A preparation method of a physical-chemical-enzyme combined modified starch, the modified starch and application thereof

Abstract

The application relates to the technical field of food additives, and discloses a preparation method of physical-chemical-enzyme combined modified starch, modified starch and application of the modified starch. The preparation method of the physical-chemical-enzyme combined modified starch comprises four steps of physical activation pretreatment, double chemical modification, composite enzyme hydrolysis and post-treatment, especially the synergistic effect of ultrasonic activation, synchronous crosslinking-hydrophobic modification and specific proportion composite enzyme preparation hydrolysis, so that the modified starch prepared by the preparation method has excellent emulsification stability, freeze-thaw stability and anti-aging property; the emulsification stability index is as high as 93.7% to 96.8%; the water analysis rate after 5 freeze-thaw cycles is as low as 7.1% to 8.9%; the retrogradation degree after 7-day cold storage at 4 DEG C is as low as 12.7% to 15.1%; and the technical problems of dry and hard taste and easy retrogradation of high-protein biomimetic food are effectively solved.

Description

Technical Field

[0001] This application relates to the field of food additives technology, and in particular to a method for preparing starch modified by a combination of physical-chemical-enzymatic methods, the modified starch and its applications. Background Technology

[0002] With the popularization of healthy eating concepts and the rapid development of the plant-based food market, the market size of high-protein biomimetic foods such as plant-based meat and low-fat salad dressings is constantly expanding. However, these products generally suffer from low consumer repurchase rates due to their dry and hard texture and easy aging, which seriously affects the large-scale production of these products and has become a key bottleneck restricting the further development of the entire industry. Adding modified starch is one of the important means to improve this problem.

[0003] Existing starch modification technologies mainly include physical modification, chemical modification, and enzymatic modification. Among them, physical modification is simple to operate and has high safety, but the degree of modification is limited, making it difficult to endow starch with new functional properties and greatly limiting its application. Chemical modification can efficiently introduce functional groups and significantly improve starch's special properties such as freeze-thaw resistance and emulsification, but the production process involves chemical reagents, posing environmental and safety hazards such as solvent toxicity, acid and alkali corrosion, and wastewater treatment. Moreover, the problem of chemical reagent residues in the product also restricts its application in high-end foods. Enzymatic modification has mild conditions, high specificity, and is green and safe, but it is expensive and has a limited range of functional groups that can be introduced, resulting in a single modification effect.

[0004] In recent years, researchers have attempted to combine various modification methods to overcome the limitations of single modification, mainly including physical-enzymatic methods, cross-linking-esterification, physical-chemical combined modification, and diversified premixing methods. Among these, physical-enzymatic combined modification, while green and efficient, does not introduce chemical functional groups, resulting in insufficient lipophilic emulsification; cross-linking-esterification chemical combined modification, while imparting good freeze-thaw resistance and lipophilicity to starch, does not involve enzymatic reconstruction, limiting its anti-aging properties; physical-chemical combined modification often uses dodecenyl succinic anhydride instead of octenyl succinic anhydride and does not involve cross-linking and enzymatic reconstruction, thus its freeze-thaw stability needs improvement; diversified premixing methods are merely physical mixing, making it difficult to achieve chemical coupling and synergistic optimization of multiple functions. In summary, although existing technologies have explored combinations of various modification methods, they still suffer from technical problems such as single modification pathways, lack of deep coupling, significant chemical reagent residue issues, and the inability to achieve synergistic optimization of multiple functions. Summary of the Invention

[0005] To overcome at least one of the problems existing in the prior art, one objective of this application is to provide a method for preparing starch modified by a combination of physical, chemical, and enzymatic methods. This method involves four main steps: physical activation pretreatment, dual chemical modification, compound enzymatic hydrolysis, and post-treatment. In particular, the compound enzymatic hydrolysis step introduces a compound enzyme preparation composed of starch branching enzyme, β-amylase, and pullulanase in a specific ratio of active units. This achieves directional shearing and reconstruction of the branched structure of starch molecular chains, thereby obtaining modified starch that combines the excellent emulsifying stability imparted by chemical modification with the superior anti-aging properties imparted by enzymatic modification. This solves the technical problems of insufficient starch structure regulation and poor shear resistance and emulsification effect caused by using only physical, chemical, or enzymatic modifications. A second objective of this application is to provide the modified starch prepared by the aforementioned method of combining physical, chemical, and enzymatic methods. A third objective of this application is to provide applications for the modified starch.

[0006] Therefore, this application adopts the following technical solution: The first aspect of this application provides a method for preparing starch modified by a combination of physical, chemical, and enzymatic methods, comprising the following steps: S1. Physical activation pretreatment: Disperse the raw starch in water and perform ultrasonic treatment to obtain an activated starch suspension with a mass fraction of 5%~15%. S2, Dual Chemical Modification: The pH of the activated starch suspension is adjusted to alkaline, a crosslinking agent and a hydrophobic modifier are added, and a chemical reaction is carried out to obtain a dual chemically modified starch suspension. S3. Compound Enzymatic Hydrolysis: Adjust the pH of the double chemically modified starch suspension to 6.0~6.5, set the temperature to 48~52℃, and add compound enzyme preparation to carry out enzymatic hydrolysis reaction; S4. Post-processing: The complex enzyme preparation is inactivated by heating, and then centrifuged, washed and dried to obtain starch modified by physical-chemical-enzymatic method. The compound enzyme preparation comprises starch branching enzyme, β-amylase and pullulanase in an activity unit ratio of 1:(1~2):(0.3~0.8).

[0007] In the preparation method of the physical-chemical-enzymatic combined modified starch of this application, the modified starch is prepared by physical activation pretreatment, dual chemical modification and compound enzymatic hydrolysis. First, ultrasonic physical activation pretreatment is used to loosen the structure of the raw starch granules and increase the specific surface area, which is conducive to the full contact and action of subsequent chemical reagents and compound enzyme preparations, thereby improving the uniformity of modification and reaction efficiency. Then, dual chemical modification is carried out by cross-linking agent and hydrophobic modifier, which simultaneously improves the stability of the cross-linking structure and hydrophobicity of starch, giving starch basic shear resistance and emulsification properties. Finally, based on chemical modification, enzymatic hydrolysis is carried out by a compound enzyme preparation consisting of starch branching enzyme, β-amylase and pullulanase in a specific ratio, which is conducive to the directional shearing of starch molecular chains and reconstruction of branched structure, making the starch gelatinization characteristics, gel strength and solubility more controllable. The modified starch has strong emulsification stability and good anti-aging properties, and fundamentally inhibits the retrogradation of starch.

[0008] Preferably, in step S1, the frequency of the ultrasonic treatment is 25~40kHz, the power is 80~150W, and the time is 10~15min. More preferably, in step S1, the frequency of the ultrasonic treatment is 30~40kHz, the power is 89~150W, and the time is 10~15min.

[0009] The aforementioned ultrasonic frequencies help to achieve appropriate swelling of particles and loosening of crystal regions, thereby improving the efficiency of subsequent reactions. At the same time, the power and time can be controlled to avoid excessive starch degradation or excessive energy consumption caused by excessive ultrasonic strength, thus stabilizing the activation degree of the raw starch.

[0010] Preferably, in step S1, the raw starch is selected from at least one of corn starch, potato starch, cassava starch, and wheat starch. More preferably, in step S1, the raw starch is selected from at least one of corn starch, potato starch, and cassava starch.

[0011] Preferably, in step S2, the pH is 8.0~9.0, the temperature of the chemical reaction is 35~50℃, and the reaction time is 45~60 min. More preferably, in step S2, the pH is 8.2~9.0, the temperature of the chemical reaction is 35~45℃, and the reaction time is 45~60 min.

[0012] Preferably, in step S2, the crosslinking agent is sodium trimetaphosphate, and the amount of the crosslinking agent added is 0.5% to 1.0% of the dry weight of the raw starch; the hydrophobic modifier is octenyl succinic anhydride, and the amount of the hydrophobic modifier added is 2.0% to 4.0% of the dry weight of the raw starch. More preferably, in step S2, the crosslinking agent is sodium trimetaphosphate, and the amount of the crosslinking agent added is 0.6% to 1.0% of the dry weight of the raw starch; the hydrophobic modifier is octenyl succinic anhydride, and the amount of the hydrophobic modifier added is 2.5% to 4.0% of the dry weight of the raw starch. Even more preferably, in step S2, the crosslinking agent is sodium trimetaphosphate, and the amount of the crosslinking agent added is 0.7% to 1.0% of the dry weight of the raw starch; the hydrophobic modifier is octenyl succinic anhydride, and the amount of the hydrophobic modifier added is 2.8% to 4.0% of the dry weight of the raw starch.

[0013] The weakly alkaline conditions in step S2 facilitate the full reaction of the crosslinking agent, hydrophobic modifier, and starch, improving the degree of crosslinking and uniformity of substitution. Simultaneously, they prevent starch degradation or increased side reactions caused by strong alkali. The temperature of 35-50°C allows the reaction to occur in the non-gelatinized state of the starch, resulting in a more uniform distribution of substituents. Furthermore, the phosphate crosslinking bonds introduced by sodium metaphosphate enhance the starch's heat resistance, acid resistance, and shear resistance, while the hydrophobic groups introduced by octenyl succinic anhydride impart excellent emulsifying stability to the starch. Within the range of crosslinking agent and hydrophobic modifier additions in this application, a suitable degree of crosslinking and substitution is ensured to achieve the modification effect, while avoiding gelatinization difficulties caused by excessive crosslinking or off-flavor problems caused by excessive hydrophobic groups.

[0014] Preferably, in step S3, the ratio of the activity units of starch branching enzyme, β-amylase and pullulanase in the compound enzyme preparation is 1:(1.4~2):(0.5~0.8).

[0015] At this ratio, the linear short chains generated by pullulanase debranching provide sufficient substrate for β-amylase. The non-reducing ends exposed after orderly cleavage by β-amylase are conducive to the transglycosylation of starch branching enzyme. Meanwhile, the newly generated branch points of starch branching enzyme can resist excessive hydrolysis by pullulanase. A dynamic equilibrium enzymatic hydrolysis system is formed among starch branching enzyme, β-amylase, and pullulanase, thereby achieving effective regulation of starch molecular chain length distribution, branching degree, and molecular weight, so that the modified starch achieves a better level of anti-aging properties, gel properties, and emulsification stability.

[0016] Preferably, in step S3, the amount of the compound enzyme preparation added is 0.3% to 0.6% of the dry weight of the raw starch. More preferably, in step S3, the amount of the compound enzyme preparation added is 0.4% to 0.6% of the dry weight of the raw starch.

[0017] In step S3, the amount of compound enzyme preparation added is controlled to be 0.3% to 0.6% of the dry weight of raw starch. This is conducive to achieving full enzymatic hydrolysis of starch. If the amount added is less than 0.3% of the dry weight of raw starch, the enzymatic hydrolysis is insufficient, making it difficult to achieve effective modification of the molecular structure. If it is higher than 0.6%, it may lead to excessive enzymatic hydrolysis and the generation of too many small molecule sugars, which will affect the product yield and gel performance.

[0018] Preferably, in step S3, the enzymatic hydrolysis reaction takes 90-110 minutes. More preferably, in step S3, the enzymatic hydrolysis reaction takes 100-110 minutes.

[0019] Preferably, in step S4, the drying is spray drying, the inlet air temperature of the spray drying is 150~160℃, the outlet air temperature of the spray drying is 80~90℃, and the drying is carried out until the moisture content is 12~15wt%. More preferably, in step S4, the drying is spray drying, the inlet air temperature of the spray drying is 155~160℃, the outlet air temperature of the spray drying is 80~90℃, and the drying is carried out until the moisture content is 12~13wt%.

[0020] Spray drying has the characteristics of rapid dehydration. It dries quickly and has a short heating time, avoiding the damage to the starch molecular structure and chemical or enzymatic modification effect caused by traditional long-term drying. Controlling the inlet and outlet air temperatures within the above range helps to control the heating temperature of the material while ensuring rapid evaporation of moisture, thus preventing starch from aging or degrading due to prolonged high temperature.

[0021] Preferably, in step S4, the inactivation temperature is 85-95°C, and the inactivation time is 6-10 min; the centrifugation speed is 3000-4000 rpm, and the centrifugation time is 15-20 min. More preferably, in step S4, the inactivation temperature is 90-95°C, and the inactivation time is 6-10 min; the centrifugation speed is 3200-4000 rpm, and the centrifugation time is 15-20 min.

[0022] The second aspect of this application provides modified starch prepared according to the physical-chemical-enzymatic method for modifying starch described in the first aspect of this application.

[0023] This modified starch combines the loose structure resulting from physical activation, the stability resulting from chemical cross-linking, the emulsifying properties resulting from hydrophobic modification, and the controllable structure resulting from specific enzymatic hydrolysis. It exhibits excellent comprehensive performance, with good gelatinization characteristics, gel strength, shear resistance, and emulsification stability, as well as high safety performance.

[0024] The third aspect of this application provides the use of modified starch prepared by the physical-chemical-enzymatic method for modifying starch according to the first aspect of this application, or the modified starch according to the second aspect of this application, in the preparation of high-protein biomimetic foods.

[0025] The high-protein biomimetic foods include, but are not limited to, plant-based meat, low-fat salad dressings, frozen pasta, high-protein nutrition bars, or ready-to-eat meal replacement foods.

[0026] Compared with the prior art, this application has at least the following beneficial effects: 1) The method for preparing modified starch using a combination of physical, chemical, and enzymatic methods in this application involves four major steps: physical activation pretreatment, dual chemical modification, compound enzymatic hydrolysis, and post-treatment. In particular, the compound enzymatic hydrolysis step employs a compound enzyme preparation consisting of starch branching enzyme, β-amylase, and pullulanase in a specific activity unit ratio of 1:(1~2):(0.3~0.8). This process achieves directional shearing of starch molecular chains and reconstruction of branched structure, resulting in modified starch that possesses the excellent emulsifying stability imparted by chemical modification. The excellent anti-aging properties imparted by qualitative and enzymatic modification result in an emulsification stability index as high as 93.7%~96.8%, a water leaching rate as low as 7.1%~8.9% after 5 freeze-thaw cycles, and a reversion rate as low as 12.7%~15.1% after 7 days of refrigeration at 4℃. The residual amounts of sodium trimetaphosphate and octenyl succinic acid groups are also low. In the application of high-protein biomimetic foods with a 30wt% reduction in vegetable oil, the sensory score is still as high as 8.6 points or more, effectively solving the technical problems of dry and hard texture and easy aging and reversion in high-protein biomimetic foods.

[0027] 2) In the preparation method of the physical-chemical-enzymatic modified starch of this application, the three steps of physical activation pretreatment, dual chemical modification and compound enzymatic hydrolysis are carried out continuously in the same reaction system without intermediate separation, washing and drying steps. Compared with the traditional step-by-step modification process, the production cycle is greatly shortened, energy consumption is reduced, the production process is simple and efficient, and it is suitable for industrial production. Detailed Implementation

[0028] The following detailed description of the contents of this application is provided through specific embodiments, comparative examples, and tables, but is not limited to all the arguments and data.

[0029] In the preparation method of starch modified by a combination of physical-chemical-enzymatic methods, food-grade corn starch is used. In the compound enzyme preparation, the "activity unit" of starch branching enzyme, β-amylase and pullulanase all refer to enzyme activity unit (U), that is, the amount of enzyme required to convert 1 micromolar substrate per minute is 1 activity unit (1 U). The enzyme activity unit is based on the standard determination method provided by each enzyme preparation manufacturer. In the compound enzyme preparation, the three enzymes are all commercially available freeze-dried powders. The specific activity of starch branching enzyme is 500 U / mg, the specific activity of β-amylase is 1000 U / mg, and the specific activity of pullulanase is 300 U / mg.

[0030] It is particularly important to emphasize that, unless otherwise specified, the raw materials, reagents or devices used in this application can be obtained from conventional commercial sources.

[0031] Examples of modified starch: The method for preparing modified starch using a combination of physical, chemical, and enzymatic methods, as described in this application, specifically includes the following steps: S1. Physical activation pretreatment: Disperse the raw starch in water and ultrasonically treat it for 10-15 minutes at a frequency of 25-40kHz and a power of 80-150W to obtain an activated starch suspension with a mass fraction of 5%-15%. S2. Dual chemical modification: The pH of the above activated starch suspension was adjusted to 8.0-9.0 with 1 mol / L NaOH solution, and the temperature was 35-50℃. 0.5%-1.0% of the dry weight of the raw starch and 2.0%-4.0% of the dry weight of the raw starch were added, and the chemical reaction was carried out under stirring for 45-60 min to obtain a dual chemically modified starch suspension. S3. Compound enzyme hydrolysis: The pH of the above-mentioned double chemically modified starch suspension was adjusted to 6.0~6.5 with 0.5mol / L HCl solution, the temperature was 48~52℃, and 0.3%~0.6% of the dry weight of the raw starch was added to carry out the enzymatic hydrolysis reaction for 90~110min. S4. Post-treatment: Heat to 85~95℃ and hold for 6~10 minutes to inactivate the complex enzyme preparation. Then centrifuge, wash and dry to obtain the starch modified by physical-chemical-enzymatic method.

[0032] Regarding step S1, in some specific embodiments, the frequency of ultrasonic treatment can be 25kHz, 30kHz, 35kHz, or 40kHz, the power can be 80W, 100W, 120W, or 150W, and the time can be 10min, 12min, or 15min. In the activated starch suspension, the mass fraction of the raw starch can be 5%, 7%, 10%, 13%, or 15%.

[0033] Regarding step S2, in some specific implementations, the pH can be 8.0, 8.2, 8.5, or 9.0; the chemical reaction temperature can be 35°C, 40°C, 45°C, or 50°C; and the chemical reaction time can be 45 min, 50 min, 55 min, or 60 min. The crosslinking agent is sodium trimetaphosphate, and the amount added can be 0.5%, 0.6%, 0.8%, 0.9%, or 1.0% of the dry weight of the raw starch; the hydrophobic modifier is octenyl succinic anhydride, and the amount added can be 2.0%, 3.0%, or 4.0% of the dry weight of the raw starch.

[0034] Regarding step S3, in some specific implementations, the pH is adjusted to 6.0, 6.3, or 6.5; the enzymatic hydrolysis temperature can be 48°C, 50°C, or 52°C; and the time can be 90 min, 100 min, or 110 min. The compound enzyme preparation can be composed of starch branching enzyme, β-amylase, and pullulanase in an activity unit ratio of 1:1:0.3, 1:1.5:0.5, 1:2:0.5, or 1:2:0.8. The amount of the compound enzyme preparation added accounts for 0.3%, 0.4%, or 0.6% of the dry weight of the raw starch.

[0035] Regarding step S4, in some specific embodiments, the inactivation temperature can be 85℃, 90℃, or 95℃, and the inactivation time can be 6 min, 8 min, or 10 min; the centrifugation speed can be 3000 rpm, 3200 rpm, 3600 rpm, or 4000 rpm, and the centrifugation time can be 15 min, 18 min, or 20 min. The inlet air temperature for spray drying can be 150℃, 155℃, or 160℃, and the outlet air temperature for spray drying can be 80℃, 85℃, or 90℃, drying to a moisture content of 12wt%, 13wt%, 14wt%, or 15wt%.

[0036] The following are examples and comparative examples of the preparation method of modified starch using the combined physical-chemical-enzymatic method of this application: Example 1

[0037] A method for preparing starch using a combined physical-chemical-enzymatic method, specifically including the following steps: S1. Physical activation pretreatment: 100g of raw starch is dispersed in 900g of deionized water and ultrasonically treated for 15min at a frequency of 30kHz and a power of 120W to obtain an activated starch suspension with a mass fraction of 10%. S2, Dual Chemical Modification: The pH of the above activated starch suspension was adjusted to 8.5 with 1 mol / L NaOH solution, heated to 45℃, and 0.8 g of sodium trimetaphosphate and 3 g of octenyl succinic anhydride were added. The chemical reaction was carried out for 50 min with stirring to obtain a dual chemically modified starch suspension. S3. Enzymatic hydrolysis using a compound enzyme method: Weigh the lyophilized powders of starch branching enzyme, β-amylase, and pullulanase respectively according to the activity ratio of 1:1.5:0.5 (based on the specific activity of the enzyme preparations used, the mass of the lyophilized powders of starch branching enzyme, β-amylase, and pullulanase are 193.5 mg, 145.2 mg, and 161.3 mg, respectively, with a total mass of 0.5 g). Dissolve and mix thoroughly with 5 mL of 50 mM phosphate buffer at pH 6.2. Slowly add the mixture to a double chemically modified starch suspension that has been adjusted to pH 6.5 with 0.5 mol / L HCl solution and is kept at 50 °C while stirring. After the addition is complete, perform the enzymatic hydrolysis reaction at a constant temperature of 50 °C for 100 min. S4. Post-treatment: Heat to 90℃ and hold for 10 min to inactivate the complex enzyme preparation. Centrifuge at 3500 rpm for 15 min, discard the supernatant, collect the precipitate and wash it twice with deionized water. Spray dry the washed filter cake, controlling the inlet air temperature at 155℃ and the outlet air temperature at 85℃, until the moisture content is 13wt%, to obtain the starch modified by the combined physical-chemical-enzymatic method. Example 2

[0038] A method for preparing starch modified by a combination of physical, chemical, and enzymatic methods is identical to that in Example 1, except that step S1 is performed as described below. Step S1 in Example 2 is as follows: S1. Physical activation pretreatment: 100g of raw starch was dispersed in 567g of deionized water and ultrasonically treated for 15min at a frequency of 30kHz and a power of 120W to obtain an activated starch suspension with a mass fraction of 15%. Example 3

[0039] A method for preparing modified starch using a combination of physical, chemical, and enzymatic methods is the same as in Example 1, except that the mass of sodium tripolyphosphate in step S2 of Example 3 is 0.5g. Example 4

[0040] A method for preparing modified starch using a combination of physical, chemical, and enzymatic methods is the same as in Example 1, except that in step S3 of Example 4, the pH is adjusted to 6.0 and the temperature is 48°C. Example 5

[0041] A method for preparing starch modified by a combination of physical, chemical, and enzymatic methods is identical to that in Example 1, except that step S3 is performed according to the following steps. Step S3 in Example 5 is as follows: S3. Compound Enzymatic Hydrolysis: Weigh out the lyophilized powders of starch branching enzyme, β-amylase, and pullulanase respectively according to the activity ratio of starch branching enzyme, β-amylase, and pullulanase (calculated based on the specific activity of the enzyme preparations used, the mass of starch branching enzyme, β-amylase, and pullulanase lyophilized powders are 250mg, 125mg, and 125mg respectively, with a total mass of 0.5g). Dissolve and mix thoroughly with 5mL of 50mM phosphate buffer at pH 6.2. Slowly add the mixture to a double chemically modified starch suspension adjusted to pH 6.5 with 0.5mol / L HCl solution and at a temperature of 50℃ while stirring. After the addition is complete, perform enzymatic hydrolysis at a constant temperature of 50℃ for 100min.

[0042] Comparative Example 1: A method for preparing modified starch using a combination of physical, chemical, and enzymatic methods is the same as in Example 1, except that in Comparative Example 1, step S1 is as follows: 100g of raw starch was dispersed in 900g of deionized water and stirred evenly (without ultrasonic treatment) to obtain an activated starch suspension with a mass fraction of 10%.

[0043] Comparative Example 2: A method for preparing modified starch using a combination of physical, chemical, and enzymatic methods is the same as that in Comparative Example 1, except that Comparative Example 2 does not include step S3; that is, the post-processing step S4 is performed directly after step S2.

[0044] Comparative Example 3: A method for preparing modified starch using a combination of physical, chemical, and enzymatic methods is the same as in Example 1, except that step S2 in Comparative Example 3 is as follows: The pH of the activated starch suspension was adjusted to 8.5 with 1 mol / L NaOH solution, heated to 45°C, and 0.8 g of sodium trimetaphosphate was added. The mixture was stirred and allowed to react for 25 min. Then, 3 g of octenyl succinic anhydride was added and the mixture was stirred and allowed to react for 25 min to obtain a double chemically modified starch suspension.

[0045] Comparative Example 4: A method for preparing modified starch using a combined physical-chemical-enzymatic method is identical to that in Example 1, except that step S3 is performed as described below. Step S3 in Comparative Example 4 is as follows: S3. Compound Enzymatic Hydrolysis: Weigh the lyophilized powders of starch branching enzyme, β-amylase, and pullulanase respectively according to the activity ratio of starch branching enzyme, β-amylase, and pullulanase in a ratio of 1:1:1 (based on the specific activity of the enzyme preparations used, the mass of starch branching enzyme, β-amylase, and pullulanase lyophilized powders are 157.9 mg, 78.9 mg, and 263.2 mg, respectively, with a total mass of 0.5 g). Dissolve and mix thoroughly with 5 mL of 50 mM phosphate buffer at pH 6.2. Slowly add the mixture to a double chemically modified starch suspension adjusted to pH 6.5 with 0.5 mol / L HCl solution at a temperature of 50 °C while stirring. After the addition is complete, perform enzymatic hydrolysis at a constant temperature of 50 °C for 100 min.

[0046] Material performance testing: The modified starches from Examples 1-5 and Comparative Examples 1-4 were subjected to various performance tests, and the test methods are as follows: 1. Emulsion Stability Index (ESI): Weigh 1g of modified starch, add 99mL of deionized water to prepare a starch emulsion, gelatinize in a boiling water bath for 15min, cool to room temperature, add 10mL of soybean oil, shear emulsify in a high-speed shear emulsifier at 10000rpm for 3min, immediately place in a 50℃ constant temperature oven and let stand for 24h, measure the volume of the lower aqueous phase, and calculate according to the following formula: Emulsion Stability Index (%) = (total volume of initial added aqueous phase - volume of lower aqueous phase) / total volume of initial added aqueous phase × 100%. The higher the index, the better the emulsion stability.

[0047] 2. Freeze-thaw stability (water extraction rate): Weigh 6g of modified starch, add 94mL of deionized water to prepare a starch emulsion, gelatinize in a boiling water bath for 20min to obtain starch paste, cool to room temperature, freeze at -18℃ for 24h, take it out and thaw at room temperature for 6h. This is one freeze-thaw cycle. Repeat the cycle 5 times, centrifuge at 3000rpm for 15min, separate, weigh the mass of the extracted water, and calculate according to the following formula: Water extraction rate (%) = mass of extracted water / total mass of starch paste × 100%. The lower the value, the better the freeze-thaw stability.

[0048] 3. Refrigeration Retrogradation: Weigh 10g of modified starch, add 90mL of deionized water to prepare a starch emulsion, gelatinize in a boiling water bath for 20min, cool to room temperature and refrigerate at 4℃ for 7d. Use a rapid viscosity analyzer to measure the peak viscosity of the starch paste before and after refrigeration, and calculate according to the following formula: Retrogradation (%) = (peak viscosity after refrigeration - peak viscosity before refrigeration) / peak viscosity before refrigeration × 100%. The lower the value, the better the anti-aging retrogradation performance.

[0049] 4. Sodium tripolyphosphate residue: Tested according to GB 5009.256-2016 standard.

[0050] 5. Residual octenyl succinic acid group: Tested according to GB 1886.370-2023 standard.

[0051] 6. Sensory evaluation: The control group sample was prepared according to the following formula: Weigh 100g of soy protein isolate, 10g of vegetable oil, 80g of ice water, 1.5g of salt, 1.0g of white sugar, 0.1g of sodium tripolyphosphate, and 0.1g of sodium pyrophosphate. Mix all the dry ingredients evenly, then add ice water and vegetable oil, and stir at low speed for 3 minutes to make minced meat. Take 50g of minced meat and shape it by hand into a meat patty with a diameter of 6cm and a thickness of 1cm. Steam it at 100℃ for 15 minutes and cool it to room temperature for later use. Preparation of test group samples in Examples 1-5 and Comparative Examples 1-4: Based on the control group formula, the amount of vegetable oil was reduced from 10g to 7g (equivalent to a 30wt% reduction in vegetable oil), and 5g of modified starch for the test group was added. Other ingredients and preparation processes were the same as the control group, resulting in the test group samples for Examples 1-5 and Comparative Examples 1-4. The control group and test group samples were randomly coded and placed on white plates. Ten professionally trained sensory evaluators (half male and half female, aged 25-45 years) evaluated the samples in an independent sensory evaluation room with a temperature of 25±1℃, humidity of 50±5%, and red lighting. After tasting one sample, rinse mouth with water and wait 2 minutes. Evaluation indicators included juiciness and smoothness, using a 10-point scoring system (1-2 points: very poor, dry and rough texture; 3-4 points: poor, dry and lacking smoothness; 5-6 points: average, acceptable but not juicy; 7-8 points: good, juicy and smooth texture; 9-10 points: excellent, juicy and very smooth). For each sample, the highest and lowest scores were removed, and the arithmetic mean of the remaining 8 scores was taken as the final sensory score, rounded to one decimal place. The difference in scores between the control group and the test group was compared to evaluate the oil substitution effect of the modified starch.

[0052] The test performance of Examples 1-5 and Comparative Examples 1-4 is shown in Table 1 below:

[0053] In the modified starches of Examples 1-5, the physical-chemical-enzymatic method of this application was used to prepare the modified starch. Through four major steps—physical activation pretreatment, dual chemical modification, compound enzymatic hydrolysis, and post-treatment—especially the synergistic effect of ultrasonic activation, simultaneous cross-linking-hydrophobic modification, and enzymatic hydrolysis with a specific ratio of compound enzyme preparation, the modified starches prepared using this method exhibit excellent emulsification stability, freeze-thaw stability, and anti-aging properties. The emulsification stability index is as high as 93.7%~96.8%, and the water separation rate after five freeze-thaw cycles is as low as 7.1%~8.9%. The degree of retrogradation after refrigeration at 4℃ for 7 days was as low as 12.7%~15.1%, with low residual sodium trimetaphosphate and octenyl succinic acid groups. In the application of high-protein biomimetic food with 30wt% reduced vegetable oil, the sensory score was still as high as 8.6 points or above, which was significantly better than the control group without modified starch. This shows that the modified starch obtained by the preparation method of this application has been significantly optimized in terms of emulsification performance, freeze-thaw stability, anti-aging properties, food safety and application taste, effectively solving the technical problems of dry and hard taste, easy retrogradation and high chemical residues in high-protein biomimetic food in the prior art.

[0054] Compared with Example 1, Comparative Example 1's method for preparing starch using a combined physical-chemical-enzymatic method did not involve ultrasonic activation in step S1; only ordinary stirring and dispersion were used. The results showed that the emulsification stability index of Comparative Example 1 was significantly reduced, the water separation rate after 5 freeze-thaw cycles increased to 14.8%, the degree of reversion after 7 days of refrigeration reached 19.4%, the sensory score was only 7.9, and the residue was also relatively higher. This may be because without ultrasonic activation, the starch granules have a dense structure, making it difficult for chemical reagents and compound enzyme preparations to fully penetrate and bind with starch molecules, resulting in uneven modification and insufficient reaction, which directly affects its emulsification, stability, and anti-aging properties.

[0055] Compared to Comparative Example 1, Comparative Example 2 omits step S3, proceeding directly to the post-processing step S4 after step S2. The results show that Comparative Example 2 exhibits an emulsification stability index of only 82.3%, a water leaching rate of 21.5% after five freeze-thaw cycles, a cold storage retrogradation rate of 27.3%, and a significant drop in sensory score to 6.6. This indicates that although cross-linking and hydrophobic modification are retained, the lack of directional shearing and branch reconstruction of molecular chains by the complex enzyme preparation makes starch molecules prone to recrystallization and retrogradation, failing to achieve a long-lasting anti-aging effect. Furthermore, the uneven distribution of molecular structure further weakens the emulsification and stability effects, rendering it unsuitable for use in high-protein biomimetic foods.

[0056] Compared with Example 1, Comparative Example 3 used a step-by-step sequential addition of the crosslinking agent and hydrophobic modifier in step S2, instead of simultaneous dual chemical modification. The results showed that Comparative Example 3 had lower emulsification stability index, freeze-thaw stability, and anti-aging properties than Example 1, with a cold storage regeneration degree of 16.2% and a sensory score of 8.2. This may be because the step-by-step addition of the modifying agents leads to uneven distribution of crosslinking and hydrophobic groups, resulting in local over- or under-substitution, which disrupts the synergistic stability and emulsification mechanism of the two functional groups. Simultaneously, uneven distribution affects the subsequent enzymatic hydrolysis effect, leading to a decline in overall performance and failing to achieve the superior effect of simultaneous modification as described in this application.

[0057] Compared to Example 1, Comparative Example 4, in its physical-chemical-enzymatic combined modified starch preparation method, had a 1:1:1 ratio of starch branching enzyme, β-amylase, and pullulanase activity units in step S3 of the complex enzyme preparation, which is outside the scope of this application. The results showed that Comparative Example 4 had an emulsification stability index of 90.8%, a water analysis rate of 13.2% after 5 freeze-thaw cycles, a cold storage regeneration rate of 18.7%, and a sensory score of only 8.1. This may be because the proportion of pullulanase in the complex enzyme preparation was too high, easily causing excessive debranching, while the proportion of starch branching enzyme was insufficient to form a sufficient branched structure. The imbalance of the three proportions disrupted the synergistic enzymatic hydrolysis system, making it difficult to achieve better control over molecular chain length and branching structure, thus leading to a decline in anti-aging and other related properties.

[0058] Obviously, the above embodiments of this application are merely examples for clearly illustrating this application, and are not intended to limit the implementation of this application. For those skilled in the art, other variations or modifications can be made based on the above description. Any obvious variations or modifications derived from the technical solutions of this application are still within the protection scope of this application.

Claims

1. A method for preparing starch modified by a combination of physical-chemical-enzymatic methods, characterized in that, Includes the following steps: S1. Physical activation pretreatment: Disperse the raw starch in water and perform ultrasonic treatment to obtain an activated starch suspension with a mass fraction of 5%~15%. S2, Dual Chemical Modification: The pH of the activated starch suspension is adjusted to alkaline, a crosslinking agent and a hydrophobic modifier are added, and a chemical reaction is carried out to obtain a dual chemically modified starch suspension. S3. Compound enzyme hydrolysis: Adjust the pH of the double chemically modified starch suspension to 6.0~6.5, set the temperature to 48~52℃, and add compound enzyme preparation to carry out enzymatic hydrolysis reaction; S4. Post-processing: The complex enzyme preparation is inactivated by heating, and then centrifuged, washed and dried to obtain starch modified by physical-chemical-enzymatic method. The compound enzyme preparation comprises starch branching enzyme, β-amylase and pullulanase in an activity unit ratio of 1:(1~2):(0.3~0.8).

2. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S1, the frequency of the ultrasonic treatment is 25~40kHz, the power is 80~150W, and the time is 10~15min.

3. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S1, the raw starch is selected from at least one of corn starch, potato starch, cassava starch, and wheat starch.

4. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S2, the pH is 8.0~9.0, the temperature of the chemical reaction is 35~50℃, and the time of the chemical reaction is 45~60min.

5. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S2, the crosslinking agent is sodium trimetaphosphate, and the amount of crosslinking agent added is 0.5% to 1.0% of the dry weight of the raw starch. The hydrophobic modifier is octenyl succinic anhydride, and the amount of the hydrophobic modifier added is 2.0% to 4.0% of the dry weight of the raw starch.

6. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S3, the ratio of the activity units of starch branching enzyme, β-amylase and pullulanase in the compound enzyme preparation is 1:(1.4~2):(0.5~0.8).

7. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S3, the amount of the compound enzyme preparation added is 0.3% to 0.6% of the dry weight of the raw starch.

8. The method for preparing starch using a combined physical-chemical-enzymatic method according to claim 1, characterized in that, In step S4, the drying is spray drying, the inlet air temperature of the spray drying is 150~160℃, the outlet air temperature of the spray drying is 80~90℃, and the drying is carried out until the moisture content is 12~15wt%.

9. A modified starch, characterized in that, The modified starch is prepared by the physical-chemical-enzymatic method according to any one of claims 1 to 8.

10. The application of modified starch prepared by the physical-chemical-enzymatic method as described in claims 1-8, or the modified starch as described in claim 9, in the preparation of high-protein biomimetic foods.

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