A method for regulating soybean endogenous enzyme activity to improve oil yield and prepare highly emulsifiable odorless soybean protein isolate

Abstract

The present invention discloses a method for regulating the activity of endogenous enzymes in soybeans to increase the oil yield and prepare a highly emulsifiable odorless soy protein isolate, which belongs to the field of food processing technology. The present invention mainly includes heat shocking soybeans, soaking them in a complex enzyme to control the water absorption rate, a second heat shock and drying at a controlled speed and temperature, so as to achieve the multiple purposes of destroying the cell wall structure to accelerate the water absorption of seeds, regulating the activity of endogenous enzymes in soybeans and in situ enzymatic hydrolysis of oil body proteins, and the like. The new soybeans thus obtained are subjected to conventional oil extraction to significantly improve the oil yield. In addition, the step-by-step heat shock treatment activates the activity of endogenous proteases in soybeans to the maximum extent, and by regulating the relationship between the intensity and time of heat shock and the water content in soybeans, while inhibiting the activity of lipoxygenase, the advantages of the functional characteristics of the dual-modified soy protein isolate by the in situ enzyme method of the endogenous enzyme system and the free enzyme method are fully utilized to prepare a highly emulsifiable odorless soy protein isolate product.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a method for improving soybean oil yield by utilizing endogenous soybean enzyme activity and preparing odorless and highly emulsifiable soybean protein isolate. Background Art

[0002] Soybeans are rich in protein and oil and are an important grain and oil crop. Their oil content ranges from 16% to 20%, of which over 98% is neutral fatty acids, primarily triglycerides, stored in soybean subcellular organelles called oil bodies. Oil bodies are the smallest organelles in living organisms and primarily exist as granules in plant seeds. According to the classic oil body model, the oil body core is composed of triglycerides (94% to 98%), while the surface consists of a layer of phospholipids and embedded membrane proteins (80% phospholipids and 20% embedded proteins). Oil bodies are densely packed around protein bodies and cell walls, with the majority located near the cell wall and a smaller number present within them. Their spherical particle size ranges from 0.2 to 0.5 μm. The hydrophobic acyl groups of the phospholipid layer of soybean oil bodies interact with the internal triglycerides, while the hydrophilic head groups face the cell sap. Embedded proteins wrap around the surface of the oil bodies, maintaining their stability. The commonly used solvent extraction method for soybean oil processing shows a soybean oil extraction rate of over 95% in the residue after oil extraction. However, since the oil content is also determined by solvent extraction, it is possible that the oil in some oil bodies with special morphological structures cannot be extracted by the solvent method, and the oil extraction is not thorough. However, no relevant research reports have been seen so far.

[0003] With the continuous in-depth study of the structural characteristics, functional properties, and economic value of soy protein, the demand for soy protein isolate in the food industry is showing a strong growth trend. Improving the functional properties of soy protein isolate is currently a hot topic in this field. Modification methods include enzymatic modification, physical modification, chemical modification, and bioengineering modification. Among them, enzymatic modification and chemical modification are the most widely studied and applied modification methods. Chemical modification allows it to maintain excellent functional properties in polar environments and has also been proven to be safe. Enzymatic modification conditions are mild and significantly improve the functionality of soy protein isolate. However, the main method of soy protein isolate protease modification is to use soy protein isolate powder, mix it into a solution, and then perform enzymatic hydrolysis to obtain enzymatically modified soy protein isolate, which is then dried. There are also patent studies that have found that after crushing and defatting soybeans, adding cellulase and modified enzymes can produce odorless soy protein isolate, but this method still requires exogenous enzymatic hydrolysis.

[0004] Furthermore, lipoxygenase (LOX) is widely present in plants and animals, with high activity in legumes, and the highest activity in soybeans (Huang Youru, 2007; Prigge ST, 1997). Soybean lipoxygenase, a single polypeptide chain protein, has attracted extensive attention from researchers both domestically and internationally due to its ability to react with soybean lipids to produce unpleasant flavors through lipid peroxidation (SK Arora, 1987). However, the impact of LOX on processed soybean products is not limited to producing off-flavors; it can also affect the functional properties of soy protein to a certain extent (Hua Yufei, 2003). Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the soybean oil yield by utilizing the endogenous enzyme activity of soybeans and preparing a soybean protein isolate with no fishy smell and high emulsification properties, in view of the characteristics of the existing soybean oil yield being low, the soybean protein isolate having a strong beany smell and poor emulsification properties.

[0006] The method provided by the present invention for regulating the activity of endogenous soybean enzymes to increase the oil yield and prepare a highly emulsifiable odorless soybean protein isolate comprises the following steps:

[0007] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0008] (2) Heat shock treatment of dry beans

[0009] Before soaking, the soybeans are immersed in hot water for a pre-heat shock treatment, taken out, quickly cooled to room temperature, and fished out to obtain soybeans 1, wherein the protease activity of the soybeans 1 is ≥97.8 U / g dry beans, the lipoxygenase activity is ≤1932410 U / g dry beans, and the peptide content is ≥5 mg / g dry beans;

[0010] The conditions for the primary pre-heat shock treatment are as follows: the material-liquid ratio is dry soybeans: hot water = 1 g: 10-30 mL; the hot water temperature is 80°C-100°C, and the immersion time in hot water is 4 min-30 s, preferably 80-90°C for 2-4 min or 91-100°C for 30 s-80 s, more preferably 80°C for 2 min or 100°C for 40 s; when cooling, the soybeans are quickly rinsed with room temperature tap water for 20 s to cool to room temperature;

[0011] (3) Compound enzyme soaking

[0012] adding cellulase and pectinase to room temperature tap water, adjusting the pH of the resulting soaking solution to 4-5, soaking soybeans 1 until the water absorption rate is 40%-100% of the weight of the dry beans, removing the soybeans, rinsing them with tap water, and draining the water to obtain soybeans 2, wherein the soaking material-liquid ratio is soybeans 1:room temperature tap water=1g:3-5mL, and cellulase:pectinase=1:1, and the addition amounts of each are 0.2%-0.5% (based on the weight of the dry beans);

[0013] Among them, cellulase: Beijing Solarbio Technology Co., Ltd. (Solarbio), enzyme activity 500000U / g;

[0014] Pectinase: Shandong Longkote Enzyme Preparation Co., Ltd., enzyme activity 30000U / g;

[0015] (4) Heat shock treatment of soaked beans

[0016] Soybean 2 is immersed in hot water for a second heat shock treatment, removed, and quickly cooled to room temperature to obtain soybean 3, wherein soybean 3 has a protease activity of ≥1303.2 U / g dry bean, a lipoxygenase activity of ≤97346 U / g dry bean, a peptide content of ≥12.5 mg / g dry bean, and a protein hydrolysis degree of ≥0.5%;

[0017] The second heat shock treatment conditions are as follows: the material-liquid ratio is 2 soybeans: hot water = 1 g: 10-30 mL; the hot water temperature is 80°C-100°C, and the immersion time in the hot water is 4 min-30 s, preferably 80-90°C for 1-4 min, 91-100°C for 30 s-80 s, and more preferably 80°C for 2 min or 100°C for 40 s; when cooling, the soybeans are quickly rinsed with room temperature tap water for 20 s to cool to room temperature;

[0018] (5) Soaking and double heat shock to control water loss and perform in situ enzymatic hydrolysis on soybeans

[0019] The soybeans 3 are placed in a drying oven at ≤50° C. or naturally air-dried to lose water, and enzymatically hydrolyzed in situ for 3-5 hours to reduce the moisture content to less than 25%, thereby obtaining soybeans 4, wherein the protease activity in the soybeans 4 is ≥1403.2 U / g dry beans, the lipoxygenase activity is ≤90000 U / g dry beans, the peptide content is ≥20 mg / g dry beans, and the protein hydrolysis degree is ≥1.1%;

[0020] (6) Drying of soybeans

[0021] Place the soybeans 4 in a drying oven and dry them at 40-70°C to a moisture content of ≤14%. The obtained soybeans 5 have a moisture content similar to that of untreated dry beans and can be used for oil extraction.

[0022] (7) Extraction of soybean oil

[0023] The soybean oil was extracted by conventional solvent extraction method, specifically as follows: soybean 5 was crushed and sieved, and the ratio of soybean powder to anhydrous ether was 1g:5-10mL; constant temperature water bath was used for extraction at 55℃-60℃ for 50-60min to dissolve the oil in the soybean;

[0024] (8) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0025] Drying to obtain defatted soybean meal powder, wherein the drying temperature is 45° C.-55° C.; adding 8-15 times the amount of water to the defatted soybean meal powder, the water temperature is 40-55° C., adjusting the pH value to 6.5-7.5 with a NaOH solution, stirring and extracting for 40-70 minutes at a stirring speed of 60-70 r / min, completing endogenous enzyme hydrolysis of the soybean protein, wherein the degree of hydrolysis of the soybean protein isolate is ≥2.5%;

[0026] (9) Extraction and separation of protein by alkali dissolution and acid precipitation and spray drying

[0027] The pH value is adjusted to 11 with a NaOH solution, and the mixture is stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. A supernatant is obtained by centrifugation at a speed of 4500 r / min. A 20% by mass hydrochloric acid solution is added to the supernatant at 45° C. and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture is allowed to stand for 30 minutes and an acid precipitate is separated by a centrifuge. The acid precipitate is ground and crushed with water and beaten into a homogenous slurry, and a 5% dilute alkali solution is added to neutralize the mixture to a pH of 7.2. The temperature is controlled at 25° C. and the stirring speed is 80 r / min to obtain a neutralized solution. The solution is sent to a spray drying tower and dehydrated and dried with hot air at 160° C. The tower body temperature is 90-100° C. to obtain a soy protein isolate with no fishy smell, high emulsification and oil absorption.

[0028] The soybean oil and soybean protein isolate prepared by the above method also fall within the protection scope of the present invention.

[0029] The soybean oil yield is ≥18.1%; the emulsification index of the soybean protein isolate is ≥10m 2 / g, emulsification stability ≥20min, oil absorption ≥7g / g soy protein isolate.

[0030] The present invention has the following beneficial effects:

[0031] When dried soybeans swell with water, dormant enzymes are activated, leading to increased metabolic activity. Lipoxygenase is activated, producing some beany-flavoring substances, while proteases are activated to hydrolyze soybean protein into peptides and amino acids necessary for germination. Heat shock can destroy the outer seed coat. Meanwhile, the addition of appropriate amounts of cellulase and pectinase during the soaking process effectively degrades the seed cell wall structure, accelerating water absorption in the impervious seeds and breaking physical dormancy. Furthermore, during the soaking period, the oil bodies (OBs) in the soybean seeds, composed of triglycerides and covered by a layer of phospholipids and oleoprotein, are hydrolyzed by endogenous proteases activated by the heat shock treatment, improving oil yield.

[0032] In addition, the optimum temperature of lipoxygenase in soybeans is 20-30°C, and the optimum temperature of protease is 45-55°C. The step-by-step heat shock method of the present invention inactivates the lipoxygenase as much as possible, removes the original beany smell, and improves the quality of soybean oil and soybean protein isolate; endogenous protease continues to perform in-situ enzymolysis during soaking and drying, and in the crushed soybean meal obtained after oil extraction by a solvent method when preparing soybean protein isolate, the protease contained in the crushed soybean meal becomes a free enzyme in the water phase and continues to perform soybean protein enzymolysis. No exogenous additives are added throughout the process, and the enzymatic modification of soybean protein is completed, thereby obtaining an odorless soybean protein isolate with high emulsification and oil absorption properties. The soybean protein isolate is safe and efficient and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The scanning electron microscope image (left) and transmission electron microscope image (right) of dried soybeans without heat shock and only after soaking treatment in Comparative Example 1 of the present invention are shown.

[0034] Figure 2 The scanning electron microscope image (left) and transmission electron microscope image (right) of dried soybeans after double heat shock at 80°C and soaking treatment according to Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0036] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0037] Example 1

[0038] This embodiment provides a process for preparing odorless soy protein isolate with high oil yield and high emulsification, comprising the following steps:

[0039] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0040] (2) Heat shock treatment of dry beans

[0041] Before soaking, the soybeans were immersed in 80°C hot water for 2 minutes, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; then, they were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 1. The obtained soybeans 1 had a protease activity of 97.8U / g dry beans, a lipoxygenase activity of 1932410U / g dry beans, and a peptide content of 5mg / g dry beans;

[0042] (3) Compound enzyme soaking

[0043] Cellulase and pectinase were separately added to room temperature tap water, and the pH value of the resulting soaking liquid was adjusted to 4. Soybeans 1 were soaked until the water absorption rate was 60% of the weight of the dry beans. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking liquid ratio was soybeans 1:room temperature tap water = 1 g:5 mL, and the cellulase:pectinase ratio was 1:1. The addition amounts of each were 0.25% (based on the weight of the dry beans). Cellulase was purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio), with an enzyme activity of 500,000 U / g; pectinase was purchased from Shandong Longkote Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g.

[0044] (4) Heat shock treatment of soaked beans

[0045] Soybeans 2 were immersed in 80°C hot water for 2 minutes at a material-liquid ratio of 1 g soybeans 2 to 30 mL hot water. The soybeans were then rapidly rinsed with room temperature tap water for 20 seconds and cooled to room temperature to obtain soybeans 3. The obtained soybeans 3 had a protease activity of 1303.2 U / g dry beans, a lipoxygenase activity of 97346 U / g dry beans, a peptide content of 12.5 mg / g dry beans, and a protein hydrolysis degree of 0.5%.

[0046] Figure 2 The scanning electron micrograph (left) and transmission electron micrograph (right) of dried soybeans after double heat shock at 80°C and soaking treatment;

[0047] (5) Controlled water loss after soaking and double heat shock for in situ enzymatic hydrolysis

[0048] Soybean 3 was placed in a drying oven at 40°C and dried for 5 hours to obtain soybean 4 (moisture content 20%); protease activity was 1406.1 U / g dry bean, lipoxygenase activity was 90,000 U / g dry bean, peptide content was 20 mg / g dry bean, and protein hydrolysis degree was 1.1%;

[0049] (6) Drying of soybeans

[0050] Place soybean 4 in a drying oven and dry at 40°C until the moisture content reaches 10%. The obtained soybean 5 can be used for oil extraction.

[0051] (7) Extraction of soybean oil

[0052] Soybeans 5 were crushed and sieved, and extracted with anhydrous ether at a ratio of 1 g soybean powder to 8 mL anhydrous ether. The oil in the soybeans was dissolved in a constant temperature water bath at 60°C for 50 min, and the soybean oil yield was 19.6%.

[0053] (8) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0054] Drying and removing the solvent to obtain defatted soybean meal powder at a drying temperature of 45°C; adding 12 times the amount of water to the defatted soybean meal powder at a water temperature of 50°C, adjusting the pH value to 7.0 with a NaOH solution, stirring and extracting for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes, wherein the degree of hydrolysis of the soy protein isolate is 2.5%;

[0055] (9) Continue to extract the isolated protein and spray dry

[0056] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. The pH value was adjusted to 4.2 and then allowed to stand for 30 minutes. The acid precipitate was separated by centrifuge. The acid precipitate was ground with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the slurry to a pH of 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate with an emulsification index of 10m 2 / g soy protein isolate, emulsification stability 22min, oil absorption 7g / g soy protein isolate.

[0057] Example 2

[0058] This embodiment provides a process for preparing odorless soy protein isolate with high oil yield and high emulsification, comprising the following steps:

[0059] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0060] (2) Heat shock treatment of dry beans

[0061] Before soaking, the soybeans were immersed in 100°C boiling water for 40 seconds, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; after reaching this time, the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds; and soybeans 1 were obtained. The obtained soybeans 1 had a protease activity of 102 U / g dry beans, a lipoxygenase activity of 1,750,000 U / g dry beans, and a peptide content of 6 mg / g dry beans;

[0062] (3) Compound enzyme soaking

[0063] Cellulase and pectinase were separately added to room temperature tap water, and the pH value of the soaking liquid was adjusted to 4. Soybeans 1 were soaked until the water absorption rate was 60% of the weight of the dry beans. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking liquid ratio was soybeans 1: room temperature tap water = 1 g: 5 mL, and the cellulase: pectinase ratio was 1:1. The addition amounts of each were 0.25% (based on the weight of the dry beans). Cellulase was purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio), with an enzyme activity of 500,000 U / g; pectinase was purchased from Shandong Longkote Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g.

[0064] (4) Heat shock treatment of soaked beans

[0065] Soybeans 2 were immersed in 100°C boiling water for 40 seconds, with a material-liquid ratio of 1 g soybeans 2 to 30 mL hot water. After the time reached, the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 3. The obtained soybeans 3 had a protease activity of 1516.6 U / g dry beans, a lipoxygenase activity of 74000 U / g dry beans, a peptide content of 13.2 mg / g dry beans, and a protein hydrolysis degree of 0.6%.

[0066] (5) Soaking and double heat shock to control water loss and perform in situ enzymatic hydrolysis on soybeans

[0067] Soybean 3 was placed in a drying oven at 40°C and dried for 5 hours to obtain soybean 4 (moisture content 20%); protease activity was 1600.2 U / g dry bean, lipoxygenase activity was 70000 U / g dry bean, peptide content was 20 mg / g dry bean, and degree of hydrolysis was 1.1%;

[0068] (6) Drying of soybeans

[0069] Place soybean 4 in a drying oven and dry at 40°C until the moisture content is 10%. The obtained soybean 5 can be used for oil extraction.

[0070] (7) Extraction of soybean oil

[0071] Soybeans 5 were crushed and sieved, and extracted with anhydrous ether at a ratio of 1 g soybean powder to 8 mL anhydrous ether. The oil in the soybeans was dissolved in a constant temperature water bath at 60°C for 50 min, and the soybean oil yield was 20.0%.

[0072] (8) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0073] Drying to obtain defatted soybean meal powder at a drying temperature of 45° C.; adding 12 times the amount of water to the defatted soybean meal powder at a water temperature of 50° C., adjusting the pH value to 7.0 with a NaOH solution, stirring and extracting for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes, wherein the degree of hydrolysis of the soy protein isolate is 2.5%;

[0074] (9) Continue to extract the isolated protein and spray dry

[0075] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. The pH value was adjusted to 4.2 and then allowed to stand for 30 minutes. The acid precipitate was separated by centrifuge. The acid precipitate was ground with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the slurry to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate with an emulsification index of 15m 2 / g soy protein isolate, emulsification stability 20min, oil absorption 8g / g soy protein isolate.

[0076] Comparative Example 1 (Traditional Soaking of Beans)

[0077] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0078] (2) Soaking soybeans: Soak soybeans in tap water at room temperature until the water absorption rate of the soybeans reaches 60%. At this time, the protease activity of the soaked soybeans is 629.1 U / g dry beans, the lipoxygenase content is 4849120 U / g dry beans, the peptide content is 4 mg / g dry beans, and the degree of hydrolysis is 0.15%.

[0079] (3) Drying: The soaked soybeans were placed in a drying oven at 40°C and dried within 8 hours to obtain dry soybeans (with a moisture content of 14%); the protease activity was 983.5 U / g dry beans, the lipoxygenase activity was 4215000 U / g dry beans, the peptide content was 6 mg / g dry beans, and the degree of hydrolysis was 0.17%;

[0080] Figure 1 The scanning electron micrograph (left) and transmission electron micrograph (right) of dried soybeans after soaking without heat shock in comparative example 1 are shown;

[0081] Compare Figure 1 、 Figure 2 It was found that the soybean cotyledon cells that had not been heat-shocked were arranged tightly, and the oil bodies (light-colored part) were spherical in shape, the protein bodies (dark-colored part) were distributed outside the oil bodies, and the boundaries between the two parts were clear; after 2 minutes of heat shock at 80°C, at the same magnification, the cotyledon structure became fuller and looser, the surface damage was more serious, and the oil bodies were irregular in shape, mixed with the protein bodies or discretely distributed between the protein bodies.

[0082] (4) Grinding and defatting: Grind and sieve the soybeans, add anhydrous ether to the crushed soybeans, and use a ratio of 1 g soybeans to 8 mL anhydrous ether. Extract in a constant temperature water bath at 60°C for 50 min to dissolve the oil in the soybeans; the oil extraction rate is 17.1%;

[0083] (5) Enzymatic hydrolysis of soybean meal powder by endogenous protease to extract soybean protein isolate:

[0084] Drying to obtain defatted soybean meal powder at a drying temperature of 45°C; adding 12 times the amount of water to the defatted soybean meal powder at a water temperature of 50°C, adjusting the pH value to 7.0 with a NaOH solution, and stirring and extracting for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes; wherein the soy protein isolate has a hydrolysis degree of 0.25%;

[0085] (6) Acid precipitation, neutralization and spray drying of soy protein isolate

[0086] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. The pH value was adjusted to 4.2 and then allowed to stand for 30 minutes. The acid precipitate was separated by centrifuge. The acid precipitate was ground with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a soy protein isolate with an emulsification index of 5m 2 / g soy protein isolate, emulsification stability 10min, oil absorption 3g / g soy protein isolate.

[0087] Comparative Example 2 (Traditional Soy Protein Isolate Extraction)

[0088] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0089] (2) Grinding and defatting of soybeans

[0090] The soybeans were crushed and sieved, and anhydrous ether was added to the crushed soybeans at a ratio of 1 g soybeans to 8 mL anhydrous ether. The soybeans were then extracted in a constant temperature water bath at 60°C for 50 min to dissolve the oil in the soybeans, with an oil yield of 16.8%.

[0091] (3) Enzymatic hydrolysis of soybean meal powder by endogenous protease to extract soybean protein isolate

[0092] Defatted soybean meal powder was obtained by drying at a temperature of 45°C. 12 times the amount of water was added to the defatted soybean meal powder at a temperature of 50°C. The pH value was adjusted to 7.0 with a NaOH solution. The mixture was stirred and extracted for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein with endogenous enzymes; wherein, the degree of hydrolysis of the soy protein isolate was 0.005%;

[0093] (4) Acid precipitation, neutralization and spray drying of soy protein isolate

[0094] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. The pH value was adjusted to 4.2 and then allowed to stand for 30 minutes. The acid precipitate was separated by centrifuge. The acid precipitate was ground with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a soy protein isolate with an emulsification index of 1m 2 / g soy protein isolate, emulsification stability 8min, oil absorption 1g / g soy protein isolate.

[0095] Comparative Example 3 (low heat shock temperature)

[0096] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0097] (2) Heat shock treatment of dry beans

[0098] Before soaking, the soybeans were immersed in 60°C hot water for 6 minutes, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; after reaching the time, they were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds; soybeans 1 were obtained, and the obtained soybeans 1 had a protease activity of 680.12U / g dry beans, a lipoxygenase activity of 28776213U / g dry beans, and a peptide content of 2mg / g dry beans;

[0099] (3) Compound enzyme soaking

[0100] Cellulase and pectinase were separately added to room temperature tap water, and the pH value of the soaking liquid was adjusted to 4. Soybeans 1 were soaked until the water absorption rate was 60% of the weight of the dry beans. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking liquid ratio was soybeans 1: room temperature tap water = 1 g: 5 mL, and the cellulase: pectinase ratio was 1:1. The addition amounts of each were 0.25% (based on the weight of the dry beans). Cellulase was purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio), with an enzyme activity of 500,000 U / g; pectinase was purchased from Shandong Longkote Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g.

[0101] (4) Heat shock treatment of soaked beans

[0102] Soybeans 2 were immersed in 60°C hot water for 6 minutes, with a material-liquid ratio of 1 g of soybeans 2 to 30 mL of hot water. After the time reached, the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 3. The obtained soybeans 3 had a protease activity of 958.4 U / g dry beans, a lipoxygenase activity of 3,052,340 U / g dry beans, a peptide content of 6 mg / g dry beans, and a protein hydrolysis degree of 0.23%.

[0103] (5) Soaking and double heat shock to control water loss and perform in situ enzymatic hydrolysis on soybeans

[0104] Soybeans 3 were placed in a drying oven at 40° C. and dried for 5 hours to obtain soybeans 4 (with a moisture content of 20%); the soybeans contained a protease activity of 1120 U / g dry beans, a lipoxygenase activity of 3233055 U / g dry beans, a peptide content of 7.5 mg / g dry beans, and a protein hydrolysis degree of 0.35%.

[0105] (6) Drying of soybeans

[0106] Place soybean 4 in a drying oven and dry at 40°C until its moisture content is 10%, and obtain soybean 5 which can be used for oil extraction;

[0107] (7) Extraction of soybean oil

[0108] Soybeans 5 were crushed and sieved, and extracted with anhydrous ether at a ratio of 1 g soybean powder to 8 mL anhydrous ether. The oil in the soybeans was dissolved in a constant temperature water bath at 60°C for 50 min, and the soybean oil yield was 17.3%.

[0109] (8) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0110] Drying to obtain defatted soybean meal powder at a drying temperature of 45°C; adding 12 times the amount of water to the defatted soybean meal powder at a water temperature of 50°C, adjusting the pH value to 7.0 with a NaOH solution, and stirring and extracting for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes; wherein the soy protein isolate has a hydrolysis degree of 0.5%;

[0111] (9) Continue to extract the isolated protein and spray dry

[0112] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. The pH value was adjusted to 4.2 and then allowed to stand for 30 minutes. The acid precipitate was separated by centrifuge. The acid precipitate was ground with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower temperature was 90-100°C to obtain a soy protein isolate with an emulsification index of 6.5m 2 / g soy protein isolate, emulsification stability 11min, oil absorption 3.5g / g soy protein isolate.

[0113] Comparative Example 4 (heat shock time is too long)

[0114] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0115] (2) Heat shock treatment of dry beans

[0116] Before soaking, the soybeans were immersed in 100°C boiling water for 120 seconds, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; after reaching this time, the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds; soybeans 1 were obtained, and the obtained soybeans had a protease activity of 52U / g dry beans, a lipoxygenase activity of 1345000U / g dry beans, and a peptide content of 0.5mg / g dry beans;

[0117] (3) Compound enzyme soaking

[0118] Cellulase and pectinase were separately added to room temperature tap water, and the pH of the soaking liquid was adjusted to 4. Soybeans 1 were soaked until the water absorption was 60% of the dry bean weight. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking liquid ratio was soybeans 1: room temperature tap water = 1 g: 5 mL, and the cellulase: pectinase ratio was 1:1, with the addition amount of each being 0.25% (based on the dry bean weight). Cellulase was purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio), with an enzyme activity of 500,000 U / g; pectinase was purchased from Shandong Longkote Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g.

[0119] (4) Heat shock treatment of soaked beans

[0120] Soybeans 2 were immersed in 100°C boiling water for 120 seconds, with a material-liquid ratio of 1 g soybeans 2 to 30 mL hot water. After this time, the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 3. The obtained soybeans 3 had a protease activity of 321.2 U / g dry beans, a lipoxygenase activity of 22,400 U / g dry beans, a peptide content of 2 mg / g dry beans, and a protein hydrolysis degree of 0.12%.

[0121] (5) Soaking and double heat shock to control water loss and perform in situ enzymatic hydrolysis on soybeans

[0122] Soybean 3 was placed in a drying oven at 40°C and dried for 5 hours to obtain soybean 4 (moisture content 20%); the protease activity was 360.2 U / g dry bean, the lipoxygenase activity was 23300 U / g dry bean, the peptide content was 3 mg / g dry bean, and the degree of hydrolysis was 0.18%;

[0123] (6) Drying of soybeans

[0124] Place soybean 4 in a drying oven and dry at 40°C until its moisture content is 10%, and obtain soybean 5 which can be used for oil extraction;

[0125] (7) Extraction of soybean oil

[0126] Soybeans 5 were crushed and sieved, and extracted with anhydrous ether at a ratio of 1 g soybean powder to 8 mL anhydrous ether. The oil in the soybeans was dissolved in a constant temperature water bath at 60°C for 50 min, and the soybean oil yield was 17.0%.

[0127] (8) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0128] Drying to obtain defatted soybean meal powder at a drying temperature of 45°C; adding 12 times the amount of water to the defatted soybean meal powder at a water temperature of 50°C, adjusting the pH value to 7.0 with a NaOH solution, and stirring and extracting for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes; wherein the soy protein isolate has a hydrolysis degree of 0.35%;

[0129] (9) Continue to extract the isolated protein and spray dry

[0130] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture was allowed to stand for 30 minutes and the acid precipitate was separated by centrifuge. The acid precipitate was ground and crushed with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a soy protein isolate with an emulsification index of 5m 2 / g soy protein isolate, emulsification stability 10min, oil absorption 2g / g soy protein isolate.

[0131] Comparative Example 5 (no free enzyme reaction)

[0132] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0133] (2) Heat shock treatment of dry beans

[0134] Before soaking, the soybeans were immersed in 80°C hot water for 2 minutes, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; after reaching the soaking time, they were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds; soybeans 1 were obtained, and the protease activity of the obtained soybeans was 97.8U / g dry beans, the lipoxygenase activity was 1932410U / g dry beans, and the peptide content was 5mg / g dry beans;

[0135] (3) Compound enzyme soaking

[0136] Cellulase and pectinase were separately added to room temperature tap water, and the pH value of the soaking liquid was adjusted to 4. Soybeans 1 were soaked until the water absorption rate was 60% of the weight of the dry beans. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking liquid ratio was soybeans 1: room temperature tap water = 1 g: 5 mL, and the cellulase: pectinase ratio was 1:1. The addition amounts of each were 0.25% (based on the weight of the dry beans). Cellulase was purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio), with an enzyme activity of 500,000 U / g; pectinase was purchased from Shandong Longkote Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g.

[0137] (4) Heat shock treatment of soaked beans

[0138] Soybeans 2 were immersed in 80°C hot water for 2 minutes, with a material-liquid ratio of 1 g soybeans 2 to 30 mL hot water. After the time reached, the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 3. The obtained soybeans had a protease activity of 1303.2 U / g dry beans, a lipoxygenase activity of 97346 U / g dry beans, a peptide content of 12.5 mg / g dry beans, and a protein hydrolysis degree of 0.5%.

[0139] (5) Controlled water loss after soaking and double heat shock for in situ enzymatic hydrolysis

[0140] Soybean 3 was placed in a drying oven at 40°C and dried for 5 hours to obtain soybean 4 (moisture content 20%); protease activity was 1406.1 U / g dry bean, lipoxygenase activity was 90,000 U / g dry bean, peptide content was 20 mg / g dry bean, and protein hydrolysis degree was 1.1%;

[0141] (6) Drying of soybeans

[0142] Place soybean 4 in a drying oven and dry at 40°C until its moisture content reaches 10%, obtaining soybean 5 which can be used for oil extraction;

[0143] (7) Extraction of soybean oil

[0144] Soybeans 5 were crushed and sieved, and extracted with anhydrous ether at a ratio of 1 g soybean powder to 8 mL anhydrous ether. The oil in the soybeans was dissolved in a constant temperature water bath at 60°C for 50 min, and the soybean oil yield was 19.6%.

[0145] (8) Extraction of soy protein isolate from soybean meal powder, acid precipitation, neutralization and spray drying

[0146] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture was allowed to stand for 30 minutes and the acid precipitate was separated by centrifuge. The acid precipitate was ground and crushed with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate with an emulsification index of 7m 2 / g soy protein isolate, emulsification stability 15min, oil absorption 4g / g soy protein isolate.

[0147] Comparative Example 6 (heat shock before soaking beans only)

[0148] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0149] (2) Heat shock treatment of dry beans

[0150] Before soaking, the soybeans were immersed in 80°C hot water for 2 minutes, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; after the soaking time, they were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds; soybeans 1 were obtained, and the obtained soybeans 1 had a protease activity of 97.8U / g dry beans, a lipoxygenase activity of 1932410U / g dry beans, and a peptide content of 5mg / g dry beans;

[0151] (3) Compound enzyme soaking

[0152] Cellulase and pectinase were separately added to room temperature tap water, and the pH value of the soaking liquid was adjusted to 4. Soybeans 1 were soaked until the water absorption rate was 60% of the weight of the dry beans. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking liquid ratio was soybeans 1: room temperature tap water = 1 g: 5 mL, and the cellulase: pectinase = 1: 1. The addition amounts of each were 0.25% (based on the weight of the dry beans); wherein, cellulase: Beijing Solarbio Technology Co., Ltd., enzyme activity 500,000 U / g; pectinase: Shandong Longkote Enzyme Preparation Co., Ltd., enzyme activity 30,000 U / g;

[0153] (4) Heat shock before soaking beans and control water loss after soaking to perform in situ enzymatic hydrolysis

[0154] Soybean 2 was placed in a drying oven at 40°C and dried for 5 hours to obtain soybean 3 (moisture content 20%); protease activity was 1285.7 U / g dry bean, lipoxygenase activity was 793760 U / g dry bean, peptide content was 13 mg / g dry bean, and protein hydrolysis degree was 0.8%;

[0155] (5) Drying of soybeans

[0156] Place soybean 3 in a drying oven and dry at 40°C until its moisture content reaches 10%. The obtained soybean 4 can be used for oil extraction.

[0157] (6) Extraction method for soybean oil

[0158] Soybeans 4 were crushed and sieved, and anhydrous ether was added to the crushed soybeans at a ratio of 1 g soybeans to 8 mL anhydrous ether. The soybeans were then extracted in a constant temperature water bath at 60°C for 50 min to dissolve the oil in the soybeans, with an oil yield of 17.6%.

[0159] (7) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0160] Drying to obtain defatted soybean meal powder at a drying temperature of 45°C; adding 12 times the amount of water to the defatted soybean meal powder at a water temperature of 50°C, adjusting the pH value to 7.0 with a NaOH solution, and stirring and extracting for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes; wherein the soy protein isolate has a hydrolysis degree of 1.7%;

[0161] (8) Continue to extract the isolated protein and spray dry

[0162] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture was allowed to stand for 30 minutes and the acid precipitate was separated by centrifuge. The acid precipitate was ground and crushed with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate with an emulsification index of 5m 2 / g soy protein isolate, emulsification stability 10min, oil absorption 2g / g soy protein isolate.

[0163] Comparative Example 7 (heat shock after soaking beans only)

[0164] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0165] (2) Complex enzyme soaking: Cellulase and pectinase were added to room temperature tap water, and the pH value of the resulting soaking solution was adjusted to 4. Soybeans 1 were soaked until the water absorption rate was 60% of the dry bean weight. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking material-liquid ratio was soybeans 1: room temperature tap water = 1 g: 5 mL, and the cellulase: pectinase = 1: 1. The addition amount was 0.25% (based on the dry bean weight). Cellulase: Solarbio, Beijing Solarbio Technology Co., Ltd., enzyme activity 500,000 U / g; pectinase: Shandong Longkote Enzyme Preparation Co., Ltd., enzyme activity 30,000 U / g.

[0166] (3) Heat shock: Soybean 2 was immersed in 80°C hot water for 2 min, with a material-liquid ratio of soybean 1: hot water = 1 g: 30 mL; then, the soybean 2 was quickly rinsed with room temperature tap water and cooled to room temperature within 20 s to obtain soybean 3; the obtained soybean 3 had a protease activity of 1083.9 U / g dry bean, a lipoxygenase activity of 64530 U / g dry bean, a peptide content of 10 mg / g dry bean, and a protein hydrolysis degree of 0.3%;

[0167] (4) Controlled water loss after soaking and heat shock for in-situ enzymatic hydrolysis

[0168] Soybean 3 was placed in a drying oven at 40°C and dried for 5 hours to obtain soybean 4 (moisture content 20%); protease activity was 1215.3 U / g dry bean, lipoxygenase activity was 58241 U / g dry bean, peptide content was 16.5 mg / g dry bean, and degree of hydrolysis was 0.8%;

[0169] (5) Drying of soybeans: Place soybeans 4 in a drying oven at 40°C and dry until their moisture content is 10%. The obtained soybeans 5 can be used for oil extraction.

[0170] (6) Grinding and defatting: 5% soybeans were crushed and sieved, and anhydrous ether was added to the crushed soybeans at a ratio of 1 g soybeans to 8 mL anhydrous ether. The soybeans were then extracted in a constant temperature water bath at 60°C for 50 min to dissolve the oil in the soybeans; the oil extraction rate was 17.4%;

[0171] (7) Extracting soy protein isolate by enzymatic hydrolysis of soybean meal powder with endogenous protease: drying to obtain defatted soybean meal powder; the drying temperature is 45°C; adding 12 times the amount of water to the defatted soybean meal powder, the water temperature is 50°C, and the pH value is adjusted to 7.0 with NaOH solution, stirring and extracting for 60 minutes, and the stirring speed is 60r / min, completing the enzymatic hydrolysis of soy protein with endogenous enzyme; wherein, the degree of hydrolysis of soy protein isolate is 1.7%;

[0172] (8) Continue to extract the isolated protein and spray dry

[0173] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture was allowed to stand for 30 minutes and the acid precipitate was separated by centrifuge. The acid precipitate was ground and crushed with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate with an emulsification index of 5m 2 / g soy protein isolate, emulsification stability 10min, oil absorption 5g / g soy protein isolate.

[0174] Comparative Example 8 (Soaking soybeans without adding complex enzyme)

[0175] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no diseases or insect pests as raw materials;

[0176] (2) Heat shock treatment of dry beans

[0177] Before soaking, the soybeans were immersed in 80°C hot water for 2 minutes, with a material-liquid ratio of dry soybeans: hot water = 1g:30mL; then the soybeans were quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 1. The obtained soybeans had a protease activity of 97.8U / g dry beans, a lipoxygenase activity of 1932410U / g dry beans, and a peptide content of 5mg / g dry beans;

[0178] (3) Soaking soybeans

[0179] Soak soybeans 1 in room temperature tap water until the water absorption reaches 60% of the dry bean weight, remove the soybeans, and drain the water to obtain soybeans 2; wherein the soaking material-liquid ratio is soybeans 1: room temperature tap water = 1 g: 5 mL;

[0180] (4) Heat shock treatment of soaked beans

[0181] Soybeans 2 were immersed in 80°C hot water for 2 minutes at a material-liquid ratio of 1 g soybeans 2 to 30 mL hot water. The soybeans were then quickly rinsed with room temperature tap water and cooled to room temperature within 20 seconds to obtain soybeans 3. The obtained soybeans had a protease activity of 1303.7 U / g dry beans, a lipoxygenase activity of 97342 U / g dry beans, a peptide content of 10.8 mg / g dry beans, and a protein hydrolysis degree of 0.43%.

[0182] (5) Controlled water loss after soaking and double heat shock for in situ enzymatic hydrolysis

[0183] Soybean 3 was placed in a drying oven at 40°C and dried for 4 hours to obtain soybean 4 (moisture content 20%); protease activity was 1403.3 U / g dry bean, lipoxygenase activity was 90002 U / g dry bean, peptide content was 18.3 mg / g dry bean, and degree of hydrolysis was 0.97%;

[0184] (6) Drying of soybeans

[0185] Place soybean 4 in a drying oven and dry at 40°C until its moisture content is 10%, and obtain soybean 5 which can be used for oil extraction;

[0186] (7) Soaking and double heat shock drying of soybeans and crushing and defatting

[0187] Soybeans 5 were crushed and sieved, and extracted with anhydrous ether at a ratio of 1 g soybean powder to 8 mL anhydrous ether. The oil in the soybeans was dissolved in a constant temperature water bath at 60°C for 50 min, with an oil extraction rate of 17.9%.

[0188] (8) Enzymatic hydrolysis of soybean meal powder by endogenous protease to extract soybean protein isolate

[0189] Defatted soybean meal powder was obtained by drying at a temperature of 45°C; 12 times the amount of water was added to the defatted soybean meal powder at a temperature of 50°C, the pH value was adjusted to 7.0 with a NaOH solution, and the mixture was stirred and extracted for 60 minutes at a stirring speed of 60 r / min to complete the enzymatic hydrolysis of the soy protein by endogenous enzymes; wherein the soy protein isolate had a hydrolysis degree of 2.1%;

[0190] (9) Continue to extract the isolated protein and spray dry

[0191] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture was allowed to stand for 30 minutes and the acid precipitate was separated by centrifuge. The acid precipitate was ground and crushed with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a soy protein isolate with an emulsification index of 7m 2 / g soy protein isolate, emulsification stability 18min, oil absorption 6g / g soy protein isolate.

[0192] Comparative Example 9 (no heat shock, only adding exogenous enzymes to soak soybeans)

[0193] (1) Raw material selection: Select soybeans with full, shiny, uniform size, no damage, and no pests and diseases as raw materials;

[0194] (2) Complex enzyme soaking: Cellulase and pectinase were added to room temperature tap water, and the pH value of the resulting soaking solution was adjusted to 4. The soybeans were soaked until the water absorption rate was 60% of the dry bean weight. The soybeans were removed, rinsed with tap water, and drained to obtain soybean 1. Room temperature tap water was added to soybean 1 and the pH was adjusted to 11. Alkaline protease was added and treated for 5 minutes. The soybeans were removed to obtain soybean 2.

[0195] The soaking material-liquid ratio is soybean 1 (soybean 2): room temperature tap water = 1g: 5mL, cellulase: pectinase: alkaline protease = 1:1:2, and the amount of cellulase added is 0.25% (based on the weight of dry beans);

[0196] (3) Drying of soybeans after soaking

[0197] Place soybean 2 in a 40°C drying oven and dry at 40°C until its moisture content is 10%, and obtain soybean 3 which can be used for oil extraction;

[0198] (4) Extraction method for soybean oil

[0199] Crush and sieve soybeans 3, then extract with anhydrous ether at a ratio of 1g soybean powder to 8mL anhydrous ether. Extract in a constant temperature water bath at 60°C for 50 minutes to dissolve the oil from the soybeans. The oil extraction yield can reach 17.2%.

[0200] (5) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease

[0201] Defatted soybean meal powder was obtained by drying at a temperature of 45°C. 12 times the amount of water was added to the defatted soybean meal powder at a temperature of 50°C. The pH value was adjusted to 7.0 with a NaOH solution. The mixture was stirred and extracted for 60 minutes at a stirring speed of 60 rpm to complete the enzymatic hydrolysis of the soy protein with endogenous enzymes. The degree of hydrolysis of the soy protein isolate was 1.6%.

[0202] (6) Continue to extract the isolated protein and spray dry

[0203] The pH value was adjusted to 11 with NaOH solution, and the mixture was stirred and extracted for 40 minutes at a stirring speed of 60 r / min to complete the extraction of the enzymatically hydrolyzed soy protein. The supernatant was obtained by centrifugation at a speed of 4500 r / min. A 20% mass concentration hydrochloric acid solution was added to the supernatant at 45°C and stirred at a stirring speed of 60 r / min. After adjusting the pH value to 4.2, the mixture was allowed to stand for 30 minutes and the acid precipitate was separated by centrifuge. The acid precipitate was ground and crushed with water and beaten into a homogenous slurry. A 5% dilute alkali solution was added to neutralize the mixture to pH 7.2. The temperature was controlled at 25°C and the stirring speed was 80 r / min to obtain a neutralized solution. The solution was sent to a spray drying tower and dehydrated with hot air at 160°C. The tower body temperature was 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate with an emulsification index of 5m 2 / g soy protein isolate, emulsification stability 14min, oil absorption 4g / g soy protein isolate.

[0204] The relevant detection methods and results of the present invention are as follows:

[0205] (1) Main relevant endogenous enzyme activity determination methods

[0206] ① Extraction of crude enzyme solution: Soak beans of the embodiment and comparative example, add 50mM phosphate buffer solution (pH=7.0) at a ratio of 1:6 (based on dry bean mass) for pulping, centrifuge the slurry at 2000r / m for 30min, filter the supernatant through filter paper and store at -20°C until further use.

[0207] ② Lipoxygenase activity assay

[0208] Prepare a 1.7% solution of linoleic acid with 0.2 mol / L boric acid buffer at pH 9.0. Take 2 mL of this solution and 0.95 mL of boric acid buffer in a quartz cuvette and mix them by inversion. Dilute the crude enzyme solution by an appropriate multiple, take 0.05 mL and add it to the cuvette. After quickly inverting and mixing, measure the OD value at a wavelength of 234 nm after 15 seconds and 30 seconds. The activity required to increase the absorbance by 0.01 per minute is regarded as an enzyme activity unit. The calculation method is: enzyme activity A(U) = 4×(OD 30S -OD 15S ) / 0.01, and the specific enzyme activity was recorded as U / mg.

[0209] ③Determination of protease activity

[0210] Dilute the crude enzyme solution appropriately, take 2 mL and add 2 mL of 0.5% casein solution. Shake well and incubate in a 37°C water bath for 20 minutes. Terminate the reaction by adding 10% trichloroacetic acid solution, centrifuge at 3000 rpm for 10 minutes, and measure the absorbance of the supernatant at 275 nm. For a blank experiment, add 10% trichloroacetic acid solution first, then the casein solution, incubate, centrifuge, and measure the absorbance. Calculate the enzyme activity (U / g) as follows: (H × F) / (15 × 0.05), where H is the tyrosine content (μg / mL) obtained from the standard curve based on the absorbance value; F is the final dilution factor of the enzyme solution.

[0211] (2) Determination of polypeptide content

[0212] Referring to the method of Xia Yu et al., 5 mL of enzymatically hydrolyzed soy milk and heat-scented soy milk samples were taken respectively, 5 mL of 10% TCA solution was added for precipitation, and the mixture was centrifuged at 8000 r / min for 3 min. 0.8 mL of the supernatant was taken, 3.2 mL of biuret reagent was added, and the mixture was reacted at room temperature for 30 min. The OD value was measured at 540 nm and substituted into the casein standard curve to calculate the polypeptide content.

[0213] Plotting of casein standard curve: 0.8mL of casein solution with concentrations of 0, 2, 4, 6, 8, and 10g / L was taken, and 3.2mL of biuret reagent was added to react at room temperature for 30min, and then the OD value was measured at 540nm. With casein concentration as the horizontal axis and OD value as the vertical axis, a standard curve was prepared, and the regression equation was y=0.0554x+0.0111, r 2 =0.9958.

[0214] (3) Determination of soybean protein oil absorption

[0215] Weigh 500 mg of soy protein into a 10 mL centrifuge tube, add 5 mL of deionized water (or soybean oil), and shake for 30 seconds to mix thoroughly. After standing at room temperature for 1 hour, centrifuge at 3800 rpm for 30 minutes. Discard the supernatant, invert the pellet and the centrifuge tube onto filter paper, and weigh after 30 minutes. The unit is g / g. The calculation formula is as follows:

[0216]

[0217] Wherein, m1 is the total mass of soy protein and centrifuge tube, g; m2 is the total mass of precipitate and centrifuge tube, g; m is the mass of soy protein sample, g.

[0218] (4) Determination of soy protein emulsification and emulsion stability

[0219] Refer to the method of Jamdar et al. Weigh 100mg of soy protein and dissolve it in 0.1mol / L pH 7.0 phosphate buffer to prepare a 1% soy protein solution. Take 6mL of soy protein solution, add 2mL of soybean oil and shake to mix. After high-speed shearing at 10000r / min for 1min, let it stand at room temperature. At 0min and 10min, take 50μL from the bottom of the emulsion into a test tube, add 5mL of 0.1% SDS solution, mix well, and measure the absorbance at 500nm. Use 0.1% SDS solution as blank. The calculation formula is as follows:

[0220]

[0221]

[0222] Where, N is the dilution multiple of soybean protein; C is the mass fraction of soybean protein, g / mL; is the volume fraction of oil phase, 0.25; A0, A 10 are the absorbance values of the emulsion at 0 min and 10 min respectively; Δt is 10 min.

[0223] (5) Degree of hydrolysis

[0224] The degree of hydrolysis (DH) of SPI was determined by pH-stat method under neutral and alkaline conditions. The formula is as follows:

[0225]

[0226] Where: B-amount of alkali consumed during hydrolysis (mL); N-NaOH molar concentration (mol / L); average degree of dissociation of α-soy protein amino groups; m-substrate protein mass (g); h-millimoles of peptide bonds per gram of protein substrate (mmol / L), for soy protein h = 7.75.

[0227] (6) Scanning electron microscopy and transmission electron microscopy analysis

[0228] Scanning electron microscopy analysis: Soybeans, soaked and then dried, were split in half along and perpendicular to the dorsal suture of the cotyledons. After fixation, the samples were subjected to gradient dehydration, critical point drying, and film coating. The processed sample surfaces were imaged at 200x and 1000x magnifications.

[0229] Transmission electron microscopy analysis experiment: The sample was pre-treated, that is, soybeans that had been soaked and then dried were cut into pieces with a size of about 1-2 mm in the center of the cotyledons. 3 After fixation, dehydration and embedding, the sample is sliced in an ultrathin microtome to obtain 70-90nm slices. After the slices are stained and dried, they can be observed in a transmission electron microscope.

[0230] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for regulating the activity of endogenous soybean enzymes to increase oil yield and prepare highly emulsifiable odorless soy protein isolate, comprising the following steps: (1) Raw material selection: Select soybeans that are full, shiny, uniform in size, undamaged, and free of pests and diseases as raw materials; (2) Heat shock treatment of dry beans Before soaking, the soybeans are immersed in hot water for a pre-heat shock treatment, taken out, quickly cooled to room temperature, and fished out to obtain soybeans 1, wherein, In soybean 1, the protease activity is ≥97.8 U / g dry bean, the lipoxygenase activity is ≤1932410 U / g dry bean, and the peptide content is ≥5 mg / g dry bean; The conditions for the primary heat shock treatment are as follows: the material-liquid ratio is 1g dry soybeans: 10-30 mL hot water; heat shock at 80-90°C for 2-4 minutes or 91-100°C for 30-80 seconds; when cooling, quickly rinse the soybeans with room temperature tap water for 20 seconds to cool to room temperature; (3) Compound enzyme soaking Cellulase and pectinase were added to room temperature tap water, and the pH of the resulting soaking solution was adjusted to 4-5. Soybeans 1 were soaked until the water absorption was 40%-100% of the weight of the dry beans. The soybeans were removed, rinsed with tap water, and drained to obtain soybeans 2. The soaking solution ratio was soybeans 1: room temperature tap water = 1 g: 3-5 mL, and the cellulase: pectinase ratio was 1:

1. The addition amount of each was 0.2%-0.5%, based on the weight of the dry beans. Among them, the enzyme activity of cellulase is 500000U / g; the enzyme activity of pectinase is 30000U / g; (4) Heat shock treatment of soaked beans Soybean 2 is immersed in hot water for a second heat shock treatment, removed, and quickly cooled to room temperature to obtain soybean 3, wherein soybean 3 has a protease activity of ≥1303.2 U / g dry bean, a lipoxygenase activity of ≤97346 U / g dry bean, a peptide content of ≥12.5 mg / g dry bean, and a protein hydrolysis degree of ≥0.5%; Second heat shock treatment conditions: material-liquid ratio of soybeans: hot water = 1g:10-30mL; heat shock at 80-90℃ for 1-4min or 91-100℃ for 30s-80s; when cooling, quickly rinse the soybeans with room temperature tap water for 20s to cool to room temperature; (5) Soybeans after soaking and double heat shock are dehydrated and then subjected to in situ enzymatic hydrolysis Soybean 3 is placed in a drying oven at ≤50°C or naturally air-dried to lose water, and in situ enzymatically hydrolyzed for 3-5 hours to reduce the moisture content to less than 25%, thereby obtaining soybean 4, wherein soybean 4 has a protease activity of ≥1403.2 U / g dry bean, a lipoxygenase activity of ≤90000 U / g dry bean, a peptide content of ≥20 mg / g dry bean, and a protein hydrolysis degree of ≥1.1%; (6) Drying of soybeans Place the soybeans 4 in a drying oven and dry them at 40-70°C to a moisture content of ≤14%. The obtained soybeans 5 have a moisture content similar to that of untreated dry beans and can be used for oil extraction. (7) Extraction of soybean oil Soybean oil was extracted by solvent extraction: soybeans 5 were crushed and sieved, and the ratio of soybean powder to anhydrous ether was 1g:5-10mL; the oil was dissolved in a constant temperature water bath at 55℃-60℃ for 50-60min, and the oil yield was ≥18.1%; (8) Enzymatic hydrolysis of soybean protein isolate from soybean meal powder by endogenous protease The defatted soybean meal obtained after soybean oil extraction in step (7) is dried at 45°C-55°C to obtain defatted soybean meal powder, 8-15 times the amount of water is added to the defatted soybean meal powder, the water temperature is 40-55°C, the pH value is adjusted to 6.5-7.5 with NaOH solution, and the mixture is stirred and extracted for 40-70 minutes at a stirring speed of 60-70 r / min to complete the enzymatic hydrolysis of soybean protein by endogenous enzymes, wherein the degree of hydrolysis of the soybean protein isolate is ≥2.5%; (9) Continue to extract the isolated protein and spray dry After extraction by alkali dissolution and acid precipitation, it is sent to a spray drying tower to obtain odorless, highly emulsifiable and oil-absorbing soybean protein isolate.

2. The method according to claim 1, wherein: In step (2) of the method, the conditions for the first heat shock treatment are: 80°C heat shock for 2 minutes or 100°C heat shock for 40 seconds; in step (4) of the method, the conditions for the second heat shock treatment are: 80°C heat shock for 2 minutes or 100°C heat shock for 40 seconds; in step (5) of the method, the moisture content of the soybean 4 after the in situ enzymatic hydrolysis is 20%.

3. The method according to claim 1, wherein: The operation of step (9) is as follows: adjusting the pH value to 11 with NaOH solution, stirring and extracting for 40 minutes at a stirring speed of 60 r / min to complete the extraction of enzymatic soy protein, and centrifuging to obtain a supernatant at a centrifugal speed of 4500 r / min; adding a 20% hydrochloric acid solution by mass to the supernatant at 45°C and stirring at a stirring speed of 60 r / min, adjusting the pH value to 4.2, standing for 30 minutes, and separating the acid precipitate by centrifuge; grinding and crushing the acid precipitate with water, and beating it into a homogenous slurry, adding 5% dilute alkali solution to neutralize it to pH 7.2, controlling the temperature at 25°C, and stirring at a speed of 80 r / min to obtain a neutralized solution; sending it to a spray drying tower, dehydrating and drying it with 160°C hot air, and the tower body temperature is 90-100°C to obtain a highly emulsifiable and oil-absorbing odorless soy protein isolate.

4. The soy protein isolate prepared by the method according to claim 1 or 2, characterized in that: The emulsification index of the soy protein isolate is ≥10m 2 / g, emulsification stability ≥20min, oil absorption ≥7g / g.

Images