Preparation method and application of low-sensitization high-nutrition pea protein egg custard liquid

Abstract

The invention discloses a preparation method and application of low-sensitization high-nutrition pea protein egg custard liquid. The preparation method comprises the following steps: (1) extracting pea protein A from a pea variety A; extracting pea protein B from the pea variety B; uniformly mixing the pea protein A and the pea protein B to obtain the low-sensitization high-nutrition pea protein; (2) performing compound modification on the low-sensitization high-nutrition pea protein to obtain modified low-sensitization high-nutrition pea protein; and (3) mixing and emulsifying the modified low-sensitization high-nutrition pea protein, calcium chloride, edible vegetable oil, edible polysaccharide and water to obtain the low-sensitization high-nutrition pea protein egg custard liquid. The pea protein egg custard liquid prepared by the preparation method disclosed by the invention can be kept uniform and stable after being stored at low temperature for a long time, and can be used as an egg substitute in the food field; the pea protein egg custard is used for preparing the pea protein egg custard, the formed egg custard is uniform in tissue structure, and the structure and the taste of the egg custard can be simulated to a certain extent.

Description

Technical Field

[0001] The invention relates to the technical field of pea protein egg custard processing, and specifically to a preparation method and application of low-allergenic and high-nutrition pea protein egg custard liquid. Background Art

[0002] As a nutritious source of high-quality protein, eggs are usually used in food processing in the form of steamed eggs, scrambled eggs, fried eggs, etc. or as functional raw materials because of their excellent emulsification, gelling, water retention, foaming and other properties. However, with the prevalence of multi-dimensional consumption concepts such as safety, green, health and low-carbon lifestyles, traditional animal eggs are facing more and more new controversies. Studies have shown that high cholesterol in eggs increases the risk of cardiovascular disease, and high phosphatidylcholine content is associated with the occurrence of atherosclerosis. More importantly, the health risks of allergens, cholesterol, antibiotic residues and foodborne diseases (such as Salmonella) in animal eggs cannot be ignored. In addition, the needs of vegetarians make some consumers unable to eat traditional egg products. The development of plant-based egg substitutes has become a development trend. Compared with traditional eggs, plant-based egg substitutes do not contain allergens and cholesterol, are not affected by avian influenza, and are more environmentally friendly and sustainable. They can provide consumers and egg product industry research with an additional viable option.

[0003] At present, the raw materials of plant-based egg substitutes are mainly plant proteins (soybeans, mung beans, red beans, chickpeas, potatoes, wheat, oats, etc.), polysaccharides and emulsifiers, but most of them are single-type plant protein systems, which may lead to some prominent problems, such as insufficient lysine or methionine content, so mixing multiple proteins is considered a feasible solution. In addition, soy protein, peanut protein, etc. are often troubled by allergens and are difficult to be widely used. Pea protein is rich in nutritional value, balanced in amino acid composition, high in lysine content, and has an amino acid composition similar to that of egg protein. It can not only meet human demand for high-quality protein, but also has environmental sustainability. With its environmental friendliness, non-GMO, zero cholesterol, no allergens, and good digestibility, it can be used as a viable choice for plant protein to replace egg protein. However, natural pea protein has a significant gap with animal protein in terms of solubility, emulsification, foaming and gelling, which has caused certain troubles for industrial production and limited the development and application of its related products. Therefore, it is necessary to modify natural pea protein to increase its utilization field and nutritional properties.

[0004] In addition, there are very few egg substitutes and research on the market. Most of them exist in the form of mixtures and are mainly used in cakes, mayonnaise, mousse, egg rolls, etc. There are very few products prepared as plant egg custards in the form of emulsions. The main reason is that the thermal gelation properties of plant protein are poor, which may lead to poor texture, too loose or too hard problems during processing, causing certain troubles for industrial production and limiting the development and application of related products. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a preparation method and application of a low-allergenic and high-nutrition pea protein egg custard liquid, so as to solve the technical problems such as poor thermal gelling performance of existing plant proteins and poor stability of the formed protein egg custard liquid.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for preparing low-allergenic and high-nutrition pea protein egg custard liquid comprises the following steps:

[0008] Step (1), extracting pea protein A from pea variety A having an allergenic protein content of less than or equal to 5wt%; extracting pea protein B from pea variety B having a lysine content of greater than or equal to 1.85g / 100g protein and a methionine content of greater than or equal to 0.10g / 100g protein; and uniformly mixing pea protein A and pea protein B to obtain low-allergenic and high-nutrition pea protein;

[0009] Step (2), compound-modifying the low-allergenic and high-nutrition pea protein to obtain modified low-allergenic and high-nutrition pea protein;

[0010] Step (3), emulsifying the modified low-allergenic and high-nutrition pea protein, calcium chloride, edible vegetable oil, edible polysaccharide and water to obtain a low-allergenic and high-nutrition pea protein egg custard liquid.

[0011] The pea protein egg custard liquid prepared by the invention can exist in the form of a stable emulsion, has good fluidity, and can remain uniform and stable after long-term low-temperature storage; the pea protein egg custard prepared by using the pea protein egg custard liquid has good formability and appearance color, and the formed egg custard has a uniform tissue structure, and can simulate the structure and taste of egg custard to a certain extent.

[0012] In the method for preparing the above-mentioned low-allergenic and high-nutrition pea protein egg custard liquid, in step (1), pea variety A is Zhongqin No. 1 or Zhongwan No. 8, and pea variety B is Zhongwan No. 9 or Changshoudou No. 1; the mass ratio of pea protein A to pea protein B is 1:1, 3:2 or 7:3. The low-allergenic and high-nutrition pea protein prepared under these three ratios is not only rich in nutrition and low in allergenicity, but also can be used to prepare pea protein egg custard liquid after composite modification using the method of the present invention, so as to obtain pea protein egg custard liquid with good emulsification, gelation and stability.

[0013] In the method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid, in step (1), pea protein A is extracted from pea variety A by the sophora japonica method, and the method for extracting pea protein A comprises the following steps:

[0014] Step (1-1), soaking pea variety A in water overnight, removing pea skins, and obtaining pea kernels;

[0015] Step (1-2), drying the pea kernels at a temperature of 45 to 60° C. for 8 to 12 hours, then grinding them into powder and sieving them through a 200-mesh sieve to obtain pea flour;

[0016] Step (1-3), adding pea flour to water and dispersing it evenly, wherein the mass ratio of pea flour to water is 1:3-15; then centrifuging at 4000-5000 r / min for 15-20 min, obtaining upper, middle and lower layers of separation after the centrifugation, removing the starch precipitate at the bottom layer, collecting the supernatant and the middle layer protein solution, and obtaining a crude protein solution;

[0017] Step (1-4), after the crude protein solution is sieved, the pH is adjusted to 5.8-6.2, and then lactic acid bacteria are added and allowed to stand and cool; the amount of lactic acid bacteria is 1-1.5 mL of lactic acid bacteria suspension is added to every 100 mL of crude protein solution, and the number of live lactic acid bacteria in the lactic acid bacteria suspension is (1.5-1.8)*10 8CFU / mL; the cold precipitation condition is to stand at 2-6℃ for 2-3h; since there is still a small amount of starch mixed in the crude protein solution, adding lactic acid bacteria can cause the starch to agglutinate and precipitate, and the starch precipitate obtained after agglutination with lactic acid bacteria is different from the density of pea protein precipitate, so the starch precipitate and the pea protein precipitate can be separated by stratification through centrifugation. The present invention first precipitates most of the pea protein in the crude protein solution by adjusting the pH, and then adds lactic acid bacteria to agglutinate the starch, which is more conducive to the full agglutination of starch, thereby achieving complete separation of starch and pea protein. If the pH is not adjusted first to precipitate most of the pea protein, and lactic acid bacteria are directly added to agglutinate the starch, the agglutination of the lactic acid bacteria on the starch is affected, and it is difficult to fully precipitate the starch, thereby affecting the purity of the pea protein. If the pH of the crude protein solution is too high, the acidic groups in the pea protein molecules will dissociate more negative charges, resulting in an increase in the repulsive force between the pea protein molecules and an increase in solubility; if the pH is too low, the basic groups in the pea protein molecules will dissociate more positive charges, which will also increase its solubility. The present invention adjusts the pH of the crude protein solution to within the range of 5.8 to 6.2, controls the amount of lactic acid bacteria added, and then places it at a temperature of 2 to 6°C for cold precipitation. This can not only ensure that the pea protein molecules are precipitated as much as possible from the crude protein solution due to the small range of charges dissociated from the acidic and basic groups of the pea protein molecules; it can also enable the agglutination of lactic acid bacteria on impurity starch to be fully exerted, thereby achieving the separation of pea protein from starch. In addition, under the above-mentioned cold precipitation conditions, pea protein has good stability and is not prone to denaturation and degradation.

[0018] Step (1-5), after the cold precipitation is completed, centrifuge at 4000-5000 r / min for 15-20 min to obtain upper, middle and lower layers of separation, remove the starch precipitate at the bottom, collect the supernatant and the middle layer protein precipitate and freeze-dry to obtain pea protein A;

[0019] Pea protein B is extracted from pea variety B using the completely identical method from step (1-1) to step (1-5).

[0020] In the method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid, in step (1), pea variety A is Zhongqin No. 1, and pea variety B is Zhongwan No. 9; the mass ratio of pea protein A to pea protein B is 1:1; and the method for extracting pea protein A from pea variety A comprises the following steps:

[0021] Step (1-1), soaking pea variety A in water overnight, removing pea skins, and obtaining pea kernels;

[0022] Step (1-2), drying the pea kernels at 45° C. for 12 h, grinding them into powder and sieving them through a 200-mesh sieve to obtain pea flour;

[0023] Step (1-3), adding pea flour to water and dispersing it evenly, the mass ratio of pea flour to water is 1:10; then centrifuging at 4000 r / min for 20 min, obtaining upper, middle and lower layers of separation after the centrifugation, removing the starch precipitate at the bottom layer, collecting the supernatant and the middle layer protein solution, and obtaining a crude protein solution;

[0024] Step (1-4), after the crude protein solution is passed through a 100-mesh sieve (to remove impurities and part of the starch), the pH is adjusted to 6.2, and then lactic acid bacteria are added and allowed to stand and cool; the amount of lactic acid bacteria is 1.0 mL of lactic acid bacteria suspension per 100 mL of crude protein solution, and the number of live lactic acid bacteria in the lactic acid bacteria suspension is 1.58*10 8 CFU / mL; cold precipitation condition is 4℃ for 2h;

[0025] Step (1-5), after the cold precipitation is completed, centrifuge at 4000r / min for 20min to obtain upper, middle and lower three-layer separation, remove the starch precipitate at the bottom, collect the supernatant (containing soluble protein) and the middle layer protein precipitate and freeze-dry to obtain pea protein A;

[0026] Pea protein B is extracted from pea variety B using the completely identical method from step (1-1) to step (1-5).

[0027] In the method for preparing the hypoallergenic and highly nutritious pea protein egg custard liquid, in step (2), the method for composite modification of the hypoallergenic and highly nutritious pea protein comprises the following steps:

[0028] Step (2-1), adding water to the low-allergenic and high-nutrition pea protein to prepare a pea protein dispersion;

[0029] Step (2-2), stirring the pea protein dispersion at room temperature and then performing high-pressure homogenization treatment, and after the high-pressure homogenization treatment is completed, a pea protein homogenate is obtained;

[0030] Step (2-3), adjusting the pH of the pea protein homogenate and adding TG enzyme for modification treatment, inactivating the enzyme and freeze-drying after the modification treatment to obtain modified low-allergenic and high-nutrition pea protein.

[0031] In the method for preparing the above-mentioned low-allergenic and high-nutrition pea protein egg custard liquid, in step (2-1), the total mass fraction of the low-allergenic and high-nutrition pea protein in the pea protein dispersion is 6 to 16 wt% (preferably 14 wt%, at which the viscosity and density of the pea protein dispersion are moderate, and the effect of high-pressure homogenization treatment is ideal);

[0032] In step (2-2), the stirring time at room temperature is 2 to 3 hours; the high-pressure homogenization treatment condition is 20 to 120 MPa (preferably 60 MPa), and the cycle is 1 to 5 times (preferably 3 times); under the high-pressure homogenization treatment condition, the deamidation of pea protein can be effectively promoted, providing more enzyme action sites for the subsequent TG enzyme modification treatment;

[0033] In step (2-3), the pH of the pea protein homogenate is adjusted to 5-11 (preferably 5.0-8.0, within the pH range, the activity of TG enzyme is high, and the effect of catalyzing the cross-linking reaction between pea protein molecules is better); the amount of TG enzyme is 0.02-0.12wt% of the mass of the pea protein homogenate; the modification treatment temperature is 20-60°C (preferably 45-55°C), and the modification treatment insulation time is 0.5-3h; under the modification reaction conditions, the effect of TG enzyme modification treatment is ideal, and low-allergenic and high-nutrition pea protein with good fluidity, uniformity and stability can be obtained; the enzyme inactivation conditions are: 95-100°C insulation for 10-15min; the low-allergenic and high-nutrition pea protein treated under the high-pressure homogenization and TG enzyme modification treatment conditions of the present invention has significantly improved solubility, emulsification, low-temperature stability and gelation.

[0034] The present invention adopts high-pressure homogenization to process low-allergenic and high-nutrition pea protein. Through mechanical effects such as shearing and cavitation of high-pressure homogenization, the molecular structure of the pea protein is destroyed, the size of the pea protein particles is reduced, thereby increasing its surface area, which is conducive to the full contact between the pea protein and the solvent and improving its solubility. Combined with TG enzyme cross-linking treatment, the pea protein after deamidation has good fluidity and flavor, and more TG enzyme action sites are exposed. By catalyzing the covalent cross-linking between pea protein molecules, a stable pea protein network structure is formed, thereby preventing the separation of fat and whey.

[0035] The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid, step (3) specifically comprises the following steps:

[0036] Step (3-1), adding water to the modified low-allergenic and high-nutrition pea protein to prepare a pea protein mixed solution;

[0037] Step (3-2), adding calcium chloride and edible vegetable oil to the pea protein mixture, shearing and emulsifying, to obtain an emulsion;

[0038] Step (3-3), adding edible polysaccharide to the emulsion and sequentially performing shear homogenization and high-pressure homogenization treatments, after the high-pressure homogenization treatment is completed, a low-allergenic and high-nutrition pea protein egg custard liquid is obtained.

[0039] In the method for preparing the hypoallergenic and highly nutritious pea protein egg custard liquid, in step (3-1), the mass fraction of the modified hypoallergenic and highly nutritious pea protein in the pea protein mixture is 6 to 16 wt %;

[0040] In step (3-2), the amount of calcium chloride added is 0.1-0.5wt% of the mass of the pea protein mixture (adding calcium chloride in this amount range can significantly improve the water holding capacity and tissue structure stability of the egg custard formed after the low-allergenic and high-nutrition pea protein egg custard liquid is gelled, and significantly improve its texture characteristics), and the amount of edible vegetable oil added is 10-15wt% of the mass of the pea protein mixture; the shear emulsification conditions are: emulsification at 10000-15000rpm for 2-10min; the edible vegetable oil is one or two of soybean oil, peanut oil, palm oil, rapeseed oil, sunflower seed oil and olive oil, and a mixture of more than two;

[0041] In step (3-3), the amount of edible polysaccharide added is 0.02-0.1wt% of the mass of the pea protein mixture; the shear homogenization conditions are: homogenization at 10000-15000rpm for 2-10min, the pressure of high-pressure homogenization is 20-120MPa, and the high-pressure homogenization treatment is 1-5 times; the edible polysaccharide is one or two of yeast β-glucan, curdlan, methylcellulose, carrageenan and gellan gum, and a mixture of two or more thereof.

[0042] In the method for preparing the hypoallergenic and highly nutritious pea protein egg custard liquid, in step (3-1), the mass fraction of the modified hypoallergenic and highly nutritious pea protein in the pea protein mixture is 14wt%; the egg custard formed after the pea protein mixture of this concentration is gelled has moderate hardness, uniform texture and good stability;

[0043] In step (3-2), the amount of calcium chloride added is 0.4wt% of the mass of the pea protein mixture, the edible vegetable oil is peanut oil, and the amount of peanut oil added is 10wt% of the mass of the pea protein mixture; before shearing homogenization, 0.01-0.05wt% of edible coloring is added to the mass of the pea protein mixture, and the edible coloring is one or two or more of curcumin, lutein or β-carotene; the shearing emulsification conditions are: emulsification at 12000rpm for 3min;

[0044] In step (3-3), the amount of edible polysaccharide added is 0.1wt% of the mass of the pea protein mixture; the shear homogenization conditions are: homogenization at 12000rpm for 2min, a homogenization pressure of 60MPa, and homogenization treatment for 3 times; the edible polysaccharide is yeast β-glucan.

[0045] The invention discloses an application of a low-allergenic and high-nutrition pea protein egg custard liquid, wherein the low-allergenic and high-nutrition pea protein egg custard liquid is used in the food field as an egg substitute.

[0046] A small amount of water or no water is added to the pea protein egg custard liquid prepared by the above method, and heated in a steamer for 8 to 15 minutes under medium heat to obtain a pea protein egg custard product, which has the gel property provided by egg protein, low allergenic protein content, and moderate gel strength. Since the pea protein egg custard liquid prepared by the present invention can still remain uniform and stable after long-term low-temperature storage, it is suitable for industrial production and circulation sales.

[0047] The technical solution of the present invention achieves the following beneficial technical effects:

[0048] 1. The present invention extracts pea protein from two pea varieties with low allergenic protein content and high lysine and methionine content, respectively, and mixes them evenly in proportion, which can effectively make up for the deficiency of lysine or methionine content of plant protein and improve the nutritional value of the product; then it is subjected to high-pressure homogenization combined with TG enzyme cross-linking composite modification, which can significantly improve the solubility, emulsification, low-temperature stability and gel properties of the plant-based high-protein system, and broaden its application range.

[0049] 2. The pea protein egg custard liquid prepared by the present invention using the modified low-allergenic and high-nutrition pea protein as a raw material can exist in the form of a stable emulsion, has good fluidity, and can remain uniform and stable after long-term low-temperature storage; in addition, the steamed egg prepared by using the low-allergenic and high-nutrition pea protein egg custard liquid of the present invention exhibits a uniform and dense gel network structure, which is similar to the structure of egg custard, is conducive to industrial production and circulation sales, and exerts the excellent health effects and functional characteristics of pea protein, making it an excellent substitute for animal protein resources.

[0050] 3. The pea protein egg custard liquid prepared by the preparation method of the pea protein egg custard liquid of the present invention can exist in the form of a stable emulsion, has good fluidity, and can remain uniform and stable after long-term low-temperature storage; the prepared pea protein egg custard has good formability and appearance color, and the formed egg custard has a uniform tissue structure, which can simulate the structure and taste of egg custard to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Actual photos of the appearance of the pea protein product prepared from the low-allergenic and high-nutrition pea protein egg custard liquid in the embodiment of the present invention;

[0052] Figure 2 A real shot of the organization of a pea protein product prepared from low-allergenic and high-nutrition pea protein egg custard liquid in an embodiment of the present invention;

[0053] Figure 3 Texture characteristics diagram (hardness and viscosity) of pea protein custard at different TG enzyme addition amounts in the embodiment of the present invention;

[0054] Figure 4 Texture properties diagram (elasticity and cohesion) of pea protein custard at different TG enzyme addition amounts in the embodiment of the present invention;

[0055] Figure 5 Electron microscopic image of the pea protein dispersion prepared from the low-allergenic and high-nutrition pea protein in the embodiment of the present invention;

[0056] Figure 6 Electron microscopic image of a pea protein mixture prepared from modified low-allergenic and high-nutrition pea protein in an embodiment of the present invention;

[0057] Figure 7 Electron microscopic image of the low-allergenic and highly nutritious pea protein egg custard liquid in the embodiment of the present invention;

[0058] Figure 8 Electron microscopic image of the low-allergenic and high-nutrition pea protein custard in the embodiment of the present invention;

[0059] Fig. 9 Electron microscope image of egg liquid in the embodiment of the present invention;

[0060] Fig.10 An electron microscope image of the egg custard in the embodiment of the present invention. DETAILED DESCRIPTION

[0061] 1. Test methods

[0062] (1) Determination of crude protein: refer to the first method of GB 5009.5-2016.

[0063] (2) Determination of crude fat: refer to the second method of GB 5009.6-2016.

[0064] (3) Determination of amino acids: refer to GB 5009.124-2016, 2016.

[0065] (4) Determination method of allergenic protein detection:

[0066] Reductive SDS-PAGE (polyacrylamide gel electrophoresis): The mass fractions of the separation gel and the concentrated gel are 12.5wt% and 5.02wt%, respectively, the sample load is 20μL, the sample mass concentration is 20mg / mL, the electrophoresis instrument voltage is set to 80V, and the voltage is increased to 120V after the sample leaves the concentrated gel, and the electrophoresis is terminated when the band runs to the bottom of the gel. After the electrophoresis is completed, the gel is stained in the staining solution for 1h, and placed on a shaker for decolorization and image collection. The gel staining solution uses Coomassie brilliant blue solution, and the decolorization uses a high methanol acetic acid solution, and a protein marker with a molecular weight of 11-245kDa is used as a standard.

[0067] Densitometric method: The relative contents of protein subunits 44kDa and 63kDa were calculated using the density analysis software Image Lab. The content of the allergenic protein was the sum of the contents of 44kDa and 63kDa.

[0068] (5) Determination of texture properties: The pea protein custard sample was taken out of a 4°C refrigerator and placed at 25°C for 30 min. A P / 50 probe was used, and the test parameters were: pre-test speed 1 mm / s, test speed 1 mm / s, post-test speed 1 mm / s, press down twice, trigger force 5.0 g, and compression deformation 50%. Each sample was tested 3 times, and the average value was taken to determine the hardness, elasticity, viscosity, and cohesion of the custard sample.

[0069] (6) Determination method of protein in vitro digestion simulation:

[0070] Table 1 Composition of simulated digestive fluid (oral, gastric, intestinal fluid)

[0071]

[0072] Simulated oral digestion stage: 5 g of pea protein custard and egg custard were weighed respectively, added with 5 mL of distilled water, homogenized at 12000 r / min for 1 min, mixed with 4 mL of SSF (see Table 1), 25 μL of 0.3 mol / L CaCl2 and 0.975 mL of ultrapure water, adjusted to pH 7.0 with 1 M NaOH, and digested with magnetic stirring at 37 °C for 5 min.

[0073] Simulated gastric digestion stage: After the oral digestion, add 8.0 mL of preheated SGF stock solution (see Table 1) and 5 μL of 0.3 mol / L CaCl2 solution, adjust the pH to 3.0 with 1 mol / L HCl, then add 0.5 mL of pepsin solution (2000 U / mL), add ultrapure water to make the total volume reach 30 mL, adjust the pH to 3.0, and digest the mixture in a shaking water bath (100 r / min) at 37°C for 120 min. After the end, adjust the pH of the solution to 7.0 with 6 mol / L NaOH solution to terminate the gastric digestion.

[0074] Simulated intestinal digestion stage: 8.5 mL of preheated SIF stock solution (see Table 1), 2.5 mL of 10 mmol / L ox bile salt, 0.3 mol / L CaCl2 and pancreatic enzyme solution (100 U / mL) were added to the oral and post-gastric stage reaction solution. The mixture was digested in a shaking water bath (100 r / min) at 37°C for 120 min. After the end, it was placed in a 100°C water bath and boiled for 5 min to terminate intestinal digestion.

[0075] Preservation of digestion products: After the gastrointestinal digestion experiment, all digestion products were collected and cooled in ice water, then centrifuged at 4°C for 15 min to collect the supernatant, and the total protein and digested protein content were determined by the BCA method.

[0076] Digestibility (%) = (P s -P0) / P m *100

[0077] Where P s is the protein content of the supernatant, P0 is the protein content of the blank, and P m It is the protein content before digestion.

[0078] Before digestion, all stock solutions and deionized water and other reagents were preheated to 37°C. The enzyme solution was prepared immediately and stored in ice. It was added after the sample was mixed with the stock solution. An equal mass of deionized water was used instead of food as the stomach and intestine blank group.

[0079] (7) Scanning electron microscope determination method: Pea protein custard was carefully cut into 10 mm × 10 mm × 2 mm gel blocks, pre-cooled at -80°C, and then freeze-dried. The dried sample was ion-gold plated and observed under a scanning electron microscope.

[0080] 2. Preparation of Pea Protein Custard

[0081] (1) Preparation of low-allergenic and high-nutrition pea protein

[0082] Select 10 kinds of naturally air-dried pea seeds, and remove impurities and broken grains respectively. Soak the pea seeds in deionized water overnight, peel and remove the pea skin, dry the remaining pea kernels at 45°C for 8h, grind into powder and pass through a 200-mesh sieve to obtain pea powder; mix the pea powder and ultrapure water in a mass ratio of 1:10, and then centrifuge at 4000r / min for 20min. After the centrifugation, obtain the upper, middle and lower layers of separation, remove the bottom starch precipitate, collect the supernatant and the middle protein liquid, and obtain the crude protein liquid; pass the crude protein liquid through a 100-mesh sieve, and then adjust its pH to 6.2, add an appropriate amount of lactic acid bacteria, and refrigerate at 4°C for 2h (the amount of lactic acid bacteria is 1.0mL of lactic acid bacteria suspension per 100mL of crude protein liquid, and the number of live lactic acid bacteria in the lactic acid bacteria suspension is 1.58*10 8 CFU / mL), centrifuged at 4000r / min for 20min to obtain upper, middle and lower layers of separation, removed the starch precipitate at the bottom, collected the supernatant and the middle layer protein precipitate and freeze-dried them to obtain pea protein.

[0083] The crude protein content of 10 pea varieties was measured, and the results are shown in Table 2. As can be seen from Table 2, the crude protein content of the pea varieties selected in this embodiment ranges from 22.20% to 28.45%.

[0084] Table 2 Crude protein content of 10 pea varieties

[0085]

[0086] The crude fat content of 10 pea varieties was measured, and the results are shown in Table 3. As can be seen from Table 3, the crude fat content of the pea varieties selected in this embodiment ranges from 3.20% to 4.25%.

[0087] Table 3 Crude fat content of 10 pea varieties

[0088]

[0089] The amino acid contents of 10 pea varieties were determined, and the results are shown in Table 4. As can be seen from Table 3, the amino acid composition of the selected pea varieties is rich, containing eight essential amino acids required by the human body, with high contents of lysine and arginine, among which Zhongwan No. 9 has high contents of lysine and methionine, which are 1.91g / 100g protein and 0.12g / 100g protein respectively.

[0090] Table 4 Amino acid content of 10 pea varieties

[0091]

[0092] Table 4 Amino acid content of 10 pea varieties

[0093]

[0094] The allergenic protein content of the prepared pea proteins was determined, and the results are shown in Table 5. As can be seen from Table 5, the allergenic protein content of the 10 pea proteins ranged from 4.6% to 13%, among which the allergenic protein content of Zhongqin No. 1 was the lowest at 4.6%.

[0095] Table 5 Allergenic protein content of 10 pea protein isolates

[0096]

[0097] According to the above test results, the two best pea varieties were finally selected, namely Zhongwan No. 9 and Zhongqin No. 1. Two types of pea proteins were extracted by the acid pea method, thereby forming an amino acid complementary pattern and improving the nutritional value of the product.

[0098] Take two kinds of pea proteins extracted from the selected pea varieties (Zhongwan No. 9 and Zhongqin No. 1), mix the two pea protein samples evenly in a ratio of 1:1 (w:w), and obtain low-allergenic and highly nutritious pea protein.

[0099] (2) Composite modification of low-allergenic and high-nutrition pea protein

[0100] The above-mentioned low-allergenic and high-nutrition pea protein is dispersed in deionized water to obtain a pea protein dispersion, and the total mass fraction of pea protein in the pea protein dispersion is 14wt%. The agitator is used to stir at room temperature for 2h, and the pea protein dispersion is homogenized 3 times at a pressure of 60MPa using a high-pressure homogenizer, and placed in a 4°C refrigerator overnight to obtain a pea protein homogenate. The pH value of the homogenized pea protein homogenate is adjusted to 7.0, and different concentrations of TG enzyme (0.00%, 0.02%, 0.04%, 0.06%, 0.08%, 0.10%, 0.12%) are added. After stirring evenly, the mixed system is placed in a 50°C water bath for 2.5h thermal crosslinking and enzyme crosslinking, and finally the enzyme is inactivated (the enzyme inactivation condition is: 100°C insulation for 10min), and freeze-dried to obtain a modified low-allergenic and high-nutrition pea protein.

[0101] (3) Preparation of low-allergenic and high-nutrition pea protein egg custard liquid

[0102] In this embodiment, pea protein egg custard liquid is prepared according to the following steps:

[0103] Take the modified low-allergenic and high-nutrition pea protein and add water to prepare a pea protein mixed liquid with a mass fraction of 14wt%, add 0.4wt% edible calcium chloride, 10wt% peanut oil, and 0.05wt% curcumin, mix well, and shear and emulsify at high speed (12000rpm) for 3min to obtain an emulsion; add 0.1wt% yeast β-glucan to the emulsion, mix well, shear at high speed (12000rpm) for 2min, and homogenize at 60MPa for 3 times to obtain a high-stability, low-allergenic and high-nutrition pea protein egg custard liquid.

[0104] (4) Preparation of high stability, low allergenicity and high nutrition pea protein egg custard liquid Preparation of plant egg custard

[0105] Add appropriate amount of water and 0.2wt% salt to the high stability, low allergenicity and high nutrition pea protein egg custard liquid, stir magnetically for 5 minutes to mix, and then heat it in a steamer under medium heat for 8 to 15 minutes to obtain a high stability, low allergenicity and high nutrition pea protein egg custard, the appearance and tissue structure of which are shown in FIG. Figure 1 and Figure 2 As can be seen from the figure, the prepared pea protein custard has good formability and appearance color, and is very similar to the appearance of traditional egg custard.

[0106] 3. Test result analysis

[0107] (1) Determination of the texture properties of pea protein custard prepared with different TG enzyme addition amounts

[0108] like Figure 3 and Figure 4 As shown in the figure, the hardness and viscosity of pea protein egg custard first increase and then decrease with the increase of TG enzyme addition. Except for the small elasticity at 0.08% TG enzyme addition, the elasticity difference between the other TG enzyme additions is not obvious, and the cohesion tends to decrease (P<0.05). When 0.10% TG enzyme is added, the hardness of pea protein egg custard is 80.87g, the viscosity is 0.23mJ, the elasticity is 8.75mm, and the cohesion is 0.31; its hardness, elasticity, and cohesion are similar to the texture characteristics of traditional egg custard (results in Table 6), and the viscosity is low. It can be seen that the formed egg custard has a uniform tissue structure, which can simulate the structure and taste of egg custard to a certain extent.

[0109] Table 6 Texture characteristics of egg custard

[0110]

[0111] (2) Determination of in vitro protein digestion simulation of pea protein custard prepared with 0.10% TG enzyme addition

[0112] The in vitro digestion simulation of egg custard was used as a control, and the in vitro digestion simulation of the protein of the pea protein egg custard prepared at 0.10% TG enzyme addition was measured, and the results are shown in Table 7. As shown in Table 7, the in vitro digestibility of pea protein egg custard is similar to that of egg custard, indicating that the two have similar digestive characteristics under the simulated digestion environment. For people who are allergic to eggs or other animal proteins, pea protein egg custard can be used as a safe, nutritious and healthy substitute.

[0113] Table 7 Digestibility of pea protein custard and egg custard

[0114]

[0115] (3) Determination of the microstructure of pea protein custard at each stage of processing

[0116] The microstructure of the traditional egg custard during the formation process was used as a control to determine the microstructure of the pea protein egg custard at each stage of the processing. Figures 5 to 10 As can be seen from the figure, the layered structure of the pea protein particles is significantly weakened, and the prepared pea protein custard has a uniform and dense gel network structure, which is similar to the structure of the egg custard in the control group. The gel inside the custard has a fine texture and a certain elasticity.

Claims

1. A method for preparing a low-allergenic and high-nutrition pea protein egg custard liquid, characterized in that: The steps include: Step (1), extracting pea protein A from pea variety A having an allergenic protein content of less than or equal to 5wt%; extracting pea protein B from pea variety B having a lysine content of greater than or equal to 1.85g / 100g protein and a methionine content of greater than or equal to 0.10g / 100g protein; and uniformly mixing pea protein A and pea protein B to obtain low-allergenic and high-nutrition pea protein; Step (2), compound-modifying the low-allergenic and high-nutrition pea protein to obtain modified low-allergenic and high-nutrition pea protein; Step (3), emulsifying the modified low-allergenic and high-nutrition pea protein, calcium chloride, edible vegetable oil, edible polysaccharide and water to obtain a low-allergenic and high-nutrition pea protein egg custard liquid.

2. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 1, characterized in that: In step (1), pea variety A is Zhongqin No. 1 or Zhongwan No. 8, and pea variety B is Zhongwan No. 9 or Changshoudou No. 1; the mass ratio of pea protein A to pea protein B is 1:1, 3:2 or 7:

3.

3. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 1, characterized in that: In step (1), pea protein A is extracted from pea variety A by using the acid sap method. The method for extracting pea protein A comprises the following steps: Step (1-1), soaking pea variety A in water overnight, removing pea skins, and obtaining pea kernels; Step (1-2), drying the pea kernels at a temperature of 45 to 60° C. for 8 to 12 hours, then grinding them into powder and sieving them through a 200-mesh sieve to obtain pea flour; Step (1-3), adding pea flour to water and dispersing it evenly, wherein the mass ratio of pea flour to water is 1:3-15; then centrifuging at 4000-5000 r / min for 15-20 min, obtaining upper, middle and lower layers of separation after the centrifugation, removing the starch precipitate at the bottom layer, collecting the supernatant and the middle layer protein solution, and obtaining a crude protein solution; Step (1-4), after the crude protein solution is sieved, the pH is adjusted to 5.8-6.2, and then lactic acid bacteria are added and allowed to stand and cool; the amount of lactic acid bacteria is 1-1.5 mL of lactic acid bacteria suspension is added to every 100 mL of crude protein solution, and the number of live lactic acid bacteria in the lactic acid bacteria suspension is (1.5-1.8)*10 8 CFU / mL; cold precipitation conditions are 2-6℃ for 2-3h; Step (1-5), after the cold precipitation is completed, centrifuge at 4000-5000 r / min for 15-20 min to obtain upper, middle and lower layers of separation, remove the starch precipitate at the bottom, collect the supernatant and the middle layer protein precipitate and freeze-dry to obtain pea protein A; Pea protein B is extracted from pea variety B using the completely identical method from step (1-1) to step (1-5).

4. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 3, characterized in that: In step (1), pea variety A is Zhongqin No. 1, and pea variety B is Zhongwan No. 9; the mass ratio of pea protein A to pea protein B is 1:1; the method for extracting pea protein A from pea variety A comprises the following steps: Step (1-1), soaking pea variety A in water overnight, removing pea skins, and obtaining pea kernels; Step (1-2), drying the pea kernels at 45° C. for 12 h, grinding them into powder and sieving them through a 200-mesh sieve to obtain pea flour; Step (1-3), adding pea flour to water and dispersing it evenly, the mass ratio of pea flour to water is 1:10; then centrifuging at 4000 r / min for 20 min, obtaining upper, middle and lower layers of separation after the centrifugation, removing the starch precipitate at the bottom layer, collecting the supernatant and the middle layer protein solution, and obtaining a crude protein solution; Step (1-4), after the crude protein solution is passed through a 100-mesh sieve, the pH is adjusted to 6.2, and then lactic acid bacteria are added and allowed to stand and cool; the amount of lactic acid bacteria is 1.0 mL of lactic acid bacteria suspension per 100 mL of crude protein solution, and the number of live lactic acid bacteria in the lactic acid bacteria suspension is 1.58*10 8 CFU / mL; cold precipitation condition is 4℃ for 2h; Step (1-5), after the cold precipitation is completed, centrifuge at 4000r / min for 20min to obtain upper, middle and lower layers of separation, remove the starch precipitate at the bottom, collect the supernatant and the middle layer protein precipitate and freeze-dry to obtain pea protein A; Pea protein B is extracted from pea variety B using the completely identical method from step (1-1) to step (1-5).

5. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 1, characterized in that: In step (2), the method for composite modification of low-allergenic and high-nutrition pea protein comprises the following steps: Step (2-1), adding water to the low-allergenic and high-nutrition pea protein to prepare a pea protein dispersion; Step (2-2), stirring the pea protein dispersion at room temperature and then performing high-pressure homogenization treatment, and after the high-pressure homogenization treatment is completed, a pea protein homogenate is obtained; Step (2-3), adjusting the pH of the pea protein homogenate and adding TG enzyme for modification treatment, inactivating the enzyme and freeze-drying after the modification treatment to obtain modified low-allergenic and high-nutrition pea protein.

6. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 5, characterized in that: In step (2-1), the total mass fraction of low-allergenic and high-nutrition pea protein in the pea protein dispersion is 6 to 16 wt %; In step (2-2), the stirring time at room temperature is 2 to 3 hours; the high pressure homogenization treatment condition is 20 to 120 MPa, and the cycle is 1 to 5 times; In step (2-3), the pH of the pea protein homogenate is adjusted to 5-11; the amount of TG enzyme used is 0.02-0.12wt% of the mass of the pea protein homogenate; the modification treatment temperature is 20-60°C, and the modification treatment insulation time is 0.5-3h; the enzyme inactivation condition is: insulation at 95-100°C for 10-15min.

7. The method for preparing low-allergenic and high-nutrition pea protein egg custard liquid according to claim 1, characterized in that: Step (3) specifically includes the following steps: Step (3-1), adding water to the modified low-allergenic and high-nutrition pea protein to prepare a pea protein mixed solution; Step (3-2), adding calcium chloride and edible vegetable oil to the pea protein mixture, shearing and emulsifying, to obtain an emulsion; Step (3-3), adding edible polysaccharide to the emulsion and sequentially performing shear homogenization and high-pressure homogenization treatments, after the high-pressure homogenization treatment is completed, a low-allergenic and high-nutrition pea protein egg custard liquid is obtained.

8. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 7, characterized in that: In step (3-1), the mass fraction of the modified low-allergenic and high-nutrition pea protein in the pea protein mixture is 6 to 16 wt %; In step (3-2), the amount of calcium chloride added is 0.1-0.5wt% of the mass of the pea protein mixture, and the amount of edible vegetable oil added is 10-15wt% of the mass of the pea protein mixture; the shear emulsification conditions are: emulsification at 10000-15000rpm for 2-10min; the edible vegetable oil is one or a mixture of two or more of soybean oil, peanut oil, palm oil, rapeseed oil, sunflower seed oil and olive oil; In step (3-3), the amount of edible polysaccharide added is 0.02-0.1wt% of the mass of the pea protein mixture; the shear homogenization conditions are: homogenization at 10000-15000rpm for 2-10min, the pressure of high-pressure homogenization is 20-120MPa, and the high-pressure homogenization treatment is 1-5 times; the edible polysaccharide is one or two of yeast β-glucan, curdlan, methylcellulose, carrageenan and gellan gum, and a mixture of two or more thereof.

9. The method for preparing the low-allergenic and high-nutrition pea protein egg custard liquid according to claim 8, characterized in that: In step (3-1), the mass fraction of the modified low-allergenic and high-nutrition pea protein in the pea protein mixture is 14wt%; In step (3-2), the amount of calcium chloride added is 0.4wt% of the mass of the pea protein mixture, the edible vegetable oil is peanut oil, and the amount of peanut oil added is 10wt% of the mass of the pea protein mixture; before shearing homogenization, 0.01-0.05wt% of edible coloring is added to the mass of the pea protein mixture, and the edible coloring is one or two or more of curcumin, lutein or β-carotene; the shearing emulsification conditions are: emulsification at 12000rpm for 3min; In step (3-3), the amount of edible polysaccharide added is 0.1wt% of the mass of the pea protein mixture; the shear homogenization conditions are: homogenization at 12000rpm for 2min, a homogenization pressure of 60MPa, and homogenization treatment for 3 times; the edible polysaccharide is yeast β-glucan.

10. An application of low-allergenic and high-nutrition pea protein egg custard liquid, characterized in that: The low-allergenic and high-nutrition pea protein egg custard liquid as described in any one of claims 1 to 9 is used in the food field as an egg substitute.

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