Neutral flavor pea protein isolate as well as preparation method and application thereof

Abstract

The invention provides neutral flavor pea protein isolate as well as a preparation method and application thereof, and belongs to the technical field of protein peptides. The preparation method comprises the following steps: soaking yellow peas in low-temperature deionized water, pulping, adding magnetic immobilized bacillus subtilis and bacillus licheniformis, carrying out fermentation culture, adjusting the pH value, separating to obtain a solid material 1, adding the solid material 1 into a deep eutectic solvent, introducing carbon dioxide, carrying out stirring treatment, filtering to obtain a solid material 2, carrying out high-pressure homogenization and pulsed electric field treatment, adding beta-cyclodextrin, and carrying out rapid heating sterilization to obtain the high-strength and high-toughness yellow peas. And performing spray drying to obtain the neutral flavor pea protein isolate. A plurality of groups of methods are adopted for synergistic interaction, so that the content of bad flavor substances in the pea protein isolate can be effectively reduced, the flavor is improved, the protein activity is maintained, the production cost is greatly reduced, degreasing, debitterizing and fishy smell removing are realized, the nitrogen dissolution index is increased, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of protein peptides, and in particular to a neutral-flavored pea protein isolate and a preparation method and application thereof. Background Art

[0002] Soy milk is highly nutritious and lactose-free, so it won't cause discomfort in people with lactose intolerance. However, the off-flavor components produced by the legumes themselves and during processing have limited the development of the soy milk industry. When peas are processed into pea milk, representative off-flavor components include 2-pentylfuran, n-hexanal, and n-hexanol. These volatile compounds may originate from the peas themselves, but more often they are derived from polyunsaturated fatty acids through auto-oxidation, photo-oxidation, and enzyme-catalyzed oxidation. In soybeans, the above pathways have been extensively studied. For example, the generation of off-flavor components in soybeans can be reduced by inhibiting the activity of lipoxygenase (LOX) in soybeans.

[0003] Peas have a high protein content, second only to soybeans among legumes. Pea protein is composed of globulins, albumins, prolamins, and glutenins. Globulins are the primary storage protein in peas, accounting for 65%-80% of the total. The proportions of albumins, prolamins, and glutenins are 10%-20%, 4%-5%, and 3%-4%, respectively. Pea protein has a balanced amino acid profile relative to human needs, making it particularly useful for balancing lysine deficiency in cereals. Pea protein and its hydrolysates possess antioxidant, antihypertensive, anti-inflammatory, and intestinal microbial activity modulating properties. Furthermore, the biological activity of pea protein can be further enhanced through certain chemical or combined treatments.

[0004] Volatile off-flavors associated with pea protein isolate are often described as grassy, ​​fatty, mushroom, or hay. These odor characteristics are the result of the combined action of multiple volatile small-molecule organic compounds, primarily derived from the auto-oxidation, photo-oxidation, and enzymatic reactions of unsaturated fatty acids such as linoleic and linolenic acids. Representative compounds include hexanal, 2-pentylfuran, 1-octen-3-ol, and (E,E)-2,4-decadienal. Non-volatile off-flavors associated with pea protein isolate are often described as bitter and astringent, and are associated with saponins and phenolic compounds. However, research on peas is limited, and existing methods for preparing and improving the flavor of pea protein isolate still need to be improved. Summary of the Invention

[0005] The present invention aims to provide a neutral-flavored pea protein isolate, a preparation method thereof, and an application thereof. The invention adopts a multi-group method to synergistically enhance the efficiency, effectively reduce the content of undesirable flavor substances in the pea protein isolate, improve the flavor, maintain the activity of the protein, greatly reduce the production cost, achieve degreasing, debittering, deodorizing, and improving the nitrogen solubility index, and has broad application prospects.

[0006] The technical solution of the present invention is achieved as follows: The invention provides a preparation method of neutral-flavor pea protein isolate. The method comprises the following steps: soaking yellow peas in low-temperature deionized water, beating the peas, adding magnetically immobilized Bacillus subtilis and Bacillus licheniformis for fermentation and culture, adjusting the pH value to separate and obtain a solid material 1, adding the solid material 1 into a deep eutectic solvent, introducing carbon dioxide, stirring the solid material 2, filtering the solid material 2, and subjecting the solid material 2 to high-pressure homogenization and pulse electric field treatment. Thereafter, beta-cyclodextrin is added, rapid heat sterilization is performed, and spray drying is performed to obtain the neutral-flavor pea protein isolate.

[0007] As a further improvement of the present invention, the following steps are included: S1. Soak clean yellow peas in low-temperature deionized water and beat to obtain a slurry; S2. The slurry was inoculated with magnetically immobilized Bacillus subtilis and Bacillus licheniformis, fermented, separated by a magnet, the pH of the solution was adjusted, filtered, insoluble matter was removed, the pH of the liquid was adjusted to acidic, centrifuged, the solid was collected, the solid was added to deionized water, the pH of the solution was adjusted to alkaline, centrifuged, and the solid was collected to obtain a solid material 1; S3. Solid material 1 is added to a deep eutectic solvent, carbon dioxide is introduced, stirred, and filtered to obtain solid material 2; S4. The solid material 2 was added to deionized water, homogenized under high pressure, and then treated with a pulsed electric field to obtain a protein solution; S5. The protein solution and β-cyclodextrin are mixed, sterilized by rapid heat treatment, and spray-dried to obtain a neutral-flavored pea protein isolate.

[0008] As a further improvement of the present invention, the low temperature in step S1 is 3-5° C., the soaking time is 7-10 h, and the solid-liquid ratio of the yellow peas to deionized water is 1:5-10 g / mL.

[0009] As a further improvement of the present invention, the mass ratio of the slurry, magnetically immobilized Bacillus subtilis, and Bacillus licheniformis in step S2 is 100:4-7, the fermentation culture conditions are 20-25°C, 100-200r / min, and the fermentation culture is 24-48h. The pH value is adjusted to an acidic pH value of 4-5, and the pH value of the solution is adjusted to an alkaline pH value of 7.1-7.3. The preparation method of the magnetically immobilized Bacillus subtilis and Bacillus licheniformis is as follows: T1. The nano-silica spheres were added to a Tris-HCl solution, dopamine hydrochloride was added, the reaction was heated with stirring, centrifuged, washed, and dried to obtain modified nano-silica spheres; T2. The modified nano-silica spheres were added to water, ferric chloride and ferrous chloride were added, and under inert gas protection, ammonia was added dropwise, the reaction was heated and stirred, centrifuged, washed, and dried to obtain magnetic nano-silica spheres; T3. The magnetic nano-silica spheres and β-cyclodextrin were added to water, stirred and mixed, and spray-dried to obtain a β-cyclodextrin / magnetic nano-silica sphere complex; T4. Add Bacillus subtilis and Bacillus licheniformis to water, add β-cyclodextrin / magnetic nanosilica sphere complex, stir and mix thoroughly, and separate with a magnet to produce magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0010] As a further improvement of the present invention, the pH value of the Tris-HCl solution in step T1 is 8.5-9.5, the mass ratio of the nano-silica spheres and dopamine hydrochloride is 10:2-3, the temperature of the heating and stirring reaction is 50-60°C, and the time is 3-4 hours; the mass ratio of the modified nano-silica spheres, ferric chloride and ferrous chloride in step T2 is 10-12:3.24:1.26, the temperature of the heating and stirring reaction is 80-90°C, and the time is 3-5 hours; the mass ratio of the magnetic nano-silica spheres and β-cyclodextrin in step T3 is 10:3-4, the inlet temperature of the spray drying is 170-180°C, and the outlet temperature is 70-80°C; the mass ratio of the Bacillus subtilis, Bacillus licheniformis, and β-cyclodextrin / magnetic nano-silica sphere complex in step T4 is 2-3:1-3:7-10.

[0011] As a further improvement of the present invention, the deep eutectic solvent in step S3 is prepared by mixing propylene glycol and choline chloride in a molar ratio of 1-3:1, adding water, heating to 60-70° C., and stirring and mixing evenly. The water content of the deep eutectic solvent is 15-25 wt %, the carbon dioxide ventilation rate is 5-10 mL / min, and the stirring treatment time is 1-2 h.

[0012] As a further improvement of the present invention, the high-pressure homogenization conditions in step S4 are 60-100 MPa, and the treatment is carried out at room temperature for 10-20 min. The conditions for the pulsed electric field treatment are electric field intensity: 15-40 kV / cm, pulse width: 6-10 μs, pulse frequency: 50-200 Hz, and treatment time of 500-1000 μs. The solid-liquid ratio of the solid material 2 and deionized water is 1:5-7 g / mL.

[0013] As a further improvement of the present invention, the temperature of the rapid thermal sterilization in step S5 is 125-135°C, the time is 20-40s, the inlet air temperature of the spray drying is 170-180°C, the outlet air temperature is 70-80°C, and the mass ratio of the protein solution to β-cyclodextrin is 100:3-5.

[0014] The present invention further protects a neutral-flavored pea protein isolate prepared by the above-mentioned preparation method.

[0015] The present invention further protects a use of the neutral-flavored pea protein isolate in preparing an animal protein substitute.

[0016] The present invention has the following beneficial effects: Soaking yellow peas in low-temperature deionized water can reduce the levels of off-flavors such as hexanal, hexanol, and 2-pentylfuran. The pulp obtained after beating is inoculated with magnetically immobilized Bacillus subtilis and Bacillus licheniformis. Fermentation simultaneously expresses nattokinase, protease, and lipoxygenase inhibitory peptides, significantly reducing key beany-flavoring substances such as hexanal and 2-pentylfuran in the pulp, degrading hydrophobic bitter amino acids in polypeptide chains, and maintaining protein activity.

[0017] The magnetically immobilized Bacillus subtilis and Bacillus licheniformis prepared by the present invention are coated with a sticky polydopamine layer on the surface of the nano-silica sphere, so that the magnetic ferrosoferric oxide can be subsequently deposited on the surface of the sphere. After the encapsulation reaction with cyclodextrin, the Bacillus subtilis and Bacillus licheniformis can be immobilized by forming hydrogen bonds. On the one hand, it has high bacterial fermentation activity, and on the other hand, it allows the fermented bacteria to be separated from the pea protein magnet. At the same time, it can be reused multiple times, reducing the production cost.

[0018] The present invention adds the separated crude protein solid 1 to a deep eutectic solvent and introduces carbon dioxide, which selectively extracts saponins, polyphenols, and free fatty acids. It also achieves efficient degreasing by selectively dissolving fat and disrupting protein-fat binding structures. It also offers advantages such as low residual solvent content, low toxicity, safety, environmental friendliness, high solubility, and excellent biocompatibility. Furthermore, this method can achieve degreasing, debittering, and deodorizing at room temperature, avoiding the increased fat oxidation (e.g., the formation of aldehyde and ketone odorous substances) caused by traditional high-temperature removal, while also reducing the loss of flavor substances (e.g., free amino acids) caused by thermal denaturation of proteins. The introduction of carbon dioxide synergistically enhances the extraction efficiency of fat-soluble odorants, displaces oxygen in the system, blocks lipid oxidation pathways, and prevents the loss of umami peptides (e.g., glutamine dipeptide). Furthermore, carbon dioxide can induce folding and embedding of hydrophobic regions of proteins, reducing the exposure of bitter peptides.

[0019] The present invention utilizes high-pressure homogenization to disrupt the aggregated structure of protein molecules through mechanical shearing, cavitation, and impact, improving their dispersibility and functional properties while potentially reducing the release of some unpleasant flavors. Furthermore, the synergistic pulsed electric field treatment can generate heat through localized discharge, causing protein denaturation and flavor decomposition, minimizing the generation or retention of unpleasant flavors. Finally, solid material 2 is added to water, and during the rapid thermal sterilization process, a combination of instantaneous flash evaporation and vacuum cooling achieves a one-step "sterilization, deodorization, and functional preservation" process.

[0020] The present invention adopts multiple groups of methods to synergistically enhance the efficiency, can effectively reduce the content of undesirable flavor substances in pea protein isolate, improve the flavor, maintain protein activity, reduce production costs, achieve defatting, debittering, deodorizing, and improving the nitrogen solubility index, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a GC-MS graph of the neutral-flavored pea protein isolate prepared in Example 3 of the present invention; Figure 2 This is a morphological diagram of the neutral-flavored pea protein isolate prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0024] Bacillus subtilis, 20 billion cfu / g, Bacillus licheniformis, 20 billion cfu / g. The average particle size of the nanosilica spheres is 500-600nm.

[0025] Preparation Example 1 Magnetic Immobilization of Bacillus subtilis and Bacillus licheniformis The preparation method is as follows: T1. Add 10 g of nanosilica spheres to 200 mL of Tris-HCl solution (pH 8.5), add 2 g of dopamine hydrochloride, heat to 50°C, stir for 3 h, centrifuge, wash, and dry to obtain modified nanosilica spheres. T2. Add 10g of modified nano-silica spheres to 200mL of water, along with 3.24g of ferric chloride and 1.26g of ferrous chloride. Under nitrogen, adjust the pH to 9 by dropwise addition of aqueous ammonia. Heat to 80°C and stir for 3-5h. Centrifuge, wash, and dry to obtain magnetic nano-silica spheres. T3. 10 g of magnetic nanosilica spheres and 3 g of β-cyclodextrin were added to 300 mL of water, stirred for 15 minutes, and spray-dried (inlet air temperature 170°C, outlet air temperature 70°C) to prepare a β-cyclodextrin / magnetic nanosilica sphere composite. T4. Add 2 g of Bacillus subtilis and 1 g of Bacillus licheniformis to 100 mL of water, add 7 g of β-cyclodextrin / magnetic nanosilica sphere complex, stir for 15 minutes, and separate using a magnet to prepare magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0026] Preparation Example 2 Magnetic Immobilization of Bacillus subtilis and Bacillus licheniformis The preparation method is as follows: T1. Add 10 g of nanosilica spheres to 200 mL of Tris-HCl solution (pH 9.5), add 3 g of dopamine hydrochloride, heat to 60°C, stir for 4 h, centrifuge, wash, and dry to obtain modified nanosilica spheres. T2. Add 12g of modified nanosilica spheres to 200mL of water, along with 3.24g of ferric chloride and 1.26g of ferrous chloride. Under nitrogen, adjust the pH to 9 by dropwise addition of aqueous ammonia. Heat to 90°C, stir, and react for 5h. Centrifuge, wash, and dry to obtain magnetic nanosilica spheres. T3. 10 g of magnetic nanosilica spheres and 4 g of β-cyclodextrin were added to 300 mL of water, stirred for 15 minutes, and spray-dried (inlet air temperature 180°C, outlet air temperature 80°C) to prepare a β-cyclodextrin / magnetic nanosilica sphere composite. T4. Add 3 g of Bacillus subtilis and 3 g of Bacillus licheniformis to 100 mL of water, add 10 g of β-cyclodextrin / magnetic nanosilica sphere complex, stir for 15 minutes, and separate using a magnet to prepare magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0027] Preparation Example 3 Magnetic Immobilization of Bacillus subtilis and Bacillus licheniformis The preparation method is as follows: T1. Add 10 g of nanosilica spheres to 200 mL of Tris-HCl solution (pH 9), add 2.5 g of dopamine hydrochloride, heat to 55°C, stir and react for 3.5 hours, centrifuge, wash, and dry to obtain modified nanosilica spheres. T2. Add 11 g of modified nanosilica spheres to 200 mL of water, along with 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen, adjust the pH to 9 by dropwise addition of aqueous ammonia. Heat to 85°C, stir, and react for 4 h. Centrifuge, wash, and dry to obtain magnetic nanosilica spheres. T3. 10 g of magnetic nanosilica spheres and 3.5 g of β-cyclodextrin were added to 300 mL of water, stirred for 15 minutes, and spray-dried (inlet air temperature 175°C, outlet air temperature 75°C) to prepare a β-cyclodextrin / magnetic nanosilica sphere composite. T4. Add 2.5 g of Bacillus subtilis and 2 g of Bacillus licheniformis to 100 mL of water, add 8 g of β-cyclodextrin / magnetic nanosilica sphere complex, stir for 15 minutes, and separate using a magnet to prepare magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0028] Comparative Preparation Example 1 Compared with Preparation Example 3, the difference is that step T3 is not performed.

[0029] The details are as follows: T1. Add 10 g of nanosilica spheres to 200 mL of Tris-HCl solution (pH 9), add 2.5 g of dopamine hydrochloride, heat to 55°C, stir and react for 3.5 hours, centrifuge, wash, and dry to obtain modified nanosilica spheres. T2. Add 11 g of modified nanosilica spheres to 200 mL of water, along with 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen, adjust the pH to 9 by dropwise addition of aqueous ammonia. Heat to 85°C, stir, and react for 4 h. Centrifuge, wash, and dry to obtain magnetic nanosilica spheres. T3. Add 2.5 g of Bacillus subtilis and 2 g of Bacillus licheniformis to 100 mL of water, add 8 g of magnetic nanosilica spheres, stir and mix for 15 minutes, and separate using a magnet to prepare magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0030] Comparative Preparation Example 2 Compared with Preparation Example 3, the difference is that Bacillus subtilis is not added in step T4.

[0031] The details are as follows: T1. Add 10 g of nanosilica spheres to 200 mL of Tris-HCl solution (pH 9), add 2.5 g of dopamine hydrochloride, heat to 55°C, stir and react for 3.5 hours, centrifuge, wash, and dry to obtain modified nanosilica spheres. T2. Add 11 g of modified nanosilica spheres to 200 mL of water, along with 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen, adjust the pH to 9 by dropwise addition of aqueous ammonia. Heat to 85°C, stir, and react for 4 h. Centrifuge, wash, and dry to obtain magnetic nanosilica spheres. T3. 10 g of magnetic nanosilica spheres and 3.5 g of β-cyclodextrin were added to 300 mL of water, stirred for 15 minutes, and spray-dried (inlet air temperature 175°C, outlet air temperature 75°C) to prepare a β-cyclodextrin / magnetic nanosilica sphere composite. T4. Add 4.5 g of Bacillus licheniformis to 100 mL of water, then add 8 g of β-cyclodextrin / magnetic nanosilica sphere complex. Stir and mix for 15 minutes. Separate with a magnet to obtain magnetically immobilized Bacillus licheniformis.

[0032] Comparative Preparation Example 3 The difference compared with Preparation Example 3 is that no licheniformis spore bacilli were added in step T4.

[0033] The details are as follows: T1. Add 10 g of nanosilica spheres to 200 mL of Tris-HCl solution (pH 9), add 2.5 g of dopamine hydrochloride, heat to 55°C, stir and react for 3.5 hours, centrifuge, wash, and dry to obtain modified nanosilica spheres. T2. Add 11 g of modified nanosilica spheres to 200 mL of water, along with 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen, adjust the pH to 9 by dropwise addition of aqueous ammonia. Heat to 85°C, stir, and react for 4 h. Centrifuge, wash, and dry to obtain magnetic nanosilica spheres. T3. 10 g of magnetic nanosilica spheres and 3.5 g of β-cyclodextrin were added to 300 mL of water, stirred for 15 minutes, and spray-dried (inlet air temperature 175°C, outlet air temperature 75°C) to prepare a β-cyclodextrin / magnetic nanosilica sphere composite. T4. Add 4.5 g of Bacillus subtilis to 100 mL of water, then add 8 g of β-cyclodextrin / magnetic nanosilica sphere complex. Stir and mix for 15 minutes. Separate with a magnet to prepare magnetically immobilized Bacillus subtilis. Example 1

[0034] This embodiment provides a method for preparing neutral-flavor pea protein isolate, comprising the following steps: S1. Soak 10 g of clean yellow peas in 50 mL of 3°C deionized water for 7 hours and blend for 10 minutes to obtain a uniform and fine slurry. S2. To 100 g of the slurry was added 4 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 1. The mixture was fermented at 20°C, 100 rpm, and cultured for 24 h. The solution was separated by a magnet and the pH was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 4 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH of the solution was adjusted to 7.1, and the solid was collected by centrifugation to obtain solid 1. S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, carbon dioxide was introduced at a flow rate of 5 mL / min, stirred for 1 h, and filtered to obtain solid material 2; The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 1:1, adding water, heating to 60° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 15 wt %. S4. Add 10 g of solid material 2 to 50 mL of deionized water at 60 MPa and homogenize at room temperature for 10-20 min. Then, treat with a pulsed electric field at 15 kV / cm, 6 μs pulse width, and 50 Hz pulse frequency for 500 μs to obtain a protein solution. S5. Mix 100 g of protein solution and 3 g of β-cyclodextrin, sterilize at 125°C for 20 seconds, flash-cool, and spray-dry to produce neutral-flavored pea protein isolate. Example 2

[0035] This embodiment provides a method for preparing neutral-flavor pea protein isolate, comprising the following steps: S1. Soak 10 g of clean yellow peas in 100 mL of 5°C deionized water for 10 hours and blend for 10 minutes to obtain a smooth slurry. S2. To 100 g of the slurry was added 7 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 2. The mixture was fermented at 25°C, 200 rpm, and cultured for 48 h. The solution was separated by a magnet and the pH value was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 5 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH value of the solution was adjusted to 7.3, and the solid was collected by centrifugation to obtain solid 1. S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, carbon dioxide was introduced at a flow rate of 10 mL / min, stirred for 2 h, and filtered to obtain solid material 2; The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 3:1, adding water, heating to 60-70° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 25 wt %. S4. Add 10 g of solid material 2 to 70 mL of deionized water at 100 MPa and homogenize at room temperature for 20 min. Then, treat with a pulsed electric field at 40 kV / cm, 10 μs pulse width, and 200 Hz pulse frequency for 1000 μs to obtain a protein solution. S5. Mix 100 g of protein solution and 5 g of β-cyclodextrin, sterilize at 135°C for 40 seconds, flash cool, and spray dry to produce neutral-flavored pea protein isolate. Example 3

[0036] This embodiment provides a method for preparing neutral-flavor pea protein isolate, comprising the following steps: S1. Soak 10 g of clean yellow peas in 70 mL of 4°C deionized water for 8 hours and blend for 10 minutes to obtain a smooth slurry. S2. To 100 g of the slurry was added 5 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 3. The mixture was fermented at 22°C, 150 rpm, and cultured for 36 h. The solution was separated by a magnet and the pH was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 4.5 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH of the solution was adjusted to 7.2, centrifuged, and the solid was collected to obtain solid 1. S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, and carbon dioxide was introduced at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h and filtered to obtain solid material 2. The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 2:1, adding water, heating to 65° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 20 wt %. S4. Add 10 g of solid material 2 to 60 mL of deionized water at 80 MPa and homogenize at room temperature for 15 min. Then, treat with a pulsed electric field at 25 kV / cm, 8 μs pulse width, and 100 Hz pulse frequency for 750 μs to obtain a protein solution. S5. 100 g of protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130 ° C for 30 s, flash-cooled, and spray-dried to obtain a neutral-flavored pea protein isolate. Figure 1 is the GC-MS graph of the prepared neutral flavor pea protein isolate. Figure 2 This is a product shape picture, and its color is relatively white.

[0037] Comparative Example 1 Compared with Example 3, the difference is that the magnetically immobilized Bacillus subtilis and Bacillus licheniformis are prepared by Comparative Preparation Example 1.

[0038] Comparative Example 2 Compared with Example 3, the difference is that the magnetically immobilized Bacillus subtilis and Bacillus licheniformis are prepared by Comparative Preparation Example 2.

[0039] Comparative Example 3 Compared with Example 3, the difference is that the magnetically immobilized Bacillus subtilis and Bacillus licheniformis are prepared by Comparative Preparation Example 3.

[0040] Comparative Example 4 Compared with Example 3, the difference is that the low-temperature soaking in step S1 is not performed.

[0041] The details are as follows: S1. Add 10g of clean yellow peas to 70mL of deionized water and beat for 10min to obtain a uniform and fine slurry; S2. To 100 g of the slurry was added 5 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 3. The mixture was fermented at 22°C, 150 rpm, and cultured for 36 h. The solution was separated by a magnet and the pH was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 4.5 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH of the solution was adjusted to 7.2, centrifuged, and the solid was collected to obtain solid 1. S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, and carbon dioxide was introduced at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h and filtered to obtain solid material 2. The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 2:1, adding water, heating to 65° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 20 wt %. S4. Add 10 g of solid material 2 to 60 mL of deionized water at 80 MPa and homogenize at room temperature for 15 min. Then, treat with a pulsed electric field at 25 kV / cm, 8 μs pulse width, and 100 Hz pulse frequency for 750 μs to obtain a protein solution. S5. 100 g of protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130°C for 30 seconds, flash-cooled, and spray-dried to produce neutral-flavored pea protein isolate.

[0042] Comparative Example 5 Compared with Example 3, the difference is that the fermentation treatment in step S2 is not performed.

[0043] The details are as follows: S1. Soak 10 g of clean yellow peas in 70 mL of 4°C deionized water for 8 hours and blend for 10 minutes to obtain a smooth slurry. S2. The pH value of the solution was adjusted to 7.5 by 100 g of slurry, filtered to remove insoluble matter, the liquid was adjusted to pH 4.5, centrifuged, and the solid was collected. The solid was added to 100 mL of deionized water, the pH value of the solution was adjusted to 7.2, centrifuged, and the solid was collected to obtain solid 1; S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, and carbon dioxide was introduced at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h and filtered to obtain solid material 2. The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 2:1, adding water, heating to 65° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 20 wt %. S4. Add 10 g of solid material 2 to 60 mL of deionized water at 80 MPa and homogenize at room temperature for 15 min. Then, treat with a pulsed electric field at 25 kV / cm, 8 μs pulse width, and 100 Hz pulse frequency for 750 μs to obtain a protein solution. S5. 100 g of protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130°C for 30 seconds, flash-cooled, and spray-dried to produce neutral-flavored pea protein isolate.

[0044] Comparative Example 6 Compared with embodiment 3, the difference is that step S3 is not performed.

[0045] The details are as follows: S1. Soak 10 g of clean yellow peas in 70 mL of 4°C deionized water for 8 hours and blend for 10 minutes to obtain a smooth slurry. S2. To 100 g of the slurry was added 5 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 3. The mixture was fermented at 22°C, 150 rpm, and cultured for 36 h. The solution was separated by a magnet and the pH was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 4.5 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH of the solution was adjusted to 7.2, centrifuged, and the solid was collected to obtain solid 1. S3. 10 g of solid material 1 was added to 60 mL of deionized water and homogenized at 80 MPa for 15 min at room temperature. The mixture was then treated with a pulsed electric field at 25 kV / cm, 8 μs pulse width, and 100 Hz pulse frequency for 750 μs to obtain a protein solution. S4. 100 g of protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130°C for 30 seconds, flash-cooled, and spray-dried to produce neutral-flavored pea protein isolate.

[0046] Comparative Example 7 Compared with the embodiment 3, the difference is that step S4 is not performed.

[0047] The details are as follows: S1. Soak 10 g of clean yellow peas in 70 mL of 4°C deionized water for 8 hours and blend for 10 minutes to obtain a smooth slurry. S2. To 100 g of the slurry was added 5 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 3. The mixture was fermented at 22°C, 150 rpm, and cultured for 36 h. The solution was separated by a magnet and the pH was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 4.5 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH of the solution was adjusted to 7.2, centrifuged, and the solid was collected to obtain solid 1. S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, and carbon dioxide was introduced at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h and filtered to obtain solid material 2. The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 2:1, adding water, heating to 65° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 20 wt %. S4. 10g of solid material 2 was added to 60mL of deionized water to prepare a protein solution; S5. 100 g of protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130°C for 30 seconds, flash-cooled, and spray-dried to produce neutral-flavored pea protein isolate.

[0048] Comparative Example 8 Compared with Example 3, the difference is that β-cyclodextrin is not added in step S5.

[0049] The details are as follows: S1. Soak 10 g of clean yellow peas in 70 mL of 4°C deionized water for 8 hours and blend for 10 minutes to obtain a smooth slurry. S2. To 100 g of the slurry was added 5 g of magnetically immobilized Bacillus subtilis and Bacillus licheniformis obtained in Preparation Example 3. The mixture was fermented at 22°C, 150 rpm, and cultured for 36 h. The solution was separated by a magnet and the pH was adjusted to 7.5. The insoluble matter was removed by filtration. The liquid was adjusted to pH 4.5 and centrifuged to collect the solid. The solid was added to 100 mL of deionized water, the pH of the solution was adjusted to 7.2, centrifuged, and the solid was collected to obtain solid 1. S3. 10 g of solid material 1 was added to 100 mL of a deep eutectic solvent, and carbon dioxide was introduced at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h and filtered to obtain solid material 2. The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 2:1, adding water, heating to 65° C., and stirring to mix evenly. The water content of the deep eutectic solvent is 20 wt %. S4. Add 10 g of solid material 2 to 60 mL of deionized water at 80 MPa and homogenize at room temperature for 15 min. Then, treat with a pulsed electric field at 25 kV / cm, 8 μs pulse width, and 100 Hz pulse frequency for 750 μs to obtain a protein solution. S5. 100 g of protein solution was sterilized at 130°C for 30 seconds, flash-cooled, and spray-dried to produce neutral-flavored pea protein isolate.

[0050] Test Example 1 The neutral-flavored pea protein isolates prepared in Examples 1-3 and Comparative Examples 1-8 were tested for volatile component content, saponin content, polyphenol content, and nitrogen solubility index (NSI). The results are shown in Table 1.

[0051] Table 1

[0052] As can be seen from the above table, the neutral-flavored pea protein isolates prepared in Examples 1-3 of the present invention have lower contents of volatile components, saponins, and polyphenols, and higher NSI.

[0053] The test results of the content of key volatile odor components are shown in Table 2.

[0054] Table 2

[0055] As can be seen from the above table, the neutral-flavored pea protein isolates prepared in Examples 1-3 of the present invention have low contents of key volatile off-flavor components.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a neutral-flavored pea protein isolate, characterized in that: The yellow peas were soaked in low-temperature deionized water and pulped, and magnetically immobilized Bacillus subtilis and Bacillus licheniformis were added for fermentation and culture. The pH value was adjusted to separate and obtain solid material 1, which was added to a deep eutectic solvent, and carbon dioxide was introduced. The solid material 2 was stirred and filtered to obtain solid material 2. After high-pressure homogenization and pulsed electric field treatment, β-cyclodextrin was added, rapid heat sterilization was performed, and spray drying was performed to obtain neutral-flavored pea protein isolate.

2. The preparation method according to claim 1, characterized in that The following steps are involved: S1. Soak clean yellow peas in low-temperature deionized water and beat to obtain a slurry; S2. The slurry was inoculated with magnetically immobilized Bacillus subtilis and Bacillus licheniformis, fermented, separated by a magnet, the pH of the solution was adjusted, filtered, insoluble matter was removed, the pH of the liquid was adjusted to acidic, centrifuged, the solid was collected, the solid was added to deionized water, the pH of the solution was adjusted to alkaline, centrifuged, and the solid was collected to obtain a solid material 1; S3. Solid material 1 is added to a deep eutectic solvent, carbon dioxide is introduced, stirred, and filtered to obtain solid material 2; S4. The solid material 2 was added to deionized water, homogenized under high pressure, and then treated with a pulsed electric field to obtain a protein solution; S5. The protein solution and β-cyclodextrin are mixed, sterilized by rapid heat treatment, and spray-dried to obtain a neutral-flavored pea protein isolate.

3. The preparation method according to claim 2, characterized in that The low temperature in step S1 is 3-5° C., the soaking time is 7-10 h, and the solid-liquid ratio of the yellow pea to deionized water is 1:5-10 g / mL.

4. The preparation method according to claim 2, characterized in that In step S2, the mass ratio of the slurry, magnetically immobilized Bacillus subtilis, and Bacillus licheniformis is 100:4-7, the fermentation culture conditions are 20-25°C, 100-200 r / min, and the fermentation culture is 24-48h. The pH value is adjusted to an acidic pH value of 4-5, and the pH value of the solution is adjusted to an alkaline pH value of 7.1-7.

3. The preparation method of the magnetically immobilized Bacillus subtilis and Bacillus licheniformis is as follows: T1. The nano-silica spheres were added to a Tris-HCl solution, dopamine hydrochloride was added, the reaction was heated with stirring, centrifuged, washed, and dried to obtain modified nano-silica spheres; T2. The modified nano-silica spheres were added to water, ferric chloride and ferrous chloride were added, and under inert gas protection, ammonia was added dropwise, the reaction was heated and stirred, centrifuged, washed, and dried to obtain magnetic nano-silica spheres; T3. The magnetic nano-silica spheres and β-cyclodextrin were added to water, stirred and mixed, and spray-dried to obtain a β-cyclodextrin / magnetic nano-silica sphere complex; T4. Add Bacillus subtilis and Bacillus licheniformis to water, add β-cyclodextrin / magnetic nanosilica sphere complex, stir and mix thoroughly, and separate with a magnet to produce magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

5. The preparation method according to claim 4, characterized in that The pH value of the Tris-HCl solution in step T1 is 8.5-9.5, the mass ratio of the nano-silica spheres to dopamine hydrochloride is 10:2-3, the temperature of the heating and stirring reaction is 50-60°C, and the time is 3-4 hours; the mass ratio of the modified nano-silica spheres, ferric chloride and ferrous chloride in step T2 is 10-12: 3.24:1.26, the temperature of the heating and stirring reaction is 80-90°C, and the time is 3-5h; the mass ratio of the magnetic nano-silica spheres and β-cyclodextrin in step T3 is 10:3-4, the inlet air temperature of the spray drying is 170-180°C, and the outlet air temperature is 70-80°C; the mass ratio of the Bacillus subtilis, Bacillus licheniformis, and β-cyclodextrin / magnetic nano-silica sphere complex in step T4 is 2-3:1-3:7-10.

6. The preparation method according to claim 2, characterized in that The deep eutectic solvent in step S3 is prepared by mixing propylene glycol and choline chloride in a molar ratio of 1-3:1, adding water, heating to 60-70°C, and stirring to mix uniformly. The water content of the deep eutectic solvent is 15-25wt%, the carbon dioxide ventilation rate is 5-10mL / min, and the stirring treatment time is 1-2h.

7. The preparation method according to claim 2, characterized in that The high-pressure homogenization conditions in step S4 are 60-100 MPa, room temperature treatment for 10-20 min, the pulsed electric field treatment conditions are electric field intensity: 15-40 kV / cm, pulse width: 6-10 μs, pulse frequency: 50-200 Hz, treatment for 500-1000 μs, and the solid-liquid ratio of the solid material 2 and deionized water is 1:5-7 g / mL.

8. The preparation method according to claim 2, characterized in that The temperature of the rapid thermal sterilization in step S5 is 125-135°C, the time is 20-40s, the inlet air temperature of the spray drying is 170-180°C, the outlet air temperature is 70-80°C, and the mass ratio of the protein solution to β-cyclodextrin is 100:3-5.

9. A neutral-flavored pea protein isolate prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the neutral-flavored pea protein isolate according to claim 9 in preparing an animal protein substitute.

Images