Low-denaturation low-phytic-acid plant protein and preparation method and application thereof

Abstract

The application discloses a kind of low denaturation low phytic acid plant protein and its preparation method and application.The method comprises the following steps: first, extract liquid is mixed with material, and after sufficient extraction, insoluble matter is removed by centrifugal separation and supernatant is collected.Then, membrane separation system is used to purify and concentrate protein.Subsequently, non-thermal sterilization treatment is carried out by hydrodynamic cavitation equipment, spray drying, so that low denaturation, low phytic acid soy protein product is obtained.The method has the advantages that the obtained product has high clarity and stability, and the solubility can reach 60% to 95% within a wide range of pH values.Furthermore, the protein has high purity, low beany flavor, and the phytic acid content is 0.3% to 0.7%.Compared with soy protein isolate prepared by heat sterilization treatment, the protein prepared by the application has lower denaturation degree, and can be applied to acidic food systems such as sports drinks and fruit juices, special medical use formula food, protein supplements and other easily digestible foods.

Description

Technical Field

[0001] This invention belongs to the field of high-value processing technology of plant protein, and relates to the field of membrane separation technology. Specifically, it relates to a method for preparing low-denatured, low-phytic acid soybean protein with the significance of dietary protein supplementation and its application. It can be applied to acidic beverages such as fully nutritious transparent sports drinks and fruit juices, or to fast-digestible protein in special medical foods to replace animal proteins such as demineralized whey powder, and belongs to the field of functional foods. Background Technology

[0002] Today, people are increasingly focused on the health and nutrition of food, leading to a greater demand for high-quality protein. Global population growth, environmental pressures from livestock farming, and overexploitation of marine resources are also forcing food manufacturers to seek environmentally friendly dietary protein sources. In contrast, plant-based proteins possess irreplaceable health benefits and resource advantages, thus attracting significant attention. Soybeans, as one of China's most important crops, not only rank first in both production and imports among legumes but are also a crucial raw material for oil processing and a source of dietary protein. Soy protein has significant application value in food processing. First, soy protein is rich in nutrients and is a source of high-quality protein, containing various essential amino acids to meet the body's nutritional needs. Second, soy protein possesses excellent functionality, enhancing the water retention, gelling, and emulsifying properties of food, improving taste and texture. It can serve as a plant-based substitute for meat products, increasing the plant-based protein content. Furthermore, soy protein can be used to create functional foods, making it particularly suitable as an ideal protein supplement for cancer patients and those with lactose intolerance. Therefore, soy protein has broad application potential in the food processing field.

[0003] To ensure that soy protein exhibits its corresponding functional properties, it needs to maintain its original molecular structure. However, under normal temperature (30–50°C) conditions, protein-rich slurries undergo various processing steps, leading to varying degrees of microbial growth. This is particularly true during large-scale production of soy protein isolate, where the microbial count often fails to meet relevant standards. As a typical food ingredient, commercially available soy protein requires heat sterilization to meet safety requirements. Heat treatment is an essential step in the food industry. Soy protein powder typically undergoes high-temperature flash evaporation to kill microorganisms, concentrate the protein slurry, remove some undesirable flavors, and deactivate anti-nutritional factors.

[0004] However, during high-temperature flash evaporation, protein polypeptide chains unfold, exposing hydrophobic groups. When the temperature returns to room temperature, the protein cannot recover its original structure. Under the influence of hydrophobic forces, the proteins aggregate to form insoluble aggregates. Disulfide bonds and hydrogen bonds also participate in the formation of these aggregates, leading to increased aggregate size and viscosity of the protein solution. This is the direct reason for the significant decrease in the solubility of commercial soy protein. Due to the highly hydrophobic surface of commercial soy protein, it is mostly present in a fully denatured or polymerized state. In this state, the assembly ability of soy protein is reduced, preventing it from fully realizing its functional properties in food systems. Currently, soy protein is only added to food as a nitrogen source, which severely limits its application value in plant-based foods.

[0005] Currently, many scholars have conducted extensive research on solubilization and modification technologies for soybean protein, hoping to improve its functional properties through chemical, enzymatic, and physical modifications. For example, CN112655812A utilizes ultra-high pressure homogenization combined with enzymatic modification to increase the solubility of commercial soybean protein to 85%. However, the pressure used in this process is as high as 100 MPa, making it difficult to apply in industrial production. This is because after heat treatment, most protein molecules become larger, forming a random structure that is difficult to degrade and separate using conventional physicochemical methods. Therefore, this process only transforms some insoluble large aggregates into smaller aggregates, while the protein remains in a completely denatured or polymerized state. To ensure that soybean protein meets my country's food hygiene standards and is economically viable, while simultaneously improving its solubility and related functional properties, it is necessary to mitigate the impact of heat treatment on the solubility and related functional properties of soybean protein. Summary of the Invention

[0006] Currently, the lack of a novel process for producing low-denaturation soy protein hinders its widespread use in plant-based foods. Conventional extraction processes result in soy protein containing high levels of anti-nutritional factors such as phytic acid, and commercially available soy protein often undergoes excessive heat denaturation, forming large, insoluble aggregates. To address these issues, this invention aims to provide a low-denaturation, low-phytic acid plant protein, its preparation method, and its applications. Specifically, it improves existing heat sterilization processes to produce a soy protein with superior functional properties, higher solubility, and self-assembly capabilities, while meeting the hygiene standards outlined in the National Food Safety Standard for Food Hygiene. Compared to existing soy protein production technologies, the method of this invention maintains the natural molecular structure of soy protein with a lower degree of denaturation.

[0007] Specifically, this invention relates to a modified soybean protein that exhibits extremely high solubility over a wide pH range, primarily characterized by low denaturation. The preparation method provided by this invention can reduce the degree of denaturation of soybean protein while simultaneously reducing the content of anti-nutritional factors.

[0008] The improvement made by this invention over the prior art is mainly reflected in replacing traditional thermal sterilization with hydraulic cavitation as a non-thermal sterilization technology. The hydraulic cavitation reactor used in this invention has a series of straight cylindrical sink holes of the same diameter. After the protein concentrate enters the chamber of the hydraulic cavitation reactor, it flows into the holes. However, under the centrifugal force of the rotor, the protein concentrate cannot reach the bottom of the hole but is thrown out to a certain depth; simultaneously, pressure is pumped into the hole, causing the protein concentrate to rotate and form local vortices within the hole. When the low pressure inside the hole decreases to the saturated vapor pressure of water, the water inside the hole rapidly changes from a liquid phase to a vapor phase, forming cavitation. Simultaneously, the water that has changed to a vapor phase is thrown from the bottom of the hole to the water-vapor interface under the action of centrifugal force, and the pressure rises from below the saturated vapor pressure to the saturated vapor pressure. Then, the vapor phase water reverts to a liquid phase and is thrown out of the sink hole by centrifugal force, while new water replenishes the hole, completing the water-vapor exchange process. This process generates millions of micro-cavitation bubbles, thereby increasing the contact area between the protein concentrate and the gas and solid. In the process of hydraulic cavitation, the impact of bursting air bubbles destroys microorganisms, achieving sterilization. When these bubbles burst, they generate shock waves; the powerful impact breaks the material into tiny particles, thus reducing the viscosity of the protein concentrate. This method is suitable for sterilizing high-viscosity materials and can produce low-denaturation commercial proteins.

[0009] On the other hand, non-thermal sterilization may result in issues such as anti-nutritional factors and unpleasant beany flavor. The "alkali dissolution and acid precipitation method" is a commonly used production process in my country, offering advantages such as simplicity, high extraction rate, and low production cost. However, this method cannot remove anti-nutritional factors, particularly phytic acid in soybeans. Phytic acid carries a strong charge and binds to proteins under a wide range of pH conditions, accumulating in soybean protein during the acid precipitation process. Reducing anti-nutritional factors and providing consumers with an excellent sensory experience are also issues that need to be addressed when applying soybean protein in special medical foods or sports nutrition foods.

[0010] The purpose of this invention is to replace heat sterilization with hydraulic cavitation treatment and apply it to the extraction of soybean protein. Simultaneously, membrane separation technology is used to recover the protein, reducing anti-nutritional factors in the plant protein during the extraction process. This method aims to solve the problems of unpleasant flavor and astringency in soybean protein under acidic conditions, while avoiding excessive denaturation that leads to a decline in protein functional properties. Therefore, this invention produces a low-denaturation, low-phytic acid soybean plant protein. This protein product can be widely used in the preparation of acidic food systems such as sports drinks and fruit juices, as well as easily digestible foods such as special medical purpose formulations and protein supplements.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] This invention provides a method for preparing low-denaturation, low-phytic acid plant protein, comprising the following steps:

[0013] (1) Add the extraction solution to the low-temperature defatted soybean meal powder at a material-to-liquid ratio of 1:10-15 (w / v), adjust the pH to the range of 7.0-8.0, stir and extract at room temperature to obtain protein slurry;

[0014] (2) Depending on the process conditions, the protein slurry in step (1) is maintained under acidic, neutral or alkaline conditions by adding a pH adjuster;

[0015] (3) Centrifuge the protein slurry obtained in step (2) to collect the supernatant, and filter it using an organic filter membrane to obtain crude protein extract;

[0016] (4) The crude protein extract obtained in step (3) is purified and concentrated by a membrane separation device to obtain a protein concentrate;

[0017] (5) After the protein concentrate obtained in step (4) is sterilized by non-thermal sterilization using a hydraulic cavitation device, it is dried to obtain powder, thus obtaining a plant protein product with low denaturation and low phytic acid.

[0018] Furthermore, the temperature of the low-temperature defatted soybean meal powder in step (1) is controlled at 15-25°C and passed through a 60-500 mesh sieve.

[0019] Further, the extract in step (1) is a sodium, potassium, calcium, or magnesium salt with a concentration of 0.1 to 0.8 mol / L.

[0020] Furthermore, the stirring extraction in step (1) lasts for 0.5 to 4 hours.

[0021] Further, the pH adjuster mentioned in step (2) is divided into an acidic adjuster and an alkaline adjuster, wherein the acidic adjuster is one or more of hydrochloric acid and citric acid, and the alkaline adjuster is one or more of sodium hydroxide, sodium bicarbonate and pentasodium phosphate; the protein slurry in step (1) is maintained under acidic conditions of 2.0 to 6.5, neutral conditions of 6.8 to 7.2, or alkaline conditions of 7.5 to 9.0 by the pH adjuster.

[0022] Furthermore, in step (3), the centrifugation speed is 3000g to 8000g and the centrifugation time is 10 to 20 minutes.

[0023] Furthermore, the filter membrane in step (3) is made of acid and alkali resistant polyethersulfone, and the pore size of the gauze used for filtration is 100-400 mesh.

[0024] Furthermore, the membrane separation system described in step (4) can be an ultrafiltration system, a reverse osmosis device, or a disc rotating solid-liquid separation membrane device.

[0025] Further, the specific operation of the cyclic purification and concentration in step (4) is as follows: the extract is subjected to cyclic ultrafiltration desalting between 20℃ and 30℃. When the volume of the retentate is 1 / 2 to 1 / 4 of the original volume, it is diluted with deionized water to the original volume. After repeating 3 to 8 times, it is finally concentrated to a solid content of 8% to 20%.

[0026] Furthermore, the conductivity of the protein concentrate described in step (4) should be less than 1500 μS / cm.

[0027] Furthermore, the hydraulic cavitation equipment described in step (5) includes a feed tank, a peristaltic pump, and a hydraulic cavitation generator. The feed inlet of the hydraulic cavitation generator is connected to the peristaltic pump, and the discharge outlet is connected to the feed tank. Temperature detectors are installed at both the discharge outlet and the feed inlet. The sample enters the hydraulic cavitation generator from the feed tank via the peristaltic pump for cavitation treatment and then flows back to the feed tank.

[0028] Furthermore, the non-thermal sterilization process using the hydraulic cavitation equipment described in step (5) takes 10 to 40 minutes.

[0029] Further, the drying method described in step (5) is freeze drying or spray drying. The temperature for freeze drying is -40 to -50°C, and the conditions for spray drying are an inlet temperature of 130 to 180°C and an outlet temperature of 70 to 90°C.

[0030] This invention provides a plant protein with low denaturation and low phytic acid content prepared by the above preparation method.

[0031] The plant protein with low denaturation and low phytic acid content provided by this invention has a purity of 90%–98%, a phytic acid content of 0.3%–0.6%, and a solubility of 60%–95% between pH 3.0 and 4.0. The phosphorus content of the plant protein with low denaturation and low phytic acid content provided by this invention is 0.4%–0.7%, and the percentage of hexanal relative peak area is less than 4%–12%.

[0032] The present invention also provides the application of the product prepared by the above-mentioned salt extraction-ultrafiltration separation equipment combined with hydraulic cavitation, namely low denatured low phytic acid soybean protein, as a dietary protein supplement in health foods, special medical foods or sports nutrition foods, such as transparent complete nutritional sports drinks, fruit juices and other acidic food systems, or special medical purpose formula foods, protein supplements and other fast-digesting foods.

[0033] Furthermore, the plant protein is dissolved in an acid-soluble beverage, resulting in a final protein concentration of 0.5% to 2%.

[0034] The "low denaturation" in the low-denaturation, low-phytic acid plant protein described in this invention refers to a low degree of protein denaturation, meaning that the natural molecular structure of soybean protein can be maintained relatively well overall.

[0035] In summary, the present invention has the following advantages and beneficial effects:

[0036] (1) The extraction conditions of the present invention are mild and the pH changes are small, which can maintain the natural conformation of soybean protein. The present invention is also applicable to basic scientific research.

[0037] (2) The implementation process of this invention is simple. It only requires controlling the ionic strength during the alkali dissolution process to remove phytic acid, avoiding the cumbersome process of multiple enzymatic hydrolysis in the traditional scheme, and can be widely used in the extraction of soybean protein.

[0038] (3) Under the action of salt ions, the binding of fishy substances such as hexanal and 1-octen-3-ol with protein is reduced, which can improve the flavor of soybean protein.

[0039] (4) Unlike traditional high-temperature acid hydrolysis and protease hydrolysis, which improve acid solubility, the processing technology of this invention does not alter the structure of the protein, and therefore does not increase astringency or bitterness. At the same time, the content of polyphenols can be reduced by ultrafiltration and salt, thus mitigating the source of astringency. The resulting protein product has higher purity, lower phytic acid content, and higher clarity.

[0040] (5) Membrane separation technology replaces the acid precipitation process, resulting in no whey wastewater, high protein recovery rate and complete composition, and superior nutritional value; membrane separation does not involve phase change process, consumes less energy in production conditions, and is more environmentally friendly and environmentally friendly in the production process.

[0041] (6) With the development of modern technology, the corrosion resistance of membrane equipment has been improved. With the improvement of membrane cleaning agents, ultrafiltration membrane cores are easy to clean, have a long service life, and the membrane fouling problem can be better solved; the process of this invention has high application value and is suitable for large-scale production.

[0042] (7) Soy protein isolate treated by the hydraulic cavitation process of the present invention has a low degree of denaturation and has better functional properties such as excellent solubility, thus obtaining a low-denaturation soy protein isolate powder. Attached Figure Description

[0043] Figure 1 Cluster-heatmap showing the effect of salt ion extraction on the content of volatile flavor compounds in proteins.

[0044] Figure 2 Comparison of the appearance of 1% protein solutions prepared for Comparative Example 2 and Example 1 under different pH conditions.

[0045] Figure 3Comparative images of the appearance of the high-calcium protein nutrient solutions prepared in Comparative Example 1 and Example 1 under different sterilization conditions. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited to the scope described in the embodiments.

[0047] Comparative Example 1

[0048] Low-temperature defatted soybean meal was pulverized and passed through a 60-mesh sieve. The sieved soybean powder was mixed with deionized water at a ratio of 1:10 (w / v). The pH was adjusted to 8.0, and the mixture was stirred at room temperature for 2 hours before centrifugation at 6000g for 20 minutes. The supernatant was collected, the pH was adjusted to 4.5, and the mixture was centrifuged again at 6000g for 20 minutes to obtain curd. The curd was mixed with deionized water at a ratio of 1:5 (w / v), reconstituted in water, and the pH was adjusted back to 7.0. Subsequently, the mixture was spray-dried to obtain soy protein isolate.

[0049] Comparative Example 2

[0050] Low-temperature defatted soybean meal was pulverized and passed through a 60-mesh sieve. The sieved soybean powder was mixed with deionized water at a ratio of 1:10 (w / v). The pH was adjusted to 8.0, and the mixture was stirred at room temperature for 2 hours before centrifugation at 6000g for 20 minutes. The supernatant was collected, the pH was adjusted to 4.5, and the mixture was centrifuged again at 6000g for 20 minutes to collect the curd. The curd was mixed with deionized water at a ratio of 1:10 (w / v), and after complete reconstitution, the pH was adjusted to 5.5. 5.0 g / L phytase (enzyme activity 5000 u / g) was added, and the mixture was placed in a water bath at 50–55℃ for 30 minutes. After the enzymatic hydrolysis reaction was completed, the pH of the mixture was adjusted to 7.0, and then spray-dried to obtain soy protein isolate.

[0051] Comparative Example 3

[0052] (1) Add deionized water to low-temperature defatted soybean meal powder at a ratio of 1:10 (w / v), adjust the pH, stir at 25°C for 2 hours, centrifuge at 3000g for 20 minutes, and then filter with gauze to remove soybean residue to obtain crude soybean protein extract.

[0053] (2) The extract was kept at pH=8 by adding a pH adjuster.

[0054] (3) After diluting the extract by 1 time, filter it using a 0.45um organic filter membrane to obtain the diluted crude soybean protein extract.

[0055] (4) The extract is subjected to circulating ultrafiltration desalination at 20℃~30℃. When the volume of the retentate is 1 / 2 to 1 / 4 of the original volume, it is diluted with deionized water to the original volume. After repeating 3 to 8 times, the volume is concentrated to 1 / 8 of the original volume in the last cycle.

[0056] (5) After 20 minutes of hydraulic cavitation treatment, the powder is obtained by spray drying, which is the low-denatured soybean protein product.

[0057] Example 1

[0058] The preparation method of low-denatured, low-phytic acid soybean protein is as follows:

[0059] (1) Add 0.2 mol / L NaCl extract to low-temperature defatted soybean meal powder at a material-to-liquid ratio of 1:10 (w / v), adjust the pH, stir at room temperature for 2 hours, centrifuge at 3000g for 20 minutes, and then filter with gauze to remove soybean residue to obtain crude soybean protein extract.

[0060] (2) The extract was kept at pH=8 by adding a pH adjuster.

[0061] (3) After diluting the extract by 1 time, filter it using a 0.45um organic filter membrane to obtain the diluted crude soybean protein extract.

[0062] (4) The extract is subjected to circulating ultrafiltration desalination at 20℃~30℃. When the volume of the retentate is 1 / 2 to 1 / 4 of the original volume, it is diluted with deionized water to the original volume. After repeating 3 to 8 times, the volume is concentrated to 1 / 8 of the original volume in the last cycle.

[0063] (5) The powder is obtained by spray drying after 20 minutes of hydraulic cavitation treatment. It is a low-denatured and low-phytic acid soybean protein product.

[0064] Example 2

[0065] The only difference from Example 1 is that in step (1), 0.4 mol / L NaCl extract is added to the low-temperature defatted soybean meal powder. All other steps and conditions are the same, and a soybean protein product with low denaturation and low phytic acid is prepared.

[0066] Example 3

[0067] The only difference from Example 1 is that in step (1), 0.6 mol / L NaCl extract is added to the low-temperature defatted soybean meal powder. All other steps and conditions are the same, and a soybean protein product with low denaturation and low phytic acid is prepared.

[0068] Example 4

[0069] The only difference from Example 1 is that the NaCl extraction solution was extracted in step (5) by hydraulic cavitation treatment for 10 minutes. All other steps and conditions were the same, and a low-denatured, low-phytic acid soybean protein product was prepared.

[0070] Example 5

[0071] The only difference from Example 1 is that the hydrocavitation treatment in step (5) is performed for 40 minutes with NaCl extraction of the extract. All other steps and conditions are the same, and a low-denatured, low-phytic acid soybean protein product is prepared.

[0072] Example 6

[0073] The only difference from Example 1 is that in step (1), 0.2 mol / L CaCl2 extract is added to the low-temperature defatted soybean meal powder. All other steps and conditions are the same, and a soybean protein product with low denaturation and low phytic acid is prepared.

[0074] Example 7

[0075] The only difference from Example 1 is that in step (1), 0.2 mol / L Na2SO4 extract is added to the low-temperature defatted soybean meal powder. All other steps and conditions are the same, and a soybean protein product with low denaturation and low phytic acid is prepared.

[0076] The following are the product property determination and analysis methods involved in this invention:

[0077] 1. Determination of protein content: Refer to the Kjeldahl method in the national standard GB 5009.5-2016 "Determination of Protein in Food".

[0078] 2. Determination of phytic acid content:

[0079] Sample extraction solution: Dissolve 0.25g of sample in 10mL of hydrochloric acid (1.2% w / w), extract for 2h, centrifuge at 10000g for 15min, and then take the supernatant and dilute to 10mL.

[0080] Digestion of total phosphorus: Take 1 mL of sample extract, add 1.5 mL of concentrated nitric acid and 0.5 mL of concentrated sulfuric acid, and digest using a graphite digester until the mixture is clear and colorless (using a temperature curve of 200℃ for 30 min, 320℃ for 80 min). After cooling, add deionized water, heat in a boiling water bath for 15 min, cool, and bring the volume to 25 mL. Record this as extract A.

[0081] Digestion of inorganic phosphorus: Take 5 mL of sample extract, add 5 mL of hydrochloric acid (1.2% w / w) and 6 mL of ferric chloride solution (each liter of this solution contains 2 g of ferric chloride hexahydrate and 16.3 mL of concentrated hydrochloric acid), mix well, and incubate at 97℃ for 2 h. Cool to room temperature and centrifuge to precipitate. Then take 4 mL of the supernatant after centrifugation and perform the same digestion steps (add 2.5 mL of concentrated nitric acid and 1.0 mL of concentrated sulfuric acid), heat, and make up to volume. Record this as extract B.

[0082] Colorimetric determination: Take 1 mL of extract A, add 3 mL of deionized water and 4 mL of colorimetric reagent (containing 10 mL of 10% ascorbic acid, 10 mL of 2.5% ammonium molybdate solution, 10 mL of 3M sulfuric acid and 20 mL of deionized water), incubate at 37℃ for 90 min, cool to room temperature, and measure the absorbance at 820 nm. Repeat the same method with 4 mL of extract B, add 4 mL of colorimetric reagent, and measure the absorbance. Use potassium dihydrogen phosphate (0–10 μg / mL) as a standard curve to calculate the total phosphorus and inorganic phosphorus content.

[0083] Calculation of phytic acid content: Subtract the total phosphorus content from the inorganic phosphorus content to obtain the organic phosphorus mass, then multiply by 3.546 to calculate the phytic acid content in the sample.

[0084] 3. Determination of protein solubility: The protein sample was prepared into a 1% (w / v) dispersion using deionized water. The protein content in the sample supernatant was calculated using the Lowry method. Protein solubility is the ratio of the protein content in the supernatant to the protein content in the original dispersion (before centrifugation).

[0085] 4. Analysis of Soybean Protein Denaturation: A certain mass of soy protein isolate was weighed and added to an aluminum crucible. Deionized water was added to prepare a protein dispersion system with a solid content of 20%. The crucible was pressed, sealed, and stored overnight to allow the protein to fully hydrate. The crucible was then placed in a differential scanning calorimeter for analysis. After the sample stabilized at 25℃ for 1 min, the temperature was increased to 120℃ at a rate of 5℃ / min, and the change in heat of the sample during this process was monitored. The denaturation temperature (T0) of different protein components was analyzed using TA Universal Analysis software. peak ) and denaturation enthalpy (ΔH).

[0086] 5. Analysis of volatile flavor compounds: Prepare a 10% protein solution, fully swell it, adjust the pH to 7.0, take 5 mL of the dispersion and put it into a 15 mL headspace vial, add 2-methyl-3-heptanone (0.0816 μg / mL) as an internal standard, seal the vial, equilibrate in a 50℃ water bath for 10 min, insert a solid phase microextraction fiber (50 / 30 μm CAR / DVB / PDMS) for headspace extraction for 30 min, desorb at 230℃ for 3 min, and then analyze it on the instrument.

[0087] The chromatographic column used was an Rtx-wax (30m × 0.25mm × 0.25μm) capillary column. High-purity He was used as the carrier gas at a flow rate of 1.0 mL / min. The initial column temperature was 40℃, held for 1 min, then increased to 100℃ at 3℃ / min, held for 5 min, then increased to 180℃ at 8℃ / min, and finally increased to 220℃ at 10℃ / min. The injection port temperature was 230℃, using splitless mode. The ion source was 70 eV at 230℃, and the mass acquisition range was 35-350 amu. Data were retrieved from the NIST 11S mass spectrometry library. R was used to process the data, and the clustering-heatmap results were used for visualization analysis.

[0088] 6. Analysis of the sterilization effect of using hydraulic cavitation treatment instead of thermal sterilization:

[0089] This experiment aimed to compare the effects of different hydraulic cavitation treatment times on the survival rate of *E. coli* in soy protein isolate. First, *E. coli* cultured to the logarithmic growth phase was added to a 10% soy protein isolate suspension. Next, the mixture was poured into a feed tank for hydraulic cavitation treatment, with treatment times set to 0, 10, 20, and 40 minutes. Notably, when the treatment time was 0 minutes, only the peristaltic pump was activated, without turning on the hydraulic cavitation generator. The colony count in the treated protein suspension was measured using the dilution plating method, and the total colony count obtained after 0 minutes of hydraulic cavitation treatment was used as the initial bacterial concentration of the soy protein isolate suspension to calculate the *E. coli* survival rate at each treatment time.

[0090] The experimental results are shown in Table 1. First, after 10 minutes of hydraulic cavitation treatment, the total bacterial count was 1.33 × 10⁻⁶. 6 The CFU / mL count decreased to 76.0% of the initial bacterial concentration. Subsequently, after 20 minutes of treatment, the total bacterial count decreased to 2.73 × 10⁻⁶. 3 The CFU / mL concentration was only 0.2% of the initial concentration. Finally, after 40 minutes of treatment, the total colony count further decreased to 1.50 × 10⁻⁶. 3 The CFU / mL concentration was only 0.1% of the initial bacterial concentration. This indicates that the survival rate of *E. coli* gradually decreased with prolonged hydraulic cavitation treatment time, reaching a sterilization rate of 99.9% after 40 minutes of treatment.

[0091] It is worth noting that, according to the National Food Safety Standard for Food Hygiene, the microbial testing standard for low-denatured soy protein powder should be ≤5.0×10⁻⁶. 4 CFU / g. As shown in Table 1, the total bacterial count after 20 and 40 minutes of hydraulic cavitation treatment met this hygiene standard. Therefore, it can be concluded that hydraulic cavitation treatment for more than 20 minutes can effectively replace traditional heat sterilization methods.

[0092] This phenomenon can be explained by the principle and mechanism of hydraulic cavitation. Hydraulic cavitation uses the shearing action of high-pressure water to disrupt the structure of protein molecules, thereby reducing the survival rate of *E. coli*. The cell membrane and cell wall of *E. coli* are affected by the shear force applied by hydraulic cavitation, leading to structural damage and loss of function, ultimately resulting in cell death. As the treatment time increases, the shear force intensifies, significantly reducing the survival rate of *E. coli*. After 40 minutes of treatment, the total bacterial count was only 0.1% of the initial bacterial concentration, indicating that hydraulic cavitation has a highly efficient bactericidal effect.

[0093] In conclusion, the results of this experiment show that hydraulic cavitation treatment can significantly reduce the survival rate of *E. coli* in soy protein isolate. Especially when the treatment time reaches 20 minutes or more, it meets relevant hygiene standards, proving that hydraulic cavitation treatment can replace traditional heat sterilization methods. This research provides a useful reference for the safety and quality of soy protein and offers the industry a more efficient and feasible method for bacterial colony control.

[0094] Table 1. Colony count and survival rate of protein suspension treated by hydraulic cavitation

[0095]

[0096] 7. The degree of denaturation of soy protein isolate was tested using a differential scanning calorimeter:

[0097] As shown in Table 2, the denaturation temperature of the 7S component of the soy protein isolate prepared in Comparative Example 3, Examples 1, 4, and 5 was approximately 78°C, while the denaturation temperature of the 11S component was approximately 96°C. These results indicate that different preparation methods did not significantly affect the denaturation temperature of the same component of the soy protein isolate. Furthermore, the denaturation enthalpy of the 7S component of the soy protein isolate prepared in Comparative Example 3 was approximately 1.75 ± 0.39 J / g, while the denaturation enthalpy of the same component of the soy protein isolate prepared in Examples 1, 4, and 5 was lower. In other words, hydraulic cavitation treatment leads to partial denaturation of the 7S component, and the degree of denaturation increases with increasing treatment time. In Example 5, the 7S component of the soy protein isolate was completely denatured after 40 minutes of hydraulic cavitation treatment.

[0098] This phenomenon is likely due to the characteristics of hydraulic cavitation. Hydraulic cavitation is a method of treating protein solutions using the shearing action of high-pressure water flow. This process causes changes in the protein molecular structure, particularly the denaturation of the 7S component. Because of the high shear force applied during hydraulic cavitation, prolonged cavitation disrupts the internal interactions of protein molecules, leading to structural changes and resulting in partial or complete denaturation. The low-denaturation protein proposed in this invention refers to the overall denaturation degree of the protein, including both the 7S and 11S components. After 40 minutes of hydraulic cavitation treatment, although the 7S component of the protein is completely denatured, the 11S component remains undenatured, thus meeting the definition of a low-denaturation protein.

[0099] On the other hand, extending the processing time also has a greater impact on protein molecules. With increasing processing time, the shear force and degree of disturbance applied during hydraulic cavitation also increase, thus gradually increasing the degree of protein denaturation. In Example 5, the processing time reached 40 minutes, and the shear force caused complete denaturation of the 7S component of soy protein isolate. In summary, the experimental results show that using a high-salt extract combined with hydraulic cavitation treatment to prepare soy protein isolate can reduce the denaturation enthalpy of the 7S component, and the degree of denaturation increases with the extension of hydraulic cavitation treatment time. To obtain low-denaturation soy protein isolate, the cavitation treatment time needs to be strictly controlled within a suitable range; otherwise, excessive time will lead to protein denaturation. These results are of great significance for the application and further research of soy protein isolate.

[0100] Table 2. Differential Scanning Calorimetry Results of Soy Protein Isolate

[0101]

[0102] Table 3. Basic physicochemical properties of Comparative Examples 1, 2, and 3 and Examples 1, 2, and 3

[0103]

[0104] Calculations showed that Examples 1, 2, and 3, using high-salt extract solutions, exhibited a series of advantages in soybean protein extraction compared to the control group using other methods. Firstly, Examples 1, 2, and 3 achieved higher protein purity. The high-salt extract solution accelerated protein dissolution and separation by providing suitable environmental conditions, thereby removing other impurities and non-protein substances from the solution. This effectively improved protein purity, providing a reliable foundation for subsequent applications and further research.

[0105] Furthermore, Example 3 using a high-salt extract and Comparative Example 2 treated with phytase demonstrated comparable effectiveness in phytate removal to phytase treatment. Phytic acid is a common anti-nutritional factor in soybean protein, and its presence limits protein bioavailability. Phytase treatment is a commonly used method that can effectively degrade phytates. However, phytase treatment requires additional operations and costs, while Example 3 using a high-salt extract achieves similar degradation effects in phytate removal to phytase treatment, thus simplifying the operation and reducing costs.

[0106] In addition, Figure 1 The results also provide further evidence supporting Examples 1, 2, and 3 for the high-salt extracts. GC-MS was used to determine the peak area changes of flavor compounds in the extracts under different ionic intensities, and cluster-heatmaps were plotted to obtain indices of inter-group differences, thus visualizing the data. (See Appendix...) Figure 1 The results showed that the main undesirable flavor compounds, such as hexanal, 1-octen-3-ol, and 2-pentylfuran, were present at relatively low concentrations at a 0.2 mol / L extract concentration. Overall, higher ionic strength (0.4–0.6 mol / L) reduced the adsorption of hexanal and 1-octen-3-ol, and promoted the release of flavor compounds, which is beneficial for reducing their content through subsequent heat treatment. This indicates that using a high-salt extract can effectively inhibit the formation of fishy flavor compounds and improve the taste and quality of soybean protein.

[0107] In summary, high-salt extracts offer multiple advantages in soybean protein extraction. They can improve protein purity, remove phytate with comparable efficiency to phytase, and reduce the content of characteristic fishy flavor compounds. These advantages make high-salt extracts a viable method for soybean protein extraction with broad application prospects.

[0108] Table 4. Solubility (%) of Comparative Examples 1 and 2 and Examples 1, 7, and 8

[0109]

[0110] Calculations, as shown in Table 4, indicate that under pH conditions of 7.0, 4.5, and 3.5, Example 1 exhibits superior solubility compared to Comparative Examples 1 and 2. Particularly near the isoelectric point, the solubility of Example 1 is three times that of Comparative Examples 1 and 2. (See Appendix...) Figure 2It can also be seen that Example 1 exhibits excellent clarity at pH=3.5 and pH=7.0. This is likely because, during the process of using ultrafiltration membrane separation instead of acid precipitation to recover the protein, more acid-soluble components are recovered, leading to increased solubility. Similar results were also found in Examples 7 and 8. The experimental results indicate that controlling ionic strength is key to obtaining low-denaturation, low-phytate plant proteins, and the selection of ion types in this invention has a certain universality and regularity.

[0111] Compared to Comparative Example 2, which was treated with phytase, the soybean protein obtained using salt extraction combined with membrane separation technology exhibited better hydration capacity and solubility under neutral conditions. Soybean protein is a typical colloidal particle, undergoing physicochemical processes such as acidification and centrifugation during extraction. Within the isoelectric point range of pH 4–5, the protein has a net charge of 0, eliminating the repulsive force and leading to the formation of primary particles with a size of 0.1–0.3 μm. Subsequently, during centrifugation, the concentration of primary particles increases, forming large aggregates with a size of 1–50 μm. The lower the charge, the greater the degree of aggregation.

[0112] When the pH of the centrifuged curd is adjusted back to neutral, some aggregates remain in a large particle state, requiring a longer time to deagglomerate into primary aggregates or the natural structure of soy protein. If misfolding occurs during pH adjustment, the protein solubility decreases. Therefore, Comparative Examples 1 and 2 show lower solubility than the examples recovered using the salt extraction combined with membrane separation method.

[0113] Application example: Preparing high-calcium nutrient solution using low-denatured, low-phytic acid soybean protein.

[0114] Because soy protein is sensitive to divalent cations such as calcium, its addition to foods rich in minerals (especially alkali metals) often leads to aggregation, forming flocculation and precipitation, thus affecting food quality. This problem limits the application of soy protein in special functional products such as medical clinical nutrition products and liquid foods. Although milk protein is the mainstream raw material for clinical nutrition solutions and has a higher tolerance to calcium, remaining stable even in systems with a calcium content of 100 mg / mL, it is expensive, especially casein, which has a high phosphorus content, making it unsuitable for patients with impaired kidney function, infants or the elderly with poor metabolic capacity, and those recovering from surgery. Soy protein has a digestibility and absorption rate comparable to that of milk protein and is inexpensive. Therefore, this example proposes a strategy to replace animal protein with low-denatured, low-phytic acid soy protein.

[0115] The formula for the nutritional supplement is as follows: Weigh 4 grams of soybean protein sample, 16 grams of maltodextrin, 0.2 grams of sodium citrate and 330 milligrams of anhydrous calcium chloride, add 75.7 grams of water and stir well. Adjust the pH to 3.6 with 2 mol / L citric acid, then add 4 grams of corn oil and homogenize for 2 minutes. Then, homogenize the mixture twice under a pressure of 25-50 MPa. Finally, sterilize the mixture in an autoclave at 121°C and 0.1 MPa for 15 minutes, or heat it at 100°C for 30 minutes.

[0116] Under acidic conditions, the growth of most microorganisms is limited; therefore, sterilization under mild conditions can effectively achieve commercial sterilization results. Typically, soybean protein tends to lose its electrostatic shielding effect and aggregate near its isoelectric point. Based on this, as shown in the attached... Figure 3 As shown, the nutrient solutions obtained in Comparative Example 1 and Example 1, which exhibit low denaturation and high acid solubility, both demonstrate excellent stability under acidic conditions. The effects of different sterilization methods on subsequent stability were further investigated, as shown in the attached figure. Figure 3 As shown, the high-calcium nutrient solution prepared using the sample from Example 1 was sterilized at 95℃ for 30 minutes or at 121℃ for 20 minutes. Using the vial-turning method, it was found that after sterilization, the sample did not exhibit stratification, whey separation, oil-water separation, or protein flocculation; it remained liquid, with a stable system and good fluidity. In contrast, the high-calcium nutrient solution prepared in Comparative Example 1 showed increased viscosity after sterilization, essentially becoming a semi-solid gel, which is detrimental to the sensory quality of liquid food.

[0117] Phytic acid is highly negatively charged, exhibiting varying degrees of charge across a wide pH range. This readily induces stronger hydrophobic interactions and electrostatic repulsion in proteins during gelation. Therefore, under acidic conditions, the gelling ability of proteins is enhanced, which is detrimental to high-protein liquid foods. However, the phytic acid content in the examples provided in this invention is low (see Table 3), and Example 1 remained stable in a system with a calcium ion content of 330 mg / 100 mL. This is equivalent to a daily intake of 250 mL, meeting the recommended daily calcium intake of 800 mg for adults in the Chinese Nutrition Society's "Dietary Reference Intakes for Chinese Residents." Therefore, this invention has broader application value.

[0118] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be noted that any improvements made in accordance with the principles of the present invention are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a plant protein with low denaturation and low phytic acid content, characterized in that, Includes the following steps: (1) Add 0.2 mol / L NaCl extraction solution to low-temperature defatted soybean meal powder at a material-to-liquid ratio of 1:10 w / v, adjust the pH, stir at room temperature for 2 hours, and then add 3000 g Centrifuge for 20 minutes, then filter with gauze to remove soybean residue and obtain crude soybean protein extract; (2) The extract was kept at pH=8 by adding a pH adjuster; (3) After diluting the extract by 1 time, filter it using a 0.45 μm organic filter membrane to obtain the diluted crude soybean protein extract; (4) The extract is subjected to circulating ultrafiltration desalination at 20℃~30℃. When the volume of the retentate is 1 / 2~1 / 4 of the original volume, it is diluted with deionized water to the original volume. This process is repeated 3~8 times. Finally, the volume is concentrated to 1 / 8 of the original volume. (5) After 20 minutes of hydraulic cavitation treatment, the powder is obtained by spray drying, which yields a low-denatured and low-phytic acid soybean protein product.

2. The low-denatured, low-phytate plant protein prepared by the preparation method according to claim 1.

3. The application of the low-denatured, low-phytate plant protein as described in claim 2 as a dietary protein supplement in health foods.

4. The application of the low-denatured, low-phytate plant protein of claim 2 as a dietary protein supplement in special medical foods.

5. The application of the low-denatured, low-phytate plant protein of claim 2 as a dietary protein supplement in sports nutrition foods.

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