Method for ultrasonic regulation and acidification of functional properties of cannabis sativa protein and application thereof

Abstract

The application discloses a method for ultrasonic regulation and control of functional properties of acidified cannabis sativa L. protein and application thereof. The application fully hydrates the cannabis sativa L. protein under acidification conditions, combines ultrasonic pretreatment, and performs heat induction to make the protein have ordered aggregation with enriched beta-folding, and meanwhile, improve the functional properties of the protein: the gel strength is 12.37 g under 60% ultrasonic power, which is about 33% higher than that of a non-ultrasonic control, the cohesiveness is 8.6 g, the emulsion stability is 185.89 min, which is about 2.6 times higher than that of the non-ultrasonic control, the apparent viscosity is improved and presents shear thinning behavior (power law index n1 < 1), and the strain breakage resistance is enhanced. The method can simultaneously regulate and control the ordered aggregation degree, gel property, emulsification and rheological property of the cannabis sativa L. protein, is suitable for strong acid type plant protein beverages, acid emulsions and high-acid gel food systems, and has the advantages of simple process, green safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of plant protein and acidic food ingredients, specifically relating to a method and application of ultrasonic regulation of the functional properties of acidified hemp seed protein. Background Technology

[0002] Hemp protein isolate (HPI), as an emerging high-quality plant protein resource, boasts a balanced essential amino acid composition, high digestibility (88%-91%), low allergenicity, and is rich in arginine and branched-chain amino acids, showing great promise in the development of plant-based foods. However, natural hemp protein exhibits poor solubility and weak gelling properties due to disulfide bond-induced aggregation and precipitation under neutral conditions, limiting its widespread application in the food industry.

[0003] In acidic food systems (such as fruit juices, acidic milk drinks, jellies, puddings, etc.), proteins are often near their isoelectric point or under acidic conditions, making them prone to aggregation and precipitation, resulting in poor product stability and a rough texture. Existing methods for improving the functional properties of plant proteins mainly include enzymatic modification, chemical modification, and physical modification. Among these, ultrasonic modification, as a green, non-thermal processing technology, can induce protein structure unfolding through cavitation effects, improving its solubility, emulsification, and gelling properties. However, current research mostly focuses on neutral or slightly acidic conditions, and systematic regulation of the multifunctional properties (ordered aggregation, gelling, emulsification, and rheological properties) of hemp seed protein under strongly acidic conditions (pH 2.0-3.0) using ultrasound-assisted acid induction remains a gap. Furthermore, there is no method that can simultaneously regulate the degree of ordered aggregation, gel strength, emulsification stability, and apparent viscosity of hemp seed protein and directly apply it to strongly acidic plant protein beverages, acidic emulsions, and high-acid gel food systems. Summary of the Invention

[0004] Objective: To address the technical limitations of existing technologies for hemp seed protein (HPI), which suffers from poor solubility and easy aggregation, resulting in weak gelation properties and requiring high concentrations and temperatures to form a gel in its natural state, thus restricting its application in acidic food systems. Furthermore, existing methods for improving the functional properties of plant proteins primarily focus on neutral or slightly acidic conditions. Systematic regulation of the multifunctional properties (ordered aggregation, gelation, emulsification, and rheological properties) of hemp seed protein under strongly acidic conditions (pH 2.0) through ultrasound-assisted acid induction remains lacking, particularly a method that simultaneously utilizes acid-induced ordered aggregation and ultrasound-assisted regulation to improve multiple functional properties of hemp seed protein. This invention provides a method for ultrasound-regulated acidified hemp seed protein functional properties. This invention utilizes acidification treatment and ultrasound pretreatment to synergistically regulate the molecular conformation and intermolecular forces of hemp seed protein, and provides protein gel or protein solution products with improved functional properties prepared using this method.

[0005] This invention fully hydrates hemp seed protein under pH 2.0 acidification conditions, followed by ultrasonic pretreatment with specific power to induce appropriate protein unfolding. Acid-induced electrostatic repulsion promotes the orderly aggregation of β-sheets, and thermal induction forms a protein gel with suitable gel strength, good emulsion stability, shear-thinning behavior, and a dense network structure. This invention solves the technical problems of poor functional properties of hemp seed protein under acidic conditions, limited effects of single ultrasonic modification, and the lack of systematic multi-index regulation methods in existing technologies.

[0006] Technical Solution: To achieve the above objectives, the present invention adopts the following technical solution: The method for ultrasound-regulated functional properties of acidified hemp seed protein according to the present invention is characterized by comprising the following steps:

[0007] (1) Disperse hemp seed protein in water, adjust the pH to strong acidity, and stir to obtain a dispersion;

[0008] (2) The dispersion obtained in step (1) is subjected to ultrasonic pretreatment. The ultrasonic frequency is 20-30 kHz, the power is 50%-60%, the treatment time is 10-15 min, and the pulse mode is on for 4-5 s and off for 4-5 s.

[0009] (3) Heat the dispersion obtained in step (2) at 80-90℃ for 2-3 h, and then cool it to obtain hemp seed protein gel or protein solution with improved functional properties.

[0010] The concentration of hemp seed protein in the dispersion is 90-100 mg / mL, and the pH is adjusted to 2.0-3.0.

[0011] The ultrasonic pretreatment power is 55-60%, the treatment time is 10-12 min, and the pulse mode is on for 5 s and off for 5 s.

[0012] The heating reaction is carried out at 85-90℃ for 2-2.5 h, followed by cooling in an ice bath and standing at 4℃ for 10-12 h.

[0013] The functional characteristics include at least one of the following:

[0014] (a) Increased degree of ordered aggregation of β-sheets: Th T fluorescence intensity reaches its peak at 60% power;

[0015] (b) The gel strength is increased to 10-15 g, and the adhesiveness is increased to 5-10 g;

[0016] (c) Emulsion stability (ESI) improved to ≥ 180 min;

[0017] (d) The apparent viscosity increases and exhibits shear-thinning behavior, with a power-law exponent n1 < 1;

[0018] (e) Resistance to strain failure is optimal at 60% power.

[0019] The acidified hemp seed protein gel or protein solution prepared by the method described in this invention.

[0020] The application of the acidified hemp seed protein gel or protein solution described in this invention in the preparation of acidic foods.

[0021] The pH of the gel or protein solution is 2.0-3.0, which can be used directly in acidic food systems, or after preparation, the pH can be adjusted to 4.0-6.5 before use.

[0022] The acidic foods include strongly acidic plant protein beverages, acidic emulsions, and high-acid gel foods.

[0023] The strong acid plant protein beverage has a pH of 2.5-4.0; the acidic emulsion is an oil-in-water emulsion with an oil phase volume fraction of 5%-30%; and the high acid gel food is jelly, pudding, or sour curd.

[0024] Furthermore, the gel or protein solution is used as an acidic food base or emulsion stabilizer.

[0025] The present invention discloses a method for ultrasonically regulating the functional properties of acidified hemp seed protein, comprising the following steps: dispersing hemp seed protein (HPI) in water, adjusting the pH to acidic, and subjecting the dispersion to ultrasonic pretreatment; heating the ultrasonically treated dispersion to react, and then cooling and allowing it to stand to obtain hemp seed protein gel or protein solution with improved functional properties.

[0026] Preferably, 2 M HCl is used to adjust the pH to 2.0.

[0027] Preferably, the ultrasonic pretreatment conditions are: frequency 20 kHz, power 60% (corresponding to a power density of approximately 40 W / cm²), treatment time 10 min, and pulse mode on for 5 s and off for 5 s.

[0028] Preferably, the concentration of hemp seed protein in the dispersion is 95 mg / mL, and the heating reaction conditions are heating at 85°C for 2 h, followed by cooling in an ice bath and standing at 4°C for 12 h.

[0029] The acidified hemp seed protein gel or protein solution prepared by the preparation method described in this invention.

[0030] The pH of the gel or protein solution is 2.0-3.0, which can be used directly in acidic food systems, or after preparation, the pH can be adjusted to 4.0-6.5 before use.

[0031] Among them, the gel strength of the gel at 60% ultrasonic power is ≥12 g, the adhesiveness is ≥8 g, the emulsification stability (ESI) is ≥180 min, the apparent viscosity exhibits shear thinning behavior, and the power law exponent n1 < 1; the resistance to strain failure is optimal at 60% power.

[0032] Among them, the denaturation temperature (T) of the gel at 60% ultrasonic power d The initial temperature (T0 = 113.08℃) and the starting temperature (T0 = 67.97℃) were both higher than those of the control group without ultrasound (T = 113.08℃). d =108.5℃, T0=61.34℃), indicating that the protein was moderately unfolded and had moderate thermal stability, which is beneficial to functional improvement. Thermal stability decreased at 30% power (T0=61.34℃). d =90.1℃), insufficient protein development; thermal stability further improved at 90% power (T d =126.4℃), but excessive ultrasounding can actually reduce gel strength and emulsification. Therefore, 60% power strikes the best balance between "moderate unfolding" and "functional optimization".

[0033] The application of ultrasound in the preparation of acidified hemp seed protein gel or protein solution of the present invention is that ultrasound induces the appropriate unfolding of protein through cavitation effect, and synergistically regulates the degree of orderly aggregation of protein β-sheet, gelation, emulsification and rheological properties with acidification conditions.

[0034] Furthermore, the method for ultrasound-regulated functional properties of acidified hemp seed protein includes the following steps:

[0035] (1) Acidification hydration: Disperse hemp seed protein (HPI) powder in ultrapure water to prepare a dispersion with a concentration of 95 mg / mL. Adjust the pH to 2.0 with 2 M HCl and stir magnetically at room temperature for 12 h to fully hydrate it.

[0036] (2) Ultrasonic pretreatment: The dispersion obtained in step (1) was placed in an ice bath and ultrasonically pretreated using an ultrasonic cell disruptor. The ultrasonic probe diameter was 6 mm and the immersion depth was 1-2 cm. The ultrasonic parameters were: frequency 20 kHz, power 60% (corresponding to a power density of approximately 40 W / cm²), treatment time 10 min, and pulse mode (on for 5 s, off for 5 s). The ultrasonic pretreatment disrupted the non-covalent forces between protein molecules through the shear force generated by the cavitation effect, causing the hemp seed protein molecules to unfold appropriately, exposing more binding sites and promoting the subsequent orderly aggregation of β-sheets.

[0037] (3) Thermally induced gelation: The dispersion obtained in step (2) is heated in an 85°C water bath for 2 hours, then immediately cooled in an ice bath and placed in a 4°C refrigerator for 12 hours to obtain acidified hemp seed protein gel or protein solution with improved functional properties.

[0038] The pH 2.0 acidification condition described in step (1) of this invention is one of the key process parameters. This acidic condition causes the hemp seed protein molecules to carry a positive charge, generating strong electrostatic repulsion between molecules, thereby preventing random aggregation and promoting the orderly arrangement of protein molecules along a specific direction, forming an ordered structure enriched with β-sheets.

[0039] The 60% ultrasonic power mentioned in step (2) of this invention is the optimal control power selected through experiments. At this power, ultrasound can induce protein molecules to "moderately unfold"—enough to fully open the protein structure to expose binding sites without destroying the protein conformation due to excessive shearing. Below this power (e.g., 30%), the modification effect is not significant, while above this power (e.g., 90%), excessive shearing leads to protein conformational destruction, resulting in irregular macropores and decreased functional properties.

[0040] The heating conditions (85°C, 2 h) described in step (3) of this invention are the optimal gelation conditions selected through experiments. Under these conditions, the moderately expanded protein molecules further crosslink to form a dense and uniform weak gel network, while maintaining suitable gel strength, good emulsification stability, and shear thinning behavior.

[0041] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0042] (1) Simultaneous improvement of multifunctional properties: This invention achieves multi-dimensional synergistic regulation of hemp seed protein’s “β-sheet ordered aggregation—gelation—emulsification—rheology—thermal stability” through ultrasound-assisted acid-induced treatment. Specifically, at 60% ultrasound power, the gel strength increased by about 33% (reaching 12.37 g) compared to the control without ultrasound (9.25 g), and the adhesiveness was improved to 8.6 g; the emulsification stability (ESI) increased significantly from 71.10 min to 185.89 min, an increase of about 160%; the apparent viscosity increased significantly and exhibited shear thinning behavior (power law exponent n1 < 1), and the resistance to strain damage was best at 60% power; thermal stability data showed that the denaturation temperature T at 60% power was d =113.08℃ and initial temperature T0=67.97℃, both higher than the non-ultrasound control, indicating that the protein is in a "moderately unfolded" state, which is beneficial for functional optimization. However, the thermal stability at 30% power is insufficient, and the thermal stability at 90% power is too high but the function deteriorates. This invention avoids the defect of improving a single indicator while degrading other properties.

[0043] (2) Precise and controllable regulation methods: By adjusting the ultrasonic power (especially 60%) and acidification conditions (pH 2.0-3.0), this invention can precisely control the degree of orderly aggregation of β-sheets, gel strength, adhesiveness, emulsification stability, apparent viscosity, shear thinning behavior and thermal stability of proteins, thereby achieving directional regulation of the functional properties of hemp seed protein.

[0044] (3) Applicable to strongly acidic systems: The present invention completes protein functionalization modification under pH 2.0-3.0 conditions. The product can be directly used in strongly acidic plant protein beverages (pH 2.5-4.0), acidic emulsions (oil phase 5%-30%) and high acid gel foods (such as jelly, pudding, and acid curd), without the need for additional neutralization steps, thus avoiding the risk of protein aggregation and precipitation under neutral conditions.

[0045] (4) Simple process, green and safe: It only uses three steps: acidification, ultrasound and heating, without using any chemical cross-linking agents or organic solvents, which is in line with the development trend of clean label and green and healthy food.

[0046] (5) High-value utilization: This invention transforms hemp seed protein, which originally had poor gelation properties and limited applications, into an acidic food ingredient with excellent multifunctionality, significantly improving the economic value of hemp seed protein and providing technical support for the high-value utilization of plant protein in acidic food systems. Attached Figure Description

[0047] Figure 1 Gel strength (A) and adhesiveness (B) of acidified hemp seed protein gel under different ultrasonic powers (0%, 30%, 60%, 90%). The optimal gel performance is shown at 60% power.

[0048] Figure 2 Emulsification stability (ESI) of acidified hemp seed protein solution under different ultrasonic powers. The highest ESI value (185.89 min) was observed at 60% power, which was approximately 160% higher than the control without ultrasonication (71.10 min).

[0049] Figure 3 Th and T fluorescence intensities of acidified hemp seed protein dispersions under different ultrasonic powers. The highest fluorescence intensity is observed at 60% power, indicating the highest degree of ordered aggregation of β-sheets.

[0050] Figure 4 DSC thermal analysis images of acidified hemp seed protein gels under different ultrasonic powers. The image shows the temperature at 60% power. d =113.08℃, T0=67.97℃, indicating moderate thermal stability.

[0051] Figure 5Apparent viscosity curves of acidified hemp seed protein gels under different ultrasonic powers (shear rates 0.01-100 s⁻¹). -1 The results show that all samples exhibit shear-thinning behavior, with the consistency index K1 being the highest at 60% power.

[0052] Figure 6 Strain scanning results (critical strain values) of acidified hemp seed protein gel under different ultrasonic powers. The results show that the critical strain is highest at 60% power, indicating the strongest resistance to strain damage. Detailed Implementation

[0053] Example 1

[0054] Basic preparation method for ultrasound-regulated functional properties of acidified hemp seed protein

[0055] (1) Experimental materials and instruments

[0056] Hemp seeds (commercially available); hemp seed protein can be commercially available or extracted from hemp seeds; hydrochloric acid (analytical grade, Shanghai Sinopharm Chemical Reagent Co., Ltd.); ultrapure water (laboratory-made).

[0057] In this embodiment of the invention, hemp seed protein is extracted using the following method:

[0058] Hemp seeds were mechanically pressed (shelled) to remove oil. The degreased hemp seed powder was then mixed with n-hexane at a ratio of 1:20 (g / mL), and stirred thoroughly at 25 °C to remove remaining oil. The defatted sample was placed in a fume hood to evaporate residual solvent, yielding defatted hemp seed powder (HPM). The defatted hemp seed powder was dispersed in deionized water at a ratio of 1:20 (g / mL), and the pH was adjusted to 9.5. The mixture was stirred thoroughly at 50 °C, and the pH of the supernatant was adjusted to 5.0 to allow for protein precipitation. The precipitate was obtained by centrifugation at 4000 r / min for 10 min. The precipitate was dispersed in deionized water, the pH was adjusted to 7.0, and then freeze-dried to obtain hemp seed protein (HPI).

[0059] Ultrasonic cell disruptor (JY92-IIN, Ningbo Xinzhi Biotechnology Co., Ltd.); Electronic analytical balance (XB120A, Shanghai Tianmei Balance Instrument Co., Ltd.); Multi-unit magnetic stirrer (MS-4-500ML, Bickman Biotechnology); pH meter (PHS-2F, Shanghai Instrument & Electronics Scientific Instrument Co., Ltd.); Electric thermostatic water bath (HWS-11, Shanghai Shangdao Instrument Manufacturing Co., Ltd.); Texture analyzer (TA.XTC, Shanghai Baosheng Technology Co., Ltd.); UV-Vis spectrophotometer (TU-1810P, Beijing Puxi General Instrument Co., Ltd.); Fluorescence spectrophotometer (F-380, Tianjin Gangdong Technology Co., Ltd.); Differential scanning calorimeter (Q2000, TA Instruments, USA); Harker rheometer (Thermofisher / MARSⅢ, TA Instruments, USA).

[0060] (2) Experimental steps

[0061] ① Weigh 9.5 g of hemp seed protein (HPI), disperse it in ultrapure water, and bring the volume to 100 mL. Stir magnetically at room temperature for 12 h (no splashing, no bubbles, uniform stirring and stable vortex formation).

[0062] ② Adjust the pH of the above dispersion to 2.0 with 2 M HCl.

[0063] ③ Place the HPI dispersion at pH 2.0 in an ice bath and sonicate it using an ultrasonic cell disruptor (probe diameter 6 mm, immersion depth 1.5 cm). The ultrasonic parameters are: frequency 20 kHz, power 60%, processing time 10 min, pulse mode on for 5 s, off for 5 s.

[0064] ④ The ultrasonically treated dispersion was heated in an 85°C water bath for 2 hours, then immediately cooled in an ice bath and placed in a 4°C refrigerator for 12 hours to obtain the acidified hemp seed protein gel. This sample was labeled as US-60%.

[0065] (3) Experimental results

[0066] The resulting gel had a smooth surface. The gel strength was measured to be 12.37 g and the adhesiveness to be 8.6 g using a texture analyzer (TA.XTC, P / 0.5 probe, compression ratio 50%, 5 s interval between compressions). Figure 1 The emulsification stability (ESI) was measured at 185.89 min ( ). Figure 2 The relative fluorescence intensity is relatively high (). Figure 3 Differential scanning calorimetry (DSC, heating rate 10 °C / min) showed that the denaturation temperature T... d =113.08℃, initial temperature T0=67.97℃ ( Figure 4Rheological tests showed that all samples exhibited shear thinning (n1 < 1), with the 60% power sample showing the highest apparent viscosity (K1 = 12.5 Pa·s). n Shear thinning is moderate at 30% power; moderate at 30% power, and viscosity decreases at 90% power. Figure 5 and Figure 6 ).

[0067] Example 2

[0068] Effect of different ultrasonic powers on gel strength and adhesiveness

[0069] (1) Experimental materials and instruments

[0070] Same as Example 1.

[0071] (2) Experimental steps

[0072] The procedure is essentially the same as in Example 1, except that the ultrasonic power in step ③ is set to 0%, 30%, 60%, and 90%, respectively. All other steps are the same. Gels were prepared and labeled as US-0%, US-30%, US-60%, and US-90%.

[0073] (3) Experimental results

[0074] The gel strength and adhesiveness of each gel were determined using a texture analyzer (P / 0.5 probe, compression ratio 50%, 5 s interval between two compressions). Figure 1 The results showed that the gel strength of the unultrasonicated control (0% power) gel was 9.25 g, and the adhesiveness was 8.1 g. When the ultrasonic power was 30%, the gel strength decreased to 6.1 g, and the adhesiveness decreased to 4.9 g, indicating that low-power ultrasonication was detrimental to gel performance. When the ultrasonic power was increased to 60%, the gel strength significantly increased to 12.37 g, an improvement of approximately 33% compared to the unultrasonicated control, and the adhesiveness recovered to 8.6 g, reaching the optimal level. Further increasing the ultrasonic power to 90% resulted in a decrease in gel strength to 8.4 g and adhesiveness to 4.5 g, indicating that excessive ultrasonication led to deterioration of gel performance. Therefore, 60% ultrasonic power is the optimal condition for improving gel strength and adhesiveness.

[0075] Example 3

[0076] Effect of different ultrasonic powers on emulsion stability

[0077] (1) Experimental materials and instruments

[0078] Same as Example 1.

[0079] (2) Experimental steps

[0080] The gels prepared in Example 2 under different ultrasonic powers were reheated to 85°C to restore them to a solution state, resulting in acidified HPI solutions (concentration 95 mg / mL) for later use.

[0081] (3) Experimental results

[0082] The emulsification stability test results showed that the ESI of the unultrasonicated control gel (0% power) was 71.10 min. At 30% ultrasonic power, the ESI increased to 140.2 min, indicating a significant improvement in emulsification stability. When the ultrasonic power reached 60%, the ESI further increased to 185.89 min, an increase of approximately 160% compared to the unultrasonicated control, reaching its maximum value. Further increasing the ultrasonic power to 90% resulted in a decrease in ESI to 150.3 min, indicating a decline in emulsification stability. Figure 2 Therefore, 60% ultrasonic power is the optimal condition for improving the emulsification stability of hemp seed protein.

[0083] Example 4

[0084] Effects of different ultrasound powers on protein orderly aggregation behavior

[0085] (1) Experimental materials and instruments: Same as in Example 1, except for a fluorescence spectrophotometer.

[0086] (2) Experimental Procedure: The gels prepared in Example 2 under different ultrasonic powers were reheated to 85°C to restore the solution state, resulting in an acidified HPI solution (concentration 95 mg / mL), which was diluted to 0.1 mg / mL for later use. Measurements were performed according to the literature method with slight modifications (Gao Y, Xu Z, Jin M, et al. Ability of soy protein derived amyloid fibrils to stabilize aqueous two-phase system and effect of pH on the system[J]. Food Hydrocolloids, 2023, 145: 109084.). Thioflavin T (Th T) powder (8 mg) was dissolved in 10 mL of phosphate buffer (pH 7.0) containing 150 mM NaCl to prepare the Th T stock solution. The Th T stock solution was filtered through a 0.2 μM filter membrane to remove undissolved powder. It was then diluted 50 times with phosphate buffer. When measuring the sample, 4 mL of Th T diluent was mixed with 50 μL of sample, and the fluorescence intensity was measured using a fluorescence spectrophotometer at an excitation wavelength of 440 nm and an emission wavelength of 490 nm.

[0087] (3) Experimental results

[0088] Using the fluorescence intensity of the unultrasound control as a baseline, a slight increase in fluorescence intensity at 30% power indicates an improved degree of ordered β-sheet aggregation. The fluorescence intensity reaches its highest value at 60% power, indicating the most significant formation of ordered β-sheet-enriched structures. A significant decrease in fluorescence intensity at 90% power indicates that excessive sonication disrupts the already formed ordered structures. Therefore, 60% sonication power is most conducive to promoting the formation of ordered β-sheet aggregation of acidified hemp seed protein. Figure 3 ).

[0089] Example 5

[0090] Effect of different ultrasonic powers on thermal stability

[0091] (1) Experimental materials and instruments: Same as in Example 1, except for differential scanning calorimeter (DSC).

[0092] (2) Experimental steps: After freeze-drying the gels prepared in Example 2 under different ultrasonic powers, accurately weigh 10 mg of the sample and place it in an aluminum crucible. With an empty crucible as a control, scan from 20℃ to 180℃ at a heating rate of 10℃ / min under a nitrogen atmosphere.

[0093] (3) Experimental results

[0094] DSC analysis shows (see) Figure 4 Denaturation temperature T of the non-ultrasonic control (0% power) d The initial temperature T0 is 61.34℃, and the temperature is 108.5℃. Thermal stability decreases significantly at 30% power. d Temperature dropped to 90.1℃, and T0 dropped to 43.2℃. At 60% power, T... d The temperature rose to 113.08℃, and T0 was 67.97℃, both higher than the control group without ultrasound, indicating moderate thermal stability. At 90% power, T0... d The temperature was further increased to 126.4℃, and T0 increased to 103.02℃. However, excessive sonication led to an overly compact protein conformation, which in turn reduced gel strength and emulsion stability. Therefore, 60% sonication power achieved "moderate unfolding" in terms of thermal stability, which is beneficial for the overall improvement of functional properties.

[0095] Example 6

[0096] Effect of different ultrasonic powers on rheological properties

[0097] (1) Experimental methods

[0098] The rheological properties of the acidified hemp seed protein gel (60% ultrasonic power) prepared in Example 1 were characterized using a Hacker rheometer, following the rheological analysis methods. Shear rates ranging from 0.01 to 100 s⁻¹ were measured at 4°C. -1The apparent viscosity curves within the range were obtained, and the consistency index (K1) and flow behavior index (n1) were fitted using a power-law model. Strain scans (0.1%-1000%) were performed at 25℃ and 1 Hz to determine the critical strain value, in order to evaluate the strain resistance of the gel network.

[0099] (2) Experimental results

[0100] The flow behavior index n1 of the sample with 60% ultrasonic power is < 1, exhibiting typical shear-thinning behavior. It has a high apparent viscosity and a moderate degree of shear-thinning, which is beneficial for processing operations. Figure 5 Strain scanning results show that the sample has a large critical strain value, the gel network has strong resistance to strain failure, and the structure is dense and stable. Figure 6 In conjunction with other functional indicators of this invention: gel strength reaches 12.37 g, adhesiveness is 8.6 g (…). Figure 1 Emulsification stability 185.89 min ( Figure 2 ), Th T fluorescence intensity was the highest (β-sheet ordered aggregation was the most significant) Figure 3 DSC showed moderate thermal stability (T). d =113.08℃) Figure 4 It has excellent overall performance.

[0101] Comparative Example 1 (acidification only, no sonication)

[0102] (1) Experimental materials and instruments: same as in Example 1.

[0103] (2) Experimental steps: basically the same as in Example 1, except that ultrasonic pretreatment is not performed in step ③ (i.e., ultrasonic power is 0%). Other steps are the same.

[0104] (3) Experimental results

[0105] The unultrasonicated sample had a lower apparent viscosity, a smaller consistency index, and weaker shear thinning behavior; its critical strain value was also lower, and its resistance to strain failure was poor (see...). Figure 5 , Figure 6 The sample exhibited a gel strength of only 9.25 g and an adhesiveness of 8.1 g; emulsification stability of only 71.10 min; low ThT fluorescence intensity and insufficient β-sheet ordered aggregation; and DSC showed low thermal stability (T). d =108.5℃) (see) Figure 1-4 )

[0106] All indicators of the untreated group were significantly worse than those of the 60% ultrasonic power treated group, indicating that ultrasonic pretreatment is a key step to simultaneously improve gel strength, adhesiveness, emulsification stability, β-sheet ordered aggregation, thermal stability and rheological properties.

[0107] Comparative Example 2 (30% ultrasound power)

[0108] (1) Experimental materials and instruments: same as in Example 1.

[0109] (2) Experimental steps: basically the same as in Example 1, except that the ultrasonic power is set to 30% in step ③. Other steps are the same.

[0110] (3) Experimental results:

[0111] The apparent viscosity and consistency index of the sample with 30% ultrasonic power were slightly improved compared to the sample without ultrasonication, but were still significantly lower than those of the 60% power group; the shear thinning behavior was enhanced, but the critical strain value was still low, and the resistance to strain failure was insufficient (see...). Figure 5 , Figure 6 ).

[0112] The sample exhibited a gel strength of 6.4 g and an adhesiveness of 4.8 g, both lower than the 60% power group; its emulsification stability was 127.7 min, lower than the 60% power group (185.9 min); although the ThT fluorescence intensity was slightly higher than the unultrasonicated sample, it did not reach the peak at 60% power, indicating insufficient ordered aggregation of β-sheets; surface hydrophobicity decreased to its lowest level at 30% power, with the molecular conformation exhibiting an overly hydrophilic state, which is unfavorable for the formation of a gel network dominated by hydrophobic interactions; DSC showed the thermal denaturation temperature (T... d =90.1℃) was significantly lower than the control group (108.5℃), indicating a decrease in thermal stability (see Figure 1-4 ).

[0113] Although 30% ultrasound power improved some indicators (solubility, particle size reduction) to a certain extent, it failed to effectively induce the protein to unfold appropriately to form the optimal β-sheet ordered structure. As a result, the gel strength, adhesiveness and rheological properties were significantly worse than those of the 60% power treatment group, indicating that low power ultrasound is not enough to fully activate the ordered aggregation of proteins and the construction of gel networks.

[0114] Comparative Example 3 (excessive ultrasound, 90% power)

[0115] (1) Experimental materials and instruments: same as in Example 1.

[0116] (2) Experimental steps: basically the same as in Example 1, except that the ultrasonic power is set to 90% in step ③. Other steps are the same.

[0117] (3) Experimental results

[0118] The apparent viscosity of the sample at 90% ultrasonic power was significantly reduced, the consistency index decreased compared to the 60% power sample, and the shear thinning behavior was significantly weakened; its critical strain value was also lower than that of the 60% power sample, and its resistance to strain failure decreased (see...). Figure 5 , 6 ).

[0119] The gel strength of this sample decreased to 8.4 g, and the adhesiveness decreased to 4.5 g; the emulsification stability decreased to 150.3 min; the ThT fluorescence intensity decreased significantly compared to 60% power, indicating that the ordered β-sheet structure was disrupted; DSC showed excessively high thermal stability (T d =126.4℃), protein conformation is overly compact (see Figure 1-4 ).

[0120] Excessive sonication causes irreversible damage to protein conformation, significantly deteriorating various functional indicators (gel strength, adhesiveness, emulsion stability, ordered aggregation of β-sheets, and rheological properties). While thermal stability improves, it is accompanied by a decline in other properties. This further confirms that 60% power is the optimal control condition.

[0121] In summary, the acidified hemp seed protein gel or protein solution prepared in Example 1 of this invention (especially the sample treated with 60% ultrasonic power) exhibits excellent gel strength (12.37 g), emulsification stability (185.89 min), β-sheet ordered aggregation, and moderate thermal stability (T). d =113.08℃) and exhibits good shear thinning behavior and resistance to strain failure. Based on these multifunctional properties, this material is particularly suitable for the following acidic food systems:

[0122] (1) Strong acid plant protein beverage (pH 2.5-4.0): The product of this invention is prepared directly at pH 2.0-3.0 without neutralization. It can be stably dispersed in a high acid environment, preventing protein precipitation, while providing a smooth taste and suspension stability.

[0123] (2) Acidic emulsions (oil-in-water type, oil phase volume fraction 5%-30%): Due to the high emulsification stability of the product (ESI ≥180 min), it can be used as a natural emulsifier or emulsification stabilizer for acidic flavored milk, salad dressing, acidic beverage emulsions, etc.

[0124] (3) High-acid gel foods (such as jelly, pudding, and sour curd): Utilizing their acid-induced gelation properties, they can be directly used as gel base materials or texture modifiers to give products suitable hardness and adhesiveness, without the need to add calcium salts or gelling agents.

[0125] In summary, the product prepared by this invention provides a practical and feasible technical solution for the high-value utilization of hemp seed protein in the field of acidic foods.

Claims

1. A method for ultrasound-regulated functional properties of acidified hemp seed protein, characterized in that, Includes the following steps: (1) Disperse hemp seed protein in water, adjust the pH to strong acidity, and stir to obtain a dispersion; (2) The dispersion obtained in step (1) is subjected to ultrasonic pretreatment. The ultrasonic frequency is 20-30 kHz, the power is 50%-60%, the treatment time is 10-15 min, and the pulse mode is on for 4-5 s and off for 4-5 s. (3) Heat the dispersion obtained in step (2) at 80-90℃ for 2-3 h, and then cool it to obtain hemp seed protein gel or protein solution with improved functional properties.

2. The method according to claim 1, characterized in that, The concentration of hemp seed protein in the dispersion is 90-100 mg / mL, and the pH is adjusted to 2.0-3.

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3. The method according to claim 1, characterized in that, The ultrasonic pretreatment power is 55-60%, the treatment time is 10-12 min, and the pulse mode is on for 5 s and off for 5 s.

4. The method according to claim 1, characterized in that, The heating reaction is carried out at 85-90℃ for 2-2.5 hours, followed by cooling in an ice bath and standing at 4℃ for 10-12 hours.

5. The method according to claim 1, characterized in that, The functional characteristics include at least one of the following: (a) Increased degree of ordered aggregation of β-sheets: Th T fluorescence intensity reaches its peak at 60% power; (b) The gel strength is increased to 10-15 g, and the adhesiveness is increased to 5-10 g; (c) Emulsion stability (ESI) improved to ≥ 180 min; (d) The apparent viscosity increases and exhibits shear-thinning behavior, with a power-law exponent n1 < 1; (e) Resistance to strain failure is optimal at 60% power.

6. An acidified hemp seed protein gel or protein solution prepared by the method according to any one of claims 1-5.

7. The application of the acidified hemp seed protein gel or protein solution according to claim 6 in the preparation of acidic foods.

8. The application according to claim 7, characterized in that, The pH of the gel or protein solution is 2.0-3.0, which can be used directly in acidic food systems, or after preparation, the pH can be adjusted to 4.0-6.5 before use.

9. The application according to claim 8, characterized in that, The acidic foods preferably include strongly acidic plant protein beverages, acidic emulsions, and high-acid gel foods.

10. The application according to claim 9, characterized in that, The pH of the strongly acidic plant protein beverage is 2.5-4.0; the acidic emulsion is an oil-in-water emulsion with an oil phase volume fraction of 5%-30%; and the high-acid gel food is jelly, pudding, or sour curd.

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