Preparation process of high-purity low-antinutritional-factor soybean protein

Abstract

The invention belongs to the field of food, and particularly relates to a preparation process of high-purity low-antinutritional-factor soybean protein. Specifically, the invention provides a method for extracting soybean protein or a method for reducing the phytic acid content of soybean protein, and the method comprises the following steps: (1) equally dividing a soybean raw material into n batches for extraction at 15-35 DEG C, extracting for m times in each batch, n being a positive integer greater than or equal to 2, and m being a positive integer greater than or equal to 3; (2) mixing the soybean protein-containing protein slurry obtained by the first extraction and separation of each batch; wherein in the first batch of extraction, the soybean raw material is extracted by using water, and in the second batch of extraction and each batch of extraction, the soybean raw material is extracted by using a liquid phase obtained by the previous batch of extraction. The invention also provides a method for extracting soybean protein. The method provided by the invention can effectively reduce the content of anti-nutritional factors such as trypsin inhibitors and phytic acid in the protein powder, improve the digestibility of protein and the utilization rate of mineral substances, and optimize the nutritional efficacy of the product.

Description

Technical Field

[0001] This invention belongs to the food field, specifically relating to the preparation process of high-purity soybean protein with low anti-nutritional factors. Background Technology

[0002] Most soybean protein powders currently on the market are produced using traditional extraction processes. These processes involve extracting protein from plant materials through aqueous solution extraction and centrifugation, followed by sterilization, flash evaporation, and spray drying to create a powder product. These products retain the natural components of the plant materials, including protein and various anti-nutritional factors, resulting in low absorption and utilization rates, and the product purity cannot reach over 90% on a wet basis.

[0003] Negative effects of trypsin inhibitors: Trypsin inhibitors, which are naturally present in plant proteins, can inhibit the activity of trypsin in the human intestine, hindering the digestion and absorption of protein. Long-term intake may lead to decreased protein utilization and even cause indigestion, bloating and other problems.

[0004] Adverse effects of phytic acid: As a common anti-nutritional factor, phytic acid can bind with proteins to form indigestible complexes. It can also chelate minerals such as calcium, iron, and zinc, reducing the bioavailability of these nutrients. This has a significant impact, especially on people who need mineral supplementation (such as children, pregnant women, and the elderly). Summary of the Invention

[0005] This technical solution, through a special process, can effectively reduce the content of anti-nutritional factors such as trypsin inhibitors and phytic acid in protein powder, thereby specifically overcoming the above-mentioned defects, improving the digestibility and absorption rate of protein and the utilization rate of minerals, and optimizing the nutritional efficacy of the product.

[0006] The first aspect of the present invention provides a method for extracting soybean protein or a method for reducing the phytic acid content of soybean protein, the method comprising the following steps: (1) extracting soybean raw material into n equal batches at 15-35℃, each batch being extracted m times, wherein n is a positive integer greater than or equal to 2, and m is a positive integer greater than or equal to 3; (2) mixing the protein slurry containing soybean protein obtained from the first extraction of each batch; wherein: in the first extraction, water is used to extract the soybean raw material, and in the second and subsequent extractions, the liquid obtained from the previous extraction is used to extract the soybean raw material; in the first extraction, water is used for the first extraction of the soybean raw material, and the soybean residue is filtered out to obtain protein. The slurry is further extracted by adding water to the soybean residue for m-1 extractions. For the nth batch extraction, the liquid phase obtained from the second extraction in the n-1th batch is used for the first extraction of the soybean raw material. The soybean residue is filtered out to obtain a protein slurry. The liquid phase obtained from the mth extraction in the n-1th batch is added to the soybean residue for the m-1th extraction. The liquid phase is filtered out, and water is added to the soybean residue for the mth extraction. In each batch extraction, during the first extraction step, soluble calcium salts are added to adjust the pH to 7.0-7.2. During the mth extraction step, soluble sodium or potassium salts are added to adjust the pH to 6.8-7.0, and then soluble calcium salts are added to adjust the pH to 7.5-8.0. In one or more embodiments, the extraction temperature is 25-35°C or 30-35°C. In one or more embodiments, the soybean raw material is defatted soybeans, including soybean meal and / or soybean cake. In one or more embodiments, if the protein content of the soybean residue obtained from the m-th extraction step is less than or equal to 10% during the n-th batch extraction, the (m+1)-th batch extraction is not performed. In one or more embodiments, n is a positive integer greater than or equal to 3, such as n≥5, n≥10, n≥15, n≥20, or n≥50, or any positive integer between 3-5, 50-100, 100-500, 100-300, or 300-500. In one or more embodiments, m is a positive integer greater than or equal to 4, such as m≥5, m≥7, m≥9, or m≥10, or m=4-6, 4-8, 5-8, 6-10, or 10-15. In one or more embodiments, during each batch extraction, the extraction time in the first extraction step is greater than or equal to the extraction time in the second or m-th extraction step. In one or more embodiments, in the first extraction step, the pH value is adjusted to be within the range of 7.0-7.2, and the extraction time is 10-20 minutes or 15-20 minutes. In one or more embodiments, in the m-th extraction step, the pH value is adjusted to be in the range of 7.5-8.0, and the extraction time is 10-15 minutes. In one or more embodiments, the first extraction time is 15-20 minutes, and the m-th extraction time is 10-12 minutes.In one or more embodiments, in the nth batch extraction step, the ratio of the soybean raw material or soybean residue to the added water is 1:15-1:20. In one or more embodiments, in the extraction step, stirring is performed to ensure uniform mixing. In one or more embodiments, the stirring rate is 100-200 r / min or 150-200 r / min. In one or more embodiments, the water includes soft water, drinking water, mineral water, distilled water, or pure water, preferably pure water. In one or more embodiments, in the extraction step, the amount of soluble calcium salt added is 0.5-1 wt% of the solid phase mass. In one or more embodiments, the soluble calcium salt includes calcium hydroxide and its aqueous solution. In one or more embodiments, the soluble sodium salt or soluble potassium salt includes sodium hydroxide, potassium hydroxide, or their aqueous solutions.

[0007] In some implementations, n is a positive integer from 10 to 100 and m is a positive integer from 3 to 10, or n is a positive integer from 10 to 50 and m is a positive integer from 3 to 8, or n is a positive integer from 20 to 30 and m is a positive integer from 3 to 5, or n is a positive integer from 15 to 20 and m is a positive integer from 4 to 6.

[0008] A second aspect of this invention provides a method for preparing soybean protein, comprising the extraction step as described herein, and further comprising the steps of acid precipitation, water washing, optional enzymatic hydrolysis, neutralization, nitrogen homogenization, optional sterilization, and optional drying to obtain the soybean protein. The acid precipitation step comprises: first adjusting the pH of the extracted protein slurry to a range of 2.0-3.0, then adjusting the pH of the protein slurry to a range of 4.5-5.0 to obtain a protein suspension, and separating the protein precipitate. The water washing step comprises: washing the protein precipitate to obtain wet-based protein. The enzymatic hydrolysis step comprises: enzymatically hydrolyzing the wet-based protein to obtain a protein solution. The neutralization step comprises: adjusting the pH of the protein solution to neutral. The nitrogen homogenization step comprises: purging the protein solution with nitrogen and homogenizing it in a sealed environment. In one or more embodiments, the protein content in the protein slurry is greater than or equal to 94.5 wt%, preferably 94.5-99 wt% or 94.5-95.5 wt%. In one or more embodiments, the mass of water in the washing step is 4-5 times the mass of the protein precipitate. In one or more embodiments, the protein precipitate is washed twice: in the first washing step, 4-5 times the mass of the liquid phase obtained from centrifugation in the second washing step is added to the protein precipitate; after washing, the liquid phase is filtered off to obtain a solid phase; in the second washing step, 4-5 times the mass of soft water is added to the solid phase after the first washing; after washing, the liquid phase is filtered off to separate the solid phase, which is the wet-based protein.

[0009] In one or more embodiments, the protein precipitate is washed a times, where a is a positive integer greater than or equal to 3: In the first washing step, 4-5 times the mass of the liquid phase obtained from the second washing step by centrifugation is added to the protein precipitate. After washing, the liquid phase is filtered off to obtain a solid phase; In the (a-1)th washing step, 4-5 times the mass of the liquid phase obtained from the ath washing step is added to the solid phase after the (a-2)th washing step. After washing, the separated liquid phase is refluxed for the second washing, and the separated solid phase is washed again, and this cycle is repeated; In the ath washing step, 4-5 times the mass of soft water is added to the solid phase after the (a-1)th washing step. After washing, the liquid phase is filtered off, and the separated solid phase is the wet-based protein.

[0010] In one or more embodiments, 'a' is a positive integer from 3 to 5. In one or more embodiments, in the acid precipitation step, acid is added to adjust the pH of the protein slurry to the range of 2-3, maintained at 40-50°C for 30-60 minutes, and then the pH of the protein slurry is adjusted to the range of 4.5-5.0 to obtain the protein suspension, from which the protein precipitate is separated. In one or more embodiments, the amount of enzyme added is 0.01-0.5 wt% of the substrate for the enzymatic hydrolysis reaction. In one or more embodiments, the nitrogen homogenization pressure is 10-20 MPa, the homogenization pressure is controlled at 10-50 MPa, and the homogenization temperature is 50-55°C. In one or more embodiments, the sterilization temperature is 150-170°C, maintained for 20-60 seconds. In one or more embodiments, the drying is freeze-drying, vacuum drying, or spray drying.

[0011] A third aspect of the present invention provides soy protein isolate prepared as described herein, or a composition containing the soy protein isolate.

[0012] A fourth aspect of this invention provides soy protein isolate or compositions containing such soy protein isolate as described herein, said compositions being nutritional supplements, pharmaceutical excipients, health products, feed, quality improvers, dairy substitutes / plant-based foods, baked / snack foods, beverages / seasonings. In one or more embodiments, the composition is a meat product or aquatic product, such as sausage, ham, meatballs, fish balls, or luncheon meat. In one or more embodiments, the composition is a dairy product or plant-based food, such as plant-based milk, high-protein soy milk, plant-based yogurt, plant-based meat patties, or artificial meat. In one or more embodiments, the composition is a baked good or snack food, such as bread, cake, protein bars, meal replacement powder, or sports nutrition products. In one or more embodiments, the composition is a beverage or seasoning, such as functional beverages, soy sauce, or condiments. In one or more embodiments, the composition is feed. In one or more embodiments, the composition is a nutrient or health product. In one or more embodiments, the soy protein isolate is in powder, emulsion, or liquid form. In one or more embodiments, the composition containing soy protein isolate is in the form of an aqueous suspension, powder, solid, or semi-solid.

[0013] The fifth aspect of the present invention provides for the application of the methods described herein in one or more of the following aspects: (1) preparing a composition containing soy products, (2) increasing the protein content in soy protein isolate, (3) decreasing the phytic acid content in soy protein isolate, (4) decreasing the trypsin inhibitor content in soy protein isolate, and (5) increasing the yield of soy protein isolate. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the extraction process in Embodiment 1 of the present invention.

[0015] Figure 2 This is a schematic diagram showing the breakdown of the extraction process in Embodiment 2 of the present invention. Detailed Implementation

[0016] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0017] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0018] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0019] In this document, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0020] Unless otherwise specified, percentages refer to mass percentages, and proportions refer to mass ratios. In this document, the sum of the percentage contents of all components in the composition is 100%.

[0021] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0022] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0023] The inventors have discovered that the method of this invention can improve the protein content, trypsin inhibitor content, and phytic acid content of soy protein isolate. This completes the present invention.

[0024] Soy protein and its preparation method

[0025] Soybean raw materials are typically extracted using alkaline solutions at temperatures of 40-50℃. However, the method described in this paper employs low-temperature extraction, with temperatures ranging from 15-35℃, preferably 25-35℃ or 30-35℃. This method allows for multiple or multi-stage extraction of the soybean residue solid phase from pulverized soybean raw materials (such as soybean meal) to improve protein extraction efficiency.

[0026] Therefore, this article provides a method for extracting soybean protein or reducing the phytic acid content of soybean protein. The method includes the following steps: (1) extracting soybean raw materials into n equal batches at 15-35℃, with each batch being extracted m times, where n is a positive integer greater than or equal to 2 and m is a positive integer greater than or equal to 3; (2) mixing the protein slurry containing soybean protein obtained from the first extraction of each batch; wherein: in the first extraction, water is used to extract the soybean raw materials, and in the second and subsequent extractions, the liquid obtained from the previous extraction is used to extract the soybean raw materials.

[0027] In the first batch extraction, water is used to perform the first extraction on the soybean raw material. The soybean residue is filtered out to obtain a protein slurry. Water is added to the soybean residue to continue the m-1 extraction. In the nth batch extraction, the liquid phase obtained from the second extraction in the n-1th batch extraction is used to perform the first extraction on the soybean raw material. The soybean residue is filtered out to obtain a protein slurry. The liquid phase obtained from the mth extraction in the n-1th batch extraction is added to the soybean residue to perform the m-1st extraction. The liquid phase is filtered out, and water is added to the soybean residue to perform the mth extraction. In each batch extraction, in the first extraction step, soluble calcium salt is added to adjust the pH to the range of 7.0-7.2. In the mth extraction step, soluble sodium salt or soluble potassium salt is added to adjust the pH to the range of 6.8-7.0. Then, soluble calcium salt is added to adjust the pH to the range of 7.5-8.0.

[0028] It should be understood that industrial production typically employs continuous batch feeding, as described in this article. Figure 2 As shown, the greater the amount of soybean raw material (i.e., the greater the n) and the more extraction batches (i.e., the greater the m), the higher the protein content and the lower the phytic acid content in the protein slurry obtained by the method in this paper. It should be understood that each batch of extracted soybean raw material is of the same quality and origin.

[0029] An exemplary embodiment of the method includes the following steps: Soybean raw material is divided into three batches and extracted three times at 15-35℃; the protein slurry containing soybean protein obtained from the first extraction of each batch is mixed; in the first extraction, water is used to perform a first extraction of the soybean raw material, the soybean residue is filtered out to obtain a protein slurry, water is added to the soybean residue for a second extraction, the liquid phase is filtered out, and water is added to the soybean residue for a third extraction; in the second extraction, the liquid phase obtained from the second extraction of the first batch is used to perform a first extraction of the soybean raw material, the soybean residue is filtered out to obtain a protein slurry, and the liquid phase obtained from the third extraction of the first batch is added to the soybean residue for a second extraction. The liquid phase is filtered out, and water is added to the soybean residue for a third extraction. For the third batch extraction, the liquid phase obtained from the second extraction in the second batch is used for the first extraction of the soybean raw material. The soybean residue is filtered out to obtain a protein slurry. The liquid phase obtained from the third extraction in the second batch is added to the soybean residue for a second extraction. The liquid phase is filtered out, and water is added to the soybean residue for a third extraction. For each batch extraction, in the first extraction step, soluble calcium salt is added to adjust the pH to the range of 7.0-7.2. In the third extraction step, soluble sodium salt or soluble potassium salt is added to adjust the pH to the range of 6.8-7.0, and then soluble calcium salt is added to adjust the pH to the range of 7.5-8.0.

[0030] In an exemplary implementation, the method described herein includes performing at least three batches of soybean meal on three separate extractions (i.e., n is a positive integer greater than or equal to 3, and m is a positive integer greater than or equal to 3):

[0031] 1) First batch of soybean meal extraction: a) First extraction: Peel and grind soybean meal to 60-80 mesh for the first extraction. Add 15-20 times its weight of water, mix thoroughly, and slowly add calcium hydroxide solution while stirring at 120-150 rpm. Adjust the pH of the mixture to 7.0-7.2, and extract at 15-35℃ or 25-35℃ for 15-20 minutes, separating the soybean residue and protein slurry; b) Second extraction: Add 15-20 times its weight of water to the soybean residue, mix thoroughly, and extract at 15-35℃ or 25-35℃ for 15-20 minutes, separating the soybean residue and protein slurry; Extract at 25-35℃ for 15-20 min to separate soybean residue and liquid phase. Continue the third extraction of soybean residue; c. Third extraction: Add 15-20 times the weight of soybean meal water to the soybean residue obtained from the second extraction, mix well, and slowly add sodium hydroxide solution at 120-150 r / min to adjust the pH of the mixture to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 15-35℃ or 25-35℃ for 15-20 min to separate soybean residue and liquid phase.

[0032] 2) Second batch soybean meal extraction: a) First extraction: Pour the peeled and pulverized soybean meal (60-80 mesh) into the first-stage extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, and slowly add calcium hydroxide solution at 120-150 r / min to adjust the pH of the mixed slurry to 7.0-7.2. Extract at 15-35℃ or 25-35℃ for 15-20 min, separating the soybean residue solid phase and protein slurry; b) Second extraction: Add the liquid phase obtained from the third extraction during the first batch of soybean meal extraction to the solid phase, mix evenly, and then... Extract at 5-35℃ or 25-35℃ for 15-20 min to separate the soybean residue solid phase and soybean milk liquid phase. The solid phase is then subjected to a third extraction. c. Third extraction: Add 15-20 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution at 120-150 r / min to adjust the pH of the mixture to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 15-35℃ or 25-35℃ for 15-20 min to separate the solid and liquid phases.

[0033] 3) Third batch soybean meal extraction: a) First extraction: Pour the peeled and pulverized soybean meal (60-80 mesh) into the first-stage extraction tank, add the liquid phase obtained from the second extraction during the second batch soybean meal extraction, mix evenly, and slowly add calcium hydroxide solution at 120-150 r / min to adjust the pH of the mixed slurry to 7.0-7.2. Extract at 15-35℃ or 25-35℃ for 15-20 min, separating the soybean residue solid phase and protein slurry; b) Second extraction: Add the liquid phase obtained from the third extraction during the second batch soybean meal extraction to the solid phase, mix evenly, and extract at 15-35℃ or 25-35℃. Extract for 15-20 min to separate soybean residue and soybean milk liquid phase. Continue to extract the soybean residue three times. c. Third extraction: Add 15-20 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution at 120-150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 15-35℃ or 25-35℃ for 15-20 min to separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10wt%, no fourth extraction is performed.

[0034] 4) Take the protein slurry obtained from the first to the third batch of soybean meal extraction and mix them together. This mixture is the protein slurry described in this article.

[0035] In the first extraction step, a soluble calcium salt (such as calcium hydroxide or its aqueous solution) is added to maintain the pH value within the range of 7.0-7.2. The amount of soluble calcium salt added is based on the pH value, and the amount of calcium hydroxide added is usually 0.5-2 wt% of the soybean meal mass. Extraction time is 10-20 minutes or 15-20 minutes. In the m-th extraction step, water is added to the soybean residue for extraction. Soft water, drinking water, mineral water, distilled water, or pure water is preferred. Then, food-grade alkali solution (such as sodium hydroxide) is added to adjust the pH to the range of 6.8-7.0. Calcium hydroxide or its aqueous solution is then added to adjust the pH to the range of 7.5-8.0. Extraction time is 10-15 minutes. The mass of water added is 15-20 times the mass of soybean meal. The weight of the alkali solution is based on the pH range, and the addition ratio is approximately 0.5-1 wt% of the soybean meal mass. During the extraction step, stirring can be used to ensure uniform mixing. The stirring speed can be 100-200 rpm or 150-200 rpm. In the extraction steps of this paper, if the protein content of the solid soybean residue separated in the m-th extraction step is less than or equal to 10%, then the next extraction step (i.e., the m+1th extraction) will not be performed.

[0036] The method described in this paper does not have any particular restrictions on soybean raw materials. Conventional low-denatured defatted soybean meal can be used as raw material, with a protein content of not less than 53.5% and an NSI of not less than 80%. Low-denatured defatted soybean meal obtained by washing, dehulling, rolling, and solvent extraction (such as n-hexane) can be selected. The soybean meal should be pulverized to a fineness of 40-80 mesh to increase the surface area and improve the extraction efficiency.

[0037] During extraction, calcium ions can chelate with phytic acid in the protein slurry to form insoluble calcium phytate, thus reducing its phytic acid content. The amount of calcium salt (such as food-grade calcium hydroxide solution) added can be 0.5-1 wt% of the solid phase mass.

[0038] This article also provides a method for preparing soybean protein, which includes the steps of extraction, acid precipitation, water washing, optional enzymatic hydrolysis, neutralization, nitrogen homogenization, and optional sterilization.

[0039] In the acid precipitation process described in this paper, excess acid is first added to reduce the activity of trypsin inhibitors, and then the pH is adjusted back to the isoelectric point of the protein to separate the protein precipitate. In the deactivation step, the acid can be conventional hydrochloric acid, citric acid, or phosphoric acid. The pH of the protein purification solution is adjusted to within the range of 2-3. During acid precipitation, the temperature can be maintained at 40-50℃, and the precipitation time can be 30-60 minutes. Stirring can be used to assist in uniform mixing. After deactivation, an alkaline solution (such as sodium calcium hydroxide solution) can be used to adjust the pH of the protein solution back to around 4.5, causing the protein to aggregate into large particles and precipitate fully. The precipitation time can be 20-30 minutes. Separation can be performed using conventional equipment, such as a horizontal decanter centrifuge.

[0040] In the washing process described in this paper, a countercurrent washing process can be used, in which the protein precipitate and water flow in opposite directions to maximize the concentration gradient and remove residual impurities such as acid, soluble sugars, salts, and minerals from the protein precipitate. The washing water is typically soft water, with the protein precipitate moving from left to right and the soft water moving from right to left. In this paper, the protein precipitate can be washed countercurrently at least twice, with the water mass being 4-5 times the mass of the protein precipitate. In the first washing step, 4-5 times the mass of the liquid phase obtained from the second washing step (obtained by centrifugation) is added to the protein precipitate obtained from acid precipitation. After washing, the liquid phase is filtered off, yielding a solid phase. In the second washing step, 4-5 times the mass of the liquid phase obtained from the third washing step (obtained by centrifugation) is added to the solid phase obtained from the first washing step. After washing, the separated liquid phase is refluxed for the first washing, and the separated solid phase is further washed. In the third washing step, 4-5 times the mass of soft water is added to the solid phase. After washing, the separated liquid phase is refluxed for the second washing, and the separated solid phase is further washed, thus completing the cycle. In the final water washing step, after washing is completed, the liquid phase is filtered out, and the solid phase obtained is the wet protein.

[0041] In an exemplary embodiment, the method described herein employs two countercurrent washing steps. In the first washing step, 4-5 times the volume of the liquid phase obtained from centrifugation in the second washing step is added to the protein precipitate obtained from acid precipitation. After washing, the liquid phase is filtered off to obtain a solid phase. In the second washing step, 4-5 times the volume of soft water is added to the solid phase after the first washing. After washing, the separated liquid phase is refluxed for the first washing. The separated solid phase is the wet-based protein.

[0042] In this method, the wet-based protein after washing is enzymatically hydrolyzed. Depending on the enzyme used, such as phytase, the hydrolysis system can be adjusted to a suitable concentration (e.g., 10-15%), temperature, pH, and water content for neutralization. The sugar content is adjusted to 15-18, and food-grade sodium chloride or its aqueous solution and sodium hydroxide are added to adjust the pH to within the range of 6-7. For example, using phytase with an activity of 40,000-50,000 U / g, hydrolysis can be performed at 40-60℃ for 40-60 min or 1-3 hours, with the hydrolysis effect monitored every few minutes. After hydrolysis, the enzyme is inactivated at 80-85℃ for 10-20 min. In this enzymatic hydrolysis process, the wet-based protein obtained from washing can be prepared using sodium hydroxide solution to a protein solution with a pH of 5-6, a sugar content of 15-18, and a concentration of 5-15 wt%. Excessive protein concentration can lead to aggregation, while insufficient concentration will reduce the efficiency of the hydrolysis reaction. Appropriate acids or bases can be used to adjust the pH of the enzyme solution. The amount of enzyme added can be adjusted according to the actual substrate mass of the enzymatic hydrolysis reaction. For example, the amount of enzyme added can be 0.01-0.5 wt% of the substrate mass. The enzyme can be added in liquid form, for example, by dissolving the solid enzyme to the required concentration using a buffer solution. During the enzymatic hydrolysis reaction, stirring can be used to ensure that the enzyme and the reaction substrate are mixed evenly and in full contact, for example, at a speed of 50-100 rpm. After enzymatic hydrolysis, the pH of the hydrolysate usually needs to be adjusted to neutral.

[0043] In the homogenization process described in this paper, food-grade nitrogen (purity greater than or equal to 99.99%) is introduced into the enzymatically hydrolyzed protein at a pressure of 10-20 MPa. The homogenization pressure is controlled at 10-50 MPa, and the homogenization temperature can be 50-55℃. This generates strong shear force, breaking the protein down to the required size.

[0044] After homogenization, sterilization and drying steps are usually included to obtain soy protein isolate. The sterilization temperature described in this article is 150-170℃, held for 20-60 seconds. Appropriate sterilization temperature and time help reduce trypsin inhibitor activity. This temperature and time effectively inactivate residual trypsin inhibitors in the raw materials (they are heat-sensitive; activity is reduced at 121℃ for 15 seconds), while also killing E. coli, mold, and other microorganisms, ensuring that the product's microbiological indicators meet food safety standards (total bacterial count ≤10000 CFU / g, pathogenic bacteria not detectable). After sterilization, the protein solution is usually cooled at a relatively high temperature before drying. A common cooling step is flash cooling, with a vacuum of approximately 80-100 kPa, which can cool to 60-65℃. Conventional drying methods, such as freeze-drying, vacuum drying, or spray drying, can also be used.

[0045] In some embodiments, the method described herein includes one or more of the following steps: extraction, acid precipitation, water washing, enzymatic hydrolysis, neutralization, homogenization, and sterilization, wherein: in the extraction step, the soybean raw material is extracted three times (e.g., three, four, five, six, or more times) in at least three batches (e.g., three, four, five, six, or more batches) to obtain a protein slurry; in the acid precipitation step, acid is added to the protein slurry to deactivate it, followed by the addition of alkali, and the liquid phase is filtered off to obtain a protein precipitate; in the water washing step, the protein precipitate is washed at least twice (e.g., two, three, four, five, six, or more times) to obtain a wet-based protein.

[0046] In some embodiments, this document also provides soy protein isolate prepared as described in any embodiment herein. In some embodiments, the soy protein isolate herein may be in powder, emulsion, or liquid form.

[0047] Compositions containing soy protein isolate

[0048] The soy protein isolate of the present invention can be used as a nutritional supplement, pharmaceutical excipient, health product, feed, quality improver, dairy substitute / plant-based food, baked / snack food, beverage / seasoning, etc. Therefore, this document also provides compositions containing soy protein isolate as described in any embodiment herein.

[0049] The compositions described herein can be meat products and aquatic products, such as sausages, ham, meatballs, fish balls, luncheon meat, etc.; dairy products and plant-based foods, such as plant-based milk, high-protein soy milk, plant-based yogurt, plant-based meat patties, artificial meat, etc.; baked goods and snack foods, such as bread, cakes, protein bars, meal replacement powders, sports nutrition products, etc.; beverages and condiments, such as functional beverages, soy sauce, seasoning sauces, etc.; animal feed; and also nutritional supplements and health products. The compositions containing soy protein isolate described herein can be in various forms, such as aqueous suspensions, powders, solids, semi-solids, etc.

[0050] The soy protein isolate of the present invention can be applied in the fields of food, agricultural products, feed, health products, cosmetics, and pharmaceuticals. Therefore, this document also provides the application of the method described in any embodiment herein in the preparation of compositions containing soy products, increasing the protein content in soy protein isolate, decreasing the phytic acid content in soy protein isolate, decreasing the trypsin inhibitor content in soy protein isolate, and increasing the yield of soy protein isolate.

[0051] In some embodiments, this document also provides the use of methods incorporating extraction steps as described herein in the preparation of soy protein, particularly soy protein isolate. In some embodiments, this document also provides the use of methods as described in any embodiment herein in increasing the protein content of soy protein, decreasing the phytic acid content of soy protein, and / or decreasing the trypsin inhibitor content.

[0052] The present invention has the following beneficial effects:

[0053] 1) This invention reduces the phytic acid content of the finished product from the source, solving the problem of incomplete phytic acid removal in traditional processes; 2) This application uses an acid treatment combined with isoelectric point adjustment technology, first adjusting to pH 2~3 and 40~60℃, and then adjusting the isoelectric point to 4.5 to separate the protein, which not only ensures protein precipitation efficiency but also further reduces inhibitor activity; 3) This application uses a countercurrent two-stage water washing process to improve pure water utilization and can specifically remove residual phytic acid, trypsin inhibitors, and inorganic salts, making the wet-based protein content reach more than 90%, while reducing the residual amount of anti-nutritional factors compared to traditional processes; 4) This application uses high-pressure nitrogen homogenization combined with high-temperature sterilization technology to refine protein particles, reduce protein contact with oxygen, reduce oxidation risk, and increase the contact area with high-temperature sterilization, making trypsin inhibitor inactivation more thorough. Compared with simple high-temperature sterilization, the residual activity of inhibitors is reduced.

[0054] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention. The methods and reagents used in the embodiments are conventional methods and reagents in the art, unless otherwise stated. In the embodiments and comparative examples herein, the soybean meal has a protein content >53.5% and a nitrogen solubility index (NSI) of not less than 80%.

[0055] Detection methods

[0056] Protein content: Determined according to GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food" - Kjeldahl method. Trypsin inhibitor content: Determined according to GB 5009.224-2016 "National Food Safety Standard - Determination of Trypsin Inhibitor Activity in Soybean Products". Phytic acid content: Determined according to GB 5009.153-2016 "National Food Safety Standard - Determination of Phytic Acid in Food". Color detection: R457 value measured using a whiteness colorimeter. Yield: Yield = (mass of protein powder / mass of soybean meal) × 100%.

[0057] Example 1

[0058] 1. Extraction process (each batch of soybean meal is of the same quality and from the same origin)

[0059] 1) First batch of soybean meal extraction:

[0060] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add 15 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate soybean residue and protein slurry.

[0061] b. Second extraction: Add 15 times the weight of the soybean meal in soft water to the soybean residue, mix well, and extract at 25°C for 15 minutes to separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0062] c. Third extraction: Add 15 times the weight of soft water to the soybean residue obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the soybean residue and liquid phase.

[0063] 2) Second batch of soybean meal extraction:

[0064] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0065] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate the soybean residue solid phase and soybean milk liquid phase. The solid phase is then subjected to a third extraction.

[0066] c. Three-stage extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the solid phase and liquid phase.

[0067] 3) Third batch of soybean meal extraction:

[0068] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0069] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0070] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min, and separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction will be performed.

[0071] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation;

[0072] 2. Acid precipitation process

[0073] 1) Deactivation by acidification: Pump the protein slurry obtained in the extraction process into the acid precipitation tank, stir at 120 r / min, slowly add 1 mol / L hydrochloric acid solution, adjust the pH value of the protein slurry (soy milk) to the range of 2-3, and at the same time raise the temperature inside the tank to 60℃ and keep stirring for 30 min;

[0074] 2) Isoelectric point adjustment separation: After deactivation, use 1 mol / L sodium hydroxide solution to adjust the pH of the soy milk back to 4.5, reduce the stirring speed to 80 r / min, and let it stand for 20 min to allow the protein to precipitate fully; centrifuge at 6000 r / min for 25 min using a horizontal screw centrifuge, discard the supernatant, and separate the protein precipitate;

[0075] 3. Countercurrent washing process

[0076] The two-stage cleaning units are connected in series. The first stage is the material inlet, which uses the liquid phase obtained from the separation after the second stage cleaning. The second stage is the material outlet, which uses clean water.

[0077] 1) First wash: Add 3 times the volume of the liquid phase obtained from the second wash step to the protein precipitate obtained by acid precipitation. After stirring and washing at 120 r / min, discard the washing liquid and centrifuge (5000 r / min, 10 min) to separate the solid phase.

[0078] 2) Second wash: Add 2 times the volume of soft water to the solid phase after the first wash, stir and wash at 120 r / min, discard the washing solution, and separate the wet protein.

[0079] 4. Enzymatic hydrolysis and neutralization process

[0080] 1) Enzymatic hydrolysis preparation: Put the washed wet protein into the enzymatic hydrolysis tank, add an appropriate amount of pure water to adjust the protein concentration to 15%, turn on the stirring (180r / min), raise the temperature inside the tank to 52℃, and add sodium hydroxide to adjust the pH value to 6.0;

[0081] 2) Addition and reaction of compound enzymes: Add 0.1wt% of food-grade phytase (activity 10000U / g) and 0.1wt% of acidic protease (activity 50000U / g) of the wet basis protein of the raw material, respectively, and enzymatically hydrolyze at 50℃ for 30 min; during the process, samples are taken every 10 min to ensure the enzymatic hydrolysis effect;

[0082] 3) Enzyme inactivation and neutralization: After the enzymatic hydrolysis is completed, raise the temperature inside the tank to 85°C and keep it constant for 15 minutes to completely inactivate phytase and protease, and adjust the protein solution to neutral.

[0083] 5. Combined treatment of high-pressure nitrogen homogenization and high-temperature sterilization

[0084] 1) High-pressure nitrogen homogenization: The neutralized protein solution is sent into a high-pressure homogenizer, and food-grade nitrogen (purity ≥99.99%) is introduced at the same time. The nitrogen pressure is 10MPa, the homogenization pressure is controlled at 10MPa, the homogenization temperature is 55℃, and the homogenization process is carried out twice.

[0085] 2) High-temperature sterilization: The homogenized protein solution is sent to an ultra-high temperature instantaneous sterilizer (UHT), the sterilization temperature is controlled at 150℃, the holding time is 20s, and then it is put into a flash tank (80kpa) for rapid cooling to 65℃ to obtain soybean protein isolate.

[0086] Example 2

[0087] Except for the extraction process in step 1, which is carried out according to the following steps, the remaining process steps are the same as in Example 1:

[0088] 1. Extraction process (each batch of soybean meal is of the same quality and origin)

[0089] 1) First batch of soybean meal extraction:

[0090] a. First extraction: Pour the peeled and crushed soybean meal to 60 mesh into the first extraction tank, add 15 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate soybean residue and protein slurry.

[0091] b. Second extraction: Add 15 times the weight of the soybean meal in soft water to the soybean residue, mix well, and extract at 25°C for 15 minutes to separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0092] c. Third extraction: Add 15 times the weight of soft water to the soybean residue obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the soybean residue and liquid phase.

[0093] 2) Second batch of soybean meal extraction:

[0094] a. First extraction: Pour the peeled and crushed soybean meal to 60 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0095] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 min to separate the solid phase and the soybean milk liquid phase.

[0096] c. Third extraction: Add 15 times the weight of soft water to the solid phase obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-7.8. Extract at 25℃ for 15 min to separate the solid phase and liquid phase.

[0097] 3) Third batch of soybean meal extraction:

[0098] a. First extraction: Pour the peeled and crushed soybean meal to 60 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0099] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0100] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the solid phase and liquid phase.

[0101] 4) Fourth batch of soybean meal extraction:

[0102] a. First extraction: Pour the peeled and crushed soybean meal to 60 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the third batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0103] b. Second extraction: Add the liquid phase obtained from the third extraction of soybean meal to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0104] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase obtained from the second extraction, mix well, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction will be performed.

[0105] 5) Take the protein slurry obtained from the first to fourth batches of soybean meal extraction, mix them together, and perform acid precipitation.

[0106] Example 3

[0107] 1. Extraction process (each batch of soybean meal is of the same quality and origin)

[0108] 1) First batch of soybean meal extraction:

[0109] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add 20 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 30℃ for 15 min, and separate soybean residue and protein slurry.

[0110] b. Second extraction: Add 20 times the weight of the soybean meal in soft water to the soybean residue, mix well, and extract at 30°C for 15 minutes to separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0111] c. Third extraction: Add 20 times the weight of the soybean meal in soft water to the soybean residue obtained from the second extraction, mix well, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 30℃ for 15 min to separate the soybean residue and liquid phase.

[0112] 2) Second batch of soybean meal extraction:

[0113] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 30℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0114] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, and extract at 30°C for 15 minutes to separate the solid phase and the soybean milk liquid phase.

[0115] c. Third extraction: Add 20 times the weight of soybean meal in soft water to the solid phase obtained from the second extraction, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.8-8.0. Extract at 30℃ for 15 min to separate the solid phase and liquid phase.

[0116] 3) Third batch of soybean meal extraction:

[0117] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 30℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0118] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 30°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0119] c. Third extraction: Add 20 times the weight of soybean meal in soft water to the solid phase obtained from the second extraction, mix well, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 30℃ for 15 min, and separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction will be performed.

[0120] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation;

[0121] 2. Acid precipitation process

[0122] 1) Deactivation by over-acidification: Pump the protein slurry obtained in the extraction process into the acid precipitation tank, stir at 120 r / min, slowly add 1 mol / L hydrochloric acid solution, adjust the pH value of the soy milk to the range of 2-3, and at the same time raise the temperature inside the tank to 50℃ and keep stirring for 30 min.

[0123] 2) Isoelectric point adjustment separation: After deactivation, use 1 mol / L sodium hydroxide solution to adjust the pH of the soy milk back to 4.5, reduce the stirring speed to 80 r / min, and let it stand at a constant temperature for 20 min to allow the protein to precipitate fully; use a horizontal screw centrifuge at 6000 r / min for 25 min to separate the protein precipitate, and discard the supernatant;

[0124] 3. Countercurrent washing process

[0125] The three-stage cleaning units are connected in series. The first stage is the material inlet, which uses the liquid phase obtained from the separation after the second stage of cleaning. The second stage uses the liquid phase obtained from the separation after the third stage of cleaning. The third stage is the material outlet, which uses clean water.

[0126] 1) First wash: Add 3 times the volume of the liquid phase obtained from the second wash step to the protein precipitate obtained by acid precipitation, stir and wash at 100 r / min for 15 min, discard the washing liquid, and centrifuge (5000 r / min, 10 min) to obtain the solid phase;

[0127] 2) Second wash: Add twice the volume of the liquid phase obtained from the third wash step to the solid phase after the first wash. After stirring and washing at 120 r / min, discard the washing liquid and centrifuge (5000 r / min, 10 min) to separate the solid phase.

[0128] 3) Third wash: Add twice the volume of soft water to the solid phase after the second wash, and repeat the washing and centrifugation process to obtain the wet protein after the third wash.

[0129] 4. Enzymatic hydrolysis and neutralization process

[0130] 1) Enzymatic hydrolysis preparation: Put the washed wet protein into the enzymatic hydrolysis tank, add an appropriate amount of pure water to adjust the protein concentration to 15%, turn on the stirring (180r / min), raise the temperature inside the tank to 52℃, and add sodium hydroxide to adjust the pH value to 7.0;

[0131] 2) Addition and reaction of compound enzyme: Add 0.3wt% of food-grade phytase (activity 10000U / g) of the wet basis protein of the raw material and enzymatically hydrolyze at a constant temperature for 45 min; take samples every 10 min during the process to ensure the effect of enzymatic hydrolysis.

[0132] 3) Enzyme inactivation: After the enzymatic hydrolysis is completed, raise the temperature inside the tank to 85°C and keep it constant for 15 minutes to completely inactivate the phytase. Adjust the protein solution to neutral.

[0133] 5. Combined treatment of high-pressure nitrogen homogenization and high-temperature sterilization

[0134] 1) High-pressure nitrogen homogenization: The neutralized and enzymatically hydrolyzed protein solution is sent into a high-pressure homogenizer, and food-grade nitrogen (purity ≥99.99%) is introduced at the same time. The nitrogen pressure is 15MPa, the homogenization pressure is controlled at 30MPa, and the homogenization temperature is 55℃. The homogenization process is carried out twice.

[0135] 2) High-temperature sterilization: The homogenized protein solution is sent to an ultra-high temperature instantaneous sterilizer (UHT), the sterilization temperature is controlled at 170℃, the holding time is 20s, and then it is put into a flash tank (80kpa) for rapid cooling to 65℃ to obtain soybean protein isolate.

[0136] Example 4

[0137] 1. Extraction process (each batch of soybean meal is of the same quality and origin)

[0138] 1) First batch of soybean meal extraction:

[0139] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add 20 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 35℃ for 15 min, and separate soybean residue and protein slurry.

[0140] b. Second extraction: Add 20 times the weight of the soybean meal in soft water to the soybean residue, mix well, and extract at 35°C for 15 minutes to separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0141] c. Third extraction: Add 20 times the weight of the soybean meal in soft water to the soybean residue obtained from the second extraction, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 35℃ for 15 min to separate the soybean residue and liquid phase.

[0142] 2) Second batch of soybean meal extraction:

[0143] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 35℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0144] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, and extract at 35℃ for 15 minutes to separate the solid phase and the soybean milk liquid phase. The soybean residue is used for the third extraction.

[0145] c. Third extraction: Add 20 times the weight of soybean meal in soft water to the solid phase obtained from the second extraction, mix well, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.8-8.0. Extract at 35℃ for 15 min to separate the solid phase and liquid phase.

[0146] 3) Third batch of soybean meal extraction:

[0147] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 35℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0148] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0149] c. Third extraction: Add 20 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 35℃ for 15 min, and separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction is performed.

[0150] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation;

[0151] 2. Acid precipitation process

[0152] 1) Deactivation by over-acidification: Pump the protein slurry obtained in the extraction process into the acid precipitation tank, stir at 120 r / min, slowly add 1 mol / L hydrochloric acid solution, adjust the pH value of the soy milk to the range of 2-3, and at the same time raise the temperature inside the tank to 50℃ and keep stirring for 30 min.

[0153] 2) Isoelectric point adjustment separation: After deactivation, use 1 mol / L sodium hydroxide solution to adjust the pH of the soy milk back to 4.5, reduce the stirring speed to 80 r / min, and let it stand at a constant temperature for 20 min to allow the protein to precipitate fully; use a horizontal screw centrifuge at 6000 r / min for 25 min to separate the protein precipitate, and discard the supernatant;

[0154] 3. Countercurrent washing process

[0155] The three-stage cleaning units are connected in series. The first stage is the material inlet, which uses the liquid phase obtained from the separation after the second stage of cleaning. The second stage uses the liquid phase obtained from the separation after the third stage of cleaning. The third stage is the material outlet, which uses clean water.

[0156] 1) First wash: Add 3 times the volume of the liquid phase obtained from the second wash step to the protein precipitate obtained by acid precipitation, stir and wash at 100 r / min for 15 min, discard the washing liquid, and centrifuge (5000 r / min, 10 min) to obtain the solid phase;

[0157] 2) Second wash: Add twice the volume of the liquid phase obtained from the third wash step to the solid phase after the first wash. After stirring and washing at 120 r / min, discard the washing liquid and centrifuge (5000 r / min, 10 min) to separate the solid phase.

[0158] 3) Third wash: Add twice the volume of soft water to the solid phase after the second wash, and repeat the washing and centrifugation process to obtain the wet protein after the third wash.

[0159] 4. Enzymatic hydrolysis and neutralization process

[0160] 1) Enzymatic hydrolysis preparation: Put the washed wet protein into the enzymatic hydrolysis tank, add an appropriate amount of pure water to adjust the protein concentration to 15%, turn on the stirring (180r / min), raise the temperature inside the tank to 52℃, and add sodium hydroxide to adjust the pH value to 7.0;

[0161] 2) Addition and reaction of compound enzyme: Add 0.3wt% of food-grade phytase (activity 10000U / g) of the wet basis protein of the raw material and enzymatically hydrolyze at a constant temperature for 45 min; take samples every 10 min during the process to ensure the effect of enzymatic hydrolysis.

[0162] 3) Enzyme inactivation and neutralization: After the enzymatic hydrolysis is completed, raise the temperature inside the tank to 85°C and keep it constant for 15 minutes to completely inactivate the phytase and adjust the protein solution to neutral.

[0163] 5. Combined treatment of high-pressure nitrogen homogenization and high-temperature sterilization

[0164] 1) High-pressure nitrogen homogenization: The neutralized and enzymatically hydrolyzed protein solution is sent into a high-pressure homogenizer, and food-grade nitrogen (purity ≥99.99%) is introduced at the same time. The nitrogen pressure is 15MPa, the homogenization pressure is controlled at 30MPa, and the homogenization temperature is 55℃. The homogenization process is carried out twice.

[0165] 2) High-temperature sterilization: The homogenized protein solution is sent to an ultra-high temperature instantaneous sterilizer (UHT), the sterilization temperature is controlled at 170℃, the holding time is 20s, and then it is put into a flash tank (80kpa) for rapid cooling to 65℃ to obtain soybean protein isolate.

[0166] Example 5

[0167] 1. Extraction process (each batch of soybean meal is of the same quality and from the same origin)

[0168] 1) First batch of soybean meal extraction:

[0169] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add 15 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate soybean residue and protein slurry.

[0170] b. Second extraction: Add 15 times the weight of the soybean meal in soft water to the soybean residue, mix well, and extract at 25°C for 15 minutes to separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0171] c. Third extraction: Add 15 times the weight of soft water to the soybean residue obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the soybean residue and liquid phase.

[0172] 2) Second batch of soybean meal extraction:

[0173] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0174] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 min to separate the solid phase and the soybean milk liquid phase.

[0175] c. Third extraction: Add 15 times the weight of soft water of soybean meal to the solid phase obtained from the second extraction, mix evenly, slowly add sodium hydroxide solution at 150 r / min, adjust the pH of the mixed slurry to the range of 6.8-7.0, then add calcium hydroxide to adjust the pH to the range of 7.5-8.0, extract at 25℃ for 15 min, and separate the solid phase and liquid phase.

[0176] 3) Third batch of soybean meal extraction:

[0177] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0178] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0179] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min, and separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction will be performed.

[0180] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation;

[0181] 2. Acid precipitation process

[0182] 1) Deactivation by acidification: Pump the protein slurry obtained in the extraction process into the acid precipitation tank, stir at 120 r / min, slowly add 1 mol / L hydrochloric acid solution, adjust the pH value of the protein slurry (soy milk) to the range of 2-3, and at the same time raise the temperature inside the tank to 60℃ and keep stirring for 30 min;

[0183] 2) Isoelectric point adjustment separation: After deactivation, use 1 mol / L sodium hydroxide solution to adjust the pH of the soy milk back to 4.5, reduce the stirring speed to 80 r / min, and let it stand for 20 min to allow the protein to precipitate fully; centrifuge at 6000 r / min for 25 min using a horizontal screw centrifuge, discard the supernatant, and separate the protein precipitate;

[0184] 3. Countercurrent washing process

[0185] The two-stage cleaning units are connected in series. The first stage is the material inlet, which uses the liquid phase obtained from the separation after the second stage cleaning. The second stage is the material outlet, which uses clean water.

[0186] 1) First wash: Add 3 times the volume of the liquid phase obtained from the second wash step to the protein precipitate obtained by acid precipitation. After stirring and washing at 120 r / min, discard the washing liquid and centrifuge (5000 r / min, 10 min) to separate the solid phase.

[0187] 2) Second wash: Add 2 times the volume of pure water to the solid phase after the first wash, stir and wash at 120 r / min, discard the washing liquid, and separate the wet protein.

[0188] 4. Enzymatic hydrolysis and neutralization process

[0189] 1) Enzymatic hydrolysis preparation: Put the washed wet protein into the enzymatic hydrolysis tank, add an appropriate amount of pure water to adjust the protein concentration to 15%, turn on the stirring (180r / min), raise the temperature inside the tank to 52℃, and add sodium hydroxide to adjust the pH value to 6.0;

[0190] 2) Addition and reaction of compound enzyme: Add 0.1wt% of food-grade phytase (activity 10000U / g) of the wet basis protein of the raw material, and enzymatically hydrolyze at 50℃ for 30 min; take samples every 10 min during the process to ensure the effect of enzymatic hydrolysis.

[0191] 3) Enzyme inactivation: After the enzymatic hydrolysis is completed, raise the temperature inside the tank to 85°C and keep it constant for 15 minutes to completely inactivate the phytase. Adjust the protein solution to neutral.

[0192] 5. Combined treatment of high-pressure nitrogen homogenization and high-temperature sterilization

[0193] 1) High-pressure nitrogen homogenization: The neutralized protein solution is sent into a high-pressure homogenizer, and food-grade nitrogen (purity ≥99.99%) is introduced at the same time. The nitrogen pressure is 20MPa, the homogenization pressure is controlled at 10MPa, and the homogenization temperature is 55℃. The homogenization process is carried out twice.

[0194] 2) High-temperature sterilization: The homogenized protein solution is sent to an ultra-high temperature instantaneous sterilizer (UHT), the sterilization temperature is controlled at 150℃, the holding time is 20s, and then it is put into a flash tank (80kpa) for rapid cooling to 65℃ to obtain soybean protein isolate.

[0195] Example 6

[0196] 1. Extraction process (each batch of soybean meal is of the same quality and origin)

[0197] 1) First batch of soybean meal extraction:

[0198] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add 15 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate soybean residue and protein slurry.

[0199] b. Second extraction: Add 15 times the weight of the soybean meal in soft water to the soybean residue, mix well, and extract at 25°C for 15 minutes to separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0200] c. Third extraction: Add 15 times the weight of soft water to the soybean residue obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min to separate the soybean residue and liquid phase.

[0201] 2) Second batch of soybean meal extraction:

[0202] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0203] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, extract at 25℃ for 15 minutes, and separate the solid phase and soybean milk liquid phase. The soybean residue is then subjected to the third extraction.

[0204] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min, and separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction will be performed.

[0205] 3) Third batch of soybean meal extraction:

[0206] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 25℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0207] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 25°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0208] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 25℃ for 15 min, and separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, no fourth extraction will be performed.

[0209] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation;

[0210] 2. Acid precipitation process

[0211] 1) Deactivation by acidification: Pump the protein slurry obtained in the extraction process into the acid precipitation tank, stir at 120 r / min, slowly add 1 mol / L hydrochloric acid solution, adjust the pH value of the protein slurry (soy milk) to the range of 2-3, and at the same time raise the temperature inside the tank to 60℃ and keep stirring for 30 min;

[0212] 2) Isoelectric point adjustment separation: After deactivation, use 1 mol / L sodium hydroxide solution to adjust the pH of the soy milk back to 4.5, reduce the stirring speed to 80 r / min, and let it stand for 20 min to allow the protein to precipitate fully; centrifuge at 6000 r / min for 25 min using a horizontal screw centrifuge, discard the supernatant, and separate the protein precipitate;

[0213] 3. Countercurrent washing process

[0214] The two-stage cleaning units are connected in series. The first stage is the material inlet, which uses the liquid phase obtained from the separation after the second stage cleaning. The second stage is the material outlet, which uses clean water.

[0215] 1) First wash: Add 3 times the volume of the liquid phase obtained from the second wash step to the protein precipitate obtained by acid precipitation. After stirring and washing at 120 r / min, discard the washing liquid and centrifuge (5000 r / min, 10 min) to separate the solid phase.

[0216] 2) Second wash: Add 2 times the volume of pure water to the solid phase after the first wash, stir and wash at 120 r / min, discard the washing liquid, and separate the wet protein.

[0217] 4. Enzymatic hydrolysis and neutralization process

[0218] 1) Enzymatic hydrolysis preparation: Put the washed wet protein into the enzymatic hydrolysis tank, add an appropriate amount of pure water to adjust the protein concentration to 15%, turn on the stirring (180r / min), raise the temperature inside the tank to 52℃, and add sodium hydroxide to adjust the pH value to 6.0;

[0219] 2) Addition and reaction of compound enzyme: Add 0.1wt% of food-grade phytase (activity 10000U / g) of the wet basis protein of the raw material, and enzymatically hydrolyze at 50℃ for 30 min; take samples every 10 min during the process to ensure the effect of enzymatic hydrolysis.

[0220] 3) Enzyme inactivation and neutralization: After the enzymatic hydrolysis is completed, raise the temperature inside the tank to 85°C and keep it constant for 15 minutes to completely inactivate the phytase and adjust the protein solution to neutral.

[0221] 5. Combined treatment of high-pressure nitrogen homogenization and high-temperature sterilization

[0222] 1) High-pressure nitrogen homogenization: The neutralized protein solution is sent into a high-pressure homogenizer, and food-grade nitrogen (purity ≥99.99%) is introduced at the same time. The nitrogen pressure is 20MPa, the homogenization pressure is controlled at 50MPa, and the homogenization temperature is 50℃. The homogenization process is carried out twice.

[0223] 2) High-temperature sterilization: The homogenized protein solution is sent to an ultra-high temperature instantaneous sterilizer (UHT), the sterilization temperature is controlled at 150℃, the holding time is 20s, and then it is put into a flash tank (80kpa) to be rapidly cooled to 60℃ to obtain soybean protein isolate.

[0224] Example 7

[0225] Except for the extraction step, which is performed according to the following method, the other steps are the same as in Example 1:

[0226] 1) First batch of soybean meal extraction:

[0227] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add 15 times its weight of soft water, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 15℃ for 15 min, and separate soybean residue and protein slurry.

[0228] b. Second extraction: Add 15 times the weight of the soybean meal in soft water to the soybean residue, mix well, extract at 15℃ for 15 minutes, and separate the soybean residue and liquid phase. The soybean residue is then subjected to a third extraction.

[0229] c. Third extraction: Add 15 times the weight of soft water to the soybean residue obtained from the second extraction, mix evenly, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 15℃ for 15 min to separate the soybean residue and liquid phase.

[0230] 2) Second batch of soybean meal extraction:

[0231] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 15℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0232] b. Second extraction: Add the liquid phase obtained from the third extraction during the first soybean meal extraction to the solid phase, mix evenly, and extract at 15°C for 15 minutes to separate the soybean residue solid phase and the soybean milk liquid phase. The solid phase is then subjected to a third extraction.

[0233] c. Three-stage extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 15℃ for 15 min to separate the solid phase and liquid phase.

[0234] 3) Third batch of soybean meal extraction:

[0235] a. First extraction: Pour the peeled and crushed soybean meal to 80 mesh into the first extraction tank, add the liquid phase obtained from the second extraction of the second batch of soybean meal, mix evenly, slowly stir at 150 r / min and add calcium hydroxide solution to adjust the pH value of the mixed slurry to 7.0-7.2, extract at 15℃ for 15 min, and separate the soybean residue solid phase and protein slurry.

[0236] b. Second extraction: Add the liquid phase obtained from the third extraction during the second batch of soybean meal extraction to the solid phase, mix evenly, and extract at 15°C for 15 minutes to separate soybean residue and soybean milk liquid phase. The soybean residue is then subjected to a third extraction.

[0237] c. Third extraction: Add 15 times the weight of soybean meal in soft water to the solid phase, mix well, and slowly add sodium hydroxide solution while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0. Extract at 15℃ for 15 min to separate the solid phase and liquid phase. If the protein content in the solid phase is less than or equal to 10 wt%, a fourth extraction is not performed.

[0238] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation;

[0239] Comparative Example 1

[0240] Except for the extraction step, the other steps are the same as in Example 1: For each batch of soybean meal extraction, the three extractions are as follows: add 15 times the weight of soft water to the solid phase obtained from the second extraction, mix evenly, slowly stir at 150 r / min and add sodium hydroxide solution to adjust the pH value of the mixed slurry to the range of 6.8-7.0, then add calcium hydroxide to adjust the pH value of the mixed slurry to 8.5-9.0, extract at 25℃ for 15 min, and separate the solid phase and liquid phase. The liquid phase is used for the second extraction, and the solid phase is transported to the soybean residue tank.

[0241] Comparative Example 2

[0242] Except for the extraction step, the other steps are the same as in Example 1: For each batch of soybean meal extraction, the first extraction is as follows: the peeled and crushed soybean meal to 80 mesh is poured into the first extraction tank, and 15 times the weight of the soybean meal is added to the liquid phase obtained from the second extraction separation. After mixing evenly, calcium hydroxide solution is slowly added while stirring at 150 r / min. The pH value of the mixed slurry is adjusted to 8.0-8.5, and the extraction is carried out at 25℃ for 15 min to separate the soybean residue solid phase and protein slurry.

[0243] Comparative Example 3

[0244] Except for the extraction step, which is performed in the following manner, the other steps are the same as in Example 1: the extraction temperature is 50°C for each batch of soybean meal.

[0245] Comparative Example 4

[0246] Except for the extraction step, which is performed according to the following method, the other steps are the same as in Example 1:

[0247] 1) First batch of soybean meal extraction: a) First extraction: Pour the peeled and pulverized soybean meal (80 mesh) into the first extraction tank, add 8 times its weight of soft water, mix well, and slowly add calcium hydroxide solution at 150 r / min to adjust the pH of the mixture to 7.0-7.2. Extract at 25℃ for 15 min, and separate soybean residue and protein slurry; b) Second extraction: Add 8 times its weight of soft water to the soybean residue, mix well, and extract at 25℃ for 15 min, separating soybean residue and liquid phase. The soybean residue is then subjected to a third extraction; c) Third extraction: Add 8 times its weight of soft water to the soybean residue obtained from the second extraction, mix well, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixture to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0, and extract at 25℃ for 15 min, separating soybean residue and liquid phase.

[0248] 2) Second batch soybean meal extraction: a) First extraction: Pour the peeled and pulverized soybean meal (80 mesh) into the first-stage extraction tank, add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction, mix evenly, and slowly add calcium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to 7.0-7.2. Extract at 25℃ for 15 min to separate the soybean residue solid phase and protein slurry; b) Second extraction: Add the liquid phase obtained from the third extraction during the first batch of soybean meal extraction to the solid phase, mix evenly, and extract at 25℃ for 15 min to separate the soybean residue solid phase and soybean milk liquid phase. The solid phase is then subjected to a third extraction; c) Third extraction: Add 8 times the weight of the soybean meal in soft water to the solid phase, mix evenly, and slowly add sodium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then add calcium hydroxide to adjust the pH to the range of 7.5-8.0, and extract at 25℃ for 15 min to separate the solid and liquid phases;

[0249] 3) Third batch soybean meal extraction: a) First extraction: Pour the peeled and pulverized soybean meal (80 mesh) into the first-stage extraction tank, add the liquid phase obtained from the second extraction during the second batch soybean meal extraction, mix well, and slowly add calcium hydroxide solution at 150 r / min to adjust the pH of the mixed slurry to 7.0-7.2. Extract at 25℃ for 15 min, separating the soybean residue solid phase and protein slurry; b) Second extraction: Add the liquid phase obtained from the third extraction during the second batch soybean meal extraction to the solid phase, mix well, and extract at 25℃ for 1 minute. After 5 minutes, the soybean residue and soybean milk liquid phases were separated. The soybean residue was then subjected to a third extraction. c. Third extraction: Soft water with a mass of 8 times that of soybean meal was added to the solid phase. After mixing evenly, sodium hydroxide solution was slowly added while stirring at 150 r / min to adjust the pH of the mixed slurry to the range of 6.8-7.0. Then, calcium hydroxide was added to adjust the pH to the range of 7.5-8.0. Extraction was carried out at 25℃ for 15 minutes. The solid phase and liquid phase were separated. The protein content in the solid phase was found to be less than or equal to 10 wt%. A fourth extraction was not performed.

[0250] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation.

[0251] Comparative Example 5

[0252] Except for step 5, which is carried out according to the following process, the remaining steps are the same as in Example 1: 5. High temperature sterilization combined treatment: High temperature sterilization: The protein liquid is sent into an ultra-high temperature instantaneous sterilizer (UHT), the sterilization temperature is controlled at 150°C, the holding time is 20s, and then it is put into a flash tank (80kpa) to be rapidly cooled to 65°C to obtain soybean protein isolate liquid.

[0253] Comparative Example 6

[0254] Except for the extraction step, which is performed according to the following method, the other steps are the same as in Example 1:

[0255] 1. Extraction process (each batch of soybean meal is of the same quality and from the same origin)

[0256] 1) First batch of soybean meal extraction:

[0257] a) First extraction: Pour the peeled and pulverized soybean meal (80 mesh) into the first extraction tank, add 15 times its weight of soft water, mix well, and slowly add sodium hydroxide solution while stirring at 150 rpm to adjust the pH of the mixture to 7.0-7.2. Extract at 25°C for 15 minutes to separate soybean residue and protein slurry. b) Second extraction: Add 15 times its weight of soft water to the soybean residue, mix well, and extract at 25°C for 15 minutes to separate soybean residue and liquid phase. The soybean residue is then subjected to a third extraction. c) Third extraction: Add 15 times its weight of soft water to the soybean residue obtained from the second extraction, mix well, and slowly add sodium hydroxide solution while stirring at 150 rpm to adjust the pH of the mixture to 6.8-7.0. Then add more sodium hydroxide to adjust the pH to 7.5-8.0, and extract at 25°C for 15 minutes to separate soybean residue and liquid phase.

[0258] 2) Second batch of soybean meal extraction:

[0259] a) First Extraction: Pour the peeled and pulverized soybean meal (80 mesh) into the first-stage extraction tank. Add the liquid phase obtained from the second extraction during the first batch of soybean meal extraction. Mix well, then slowly add sodium hydroxide solution at 150 rpm to adjust the pH of the mixture to 7.0-7.2. Extract at 25°C for 15 minutes to separate the soybean residue solid phase and the protein slurry. b) Second Extraction: Add the liquid phase obtained from the third extraction during the first batch of soybean meal extraction to the solid phase. Mix well, then extract at 25°C for 15 minutes to separate the soybean residue solid phase and the soybean milk liquid phase. The solid phase is then subjected to a third extraction. c) Third Extraction: Add 15 times the weight of the soybean meal in soft water to the solid phase. Mix well, then slowly add sodium hydroxide solution at 150 rpm to adjust the pH of the mixture to 6.8-7.0. Add more sodium hydroxide to adjust the pH to 7.5-8.0. Extract at 25°C for 15 minutes to separate the solid and liquid phases.

[0260] 3) Third batch of soybean meal extraction:

[0261] a) First Extraction: Peel and grind soybean meal to 80 mesh into the first-stage extraction tank. Add the liquid phase obtained from the second extraction of the second batch of soybean meal. Mix well and slowly add sodium hydroxide solution at 150 rpm to adjust the pH of the mixture to 7.0-7.2. Extract at 25°C for 15 minutes to separate the soybean residue solid phase and protein slurry. b) Second Extraction: Add the liquid phase obtained from the third extraction of the second batch of soybean meal to the solid phase. Mix well and extract at 25°C for 15 minutes to separate the soybean residue and soy milk liquid phase. The soybean residue is then subjected to a third extraction. c) Third Extraction: Add 15 times the weight of the soybean meal in soft water to the solid phase. Mix well and slowly add sodium hydroxide solution at 150 rpm to adjust the pH of the mixture to 6.8-7.0. Add more sodium hydroxide to adjust the pH to 7.5-8.0. Extract at 25°C for 15 minutes to separate the solid and liquid phases. If the protein content in the solid phase is less than or equal to 10 wt%, a fourth extraction is not performed.

[0262] 4) Take the protein slurry obtained from the first to third batches of soybean meal extraction, mix them together, and perform acid precipitation.

[0263] Comparative Example 7

[0264] 1. Extraction process

[0265] Peeled and pulverized soybean meal to 80 mesh was poured into an extraction tank, and soft water with 15 times the weight of the soybean meal was added. After mixing evenly, sodium hydroxide solution was slowly added while stirring at 150 r / min. The pH of the mixed slurry was adjusted to 7.0-7.2, and the mixture was extracted at 25℃ for 15 min to separate the soybean residue solid phase and protein slurry.

[0266] 2. Acid precipitation process

[0267] The protein slurry obtained from the extraction process was pumped into an acid precipitation tank and stirred at 120 r / min. A 1 mol / L hydrochloric acid solution was slowly added dropwise to adjust the pH of the protein slurry (soy milk) to 4.5. The stirring speed was reduced to 80 r / min, and the mixture was allowed to stand for 20 min to allow the protein to precipitate fully. The mixture was then centrifuged at 6000 r / min for 25 min using a horizontal screw centrifuge. The supernatant was discarded, and the protein precipitate was obtained.

[0268] 3. Neutralization process

[0269] Add the wet-based protein to the neutralization and dissolution tank, add an appropriate amount of pure water to adjust the protein concentration to 15%, turn on the stirrer (180 r / min), and adjust the pH value to 7.0;

[0270] 5. High-temperature sterilization treatment

[0271] The protein solution was fed into an ultra-high temperature instantaneous sterilizer (UHT), where the sterilization temperature was controlled at 150°C and the holding time was 20 seconds. Then it was put into a flash tank (80 kPa) for rapid cooling to 65°C to obtain soybean protein isolate.

[0272] Comparative Example 8

[0273] The acid precipitation step is performed as follows, with other steps being the same as in Example 1: the protein slurry obtained in the extraction process is pumped into the acid precipitation tank, stirred at 120 r / min, and 1 mol / L hydrochloric acid solution is slowly added dropwise to adjust the pH of the protein slurry (soy milk) to 4.5. The stirring speed is reduced to 80 r / min, and the mixture is allowed to stand for 20 min to allow the protein to precipitate fully. The mixture is then centrifuged at 6000 r / min for 25 min using a horizontal screw centrifuge, and the supernatant is discarded to separate the protein precipitate.

[0274] Comparative Example 9

[0275] Except for the countercurrent water washing method, the other steps are the same as in Example 1: Add 3 times the volume of pure water to the protein precipitate obtained by acid precipitation, stir and wash at 120 r / min, discard the washing liquid, and centrifuge (5000 r / min, 10 min) to separate the wet protein.

[0276] The protein content, trypsin inhibitor content, and phytic acid content of the soybean protein isolate prepared in each example and comparative example were tested, and the results are shown in Table 1 below.

[0277] Table 1: Detection results of various parameters of soybean protein isolate liquid

[0278] Protein content (dry basis) % Phytic acid (mg / g) Trypsin inhibitor (mg / g) Yield Sensory color Color value Example 1 94.5 9.0 1.3 45.1 cream 70.0 Example 2 95.5 8.5 1.5 43.2 cream 69.0 Example 3 95.3 7.5 1.0 43.5 cream 69.5 Example 4 94.8 7.5 1.1 44.0 pale yellow 60.5 Example 5 94.4 9.0 1.3 44.8 cream 70.1 Example 6 94.4 8.8 0.9 44.8 cream 69.7 Example 7 94.7 10.5 1.5 42.1 cream 71.1 Comparative Example 1 89.8 9.4 1.5 46.1 pale yellow 63.5 Comparative Example 2 90.2 10.2 1.4 46.0 pale yellow 61.1 Comparative Example 3 94.2 9.5 1.2 46.5 Slightly red 55.8 Comparative Example 4 90.5 15.5 2.2 41.3 cream 69.3 Comparative Example 5 94.5 9.0 3.2 45.0 cream 70.2 Comparative Example 6 94.4 18.6 1.2 46.0 pale yellow 64.3 Comparative Example 7 90.5 25.3 4.9 46.3 pale yellow 63.7 Comparative Example 8 94.5 17.1 3.1 45.0 cream 68.1 Comparative Example 9 93.0 12.0 2.0 45.5 cream 70.6

Claims

1. A method for extracting soybean protein or reducing the phytic acid content of soybean protein, characterized in that, The method includes the following steps: (1) The soybean raw material is divided into n batches at 15-35℃ and extracted, with each batch being extracted m times, where n is a positive integer greater than or equal to 2 and m is a positive integer greater than or equal to 3; (2) Mix the protein slurry containing soybean protein obtained from the first extraction and separation of each batch; wherein: For the first batch of extraction, water was used to extract the soybean raw material. For the second and subsequent batches, the liquid obtained from the previous batch was used to extract the soybean raw material. In the first batch of extraction, water was used to extract the soybean raw material for the first time, and the soybean residue was filtered out to obtain a protein slurry. Water was added to the soybean residue to continue the m-1 extraction. In the nth batch of extraction, the liquid phase obtained from the second extraction in the n-1th batch of extraction is used to perform the first extraction on the soybean raw material. The soybean residue is filtered out to obtain a protein slurry. The liquid phase obtained from the mth extraction in the n-1th batch of extraction is added to the soybean residue for the m-1st extraction. The liquid phase is filtered out. Water is added to the soybean residue for the mth extraction. During each batch of extraction, in the first extraction step, soluble calcium salt is added to adjust the pH to the range of 7.0-7.

2. In the m-th extraction step, soluble sodium salt or soluble potassium salt is added to adjust the pH to the range of 6.8-7.0, and then soluble calcium salt is added to adjust the pH to the range of 7.5-8.

0.

2. The soybean protein prepared by the method according to claim 1, characterized in that, The method includes one or more of the following features: The extraction temperature is 25-35℃ or 30-35℃; The soybean raw material is defatted soybean, including soybean meal and / or soybean cake; When extracting the nth batch, if the protein content of the soybean residue obtained in the mth extraction step is less than or equal to 10%, the (m+1)th batch extraction will not be performed; preferably, n is a positive integer greater than or equal to 3, such as n≥5, n≥10, n≥15, n≥20 or n≥50; preferably, m is a positive integer greater than or equal to 4, such as m≥5, m≥7, m≥9 or m≥10; For each batch of extraction, the extraction time in the first extraction step is greater than or equal to the extraction time in the second or m-th extraction step; preferably, in the first extraction step, the pH value is adjusted to be within the range of 7.0-7.2, and the extraction time is 10-20 minutes or 15-20 minutes; preferably, in the m-th extraction step, the pH value is adjusted to be within the range of 7.5-8.0, and the extraction time is 10-15 minutes. In the nth batch extraction step, the ratio of soybean raw material or soybean residue to added water is 1:15-1:

20. In the extraction step, stirring is performed to ensure uniform mixing; preferably, the stirring rate is 100-200 r / min or 150-200 r / min. The water used in the extraction step includes soft water, drinking water, mineral water, distilled water, or pure water, preferably pure water; The soluble calcium salts include calcium hydroxide and its aqueous solution; and The soluble sodium or potassium salt includes sodium hydroxide, potassium hydroxide, or an aqueous solution thereof.

3. A method for preparing soybean protein, characterized in that, The method includes the extraction step of the method as described in claim 1 or 2, and further includes the steps of acid precipitation, water washing, optional enzymatic hydrolysis, neutralization, nitrogen homogenization, optional sterilization, and optional drying to obtain the soy protein; wherein: The acid precipitation step includes: first adjusting the pH of the extracted protein slurry to the range of 2.0-3.0, then adjusting the pH of the protein slurry to the range of 4.5-5.0 to obtain a protein suspension, and then separating the protein precipitate. The washing step includes: washing the protein precipitate with 4-5 times the mass of the protein precipitate with water to obtain wet-based protein; The enzymatic hydrolysis step includes: enzymatically hydrolyzing the wet-based protein to obtain a protein solution; The neutralization step includes: adjusting the pH of the protein solution to neutral; The nitrogen-filling and homogenization step includes: filling the protein solution with nitrogen and homogenizing it in a sealed environment; Preferably, the soy protein has a protein content of 94.5 wt%, more preferably 94.5-96 wt% or 94.5-95.5 wt%.

4. The method as described in claim 3, characterized in that, In the water washing step, the protein precipitate is washed twice. In the first water washing step, 4-5 times the mass of the liquid phase obtained from the centrifugation step in the second water washing step is added to the protein precipitate. After washing, the liquid phase is filtered off to obtain the solid phase. In the second water washing step, 4-5 times the mass of water is added to the solid phase after the first water washing. After washing, the liquid phase is filtered off and the solid phase is obtained, which is the wet protein. Preferably, The protein precipitate is washed with water a times, where a is a positive integer greater than or equal to 3: In the first water washing step, 4-5 times the mass of the liquid phase obtained from the centrifugation step in the second water washing step is added to the protein precipitate. After washing, the liquid phase is filtered off to obtain the solid phase. In the a-1th water washing step, 4-5 times the mass of the liquid phase obtained in the fourth water washing step is added to the solid phase after the a-2th water washing step. After washing, the separated liquid phase is refluxed for the second water washing, and the separated solid phase is washed again, and so on in this cycle. In the a-th water washing step, soft water with a mass of 4-5 times is added to the solid phase after the a-1th water washing. After washing, the liquid phase is filtered out, and the separated solid phase is the wet-based protein. Preferably, a is a positive integer from 3 to 5.

5. The method as described in claim 3, characterized in that, The method includes one or more of the following features: In the acid precipitation step, acid is added to bring the pH of the protein slurry to the range of 2-3, and the temperature is maintained at 40-50°C for 30-60 minutes. Then, the pH of the protein slurry is adjusted to the range of 4.5-5.0 to obtain the protein suspension, and the protein precipitate is separated. The amount of enzyme added is 0.01-0.5 wt% of the substrate mass in the enzymatic hydrolysis reaction; The nitrogen purging pressure is 10-20 MPa, the homogenization pressure is 10-50 MPa, and the homogenization temperature is 50-55℃. The sterilization temperature is 150-170℃, maintained for 20-60 seconds; and The drying process includes freeze drying, vacuum drying, or spray drying.

6. Soy protein isolate prepared by any one of claims 3-5, or a composition containing the soy protein isolate.

7. The soy protein isolate or a composition containing the soy protein isolate as described in claim 6, characterized in that, The composition is a nutritional supplement, pharmaceutical excipient, health product, feed, quality improver, dairy substitute / plant-based food, baked / snack food, beverage / seasoning.

8. The soy protein isolate or a composition containing the soy protein isolate as described in claim 6 or 7, characterized in that, The composition is a meat product or aquatic product, such as sausage, ham, meatball, fish ball, luncheon meat; preferably, the composition is a dairy product or plant-based food, such as plant milk, high-protein soy milk, plant yogurt, plant meat patties, artificial meat; preferably, the composition is a baked good or snack food, such as bread, cake, protein bar, meal replacement powder, sports nutrition product; preferably, the composition is a beverage or condiment, such as functional beverage, soy sauce, seasoning sauce; preferably, the composition is feed; preferably, the composition is a nutrient or health product.

9. The soy protein isolate as claimed in claim 6, or a composition containing the soy protein isolate, characterized in that, The soy protein isolate is in powder, emulsion, or liquid form; and / or the composition containing soy protein isolate is in the form of an aqueous suspension, powder, solid, or semi-solid.

10. The application of the method according to any one of claims 1-5 in one or more of the following aspects: (1) preparing a composition containing soy products, (2) increasing the protein content in soy protein isolate, (3) decreasing the phytic acid content in soy protein isolate, (4) decreasing the trypsin inhibitor content in soy protein isolate, and (5) increasing the yield of soy protein isolate.

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