Method for synergistically improving bleeding problem in storage process of pea starch-quinoa protein gel through proteolysis and addition of gellan gum

Abstract

The invention discloses a method for synergistically improving the bleeding problem in the storage process of pea starch-quinoa protein gel through proteolysis and gellan gum addition. The invention provides a method for preparing gel. The method comprises the following steps: (1) mixing enzymolysis quinoa protein, pea starch, gellan gum and water to obtain a mixed suspension; and (2) heating and gelatinizing the mixed suspension, and cooling and molding to obtain the gel. The gel prepared by the method has the beneficial effects that the water binding capacity is high, and the bleeding rate is low; microscopic structure observation shows that a highly compact and uniform network is formed, and no obvious bubbles exist; low-field nuclear magnetic resonance analysis shows that the bound water proportion is obviously increased; a dynamic rheological test proves that the gel has excellent gel stability; and the process is simple and safe and easy for industrial production. The invention provides a reliable technical scheme for the development of high-protein nutritional soft food for old people.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for improving the water separation problem during the storage of pea starch-quinoa protein gel by synergistically combining proteolytic hydrolysis with the addition of gellan gum. Background Technology

[0002] Pea starch-quinoa protein gel is particularly suitable as a matrix for nutritional foods for the elderly due to its soft texture, ease of swallowing, and balanced nutrition, especially for seniors with swallowing difficulties or those requiring soft diets. This system combines the excellent gelling properties of pea starch with the complete amino acid composition of quinoa protein, showing great potential for developing high-protein, easily digestible functional foods for the elderly (Chinese invention patent application publication number CN 119969576 A). However, such soft gels commonly suffer from severe water separation during storage, significantly affecting product stability and sensory quality.

[0003] Water separation not only leads to gel structure collapse and deterioration in appearance, but also reduces water retention and taste, thus diminishing consumer acceptance. The main causes are the retrogradation of pea starch to form a dense but brittle network, and the loose structure and poor compatibility of the intact quinoa protein gel network with starch. To improve the water separation problem of plant-based gels, existing technologies primarily employ physical modification (such as heat treatment or high-pressure treatment), chemical modification, or the addition of hydrophilic colloids. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the water separation problem during the storage of pea starch-quinoa protein gels through proteolysis and the synergistic addition of gellan gum. The method provided by this invention is particularly suitable for the development of high-protein soft nutritional foods for the elderly. The gel prepared by the method provided by this invention is especially suitable for elderly people with swallowing difficulties or those requiring soft diets.

[0005] This invention provides a method for improving the water separation problem of pea starch-quinoa protein gel; the pea starch-quinoa protein gel is a gel made of quinoa protein and pea starch; the method for improving the water separation problem of pea starch-quinoa protein gel is a method for preparing a gel with a low water separation rate;

[0006] The method for preparing a gel with low water separation rate includes the following steps: (1) Mix enzymatically hydrolyzed quinoa protein, pea starch, gellan gum and water to obtain a mixed suspension; the enzymatically hydrolyzed quinoa protein is obtained by enzymatic hydrolysis of quinoa protein with alkaline protease; (2) The mixed suspension is heated to gelatinize and then cooled to form a gel with low water separation rate.

[0007] The low water separation rate gel has a lower water separation rate than the pea starch-quinoa protein gel.

[0008] The low water separation rate gel has at least one of the following properties: (a1) Water holding capacity not less than 90%; (a2) The water separation rate is not higher than 0.5% (e.g., not higher than 0.2%).

[0009] The low water separation rate gel also has at least one of the following properties: (a3) The T2 relaxation time in low-field nuclear magnetic resonance (LF-NMR) measurements is not greater than 160 ms; (a4) Under the conditions of 25°C and 1Hz, the storage modulus (G') of the low water separation rate gel is not less than 200Pa; (a5) The low water separation rate gel is a pseudoplastic fluid, and its properties are within 0.1-100 s. -1 It exhibits shear-thinning behavior within the shear rate range.

[0010] The present invention also provides a method for preparing a gel, comprising the following steps: (1) Mix enzymatically hydrolyzed quinoa protein, pea starch, gellan gum and water to obtain a mixed suspension; the enzymatically hydrolyzed quinoa protein is obtained by enzymatic hydrolysis of quinoa protein with alkaline protease; (2) The mixed suspension is heated to gelatinize and then cooled to form a gel.

[0011] The gel has at least one of the following properties: (a1) Water holding capacity not less than 90%; (a2) The water separation rate is not higher than 0.5% (e.g., not higher than 0.2%).

[0012] The gel also has at least one of the following properties: (a3) The T2 relaxation time in low-field nuclear magnetic resonance (LF-NMR) measurements is not greater than 160 ms; (a4) At 25°C and 1 Hz, the storage modulus (G') of the low water separation rate gel is not less than 200 Pa; (a5) The low water separation rate gel is a pseudoplastic fluid, and its properties are within 0.1-100 s. -1 It exhibits shear-thinning behavior within the shear rate range.

[0013] In any of the steps (1) described above, the mass ratio of the enzymatically hydrolyzed quinoa protein, the pea starch, and the gellan gum is 2.4:10:0.12.

[0014] The heating and gelatinization process involves heating the mixed suspension to form a viscous, homogeneous colloid.

[0015] In any of the steps (2) described above, the heating and gelatinization conditions are: incubation in a water bath at 98°C for 35 minutes.

[0016] In any of the steps (2) described above, the cooling and molding conditions are: standing at 4°C.

[0017] In any of the steps (2) described above, the cooling and molding conditions are: standing at 4°C for 6-12 hours.

[0018] In any of the steps (2) described above, the cooling and molding conditions are: standing at 4°C for 12 hours.

[0019] In any of the above-described mixed suspensions, the pea starch content is 5 g / 100 mL.

[0020] In any of the above-described mixed suspensions, the content of enzymatically hydrolyzed quinoa protein is 1.2 g / 100 mL.

[0021] In any of the above-described mixed suspensions, the content of gellan gum is 0.06 g / 100 mL.

[0022] As an example, the preparation method of the low water separation rate gel is as follows: ① Weigh 10.0g of pea starch, add deionized water to a volume of 100mL, and stir magnetically for 1min at room temperature to obtain a suspension, which is a 10% PS suspension; ② Weigh 2.4g of enzymatically hydrolyzed quinoa protein, add deionized water to a volume of 100mL, and stir magnetically at room temperature for 2h to obtain a suspension, which is the 2.4% HQP suspension. ③ Mix the 10% PS suspension prepared in step ① with the 2.4% HQP suspension prepared in step ② in equal volumes, then add gellan gum and make its concentration in the system 0.06 g / 100 mL, stir magnetically at room temperature for 30 min, then incubate in a 98℃ water bath for 35 min, cool at room temperature, and then stand at 4℃ for 12 h to obtain a gel with low water separation rate.

[0023] As an example, the gel is prepared as follows: ① Weigh 10.0g of pea starch, add deionized water to a volume of 100mL, and stir magnetically for 1min at room temperature to obtain a suspension, which is a 10% PS suspension; ② Weigh 2.4g of enzymatically hydrolyzed quinoa protein, add deionized water to a volume of 100mL, and stir magnetically at room temperature for 2h to obtain a suspension, which is the 2.4% HQP suspension. ③ Mix the 10% PS suspension prepared in step ① with the 2.4% HQP suspension prepared in step ② in equal volumes, then add gellan gum and make its concentration in the system 0.06 g / 100 mL, stir magnetically at room temperature for 30 min, then incubate in a 98℃ water bath for 35 min, cool at room temperature, and then stand at 4℃ for 12 h to obtain gel.

[0024] The present invention also protects compositions for preparing gels, including enzymatically hydrolyzed quinoa protein, pea starch, and gellan gum; wherein the enzymatically hydrolyzed quinoa protein is obtained by enzymatic hydrolysis of quinoa protein with alkaline protease.

[0025] In the composition, the mass ratio of the enzymatically hydrolyzed quinoa protein, the pea starch, and the gellan gum is 2.4:10:0.12.

[0026] The gel has at least one of the following properties: (a1) Water holding capacity not less than 90%; (a2) The water separation rate is not higher than 0.5% (e.g., not higher than 0.2%).

[0027] The gel also has at least one of the following properties: (a3) The T2 relaxation time in low-field nuclear magnetic resonance (LF-NMR) measurements is not greater than 160 ms; (a4) At 25°C and 1 Hz, the storage modulus (G') of the low water separation rate gel is not less than 200 Pa; (a5) The low water separation rate gel is a pseudoplastic fluid, and its properties are within 0.1-100 s. -1 It exhibits shear-thinning behavior within the shear rate range.

[0028] The composition also includes water.

[0029] This invention also protects the use of any of the methods or compositions described above in the preparation of soft nutritional foods for the elderly.

[0030] The aforementioned soft nutritional food for the elderly is a high-protein gel food suitable for elderly people with swallowing difficulties or who require a soft diet.

[0031] The gel in this application is a gel-like substance made from components extracted from plants.

[0032] The quinoa protein is a protein extracted from quinoa rice.

[0033] The method for preparing quinoa protein includes the following steps: taking quinoa rice, first defatting it with petroleum ether, and then extracting the protein using an alkali-dissolving and acid-precipitating method.

[0034] For example, the preparation method of quinoa protein includes the following steps: grinding quinoa rice and passing it through an 80-mesh sieve to obtain quinoa powder; mixing 500g of quinoa powder with 1500mL of petroleum ether at room temperature for 24h, then evaporating the petroleum ether to obtain powder, which is defatted quinoa powder; dispersing the defatted quinoa powder in 3133.33mL of distilled water, then adding 2M NaOH aqueous solution to adjust the pH to 9, then stirring at room temperature for 2h, then centrifuging at 20℃ and 3500×g for 20min, and collecting the supernatant; taking the supernatant and using 2M... The pH was adjusted to 4.5 with HCl aqueous solution, and the mixture was allowed to stand at 4℃ for 2 hours to promote protein aggregation. The supernatant was discarded, and the remaining liquid phase was centrifuged at 5℃ and 3500×g for 20 minutes. The supernatant was discarded, and the precipitate was retained. The precipitate was washed three times with distilled water (each wash consisted of adding distilled water, resuspending the precipitate, centrifuging at 5℃ and 3500×g for 20 minutes, and discarding the supernatant). The washed precipitate was then suspended in distilled water, and the pH was adjusted to 7 with 2M NaOH aqueous solution. The precipitate was then transferred to a freezer at -80℃ and frozen. Subsequently, it was freeze-dried to obtain a solid product. The solid product was ground into powder and passed through a 100-mesh sieve. The powder obtained was quinoa protein.

[0035] The pea starch is starch extracted from pea seeds.

[0036] The enzymatically hydrolyzed quinoa protein is a substance obtained by enzymatically hydrolyzing quinoa protein using alkaline protease.

[0037] The alkaline protease is an enzyme that hydrolyzes protein peptide bonds under alkaline conditions with a pH of 8-11.

[0038] The alkaline protease is an enzyme with an optimal temperature of 50-60℃ (e.g., 55℃) and an optimal pH of 8.0-9.0 (e.g., 8.0).

[0039] In one specific embodiment of this application, the alkaline protease is Alcalase.

[0040] The conditions for enzymatic hydrolysis with alkaline protease as described above are: pH 8.0-9.0 and temperature 50-60℃.

[0041] As an example, the conditions for enzymatic hydrolysis with alkaline protease described above are: pH 8.0 and temperature 55°C.

[0042] As an example, the mass ratio of quinoa protein to alkaline protease is 200:3.

[0043] As an example, the method for preparing enzymatically hydrolyzed quinoa protein includes the following steps: enzymatically hydrolyzing quinoa protein with alkaline protease and then drying it to obtain a powdered product, which is enzymatically hydrolyzed quinoa protein.

[0044] As an example, the method for preparing enzymatically hydrolyzed quinoa protein includes the following steps: enzymatically hydrolyzing quinoa protein with alkaline protease, cooling to room temperature, then freezing at -80°C, followed by freeze-drying to obtain a powdered product, which is enzymatically hydrolyzed quinoa protein.

[0045] The method for enzymatically hydrolyzing quinoa protein with alkaline protease includes the following steps: 50g of quinoa protein is suspended in 1000mL of deionized water in a 55℃ water bath, then 0.75g of alkaline protease is added and the reaction is carried out for 180min (the pH value of the system is continuously monitored during the reaction, and the pH value of the system is maintained at 8.0 by adding 2M NaOH aqueous solution).

[0046] Compared with the preparation method of the low water separation rate gel, the preparation method of the pea starch-quinoa protein gel differs only in the following aspects: quinoa protein is used instead of enzymatically hydrolyzed quinoa protein, and gellan gum is not added.

[0047] For example, in pea starch, starch accounts for 98.61% of the dry weight (amylose accounts for 38.11% of the dry weight), protein accounts for 0.40% of the dry weight, fat accounts for 0.34% of the dry weight, and ash accounts for 0.07% of the dry weight.

[0048] For example, the alkaline protease activity is 2.4 AU / g.

[0049] For example, the quinoa is white quinoa.

[0050] Alkaline protease hydrolysis of quinoa protein generates self-assembling peptides (average molecular weight 5.01 kDa), which can form a regular porous hydrogel under mild conditions (pH 7.0) with a hardness of 487.52 mN, 5.3 times that of the original protein. Gellan gum can significantly enhance the water-holding capacity of the gel network with extremely low dosage. In this invention, enzymatically hydrolyzed quinoa protein is combined with gellan gum to synergistically inhibit water separation and improve gel storage stability. This invention discovers the significant synergistic advantages of enzymatically hydrolyzed quinoa protein combined with gellan gum treatment, providing an efficient and feasible technical solution that can fundamentally solve the problem of water separation during the storage of PS-QP soft gel, significantly improving product quality stability and market application potential.

[0051] The beneficial effects of the gel prepared by this invention are as follows: water retention capacity is as high as 90.35%, and water separation rate is only 0.11%; microstructure observation shows the formation of a highly dense and uniform network without obvious air bubbles; low-field nuclear magnetic resonance analysis shows a significant increase in the proportion of bound water; dynamic rheological testing confirms excellent gel stability; and the process is simple, safe, and easy for industrial production. This invention provides a reliable technical solution for the development of high-protein soft nutritional foods for the elderly. Attached Figure Description

[0052] Figure 1 The effect of different types of polysaccharides on the water-holding capacity of gels.

[0053] Figure 2 To investigate the effects of different treatments on the water-holding capacity of the gel.

[0054] Figure 3 The effect of different treatments on the gel water separation rate.

[0055] Figure 4 The macroscopic and microscopic morphologies of the gels under different treatments are shown.

[0056] Figure 5 The effect of different treatments on the moisture distribution of the gel.

[0057] Figure 6 The effect of different treatments on the apparent viscosity of the gel.

[0058] Figure 7 The effect of different treatments on the gel frequency scanning storage modulus (G′).

[0059] Figure 8 The effect of different treatments on the loss modulus (G″) of gel frequency scanning. Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, room temperature in the examples refers to 20-25°C. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the examples are performed in triplicate, and the results are averaged. In Example 2, gels of any shape and size can be prepared depending on the container used.

[0062] Quinoa is an annual crop belonging to the genus *Chenopodium* of the family Amaranthaceae. The mature grains of quinoa, after being hulled, become quinoa rice. The quinoa rice in this example is white quinoa rice, a product of Guyuan County Beimai Ecological Agriculture Co., Ltd. The pea starch (PS) in this example is a product of Yantai Shuangta Food Co., Ltd.; this pea starch contains 98.61% starch (38.11% amylose), 0.40% protein, 0.34% fat, and 0.07% ash on a dry basis. Gellan gum (GG) (CAS No.: 71010-52-1): Xinjiang Fufeng Biotechnology Co., Ltd. Petroleum ether (CAS No.: 8032-32-4): Beijing Mairuida Technology Co., Ltd., product number M042760. Alcalase (nominal activity 2.4 AU-A / g): Novozymes, catalog number Alcalase 2.4 L FG. Carrageenan: Linyi Aidesen Biotechnology Co., Ltd. Xanthan Gum: Jianlong Biotechnology Co., Ltd. Konjac Powder: Hubei Qiangsen Konjac Technology Co., Ltd. Sodium Alginate: Qingdao Mingyue Seaweed Group Co., Ltd. Locust Bean Gum: Taoxuan Trading (Shanghai) Co., Ltd. β-Glucan: Nanjing Taixin Biotechnology Co., Ltd. Apple Pectin: Sigma-Aldrich, catalog number 93854.

[0063] The method for detecting the water-holding capacity (WHC) of the gel in the example is as follows: 5.0 g of the test gel (i.e., W1=5) is placed in a centrifuge tube, centrifuged at 4°C and 2000×g for 30 min, then the water is removed, the gel is taken out and weighed (unit: g, value: W2). Water-holding capacity (%) = W2 ÷ W1 × 100%.

[0064] The method for observing the macroscopic morphology of the gel in the examples is as follows: the test gel is placed on a black background, and a macroscopic photograph is taken under the same lighting conditions using a high-resolution digital camera to observe the surface smoothness, bubble distribution and overall uniformity of the sample.

[0065] The method for observing the microstructure of the gel in the examples is as follows (using an inverted fluorescence microscope): the test gel is cut into 5 mm thick slices, placed on a glass slide, and the microscope objective lenses are 5× and 40×. Images are taken under the same exposure conditions to analyze the compactness, uniformity and bubble distribution of the internal network structure of the sample.

[0066] The method for detecting the TPA characteristic in the textural properties of the gel in the embodiment is as follows: A cylindrical test gel (diameter 20 mm, height 15 mm) is placed in a texture analyzer for TPA detection. A double-cycle compression test is performed using a 5 mm diameter cylindrical probe (P / 5), a pre-test speed of 0.5 mm / s, a compression speed of 0.5 mm / s, a target distance of 5.0 mm, and a time of 5 s. The software records the force-time curve and required parameters during the double-cycle compression-decompression test and performs analysis to obtain the hardness, elasticity, cohesiveness, adhesiveness, chewiness, and resilience of the test gel.

[0067] The method for detecting gel rupture characteristics, among the textural properties of the gel, in this embodiment is as follows: A cylindrical test gel (60 mm in diameter and 30 mm in height) is placed in a texture analyzer for a gel puncture test. A 12.7 mm black cylindrical probe is used, with a testing rate of 30 mm / min during the test, a post-test rate of 600 mm / min, and a pressing distance of 15.0 mm. The rupture strength and rupture distance of the test gel are obtained. The gel rupture strength is the initial pressure (N) required to penetrate the gel. The gel rupture distance is the distance (mm) corresponding to the maximum force.

[0068] The method for detecting the water distribution of the gel in this embodiment is as follows (using low-field nuclear magnetic resonance): 1.0 g of the test gel was placed in a low-field nuclear magnetic resonance (LF-NMR) detection tube, and the T2 relaxation time distribution was determined using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence mode. The spectral width (SW) was 200 kHz, the echo count (EC) was 15000, the number of scan cycles (NS) was 2, the sampling repetition time (TW) was 3500 ms, and the magnetic field strength was 0.5 T. The water distribution (T2 relaxation time) inside the gel was obtained. The shorter the T2 relaxation time, the more tightly the water molecules are bound to the gel matrix, mainly as bound water or non-flowing water; the longer the T2 relaxation time, the higher the proportion of free water, which is more prone to migration and precipitation.

[0069] The method for detecting the static rheological properties of the gel in this embodiment is as follows (using a PP50 plate for measurement): The test gel is placed between the rheometer plates, with a plate clamp diameter of 40 mm and a plate spacing of 1 mm. First, strain correlation measurements are performed to obtain the linear viscoelastic region of the hydrogel. Then, the rheological properties are measured at a small constant strain (1.0%), which is within the linear region of all samples. The frequency is set to 1 Hz, the test temperature to 25 °C, and the shear rate to 0.1 s⁻¹. -1 -100s -1 Plot the apparent viscosity-shear rate curve within the range.

[0070] The method for detecting the dynamic rheological properties of the gel in the embodiment is as follows (measurement was performed using a PP50 plate): The test gel was placed between the rheometer plates with a plate clamp diameter of 40 mm and a plate spacing of 1 mm. A dynamic frequency scan of 0.6283-62.83 rad / s was performed at 25 °C, and the storage modulus (G′) and loss modulus (G″) were recorded as functions of frequency.

[0071] Example 1: Preparation of quinoa protein powder and preparation of enzymatically hydrolyzed quinoa protein I. Preparation of Quinoa Protein Powder Quinoa rice was ground and passed through an 80-mesh sieve to obtain quinoa flour. 500g of quinoa flour was mixed with 1500mL of petroleum ether at room temperature for 24 hours, then the petroleum ether was evaporated to obtain 470g of powder, which is defatted quinoa flour. 470g of defatted quinoa flour was dispersed in 3133.33mL of distilled water, then 2M NaOH aqueous solution was added to adjust the pH to 9, and the mixture was stirred at room temperature for 2 hours. The mixture was then centrifuged at 20°C and 3500×g for 20 minutes, and the supernatant was collected. The supernatant was adjusted to pH 4.5 with 2M HCl aqueous solution and allowed to stand at 4°C for 2 hours to promote protein aggregation. The supernatant was discarded, and the remaining liquid phase was centrifuged at 5°C and 3500×g for 20 minutes. The supernatant was discarded, and the precipitate was retained. The precipitate was washed three times with distilled water (each wash consisting of adding distilled water, resuspending the precipitate, centrifuging at 5°C and 3500×g for 20 minutes, and discarding the supernatant). The washed precipitate was then resuspended in distilled water, and 2M NaOH aqueous solution was added to adjust the pH to 9. The pH was adjusted to 7 with NaOH aqueous solution, then transferred to a -80℃ freezer for freezing, followed by freeze-drying to obtain a solid product. This solid product was then ground into powder and passed through a 100-mesh sieve, yielding 31±1g of powder, which is quinoa protein powder (QP). Kjeldahl nitrogen determination showed that the protein content of the quinoa protein powder was 91.44% by weight on a dry basis.

[0072] II. Preparation of enzymatically hydrolyzed quinoa protein 1. Preparation of hydrolysis products The following steps were performed in a 55℃ water bath: 50g of QP was suspended in 1000mL of deionized water, then 0.75g of alkaline protease was added and the reaction was carried out for 180min (the pH value of the system was continuously monitored during the reaction, and the pH value of the system was maintained at 8.0 by adding 2M NaOH aqueous solution), and the hydrolysis product was obtained.

[0073] 2. Cool the hydrolysate to room temperature naturally, then transfer it to a -80°C freezer and freeze-dry it until you get a powdered product, which is enzymatically hydrolyzed quinoa protein (HQP).

[0074] Example 2: Preparation of Gel I. Preparation of PS suspension Weigh 10.0g of pea starch, add deionized water to a volume of 100mL, and stir magnetically for 1min at room temperature to obtain a suspension, which is the 10% PS suspension.

[0075] II. Preparation of QP Suspension Weigh 2.4g of the QP prepared in step one of Example 1, add deionized water to a volume of 100mL, and stir magnetically at room temperature for 2h to obtain a suspension, which is the 2.4% QP suspension.

[0076] III. Preparation of HQP Suspension Weigh 2.4g of the HQP prepared in step two of Example 1, add deionized water to a volume of 100mL, and stir magnetically at room temperature for 2h to obtain a suspension, which is the 2.4% HQP suspension.

[0077] IV. Preparation of PS-QP Gel The 10% PS suspension prepared in step one and the 2.4% QP suspension prepared in step two were mixed in equal volumes, magnetically stirred at room temperature for 30 min, then incubated in a 98℃ water bath for 35 min, cooled to room temperature, and then allowed to stand at 4℃ for 12 h to obtain PS-QP gel.

[0078] V. Preparation of PS-HQP Gel The 10% PS suspension prepared in step one and the 2.4% HQP suspension prepared in step three were mixed in equal volumes, magnetically stirred at room temperature for 30 min, then incubated in a 98℃ water bath for 35 min, cooled to room temperature, and then allowed to stand at 4℃ for 12 h to obtain PS-HQP gel.

[0079] VI. Preparation of PS-QP-polysaccharide gel The 10% PS suspension prepared in step one and the 2.4% QP suspension prepared in step two were mixed in equal volumes. Then, polysaccharide was added to make the concentration in the system 0.06 g / 100 mL. The mixture was magnetically stirred at room temperature for 30 min, then incubated in a 98 °C water bath for 35 min, cooled to room temperature, and then allowed to stand at 4 °C for 12 h to obtain PS-QP-polysaccharide gel.

[0080] The polysaccharides used are carrageenan, gellan gum, xanthan gum, konjac flour, sodium alginate, locust bean gum, β-glucan, or apple pectin, and the corresponding PS-QP-polysaccharide gels are PS-QP-carrageenan gel, PS-QP-gellan gum gel, PS-QP-xanthan gum gel, PS-QP-konjac flour gel, PS-QP-sodium alginate gel, PS-QP-locust bean gum gel, PS-QP-β-glucan gel, or PS-QP-apple pectin gel.

[0081] VII. Preparation of PS-HQP-Gellan gel The 10% PS suspension prepared in step one and the 2.4% HQP suspension prepared in step three were mixed in equal volumes. Then, gellan gum was added to make its concentration in the system 0.06 g / 100 mL. The mixture was magnetically stirred at room temperature for 30 min, then incubated in a 98 °C water bath for 35 min, cooled to room temperature, and then allowed to stand at 4 °C for 12 h to obtain PS-HQP-gellan gum gel.

[0082] Example 3: Effect of adding different polysaccharides on gel water retention The tested gels were: PS-QP gel (represented by CK), PS-QP-carrageenan gel, PS-QP-gellan gum gel, PS-QP-xanthan gum gel, PS-QP-konjac flour gel, PS-QP-sodium alginate gel, PS-QP-locust bean gum gel, PS-QP-β-glucan gel, or PS-QP-apple pectin gel. All were prepared in Example 2.

[0083] Take the test gel and test its water-holding capacity. Set up three replicate samples and take the average of the results.

[0084] See results Figure 1 .

[0085] The water-holding capacity of PS-QP gel was 76.61%, that of PS-QP-gellan gum gel was 84.33%, that of PS-QP-konjac flour gel was 70.99%, that of PS-QP-locust bean gum gel was 70.86%, that of PS-QP-xanthan gum gel was 69.33%, that of PS-QP-sodium alginate gel was 69.68%, that of PS-QP-carrageenan gel was 63.71%, that of PS-QP-β-glucan gel was 65.67%, and that of PS-QP-apple pectin gel was 65.91%. The results indicate that the addition of different polysaccharides during the preparation process significantly affected the water-holding capacity of the gels. The addition of gellan gum significantly improved the water-holding capacity, showing a significant difference compared to the control (CK) or compared to the addition of other polysaccharides.

[0086] Example 4: Comparison of water retention and water separation rate of gels prepared by different methods Water retention and water separation rate are key indicators for evaluating the stability and moisture retention capacity of hydrogel network structures, which directly affect the texture, stability and shelf life of products.

[0087] I. Comparison of the water-holding capacity of gels prepared by different methods The tested gels were: PS-QP gel, PS-HQP gel, PS-QP-gellan gel, or PS-HQP-gellan gel. All were prepared in Example 2.

[0088] Take the test gel and test its water-holding capacity. Set up three replicate samples and take the average of the results.

[0089] See results Figure 2 .

[0090] II. Comparison of the water-separating properties of gels prepared by different methods Preparation of PS-QP gel: The 10% PS suspension prepared in step one of Example 2 and the 2.4% QP suspension prepared in step two of Example 2 were mixed in equal volumes and magnetically stirred at room temperature for 30 min. Then, the mixture was incubated in a 98°C water bath for 35 min. After incubation, 10.0 g of the mixture was placed in a centrifuge tube (i.e., W3=10), the centrifuge tube was capped and sealed, and the mixture was allowed to stand at 4°C for 12 hours. Then, the centrifuge tube cap was removed, the centrifuge tube was inverted on a 200-mesh sieve and allowed to stand for 10 min. The filtrate was collected and weighed (in g, value is W4).

[0091] Preparation of PS-HQP gel: The 10% PS suspension prepared in step one of Example 2 and the 2.4% HQP suspension prepared in step three of Example 2 were mixed in equal volumes and magnetically stirred at room temperature for 30 min. Then, the mixture was incubated in a 98°C water bath for 35 min. After incubation, 10.0 g of the mixture was placed in a centrifuge tube (i.e., W3=10), the centrifuge tube was capped and sealed, and the mixture was allowed to stand at 4°C for 12 hours. Then, the centrifuge tube cap was removed, the centrifuge tube was inverted on a 200-mesh sieve and allowed to stand for 10 min. The filtrate was collected and weighed (in g, value is W4).

[0092] Preparation of PS-QP-Gellan Gel: The 10% PS suspension prepared in step one of Example 2 and the 2.4% QP suspension prepared in step two of Example 2 were mixed in equal volumes. Then, gellan gum was added to make the concentration in the system 0.06 g / 100 mL. The mixture was magnetically stirred at room temperature for 30 min and then incubated in a 98 °C water bath for 35 min. After incubation, 10.0 g was placed in a centrifuge tube (i.e., W3=10), the centrifuge tube was capped and sealed, and the mixture was allowed to stand at 4 °C for 12 hours. Then, the centrifuge tube cap was removed, the centrifuge tube was inverted on a 200-mesh sieve and allowed to stand for 10 min. The filtrate was collected and weighed (in g, value is W4).

[0093] Preparation of PS-HQP-Gellan Gel: The 10% PS suspension prepared in step one of Example 2 and the 2.4% HQP suspension prepared in step three of Example 2 were mixed in equal volumes. Then, gellan gum was added to make the concentration in the system 0.06 g / 100 mL. The mixture was magnetically stirred at room temperature for 30 min and then incubated in a 98 °C water bath for 35 min. After incubation, 10.0 g was placed in a centrifuge tube (i.e., W3=10), the centrifuge tube was capped and sealed, and the mixture was allowed to stand at 4 °C for 12 hours. Then, the centrifuge tube cap was removed, the centrifuge tube was inverted on a 200-mesh sieve and allowed to stand for 10 min. The filtrate was collected and weighed (in g, value is W4).

[0094] Water separation rate (%) = W4 ÷ W3 × 100%.

[0095] See results Figure 3 .

[0096] III. Results Analysis The PS-QP gel exhibited a water-holding capacity of 76.71% and a water separation rate of 10.88%, indicating a relatively loose gel network structure and limited water-holding capacity. The PS-HQP gel had a water-holding capacity of 72.59% and a water separation rate of 3.39%, suggesting that enzymatic hydrolysis further disrupted the gel network structure; however, the resulting small peptide fragments could self-assemble into a denser micronetwork, thereby enhancing the physical water retention capacity. The PS-QP-gellan gel had a water-holding capacity of 84.33% and a water separation rate of 2.65%, indicating that gellan gel molecules effectively strengthened the gel matrix. The PS-HQP-gellan gel achieved the optimal water-holding capacity of 90.35% and a water separation rate of 0.11%, showing a significant difference compared to the other three gels. This indicates that the active peptides generated by enzymatic hydrolysis synergistically work with gellan gel molecules to construct an extremely stable and highly efficient water-locking three-dimensional network structure.

[0097] Example 5: Comparison of the macroscopic morphology and microstructure of gels prepared by different methods The tested gels were: PS-QP gel, PS-HQP gel, PS-QP-gellan gel, or PS-HQP-gellan gel. All were prepared in Example 2.

[0098] Take the test gel and examine its macroscopic morphology and microstructure.

[0099] See example photos Figure 4 .

[0100] Macroscopic images of the PS-QP gel show a relatively smooth surface with slight inhomogeneities; microscopic images clearly reveal numerous dispersed dark areas and irregular structures, indicating the presence of many bubbles and a loose network within the system. This structural characteristic is consistent with the gel's water separation rate (10.88%) and water retention capacity (76.71%), suggesting that the loose network cannot effectively retain water. Macroscopic images of the PS-HQP gel show surface wrinkles and irregular textures, which are related to changes in protein structure during enzymatic hydrolysis; microscopic images show that this group of structures is relatively uniform, with fewer and more dispersed dark areas, indicating that the small peptides produced by enzymatic hydrolysis form a relatively dense network, reducing the formation of large bubbles. This structural characteristic is consistent with the gel's water separation rate (3.39%), confirming the optimization effect of enzymatic hydrolysis on the network structure. Macroscopic images of PS-QP-gellan gel show a relatively smooth surface, but microscopic images reveal numerous dispersed dark areas and irregular structures. This inhomogeneity may stem from the local aggregation of gellan gel within the intact protein system, resulting in a network structure that is dense overall but contains microscopic defects. This structural characteristic is consistent with the gel's water-holding capacity (84.33%) and anomalous textural properties (resilience 3.0%, cohesiveness 13.8%), indicating that while gellan gel enhances water-holding capacity, it fails to form a completely uniform network structure. PS-HQP-gellan gel exhibits superior structural properties. Macroscopic images show a smooth, uniform surface with no obvious defects; microscopic images reveal a highly dense and uniform structure with almost no obvious dark areas or voids. This highly uniform network structure is perfectly consistent with the group's ultra-high water-holding capacity of 90.35% and extremely low water separation rate of only 0.11%, indicating that the enzymatically hydrolyzed peptides and gellan gel work synergistically to successfully construct a highly uniform gel network without obvious air bubbles. This precisely meets the special requirements of elderly nutritional foods for "easy to swallow and non-water separation." Based on comprehensive morphological analysis and functional property data, it can be clearly concluded that the composite treatment fundamentally solves the water separation problem of PS-QP gel by eliminating air bubbles and improving structural uniformity, while simultaneously endowing the product with ideal texture characteristics.

[0101] Example 6: Comparison of the textural and rupture properties of gels prepared by different methods The tested gels were: PS-QP gel, PS-HQP gel, PS-QP-gellan gel, or PS-HQP-gellan gel. All were prepared in Example 2.

[0102] Take the test gel and test its textural and fracture properties. Set up three replicate samples and take the average of the results.

[0103] The results of the textural properties are shown in Table 1. All textural parameters of the PS-HQP-gellan gel were undetectable (ND), which is closely related to its unique physical structure. This group of samples exhibited an ultra-high water retention of 90.35% and a water separation rate of only 0.11%. Low-field NMR analysis showed that its T2 relaxation time was only 157.87 ms, confirming that water molecules were tightly bound in a highly hydrated gel network. This structural feature resulted in an extremely soft gel texture, with its mechanical response strength below the detection threshold of the texture analyzer. Although the PS-QP-gellan gel had good water retention (84.33%), its textural parameters were significantly reduced (resilience 3.0%, cohesiveness 13.8%), indicating that gellan gel formed a network structure with excessive rigidity and insufficient cohesion within the intact protein system, consistent with the non-uniform structure observed in its microstructure. PS-HQP gel exhibits low hardness (0.032N) and good water retention (72.59%), reflecting that the network formed by enzymatically hydrolyzed peptides has both flexibility and cohesion.

[0104] Table 1

[0105] Note: ND indicates not detected because the sample is too soft and the mechanical response intensity is below the threshold of the detection equipment.

[0106] The results of the fracture characteristics are shown in Table 2. No data were detected for PS-HQP-gellan gel. Combined with its excellent microstructure and extremely low water separation rate in its rheological properties, this indicates that the composite system formed a continuous, homogeneous, and highly hydrated ultra-soft gel structure. Although this structure has relatively low textural strength, its uniform network structure and stable water retention capacity perfectly meet the core requirements of elderly people with swallowing disorders for "easy to swallow and non-water separation" in nutritional foods, demonstrating a balance between functionality and safety. Table 2

[0107] Note: ND indicates not detected because the sample is too soft and the mechanical response intensity is below the threshold of the detection equipment.

[0108] Example 7: Comparison of moisture distribution of gels prepared by different methods The tested gels were: PS-QP gel, PS-HQP gel, PS-QP-gellan gel, or PS-HQP-gellan gel. All were prepared in Example 2.

[0109] Take the test gel and determine the moisture distribution. Set up three replicate samples and take the average of the results.

[0110] See results Figure 5 .

[0111] The T2 relaxation time of the PS-QP gel was 778.596 ms, with a high peak intensity and wide distribution, indicating that its internal water mainly exists in the form of free water in the large gel pores, consistent with the high water separation rate. The T2 relaxation time of the PS-HQP gel was 154.904 ms, with a significant decrease in peak intensity, indicating that the dense network formed by the enzymatically hydrolyzed peptides effectively converted free water into bound water with limited mobility. The T2 relaxation time of the PS-QP-gellan gel was 683.158 ms, which was better than that of the PS-QP gel, but still contained a relatively large amount of free water, reflecting that gellan gel may have limited the densification of the network to some extent. The T2 relaxation time of the PS-HQP-gellan gel was 157.868 ms, almost the same as that of the PS-HQP gel, indicating that the addition of gellan gel did not destroy the fine network constructed by the enzymatically hydrolyzed peptides, but instead maintained its strong water-holding capacity through synergistic effects. In summary, the composite treatment significantly increases the proportion of bound water and reduces the content of free water, explaining its excellent macroscopic water-holding performance at the microscopic level and providing intrinsic mechanistic support for the effectiveness of the technical solution.

[0112] Example 8: Comparison of the static rheological properties of gels prepared by different methods The tested gels were: PS-QP gel, PS-HQP gel, PS-QP-gellan gel, or PS-HQP-gellan gel. All were prepared in Example 2.

[0113] Take the test gel and measure its static rheological properties. Set up three replicate samples and take the average of the results.

[0114] The apparent viscosity-shear rate curve is shown in [reference]. Figure 6 .

[0115] All tested gels exhibited typical pseudoplastic fluid behavior, meaning their viscosity decreased with increasing shear rate, reflecting the partial disintegration of their internal three-dimensional network under shear stress. The PS-QP gel showed the highest initial viscosity, attributed to the synergistic network formed by intact quinoa protein and retrograded starch. The PS-HQP gel exhibited a significant decrease in viscosity due to enzymatic hydrolysis disrupting the higher-order structure of the protein and weakening protein-starch interactions. The PS-QP-gellan gum gel had a significantly lower viscosity than the PS-QP gel but higher viscosity than the PS-HQP gel, indicating that while gellan gum can form weak gels, its weakening effect on the network structure is slightly less than that of the enzymatically treated group. The apparent viscosity of the PS-HQP-gellan gum gel was lower than that of the PS-QP gel but significantly higher than both the PS-HQP and PS-QP-gellan gum gels, suggesting that in systems where the protein network is weakened by enzymatic hydrolysis, gellan gum molecular chains can effectively entangle through hydrogen bonds and hydrophobic interactions, forming a support network with a certain strength. The results indicate that the composite treatment maintains the soft texture of the gel while still possessing sufficient structural strength to resist moisture migration during storage, thereby helping to suppress water separation.

[0116] Example 9: Comparison of the dynamic rheological properties of gels prepared by different methods The tested gels were: PS-QP gel, PS-HQP gel, PS-QP-gellan gel, or PS-HQP-gellan gel. All were prepared in Example 2.

[0117] Collect the test gel and measure its dynamic rheological properties. Set up three replicate samples and take the average of the results.

[0118] The storage modulus (G′) recorded as a function of angular frequency is shown in the following results. Figure 7 .

[0119] The result of recording the loss modulus (G) as a function of angular frequency is shown in [the original text]. Figure 8 .

[0120] The G′ of all tested gels was always greater than G″, indicating that each group exhibited typical elastic gel characteristics.

[0121] Compared to PS-QP gel, the G′ and G″ values ​​of all treatment groups changed more gradually with frequency, indicating the formation of a highly stable gel network structure with excellent viscoelasticity. The G′ value of PS-HQP-gellan gel was slightly higher than that of PS-HQP gel and slightly lower than that of PS-QP-gellan gel, demonstrating that gellan gel effectively compensated for the structural weakening caused by enzymatic hydrolysis. In summary, the dynamic rheological data confirm that the combined treatment of enzymatic hydrolysis of quinoa protein and gellan gel, through synergistic effects, endows it with superior elasticity and viscosity modulus, stability, and structural integrity, providing a solid foundation for maintaining a low water separation rate during long-term cold storage.

[0122] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for improving the water separation problem of pea starch-quinoa protein gel; wherein the pea starch-quinoa protein gel is a gel made of quinoa protein and pea starch; the method for improving the water separation problem of pea starch-quinoa protein gel is a method for preparing a gel with a low water separation rate; The method for preparing a gel with low water separation rate includes the following steps: (1) Mix enzymatically hydrolyzed quinoa protein, pea starch, gellan gum and water to obtain a mixed suspension; the enzymatically hydrolyzed quinoa protein is obtained by enzymatic hydrolysis of quinoa protein with alkaline protease; (2) The mixed suspension is heated to gelatinize and then cooled to form a gel with low water separation rate.

2. The method according to claim 1, characterized in that: The low water separation rate gel has at least one of the following properties: (a1) Water holding capacity not less than 90%; (a2) The water separation rate shall not exceed 0.5%.

3. A method for preparing a gel, comprising the following steps: (1) Mix enzymatically hydrolyzed quinoa protein, pea starch, gellan gum and water to obtain a mixed suspension; the enzymatically hydrolyzed quinoa protein is obtained by enzymatic hydrolysis of quinoa protein with alkaline protease; (2) The mixed suspension is heated to gelatinize and then cooled to form a gel.

4. The method according to claim 3, characterized in that: The gel has at least one of the following properties: (a1) Water holding capacity not less than 90%; (a2) The water separation rate shall not exceed 0.5%.

5. The method according to any one of claims 1 to 4, characterized in that: The conditions for enzymatic hydrolysis using alkaline protease are: pH 8.0-9.0 and temperature 50-60℃.

6. The method according to any one of claims 1 to 5, characterized in that: In step (1), the mass ratio of the enzymatically hydrolyzed quinoa protein, the pea starch, and the gellan gum is 2.4: 10：0.12。 7. The method according to any one of claims 1 to 6, characterized in that: In step (2), the heating and gelatinization conditions are: incubation in a 98°C water bath for 35 minutes.

8. A composition for preparing a gel, comprising enzymatically hydrolyzed quinoa protein, pea starch, and gellan gum; wherein the enzymatically hydrolyzed quinoa protein is obtained by enzymatic hydrolysis of quinoa protein with an alkaline protease.

9. The composition according to claim 8, characterized in that: In the composition, the mass ratio of the enzymatically hydrolyzed quinoa protein, the pea starch, and the gellan gum is 2.4: 10：0.12。 10. The use of the method of any one of claims 1 to 7 or the composition of claim 8 or 9 in the preparation of soft nutritional foods for the elderly.

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