A neutral flavor pea protein isolate, and a preparation method and application thereof

Abstract

The application provides a neutral flavor pea protein isolate and a preparation method and application thereof, and belongs to the technical field of proteins and peptides. Yellow peas are soaked in low-temperature deionized water, are beaten, and are subjected to fermentation culture by adding magnetically immobilized Bacillus subtilis and Bacillus licheniformis, so that solid material 1 is obtained by adjusting pH value; the solid material 1 is added into a deep eutectic solvent, carbon dioxide is introduced, stirring treatment is performed, and then filtration is performed, so that solid material 2 is obtained; after high-pressure homogenization and pulse electric field treatment, beta-cyclodextrin is added, rapid heat sterilization is performed, and spray drying is performed, so that the neutral flavor pea protein isolate is prepared. The application adopts multiple methods for synergistic effect, can effectively reduce the content of undesirable flavor substances in the pea protein isolate, improves the flavor, maintains the protein activity, greatly reduces the production cost, realizes defatting, debittering and deodorization, improves the nitrogen solubility index, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein peptides, and particularly relates to a neutral flavor pea protein isolate and a preparation method and application thereof. BACKGROUND

[0002] Soy milk has high nutritional value and no lactose, and will not cause discomfort in people with lactose intolerance. However, the peculiar smell components produced by legumes themselves and in the processing process limit the industrial development of soy milk. After pea is processed into pea milk, the representative peculiar smell components include 2-pentyl furan, n-hexanal and n-hexanol, etc. These volatile compounds may be derived from pea itself, but more are derived from polyunsaturated fatty acids through auto-oxidation, photo-oxidation and enzyme-catalyzed oxidation, etc. In soybeans, the above-mentioned pathways have been widely studied, for example, the generation of peculiar smell components in soybeans can be reduced by inhibiting the activity of lipoxygenase (LOX) in soybeans.

[0003] Pea has a high protein content, second only to soybeans among legume crops. Pea protein is composed of globulin, albumin, prolamin and glutelin, among which globulin is the main storage protein of pea, accounting for 65-80% of the total. The proportions of albumin, prolamin and glutelin are 10-20%, 4-5% and 3-4%, respectively. Relative to the required amount of the human body, the amino acid composition of pea protein is balanced, and it can be used to balance the lack of lysine in cereal foods. Pea protein and its hydrolysate have functions such as antioxidant, antihypertensive, anti-inflammatory and regulation of intestinal flora activity, in addition, the biological activity of pea protein can be further enhanced by certain chemical or combined treatment methods.

[0004] The volatile peculiar smell related to pea protein isolate is usually described as grassy, fatty, mushroom, hay, etc., and the odor characteristics are the result of the joint action of a variety of volatile small-molecule organic compounds, mainly derived from the auto-oxidation, photo-oxidation and enzymatic reaction of unsaturated fatty acids such as linoleic acid and linolenic acid, and the representative compounds are hexanal, 2-pentyl furan, 1-octen-3-ol and (E, E)-2, 4-decadienal, etc. The non-volatile peculiar smell related to pea protein isolate is usually described as bitter, which is related to saponins and phenolic compounds. However, there are few related researches on pea, and the existing preparation and flavor improvement methods of pea protein isolate still need to be improved. SUMMARY

[0005] The present application aims to provide a neutral flavor pea protein isolate and a preparation method and application thereof, which can effectively reduce the content of undesirable flavor substances in pea protein isolate by using multiple methods in synergy, improve the flavor, maintain the protein activity, greatly reduce the production cost, realize defatting, debittering and deodorization, and improve the nitrogen solubility index, and has a broad application prospect.

[0006] The technical scheme of the present application is implemented as follows:

[0007] The present application provides a preparation method of neutral flavor pea protein isolate, wherein yellow peas are soaked in low-temperature deionized water, slurried, and then magnetic immobilized Bacillus subtilis and Bacillus licheniformis are added for fermentation culture, and solid material 1 is obtained by adjusting the pH value, and then the solid material 1 is added into a deep eutectic solvent, carbon dioxide is introduced, stirring treatment is performed, filtration is performed, and solid material 2 is obtained, and then after high-pressure homogenization and pulse electric field treatment, beta-cyclodextrin is added, rapid heat sterilization is performed, and spray drying is performed to obtain the neutral flavor pea protein isolate.

[0008] As a further improvement of the present application, the following steps are included:

[0009] S1. Clean yellow peas are soaked in low-temperature deionized water, slurried, and then magnetic immobilized Bacillus subtilis and Bacillus licheniformis are added for fermentation culture, and solid material 1 is obtained by adjusting the pH value, and then the solid material 1 is added into a deep eutectic solvent, carbon dioxide is introduced, stirring treatment is performed, filtration is performed, and solid material 2 is obtained, and then after high-pressure homogenization and pulse electric field treatment, beta-cyclodextrin is added, rapid heat sterilization is performed, and spray drying is performed to obtain the neutral flavor pea protein isolate.

[0010] S2. Magnetic immobilized Bacillus subtilis and Bacillus licheniformis are inoculated into the slurry, fermentation culture is performed, magnetic separation is performed, the solution pH value is adjusted, filtration is performed to remove insoluble substances, the liquid pH value is adjusted to be acidic, centrifugation is performed, the solid is collected, the solid is added into deionized water, the solution pH value is adjusted to be alkaline, centrifugation is performed, the solid is collected, and solid material 1 is obtained.

[0011] S3. The solid material 1 is added into a deep eutectic solvent, carbon dioxide is introduced, stirring treatment is performed, filtration is performed, and solid material 2 is obtained.

[0012] S4. The solid material 2 is added into deionized water, high-pressure homogenization is performed, and then pulse electric field treatment is performed to obtain a protein liquid.

[0013] S5. The protein liquid and beta-cyclodextrin are mixed, rapid heat sterilization is performed, and spray drying is performed to obtain the neutral flavor pea protein isolate.

[0014] As a further improvement of the present application, the temperature of the low temperature in step S1 is 3-5℃, the soaking time is 7-10h, and the solid-liquid ratio of the yellow peas and deionized water is 1:5-10g / mL.

[0015] As a further improvement of the present application, the mass ratio of the slurry, magnetic immobilized Bacillus subtilis and Bacillus licheniformis in step S2 is 100:4-7, the fermentation culture conditions are 20-25℃, 100-200r / min, and the fermentation culture time is 24-48h, the pH value adjusted to be acidic is 4-5, the pH value adjusted to be alkaline is 7.1-7.3, and the preparation method of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis is as follows:

[0016] T1. Add nano-silica balls into Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, centrifugal, wash, dry, and modify nano-silica balls are prepared;

[0017] T2. Add modified nano-silica balls into water, add ferric chloride and ferrous chloride, drop ammonia water under inert gas protection, heat and stir to react, centrifugal, wash, dry, and magnetic nano-silica balls are prepared;

[0018] T3. Add magnetic nano-silica balls and β-cyclodextrin into water, stir to mix uniformly, spray dry, and β-cyclodextrin / magnetic nano-silica ball complex is prepared;

[0019] T4. Add Bacillus subtilis and Bacillus licheniformis into water, add β-cyclodextrin / magnetic nano-silica ball complex, stir to mix uniformly, and magnetic immobilized Bacillus subtilis and Bacillus licheniformis are prepared.

[0020] As a further improvement of the present application, the pH value of the Tris-HCl solution in step T1 is 8.5-9.5, the mass ratio of the nano-silica balls to dopamine hydrochloride is 10:2-3, the temperature of the heat and stirring reaction is 50-60℃, and the time is 3-4h; in step T2, the mass ratio of the modified nano-silica balls to ferric chloride and ferrous chloride is 10-12:3.24:1.26, the temperature of the heat and stirring reaction is 80-90℃, and the time is 3-5h; in step T3, the mass ratio of the magnetic nano-silica balls to β-cyclodextrin is 10:3-4, the inlet air temperature of the spray drying is 170-180℃, and the outlet air temperature is 70-80℃; in step T4, the mass ratio of the Bacillus subtilis, Bacillus licheniformis, and β-cyclodextrin / magnetic nano-silica ball complex is 2-3:1-3:7-10.

[0021] As a further improvement of the present application, 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, and stirring to mix uniformly at 60-70℃, the water content of the deep eutectic solvent is 15-25wt%, the ventilation amount of the carbon dioxide is 5-10mL / min, and the stirring treatment time is 1-2h.

[0022] As a further improvement of the present application, the high-pressure homogenization condition in step S4 is 60-100MPa at room temperature for 10-20min, the pulse electric field treatment condition is electric field strength: 15-40kV / cm, pulse width: 6-10μs, pulse frequency: 50-200Hz, and the treatment time is 500-1000μs, and the solid-liquid ratio of the solid material 2 to deionized water is 1:5-7g / mL.

[0023] As a further improvement of the present application, the temperature of the fast heat sterilization in step S5 is 125-135 DEG C, the time is 20-40s, the inlet air temperature of the spray drying is 170-180 DEG C, the outlet air temperature is 70-80 DEG C, and the mass ratio of the protein solution and the beta-cyclodextrin is 100:3-5.

[0024] The present application further protects a neutral flavor pea protein isolate prepared by the above preparation method.

[0025] The present application further protects the application of the above neutral flavor pea protein isolate in the preparation of animal protein substitutes.

[0026] The present application has the following beneficial effects:

[0027] Yellow peas are soaked in low-temperature deionized water to reduce the content of odor substances such as hexanal, hexanol and 2-pentyl furan. After beating, the slurry is inoculated with magnetically immobilized Bacillus subtilis and Bacillus licheniformis, and fermentation can simultaneously express nattokinase, protease and lipoxygenase inhibitory peptide. The key beany flavor substances such as hexanal and 2-pentyl furan in the slurry can be significantly reduced, the hydrophobic bitter amino acids in the polypeptide chain can be degraded, and the protein activity can be maintained.

[0028] The magnetically immobilized Bacillus subtilis and Bacillus licheniformis prepared by the present application are coated with a sticky polydopamine layer on the surface of nano-silicon dioxide balls, so that the magnetic magnetite can be subsequently deposited on the surface of the balls. After the ring cyclodextrin embedding reaction, the Bacillus subtilis and Bacillus licheniformis can be fixed by forming hydrogen bonds. On the one hand, the fermentation activity of the bacteria is high. On the other hand, the fermentation bacteria can be separated from the pea protein magnet. At the same time, it can be repeatedly used for many times, reducing the production cost.

[0029] The present application adds the separated crude protein solid 1 into the deep eutectic solvent, and passes carbon dioxide to selectively extract saponins, polyphenols and free fatty acids, etc. High-efficiency degreasing can also be achieved by selectively dissolving fat and destroying the protein-fat combination structure. The method has the advantages of less residual solvent, low toxicity, safety, environmental protection, high solubility and good biocompatibility. At the same time, the method can realize degreasing, debittering and deodorization at room temperature, avoiding the intensification of lipid oxidation caused by traditional high-temperature removal (such as the generation of aldehyde and ketone odor substances), and reducing the loss of flavor substances (such as free amino acids) caused by protein thermal denaturation. The passage of carbon dioxide can synergistically enhance the extraction efficiency of lipid-soluble odor substances, replace the oxygen in the system, block the lipid oxidation path, avoid the loss of umami peptides (such as glutamine dipeptides), and at the same time, carbon dioxide can also induce the folding and embedding of the hydrophobic region of the protein, reducing the exposure of bitter peptides.

[0030] In the present application, high pressure homogenization is adopted to destroy the aggregation structure among protein molecules by mechanical shearing, cavitation effect and impact force, improve the dispersibility and functional properties, and possibly reduce the release of part of the undesirable flavor substances. At the same time, in cooperation with the pulse electric field treatment, local discharge may generate heat, causing protein denaturation and flavor substance decomposition, and minimizing the production or retention of undesirable flavors. Finally, solid material 2 is added to water, and in the process of fast heat sterilization, the combination of instantaneous flash evaporation and vacuum cooling realizes the one-step completion of "sterilization-flavor removal-function preservation".

[0031] The present application adopts multiple methods for synergistic effect, which can effectively reduce the content of undesirable flavor substances in pea protein isolate, improve the flavor, maintain the activity of protein, reduce the production cost, realize defatting, debittering and deodorization, and improve the nitrogen solubility index, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only represent some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0033] Figure 1 The GC-MS diagram of the neutral flavor pea protein isolate prepared in Example 3 of the present application;

[0034] Figure 2 The morphology diagram of the neutral flavor pea protein isolate prepared in Example 3 of the present application. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments only represent some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] Bacillus subtilis, 20 billion cfu / g, Bacillus licheniformis, 20 billion cfu / g. The average particle size of the nano-silicon dioxide spheres is 500-600 nm.

[0037] Preparation Example 1: Magnetic immobilization of Bacillus subtilis and Bacillus licheniformis

[0038] The preparation method is as follows:

[0039] T1. 10 g of nanometer silica balls were added to 200 mL of Tris-HCl solution with pH value of 8.5, 2 g of dopamine hydrochloride was added, heated to 50℃, stirred for 3 h, centrifuged, washed, dried, and modified nanometer silica balls were prepared;

[0040] T2. 10 g of modified nanometer silica balls were added to 200 mL of water, 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added, ammonia water was added dropwise under nitrogen protection to adjust the pH value to 9, heated to 80℃, stirred for 3-5 h, centrifuged, washed, dried, and magnetic nanometer silica balls were prepared;

[0041] T3. 10 g of magnetic nanometer silica balls and 3 g of β-cyclodextrin were mixed and added to 300 mL of water, stirred for 15 min, and spray dried (inlet temperature 170℃, outlet temperature 70℃), and β-cyclodextrin / magnetic nanometer silica ball complex was prepared;

[0042] T4. 2 g of Bacillus subtilis and 1 g of Bacillus licheniformis were added to 100 mL of water, 7 g of β-cyclodextrin / magnetic nanometer silica ball complex was added, stirred for 15 min, and magnetic immobilized Bacillus subtilis and Bacillus licheniformis were prepared.

[0043] Preparation Example 2 Magnetic immobilized Bacillus subtilis and Bacillus licheniformis

[0044] The preparation method is as follows:

[0045] T1. 10 g of nanometer silica balls were added to 200 mL of Tris-HCl solution with pH value of 9.5, 3 g of dopamine hydrochloride was added, heated to 60℃, stirred for 4 h, centrifuged, washed, dried, and modified nanometer silica balls were prepared;

[0046] T2. 12 g of modified nanometer silica balls were added to 200 mL of water, 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added, ammonia water was added dropwise under nitrogen protection to adjust the pH value to 9, heated to 90℃, stirred for 5 h, centrifuged, washed, dried, and magnetic nanometer silica balls were prepared;

[0047] T3. 10 g of magnetic nanometer silica balls and 4 g of β-cyclodextrin were mixed and added to 300 mL of water, stirred for 15 min, and spray dried (inlet temperature 180℃, outlet temperature 80℃), and β-cyclodextrin / magnetic nanometer silica ball complex was prepared;

[0048] T4. 2.5g Bacillus subtilis and 2g Bacillus licheniformis were added to 100mL water, 8g β-cyclodextrin / magnetic nanosilica ball complex was added, stirred and mixed for 15min, and then separated by magnet to obtain magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0049] Preparation Example 3: Magnetically immobilized Bacillus subtilis and Bacillus licheniformis

[0050] The preparation method is as follows:

[0051] T1. 10g nanosilica balls were added to 200mL Tris-HCl solution with pH value of 9, 2.5g dopamine hydrochloride was added, heated to 55℃, stirred for 3.5h, centrifuged, washed, and dried to obtain modified nanosilica balls;

[0052] T2. 11g modified nanosilica balls were added to 200mL water, 3.24g ferric chloride and 1.26g ferrous chloride were added, and the pH value was adjusted to 9 by dropwise addition of ammonia under nitrogen protection, heated to 85℃, stirred for 4h, centrifuged, washed, and dried to obtain magnetic nanosilica balls;

[0053] T3. 10g magnetic nanosilica balls and 3.5g β-cyclodextrin were mixed and added to 300mL water, stirred and mixed for 15min, and then spray dried (inlet temperature 175℃, outlet temperature 75℃) to obtain β-cyclodextrin / magnetic nanosilica ball complex;

[0054] T4. 2.5g Bacillus subtilis and 2g Bacillus licheniformis were added to 100mL water, 8g β-cyclodextrin / magnetic nanosilica ball complex was added, stirred and mixed for 15min, and then separated by magnet to obtain magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0055] Comparative Preparation Example 1

[0056] Compared with Preparation Example 3, the difference is that step T3 is not performed.

[0057] The specific process is as follows:

[0058] T1. 10g nanosilica balls were added to 200mL Tris-HCl solution with pH value of 9, 2.5g dopamine hydrochloride was added, heated to 55℃, stirred for 3.5h, centrifuged, washed, and dried to obtain modified nanosilica balls;

[0059] T2. 11 g of modified nanosilica spheres were added to 200 mL of water, 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added, ammonia water was added dropwise under nitrogen protection to adjust the pH value to 9, heated to 85°C, stirred for 4 h, centrifuged, washed, dried, and magnetic nanosilica spheres were prepared;

[0060] T3. 2.5 g of Bacillus subtilis and 2 g of Bacillus licheniformis were added to 100 mL of water, 8 g of magnetic nanosilica spheres were added, stirred and mixed for 15 min, and magnetic separation was performed to prepare magnetically immobilized Bacillus subtilis and Bacillus licheniformis.

[0061] Comparative Preparation Example 2

[0062] Compared with Preparation Example 3, the difference is that no Bacillus subtilis is added in step T4.

[0063] Specifically as follows:

[0064] T1. 10 g of nanosilica spheres were added to 200 mL of Tris-HCl solution with a pH value of 9, 2.5 g of dopamine hydrochloride was added, heated to 55°C, stirred for 3.5 h, centrifuged, washed, dried, and modified nanosilica spheres were prepared;

[0065] T2. 11 g of modified nanosilica spheres were added to 200 mL of water, 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added, ammonia water was added dropwise under nitrogen protection to adjust the pH value to 9, heated to 85°C, stirred for 4 h, centrifuged, washed, dried, and magnetic nanosilica spheres were prepared;

[0066] T3. 10 g of magnetic nanosilica spheres and 3.5 g of β-cyclodextrin were mixed and added to 300 mL of water, stirred and mixed for 15 min, and spray dried (inlet temperature 175°C, outlet temperature 75°C) to prepare β-cyclodextrin / magnetic nanosilica sphere complexes;

[0067] T4. 4.5 g of Bacillus licheniformis was added to 100 mL of water, 8 g of β-cyclodextrin / magnetic nanosilica sphere complexes were added, stirred and mixed for 15 min, and magnetic separation was performed to prepare magnetically immobilized Bacillus licheniformis.

[0068] Comparative Preparation Example 3

[0069] Compared with Preparation Example 3, the difference is that no Bacillus licheniformis is added in step T4.

[0070] Specifically as follows:

[0071] T1. 10 g of nanometer silica balls were added to 200 mL of Tris-HCl solution with pH value of 9, 2.5 g of dopamine hydrochloride was added, heated to 55℃, stirred for 3.5 h, centrifuged, washed, dried, and modified nanometer silica balls were prepared;

[0072] T2. 11 g of modified nanometer silica balls were added to 200 mL of water, 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added, ammonia water was added dropwise under nitrogen protection to adjust the pH value to 9, heated to 85℃, stirred for 4 h, centrifuged, washed, dried, and magnetic nanometer silica balls were prepared;

[0073] T3. 10 g of magnetic nanometer silica balls and 3.5 g of β-cyclodextrin were mixed and added to 300 mL of water, stirred for 15 min, and spray dried (inlet temperature 175℃, outlet temperature 75℃), and β-cyclodextrin / magnetic nanometer silica ball complex was prepared;

[0074] T4. 4.5 g of Bacillus subtilis was added to 100 mL of water, 8 g of β-cyclodextrin / magnetic nanometer silica ball complex was added, stirred for 15 min, and magnetic immobilized Bacillus subtilis was prepared. Example 1

[0075] The present embodiment provides a preparation method of neutral flavor pea protein isolate, comprising the following steps:

[0076] S1. 10 g of clean yellow peas were soaked in 50 mL of deionized water at 3℃ for 7 h, and the slurry was beaten for 10 min to obtain a uniform and fine slurry;

[0077] S2. 4 g of magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 1 were added to 100 g of the slurry, and the mixture was fermented at 20℃ and 100 r / min for 24 h, then separated by a magnet, the solution pH value was adjusted to 7.5, filtered to remove insoluble substances, the liquid pH value was adjusted to 4, centrifuged to collect solids, and the solids were added to 100 mL of deionized water, the solution pH value was adjusted to 7.1, and the solids were collected by centrifugation to obtain solid material 1;

[0078] S3. 10 g of solid material 1 was added to 100 mL of deep eutectic solvent, carbon dioxide was introduced at a flow rate of 5 mL / min, and the mixture was stirred for 1 h, then filtered to obtain solid material 2;

[0079] The deep eutectic solvent was prepared by mixing propylene glycol and choline chloride at a molar ratio of 1:1, adding water, and stirring uniformly at 60℃, and the water content of the deep eutectic solvent was 15 wt%;

[0080] S4. 10g solid material 2 was added to 50mL deionized water, homogenized at 60MPa and room temperature for 10-20min, treated by pulsed electric field, electric field strength: 15kV / cm, pulse width: 6μs, pulse frequency: 50Hz, treatment time: 500μs, to obtain a protein solution;

[0081] S5. 100g of the protein solution and 3g of β-cyclodextrin were mixed, sterilized at 125℃ for 20s, flash cooled, and spray dried to obtain neutral flavor pea protein isolate. Example 2

[0082] The embodiment provides a preparation method of neutral flavor pea protein isolate, comprising the following steps:

[0083] S1. 10g of clean yellow peas were soaked in 100mL of deionized water at 5℃ for 10h, and then slurry was obtained by slurry beating for 10min;

[0084] S2. 7g of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 2 was added to 100g of the slurry, and then fermentation culture was carried out at 25℃ and 200r / min for 48h; the slurry was separated by a magnet, the pH value of the solution was adjusted to 7.5, and then the solution was filtered to remove insoluble substances; the pH value of the liquid was adjusted to 5, and then the liquid was centrifuged to collect solids; the solids were added to 100mL of deionized water, the pH value of the solution was adjusted to 7.3, and then the solution was centrifuged to collect solids, thereby obtaining solid material 1;

[0085] S3. 10g of the solid material 1 was added to 100mL of deep eutectic solvent, carbon dioxide was introduced at a flow rate of 10mL / min, and then stirring treatment was carried out for 2h; the solution was filtered to obtain solid material 2;

[0086] The deep eutectic solvent was prepared by mixing propylene glycol and choline chloride at a molar ratio of 3:1, adding water, and then stirring and uniformly mixing the mixture at 60-70℃; the water content of the deep eutectic solvent was 25wt%;

[0087] S4. 10g of the solid material 2 was added to 70mL of deionized water, homogenized at 100MPa and room temperature for 20min, treated by pulsed electric field, electric field strength: 40kV / cm, pulse width: 10μs, pulse frequency: 200Hz, treatment time: 1000μs, to obtain a protein solution;

[0088] S5. 100g of the protein solution and 5g of β-cyclodextrin were mixed, sterilized at 135℃ for 40s, flash cooled, and spray dried to obtain neutral flavor pea protein isolate. Example 3

[0089] The embodiment provides a preparation method of neutral flavor pea protein isolate, comprising the following steps:

[0090] S1. 10 g of clean yellow peas were soaked in 70 mL of deionized water at 4°C for 8 h, and then slurried for 10 min to obtain a uniform and fine slurry;

[0091] S2. 5 g of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 3 were added to 100 g of the slurry, and then the mixture was fermented at 22°C and 150 r / min for 36 h. The mixture was separated by a magnet, the pH of the solution was adjusted to 7.5, and then the mixture was filtered to remove insoluble substances. The pH of the liquid was adjusted to 4.5, and then the mixture was centrifuged to collect solids. The solids were added to 100 mL of deionized water, the pH of the solution was adjusted to 7.2, and then the mixture was centrifuged to collect solids, thereby obtaining solid material 1;

[0092] S3. 10 g of the solid material 1 was added to 100 mL of a deep eutectic solvent, and then carbon dioxide was bubbled into the mixture at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h, and then filtered to obtain solid material 2;

[0093] The deep eutectic solvent was prepared by mixing propylene glycol and choline chloride at a molar ratio of 2:1, adding water, and then stirring and mixing uniformly at 65°C until the mixture was homogeneous. The water content of the deep eutectic solvent was 20 wt%.

[0094] S4. 10 g of the solid material 2 was added to 60 mL of deionized water, and then the mixture was homogenized at 80 MPa and room temperature for 15 min. The mixture was treated by a pulsed electric field at an electric field strength of 25 kV / cm, a pulse width of 8 μs, and a pulse frequency of 100 Hz for 750 μs to obtain a protein solution;

[0095] S5. 100 g of the protein solution and 4 g of β-cyclodextrin were mixed, and then sterilized at 130°C for 30 s. The mixture was flash-cooled, and then spray-dried to obtain neutral-flavored pea protein isolate. Figure 1 The GC-MS diagram of the neutral-flavored pea protein isolate prepared is shown in FIG. 1. Figure 2 The product morphology diagram is shown in FIG. 2. The color of the product was relatively white.

[0096] Comparative Example 1

[0097] Compared with Example 3, the difference lies in that the magnetic immobilized Bacillus subtilis and Bacillus licheniformis were prepared by Comparative Preparation Example 1.

[0098] Comparative Example 2

[0099] Compared with Example 3, the difference lies in that the magnetic immobilized Bacillus subtilis and Bacillus licheniformis were prepared by Comparative Preparation Example 2.

[0100] Comparative Example 3

[0101] Compared with Example 3, the difference lies in that the magnetic immobilized Bacillus subtilis and Bacillus licheniformis were prepared by Comparative Preparation Example 3.

[0102] Comparative Example 4

[0103] The difference compared with Example 3 is that the low-temperature soaking in step S1 is not performed.

[0104] The details are as follows:

[0105] S1. 10 g of clean yellow peas were added to 70 mL of deionized water, and the slurry was beaten for 10 min to obtain a uniform and fine slurry;

[0106] S2. 5 g of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 3 were added to 100 g of the slurry, and the solution was fermented at 22°C and 150 r / min for 36 h. The solution was separated by a magnet, the pH value of the solution was adjusted to 7.5, the insoluble substances were removed by filtration, the pH value of the liquid was adjusted to 4.5, and the solid was collected by centrifugation. The solid was added to 100 mL of deionized water, the pH value of the solution was adjusted to 7.2, and the solid was collected by centrifugation to obtain solid material 1;

[0107] S3. 10 g of solid material 1 was added to 100 mL of deep eutectic solvent, and carbon dioxide was bubbled at a flow rate of 7 mL / min. The mixture was stirred for 1.5 h, and then filtered to obtain solid material 2;

[0108] The deep eutectic solvent was prepared by mixing propylene glycol and choline chloride at a molar ratio of 2:1, adding water, and stirring until uniform at 65°C. The water content of the deep eutectic solvent was 20 wt%;

[0109] S4. 10 g of solid material 2 was added to 60 mL of deionized water, and the mixture was homogenized at 80 MPa and room temperature for 15 min. The mixture was treated by a pulsed electric field at an electric field strength of 25 kV / cm, a pulse width of 8 μs, and a pulse frequency of 100 Hz for 750 μs to obtain a protein solution;

[0110] S5. 100 g of the protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130°C for 30 s, flash-cooled, and spray-dried to obtain neutral-flavored pea protein isolate.

[0111] Comparative Example 5

[0112] The difference compared with Example 3 is that the fermentation treatment in step S2 is not performed.

[0113] The details are as follows:

[0114] S1. 10 g of clean yellow peas were soaked in 70 mL of deionized water at 4°C for 8 h, and the slurry was beaten for 10 min to obtain a uniform and fine slurry;

[0115] S2. Adjust the pH value of 100 g of the slurry solution to 7.5, filter, remove insoluble substances, adjust the pH value of the liquid to 4.5, centrifuge, collect the solids, add the solids to 100 mL of deionized water, adjust the pH value of the solution to 7.2, centrifuge, collect the solids, and obtain solid material 1;

[0116] S3. Add 10 g of solid material 1 to 100 mL of deep eutectic solvent, pass in carbon dioxide, the aeration amount is 7 mL / min, stir for 1.5 h, filter, and obtain solid material 2;

[0117] The deep eutectic solvent is prepared by mixing propylene glycol and choline chloride at a molar ratio of 2:1, adding water, heating to 65°C, and stirring until uniform. The water content of the deep eutectic solvent is 20 wt%;

[0118] S4. Add 10 g of solid material 2 to 60 mL of deionized water, homogenize at 80 MPa and room temperature for 15 min, and then treat with a pulsed electric field at an electric field strength of 25 kV / cm, a pulse width of 8 μs, and a pulse frequency of 100 Hz for 750 μs to obtain a protein solution;

[0119] S5. Mix 100 g of the protein solution and 4 g of β-cyclodextrin, sterilize at 130°C for 30 s, flash cool, and spray dry to obtain neutral-flavored pea protein isolate.

[0120] Comparative Example 6

[0121] The difference compared with Example 3 is that step S3 is not performed.

[0122] The details are as follows:

[0123] S1. Soak 10 g of clean yellow peas in 70 mL of deionized water at 4°C for 8 h, and grind the slurry for 10 min to obtain a uniform and fine slurry;

[0124] S2. Add 5 g of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 3 to 100 g of the slurry, and cultivate at 22°C and 150 r / min for 36 h. Separate the magnetic particles, adjust the pH value of the solution to 7.5, filter, remove insoluble substances, adjust the pH value of the liquid to 4.5, centrifuge, collect the solids, add the solids to 100 mL of deionized water, adjust the pH value of the solution to 7.2, centrifuge, collect the solids, and obtain solid material 1;

[0125] S3. Add 10 g of solid material 1 to 60 mL of deionized water, homogenize at 80 MPa and room temperature for 15 min, and then treat with a pulsed electric field at an electric field strength of 25 kV / cm, a pulse width of 8 μs, and a pulse frequency of 100 Hz for 750 μs to obtain a protein solution;

[0126] S4. 100 g of the protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130 °C for 30 s, flash cooled, and spray dried to obtain neutral flavoring pea protein isolate.

[0127] Comparative Example 7

[0128] Compared with Example 3, the difference is that step S4 is not performed.

[0129] The specific process is as follows:

[0130] S1. 10 g of clean yellow peas were soaked in 70 mL of deionized water at 4 °C for 8 h, and then slurry was prepared by slurry for 10 min to obtain uniform and fine slurry;

[0131] S2. 5 g of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 3 were added to 100 g of the slurry, and then the slurry was fermented at 22 °C and 150 r / min for 36 h. The slurry was separated by a magnet, the pH of the solution was adjusted to 7.5, and then the solution was filtered to remove insoluble substances. The pH of the liquid was adjusted to 4.5, and then the liquid was 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, and then the solution was centrifuged to collect the solid. Solid material 1 was obtained;

[0132] S3. 10 g of solid material 1 was added to 100 mL of deep eutectic solvent, and then carbon dioxide was bubbled into the solution at a flow rate of 7 mL / min. The solution was stirred for 1.5 h, and then the solution was filtered to obtain solid material 2;

[0133] The deep eutectic solvent was prepared by mixing propylene glycol and choline chloride at a molar ratio of 2:1, adding water, and stirring until the mixture was uniform. The water content of the deep eutectic solvent was 20 wt%.

[0134] S4. 10 g of solid material 2 was added to 60 mL of deionized water to prepare a protein solution;

[0135] S5. 100 g of the protein solution and 4 g of β-cyclodextrin were mixed, sterilized at 130 °C for 30 s, flash cooled, and spray dried to obtain neutral flavoring pea protein isolate.

[0136] Comparative Example 8

[0137] Compared with Example 3, the difference is that β-cyclodextrin is not added in step S5.

[0138] The specific process is as follows:

[0139] S1. 10 g of clean yellow peas were soaked in 70 mL of deionized water at 4 °C for 8 h, and then slurry was prepared by slurry for 10 min to obtain uniform and fine slurry;

[0140] S2. 5 g of the magnetic immobilized Bacillus subtilis and Bacillus licheniformis prepared in Preparation Example 3 was added into 100 g of the slurry, and fermentation culture was carried out at 22℃ and 150 r / min for 36 h, and then the slurry was separated by a magnet, the pH value of the solution was adjusted to 7.5, filtration was performed, the insoluble substances were removed, the pH value of the liquid was adjusted to 4.5, centrifugation was performed, the solid was collected, the solid was added into 100 mL of deionized water, the pH value of the solution was adjusted to 7.2, centrifugation was performed, and the solid was collected to obtain solid material 1;

[0141] S3. 10 g of the solid material 1 was added into 100 mL of the deep eutectic solvent, carbon dioxide was introduced at a flow rate of 7 mL / min, stirring treatment was performed for 1.5 h, and then filtration was performed to obtain solid material 2;

[0142] The deep eutectic solvent was prepared by mixing propylene glycol and choline chloride at a molar ratio of 2:1, adding water, and stirring and uniformly mixing at 65℃, and the water content of the deep eutectic solvent was 20 wt%;

[0143] S4. 10 g of the solid material 2 was added into 60 mL of deionized water, and homogenization treatment was performed at 80 MPa and room temperature for 15 min, and then pulse electric field treatment was performed at an electric field strength of 25 kV / cm, a pulse width of 8 μs, and a pulse frequency of 100 Hz for 750 μs to obtain a protein solution;

[0144] S5. 100 g of the protein solution was sterilized at 130℃ for 30 s, flash cooling was performed, and then spray drying was performed to obtain the neutral flavor pea protein isolate.

[0145] Test Example 1

[0146] The neutral flavor 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), and the results are shown in Table 1.

[0147] Table 1

[0148]

[0149] As shown in the above table, the neutral flavor pea protein isolates prepared in Examples 1-3 have lower volatile component content, saponin content, and polyphenol content, and higher NSI.

[0150] The content of key volatile off-flavor components was tested, and the results are shown in Table 2.

[0151] Table 2

[0152]

[0153] As shown in the above table, the neutral flavor pea protein isolates prepared in Examples 1-3 have lower content of key volatile off-flavor components.

[0154] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing neutral-flavored pea protein isolate, characterized in that, Includes the following steps: S1. Soak clean yellow peas in low-temperature deionized water, then blend them to obtain a slurry; the low temperature is 3-5℃, the soaking time is 7-10h, and the solid-liquid ratio of yellow peas to deionized water is 1:5-10g / mL. S2. Inoculate the slurry with magnetically immobilized Bacillus subtilis and Bacillus licheniformis, ferment and culture, separate using magnets, adjust the pH of the solution, filter to remove insoluble matter, adjust the pH of the liquid to acidic, centrifuge, collect the solid, add the solid to deionized water, adjust the pH of the solution to alkaline, centrifuge, collect the solid, and obtain solid material 1; the mass ratio of the slurry, magnetically immobilized Bacillus subtilis, and Bacillus licheniformis is 100:4-7, the fermentation conditions are 20-25℃, 100-200 r / min, fermentation culture for 24-48 h, the pH of the acidic solution is 4-5, and the pH of the alkaline solution is 7.1-7.3; the preparation method of the magnetically immobilized Bacillus subtilis and Bacillus licheniformis is as follows: T1. Add nano-silica spheres to Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, centrifuge, wash, and dry to obtain modified nano-silica spheres; T2. Modified nano-silica spheres were added to water, along with ferric chloride and ferrous chloride. Under inert gas protection, ammonia was added dropwise, and the mixture was heated and stirred to react. After centrifugation, washing, and drying, magnetic nano-silica spheres were obtained. T3. Magnetic nano silica spheres and β-cyclodextrin are mixed and added to water, stirred and mixed evenly, and spray-dried to obtain β-cyclodextrin / magnetic nano silica spheres composite. T4. Add Bacillus subtilis and Bacillus licheniformis to water, add β-cyclodextrin / magnetic nano silica spheres complex, stir and mix evenly, and separate with a magnet to obtain magnetically immobilized Bacillus subtilis and Bacillus licheniformis; S3. Add solid material 1 to a deep eutectic solvent, introduce carbon dioxide, stir, and filter to obtain solid material 2; the deep eutectic solvent is prepared by mixing propylene glycol and choline chloride in a molar ratio of 1-3:1, adding water, heating to 60-70℃, and stirring until uniformly mixed; the water content of the deep eutectic solvent is 15-25wt%, the carbon dioxide aeration rate is 5-10mL / min, and the stirring time is 1-2h; S4. Add solid material 2 to deionized water, homogenize under high pressure, and then treat with a pulsed electric field to obtain a protein solution; the conditions for high-pressure homogenization are 60-100MPa, room temperature for 10-20min, and the conditions for pulsed electric field treatment are electric field strength: 15-40kV / cm, pulse width: 6-10μs, pulse frequency: 50-200Hz, treatment for 500-1000μs, and the solid-liquid ratio of solid material 2 to deionized water is 1:5-7g / mL; S5. Mix the protein solution and β-cyclodextrin, sterilize by rapid heat treatment, and spray dry to obtain neutral-flavored pea protein isolate.

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

3. The preparation method according to claim 1, characterized in that, The rapid heat sterilization in step S5 is performed at a temperature of 125-135℃ for 20-40 seconds. The inlet air temperature of the spray dryer is 170-180℃, and the outlet air temperature is 70-80℃. The mass ratio of the protein solution to β-cyclodextrin is 100:3-5.

4. A neutral-flavored pea protein isolate prepared by the method according to any one of claims 1-3.

5. The use of the neutral-flavored pea protein isolate as described in claim 4 in the preparation of animal protein substitutes.

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