A method for improving the quality of Maillard products in high-temperature soybean meal using ultrasound-microwave time-series assistance

Abstract

This invention discloses a method for improving the quality of Maillard reaction products from high-temperature soybean meal using a time-series ultrasound-microwave assisted process. The method uses high-temperature soybean meal protease hydrolysate as raw material, which, after ultrafiltration separation, employs a time-series combined with physical-assisted technology of "ultrasound followed by microwave" to optimize the Maillard reaction products. This process not only significantly shortens the Maillard reaction time but also greatly improves the flavor quality and functional properties of the reaction products. Studies have shown that this combined treatment technology effectively enhances the umami flavor of the products, significantly inhibits undesirable flavors, promotes the directional formation of characteristic aroma compounds, and simultaneously enhances the antioxidant activity of the system. This invention provides a process basis for physical-assisted Maillard reactions, offers new ideas for the development of high-quality plant-based flavor ingredients, and also provides an effective strategy for the efficient conversion and value-added processing of plant protein resources, which has significant application value for promoting green manufacturing and sustainable development in the food industry.

Description

Technical Field

[0001] This invention relates to the field of food additives, and in particular to a method for improving the quality of Maillard products in high-temperature soybean meal using ultrasound-microwave time-series assistance. Background Technology

[0002] Soybean meal is an important and abundant plant protein resource after soybean oil extraction, typically containing 44%-49% protein and rich in various amino acids and minerals. It is a high-quality raw material for producing protein hydrolysates and high-value-added flavorings. However, currently, over 95% of soybean meal produced in industrial production is high-temperature soybean meal. During oil extraction, the high temperatures cause significant thermal denaturation of the protein. This deep thermal denaturation not only limits the effective utilization of the protein but also results in high-temperature soybean meal generally having a strong bitter and beany taste, greatly restricting its direct application in the food industry.

[0003] To improve the unpleasant flavor of high-temperature soybean meal, the industry often uses enzymatic hydrolysis to convert large protein molecules into small peptides and amino acids with umami characteristics. This is further combined with Maillard reactions to form richer flavor compounds such as furans, pyrazines, and aldehydes, thereby developing flavor bases or meat flavorings. However, traditional thermo-Maillard reactions have drawbacks such as long reaction cycles, high energy consumption, and low efficiency.

[0004] In recent years, physical-assisted technologies such as ultrasound and microwave have begun to be applied to Maillard reactions in an attempt to improve efficiency. For example, invention patent CN110547410A discloses a method for preparing Maillard-flavored meat products, in which xylose, glucose, and enzymatic hydrolysate are mixed by mass, placed in a microwave heating device, heated to boiling and kept warm to obtain a Maillard reaction solution. Invention patent CN116369491A discloses a method and application for preparing Maillard-characteristic meat flavor peptides from compound meal, in which the secondary enzymatic hydrolysate is centrifuged and the supernatant is collected to obtain a mixed meal enzymatic hydrolysate; by weight, 1.0%–3.0% cysteine, 2.0%–4.0% xylose, 0.03%–0.07% VB1, and 0.5%–1.5% lyophilized lean pork enzymatic hydrolysate powder are dissolved in the above mixed meal enzymatic hydrolysate, and then MRPs are prepared by ultrasound-assisted heating. However, existing technologies often focus only on the application of a single physical technology, lacking research on the synergistic effect of multiple physical technologies in regulating flavor formation pathways and product composition. This results in poor product flavor quality, and the Maillard reaction cycle is long (several hours or even days), with extremely high energy consumption. Prolonged high-temperature cooking can easily lead to the loss of heat-sensitive nutrients and even increase the risk of generating harmful substances such as acrylamide.

[0005] Therefore, when processing high-temperature soybean meal protease hydrolysates, how to accurately improve the flavor quality of the product (such as enhancing umami and suppressing bitterness) through efficient and controllable means, and simultaneously enhance its functional properties (such as antioxidant capacity), remains a key technical problem that urgently needs to be solved in the high-value processing of plant protein resources. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is: how to provide an ultrasonic-microwave time-series assisted method to improve the quality of Maillard products in high-temperature soybean meal, and solve the problems of low reaction efficiency, long reaction time, high energy consumption, and poor flavor quality of high-temperature soybean meal protein reactants in traditional heat treatment for inducing Maillard reaction.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-series assistance, comprising the following steps: (1) The soybean meal protein hydrolysate prepared from high-temperature soybean meal was centrifuged and then separated by ultrafiltration. The permeate was collected and dried to obtain soybean meal protein hydrolysate powder.

[0008] (2) Prepare a solution by mixing soybean meal protein hydrolysate powder obtained in step (1) with xylose, and adjust the pH of the system to 6-8.

[0009] (3) The reaction system prepared in step (2) is first subjected to ultrasonic treatment, and then immediately subjected to microwave treatment; wherein the ultrasonic treatment power is 100~600 W, the pulse mode is adopted, the on time is 2~20 s, the interval time is 2~20 s, and the total treatment time is 10~50 min; the microwave treatment power is 100~600 W, and the treatment time is 1~20 min.

[0010] (4) The reaction system after joint pretreatment in step (3) is transferred to a closed reaction vessel and heated to carry out Maillard reaction. After the reaction is completed, the Maillard reaction product of high temperature soybean meal protein hydrolysate is obtained.

[0011] In this way, firstly, by combining enzymatic hydrolysate with ultrafiltration technology, the severely heat-denatured high-temperature soybean meal macromolecular proteins are targeted for degradation and sieved into small-molecule free amino acids and peptides with flavor potential. Then, a specific temporal physical assistance process of "ultrasound first, then microwave" is applied: ultrasound, through its strong cavitation effect, disrupts the aggregated protein peptide structure, exposing internally hidden active sites such as free amino groups, improving system homogeneity and accelerating the initial stage of the Maillard reaction. Immediately afterwards, microwave dielectric heating is introduced, causing polar molecules to generate intense directional vibrations and frictional heat at the exposed active sites, instantly activating the reaction and accelerating the Maillard reaction process. This ultrasound-microwave sequence not only significantly accelerates the Maillard reaction process but also directionally promotes the formation of key characteristic aroma substances and antioxidant intermediates, fundamentally achieving a simultaneous leapfrog improvement in the flavor and functional quality of the high-temperature soybean meal enzymatic hydrolysate.

[0012] Preferably, the soybean meal protein hydrolysate is obtained through the following methods: compound enzyme hydrolysis, microwave-assisted hydrolysis, or solid-state fermentation combined with hydrolysis. Compound enzyme hydrolysis involves pulverizing high-temperature soybean meal, adding water to form a suspension, and then sequentially adding alkaline protease, flavor protease, glutamine, and / or glutaminase. Hydrolysis is performed stepwise or simultaneously under their respective optimal pH and temperature conditions, degrading soybean meal protein into small peptides and free amino acids. Finally, the enzymes are inactivated and filtered to obtain the hydrolysate. Microwave-assisted hydrolysis utilizes the synergistic effect of microwaves significantly enhancing the thermal kinetic energy of protein molecules and the frequency of intermolecular collisions, increasing the effective contact between enzyme and substrate molecules, thereby enhancing hydrolysis efficiency and obtaining the hydrolysate. Solid-state fermentation combined with hydrolysis involves inoculating a high-temperature soybean meal powder suspension with enzyme-producing strains such as Aspergillus oryzae and Bacillus subtilis for solid-state fermentation. After fermentation, flavor protease and / or glutaminase are added for liquid hydrolysis. Finally, the enzymes are inactivated and separated to obtain the hydrolysate.

[0013] Preferably, the enzyme used in the enzymatic hydrolysis includes at least one of alkaline protease, flavor protease, and glutaminase.

[0014] Preferably, the ultrafiltration separation has a molecular weight cutoff of 4-6 kDa and an operating pressure of 0.3-0.4 MPa. This solves the problem of complex composition in the crude enzymatic hydrolysate and the tendency of large molecules to undergo excessive coking or precipitation in subsequent high-temperature reactions.

[0015] Preferably, in step (2), the mass ratio of soybean meal protein hydrolysate powder to xylose is 5:(2~5); and the mass concentration of the solution is 2%~15%. This range optimizes the pathway for characteristic flavor formation, specifically promoting the generation of pyrazine and furfural volatiles with meaty and caramel aromas, while avoiding problems such as incomplete reaction, excessive browning, or off-flavors caused by substrate imbalance or improper concentration.

[0016] Preferably, the ultrasonic treatment process uses a power of 200-500 W and a total treatment time of 10-35 min. Ultrasonic treatment within this range significantly enhances the sweetness, umami, richness, and overall acceptability of the product. Furthermore, it promotes the formation of rich and harmonious flavor and aroma compounds through Maillard reactions, resulting in a bright color and moderate yellowness, achieving a good balance between appearance and flavor. Conversely, excessively high ultrasonic power exacerbates cavitation and mechanical effects, leading to excessive unfolding and aggregation of soybean meal proteins, hindering the normal Maillard reaction pathway. Excessively long ultrasonic treatment times can damage existing flavor structures or produce undesirable byproducts, both of which degrade the product's flavor and quality.

[0017] Preferably, the microwave treatment process uses a power of 200-400 W and a treatment time of 3-15 min. This microwave treatment within the above range can induce changes in protein structure to increase the contact sites between soybean meal protein hydrolysate and xylose, effectively accelerating the Amadori rearrangement and subsequent intermediate cleavage reactions, thereby significantly promoting the Maillard reaction process.

[0018] Preferably, the heating temperature in step (4) is 80~150℃, and the reaction time is 30~90min. This allows the high-energy intermediate activated by ultrasound and microwave to fully and stably complete deep condensation, driving the Maillard reaction into the middle and late stages, thereby effectively improving the Maillard reaction efficiency and shortening the reaction cycle.

[0019] Another objective of this invention is to provide a Maillard reaction product of high-temperature soybean meal protease hydrolysate prepared by the above method. In the molecular weight distribution of the reaction product, flavor-active peptides with a mass ratio of 180-500 Da account for more than 50%. This product eliminates the beany and bitter tastes, exhibiting a prominent fresh and rich flavor and a harmonious and full aroma. Simultaneously, it possesses a DPPH free radical scavenging capacity of over 86%, achieving a perfect combination of excellent flavor and strong antioxidant properties.

[0020] Another objective of this invention is to provide the application of the Maillard reaction product of the above-mentioned high-temperature soybean meal protease hydrolysate in the preparation of food flavorings. Based on the excellent sensory characteristics (such as umami and caramel flavor) and physicochemical properties (such as antioxidant stability) of this product, it can be used as a natural flavoring base, meat flavor precursor, or antioxidant functional ingredient, and incorporated into various food matrices (such as seasonings, artificial meat, soup bases, and snack foods) through formulation compounding, emulsification, or direct addition.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention innovatively employs a combined ultrasonic-microwave pretreatment technique, achieving highly efficient synergistic coupling in the Maillard reaction process. The initial ultrasonic wave utilizes a strong mechanical cavitation effect to effectively disrupt the dense aggregated structure of the high-temperature soybean meal protein hydrolysate, exposing numerous masked reactive sites and significantly improving the system's homogeneity. Subsequently, microwave heating via dielectric heating induces high-frequency directional vibration and frictional heat generation in polar molecules, instantly overcoming the reaction energy barrier and greatly accelerating the mid-to-late stages of the Maillard reaction. The sequential synergistic effect of ultrasonic-microwave not only accelerates the Maillard reaction process and reduces the bitterness background, but also accumulates abundant low-molecular-weight active peptides and free amino acids. Furthermore, it directionally promotes the formation of melanoidin-like macromolecules and reductone intermediates with strong antioxidant activity within the system, significantly improving the flavor quality and antioxidant function of the high-temperature soybean meal protein hydrolysate Maillard reaction product at both molecular and sensory levels.

[0022] 2. The Maillard reaction product of the high-temperature soybean meal protease hydrolysate prepared by this invention precisely regulates the protein degradation and recombination pathways. In terms of flavor, the proportion of the core components contributing to the umami flavor (flavor-active peptides of 180-500 Da) jumps to 52.35%, while the total content of umami amino acids (glutamic acid and aspartic acid) increases by 1.35 times. In terms of aroma, it specifically promotes the large-scale generation of key caramel / roast meat characteristic flavor substances such as 2,5-dimethylpyrazine and 3-furfural. This process not only endows the product with a rich and full-bodied umami flavor and caramel aroma, but also effectively reduces the total amount of bitter amino acids and reduces unpleasant odors such as beany smell, greatly improving the sensory acceptance and flavor harmony of the product. Furthermore, it promotes the directed generation of more intermediates (such as reductones) and end products (such as melanoidins) with strong antioxidant activity in the Maillard reaction, resulting in a DPPH free radical scavenging rate of 86.24±0.47% and ABTS of the product prepared by this invention. + The free radical scavenging rate reached 76.42±0.18%, successfully maximizing both high-quality flavor and antioxidant function.

[0023] 3. The method of this invention is simple in process and controllable in conditions, requiring no complex equipment, and the Maillard reaction time is short with low energy consumption. Traditional Maillard reactions require several hours or even days to complete the same degree of reaction, while the method of this invention only requires 1-2 hours. This method also solves the problems of flavor degradation, loss of heat-sensitive nutrients, and even increased risk of formation of harmful substances such as acrylamide caused by prolonged high temperatures in traditional Maillard reactions. In addition, this method provides a practical industrial solution for the efficient conversion of low-value-added plant protein resources and the development of high-quality natural plant-based flavorings and antioxidant bases, and has broad prospects for industrial application. Attached Figure Description

[0024] Figure 1 The process of the Maillard reaction of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0025] Figure 2 The color of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0026] Figure 3 The images show the UV-Vis spectra of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0027] Figure 4 The fluorescence spectra of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention are shown.

[0028] Figure 5 The image shows a radar image of the sensory evaluation of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0029] Figure 6 The images show the electronic tongue (a) and principal component analysis (b) of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0030] Figure 7 The molecular weight distribution diagrams are for the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0031] Figure 8 The amino acid distribution (a) and Pearson correlation analysis (b) of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention are shown.

[0032] Figure 9 The images show OPLS-DA analysis of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0033] Figure 10 The antioxidant properties of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 of this invention. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to embodiments. Instruments and other equipment used, unless otherwise specified, are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the methods described are conventional methods, and the raw materials used are all obtainable from publicly available commercial sources. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field, or according to the product instructions.

[0035] I. A method for improving the quality of Maillard products in high-temperature soybean meal using ultrasound-microwave time-series assistance Example 1 This example uses the following steps: (1) After pulverizing the high-temperature soybean meal, pass it through a 40-mesh sieve. Take 6% (w / v) of the soybean meal powder and add it to deionized water to prepare a suspension. After microwave pretreatment (350 W, 3 min), adjust the pH to 9.0 with NaOH solution. Add alkaline protease (Alcalase 2.4 L, Novozymes) at 1.36% of the mass of the high-temperature soybean meal and hydrolyze at 55℃ for 6 h, then inactivate the enzyme at 90℃ for 10 min. Adjust the pH to 7.0, then add flavor protease (Flavourzyme 500 MG, Novozymes) at 0.8% of the mass of the high-temperature soybean meal and hydrolyze at 55℃ for 10 h. After the enzyme is inactivated twice, centrifuge (4000 r / min, 15 min) and collect the supernatant. Add 6% of the solids mass of glutamine to the supernatant, adjust the pH to 10.0, and then add 1.56% of the solids mass of glutaminase (SD-C100S, Amano Enzyme Products Co., Ltd., Japan). React at 37°C for 5.1 h. After the reaction is completed, inactivate the enzyme by boiling in a water bath for 15 min to terminate the reaction. After standing and cooling, centrifuge at 4000 r / min for 15 min and collect the supernatant to obtain the soybean meal protease hydrolysate.

[0036] (2) The obtained soybean meal protein hydrolysate was separated by ultrafiltration using a membrane-type tangential flow membrane separation system and an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The operating pressure was 0.38 MPa and the solution temperature was 5-15℃. The permeate was collected, freeze-dried to obtain soybean meal protein hydrolysate powder, and stored at -20℃ for later use.

[0037] (3) Weigh soybean meal protein hydrolysate powder and xylose at a mass ratio of 5:2 and mix with water to prepare a solution with a mass concentration of 4.67% (w / v). Adjust the pH of the solution system to 7.00 with sodium hydroxide and lactic acid standard solution.

[0038] (4) Place the reaction system prepared in step (3) in an ultrasonic cell disruptor with a frequency of 25 kHz and a power of 400 W. Use pulse mode, turn on for 5 s, pause for 5 s, and total processing time of 20 min. During the process, the sample tube opening is closed and the probe is immersed about 1 cm below the liquid surface. After ultrasonic treatment, immediately place the sample in a microwave reactor and process it at 300 W power for 4 min.

[0039] (5) Transfer the reaction system after joint pretreatment in step (4) to a closed reaction vessel, place it in an oven at 121°C and heat for 58 min to induce the Maillard reaction. After the reaction is completed, cool to room temperature, centrifuge to collect the supernatant, and freeze dry to obtain the Maillard reaction product of high temperature soybean meal protein hydrolysate.

[0040] Example 2 This example specifically includes the following steps: (1) After pulverizing the high-temperature soybean meal, pass it through a 40-mesh sieve. Take 6% (w / v) of the soybean meal powder and add it to deionized water to prepare a suspension. After microwave pretreatment (350 W, 3 min), adjust the pH to 9.0 with NaOH solution. Add alkaline protease (Alcalase 2.4 L, Novozymes) at 1.36% of the mass of the high-temperature soybean meal and hydrolyze at 55℃ for 6 h, then inactivate the enzyme at 90℃ for 10 min. Adjust the pH to 7.0, then add flavor protease (Flavourzyme 500 MG, Novozymes) at 0.8% of the mass of the high-temperature soybean meal and hydrolyze at 55℃ for 10 h. After the enzyme is inactivated twice, centrifuge (4000 r / min, 15 min) and collect the supernatant. Add 6% of the solids mass of glutamine to the supernatant, adjust the pH to 10.0, and then add 1.56% of the solids mass of glutaminase (SD-C100S, Amano Enzyme Products Co., Ltd., Japan). React at 37°C for 5.1 h. After the reaction is completed, inactivate the enzyme by boiling in a water bath for 15 min to terminate the reaction. After standing and cooling, centrifuge at 4000 r / min for 15 min and collect the supernatant to obtain the soybean meal protease hydrolysate.

[0041] (2) The obtained soybean meal protein hydrolysate was separated by ultrafiltration using a membrane-type tangential flow membrane separation system and an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The operating pressure was 0.38 MPa and the solution temperature was 5-15℃. The permeate was collected, freeze-dried to obtain soybean meal protein hydrolysate powder, and stored at -20℃ for later use.

[0042] (3) Weigh soybean meal protein hydrolysate powder and xylose at a mass ratio of 5:2 and mix with water to prepare a solution with a mass concentration of 4.67% (w / v). Adjust the pH of the solution system to 7.00 with sodium hydroxide and lactic acid standard solution.

[0043] (4) Place the reaction system prepared in step (3) in an ultrasonic cell disruptor with a frequency of 25 kHz and a power of 200 W. Use pulse mode, turn on for 2 s, pause for 6 s, and total processing time of 35 min. During the process, the sample tube opening is closed and the probe is immersed about 1 cm below the liquid surface. After ultrasonic treatment, immediately place the sample in a microwave reactor and process it at 200 W power for 15 min.

[0044] (5) Transfer the reaction system after joint pretreatment in step (4) to a closed reaction vessel, place it in an oven at 121°C and heat for 58 min to induce the Maillard reaction. After the reaction is completed, cool to room temperature, centrifuge to collect the supernatant, and freeze dry to obtain the Maillard reaction product of high temperature soybean meal protein hydrolysate.

[0045] Example 3 This example includes the following steps: (1) After pulverizing the high-temperature soybean meal, pass it through a 40-mesh sieve. Take 6% (w / v) of the soybean meal powder and add it to deionized water to prepare a suspension. After microwave pretreatment (350 W, 3 min), adjust the pH to 9.0 with NaOH solution. Add alkaline protease (Alcalase 2.4 L, Novozymes) at 1.36% of the mass of the high-temperature soybean meal and hydrolyze at 55℃ for 6 h, then inactivate the enzyme at 90℃ for 10 min. Adjust the pH to 7.0, then add flavor protease (Flavourzyme 500 MG, Novozymes) at 0.8% of the mass of the high-temperature soybean meal and hydrolyze at 55℃ for 10 h. After the enzyme is inactivated twice, centrifuge (4000 r / min, 15 min) and collect the supernatant. Add 6% of the solids mass of glutamine to the supernatant, adjust the pH to 10.0, and then add 1.56% of the solids mass of glutaminase (SD-C100S, Amano Enzyme Products Co., Ltd., Japan). React at 37°C for 5.1 h. After the reaction is completed, inactivate the enzyme by boiling in a water bath for 15 min to terminate the reaction. After standing and cooling, centrifuge at 4000 r / min for 15 min and collect the supernatant to obtain the soybean meal protease hydrolysate.

[0046] (2) The obtained soybean meal protein hydrolysate was separated by ultrafiltration using a membrane-type tangential flow membrane separation system and an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The operating pressure was 0.38 MPa and the solution temperature was 5-15℃. The permeate was collected, freeze-dried to obtain soybean meal protein hydrolysate powder, and stored at -20℃ for later use.

[0047] (3) Weigh soybean meal protein hydrolysate powder and xylose at a mass ratio of 5:2 and mix with water to prepare a solution with a mass concentration of 4.67% (w / v). Adjust the pH of the solution system to 7.00 with sodium hydroxide and lactic acid standard solution.

[0048] (4) Place the reaction system prepared in step (3) in an ultrasonic cell disruptor with a frequency of 25 kHz and a power of 500 W. Use pulse mode, turn on for 6 s, pause for 10 s, and total processing time of 10 min. During the processing, the sample tube opening is closed and the probe is immersed about 1 cm below the liquid surface. After ultrasonic treatment, immediately place the sample in a microwave reactor and process it at 400 W power for 10 min.

[0049] (5) Transfer the reaction system after joint pretreatment in step (4) to a closed reaction vessel, place it in an oven at 121°C and heat for 58 min to induce the Maillard reaction. After the reaction is completed, cool to room temperature, centrifuge to collect the supernatant, and freeze dry to obtain the Maillard reaction product of high temperature soybean meal protein hydrolysate.

[0050] Comparative Example 1: No ultrasonic or microwave treatment was performed; other steps were the same as in Example 1.

[0051] Comparative Example 2: No microwave treatment was performed; other steps were the same as in Example 1.

[0052] Comparative Example 3: No ultrasonic treatment was performed; other steps were the same as in Example 1.

[0053] Comparative Example 4: Microwave treatment was performed first, followed by ultrasonic treatment, with other steps the same as in Example 1.

[0054] II. Performance Verification 1. The products prepared in Examples 1 and Comparative Examples 1-4 were monitored for the Maillard reaction process by measuring pH value, intermediate products, and browning degree. The experimental methods are as follows: The pH value of each sample was directly measured using a pH meter, and the average value was taken from three parallel measurements. The sample solution was diluted 50 times, and the absorbance was measured at a wavelength of 294 nm to reflect the amount of Amadori rearrangement intermediate products generated. The sample solution was diluted 10 times, and the absorbance was measured at a wavelength of 420 nm to indicate the formation of brown polymers such as melanoidins in the Maillard reaction and the degree of browning of the system. The results are as follows: Figure 1 As shown.

[0055] from Figure 1 As can be seen, during the Maillard reaction, the formation of intermediate products from the Amadori reaction and the brown polymer are related to browning in the system, exhibiting obvious strong absorption peaks at ultraviolet wavelengths of 294 nm and 420 nm, respectively. Figure 1 As can be seen from b, compared with the comparative example, Example 1 (UMUF-MRP), which adopted the combined treatment of "ultrasound followed by microwave", showed the most significant promoting effect: the pH value of the system decreased the most (to 6.21±0.02), and the degree of browning and the amount of intermediate product formation both reached their peaks, with absorbance as high as 0.87±0.02 (420 nm) and 1.34±0.05 (294 nm), respectively. This indicates that the combined physical assistance strategy of ultrasound-microwave has a significant synergistic effect, and its promoting effect on the Maillard reaction is significantly better than that of a single physical assistance technique. At the same time, the treatment group of microwave followed by ultrasound (comparative example 4) was significantly slower than the Maillard reaction process of Example 1. This may be because microwave preferential treatment easily induces non-specific caramelization of the substrate, consumes the active precursor and generates by-products, and subsequent ultrasound is difficult to reverse the unbalanced reaction. In contrast, Example 1, which first ultrasound and then microwave, utilizes the ultrasonic cavitation effect to promote the premixing and dispersion of substrate molecules, and then uses microwaves to uniformly supply energy to drive the reaction in a directional and efficient manner. Therefore, sonication followed by microwave has a more significant advantage in promoting the Maillard reaction in this system.

[0056] 2. The color of the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 was measured using the following method: A WSF spectrophotometer was used under a D65 standard light source in transmission mode to measure the color of the samples. The instrument was calibrated before measurement. The lightness values ​​(L) were recorded respectively. * ), red-green value (a * ) and yellow-blue value (b * The result is as follows: Figure 2 As shown.

[0057] from Figure 2 It can be seen that, compared with the untreated group (Comparative Example 1), the L of the other treated samples... * The values ​​(brightness) all decreased significantly, while a * Value (redness), b * The values ​​(yellowness) all increased significantly. This is a typical indicator of a deeper Maillard reaction, indicating that treatment can promote the formation of brown pigments such as melanoidins and yellow intermediates (such as reductones). In Example 1, which used a combined ultrasonic-microwave treatment, the L... * The value drops to its minimum, and a * and b * The increase in value was most significant, mainly attributed to the large accumulation of melanoidins and the efficient caramelization reaction within the synergistic system. Mechanistically, the initial ultrasonic treatment significantly improved the homogeneity of the reaction system through mechanical cavitation, allowing sugar molecules to be more fully exposed to the reaction microenvironment; the subsequent microwave treatment utilized efficient dielectric heating to induce vigorous dehydration and degradation of xylose (a pentose) in the system, rapidly generating furfural and its derivatives. These compounds themselves are yellow to brown and are the driving force behind the system's degradation. * The significant increase in values ​​is a key contributor. In summary, the temporal synergy of ultrasound and microwave significantly accelerates the browning process of the Maillard reaction.

[0058] 3. The Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 were subjected to UV-Vis spectroscopy. Specifically, each sample solution was diluted 50 times with distilled water, and then scanned across the entire wavelength range of 200-700 nm using a UV-Vis spectrophotometer. The results are as follows: Figure 3 As shown.

[0059] from Figure 3It can be seen that, within the same reaction time, the untreated group (Comparative Example 1) exhibited a significant UV absorption peak at 264 nm; while the Maillard reaction products (MRPs) of the other treated groups not only showed a red shift in their absorption peak (to 270 nm), but also exhibited a stronger UV absorption signal at 294 nm. This phenomenon is highly consistent with the aforementioned research results on the accumulation of Amadori intermediates and the deepening of browning in the system. In particular, Example 1, which used a combined ultrasonic-microwave treatment, achieved the highest UV absorption intensity. This is mainly attributed to the specific sequential stepwise optimization of the molecular structure and mass and heat transfer kinetics of the reaction system by "ultrasonic treatment followed by microwave treatment," and the synergistic physical effect of the two made the Maillard reaction more vigorous and complete.

[0060] 4. The Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 were subjected to fluorescence spectroscopy. The specific method was as follows: the sample solution was diluted 50 times with distilled water, and then scanned using a fluorescence spectrometer. The test parameters were set as follows: excitation wavelength 347 nm, emission wavelength scanning range 350-550 nm, excitation and emission slit widths both 5 nm, and scanning interval 1 nm. The results are as follows. Figure 4 As shown.

[0061] from Figure 4 As can be seen, compared with Comparative Example 1, the fluorescence signals of all treatment groups were enhanced, with Example 1 exhibiting the strongest fluorescence emission intensity. This is mainly because the pre-ultrasonic treatment utilizes a strong mechanical cavitation effect to effectively disrupt the dense aggregated structure of denatured soybean meal protein, exposing a large number of reactive sites (such as lysine ε-amino groups) that were originally hidden inside the molecule. This "structural depolymerization" effect provides an extremely sufficient reaction interface and highly active substrate for the subsequent microwave dielectric heating stage, thereby greatly promoting the formation of fluorescent Maillard reaction intermediates and complex polymers.

[0062] 5. Sensory evaluation was conducted on the Maillard reaction products prepared in Example 1 and Comparative Examples 1-4. The experimental method is as follows: Sensory evaluation was carried out in a constant temperature environment of 23±2℃. A sensory evaluation team composed of 8 professional evaluators (20-30 years old) who had undergone systematic training was formed. Standard calibration was performed before evaluation: standard solutions of 0.08% citric acid, 1% sucrose, 0.08% caffeine, 0.35% sodium chloride, and 0.35% monosodium glutamate were used to represent the five basic tastes of sour, sweet, bitter, salty, and umami, and the highest intensity of each was defined as 10 points. During evaluation, the evaluators tasted each sample for about 10 seconds in turn and scored them using a 10-point scale (0 points represent no taste, and 10 points represent extremely strong taste). To avoid taste fatigue and residual interference, the mouths were rinsed with ultrapure water and rested for 5 minutes between tasting each two samples. The final results were averaged and plotted as a radar chart. The results are as follows. Figure 5 As shown.

[0063] from Figure 5 As can be seen, compared with the untreated group (Comparative Example 1), the physical-assisted treatment significantly improved the overall flavor profile of Maillard reaction products (MRPs). Each pretreated group showed varying degrees of improvement in umami, richness, caramel flavor, and overall acceptability, while the scores for unpleasant odors were significantly reduced. Among them, Example 1 (ultrasound followed by microwave) exhibited the best overall sensory performance. This sequential synergistic treatment effectively promoted the Maillard reaction process, significantly enhancing the product's umami and caramel aroma, while strongly suppressing unpleasant flavors inherent in plant proteins, such as beany odor, greatly improving the product's flavor quality and sensory acceptability.

[0064] 6. The Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 were analyzed using an electronic tongue. The experimental method is as follows: Take 4 mL of the sample solution to be tested and dilute to 100 mL with deionized water. Accurately transfer approximately 80 mL of the diluent into a sample cup specifically for the electronic tongue and perform the measurement at room temperature (the instrument has completed self-testing and calibration before testing). The taste indicators detected include: sourness, astringency, astringent aftertaste (Aftertaste-A), bitterness, bitter aftertaste (Aftertaste-B), saltiness, umami, and richness. Each sample was measured continuously for 4 cycles. The data from the first cycle were discarded, and the stable readings from the last 3 cycles were used for subsequent analysis. The results are as follows: Figure 6 As shown.

[0065] from Figure 6 As can be seen, compared with Comparative Example 1, the umami, richness, and saltiness of the samples in each physical treatment group generally increased significantly, while the sourness, bitterness, and astringency showed a decreasing trend, comprehensively optimizing the flavor characteristics of MRPs. Among them, Example 1 showed the most significant flavor improvement effect, with both bitter and astringent aftertastes reduced to extremely low levels, indicating that this combined treatment can effectively improve the persistence of the product's taste. This is mainly attributed to the synergistic effect of ultrasound and microwaves deeply regulating the structure and degradation pathway of soybean meal protein, promoting the generation of more flavor compounds. Further principal component analysis (PCA) was performed on the electronic tongue data, such as... Figure 6 As shown in b, the variance contribution rates of PC1 and PC2 were 93.8% and 4.0%, respectively, with a cumulative contribution rate of 97.8%. This indicates that the two extracted principal components can fully represent the overall information of the sample and effectively distinguish the differential effects of different physical pretreatments on the flavor profile of MRPs. In the PCA analysis results, Example 1 showed a clear separation from Comparative Example 1 and other physical pretreatment groups in the principal component space, demonstrating its unique flavor profile. This further confirms the unique advantages of ultrasonic and microwave time-series processing in optimizing the flavor characteristics of MRPs.

[0066] 7. The molecular weight distribution of the Maillard reaction products prepared in Examples 1 and Comparative Examples 1-4 was determined using the following methods: High-performance liquid chromatography (HPLC) combined with gel permeation chromatography software (Empower GPC) was used. The sample solution was filtered through a 0.22 μm microporous membrane before injection (injection volume 10 μL). Chromatographic conditions: mobile phase was acetonitrile / water / trifluoroacetic acid (volume ratio 45:55:0.1), elution flow rate was 0.5 mL / min, column temperature was 30℃, and detection was performed at 220 nm using a UV detector. A molecular weight standard curve was plotted using disaccharide peptide (132 Da), unmodified tetrapeptide GGYR (451 Da), Bacillus subtilis peptide (1,450 Da), aprotinin (6,500 Da), and cytochrome C (12,500 Da) as standards. The results are as follows: Figure 7 As shown.

[0067] from Figure 7 As can be seen, compared with Comparative Example 1, the other physical pretreatments significantly reshaped the molecular weight distribution profile of the products. Specifically, the proportion of low molecular weight components (<180 Da and 180~500 Da) increased significantly, while the proportion of high molecular weight components (>1000 Da) decreased accordingly. In particular, in Example 1, the proportion of molecules in the 180~500 Da range surged to 52.35%. This range was mainly enriched with flavor-active oligopeptides (such as dipeptides and tripeptides) that play a key role in enhancing flavor. This result explains, at the molecular level, the phenomenon that "Example 1 has the strongest umami flavor" in the aforementioned sensory evaluation and electronic tongue test, confirming that time-series physical-assisted technology can directionally generate more flavor-active precursors by precisely regulating the protein degradation pathway.

[0068] 8. The Maillard reaction products prepared in Examples 1 and Comparative Examples 1-4 were subjected to determination of free amino acids. The experimental method is as follows: Quantitative determination was performed using a fully automated amino acid analyzer. 1 mL of sample solution was mixed with 1 mL of 5% sulfosalicylic acid to precipitate large protein molecules; after standing at 4℃ for 1 h, it was centrifuged at 10000 r / min for 20 min; the supernatant was filtered through a 0.22 μm filter membrane and then injected for detection. Qualitative and quantitative analysis was performed based on the retention time and peak area of ​​standard amino acids. The results are as follows: Figure 8 As shown.

[0069] from Figure 8As can be seen, compared with Comparative Example 1, the physical pretreatment significantly altered the amino acid profile of the reaction system. The total content of umami amino acids (Glu and Asp) in Example 1 and Comparative Example 4 increased significantly, jumping from the initial 34.42 mg / 100 mL to 46.66 mg / 100 mL and 45.83 mg / 100 mL, respectively. This improvement in chemical indicators is highly consistent with the enhanced umami flavor observed in sensory and electronic tongue analyses. These enriched umami amino acids not only directly impart umami flavor to the product but also efficiently condense with xylose during subsequent microwave heating, serving as important precursors to generate aromatic heterocyclic compounds such as pyrazines and furans, thereby enhancing the caramel aroma. Furthermore, combined with... Figure 8 Pearson correlation analysis (P<0.05) for b showed that (red indicates positive correlation, blue indicates negative correlation, × indicates a significant difference of 0.05 level (P<0.05)), there was a significant structure-activity relationship between the sensory indicators and physicochemical properties of the samples: samples with higher umami and richness scores also showed a significant positive correlation with the content of low molecular weight peptides <500Da (especially in the 180~500Da range) and umami amino acids; conversely, bitterness and astringency indicators showed a significant positive correlation with the content of macromolecular components >3kDa and bitter amino acids. This fully confirms the mechanism logic of Example 1: that is, the "ultrasound-microwave" synergistic pretreatment efficiently degrades macromolecular proteins, reduces the background of bitterness and astringency, and accumulates abundant low molecular weight active peptides and free amino acids. These high-quality precursors are deeply transformed into aroma and flavor substances in the subsequent Maillard reaction, ultimately achieving a leapfrog improvement in the overall flavor quality of the product.

[0070] 9. The Maillard reaction products prepared in Examples 1 and Comparative Examples 1-4 were analyzed for volatile substances using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The experimental method is as follows: Accurately pipette 5 mL of sample solution into a 20 mL headspace vial, add 1 g of NaCl and a magnetically stirred rotor, and immediately seal with a polytetrafluoroethylene (PTFE) septum. After equilibration at 60 °C for 15 min with magnetic stirring, insert a 50 / 30 μm DVB / CAR / PDMS extraction head (activated at 280 °C for 30 min before use) through the septum into the headspace vial for headspace adsorption for 30 min. Subsequently, quickly insert the extraction head into the gas chromatograph inlet and desorb at 250 °C for 5 min. The relative contents of volatile compounds in different pretreatment groups are shown in Table 1. A total of 60 volatile compounds were identified, mainly including 18 aldehydes, 13 ketones, 9 alcohols, 7 esters, 2 pyrazines, and 11 other compounds.

[0071] Gas chromatography (GC) conditions: HP-5MS capillary column (30m × 0.25mm × 0.25μm); injection port temperature 250℃; carrier gas high-purity helium (He), constant flow rate 1.0mL / min; splitless injection mode. Temperature program: initial column temperature 40℃, hold for 4 min; increase to 120℃ at 5℃ / min, hold for 1 min; then increase to 250℃ at 6℃ / min, hold for 6 min.

[0072] Mass spectrometry (MS) conditions: ionization mode was electron impact source (EI), electron energy 70 eV; chromatographic-mass spectrometry interface temperature 280℃, ion source temperature 230℃, quadrupole temperature 150℃; mass scan range (m / z) was 30~550.

[0073] Table 1 As shown in Table 1, benzaldehyde and isophorone were the main volatile components in the untreated group (Comparative Example 1). In contrast, the treatment in Example 1 significantly promoted the formation of key aroma compounds such as pyrazines and furans in the Maillard reaction. This change in microstructure is highly consistent with the previously mentioned "enrichment of flavor peptides" in the molecular weight distribution, and also corroborates the enhancement of "caramel aroma" in sensory evaluation and the improvement of taste profile in electronic tongue analysis, jointly confirming that Example 1 can significantly improve the flavor quality of soybean meal Maillard reaction products.

[0074] To further evaluate the impact of different physical pretreatments on the overall aroma profile of Maillard reaction products, an orthogonal partial least squares discriminant analysis (OPLS-DA) model was constructed, using the relative content of 60 common volatile compounds as variables and different pretreatment methods as the classification criteria. Figure 9 As shown in the score plot (a), sample clusters under different physical preprocessing conditions can be effectively distinguished. The model exhibits excellent goodness-of-fit indices: R0 2 The x-fit index is 0.99, and R0 is... 2 The y-fit index is 0.999, and the model prediction index (Q) is... 2 The coefficient of performance (COP) is 0.98, indicating a good model fit. Further verification of model reliability was conducted using a permutation test with 200 iterations, and the results are as follows: Figure 9 As shown in (b), Q is generated by all permutations. 2 The intersections of the regression lines with the vertical axis were all less than 0, confirming that the model did not overfit. The model was validated and demonstrated high robustness and reliability. It is believed that the model can be used to effectively identify and analyze the effects of different physical pretreatments on the aroma profile of the Maillard reaction products of soybean meal.

[0075] 10. The Maillard reaction products prepared in Example 1 and Comparative Examples 1-4 were analyzed for antioxidant activity. The results are as follows: Figure 10 As shown. The experimental method is as follows: The DPPH free radical scavenging rate of each sample was determined. The enzymatic hydrolysate and Maillard reaction product were diluted 50 times respectively. 2.0 mL of each sample solution was mixed with 2.0 mL of 0.1 mol / L DPPH solution and reacted at room temperature in the dark for 20 min. The absorbance was measured at 517 nm. 2.0 mL of 95% anhydrous ethanol solution was used as the control group instead of DPPH solution, and 2.0 mL of distilled water was used as the blank control instead of each sample solution. The DPPH free radical scavenging rate of each sample solution was calculated using formula (1): (1) In the formula, A1 represents the absorbance of each sample liquid; A2 represents the absorbance of the control group; and A0 represents the absorbance of the blank control group.

[0076] ABTS of each sample was measured + Free radicals are generated by mixing equal volumes of 7 mM ABTS solution and 2.45 mM potassium persulfate solution to form ABTS. + The working solution should be stored at room temperature, protected from light, for 12-16 hours. Before starting the test, prepare ABTS with phosphate buffer (pH 7.4, 5 mM). + The absorbance of the working solution was diluted to 0.70 ± 0.02. 2.5 mL of ABTS was added to 2.5 mL of the sample solution. + The working solution was thoroughly mixed and reacted at room temperature for 25 min. The absorbance was then measured at 734 nm. A buffer solution was used instead of the ABTS solution as a blank control, and distilled water was used instead of the sample solution as a control. + The free radical scavenging rate is calculated using formula (2).

[0077] (2) In the formula, B1 represents the absorbance of each sample liquid; B2 represents the absorbance of the control group; and B0 represents the absorbance of the blank group.

[0078] from Figure 10It can be seen that the antioxidant activity of the combined treatment groups (Example 1 and Comparative Example 4) was significantly enhanced compared with the untreated group (Comparative Example 1) (P<0.05). Furthermore, Example 1 showed significantly better performance than other single treatments and reverse-order treatments, achieving a DPPH radical scavenging rate of 86.24±0.47% and an ABTS radical scavenging rate of 76.42±0.18%. This leap in antioxidant capacity is mainly attributed to the effective depolymerization of protein structures by physical pretreatment, exposing more reaction sites and thus powerfully driving the in-depth development of the Maillard reaction. Example 1 exhibited the strongest antioxidant activity, which is highly consistent with the aforementioned molecular weight analysis result that "Example 1 had the highest proportion of low molecular weight active peptides." The temporal synergistic effect of ultrasound-microwave not only accelerated the reaction process but also directionally promoted the generation of melanoidin-like macromolecules and reductone intermediates with extremely strong antioxidant activity within the system. The results confirm that the specific temporal combined pretreatment of "ultrasound first, then microwave" is the optimal strategy for enhancing the antioxidant function of high-temperature soybean meal Maillard reaction products.

[0079] It should be noted that the experimental results of Examples 2 and 3 are basically the same as those of Example 1, and will not be repeated here.

[0080] In summary, this invention, by establishing a combined pretreatment sequence of "ultrasound followed by microwave" and a synergistic mechanism with the Maillard reaction, can significantly improve the flavor quality and antioxidant function of the Maillard reaction products of high-temperature soybean meal protease hydrolysates at the molecular and sensory levels within a shorter process cycle. This method provides a practical and feasible technical path to solve the problem of low utilization rate of low-value bulk plant protein resources, and offers a new process reference for developing high-quality, natural plant-based flavor ingredients. It has significant theoretical and applied value for promoting green manufacturing, energy conservation, and sustainable development in the modern food industry.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction, characterized in that, Includes the following steps: (1) The soybean meal protein hydrolysate prepared from high-temperature soybean meal was centrifuged and then subjected to ultrafiltration separation. The permeate was collected and dried to obtain soybean meal protein hydrolysate powder. (2) Prepare a solution by mixing soybean meal protein hydrolysate powder obtained in step (1) with xylose, and adjust the pH of the system to 6-8; (3) The reaction system prepared in step (2) is first subjected to ultrasonic treatment, and then immediately subjected to microwave treatment; wherein, the power of ultrasonic treatment is 100~600 W, pulse mode is adopted, the on time is 2~20 s, the interval time is 2~20 s, and the total treatment time is 10~50 min; the power of microwave treatment is 100~600 W, and the treatment time is 1~20 min; (4) The reaction system after the combined treatment in step (3) is transferred to a closed reaction vessel and heated to carry out the Maillard reaction. After the reaction is completed, the Maillard reaction product of high temperature soybean meal protein hydrolysate is obtained.

2. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 1, characterized in that, The soybean meal protein hydrolysate is obtained by the following methods: compound enzyme hydrolysis, microwave-assisted hydrolysis, or solid-state fermentation combined with hydrolysis.

3. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 2, characterized in that, The enzymes used in the enzymatic hydrolysis include at least one of alkaline protease, flavor protease, and glutaminase.

4. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 1, characterized in that, The ultrafiltration separation process has a molecular weight cutoff of 4-6 kDa and an operating pressure of 0.3-0.4 MPa.

5. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 1, characterized in that, The mass ratio of soybean meal protein hydrolysate powder to xylose in step (2) is 5:(2~5); the mass concentration of the solution is 2%~15%.

6. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 1, characterized in that, The ultrasonic treatment process uses a power of 200-500 W and a total treatment time of 10-35 min.

7. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 1, characterized in that, The microwave processing power is 200~400 W, and the processing time is 3~15 min.

8. The method for improving the quality of Maillard products in high-temperature soybean meal with ultrasound-microwave time-assisted extraction according to claim 1, characterized in that, The heating temperature in step (4) is 80~150℃, and the reaction time is 30~90min.

9. The Maillard reaction product of high-temperature soybean meal protease hydrolysate prepared by any one of claims 1 to 8.

10. The application of the Maillard reaction product of the high-temperature soybean meal protease hydrolysate according to claim 9 in the preparation of food flavoring agents.

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