A preparation process for chickpea plant-based milk

Abstract

This invention discloses a preparation process for chickpea plant-based milk, relating to the field of food processing technology. The process includes the following steps: cooking → crushing → homogenization → first enzymatic hydrolysis → pH adjustment → second enzymatic hydrolysis → enzyme inactivation → filtration → ingredient blending → high-speed shearing → homogenization → sterilization → bottling. The chickpea plant-based milk prepared by this invention based on enzymatic hydrolysis technology has a protein content of 1.86±0.05g / 100g, with a protein utilization rate increased by 38.4%, a fat content of 0.85±0.13g / 100g, a dietary fiber content of 1.51±0.29g / 100g, an eGI value of 57.27±1.35, an average particle size of 124.49μm, and an absolute zeta potential of 55.67±2.16mV. Eleven flavor compounds that contribute to sensory evaluation were detected in the plant milk. The content of beany substances such as hexanal was reduced, while other flavor compounds were enhanced, giving the plant milk a mellow aroma, a subtle fruity fragrance, and a more distinct sense of layering.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a preparation process for chickpea plant-based milk. Background Technology

[0002] Milk is favored by the public for its high protein content, but some people cannot drink it due to lactose intolerance. In addition, dairy farming has a certain negative impact on the ecological environment, including the occupation of land resources and pollution caused by feed and excrement.

[0003] However, current plant-based milks suffer from low protein content and poor stability, often requiring the addition of food additives to improve their quality. This contradicts consumers' pursuit of natural and healthy products and fails to meet their daily nutritional needs. Many companies are actively investing in plant-based milk research and development. Using plants as raw materials, plant-based milk is both environmentally friendly and meets the needs of lactose-intolerant individuals.

[0004] Therefore, there is an urgent need for innovative technological solutions to resolve this dilemma. The chickpea plant-based milk preparation process has emerged as a result. It is expected to overcome the challenges of protein and stability by optimizing chickpeas, processing technology and product formula, enriching product flavor, providing consumers with high-quality plant-based beverages and promoting the healthy development of the plant-based milk industry. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of low protein content, poor stability and monotonous flavor in existing plant-based milk, and to provide a preparation process for chickpea plant-based milk.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a preparation process for chickpea plant-based milk, characterized by comprising the following steps:

[0007] S1. Preparation of raw materials

[0008] S11. Take chickpeas, wash and remove impurities, soak and pressure cook them;

[0009] S12. Crush the steamed chickpeas and homogenize them in an ultrasonic cell disruptor;

[0010] S2. Stepwise enzymatic hydrolysis to prepare enzymatic hydrolysate

[0011] S21. Add α-amylase and β-amylase to the homogenized mixture, and perform enzymatic hydrolysis for 25-40 min with magnetic stirring in a water bath at 50-55℃ to carry out the first stage of enzymatic hydrolysis. Add sodium bicarbonate to adjust the pH to 8.0.

[0012] S22. After the first stage of enzymatic hydrolysis, add alkaline protease and hydrolyze with magnetic stirring in a water bath at 40-50℃ for 25-40 minutes.

[0013] S23. The enzyme-inactivated mixture after enzymatic hydrolysis is placed in a water bath and filtered to obtain the enzymatic hydrolysate;

[0014] S3. Blending plant-based milk products

[0015] S31. Heat the enzymatic hydrolysate and add the weighed xylitol, sucralose, acesulfame potassium, stabilizer, perilla oil, and dietary fiber evenly.

[0016] S4. Shear and homogenize the prepared mixture;

[0017] S5. Sterilize the homogenized mixture by ultra-high temperature instantaneous sterilization;

[0018] S6. Cool and fill the sterilized product.

[0019] Preferably, the formulation of the enzymatic hydrolysate prepared in step S2 includes: 8-12% chickpeas, 0.2%-1.0% β-amylase, 0.2%-1.2% α-amylase, 0.1%-0.3% alkaline protease, and the remainder being purified water.

[0020] Preferably, the formulation of the plant-based milk obtained in step S3 includes: 93%-95% enzymatic hydrolysate, 0.2%-0.8% compound stabilizer, 0.4%-0.6% xylitol, 0.001%-0.004% sucralose, 0.006%-0.01% acesulfame potassium, 0.2%-0.8% perilla oil, and 3%-3.5% dietary fiber.

[0021] Preferably, in step S22, the alkaline protease is enzymatically hydrolyzed in a water bath at 46°C with magnetic ion stirring for 25-40 minutes.

[0022] Preferably, the specific steps of preparation in step S31 are as follows: heating the enzymatic hydrolysate to 55-60°C, adding the well-mixed xylitol, sucralose, acesulfame potassium, stabilizer, and dietary fiber while stirring, and finally adding perilla oil.

[0023] Preferably, the specific steps of soaking and high-pressure cooking in step S11 are as follows: add water with a weight of 9 times that of the raw material and soak for 2 hours, then high-pressure cook for 50 minutes.

[0024] Preferably, the specific steps of crushing and homogenizing in step S12 are as follows: crushing chickpeas in a high-speed blender for 8-15 minutes at a power of 800W; homogenizing the resulting mixture in an ultrasonic cell disruptor at 420W for 20-40 minutes.

[0025] Preferably, the ratio of α-amylase, β-amylase and alkaline protease added in step S2 is 5:4:1.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention incorporates 3%-3.5% dietary fiber, which improves the gut microenvironment. The composition of the gut microbiota determines the efficiency of energy acquisition from food, and changes in dietary composition are related to changes in the gut microbiota composition. Diet can regulate the composition and function of the gut microbiota; when dietary changes are significant and rapid, the microbiota changes rapidly, and long-term dietary habits can shape the gut microbiota composition. Fiber-rich diets have been shown to improve insulin resistance in lean and obese diabetic patients.

[0028] This invention uses chickpeas as raw material. Chickpeas, as a low-glycemic food, possess health benefits such as lowering blood sugar and lipids, anti-oxidation, and regulating intestinal flora, making them suitable as an adjunct treatment food for diabetes and other conditions. Based on the glycemic index theory and using low glycemic index (GI) raw materials, this invention produces a stable chickpea enzymatic hydrolysate with a protein content of 1.9g / 100g, which serves as a base for subsequent product development. The chickpea plant-based milk prepared by this invention incorporates stabilizers to improve product stability and vegetable oils to enhance the saturation of the plant-based milk's flavor, thus achieving a similar effect to animal milk. Furthermore, by selecting low glycemic index (GI) raw materials, this invention can, to some extent, delay the rapid rise in postprandial blood sugar. The chickpea plant-based milk prepared using the formula and method of this invention has a glycemic index of 50-58, making it suitable for diabetic patients.

[0029] The enzymatic hydrolysis process in this invention has a significant impact on odor compounds. The contents of substances such as ethyl acetate, (E)-2-butenal, n-butanol, and β-pinene show an increasing trend as the enzymatic hydrolysis process proceeds. These flavor compounds impart an mellow aroma, a subtle fruity fragrance, and a more distinct sense of layering to the chickpea hydrolysate. The enzymatic hydrolysis process also helps to improve product stability and extend its shelf life. Attached Figure Description

[0030] Figure 1 This is a flowchart of the preparation process of the present invention.

[0031] Figure 2 The figure shows the experimental results of the effect of different enzyme preparation combinations on the enzymatic hydrolysis effect in the preparation process of the present invention.

[0032] Figure 3 The figure shows the experimental results of the effect of different material-to-liquid ratios on the enzymatic hydrolysis effect in the preparation process of this invention.

[0033] Figure 4 The figure shows the experimental results of the effect of different enzyme-to-base ratios on the enzymatic hydrolysis effect in the preparation process of this invention.

[0034] Figure 5The figure shows the experimental results of the effect of different enzymatic hydrolysis times on the enzymatic hydrolysis effect in the preparation process of the present invention.

[0035] Figure 6 The figure shows the experimental results of the effect of different enzymatic hydrolysis temperatures on the enzymatic hydrolysate in the preparation process of this invention.

[0036] Figure 7 This is a graph showing the experimental results of comparing the predicted values ​​with the actual values ​​in the RSM model during process selection in this invention.

[0037] Figure 8 The figure shows the experimental results of the interaction between various factors in the process selection of this invention on the effect of turbidity.

[0038] Figure 9 The figure shows the experimental results of the interaction between various factors in the process selection of this invention on the protein content.

[0039] Figure 10 Differential caloric scanning (DSC) spectrum of the chickpea plant milk prepared according to the present invention.

[0040] Figure 11 This is a principal component analysis diagram of the chickpea flour, chickpea pulp, and enzymatic hydrolysate samples of the present invention.

[0041] Figure 12 This is a bar chart showing the differential compound content of chickpea flour, pulp, and enzymatic hydrolysate samples from this invention.

[0042] Figure 13 This is a diagram showing the particle size comparison results of the chickpea enzymatic hydrolysate and the original pulp of this invention.

[0043] Figure 14 This is a photograph comparing the layering of the chickpea enzymatic hydrolysate and the original pulp of this invention.

[0044] Figure 15 This is a comparison diagram of the microstructure of the chickpea enzymatic hydrolysate and the original pulp of the present invention.

[0045] Figure 16 This is a comparison chart of the coagulation index of the chickpea enzymatic hydrolysate and the original pulp of this invention.

[0046] Figure 17 This is a comparison chart of the flocculation index of chickpea enzymatic hydrolysate and raw pulp according to the present invention.

[0047] Figure 18 This is a comparison diagram of the protein concentration at the interface between the chickpea enzymatic hydrolysate and the original pulp of this invention.

[0048] Figure 19 This is a comparison chart of the protein content at the interface between the chickpea enzymatic hydrolysate and the original pulp of this invention.

[0049] Figure 20This is a comparison diagram of the rheological properties of chickpea enzymatic hydrolysate and original pulp in this invention. Detailed Implementation

[0050] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0051] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0052] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0053] I. Chickpea Plant-Based Milk and its Preparation Process

[0054] A low glycemic index chickpea plant-based milk is composed of the following substances in weight percentage:

[0055] The formulation of the enzymatic hydrolysate is as follows: 8-12% chickpeas, 0.2%-1.0% β-amylase, 0.2%-1.2% α-amylase, 0.1%-0.3% alkaline protease, and the remainder is purified water; the pH is adjusted to 8.0 using sodium bicarbonate.

[0056] The formula for chickpea plant-based milk includes: 93%-95% chickpea hydrolysate (protein content 1.89±0.12g / 100g), 0.2%-0.8% compound stabilizer, 0.4%-0.6% xylitol, 0.001%-0.004% sucralose, 0.006%-0.01% acesulfame potassium, 0.2%-0.8% perilla oil, and 3%-3.5% cellulose.

[0057] The preparation process of the chickpea plant-based milk involves using the above-mentioned substances as raw materials in the stated percentage proportions. The enzymatic hydrolysate process includes: washing, impurity removal, soaking, cooking, crushing, homogenization, enzymatic hydrolysis, pH adjustment, enzymatic hydrolysis, enzyme inactivation, and filtration; cooking → crushing → homogenization → first enzymatic hydrolysis → pH adjustment → second enzymatic hydrolysis → enzyme inactivation → filtration; and the chickpea plant-based milk process includes: ingredient blending → high-speed shearing → homogenization → sterilization → bottling.

[0058] Specifically, the preparation steps for the chickpea plant-based milk are as follows:

[0059] Step 1: Steaming: Wash the chickpeas and remove impurities. Soak them in water with 9 times their weight for 2 hours. Steam them under high pressure (using the SY-50YC9086 electric pressure cooker) for 50 minutes. The steaming process is to remove the raw bean smell from the chickpeas.

[0060] Step 2: Crushing: Crush the chickpeas in a high-speed blender (the equipment can be the SP605R high-speed blender produced by SUPOR Group Co., Ltd.) for 8-15 minutes with a power of 800W; crush the cooked chickpeas to prepare for the extraction of internal nutrients.

[0061] Step 3: Homogenize the mixture obtained in the previous step in an ultrasonic cell disruptor at 420W for 20-40 minutes.

[0062] Step 4: Enzymatic hydrolysis: Enzymatic hydrolysis consists of two stages:

[0063] The first stage of enzymatic hydrolysis: Add α-amylase and β-amylase as shown in the enzymatic hydrolysate ingredients to the mixture obtained in the previous step, and perform enzymatic hydrolysis with magnetic stirring in a water bath at 50-55℃ for 25-40 minutes. The addition of α-amylase and β-amylase in this step can effectively improve the stability and rheological properties of chickpea plant-based milk, degrade α-galactose into small glucose molecules, thereby improving product stability, solving the bottleneck problem of easy precipitation in plant-based milk, and effectively controlling the glycemic index of the final product; then adjust the pH to 8.0 using sodium bicarbonate.

[0064] The second stage of enzymatic hydrolysis: Add the alkaline protease as shown in the enzymatic hydrolysate ingredients, and perform magnetic ion stirring enzymatic hydrolysis in a water bath at 46℃ for 25-40 minutes. Chickpea protein accounts for 25%–30% of the total dry weight of the kernels, twice that of oat protein. Chickpea protein is mainly composed of globulin (56%), glutenin (15%–25%), albumin (12%), and prolamins (2.8%). Glutenin is an insoluble protein, and the alkaline protease effectively enhances its solubility, thereby increasing the protein content in plant-based milk to as high as 1.9g / 100g.

[0065] Step 5: Enzyme inactivation: Start timing when the mixture reaches 90°C in a water bath and maintain for 10 minutes.

[0066] Step 6: Filtration: Filter through a 100-mesh filter and retain the filtrate as the enzymatic hydrolysate.

[0067] The above steps involve adding the appropriate amounts of substances according to the formula of the enzymatic hydrolysate.

[0068] Step 7: Preparation: Weigh out xylitol, sucralose, acesulfame potassium, stabilizer, perilla oil, and dietary fiber according to the chickpea plant-based milk formula. First, heat the enzymatic hydrolysate to 55-60℃, then add the well-mixed xylitol, sucralose, acesulfame potassium, stabilizer, and dietary fiber while stirring. Finally, add the perilla oil. Xylitol, sucralose, and acesulfame potassium are added to increase the sweetness of the plant-based milk; the stabilizer is added to further increase the stability of the final plant-based milk product based on the enzymatic hydrolysate, extending its shelf life; and dietary fiber is added to improve the intestinal microenvironment.

[0069] Step 8: Shearing: Shear the prepared mixture at 1800 rpm / min for 10 min.

[0070] Step 9: Homogenization: Homogenize the mixture obtained in the previous step in an ultrasonic cell disruptor at 420W for 30 minutes;

[0071] Step 10: Sterilization: Use ultra-high temperature instantaneous sterilization at 123℃ for 3-5 seconds;

[0072] Step 11: Cooling and Packaging: Cool to room temperature before packaging.

[0073] II. Creative Analysis of Reaction Conditions in the Process of this Invention

[0074] The selection of enzyme preparation combination, material-to-liquid ratio (amount of chickpeas added), enzyme-to-base ratio, total hydrolysis time, and hydrolysis temperature are all innovative designs adopted for this invention. Corresponding experiments were conducted to screen, determine, and verify these aspects. The specific screening and verification methods are as follows:

[0075] 1. Single-factor experiment

[0076] Based on the preliminary experimental results, single-factor experiments were conducted on enzyme preparation combination, material-to-liquid ratio, enzyme-to-bottom ratio, total enzymatic hydrolysis time, and enzymatic hydrolysis temperature, using turbidity and protein content as indicators.

[0077] 1.1 Effect of different enzyme combinations on enzymatic hydrolysis efficiency

[0078] Weigh and mix chickpeas with distilled water at a ratio of 1:9 (w / w), soak for 4 hours, then place in a juicer and grind at speed 8 (800W) for 10 minutes. Then homogenize using an ultrasonic cell disruptor (420W, 30 minutes). The liquid obtained in this step is chickpea puree, which is ready for use.

[0079] Combination I: Amylase, hemicellulase, and alkaline protease. Chickpea pulp was kept warm in a 55°C water bath. 1% amylase and 0.7% hemicellulase were added, mixed well, and then incubated in a 55°C water bath with stirring for 30 minutes. The pH of the solution was adjusted to 8 with sodium bicarbonate. 0.20% alkaline protease was added and incubated in a 53°C water bath with stirring for 30 minutes. The temperature was then raised to 90°C and maintained for 10 minutes to inactivate the enzyme. After cooling to room temperature, the solution was filtered through a 100-mesh filter. The resulting filtrate was used as the aqueous phase for later use.

[0080] Combination II: Hemicellulase, pectinase, amylase, and alkaline protease. Chickpea pulp was kept warm in a 55°C water bath. 1% amylase, 0.7% hemicellulase, and 0.2% pectinase were added, mixed well, and then placed in a 53°C water bath for enzymatic hydrolysis for 30 minutes. Subsequent treatment was the same as in Combination I.

[0081] Combination III: Hemicellulase, pectinase, α-amylase, β-amylase, and alkaline protease. Chickpea pulp was incubated in a 55°C water bath. 1% amylase, 0.8% β-amylase, 0.7% hemicellulase, and 0.2% pectinase were added, mixed well, and then placed in a 53°C water bath for enzymatic hydrolysis for 30 minutes. Subsequent treatment was the same as in Combination I.

[0082] Combination IV: Hemicellulase, α-amylase, β-amylase, and alkaline protease. Chickpea pulp was incubated in a 55°C water bath. 1% α-amylase, 0.8% β-amylase, and 0.7% hemicellulase were added, mixed well, and then placed in a 53°C water bath for enzymatic hydrolysis for 30 minutes. Subsequent treatment was the same as in Combination I.

[0083] Combination V: α-amylase, β-amylase, and alkaline protease. The treated mixture was incubated in a water bath at 55°C. 1% α-amylase and 0.8% β-starch were added, mixed thoroughly, and then placed in a water bath at 53°C for enzymatic hydrolysis for 30 minutes. Subsequent treatment was performed using combination I.

[0084] 1.2 Effect of material-to-liquid ratio on the enzymatic hydrolysis effect of chickpeas

[0085] The material-to-liquid ratio was 6:100, 8:100, 10:100, 12:100, 14:100, and 16:100. The fixed enzyme-to-substrate ratio was 2:100 (α-amylase:β-amylase:alkaline protease = 5:4:1, i.e., the addition amounts of α-amylase, β-amylase, and alkaline protease were 1%, 0.8%, and 0.2%, respectively). The total enzymatic hydrolysis time was 60 min (the enzymatic hydrolysis time of stage I: stage II = 1:1, i.e., the enzymatic hydrolysis time of stage I and stage II were 30 min each). The enzymatic hydrolysis temperature of stage II was 53℃.

[0086] 1.3 Effect of enzyme-to-base ratio on the enzymatic hydrolysis efficiency of chickpeas

[0087] The enzyme-to-solid ratio was 0:100, 1:100, 2:100, 3:100, and 4:100, with a fixed material-to-liquid ratio of 10:100. The total enzymatic hydrolysis time was 60 min, and the enzymatic hydrolysis temperature in stage II was 53℃.

[0088] 1.4 Effect of hydrolysis time on the hydrolysis efficiency of chickpeas

[0089] Preliminary experiments showed that the optimal enzymatic hydrolysis effect was achieved when the ratio of enzymatic hydrolysis time in stage I to stage II was 1:1. The total enzymatic hydrolysis time was 40 min, 50 min, 60 min, 70 min, and 80 min, with a fixed material-to-liquid ratio of 10:100, an enzyme-to-substrate ratio of 2:100, and a stage II enzymatic hydrolysis temperature of 53℃.

[0090] 1.5 Effect of enzymatic hydrolysis temperature on the enzymatic hydrolysis effect of chickpeas

[0091] The enzymatic hydrolysis temperatures for stage II were 43℃, 46℃, 49℃, 52℃, and 55℃, with a fixed material-to-liquid ratio of 10:100, an enzyme-to-substrate ratio of 2:100, and a total hydrolysis time of 70 min.

[0092] 2. Single-factor experimental verification and analysis

[0093] 2.1 Effect of different enzyme combinations on enzymatic hydrolysis efficiency

[0094] With the same substrate, different enzyme preparations act on different sites. Turbidity can effectively reflect the effect of enzyme preparations on chickpea dissolution; higher turbidity indicates higher chickpea dissolution and less precipitation. Stability is also an important indicator for evaluating a food base. Furthermore, gluten in chickpeas is an insoluble protein, and alkaline protease can act on the peptide bonds of amino acids such as alanine and leucine in gluten, thereby increasing the content of soluble proteins in the enzymatic hydrolysate. Therefore, under the same conditions, turbidity, protein content, and static standing tests were performed on chickpea hydrolysates hydrolyzed with different enzyme combinations. Figure 2 As shown.

[0095] like Figure 2 It was found that the protein content in the chickpea hydrolysate was greater than 0.76±0.0245 g / 100 g, with the highest value being 1.32±0.0102 g / 100 g. Alkaline protease significantly increased the soluble protein content (p<0.05). Among the five enzyme combinations, Group I had the lowest turbidity (0.86±0.0078 NTU); Groups III and V had turbidities of 1.34±0.0078 NTU and 1.43±0.0137 NTU, respectively, showing no significant difference, but significantly higher than the other combinations. This indicates that Groups III and V had better enzymatic hydrolysis effects.

[0096] Chickpea hydrolysates prepared by five different combinations of enzyme preparations were left to stand at room temperature for 24 hours. Groups II and IV showed a small amount of precipitation, Group I showed slight stratification, and Groups III and V showed stable and homogeneous hydrolysates.

[0097] In summary, Group V enzyme preparations showed the best enzymatic hydrolysis effect, the highest protein content, the most dissolved substances, and the solution was homogeneous and stable, without precipitation or stratification.

[0098] 2.2 Effect of material-to-liquid ratio on enzymatic hydrolysis efficiency

[0099] The enzyme-to-solvent ratio was selected as 2:100, the hydrolysis time was 60 min, and the second-stage hydrolysis temperature was 53℃. The effect of the solid-liquid ratio on turbidity and protein content was investigated, and the results are as follows: Figure 3 As shown.

[0100] Depend on Figure 3 It can be seen that as the chickpea content increases, the turbidity initially increases and then levels off. This may be because the increased material-to-liquid ratio and enzymatic hydrolysis lead to an increase in the amount of substances in the hydrolysate. However, when the chickpea content reaches a certain level, the enzymatic hydrolysis effect of the enzyme reaches its maximum. When the material-to-liquid ratio is greater than 10:100, the degree of enzymatic hydrolysis decreases, and the turbidity change becomes more gradual. The protein content shows an increasing trend. Within the material-to-liquid ratio range of 6:100-10:100, alkaline protease has a significant effect on the enzymatic hydrolysis of glutenin in chickpeas, even reaching the maximum degree of hydrolysis. When the material-to-liquid ratio is greater than 10:100, only albumin and globulin in chickpeas contribute to the protein content in the chickpea hydrolysate, and the increase in protein content becomes more gradual.

[0101] 2.3 Effect of enzyme-to-base ratio on enzymatic hydrolysis efficiency

[0102] A material-to-liquid ratio of 10:100 was selected, the enzymatic hydrolysis time was 60 min, and the second-stage enzymatic hydrolysis temperature was 53℃. The effect of the enzyme-to-liquid ratio on turbidity and protein content was investigated, and the results are as follows: Figure 4 As shown.

[0103] Depend on Figure 4It can be seen that as the enzyme-to-substrate ratio increases, the turbidity first rises and then falls. This is because α-amylase acts on the α-1-4-glycosidic bonds within starch molecules, hydrolyzing starch into dextrin and small-molecule reducing sugars; alkaline protease acts on gluten, significantly reducing its molecular weight and disulfide bond content, while increasing the soluble protein content. Both of these factors contribute positively to the increase in turbidity. When the enzyme-to-substrate ratio is greater than 2:100, the turbidity decreases, possibly because the enzyme content increases, leading to over-hydrolysis and the filtration of some small molecules, resulting in less substance in the hydrolysate. When the enzyme-to-substrate ratio is less than 2:100, the enzyme reacts fully with the substrate, significantly increasing the protein content. When the enzyme-to-substrate ratio is greater than 2:100, gluten is fully hydrolyzed, and the accumulation of hydrolysis products inhibits the enzyme, resulting in a slow increase in protein content.

[0104] 2.4 Effect of enzymatic hydrolysis time on enzymatic hydrolysis efficiency

[0105] The material-to-liquid ratio was selected as 10:100, the enzyme-to-substrate ratio as 2:100, and the enzymatic hydrolysis temperature in stage II was 53℃. The effect of hydrolysis time on turbidity and protein content was investigated, and the results are as follows: Figure 5 As shown.

[0106] Depend on Figure 5 It can be seen that as the enzymatic hydrolysis time increases, the turbidity and protein content show a trend of first increasing and then decreasing. When the enzymatic hydrolysis time is 70 minutes, both turbidity and protein content reach their peak values. After 70 minutes, the contents of both begin to gradually decrease. With prolonged enzymatic hydrolysis, the hydrolysis becomes more complete, and both turbidity and protein content increase. When the enzymatic hydrolysis time exceeds 70 minutes, there is no significant effect on the hydrolysis of chickpea starch, and the accumulation of hydrolysis products also inhibits the enzyme preparation.

[0107] 2.5 Effect of enzymatic hydrolysis temperature on the enzymatic hydrolysate

[0108] The material-to-liquid ratio was selected as 10:100, the enzyme-to-substrate ratio as 2:100, and the enzymatic hydrolysis time as 70 min. The effect of the enzymatic hydrolysis temperature in stage II on turbidity and protein content was investigated, and the results are as follows: Figure 6 As shown.

[0109] from Figure 6 As the enzymatic hydrolysis temperature in stage II increases, the turbidity rises slowly without significant change. This is because the hydrolysis by α-amylase and β-amylase plays a dominant role in turbidity, while alkaline protease hydrolysis has no significant effect on turbidity. When the hydrolysis temperature exceeds 46℃, the protein content begins to decrease. This may be because the optimal hydrolysis temperature for the alkaline protease used in the experiment is around 46℃, at which point the enzyme activity is highest and the degree of hydrolysis of insoluble proteins is greater. With increasing hydrolysis temperature, the degree of hydrolysis of insoluble proteins decreases, and the protein content in the chickpea hydrolysate also decreases.

[0110] Based on the results of the single-factor experiments above, a response surface methodology (RSM) optimization experiment was designed, focusing on four factors that significantly affect the enzymatic hydrolysis effect of chickpeas: the material-to-liquid ratio, the enzyme-to-bottom ratio, the total hydrolysis time, and the second-stage hydrolysis temperature. Turbidity and protein content were used as evaluation indicators for the hydrolysis effect. The factor level table for the RSM experiment is shown in Table 1.

[0111] Table 1. Factor Level Table for Chickpea Enzymatic Hydrolysis Process

[0112]

[0113] 3. Response Surface Experiment Results and Analysis

[0114] 3.1 Response Surface Optimization Experimental Scheme and Results

[0115] Based on the results of single-factor experiments, this study used the RSM optimization method to analyze the effects of independent variables (A: material-to-liquid ratio, B: enzyme-to-base ratio, C: hydrolysis time, D: stage II hydrolysis temperature) on dependent variables (Y1: turbidity, Y2: protein content). A four-factor, three-level Box-Behnken experimental design was used to further determine the effects of hydrolysis process parameters on the turbidity and protein content of chickpea hydrolysate. The response surface optimization experiments and their results are shown in Table 2.

[0116] Table 2 Response surface design and results

[0117]

[0118] Table 2 shows that in the RSM optimized experimental group, the turbidity ranged from 1.35 NTU to 1.90 NTU, and the protein content ranged from 1.35 g / 100 g to 1.92 g / 100 g. The experimental values ​​and predicted values ​​showed good agreement (e.g., ...). Figure 7 (As shown).

[0119] 3.2 Regression Model and Analysis of Variance

[0120] Using Design Expert 13.0.5, analysis of variance and multivariate regression fitting were performed to derive a binary regression equation for turbidity and protein content. The regression equations for turbidity (Y1), protein content (Y2), and material-to-liquid ratio (A), enzyme-to-bottom ratio (B), hydrolysis time (C), and hydrolysis temperature (D) are as follows:

[0121] Y1=32.37607+1.60981A+0.956415B+0.069596C+0.979722D+0.008675AB+0.000787AC-0.0011 46AD-0.002135BC-0.010275BD+0.000881CD-0.079140A20.009860B20.000811C2-0.010962D2

[0122] (2-1)

[0123] Y2=-35.97745+0.995773A+0.254368B+0.149009C+1.16407D+0.023750AB+0.000438AC-0.0033 33AD-0.00925BC+0.013417BD+0.001873CD-0.040955A2-0.114571B2-0.001532C2-0.014054D2

[0124] (2-2)

[0125] Analysis of variance (ANOVA) was performed on the above regression equations, and the results are shown in Table 2. A response model was established, and the significance of the regression coefficients in the response surface model was evaluated using ANOVA. Larger regression coefficients and smaller p-values ​​in the model indicate a greater impact on the corresponding response variables. According to the ANOVA results (Table 2-5), the regression model p < 0.0001, indicating a good fit. The lack-of-fit term (p = 0.0721 / 0.3036) was not significant. Turbidity (Y1) and protein content (Y2) showed significant correlations with the factors, meaning the model can be used with a low error probability. The correlation coefficients for turbidity (Y1) and protein content (Y2) were R²(Y1) = 0.9726 and R²(Y2) = 0.9537, respectively, with correction coefficients AdjR²(Y1) = 0.9935 and AdjR²(Y2) = 0.9624, respectively, indicating good regression fit and strong correlation between the two models. Furthermore, the acceptable signal-to-noise ratios (Adeq Precision) for turbidity (Y1) and protein content (Y2) were 53.02 and 22.27, respectively, both greater than 4, indicating that this model can perform preliminary analysis and prediction of response surface factors.

[0126] The influence of each parameter on the dependent variable is determined based on the F-value. The influence on turbidity is in the order of A>B>C>D, and the influence on protein content is in the order of A>C>D>B.

[0127] Table 3 Analysis of variance for regression models

[0128]

[0129]

[0130] Note: * indicates a significant difference, * represents p<0.05, and ** represents p<0.01.

[0131] According to the analysis of variance (Table 3), the first and second terms of the chickpea material-to-liquid ratio (A, A2) and the first and second terms of the enzyme-to-base ratio (B, B2) had a significant effect on turbidity (p<0.01). The interaction terms between the material-to-liquid ratio, enzyme-to-base ratio, and enzymatic hydrolysis time (AB, AC) also had a significant effect on turbidity (p<0.05). This indicates that the material-to-liquid ratio and enzyme-to-base ratio are key factors affecting turbidity in the chickpea enzymatic hydrolysis process. The interaction terms between the enzyme-to-base ratio and enzymatic hydrolysis time and the second-stage enzymatic hydrolysis temperature (BC, BD), as well as the interaction term between the enzymatic hydrolysis time and the enzymatic hydrolysis temperature (CD), had significant responses to turbidity (p<0.05). Enzymatic hydrolysis can alter the structure of macromolecules in the chickpea solution. The enzyme-to-base ratio, the second-stage enzymatic hydrolysis temperature, time, and pH are all decisive factors affecting the hydrolysis effect; sufficient enzymatic hydrolysis is beneficial for improving turbidity. The linear and quadratic terms of the solid-liquid ratio (A, A2) and the linear and quadratic terms of the hydrolysis time (C, C2) had significant effects on protein content (p<0.01). The interaction terms of the solid-liquid ratio and the enzyme-to-substrate ratio (AB, AC) had a significant effect on protein content (p<0.05). Alkaline protease can effectively increase the content of soluble protein in chickpeas. Therefore, the amount of enzyme and substrate added plays a decisive role in the protein content of chickpea hydrolysate. The interaction terms among the solid-liquid ratio, hydrolysis time, and hydrolysis temperature (BC, BD, CD) had a significant effect on protein content (p<0.05).

[0132] like Figure 8 As shown in the figure, a and e represent the effects of the interactions between the material-to-liquid ratio and the enzyme-to-base ratio, the material-to-liquid ratio and the enzymatic hydrolysis time, the enzyme-to-base ratio and the enzymatic hydrolysis time, the material-to-liquid ratio and the second-stage enzymatic hydrolysis temperature, and the second-stage enzymatic hydrolysis temperature and the enzymatic hydrolysis time on turbidity, respectively. Figure 9 As shown in the figure, a and b represent the effects of the interaction between the material-to-liquid ratio and the enzyme-to-bottom ratio, the enzyme-to-bottom ratio and the enzymatic hydrolysis time, and the enzymatic hydrolysis temperature and the enzymatic hydrolysis time on the protein content, respectively.

[0133] The graph visually demonstrates the strength of the interaction between two factors. A steeper change in the response surface between the two factors indicates a greater impact on the dependent variable, while a shallower change indicates a weaker interaction. Furthermore, if the contour lines at the bottom of the graph are elliptical, a strong interaction exists, and vice versa. Figure 8(d) The contour lines mapped from the B-enzyme-base ratio and the D-II segment enzymatic hydrolysis temperature in the 3D response surface are elliptical, indicating a strong interaction between the two factors, consistent with the results of the analysis of variance (p<0.01). Figure 9 (c) The contour lines of the 3D response surface mapping between the B-enzyme-to-base ratio and the C-hydrolysis time are elliptical, indicating a strong interaction between the two factors, consistent with the variance results (p<0.01). This may be because insoluble proteins in chickpeas, under appropriate alkaline protease and suitable hydrolysis time, can bind more fully to leucine sites and undergo enzymatic hydrolysis, thus contributing to an increase in protein content in the chickpea hydrolysate.

[0134] 4. Determination and verification of optimal process

[0135] Based on the results of the single-factor experiments or response surface analysis above, and combined with the regression model, the optimal process conditions for preparing chickpea enzymatic hydrolysate are predicted as shown in Table 4.

[0136] Table 4 Optimum process and validation test of the model

[0137]

[0138] Through response surface methodology (RSM) optimization experiments, the optimal production process predicted by the model was obtained, as shown in Table 2-6. To improve the feasibility and operability of actual production, the process parameters were fine-tuned. The optimal process parameters for preparing chickpea hydrolysate were: material-to-liquid ratio of 10.5:100 (w / w), enzyme-to-substrate ratio of 2:100 (w / w), hydrolysis time of 70 min, and hydrolysis temperature of 46℃ in stage II. At this time, the turbidity was 1.85±0.21 NTU and the protein content was 1.89±0.12 g / 100 g, which were close to the predicted values. This indicates that the model optimized by RSM can obtain the optimal production process for chickpea hydrolysate.

[0139] Figure 10 This is the differential calorimetry (DSC) spectrum of chickpea plant milk. As shown in the figure, the DSC spectrum of chickpea plant milk has only one smooth endothermic peak, indicating that the purity of the chickpea plant milk components is relatively high. The denaturation temperature of chickpea plant milk is 129.92℃, which is relatively high. The enthalpy (ΔH) value reflects the denaturation state of proteins. A higher enthalpy value indicates a stronger force maintaining the protein structure, more ordered protein structure, and thus better protein thermal stability. The enthalpy value of chickpea plant milk is 1574.18 mW / g, indicating good stability and relative insensitivity to heat.

[0140] III. Performance Analysis of the Plant-Based Milk Product Prepared by the Invention

[0141] 1. Analyze the impact of enzymatic hydrolysis process on raw materials.

[0142] The following materials were selected for experimental comparison and analysis:

[0143] Chickpea flour: Ground chickpea flour, without any processing;

[0144] Original pulp: The product obtained from chickpeas through steaming, crushing, and homogenization processes, which has only undergone the method of this invention but has not undergone enzymatic hydrolysis;

[0145] Enzymatic hydrolysate: The product obtained by enzymatic hydrolysis and sterilization of raw pulp; the base material of enzymatic hydrolysate without added ingredients.

[0146] like Figure 11 The image shows a bar chart illustrating the spatial relative distances between pairs of samples. Larger distances indicate greater differences, while greater distances indicate smaller differences. Figure 11 It can be seen that the chickpea flour sample is closest to the original pulp sample, meaning the difference between the chickpea flour and the original pulp sample is the smallest, indicating that the processing steps before enzymatic hydrolysis do not significantly alter the flavor compounds. Conversely, the chickpea flour sample is furthest from the enzymatic hydrolysate sample, meaning the difference between the chickpea flour and the enzymatic hydrolysate sample is the largest, indicating that flavor development is related to the enzymatic hydrolysis process.

[0147] 2. Effects of enzymatic hydrolysis process on the differences in flavor compounds in chickpea-based milk

[0148] like Figure 12 The bar chart shows the differential compound content of chickpea flour, chickpea pulp, and enzymatic hydrolysate samples. The differences in compound content among the three samples are clearly visible. 12 and 11 flavor compounds were detected in chickpea flour and chickpea enzymatic hydrolysate, respectively, while 10 flavor compounds were detected in chickpea pulp. 2-Propane, hexanal, 2-butanone, and 2-heptanone are the compounds responsible for the beany flavor. Figure 12 It is evident that the contents of 2-propanone, 2-butanone, and hexanal increase linearly with the progress of cooking, crushing, and enzymatic hydrolysis. The contents of ethyl acetate, (E)-2-butenal, n-butanol, and β-pinene also show an increasing trend with the enzymatic hydrolysis process. These flavor compounds impart an mellow aroma, a subtle fruity fragrance, and a more distinct layering of flavor to the chickpea hydrolysate. Furthermore, after processing according to this invention, the content of 2-butanone in the hydrolysate is significantly lower than that in chickpea flour, effectively reducing the beany taste in chickpea plant milk.

[0149] 3. Product storage stability analysis

[0150] from Figure 13As can be seen, in the early stage of storage, the chickpea enzymatic hydrolysate had a particle size of d(4,3) = 51.7 μm, while the average particle size of the original pulp was 70.99 μm. The particle size of the enzymatic hydrolysate was lower than that of the original pulp, and smaller particle size is more beneficial to the stability of the product. The stability of the enzymatic hydrolysate of this invention is significantly increased compared to the original pulp.

[0151] Depend on Figure 14 , 15 It can be seen that the chickpea enzymatic hydrolysate prepared under the process conditions of the present invention is as follows: Figure 14 (a) It can be seen that the chickpea hydrolysate has good overall homogeneity, with proteins exhibiting a sheet-like structure and slight protein aggregation. Figure 14 As can be seen, the chickpea enzymatic hydrolysate is light brown. After 4 days of storage, the system exhibits good homogeneity, with no precipitation or stratification. With prolonged storage, slight stratification marks appear on day 24. The chickpea pulp is light yellow. Initially, the system is relatively stable, with uniform color and no precipitation or stratification. Slight precipitation appears after 16 days of storage, and stratification becomes more pronounced and intensifies by day 24. From... Figure 15 The microstructure diagram shows that chickpea pulp contains more macromolecules than the enzymatic hydrolysate. This is because enzymatic hydrolysis disrupts the globular structure of proteins, hydrolyzing them into smaller soluble protein monomers or oligomers, leading to the conversion of insoluble proteins into soluble proteins and increasing the soluble protein content in the system. Amylose is converted into amylopectin, addressing the sedimentation problem caused by adhesion. With prolonged storage, the aggregation rate of macromolecules in the pulp is significantly higher than that in the enzymatic hydrolysate, demonstrating that enzymatic hydrolysis significantly improves the sedimentation problem caused by macromolecules.

[0152] Figure 16 , 17 Data was recorded after storing chickpea enzymatic hydrolysate and raw pulp at 25℃ for 28 days, with monitoring every 4 days. During storage, flocculation and coagulation of droplets in the emulsion generally occurred simultaneously. The graph shows that the CI and FI of the raw pulp and enzymatic hydrolysate increased progressively: the CI of the raw pulp increased from 100.4% to 108.2%, and the FI increased from 8.9% to 102.4%; while the CI of the enzymatic hydrolysate increased from 99% to 107.5%, and the FI increased from 8.7% to 93.5%. The enzymatic hydrolysis process not only increased the soluble protein content of the chickpea emulsion system but also endowed it with better stability.

[0153] 4. Product interface protein content and concentration

[0154] Figure 18 , 19The changes in interfacial protein concentration (Γ) and interfacial protein content (AP) of chickpea hydrolysate and raw chickpea pulp during storage were recorded. The figures show that the interfacial protein concentration and content in the chickpea hydrolysate were both higher than those in the raw chickpea pulp.

[0155] 5. Comparison of product rheological properties

[0156] The effects of storage rheological properties of chickpea hydrolysate and pulp, such as Figure 20 The figures show the apparent viscosity after storage at 25℃ for 4, 8, 12, 16, 20, 24, and 28 days at shear frequencies of 0-100 s⁻¹. -1 A graph showing the changes between [times]. From [the context]... Figure 20 As can be seen, the viscosity did not change significantly with the extension of the storage time of the enzymatic hydrolysate, indicating that the rheological properties of the chickpea enzymatic hydrolysate are relatively stable, while the rheological properties of the original pulp changed significantly within 28 days.

[0157] Table 5 Basic indicators of chickpea plant-based milk Table 1 Basic indicators of CPBM

[0158] Physicochemical indicators result unit protein 1.86±0.05 g / 100g Fat 0.85±0.13 g / 100g Dietary fiber 1.51±0.29 g / 100g Total bacterial count 0 cfu / mL Acidity value 3±0.23 °T pH value 8.12±0.02 - L* / a* / b* / E 66.91±0.03 / -2.00±0.08 / 20.52±0.07 / 85.43±0.04 - eGI 57.27±1.35 - Zeta potential -55.68±2.16 mV

[0159] Note: Data in the table is expressed as "mean ± standard deviation"; - indicates no unit.

[0160] The above analysis shows that the chickpea hydrolysate prepared by this invention can be stored for more than 28 days. This storage time is solely due to the enzymatic hydrolysis process and the raw materials used in this invention, without the addition of any additives. Adding stabilizers during subsequent formulation will further enhance the stability of the finished plant-based milk. The chickpea plant-based milk prepared by this invention based on enzymatic hydrolysis technology has a protein content of 1.86±0.05g / 100g, with a protein utilization rate increased by 38.4%, a fat content of 0.85±0.13g / 100g, a dietary fiber content of 1.51±0.29g / 100g, an eGI value of 57.27±1.35, an average particle size of 124.49μm, and an absolute zeta potential of 55.67±2.16mV. Eleven flavor compounds that contribute to sensory evaluation were detected in the plant milk. The content of beany substances such as hexanal was reduced, while other flavor compounds were enhanced, giving the plant milk a mellow aroma, a subtle fruity fragrance, and a more distinct sense of layering.

[0161] In summary, the method of this invention can produce plant-based milk with a long shelf life, mellow flavor, light fruity aroma, high protein content, and low glycemic index, which can replace animal milk and is suitable for people with lactose intolerance.

Claims

1. A preparation process for chickpea plant-based milk, characterized in that, Includes the following steps: S1. Preparation of raw materials S11. After washing and removing impurities from the chickpeas, soak them in distilled water and then pressure cook them. S12. The steamed chickpeas are crushed and homogenized in an ultrasonic cell disruptor to obtain a mixture; S2. Stepwise enzymatic hydrolysis to prepare enzymatic hydrolysate S21. Add α-amylase and β-amylase to the homogenized mixture, and perform enzymatic hydrolysis for 35 min with magnetic stirring in a water bath at 50-55℃ to carry out the first stage of enzymatic hydrolysis. Add sodium bicarbonate to adjust the pH to 8.

0. S22. After the first stage of enzymatic hydrolysis, add alkaline protease and hydrolyze with magnetic stirring in a water bath at 46°C for 35 min. The ratio of α-amylase, β-amylase and alkaline protease is 5:4:

1. S23. The enzyme-inactivated mixture after enzymatic hydrolysis is placed in a water bath and filtered to obtain the enzymatic hydrolysate; The components used to prepare the enzymatic hydrolysate, by weight percentage, are: 8-12% chickpeas, 0.2%-1.0% β-amylase, 0.2%-1.2% α-amylase, 0.1%-0.3% alkaline protease, and the remainder is distilled water; S3. Blending plant-based milk products S31. Heat the enzymatic hydrolysate and add the weighed xylitol, sucralose, acesulfame potassium, compound stabilizer, perilla oil, and dietary fiber evenly. The ingredients of chickpea plant-based milk, by weight percentage, are: 3%-95% chickpea hydrolysate, 0.2%-0.8% compound stabilizer, 0.4%-0.6% xylitol, 0.001%-0.004% sucralose, 0.006%-0.01% acesulfame potassium, 0.2%-0.8% perilla oil, and 3%-3.5% dietary fiber. S4. Shear and homogenize the prepared mixture; S5. Sterilize the homogenized mixture by ultra-high temperature instantaneous sterilization; S6. Cool and fill the sterilized product.

2. The preparation process of chickpea plant-based milk as described in claim 1, characterized in that: The specific steps of preparation in step S31 are as follows: heat the enzymatic hydrolysate to 55-60°C, then add the well-mixed xylitol, sucralose, acesulfame potassium, compound stabilizer, and dietary fiber while stirring, and finally add perilla oil.

3. The preparation process of chickpea plant-based milk as described in claim 1, characterized in that: The specific steps for adding distilled water for soaking and high-pressure cooking in step S11 are as follows: add 9 times the weight of the raw material in distilled water for soaking for 2 hours, and then high-pressure cook for 50 minutes.

4. The preparation process of chickpea plant-based milk as described in claim 1, characterized in that: The specific steps for crushing and homogenizing in step S12 are as follows: crush chickpeas in a high-speed blender for 8-15 minutes at a power of 800W; homogenize the resulting mixture in an ultrasonic cell disruptor at 420W for 20-40 minutes.

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