fermentation treatment; in the first stage, add 2.5% Lactobacillus plantarum at 37°C and add 0.05% ε-polylysine hydrochloride and let it stand for anaerobic fermentation for 12 h; in the second stage, at 42°C and 120 rpm, add 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan sulfate and perform oscillating microaerobic fermentation for 12 h; in the third stage, let it stand at 30°C for after-ripening until the pH value naturally drops to 4.3, add 0.05% ε-polylysine hydrochloride and 0.5% fucoidan sulfate, and after the fermentation broth is autolyzed at 55°C for 2 h, use a 50 kDa ceramic membrane separation system to remove macromolecular impurities; the rest is the same as in Example 1.

[0045] Comparative Example 3

[0046] The difference between this comparative example and Example 1 is that only neutral protease and cellulase are added in this comparative example, specifically: Subsequently, 0.4% neutral protease and 0.3% cellulase by mass fraction are added, and ultrasonic-assisted enzymatic hydrolysis is carried out at 50 °C and pH 6.5 for 2 h, then the temperature is raised to 55 °C and pH 7.0, and enzymatic hydrolysis is continued for 1.5 h, followed by inactivation at 85 °C for 10 min; Then, a composite strain is added for three-stage fermentation treatment; In the first stage, 2.5% Lactobacillus plantarum is added at 37 °C, and 0.08% ε-polylysine hydrochloride is added and left to ferment anaerobically for 12 h; In the second stage, at 42 °C and 120 rpm, 1.25% Candida utilis, 1.25% Bacillus natto, and 0.3% fucoidan are added for oscillatory microaerobic fermentation for 12 h; In the third stage, after standing and ripening at 30 °C until the pH naturally drops to 4.3, 0.05% ε-polylysine hydrochloride and 0.5% fucoidan are added, and after the fermentation broth is autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; The rest is the same as in Example 1.

[0047] Comparative Example 4

[0048] The difference between this comparative example and Example 1 is that no supplementary substances are added during the fermentation with the addition of strains in this comparative example, specifically: Subsequently, 0.4% neutral protease, 0.2% cellulase, and 0.1% pectinase by mass fraction are added, and ultrasonic-assisted enzymatic hydrolysis is carried out at 50 °C and pH 6.5 for 2 h, then the temperature is raised to 55 °C and pH 7.0, and enzymatic hydrolysis is continued for 1.5 h, followed by inactivation at 85 °C for 10 min; Then, a composite strain is added for three-stage fermentation treatment; In the first stage, 2.5% Lactobacillus plantarum is added at 37 °C and fermented for 12 h; In the second stage, at 42 °C and 120 rpm, 1.25% Candida utilis and 1.25% Bacillus natto are added for oscillatory microaerobic fermentation for 12 h; In the third stage, after standing and ripening at 30 °C until the pH naturally drops to 4.3, the fermentation broth is autolyzed at 55 °C for 2 h, and a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; The rest is the same as in Example 1.

[0049] Comparative Example 5

[0050] The difference between this comparative example and Example 1 is that in this comparative example, the strains were added simultaneously, specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% were added. Ultrasonic-assisted enzymatic hydrolysis was carried out at 50 °C and a pH value of 6.5 for 2 h, then the temperature was raised to 55 °C and the pH value was adjusted to 7.0 for continued enzymatic hydrolysis for 1.5 h, followed by inactivation at 85 °C for 10 min; Then, a composite strain of 50% Lactobacillus plantarum, 25% Bacillus natto, and 25% Candida utilis was added according to an inoculation amount of 5% by mass for three-stage fermentation treatment; In the first stage, it was carried out at 37 °C, and 0.05% ε-polylysine hydrochloride was added for static anaerobic fermentation for 12 h; In the second stage, it was carried out at 42 °C and 120 rpm, and 0.4% fucoidan was added for oscillating microaerobic fermentation for 12 h; In the third stage, it was allowed to stand for after-ripening at 30 °C until the pH value naturally dropped to 4.3, 0.05% ε-polylysine hydrochloride and 0.5% fucoidan were added, and after the fermentation broth was autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system was used to remove macromolecular impurities; The rest was the same as in Example 1.

[0051] Comparative Example 6

[0052] The difference between this comparative example and Example 1 is that in this comparative example, the additives were different at different stages during the strain fermentation process, specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% were added. Ultrasonic-assisted enzymatic hydrolysis was carried out at 50 °C and a pH value of 6.5 for 2 h, then the temperature was raised to 55 °C and the pH value was adjusted to 7.0 for continued enzymatic hydrolysis for 1.5 h, followed by inactivation at 85 °C for 10 min; Then, a composite strain was added for three-stage fermentation treatment; In the first stage, 2.5% Lactobacillus plantarum was added at 37 °C, and 0.05% ε-polylysine hydrochloride was added for static anaerobic fermentation for 12 h; In the second stage, it was carried out at 42 °C and 120 rpm, 1.25% Candida utilis and 1.25% Bacillus natto, and 0.4% ε-polylysine hydrochloride were added for oscillating microaerobic fermentation for 12 h; In the third stage, it was allowed to stand for after-ripening at 30 °C until the pH value naturally dropped to 4.3, 0.05% ε-polylysine hydrochloride and 0.5% fucoidan were added, and after the fermentation broth was autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system was used to remove macromolecular impurities; The rest was the same as in Example 1.

[0053] Comparative Example 7

[0054] The difference between this comparative example and Example 1 is that the additives in different stages during the bacterial strain fermentation process in this comparative example are different. Specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% are added. Ultrasonic-assisted enzymatic hydrolysis is carried out at 50 °C and a pH value of 6.5 for 2 h, then the temperature is raised to 55 °C and the pH value is 7.0 for continued enzymatic hydrolysis for 1.5 h, and then inactivated at 85 °C for 10 min; then a composite bacterial strain is added for three-stage fermentation treatment; in the first stage, 2.5% Lactobacillus plantarum is added at 37 °C, and 0.05% fucoidan is added for static anaerobic fermentation for 12 h; in the second stage, at 42 °C and 120 rpm, 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan are added for oscillating micro-aerobic fermentation for 12 h; in the third stage, after standing and ripening at 30 °C until the pH value naturally drops to 4.3, 0.05% ε-polylysine hydrochloride and 0.5% fucoidan are added. After the fermentation broth is autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; the rest is the same as in Example 1.

[0055] Comparative Example 8

[0056] The difference between this comparative example and Example 1 is that the proportion of substances added in the third stage of bacterial strain fermentation in this comparative example is different. Specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% are added. Ultrasonic-assisted enzymatic hydrolysis is carried out at 50 °C and a pH value of 6.5 for 2 h, then the temperature is raised to 55 °C and the pH value is 7.0 for continued enzymatic hydrolysis for 1.5 h, and then inactivated at 85 °C for 10 min; then a composite bacterial strain is added for three-stage fermentation treatment; in the first stage, 2.5% Lactobacillus plantarum is added at 37 °C, and 0.05% ε-polylysine hydrochloride is added for static anaerobic fermentation for 12 h; in the second stage, at 42 °C and 120 rpm, 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan are added for oscillating micro-aerobic fermentation for 12 h; in the third stage, after standing and ripening at 30 °C until the pH value naturally drops to 4.3, 0.1% ε-polylysine hydrochloride and 0.45% fucoidan are added. After the fermentation broth is autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; the rest is the same as in Example 1.

[0057] Comparative Example 9

[0058] The difference between this comparative example and Example 1 lies in the different proportions of substances added in the third stage of the strain fermentation in this comparative example. Specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% are added, and ultrasonic-assisted enzymatic hydrolysis is carried out for 2 h at 50 °C and a pH value of 6.5. Then, the temperature is raised to 55 °C and the pH value is adjusted to 7.0, and enzymatic hydrolysis is continued for 1.5 h, followed by inactivation at 85 °C for 10 min; then a composite strain is added for three-stage fermentation treatment; in the first stage, 2.5% Lactobacillus plantarum is added at 37 °C, and 0.05% ε-polylysine hydrochloride is added for static anaerobic fermentation for 12 h; in the second stage, 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan are added at 42 °C and 120 rpm for oscillating microaerobic fermentation for 12 h; in the third stage, after standing and ripening at 30 °C until the pH value naturally drops to 4.3, 0.01% ε-polylysine hydrochloride and 0.54% fucoidan are added. After the fermentation broth is autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; the rest is the same as in Example 1.

[0059] Comparative Example 10

[0060] The difference between this comparative example and Example 1 lies in the different additives in different stages during the strain fermentation in this comparative example. Specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% are added, and ultrasonic-assisted enzymatic hydrolysis is carried out for 2 h at 50 °C and a pH value of 6.5. Then, the temperature is raised to 55 °C and the pH value is adjusted to 7.0, and enzymatic hydrolysis is continued for 1.5 h, followed by inactivation at 85 °C for 10 min; then a composite strain is added for three-stage fermentation treatment; in the first stage, 2.5% Lactobacillus plantarum is added at 37 °C, and 0.05% ε-polylysine hydrochloride is added for static anaerobic fermentation for 12 h; in the second stage, 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan are added at 42 °C and 120 rpm for oscillating microaerobic fermentation for 12 h; in the third stage, after standing and ripening at 30 °C until the pH value naturally drops to 4.3, 0.55% ε-polylysine hydrochloride is added. After the fermentation broth is autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; the rest is the same as in Example 1.

[0061] Comparative Example 11

[0062] The difference between this comparative example and Example 1 is that different additives are used at different stages during the bacterial strain fermentation process in this comparative example, specifically: Subsequently, neutral protease with a mass fraction of 0.4%, cellulase with a mass fraction of 0.2%, and pectinase with a mass fraction of 0.1% are added, and ultrasonic-assisted enzymatic hydrolysis is carried out at 50 °C and a pH value of 6.5 for 2 h, then the temperature is raised to 55 °C and the pH value is 7.0 for continued enzymatic hydrolysis for 1.5 h, and then inactivated at 85 °C for 10 min; Then a composite bacterial strain is added for three-stage fermentation treatment; In the first stage, 2.5% Lactobacillus plantarum is added at 37 °C, and 0.05% ε-polylysine hydrochloride is added for static anaerobic fermentation for 12 h; In the second stage, at 42 °C and 120 rpm, 1.25% Candida utilis, 1.25% Bacillus natto, and 0.4% fucoidan sulfate are added for oscillating microaerobic fermentation for 12 h; In the third stage, after standing and ripening at 30 °C until the pH value naturally drops to 4.3, 0.55% fucoidan sulfate is added, and after the fermentation broth is autolyzed at 55 °C for 2 h, a 50 kDa ceramic membrane separation system is used to remove macromolecular impurities; The rest is the same as Example 1.

[0063] Experiment 1: Protein determination

[0064] According to the standard of GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Foods", the total protein content was determined by the Kjeldahl method, and the HPCL purity was determined by the Laemmli method for the examples and comparative examples; The results are shown in Table 1 below.

[0065] Table 1

[0066]

[0067]

[0068] Experiment 2: Determination of ACE inhibition rate

[0069] The determination of the ACE inhibition rate in this experiment was evaluated by measuring the production amount of hippuric acid.

[0070] The chromatographic conditions for the determination of hippuric acid content were: chromatographic column: polaris C18, column temperature 30 °C, flow rate 1.0 mL / min, injection volume 10 μL, detection wavelength 228 nm.

[0071] The plant protein powders obtained in Examples 1-3 and Comparative Examples 1-11 were dissolved in 0.1 mol / L boric acid buffer solution with 0.3 mol / L NaCl, and the protein concentration of each portion was 10 μg / mL. The supernatant was taken by centrifugation as the inhibitor solution. When preparing the reaction system, 50 μL of buffer solution and 200 μL of 0.005 mol / L HHL substrate solution were added to the control group, 50 μL of inhibitor solution and 200 μL of HHL were added to the sample group, and 200 μL of 1 mol / L HCl was added in advance to terminate the reaction in the blank group. After preheating all groups at 37 °C for 5 min, 50 μL of 0.4 U / mL ACE solution was added to the control group and the sample group to start the reaction. After incubation at 37 °C for 30 min, HCl was immediately added to terminate the reaction. Subsequently, 1.2 mL of pre-cooled ethyl acetate was added to each tube to extract hippuric acid. After oscillation and centrifugation, 0.8 mL of the ester phase layer was taken and dried, and then redissolved in 4 mL of deionized water. The absorbance OD was measured at 228 nm. Among them, the control group was the full enzyme activity reaction without inhibitor, and the blank group deducted the substrate background interference. Finally, the inhibitory effect of the inhibitor on ACE activity and IC50 / (μg / ml) were reflected by the absorbance difference. The results are shown in Table 2 below.

[0072] Inhibition rate of inhibitor on ACE = [(OD1 - OD2) / OD1] x 100%

[0073] OD1 is the absorbance value of the control group; OD2 is the absorbance value of the sample group

[0074] Table 2

[0075] Group ACE inhibition rate / % Example 1 90.51 Example 2 88.83 Example 3 89.27 Comparative Example 1 85.92 Comparative Example 2 81.08 Comparative Example 3 80.38 Comparative Example 4 73.63 Comparative Example 5 75.64 Comparative Example 6 77.16 Comparative Example 7 78.54 Comparative Example 8 82.58 Comparative Example 9 81.63 Comparative Example 10 78.24 Comparative Example 11 79.43

Claims

1. A fermented composite plant protein, characterized in that: The preparation method is: 40-50 parts of soybeans, 25-30 parts of peas, 10-15 parts of oats, 10-15 parts of quinoa and 5-8 parts of chia seeds were used for raw material pretreatment: after being subjected to microwave puffing treatment at 800 watts for 60 seconds, the raw materials were ultra-finely crushed to a particle size of 50 microns, and then soaked in 50°C warm water at a material-water ratio of 1:3 for 2 hours; Then, 0.4% neutral protease, 0.2% cellulase and 0.1% pectinase were added, and ultrasound-assisted enzymolysis was carried out at 50°C and pH 6.5 for 2 h. The temperature was then raised to 55°C and pH 7.0, and enzymolysis was continued for 1.5 h before being inactivated at 85°C for 10 min. Then, the composite strain was added for three-stage fermentation treatment; citric acid was added to the solution obtained by the fermentation filtration to adjust the pH to 3.5, and the sample was loaded at a flow rate of 1.5 CV / h, and the strong cation exchange resin SP SepHarose 6FF was selected as the stationary phase, and the column bed was equilibrated with a sodium citrate buffer with a pH of 4.0, and the flow rate was controlled at 2CV / h for gradient elution separation: buffer A: pH 4.0 sodium citrate buffer; buffer B: 1M NaCl; at 0-10min, the mobile phase A was 90%, and the mobile phase B was 10%; at 10-15min, the mobile phase A was uniformly reduced to 70%, and the mobile phase B was uniformly increased to 30%; at 15-30min, the mobile phase A was 50%, and the mobile phase B was 50%; at 30-40min, the mobile phase A was uniformly increased to 30%, and the mobile phase B was uniformly reduced to 70%; The ion exchange eluate was passed through a 10 kDa ultrafiltration system with a transmembrane pressure of 0.2-0.4 MPa, and circulated and concentrated to 1 / 10 of the original volume. 5% trehalose was added to the concentrate, and the pH was adjusted to 6.

8. Free water was sublimated at -45°C and 50Pa vacuum for 24 hours, and then the bound water was removed at 25°C and 10Pa vacuum for 12 hours. The moisture content of the final product was ≤3%, thus obtaining a composite plant protein.

2. The method for preparing a fermented composite plant protein according to claim 1, characterized in that: The fermentation treatment is as follows: in the first stage, 2.5% Lactobacillus plantarum is added at 37° C. and allowed to stand for anaerobic fermentation for 12 hours; in the second stage, 1.25% Candida utilis and 1.25% Bacillus natto are added at 42° C. and 120 rpm for micro-aerobic fermentation for 12 hours; in the third stage, the fermentation liquid is allowed to stand at 30° C. and then matured until the pH value naturally drops to 4.3; after the fermentation liquid is subjected to autolysis treatment at 55° C. for 2 hours, a 50 kDa ceramic membrane ceramic membrane separation system is used to remove large molecular impurities.

3. A fermented composite plant protein according to claim 2, characterized in that: The fermentation process also includes the addition of additives: in the first stage, 0.03-0.08% of ε-polylysine hydrochloride is added.

4. The fermented composite plant protein according to claim 2, characterized in that: The fermentation process also includes the addition of additives: 0.3-0.5% fucoidan sulfate is added in the second stage.

5. The fermented composite plant protein according to claim 2, characterized in that: The fermentation process also includes the addition of additives: in the third stage, 0.05% ε-polylysine hydrochloride and 0.5% fucoidan sulfate are added.

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