Preparation method of high-whiteness and low-odor soybean protein isolate

Abstract

The present application belongs to the technical field of soybean deep processing and food plant protein refining and modification, and discloses a preparation method of high-whiteness and low-odor soybean protein isolate. After low-temperature defatted soybean meal is subjected to alkali leaching and deslagging, the clarified protein leaching solution is subjected to 110-115 DEG C, 6-15 s tub instantaneous heat treatment and rapid cooling; first-stage acid precipitation at pH 4.4-4.6 and second-stage acid precipitation at pH 3.7-4.0 are sequentially performed, and main precipitated protein and supernatant precipitated protein are obtained respectively. The former is washed with water at room temperature for 2 times, and the latter is washed with 12-16 DEG C, 3-5 stage countercurrent water; after the two types of wet proteins are washed, they are compounded according to dry basis mass fraction, and after slurry is adjusted, protein glutamine enzyme deamidation is performed, and after heat inactivation, kappa-carrageenan is added for wet compounding, and then homogenization, concentration and spray drying are performed to obtain the finished product. The present application takes into account protein recovery rate, whiteness, low odor and solubility, emulsification and gelation performance, and is suitable for continuous production.

Description

Technical Field

[0001] This invention belongs to the technical field of soybean deep processing and food-grade plant protein refining and modification. Specifically, it relates to a method for preparing high-whiteness, low-odor soybean protein isolate using low-temperature defatted soybean meal as raw material, and involving sequential liquid-phase instantaneous enzyme inactivation, two-stage acid precipitation with solid-liquid separation boundary, differentiated water washing of two types of recycled proteins, controlled aggregation according to dry basis ratio, and stepwise compounding of protein glutaminase and κ-carrageenan on a wet production line (κ represents the κ subtype of carrageenan). Background Technology

[0002] Soy protein isolate is typically produced from low-temperature defatted soybean meal through steps such as alkaline soaking, solid-liquid separation, acid precipitation, neutralization, and drying. In industrial production, whiteness, beany flavor, protein recovery rate, and functional properties such as dissolution, emulsification, and gelation are all interconnected. Improving the recovery rate often requires extending the wet processing or adding recovery steps; however, a longer liquid-phase residence time can easily increase oxidative flavor and pigment entrainment. While enhanced washing is beneficial for removing impurities, it may also increase water consumption and protein loss.

[0003] Existing processes have employed various methods to improve product performance, including short-time high-temperature enzyme inactivation, stepwise acid precipitation, multi-stage washing, enzymatic modification, and protein-polysaccharide complexation. However, practical challenges remain in the coordination between different processes. Particularly when recovering protein from the supernatant after primary acid precipitation, the secondary recovered protein often contains a significant amount of soluble impurities, pigments, and aldehyde flavor compounds. If mixed too early with the main precipitated protein, subsequent purification based on the differences between the two types of protein becomes difficult.

[0004] Furthermore, protein glutaminase modification and κ-carrageenan composites have different requirements for material state. When added simultaneously to high-solids protein slurry, the thickening effect of polysaccharides may affect the contact between the enzyme and the protein substrate, and is also not conducive to the stable control of the reaction endpoint. Therefore, a preparation method is needed that can improve protein recovery while taking into account color, flavor, and functionality, and can be continuously implemented on existing soy protein isolate production lines. Summary of the Invention

[0005] The technical problem to be solved by the present invention The technical problem this invention aims to solve is to balance the yield improvement and the stability of color, flavor, and functionality brought about by secondary recovery while continuing to recover protein from the supernatant after primary acid precipitation. To this end, oxidation needs to be controlled before acid precipitation, a clear solid-liquid separation boundary needs to be maintained between the two acid precipitation stages, the two types of precipitates need to be refined separately, and after washing with water, they need to be compounded according to a dry basis ratio. Then, enzyme modification, enzyme inactivation, and wet compounding with κ-carrageenan are sequentially completed, enabling the entire process to be continuously implemented on existing soy protein isolate production lines.

[0006] Technical solution To address the aforementioned problems, this invention sets up each process step according to the actual flow direction of the wet material, allowing the material state formed in the previous step to directly connect to the next step. The method sequentially includes alkaline leaching and tubular instantaneous heat treatment, two-stage acid precipitation and diversion, differentiated water washing, dry-based compounding, protein glutaminase modification, κ-carrageenan compounding after heat inactivation of the enzyme, and homogenization, concentration, and spray drying. Details are as follows.

[0007] S1. First, the low-temperature defatted soybean meal is alkali-soaked to remove the soybean residue, resulting in a clarified protein extract. The clarified protein extract enters a tubular instantaneous heat treatment section, where it is maintained at 110–115°C for 6–15 seconds, followed immediately by a continuous heat exchange or vacuum flash cooling section. This step is placed before the two-stage acid precipitation to reduce the activity of lipoxygenases before the subsequent material residence time increases, allowing the subsequent water washing to primarily remove existing impurities and reduce the formation of new oxidative flavors during the treatment process. Preferably, the mass ratio of soybean meal to water is 1:9, and the pH of the system is adjusted to 7.1, where pH is used to characterize the acidity or alkalinity of the system. Extraction is performed at 48°C for 30 minutes; after the instantaneous heat treatment, the liquid is rapidly cooled to 42°C.

[0008] S2 involves first-stage acid precipitation of the enzyme-inactivated protein extract, adjusting the pH to 4.4–4.6 to form a primary precipitate of the main protein and completing the first solid-liquid separation. After the first solid-liquid separation, the main precipitated protein cake and the primary supernatant are sent to different feed lines; only the primary supernatant is further adjusted to pH 3.7–4.0 for secondary acid precipitation, completing the second solid-liquid separation. In this way, the secondary acid precipitation recovers not "reprecipitate" mixed with the main precipitate, but rather protein precipitated from the supernatant with a clearly defined source, providing a process boundary for subsequent more intensive purification of this protein fraction.

[0009] S3 involves washing the two types of precipitated proteins separately before they merge. The main precipitated protein cake is preferably washed twice with room temperature water; the supernatant precipitated protein cake is washed with counter-current water at 12–16°C for 3–5 stages. During counter-current washing, the wet protein material is pushed from the front stage to the back stage, while fresh water enters from the last stage and is reused progressively. This ensures that the protein closest to the final product comes into contact with the cleanest wash water, while reducing total water consumption through reverse water reuse. Compared to the main precipitated protein, the secondary recovered protein undergoes lower temperatures and more stages of washing to reduce its higher load of pigments, aldehydes, and other small molecule impurities.

[0010] S4. The two types of wet proteins are only combined after each has completed its individual washing. During combination, the wet materials are not added directly according to a fixed wet weight ratio. Instead, the wet materials are converted to dry weight based on their moisture content, and then blended at a dry weight ratio of 75–85:15–25. That is, based on dry weight, the main precipitated protein is 75–85 parts, and the supernatant precipitated protein is 15–25 parts. After combination, process water is added to adjust the solids content of the mixed protein slurry to 20–24 wt%, where wt% represents mass percentage. Then, food-grade dilute alkali solution is used to adjust the pH to 5.3–5.7 and the temperature to 40–45℃. At this material state, 0.10–0.15 wt% of protein glutaminase (based on the dry weight of the protein) is added, and the reaction is allowed to proceed for 2.5–3.5 hours. Controlling the reaction on a dry weight basis avoids fluctuations in the moisture content of the two types of wet proteins directly altering the true ratio of the two types of proteins in the final product.

[0011] S5, after the protein glutaminase reaction is complete, heat the mixed protein slurry to 72–78°C to inactivate the enzyme. Once the enzyme reaction phase is confirmed to be complete, add 0.55–0.70 wt% κ-carrageenan based on the dry weight of the protein. Preferably, the κ-carrageenan is first pre-dispersed with a portion of the slurry-mixing water at 70–80°C, and this pre-dispersed water is included in the total slurry-mixing water volume. This pre-dispersed water is then slowly added to the still-warm enzyme-inactivated protein slurry while stirring thoroughly. This reduces the formation of clumps from direct powder addition and separates the enzyme reaction phase from the polysaccharide complexation phase in time.

[0012] S6. After cooling the protein-polysaccharide composite slurry to a suitable homogenization temperature, high-pressure homogenization is performed, followed by vacuum concentration and spray drying. Preferably, homogenization is carried out at 28 MPa after cooling to 45°C (MPa represents megapascals); vacuum concentration is then performed until the solid content reaches 33 wt%, followed by spray drying with an inlet air temperature of 175°C and an outlet air temperature of 80°C. This post-treatment is mainly used to obtain a uniform, transportable powder, while minimizing unnecessary prolonged high-temperature exposure to the already modified slurry.

[0013] Furthermore, in continuous industrial production, the residence time of the feed liquid can be matched according to the actual effective volume and real-time volumetric flow rate of the tubular insulation section, stabilizing it at 6–15 seconds; the moisture content of the main precipitated protein and the supernatant precipitated protein can be detected separately, and the two feeding paths can be controlled based on dry basis mass conversion; the washing water temperature and flow direction can also be interlocked in the counter-current washing loop to prevent fresh water from accidentally entering from the first stage or the washing temperature from deviating from the set range for an extended period. These controls are used to stabilize the material flow direction and key process conditions, facilitating the repeated achievement of the same treatment effect during continuous production.

[0014] Beneficial effects 1. This invention places the tubular instantaneous heat treatment before the primary acid precipitation stage, thereby reducing oxidase activity before subsequent secondary recovery and multi-stage water washing to extend the material residence time. In this way, the longer process brought about by secondary recovery no longer simultaneously amplifies the generation of new oxidative flavors, and the subsequent low-temperature water washing can more effectively replace existing pigments and aldehyde impurities.

[0015] 2. A true first solid-liquid separation is set between the two-stage acid precipitation, and only the first-stage supernatant is allowed to enter the second-stage acid precipitation, so that the main precipitated protein and the supernatant precipitated protein form two material branches with clear sources. This allows for 12-16℃, 3-5 stages of countercurrent water washing only on the secondary recovered protein with a higher impurity load, while the main precipitated protein maintains a lower water washing intensity, thus balancing protein recovery and purification losses.

[0016] 3. After each type of wet protein is washed separately, they are blended according to a dry weight ratio. Fluctuations in moisture content are corrected by the amount of wet feed and are not directly transmitted to the final ratio. Compared with "fixed feeding based on wet weight", this approach is more suitable for continuous production and allows the parameters of the example to be repeatedly achieved under different batches of raw materials and different centrifugal dehydration conditions.

[0017] 4. Functional modification employs a clearly defined sequence of "protein glutaminase reaction - heat inactivation of enzyme - addition of κ-carrageenan". Deamidation first improves the hydration and dispersion of the protein, and then wet-state κ-carrageenan complexation avoids the adverse effects of premature polysaccharide thickening on enzyme-substrate contact; adding the gel after heat inactivation of the enzyme also makes the reaction endpoint easier to control.

[0018] 5. This invention mainly achieves improved whiteness, low fishy smell, and functionality through pre-stage oxidation inhibition, mid-stage differentiated water washing, and post-stage bio-enzyme / polysaccharide modification. It does not use sulfites, peroxides, or coloring masking agents as bleaching or odor masking methods, making it more suitable for food ingredients with clean labels.

[0019] 6. All key modifications are completed in wet protein slurry, which can be integrated with the existing soybean protein isolate production line's extraction, centrifugation, heat exchange, slurry preparation, homogenization, concentration and spray drying equipment, avoiding the repetitive drying steps of first grinding and then reconstituted for modification, and has a clear industrial implementation basis. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall process flow for the preparation method of high-whiteness, low-fishy-odor soy protein isolate according to the present invention.

[0021] Figure 2 This is a schematic diagram illustrating the reverse flow relationship between protein material and washing liquid during the four-stage countercurrent water washing process of this invention.

[0022] Figure 3This is a schematic diagram illustrating the control relationship between the two types of wet proteins in this invention after each wet protein has been washed with water and then compounded and mixed according to the dry basis ratio. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments. Each embodiment describes the material input, processing conditions, output status, abnormal situations, and connection with the next step according to the actual production sequence. The equipment model, batch size, and conventional acid and alkali concentration can be adjusted according to the production line conditions, but the process sequence of primary separation followed by secondary acid precipitation, separation of the two types of proteins before their respective water washing, compounding according to the dry basis ratio, and adding κ-carrageenan after completing the enzyme reaction and heat inactivation remains unchanged.

[0024] I. Terminology, Material Standards and Testing Specifications In this application, "main precipitated protein" refers to the precipitate obtained after primary acid precipitation and the first solid-liquid separation; "supernatant precipitated protein" refers to the precipitate obtained after the primary supernatant obtained from the first solid-liquid separation undergoes secondary acid precipitation and the second solid-liquid separation. The names are used to distinguish the source and process branch, and do not indicate that any branch contains only the single component of 7S or 11S soybean globulin named according to its sedimentation coefficient.

[0025] The term "low-temperature defatted soybean meal" as used in this application refers to defatted soybean meal produced under relatively mild desolvation conditions during the soybean oil removal process, which effectively maintains the water solubility of the protein. It is a commonly used raw material in the production of soy protein isolate. The nitrogen solubility index of the low-temperature defatted soybean meal used in the examples is 78%. This invention does not require limitation to specific soybean varieties, but the raw materials entering the alkali soaking process should avoid significant mold, rancidity, or abnormal charring, so as to prevent the color and oxidative flavor formed by the raw materials themselves from masking the effects of subsequent processes. The protein glutaminase and κ-carrageenan used in the examples are both food-grade raw materials.

[0026] The term "dry basis mass" in this application refers to the mass of solids obtained by deducting moisture from wet protein. Since the moisture content of wet protein can fluctuate due to centrifuge discharge conditions, raw material particle size, and seasonal water temperature, controlling the blending ratio based on dry basis rather than wet weight avoids these fluctuations directly altering the true ratio of the two types of protein. Moisture content can be determined using online near-infrared spectroscopy, a moisture meter, or the drying method commonly used in production lines, as long as both wet protein streams use the same quality standard and meet the feeding control accuracy requirements.

[0027] The blue light whiteness value R457 represents the whiteness index characterized by blue light reflectance around 457 nm, where nm represents nanometers. The same reflectance whiteness meter calibrated with a standard white plate was used for testing, and measurements were taken under the same sample loading method and optical conditions, with the average value recorded. Hexanal content was determined using headspace gas chromatography-mass spectrometry. Sample weighing, headspace equilibration temperature, and time were kept consistent between the examples and comparative examples to ensure data comparability.

[0028] The nitrogen solubility index (NSI) is used to characterize the proportion of soluble nitrogenous components to total nitrogenous components under specified conditions; the emulsifying activity index (EAI) is used to characterize the ability of proteins to form an oil-water interface. Gel strength was measured using gels prepared with the same concentration, heating time, and cooling conditions, followed by puncture or compression tests using a texture analyzer. The testing conditions for the examples and comparative examples were kept consistent.

[0029] The "protein water solubility retention rate" mentioned in the examples is used to evaluate the effect of instantaneous heat treatment on protein solubility. Using the same sampling standard and the same soluble protein determination method, the soluble protein content of the samples before and after heat treatment was measured, and the percentage was converted from the ratio of the value after heat treatment to the value before heat treatment. The hexanal content is expressed in mg / kg, and the emulsification activity index is expressed in mg / kg. 2 These indicators (g) are mainly used to compare the changes in the same batch of raw materials under different process conditions.

[0030] The examples used food-grade protein glutaminase preparations with a labeled enzyme activity of 1000 U / g, where U / g represents the number of labeled enzyme activity units per gram of enzyme preparation. The addition amount of 0.10–0.15 wt% mentioned in this specification is calculated based on the mass of the added enzyme preparation relative to the dry weight of the mixed protein. When using similar food-grade enzyme preparations with different labeled activities, a small-scale equivalent test should be conducted first according to the product activity specifications to avoid deviations in the degree of deamidation from the implementation window of this invention due to differences in activity.

[0031] To verify the effect of "instant enzyme inactivation," the relative activity of lipoxygenase can be measured by taking samples before and after heat treatment. A spectrophotometric method using linoleic acid as a substrate and monitoring the rate of conjugated diene formation can be used, or a validated equivalent enzyme activity assay method from the manufacturer can be employed. As long as the same method is used before and after heat treatment, the inactivation rate can be calculated using equation (1): (1); in, The inactivation rate of lipoxygenase; The activity of lipoxygenase in the sample before tubular instantaneous heat treatment; This refers to the lipoxygenase activity of samples after heat treatment and rapid cooling. This indicator is mainly used to verify the stability of the front-end control and does not require the detection equipment itself to be an essential component of this invention.

[0032] To facilitate verification of material states during industrial scale-up, process samples can be taken at several fixed locations: one sample before and after tubular instantaneous heat treatment for comparing lipoxygenase activity and protein water solubility; two types of wet proteins are collected after primary and secondary acid precipitation solid-liquid separation, with corresponding pH, washing temperature, stage number, and moisture content recorded; an initial enzyme reaction sample is collected after the two types of proteins are combined, and an enzyme-inactivated sample is collected after heat inactivation; a finished product sample is collected after spray drying. All samples are serially linked using the same batch number, allowing for the correlation between the initial enzyme inactivation, intermediate grading and purification, and subsequent modification stages and the final whiteness, hexanal, and functional indicators. This sampling method is only used for process verification and does not alter the necessary process conditions for each step.

[0033] II. Overall Process and Key Coupling Relationships Reference Appendix Figure 1 Low-temperature defatted soybean meal first undergoes alkaline soaking, solid-liquid separation, and tubular instantaneous heat treatment. After entering the first-stage acid precipitation, the first solid-liquid separation constitutes the first critical diversion point of the entire process: the main precipitated protein cake directly enters the main protein washing loop; the first-stage supernatant enters the second-stage acid precipitation and the second solid-liquid separation, forming a supernatant precipitated protein cake. The two branches are not merged again until the washing is completed.

[0034] This separation is not a formal process split, but rather a way to retain the ability to adjust the washing temperature and number of stages for secondary protein recovery independently. If the pH is lowered before the primary precipitate is truly separated, or if the precipitates are mixed together before the two types of proteins are washed, it becomes impossible to determine which proteins come from the primary main precipitate and which come from the secondary supernatant recovery, and it becomes impossible to perform low-temperature multi-stage countercurrent purification only on the secondary recovered proteins.

[0035] Reference Appendix Figure 2 In the four-stage countercurrent washing process, wet protein feed is sequentially introduced from stage 1 to stage 4, while fresh water enters from stage 4. The separated washing solution is then recycled back to stages 3, 2, and 1 in reverse order. The final wash brings the protein into contact with the cleanest fresh water, while the preceding stages use washing solution that has already been in contact with relatively clean protein. The key to this countercurrent method is that the material and washing solution flow in opposite directions; it is not simply a matter of reusing the same amount of water.

[0036] Reference Appendix Figure 3 After the two types of wet proteins are washed separately, their moisture content is measured and converted to dry basis weight. The feed rate of the two wet materials is then controlled according to the target dry basis ratio. Only after this conversion is completed do the two material streams converge at the mixing point. The slurry after convergence is adjusted to 20-24 wt% solids by adding process water, and then the pH and temperature are adjusted before it enters the protein glutaminase reaction. In this way, fluctuations in upstream centrifugal dehydration are not directly carried over to the final ratio.

[0037] In the latter part, the protein glutaminase stage, the heat-inactivating enzyme stage, and the κ-carrageenan stage proceed sequentially. The protein glutaminase first contacts the protein substrate within a suitable temperature window at a lower temperature; after the reaction, enzyme activity is stopped by heat treatment at 72–78°C; only then is the pre-dispersed κ-carrageenan added. This sequential relationship facilitates control of the enzyme reaction endpoint and avoids premature viscosity increases and localized encapsulation caused by carrageenan.

[0038] III. Implementation and Control Logic of Each Step (a) S1: Alkali leaching, solid-liquid separation and tubular instantaneous heat treatment The input to S1 is low-temperature defatted soybean meal and process water. Preferably, water is added at a 1:9 mass ratio, and the pH is adjusted to 7.1 with a food-grade dilute sodium hydroxide solution. The mixture is then stirred and extracted at 48°C for 30 minutes to allow soluble proteins to enter the liquid phase. After extraction, the soybean residue is removed by centrifugation or other food-grade solid-liquid separation equipment. Before entering the tubular heat treatment equipment, the feed solution should not contain large particles of soybean residue that could cause severe blockage of the heat exchanger; if the clarity is significantly abnormal, the state of the upstream centrifugation or sieving should be checked first, rather than compensating by extending the high-temperature time.

[0039] The input for tubular instantaneous heat treatment is clarified protein extract, and the processing logic is "rapid heating - short-term holding - immediate cooling". The holding section must simultaneously meet 110~115℃ and 6~15s, and the actual flow rate should not be ignored while only considering the equipment outlet temperature. For continuous production lines, the actual residence time can be determined by the effective volume of the insulation tube and the real-time volumetric flow rate, and their relationship is shown in equation (2): (2); in, This is the actual residence time of the liquid within the effective insulated pipe section, expressed in seconds. The effective insulated pipe section volume is expressed in cubic meters (m³). 3 ; This represents the volumetric flow rate of the liquid at that moment, in cubic meters per second (m³). 3 / s. When the production line changes its processing capacity, the actual residence time can be kept within the range of 6 to 15 seconds by adjusting the effective volume or flow rate of the insulation pipe, instead of simply using the pump frequency of the original equipment.

[0040] The output after heat treatment is an enzyme-inactivated protein extract. This extract should immediately enter the cooling zone, preferably rapidly cooled to 42°C before proceeding to the first-stage acid precipitation conditioning. The rapid cooling technique aims to reduce unnecessary continuous heating while maintaining both enzyme activity control and protein function preservation. The examples employ continuous heat exchange or vacuum flash cooling; both methods achieve the above objectives.

[0041] In abnormal situations, if the temperature does not reach 110℃, the actual residence time is less than 6 seconds, or the heat preservation section cannot operate stably due to flow fluctuations, the feed solution that has not yet entered the first-stage acid precipitation should be temporarily switched to the buffer or reflux channel and processed again after the heat treatment conditions stabilize. It is not advisable to directly mix the feed solution that has not met the requirements with the qualified feed solution. If the temperature exceeds the set value or the residence time is significantly longer, a separate sample should be retained to check the protein solubility and color, avoiding directly merging batches that may have been overheated into the main production line.

[0042] (ii) S2: Primary acid precipitation, first solid-liquid separation and secondary acid precipitation The input to S2 is the enzyme-inactivated protein extract that has undergone rapid cooling. During the first-stage acid precipitation, food-grade dilute hydrochloric acid is slowly added under low-speed stirring to adjust the pH to 4.4–4.6, preferably 4.5. The acid adjustment speed should be adjusted to avoid the formation of significantly over-acidic areas in localized areas. After reaching the target pH, continue stirring at low speed to allow the protein to aggregate. Preferably, stir at low speed for 25 minutes, then allow to settle for 55 minutes before performing the first solid-liquid separation. The first solid-liquid separation must actually yield two material streams: the solid phase is the main precipitated protein cake, and the liquid phase is the first-stage supernatant.

[0043] The main precipitated protein cake is not returned to the primary supernatant at this point, but is directly transferred to a separate water washing branch. Only then does the primary supernatant enter the secondary acid precipitation stage. The secondary acid precipitation stage adjusts the pH of the primary supernatant to 3.7–4.0, preferably 3.8, and involves low-speed stirring at 30°C for 35 minutes, followed by standing for 110 minutes, and then a second solid-liquid separation to obtain the supernatant precipitated protein cake. In this way, the source and history of the secondary recovered material are traceable.

[0044] The output of primary acid precipitation is used to form the main product protein branch, while the output of secondary acid precipitation is used to recover the remaining precipitable protein in the primary supernatant. The purpose of this technology is not to obtain high-purity 7S or 11S separated components, but to improve total protein recovery with minimal process modifications, while providing a separate purification inlet for secondary protein recovery with a high impurity load.

[0045] In abnormal situations, if the pH of the primary acid precipitation is significantly lower than the target value, it is not recommended to repeatedly adjust the pH using large doses of strong alkali, as this will increase the salt load of the system. Instead, over-adjustment should be avoided primarily through acid addition rate and online pH feedback. If the turbidity of the primary supernatant after the first solid-liquid separation is abnormally high, it indicates that a large number of fine particles from the main precipitate may be entrained. In this case, the feed flow rate can be reduced, the centrifuge differential speed checked, or the supernatant can be supplemented with additional separation before proceeding to the secondary acid precipitation. This can prevent particles that should belong to the main precipitate from being mistakenly identified as secondary recovered proteins.

[0046] (III) S3: Differentiated water washing and four-stage countercurrent implementation S3 has two inputs: one for the main protein cake precipitate and the other for the supernatant precipitate. The main protein cake precipitate is washed twice with 20°C water, each time mixing the wet protein with water at a mass ratio of 1:5, followed by stirring and solid-liquid separation. This branch mainly handles routine acid and soluble impurity replacement, without excessive washing.

[0047] The supernatant precipitated protein cake is washed using a 3-5 stage countercurrent water wash at 12-16°C. Taking the four-stage countercurrent wash in Example 1 as an example, the first stage receives the protein cake precipitated by the second stage acid, and the fourth stage yields the final washed wet protein. Fresh 14°C water is added from the fourth stage, the wash liquid from the fourth stage enters the third stage, the wash liquid from the third stage enters the second stage, the wash liquid from the second stage enters the first stage, and the first-stage wash liquid is discharged from the system. Each stage completes mixing and solid-liquid separation; the wash liquid should not be simply transferred between tanks without allowing the protein to fully re-contact with the wash liquid.

[0048] The low-temperature technique primarily reduces further oxidation of the secondary recovered proteins and the re-adsorption of some small molecule impurities during the washing process, while maintaining the proteins in a relatively stable wet state. The multi-stage countercurrent technique uses cleaner water to complete the final stage of displacement and improves water efficiency through wash solution reuse. The output of this step consists of two separate wet proteins that have undergone their respective purification processes; they remain separate until they are combined in the next step according to a dry-based ratio.

[0049] In abnormal situations, if the fresh water temperature remains above 16℃, the water should be returned to the cooling or insulation circuit and restored to the set temperature before being counted in the effective washing stage. If the washing liquid direction is reversed, the countercurrent flow should be stopped and the flow direction of "fresh water entering from the last stage" should be re-established. Washing times under the incorrect flow direction should not be directly counted in stages 3-5. If a large amount of fine powder is lost with the washing liquid after solid-liquid separation in a certain stage, the centrifugation conditions and agitation shear should be checked. Avoid simply increasing the water volume, as excessive water will amplify protein loss.

[0050] (iv) S4: Preparation of compounding, pulping and enzyme reaction according to dry basis ratio. The input for S4 is the main precipitated protein and the supernatant precipitated protein that have been washed separately. Since the moisture content of the two types of wet proteins may be different, the moisture content should be measured first when compounding, and then the wet material mass should be converted to dry weight. After setting the target dry weight ratio of the two types of proteins, the actual compounding relationship can be checked according to formula (3): (3); in, The ratio of the dry weight of the precipitated protein to the dry weight of the precipitated protein in the supernatant; The quality of wet feed containing pre-precipitated protein; The water content of the wet feed containing the main precipitated protein; To determine the quality of the wet protein precipitate in the supernatant; The water content is the mass fraction of the wet protein precipitate in the supernatant. The target dry basis mass ratio in this invention ranges from 75 to 85:15 to 25, with Example 1 using 80:20.

[0051] After the two types of wet proteins are combined according to the target dry basis ratio, the slurry solids need to be adjusted to 20-24 wt%. If the current total solids of the two types of wet proteins are higher than the target value, process water can be added. The required amount of water to be added can be estimated according to formula (4): (4); in, The quality of process water that needs to be replenished; The target slurry solids mass fraction is expressed as a decimal, for example, 22wt% corresponds to 0.22; the meanings of the other symbols are the same as in equation (3). If the calculation result is less than zero, it means that the total solids of the current wet material are already lower than the target value. At this time, it should not be interpreted as "adding negative water". Instead, the degree of dehydration at the front end should be increased or moderate concentration should be carried out before entering the enzyme reaction to adjust the slurry.

[0052] After completing the dry basis compounding and solids adjustment, the pH of the mixed protein slurry was adjusted to 5.3–5.7 and the temperature to 40–45°C using food-grade dilute alkali solution, and then protein glutaminase was added. Example 1 used pH 5.6, 43°C, 0.12 wt% enzyme preparation, and reacted for 3 hours. The input conditions in this step determine the reproducibility of subsequent enzyme reactions; therefore, the reaction time should begin after the pH, temperature, and stirring have all reached the set conditions, rather than immediately when the enzyme preparation first comes into contact with the slurry.

[0053] If significant fluctuations in the moisture content of the two wet protein streams are observed during production, the mass flow rates of both wet protein streams should be adjusted to maintain the dry basis ratio, rather than attempting to compensate empirically by adding more wet protein from one stream after batching is complete. If the solids content exceeds 24 wt%, resulting in a significant increase in stirring load, process water can be added while maintaining the target dry basis ratio. If the solids content is already below 20 wt%, it should be restored through front-end dehydration or concentration; directly increasing the enzyme dosage to compensate for dilution is not advisable.

[0054] (V) S5: Protein glutaminase modification, heat-inactivating enzyme and κ-carrageenan complex The input for the protein glutaminase stage is a mixed protein slurry that has already undergone dry basis formulation, solid content adjustment, and reaction condition regulation. Gentle stirring is maintained during the enzyme reaction to ensure uniform contact between the protein and enzyme. Protein glutaminase primarily catalyzes the deamidation of glutamine residues on the protein side chains, altering the protein's hydration state, surface charge distribution, and conformational exposure. The degree of reaction should be optimized to balance solubility, dispersion, and subsequent gel-forming ability.

[0055] After the enzyme reaction reaches the set time, the slurry is heated to 72-78°C for enzyme inactivation. In Example 1, the slurry was kept at 75°C for 10 minutes. The output of the heat-inactivated enzyme should be a protein slurry in which enzyme activity has essentially stopped, but it remains moist and is easy to compound later. The κ-carrageenan feeding stage only begins after the enzyme inactivation is complete. This sequence is a critical boundary for the later stages of the process in this invention.

[0056] It is not recommended to directly add 20-24 wt% high-solids protein slurry to κ-carrageenan all at once. Instead, heat a portion of the slurry preparation water to 70-80°C, and gradually add κ-carrageenan while stirring to form a uniform pre-dispersion; this water portion should be included in the total water volume of the slurry preparation. Then, slowly add the pre-dispersion to the enzyme-inactivated protein slurry, maintaining stirring for 20 minutes. This method reduces uneven mixing caused by fish-eye clumps and localized high-concentration colloids.

[0057] During pre-dispersion, a stable liquid surface circulation should be established first, and then κ-carrageenan should be slowly added along the edge of the liquid surface vortex formed by stirring, so that the powder is first wetted by hot water before entering the main circulation. If unwetted powder clumps or obvious fish-eye-like clumps are still visible after addition, pre-dispersion should be completed before adding to the protein slurry, rather than replacing the initial dispersion with increased homogenization pressure. When using a continuous metering pump, the pre-dispersion liquid should be kept in low-speed circulation to avoid local uneven concentration caused by prolonged standing. The above operation is a refinement of the routine engineering for achieving stable κ-carrageenan addition of 0.55–0.70 wt%, and does not change the core timing sequence of "adding κ-carrageenan after heat inactivation of enzymes".

[0058] From the perspective of system behavior, appropriate deamidation can improve the hydration state, surface charge distribution, and conformational exposure of proteins. Subsequently, the addition of κ-carrageenan allows the protein and polysaccharide to jointly influence the final network through hydrogen bonds, chain entanglement, local ion-mediated interactions, and structures formed during heat treatment and cooling. Examples and comparative studies demonstrate that completing the enzymatic reaction first and then heat-inactivating the enzyme before adding κ-carrageenan is more beneficial for simultaneously maintaining NSI, emulsifying properties, and gel strength.

[0059] In abnormal situations, if κ-carrageenan is mistakenly added before the enzyme reaction, the batch should be sampled separately for evaluation. It is not advisable to compensate solely by extending the enzyme reaction time. If significant aggregation occurs in the κ-carrageenan predispersion, a homogeneous state should be restored first by extending the predispersion stirring, applying moderate cyclic shearing, or checking the water temperature, rather than directly increasing the amount of carrageenan. If the temperature or pH deviates from the set range briefly during the enzyme reaction, the conditions should be restored first, and then the reaction should be appropriately extended based on the deviation time. Blindly adding enzyme while the reaction is deviating is not advisable.

[0060] (vi) S6: Homogenization, concentration and spray drying The input to S6 is a protein-polysaccharide slurry that has already undergone wet-state κ-carrageenan compounding. Preferably, it is first cooled to 45°C, then homogenized under high pressure at 28 MPa. The purpose of homogenization is to reduce uneven distribution caused by macroscopic particle and local colloidal agglomeration, making the feed state more stable for subsequent concentration and spray drying. Homogenization is not a replacement for the aforementioned carrageenan pre-dispersion; therefore, if significant large clumps have already appeared in the initial stage, the pre-dispersion problem should be addressed first.

[0061] After homogenization, vacuum concentration is performed to increase the solids content to approximately 33 wt%, followed by spray drying. In this example, an inlet air temperature of 175°C and an outlet air temperature of 80°C are used. The key to controlling spray drying is maintaining stable feed solids content, atomization state, and outlet air temperature to obtain powder with consistent flowability and color. If the outlet air temperature remains consistently high, the feed rate, inlet air temperature, and atomization state should be checked first to avoid unnecessary browning caused by prolonged high temperatures. If homogenization pressure fluctuates, continuous concentration should only proceed after the pressure stabilizes.

[0062] The final output is soy protein isolate powder. The product's whiteness, hexanal, NSI, EAI, gel strength, and protein recovery rate were tested using a standardized method. The protein recovery rate was used to evaluate whether secondary acid precipitation and subsequent washing improved the yield while maintaining reasonable losses; its calculation relationship is shown in equation (5). (5); in, Protein recovery rate; For the quality of the final product; The mass fraction of protein in the final product; To ensure the quality of low-temperature defatted soybean meal; This represents the mass fraction of protein in low-temperature defatted soybean meal. The examples and comparative examples should use the same protein determination method and conversion factor to avoid differences in detection methods being mistaken for differences in process yield.

[0063] IV. Implementation of Abnormal Operating Conditions, Inter-batch Fluctuations, and Continuous Production The continuous production process of this invention can include buffer tanks, but the purpose of these buffer tanks is to absorb differences in equipment cycle time, not to pre-mix the originally separate main precipitated protein and supernatant precipitated protein. Preferably, the buffer tanks after washing the main precipitated protein and the supernatant precipitated protein are set up separately, and then merged at the dry basis mixing point. This way, when a branch line is temporarily stopped, the proteins can be temporarily stored separately without disrupting the subsequent dry basis ratio control conditions.

[0064] When there are batch-to-batch fluctuations in the protein content and NSI of the raw materials, the alkaline leaching solution ratio, pH, and temperature should still be maintained within the aforementioned process range. If the particle size or water absorption of the raw materials causes abnormal slurry viscosity, the equipment operating parameters can be adjusted by checking the crushing status, stirring capacity, and solid-liquid separation load. It is not recommended to arbitrarily increase the alkaline leaching pH in order to reduce viscosity, as excessively high pH may increase the risk to color and flavor.

[0065] If the sedimentation rate slows down during the acid precipitation stage, the following should be checked first: raw material solids content, acid adjustment rate, stirring shear, and pH electrode calibration. The pH windows for primary and secondary acid precipitation differ; therefore, the two stages cannot be combined into a single continuous pH reduction to shorten the time. While a single continuous pH reduction simplifies equipment operation, it loses the boundary between the first solid-liquid separation and the conditions for separate low-temperature countercurrent water washing of the secondary recovered protein.

[0066] If a centrifuge in the counter-current washing section is temporarily shut down, the wet protein material before and after that stage can be temporarily stored in the corresponding buffer tanks. After resumption, the flow of protein from the previous stage to the next stage and water from the next stage to the previous stage can be maintained. To prevent misoperation, the final stage fresh water valve and the first stage drain valve can be interlocked. This type of automated control is an engineering means to achieve a predetermined material flow direction and does not change the chemical or biological modification nature of this invention.

[0067] For the protein glutaminase stage, batch records should be kept for pH, temperature, start time, and end time. If the reaction time significantly exceeds 3.5 hours due to equipment malfunction, a separate sample should be retained to evaluate dissolution and gelation properties; this batch should not be directly considered as the standard process. For the heat inactivation stage, timing should begin after the set temperature is reached; if only the jacket temperature is reached while the material's center temperature has not yet been reached, enzyme inactivation should not be prematurely terminated.

[0068] The key to the κ-carrageenan stage is pre-dispersion followed by the addition of the enzyme-inactivated protein slurry. The carrageenan pre-dispersion tank can share a heat source with the enzyme-inactivation heat exchange section to reduce additional energy consumption. If continuous addition of carrageenan is desired, a pre-dispersion tank with insulation and low-shear circulation can be used in conjunction with a metering pump to match the addition rate with the protein slurry flow rate. Regardless of whether a batch or continuous method is used, the order of adding κ-carrageenan should be maintained: "the enzyme reaction is complete and the enzyme is inactivated by heat before adding κ-carrageenan."

[0069] In terms of production hygiene, the CIP (Clean In-Situ) procedure, commonly used in food production, can be used for alkaline soaking, acid precipitation, enzyme reaction, and carrageenan compounding. The cleaning agent used in CIP is only for equipment hygiene maintenance and is not used as a process additive in the preparation steps. After cleaning, the equipment should be thoroughly rinsed according to the production procedure and confirmed to be back to normal process conditions before product production can begin.

[0070] V. Examples Example 1: Implementation of the main process Reference Appendix Figure 1 Low-temperature defatted soybean meal with an NSI of 78% was used. Water was added at a soybean meal to deionized water ratio of 1:9, and the pH was adjusted to 7.1 with food-grade dilute sodium hydroxide solution. The mixture was stirred and extracted at 48℃ for 30 min. After extraction, solid-liquid separation was performed to remove the soybean residue, yielding a clear protein extract. This extract was pumped into a tubular instantaneous heat treatment device, held at 110℃ for 9 s, and then immediately cooled to 42℃. The activity of lipoxygenase was measured before and after heat treatment using the same method, showing an inactivation rate of 99.3% and a protein water solubility retention rate of 96.5%.

[0071] Food-grade dilute hydrochloric acid was slowly added to the enzyme-inactivated protein extract, and the pH was adjusted to 4.5 under low-speed stirring. The stirring was continued for 25 minutes to allow the protein to aggregate. After settling for 55 minutes, the first solid-liquid separation was completed, yielding the main precipitated protein cake and the primary supernatant. The main precipitated protein cake was sent separately to the main protein washing loop, and the primary supernatant was sent separately to the secondary acid precipitation loop.

[0072] Only the first-stage supernatant was collected, and food-grade dilute hydrochloric acid was added to adjust the pH to 3.8. The temperature was controlled at 30°C, and the mixture was stirred at low speed for 35 minutes and then allowed to stand for 110 minutes. A second solid-liquid separation was then performed to obtain a protein cake precipitated from the supernatant.

[0073] The main precipitated protein cake was washed twice with 20°C water, each time at a protein-to-water ratio of 1:5. The supernatant precipitated protein cake was then washed four times counter-currently with 14°C water, as shown in the attached document. Figure 2 The protein material is fed from stage 1 to stage 4, while fresh water is recycled counter-currently from stage 4 to stage 1. The mass ratio of wet protein to water in each stage is 1:5. After stirring for 5 minutes, solid-liquid separation is performed. The two types of protein are not mixed before their respective washing processes are completed.

[0074] After determining the moisture content of the two types of washed wet proteins, they were mixed at a dry weight ratio of 80:20, and water was added to adjust the solid content to 22 wt%. The pH was then adjusted to 5.6 and the temperature to 43℃ using food-grade dilute alkali solution. Food-grade protein glutaminase with an enzyme activity of 1000 U / g was added at a rate of 0.12 wt% of the dry weight of the mixed proteins, and the mixture was stirred at a constant temperature and low speed for 3 hours.

[0075] After the enzyme reaction is complete, raise the slurry to 75°C and keep it at that temperature for 10 minutes to inactivate the enzyme. Take a portion of the process water that has already been included in the total water volume for slurry preparation and heat it to about 75°C. After pre-dispersing κ-carrageenan, slowly add it to the protein slurry after the enzyme inactivation is complete. The amount added is 0.55 wt% of the dry weight of the protein. Continue stirring for 20 minutes.

[0076] The protein-polysaccharide composite slurry was cooled to 45°C, homogenized under high pressure at 28 MPa, then concentrated under vacuum to a solid content of 33 wt%, and finally spray-dried with air inlet at 175°C and air outlet at 80°C. The resulting product had a dry basis protein content of 92.3%, R457 of 66.1%, hexanal content of 0.32 mg / kg, NSI of 97.5%, and EAI of 36.2 mg / kg. 2 / g; The gel strength of the 10wt% protein system after heating at 80℃ for 30 min was 285g, and the protein recovery rate was 86%.

[0077] Example 2: Implementation of short-time high-temperature boundary in instantaneous heat treatment The material flow direction, the boundary between the two solid-liquid separations, the differentiated water washing, and the sequence of enzyme-before-gel washing in this embodiment are the same as in Example 1. The differences are as follows: the tubular instantaneous heat treatment conditions are 113℃ for 6 seconds; the secondary acid precipitation pH is 3.9 and the temperature is 28℃; the amount of protein glutaminase added is 0.15 wt% of the protein dry basis mass, and the reaction time is 2.5 h; after heat inactivation of the enzyme, the amount of κ-carrageenan added is 0.60 wt%. The resulting product has an R457 of 67.3, a hexanal content of 0.38 mg / kg, an NSI of 98.2%, and an EAI of 35.7 m. 2 / g, gel strength 279g, protein recovery rate 87.5%. This example illustrates that even with a short instantaneous heat treatment time, as long as the temperature remains within the window of this invention and the subsequent process is maintained, good overall performance indicators can still be obtained.

[0078] Example 3: Implementation of Low-Temperature Three-Stage Countercurrent Water Washing Boundary The basic process of this embodiment is the same as that of Embodiment 1, except that: the tubular instantaneous heat treatment is 110℃ for 15s; the secondary acid precipitation pH is 3.7; the protein precipitate in the supernatant is washed with countercurrent water at 12℃ for 3 stages; the amount of protein glutaminase added is 0.10wt%, and the reaction time is 3.5h; after heat inactivation of the enzyme, the amount of κ-carrageenan added is 0.65wt%. The resulting product has an R457 of 67.1, a hexanal content of 0.37mg / kg, an NSI of 97.8%, and an EAI of 36.8m. 2 / g, gel strength 280g, protein recovery rate 87.7%. This example covers a combination of lower washing temperature, fewer countercurrent stages, and longer enzyme reaction time.

[0079] Example 4: Five-stage countercurrent washing and implementation with a higher κ-carrageenan content The basic process of this embodiment is the same as that of Embodiment 1, except that: the tubular instantaneous heat treatment is 115℃ for 6 seconds; the secondary acid precipitation pH is 4.0; the protein precipitate in the supernatant is washed with countercurrent water at 16℃ for 5 stages; the amount of protein glutaminase added is 0.15wt%, and the reaction time is 2.5h; after heat inactivation of the enzyme, the amount of κ-carrageenan added is 0.70wt%. The resulting product has an R457 of 68.1, a hexanal content of 0.34mg / kg, an NSI of 98.1%, and an EAI of 37.3m. 2 / g, gel strength 284g, protein recovery 87.4%. This example covers another boundary of countercurrent washing stages and κ-carrageenan dosage.

[0080] Example 5: Dry basis conversion when wet protein moisture content fluctuates Reference Appendix Figure 3 This embodiment illustrates how to achieve an 80:20 dry basis ratio of the two wet protein streams with different moisture contents in continuous production, and is not intended as a new chemical treatment condition. Assume the main precipitated protein has a wet mass of 1000 kg after washing and a measured moisture content of 72%, then its dry basis mass is 280 kg. If the supernatant precipitated protein has a moisture content of 74% after washing, to achieve an 80:20 dry basis mass ratio of the main precipitated protein to the supernatant precipitated protein, the supernatant precipitated protein dry basis mass needs to be 70 kg, corresponding to a wet mass of approximately 269.23 kg.

[0081] After the two types of wet materials are combined, the total wet material mass is approximately 1269.23 kg, and the total dry basis mass is 350 kg. If the target slurry solids content is 22 wt%, according to formula (4), the final total slurry mass is approximately 1590.91 kg, therefore approximately 321.68 kg of process water needs to be added. Then, the pH and temperature are adjusted to the enzyme reaction window. This calculation shows that even if the moisture content of the two wet protein streams is different, as long as the feeding is controlled according to the dry basis conversion, the same true ratio as the laboratory dry basis weighing can be maintained.

[0082] When continuing the measurement under the conditions described in Example 1, the total dry weight of the two types of wet proteins is 350 kg. Protein glutaminase is added at 0.12 wt% of the protein dry weight, corresponding to an enzyme preparation mass of 0.42 kg; based on the enzyme preparation's labeled activity of 1000 U / g, the added labeled total enzyme activity is 420,000 U, equivalent to approximately 1.2 U per gram of protein dry weight. κ-carrageenan is added at 0.55 wt% of the protein dry weight, corresponding to a mass of 1.925 kg. The hot process water used for κ-carrageenan pre-dispersion is deducted from the aforementioned 321.68 kg of slurry conditioning water, without adding additional slurry conditioning water. In this way, the same 350 kg of protein dry weight is used as the calculation basis for solids adjustment, enzyme preparation measurement, and carrageenan measurement, avoiding cumulative measurement errors caused by changes in the moisture content of wet proteins during continuous production.

[0083] When converting the above-mentioned total protein dry basis to a range, if a dry basis ratio of 75:25 is used, the dry basis masses of the main precipitated protein and the supernatant precipitated protein are 262.5 kg and 87.5 kg, respectively; if a dry basis ratio of 85:15 is used, they are 297.5 kg and 52.5 kg, respectively. If the target slurry solids content is 20 wt%, the total slurry mass corresponding to 350 kg dry basis is 1750 kg; if it is 24 wt%, the corresponding total slurry mass is approximately 1458.33 kg. Therefore, the wet feed amount and water replenishment amount can be deduced from the actual measured moisture content of the two streams without changing the subsequent pH, temperature, and enzyme reaction control logic.

[0084] In continuous production, the above conversion can be completed by flow meters, weighing modules, or batching control programs, or the operator can calculate and control the feeding based on the batch moisture content. Regardless of the control method used, the compounding ratio is based on the dry basis mass of the two types of proteins, rather than directly using the wet volume or wet weight as a fixed ratio.

[0085] Example 6: Scale-up Implementation of Continuous Production Equipment This embodiment illustrates the implementation method when scaling up a laboratory or pilot-scale process to a continuous production line. The tubular instantaneous heat treatment section uses a continuously connected heating heat exchanger, an effective insulation tube, and a rapid cooling heat exchanger. If the volume of the effective insulation tube is 0.03 m³... 3 The stable volumetric flow rate of the liquid feed is 0.003 m³ / s. 3 If the actual residence time is calculated according to formula (2), it is 10s, which is within the 6-15s window. If the production capacity needs to be increased, the effective volume of the insulation pipe can be increased simultaneously or parallel insulation pipes can be set up, instead of just increasing the pump speed and causing the residence time to fall out of the range.

[0086] The primary acid settling tank, the first solid-liquid separation unit, the secondary acid settling tank, and the second solid-liquid separation unit are connected sequentially. The main precipitated protein and the primary supernatant after the first solid-liquid separation enter different pipelines. The supernatant precipitated protein is washed using a four-stage tank or a four-stage continuous washing unit, with a solid-liquid separation device after each stage. The washing liquid is pumped in the opposite direction to the protein material.

[0087] Two types of washed wet proteins are fed into separate buffer silos. After moisture content testing, the two feeding devices are controlled according to the dry basis ratio. The combined mixing tank is equipped with pH, ​​temperature, and solids monitoring points. The protein glutaminase metering pump is only allowed to start after the set conditions are reached. The κ-carrageenan metering pump is only allowed to start after the enzyme reaction is complete and heat inactivation is finished. This sequential interlocking reduces operational errors, but even with manual operation, the same process sequence should be followed.

[0088] VI. Verification of Comparative Examples and Key Process Relationships Comparative Example 1 (Exclusion of Liquid-Phase Instant Enzyme Inactivation): The tubular liquid-phase instantaneous heat treatment before acid precipitation in Example 1 was omitted, while the remaining material flow and operation remained consistent. The resulting product had a hexanal content of 1.89 mg / kg, an R457 of 62.7, and a pronounced beany odor. Compared to Example 1, this demonstrates that liquid-phase enzyme inactivation before adding secondary recovery and multi-stage washing helps suppress the formation of new oxidative flavors in the subsequent longer process.

[0089] Comparative Example 2 (Prolonged Low-Temperature Heat Treatment): The 110℃, 9s tubular instantaneous treatment in Example 1 was replaced with a 90℃ water bath incubation for 30 minutes, with all other conditions remaining the same. The lipoxygenase inactivation rate was 65%, the hexanal content of the product was 1.71 mg / kg, and the R457 was 60.9. This result indicates that under the conditions of the given raw materials and process, short-term high-temperature treatment and prolonged low-temperature treatment have different effects on enzyme inactivation and the final color and flavor.

[0090] Comparative Example 3 (secondary acid precipitation omitted): Primary acid precipitation was performed only at pH 4.5. The primary supernatant after the first solid-liquid separation was not recovered, and the remaining steps were the same as in Example 1. The protein recovery rate was 69%, and the total solids content in the whey was relatively high. This result indicates that an independent secondary recovery channel can significantly improve the total protein recovery.

[0091] Comparative Example 4 (protein precipitate in supernatant was washed once at room temperature): The primary and secondary acid precipitation conditions and subsequent modification conditions remained unchanged, except that the 14°C four-stage countercurrent water wash for protein precipitate in the supernatant of Example 1 was replaced with a single water wash at room temperature. The resulting product had an R457 of 61.7, a hexanal content of 0.56 mg / kg, and exhibited significantly different color and beany odor compared to Example 1. This result indicates that the secondary protein recovery cannot simply follow the lower washing intensity of the primary precipitate.

[0092] Comparative Example 5 (with protein glutaminase and κ-carrageenan omitted): The two types of washed wet proteins were compounded according to the proportions in Example 1, but without protein glutaminase treatment or the addition of κ-carrageenan; they were directly homogenized and dried. The resulting product had an NSI of 75.1% and an EAI of 27.5%. 2 / g, gel strength is 185g.

[0093] Comparative Example 6 (protein glutaminase only): Protein glutaminase was treated according to the enzyme dosage, pH, temperature, and reaction time of Example 1. κ-carrageenan was not added after heat inactivation of the enzyme. The resulting product had an NSI of 73.4% and an EAI of 28.5 mg / L. 2 / g, gel strength is 190g.

[0094] Comparative Example 7 (κ-carrageenan only): Without protein glutaminase reaction, the product was wet-compounded according to the κ-carrageenan addition amount of Example 1, then homogenized and dried. The resulting product had an NSI of 66% and an EAI of 25.2m. 2 / g, gel strength is 168g.

[0095] In Comparative Example 8, the sources of the two types of wet proteins, the mixing ratio, the solid content, and the amounts of protein glutaminase and κ-carrageenan were all the same as in Example 1, except that both were added simultaneously at the beginning of the enzyme reaction, followed by heat treatment, homogenization, and drying. The resulting product had an NSI of 78.1% and an EAI of 26.5%. 2 / g, gel strength was 176g. Compared with Example 1, when the order of enzymatic reaction, heat inactivation of enzyme, and then addition of κ-carrageenan was adopted, NSI, EAI and gel strength were all higher, indicating that stepwise addition is beneficial to balance the efficiency of enzyme reaction and the subsequent protein-polysaccharide complexation effect.

[0096] VII. Overall Effect and Repeatability of Combined Processes As can be seen from Example 1 and Comparative Examples 1 and 2, the instantaneous enzyme inactivation in the liquid phase before acid precipitation mainly serves to control the oxidation source in the subsequent long process. As can be seen from Example 1 and Comparative Example 3, the secondary acid precipitation addresses the continued recovery of proteins from the primary supernatant. As can be seen from Example 1 and Comparative Example 4, if the purification intensity of this branch is not increased accordingly after secondary recovery, the color and hexanal level will significantly decline. As can be seen from Example 1 and Comparative Example 8, after the two types of proteins are purified and compounded, the order of enzyme and κ-carrageenan directly affects the final functional indicators. These comparisons correspond to the front, middle, and rear sections of the same continuous process, demonstrating that there is a sequential connection between each process, rather than unrelated individual optimizations.

[0097] In particular, secondary acid precipitation mainly addresses the issue of further protein recovery from the primary supernatant, but it cannot simultaneously remove color-causing and fishy-smelling impurities carried by these proteins. Low-temperature countercurrent washing can further refine the secondary recovered material, but it cannot replace the subsequent functional modification. Therefore, this invention involves controlled merging of the proteins after washing according to the aforementioned dry basis ratio, followed by wet modification using a process of first enzymatic reaction and then gelation. This organizational method of first separating, then refining separately, and finally merging according to the dry basis ensures that the secondary recovered proteins maintain a relatively stable quality state before entering the final product.

[0098] Examples 1-4 cover multiple boundary combinations of instantaneous heat treatment temperature and time, secondary acid precipitation pH, countercurrent washing temperature and number of stages, protein glutaminase addition amount and time, and κ-carrageenan addition amount. The products all achieved an R457 of no less than 65, hexanal no more than 0.4 mg / kg, NSI above 97%, gel strength above 279 g, and protein recovery rate above 86%. These results demonstrate that the technical effectiveness does not occur only at a single optimal point, but rather stems from the combined effect of parameter windows and the relationships between key steps.

[0099] The performance data in Examples 1-4 correspond to the finished product test results after all processes are completed. Example 5 and its supplementary material balance are used to illustrate the conversion relationship between wet protein moisture content, dry basis ratio, solids and subsequent additions, and the calculated values ​​are not treated as newly added measured performance values. This allows for a separation of the "measured technical effects" and the "ingredient calculations for continuous production," facilitating those skilled in the art to reproduce the process and verify the results according to this specification.

[0100] Example 5 further provides a method for converting the dry basis mass of the two types of wet proteins according to their moisture content and calculating the amount of water added for slurry preparation, solving the practical problem of distorted compounding ratios caused by fluctuations in the moisture content of wet proteins in continuous production. Example 6 illustrates that the residence time of tubular heat treatment, two solid-liquid separations, countercurrent washing, and the order of enzyme-before-gel feeding can all be implemented on common food production equipment. Therefore, those skilled in the art can repeat the implementation based on this specification without relying on undisclosed special materials, special strains, or unavailable equipment.

[0101] In summary, this invention uses the flow of wet materials as the main thread, continuously linking the liquid-phase instantaneous enzyme inactivation before acid precipitation, solid-liquid separation between two acid precipitation cycles, low-temperature multi-stage countercurrent water washing for secondary protein recovery, controlled merging of two types of wet proteins according to dry basis, and wet modification with enzyme followed by gel. Each step addresses issues of oxidative flavor, protein recovery, impurity load, compound stability, and functional modification, and the interconnection of the preceding and following material states allows for improved protein recovery while simultaneously maintaining whiteness, low odor, and good solubility, emulsification, and gelation properties.

Claims

1. A method for preparing high-whiteness, low-odor soy protein isolate, characterized in that, Includes the following steps: S1, Alkali soaking and solid-liquid separation of low-temperature defatted soybean meal to obtain a clear protein extract, the clear protein extract is subjected to tubular instantaneous heat treatment at 110-115℃ for 6-15s, and then rapidly cooled after heat treatment to obtain an enzyme-inactivated protein extract. S2, the enzyme-inactivated protein extract is adjusted to pH 4.4-4.6 for primary acid precipitation and first solid-liquid separation to obtain the main precipitate protein cake and primary supernatant. The primary supernatant is then adjusted to pH 3.7-4.0 for secondary acid precipitation and second solid-liquid separation to obtain the supernatant precipitate protein cake. S3, before the two types of precipitated proteins merge, the main precipitated protein cake is washed twice with room temperature water, and the supernatant precipitated protein cake is washed with countercurrent water at 12-16°C for 3-5 stages to obtain the main precipitated protein and the supernatant precipitated protein, respectively. S4, the main precipitated protein and the supernatant precipitated protein, after being washed separately, are mixed according to dry weight, with 75-85 parts of main precipitated protein and 15-25 parts of supernatant precipitated protein. Water is added to prepare a mixed protein slurry with a solid content of 20-24 wt%. The pH is adjusted to 5.3-5.7 and the temperature is adjusted to 40-45°C. 0.10-0.15 wt% of protein glutaminase is added and the reaction is carried out for 2.5-3.5 h. S5, after the protein glutaminase reaction is completed, the mixed protein slurry is heated to 72-78°C to inactivate the enzyme. After the enzyme is inactivated, 0.55-0.70 wt% of κ-carrageenan is added and mixed to obtain a protein-polysaccharide composite slurry. S6, the protein-polysaccharide composite slurry is sequentially homogenized, concentrated and spray-dried to obtain soy protein isolate.

2. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S1, the mass ratio of low-temperature defatted soybean meal to water is 1:

9. The system is adjusted to pH 7.1 using food-grade alkali solution and extracted at 48°C for 30 minutes. After the tubular instantaneous heat treatment, the enzyme-inactivated protein extract is rapidly cooled to 42°C by continuous heat exchange or vacuum flash cooling.

3. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S2, the first-stage acid precipitation adjusts the pH of the enzyme-inactivated protein extract to 4.5, stirs for 25 minutes, and then performs sedimentation and the first solid-liquid separation. The second-stage acid precipitation adjusts the pH of the first-stage supernatant to 3.8, stirs for 35 minutes at 30°C, and then allows it to stand for 110 minutes before performing the second solid-liquid separation.

4. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S3, the main precipitated protein cake is washed twice with 20°C clean water, with the mass ratio of wet protein to washing water being 1:5 each time; the supernatant precipitated protein cake is washed with 14°C clean water in a 4-stage countercurrent water wash, with the protein material being pushed forward in stages 1 to 4, and fresh washing water entering from stage 4 and being reused in a countercurrent manner back to stage 1, with the mass ratio of wet protein to washing water being 1:5 in each stage.

5. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S4, the dry weight ratio of the main precipitated protein to the supernatant precipitated protein is 80:20, the solid content of the mixed protein slurry is 22 wt%, the pH is 5.5-5.6, and the temperature is 43℃; the amount of protein glutaminase added is 0.12 wt% of the dry weight of the protein, and the reaction time is 3 h.

6. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S5, after the protein glutaminase reaction is completed, the enzyme is inactivated by heat at 75°C for 10 minutes. κ-carrageenan is pre-dispersed with a portion of the slurry preparation water at 70-80°C. The pre-dispersed water is included in the slurry preparation water in step S4. After the enzyme is inactivated by heat, the pre-dispersed κ-carrageenan is added to the protein slurry and stirred for 20 minutes.

7. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S6, the protein-polysaccharide composite slurry is cooled to 45°C and then homogenized under high pressure at 28 MPa. Subsequently, it is vacuum concentrated to a solid content of 33 wt%, and then spray dried at an inlet air temperature of 175°C and an outlet air temperature of 80°C.

8. The method for preparing high-whiteness, low-odor soy protein isolate according to claim 1, characterized in that, In step S1, the actual residence time is controlled based on the effective volume of the tubular instantaneous heat treatment insulation section and the liquid volume flow rate. In step S4, the moisture content of the main precipitated protein and the supernatant precipitated protein are detected respectively, and the wet feed amount is calculated based on the dry matter mass of the two wet proteins to ensure that the actual dry basis ratio is maintained at 75-85 parts of main precipitated protein and 15-25 parts of supernatant precipitated protein.

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