Soybean protein small peptide capable of generating meat flavor substance through Maillard reaction as well as preparation method and application of soybean protein small peptide

Abstract

The invention relates to a soybean protein small peptide capable of generating a meat flavor substance through a Maillard reaction, and a preparation method and application thereof. The preparation method comprises the following specific steps: drying, crushing, sieving, degreasing and drying soybean cake meal to obtain degreased soybean cake meal powder; the method comprises the following steps: carrying out serial enzymolysis on soybean meal by using alkaline protease and flavourzyme to obtain soybean meal protein enzymatic hydrolysate, carrying out ultrafiltration purification on the soybean meal protein enzymatic hydrolysate, collecting ultrafiltrate, and freeze-drying; the functional peptide is identified through a mass spectrum analysis result, and the sequence of the functional peptide is as follows: leucine-glycine-methionine-glycine-serine-tyrosine-arginine-serine-alanine-leucine (LGMGSYRSAL). Then preparing a roasted steak flavor substance through a Maillard reaction, and analyzing flavor components of the product through GC-MS (Gas Chromatography-Mass Spectrometer). The small peptide prepared by the invention is clear in amino acid sequence structure, a Maillard reaction product roasted beef steak is aromatic in flavor, and the flavor functional substances of roasted beef steak meat are analyzed; the process can be put into industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of agricultural products, and in particular to a soybean protein peptide that can generate meaty flavor through Maillard reaction, and a preparation method and application thereof. Background Art

[0002] Soybean meal, produced after soybean oil removal or oil extraction, contains a high protein content. Enzymatic hydrolysis of these proteins yields numerous small peptides, some of which possess unique biological activities, while others can further react to produce functional substances. The Maillard reaction, also known as the "non-enzymatic browning reaction," is a complex reaction between carbonyl compounds (reducing sugars) and amino compounds (such as amino acids, peptides, proteins, and amines) in foods at a specific temperature. The Maillard reaction produces a large number of volatile heterocyclic compounds and unique volatile flavoring substances, which are one of the primary sources of food color and aroma. The complex and diverse structural compositions of soybean meal small peptides suggest that utilizing soybean meal small peptides via the Maillard reaction to prepare meat-flavored seasonings has broad application prospects and research potential. Summary of the Invention

[0003] The present invention aims to provide a method for preparing a functional peptide with a specific amino acid sequence from soybean meal through enzymatic hydrolysis and ultrafiltration, and further generating a meaty flavor through the Maillard reaction. The functional peptide has the following sequence: Leucine-Glycine-Methionine-Glycine-Serine-Tyrosine-Arginine-Serine-Alanine-Leucine (LGMGSYRSAL).

[0004] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a soybean protein peptide capable of generating a meaty flavor through the Maillard reaction, the peptide being composed of the following amino acid residues: leucine-glycine-methionine-glycine-serine-tyrosine-arginine-serine-alanine-leucine.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: the method for preparing soybean protein peptides capable of generating meaty flavor through the Maillard reaction comprises the following steps:

[0006] Step 1: Raw material pretreatment

[0007] drying, crushing and defatting the soybean meal to obtain defatted soybean meal powder;

[0008] Step 2: Enzymatic hydrolysis of soybean meal

[0009] Defatted soybean meal powder is added to water, and alkaline protease and flavor protease are used for tandem enzymatic hydrolysis, and soybean meal protease hydrolyzate is obtained after the enzymatic hydrolysis is completed.

[0010] Step 3: Separation and purification of soybean meal flavor peptides

[0011] The soybean meal protein hydrolysate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the 2000 Da ultrafiltration permeate was collected; the 2000 Da ultrafiltration permeate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean protein peptides;

[0012] Step 4: Mass spectrometry analysis and identification

[0013] It has been identified that the amino acid sequence of the soybean protein peptide is composed of: leucine-glycine-methionine-glycine-serine-tyrosine-arginine-serine-alanine-leucine.

[0014] The preferred technical solution is: in step 1, the soybean cake is dried and then crushed, and the soybean cake powder is sieved through a 60-mesh sieve, and petroleum ether is added at a ratio of 10-15:1 v / w to soak the soybean cake powder, stirring once every 10-20 minutes, soaking for 1.5-3 hours, filtering to remove the solvent, and drying to obtain defatted soybean cake powder.

[0015] The preferred technical solution is: in step 2, adding 85-90°C hot water at a ratio of 15-20:1 v / w to soak defatted soybean meal powder and keeping warm for 20-60 minutes, then cooling to 40-50°C, and adjusting the pH value of the solution to 8.8-9.2; adding alkaline protease at a rate of 3000-4000U per gram of substrate, keeping warm for enzymatic hydrolysis for 3.5-4.0 hours, and keeping warm at 85-95°C for 5-15 minutes to inactivate the enzyme; adjusting the pH value of the solution to 6.3-6.7, and then adding flavor protease at a rate of 650-700U per gram of substrate, keeping warm at 45-55°C for enzymatic hydrolysis for 4.5-5.0 hours, and keeping warm at 85-95°C for 5-15 minutes to inactivate the enzyme, to obtain soybean meal protease hydrolyzate.

[0016] The preferred technical solution is: in step 4, the soy protein peptide is desalted using a ZiptipC18 microchromatographic column, the flavor peptide solution is collected, and freeze-dried. The dried powder is dissolved with 0.1% formic acid by mass, centrifuged at 3-5°C for 15-25 minutes, and the supernatant is transferred to a sample tube for LC-MS / MS identification; the amino acid sequence of the substance with a retention time of 22.27 minutes is: Leucine-Glycine-Methionine-Glycine-Serine-Tyrosine-Arginine-Serine-Alanine-Leucine.

[0017] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the use of the soy protein peptide that can produce meat flavor through the Maillard reaction.

[0018] The preferred technical solution is: soy protein peptides and xylose are mixed in a weight ratio of 5-15:3, the mixture is dissolved in ultrapure water to prepare a solution with a concentration of 5-15% w / v, the pH value of the solution is adjusted to 7.5, and it is transferred to a reaction vessel, the reaction temperature is controlled at 105-115°C, the reaction time is 25-35 min, and then the reaction temperature is changed to 115-125°C, the reaction time is 85-100 min, and a grilled steak flavor Maillard reaction product is obtained.

[0019] Due to the use of the above technical solution, the present invention has the following advantages compared with the prior art:

[0020] The small peptide prepared by the invention has a clear amino acid sequence structure, the Maillard reaction product has a strong grilled steak aroma, and grilled steak aroma functional substances are analyzed; and the process of the invention can be put into industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Mass spectrometric peak patterns of Maillard reaction products from soybean meal by GC-MS analysis.

[0022] Figure 2 Characteristic fragment ion peaks of soybean cake flavor functional peptides.

[0023] Figure 3 Example 1 Mass spectrum peak pattern of GC-MS analysis of Maillard reaction products.

[0024] Figure 4 Example 2 Radar chart of sensory evaluation of Maillard reaction products. DETAILED DESCRIPTION

[0025] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0026] See also Figure 1-4 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no technical substantive significance. Any modification of the structure, change in the proportional relationship or adjustment of the size. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional consumables and biochemical reagent stores.

[0027] Example 1: A soybean protein peptide capable of generating meaty flavor through the Maillard reaction, and its preparation method and application

[0028] (1) Pretreatment: 150 g of soybean meal was dried, crushed, and passed through a 60-mesh sieve to obtain soybean meal powder.

[0029] (2) Defatting: Take 100 g of soybean meal powder, add 1 L of petroleum ether and soak it, stirring once every 15 minutes, soak it for 2 hours, filter out the solvent, and dry it to obtain defatted soybean meal powder.

[0030] (3) Enzymatic hydrolysis of soybean meal: 50 g of soybean meal was added to 750 mL of hot water at 85°C, kept warm for 30 min, cooled to 45°C, and the pH of the solution was adjusted to 8.8 with 0.1 mol / L NaOH. 175,000 U of alkaline protease was added for hydrolysis for 3.5 h, and then kept warm at 90°C for 10 min to inactivate the enzyme. The pH of the solution was adjusted to 6.3 with 0.1 mol / L hydrochloric acid, and 34,000 U of flavor protease was added. The solution was kept warm at 50°C for 4.5 h, and then kept warm at 90°C for 10 min to inactivate the enzyme. The soybean meal enzymatic hydrolyzate was obtained.

[0031] (4) Collection of soybean meal flavor functional peptides: The soybean meal enzymatic hydrolysate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the permeate was collected; the 2000 Da ultrafiltration permeate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean meal flavor functional peptides.

[0032] (5) Preparation of meat flavor seasoning: 5.0 g of soybean cake flavor functional peptide was mixed with 1.5 g of D-xylose, and ultrapure water was added to adjust the mixture concentration to 10% (w / v). The pH of the solution was adjusted to 7.5, and the mixture was placed in a 150 mL beaker. The oil bath reaction temperature was 110 °C, the reaction time was 30 min, and then the reaction temperature was changed to 120 °C, the reaction time was 90 min, and the Maillard reaction product was obtained.

[0033] (6) GC-MS analysis of the composition of Maillard reaction products: Solid phase microextraction (SPME) combined with GC-MS was used to determine the volatile compounds in the Maillard reaction products. The sample (5.0 mL) was placed in a headspace vial (15 mL) and 2.0 μL of 1,2-dichlorobiphenyl was added and mixed thoroughly, then sealed. The SPME head was inserted into the headspace vial in a 55°C water bath for extraction for 40 min, and then transferred to the inlet of the gas chromatography-mass spectrometry (GC-MS) and resolved at 250°C for 5 min. GC-MS conditions were as follows: a WAX column (30 m × 0.25 mm × 0.25 μm); high-purity helium carrier gas at a flow rate of 1 mL / min, splitless; temperature settings were as follows: 40°C for 3 min, then increased to 100°C at 2°C / min, then to 150°C at 4°C / min, and then to 280°C at 20°C / min for 10 min; the detector port temperature was 250°C; the ion source and interface temperatures were set at 280°C and 250°C, respectively; the filament emission current was 35 μA, the detector voltage was 1000 V, the electron energy was 70 eV, the scan mass range was 35–450 amu, and the scan rate was 4.45 amu / s. The NIST 20.L database was used for database searching and matching. Figure 3 Figure 1 is the mass spectrum peak spectrum of the Maillard reaction product GC-MS analysis, and Table 1 is the analysis results of its important volatile substances.

[0034] Table 1 Analysis results of flavor composition of Maillard reaction products in Example 1

[0035] Detected flavors Molecular formula Content (ng / g) CAS number 2-Methyl-3-furanthiol <![CDATA[C5H6OS]]> 77.16 28588-74-1 Bis(2-methyl-3-furyl) disulfide <![CDATA[C 10 H 10 OS2]]> 3.861 28588-75-2 2-Furylmethylthiol <![CDATA[C5H6OS]]> 23.36 35828 2-Methyl-3-methylthiopyrazine <![CDATA[C6H8N2S]]> 40.28 67952-65-2 2,5-Dimethylpyrazine <![CDATA[C6H8N2]]> 18.57 123-32-0 2,3,5-Trimethylpyrazine <![CDATA[C7H 10 N2]]> 5.23 14667-55-1 2-Acetylpyrazine <![CDATA[C6H6N2O]]> 9.86 22047-25-2 3-Mercapto-2-butanone <![CDATA[C4H8OS]]> 8.86 40789-98-8 Methylthiopropionaldehyde <![CDATA[C4H8OS]]> 4.86 3268-49-3 3-Methylbutanal <![CDATA[C5H 10 The]]> 25.36 590-86-3 2-Methylpropionaldehyde <![CDATA[C4H8O]]> 20.08 78-84-2 Phenylacetaldehyde <![CDATA[C8H8O]]> 7.77 122-78-1 p-Cresol <![CDATA[C7H8O]]> 4.66 106-44-5 2-Acetylthiazole <![CDATA[C5H5NOS]]> 32.56 24295-03-2 2-Acetyl-2-thiazoline <![CDATA[C5H7NOS]]> 45.24 29926-41-8 4-Hydroxy-2,5-dimethyl-3(2H)-furanone <![CDATA[C6H8O3]]> 2.28 3658-77-3 2-Ethyl-3,5-dimethylpyrazine <![CDATA[C8H 12 N2]]> ND 27043-05-6 Furfurylthiol <![CDATA[C4H4OS]]> 85.02 98-02-2 2-Methyl-3-sulfide <![CDATA[C2H6S3]]> 15.29 3658-80-8 γ-Valerolactone <![CDATA[C5H8O2]]> 45.76 108-29-2

[0036] Example 2: A soybean protein peptide capable of generating meaty flavor through the Maillard reaction, and its preparation method and application

[0037] (1) Pretreatment: 300 g of soybean meal was dried, crushed, and passed through a 60-mesh sieve to obtain soybean meal powder.

[0038] (2) Defatting: Take 200 g of soybean meal powder, add 3 L of petroleum ether and soak it, stirring once every 15 minutes, soak it for 2 hours, filter out the solvent, and dry it to obtain soybean meal.

[0039] (3) Enzymatic hydrolysis of soybean meal: 100 g of soybean meal was added to 2 L of hot water at 85°C, kept warm for 30 min, cooled to 45°C, and the pH of the solution was adjusted to 9.2 with 0.1 mol / L NaOH. 350,000 U of alkaline protease was added for hydrolysis for 4 h, and then kept warm at 90°C for 10 min to inactivate the enzyme. The pH of the solution was adjusted to 6.7 with 0.1 mol / L hydrochloric acid, and 68,000 U of flavor protease was added. The solution was kept warm at 50°C for 5 h, and then kept warm at 90°C for 10 min to inactivate the enzyme. The soybean meal enzymatic hydrolyzate was obtained.

[0040] (4) Collection of soybean meal flavor functional peptides: The soybean meal enzymatic hydrolysate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the permeate was collected; the 2000 Da ultrafiltration permeate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean meal flavor functional peptides.

[0041] (5) Preparation of meat flavor seasoning: 50 g of soybean cake flavor functional peptide was mixed with 15 g of D-xylose, and ultrapure water was added to adjust the mixture concentration to 10% (w / v). The pH of the solution was adjusted to 7.5, and the mixture was placed in a 1000 mL beaker. The beaker was placed in an autoclave, and the reaction temperature was adjusted to 110 °C and the reaction time was 30 min. The reaction temperature was then changed to 120 °C and the reaction time was 90 min to obtain the Maillard reaction product.

[0042] (6) Sensory evaluation: 14 assessors (6 males and 8 females) with experience in sensory evaluation evaluated the samples. Before evaluating the samples, the assessors first conducted a sensory evaluation on the reference samples to familiarize themselves with the various indicators. The umami soup was prepared with 1.0% (w / v) sodium glutamate and 0.5% (w / v) salt. The Maillard reaction product was added to the umami soup at a concentration of 0.5% (w / v) and mixed. After heating in a 60°C water bath for 10 minutes, the umami, meaty, salty, bitter, caramelized, and palatability were scored on a scale of 0 to 10. Each sample was evaluated three times, and the average value was used as the final score for each indicator. The stronger and more comfortable the taste, the higher the score. The scoring results are shown in Figure 4 .

[0043] Example 3: A soybean protein peptide capable of generating meaty flavor through the Maillard reaction, and its preparation method and application

[0044] A method for producing flavor functional peptides from soybean meal and further producing meaty flavor substances comprises the following steps:

[0045] (1) Pretreatment: Dry the soybean meal and crush it. Sieve through a 60-mesh sieve to obtain soybean meal powder. Add petroleum ether at a ratio of 10-15:1 (v / w) to soak the soybean meal powder. Stir once every 15 minutes and soak for 2 hours. Filter to remove the solvent and dry to obtain defatted soybean meal.

[0046] (2) Enzymatic hydrolysis of soybean meal: Soak soybean meal powder in 85-90℃ hot water at a ratio of 15-20:1 (v / w) and keep warm for 30 min. Then cool to 45℃ and adjust the pH of the solution to 8.8-9.2. Add 3500 U of alkaline protease per gram of substrate and keep warm for 3.5-4.0 h. Keep warm at 90℃ for 10 min to inactivate the enzyme. Adjust the pH of the solution to 6.3-6.7, then add 680 U of flavor protease per gram of substrate and keep warm at 50℃ for 4.5-5.0 h. Keep warm at 90℃ for 10 min to inactivate the enzyme. The soybean meal enzymatic hydrolyzate is obtained.

[0047] (3) Collection of soybean meal flavor functional peptides: The soybean meal hydrolyzate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the 2000 Da ultrafiltration permeate was collected; the 2000 Da ultrafiltration permeate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the intercepted liquid was collected and freeze-dried to obtain the soybean meal flavor functional peptides.

[0048] (4) Preparation of meat flavor by flavor functional peptides: soybean cake flavor functional peptides and xylose were mixed in a weight ratio of 10:3, and the mixture was dissolved in ultrapure water to prepare a 10% (w / v) solution. The pH of the solution was adjusted to 7.5 with 0.1 mol / L NaOH and HCl, and the mixture was transferred to a reaction vessel. The reaction temperature was controlled at 110 °C and the reaction time was 30 min. The reaction temperature was then changed to 120 °C and the reaction time was 90 min to obtain a grilled steak flavor Maillard reactant.

[0049] (5) Identification of the components of the meaty Maillard reaction: Solid phase microextraction (SPME) combined with GC-MS was used to determine the volatile compounds in the meaty Maillard reaction products. The sample (5.0 mL) was placed in a headspace vial (15 mL) and 2.0 μL of 1,2-dichlorobiphenyl was added and mixed thoroughly. The vial was sealed and the SPME tip was inserted into the headspace vial in a 55°C water bath for 40 min. The sample was then transferred to the inlet of a gas chromatography-mass spectrometer (GC-MS) and resolved at 250°C for 5 min. GC-MS conditions were as follows: a WAX column (30 m × 0.25 mm × 0.25 μm); high-purity helium carrier gas at a flow rate of 1 mL / min, splitless; temperature settings were as follows: 40°C for 3 min, ramped to 100°C at 2°C / min, then to 150°C at 4°C / min, and then to 280°C at 20°C / min for 10 min; detector port temperature was 250°C; ion source and interface temperatures were set at 280°C and 250°C, respectively; filament emission current was 35 μA, detector voltage was 1000 V, electron energy was 70 eV, and the scan mass range was 35–450 amu at a scan rate of 4.45 amu / s. The NIST 20.L database was used for database searching and matching. Figure 1 Table 2 shows the mass spectrum peaks of the GC-MS analysis of MRPs. Table 3 shows the analysis results of the important volatile substances.

[0050] Table 2 Analysis results of flavor components of Maillard reaction products of soybean meal

[0051] Detected flavors Molecular formula Content (ng / g) CAS number 2-Methyl-3-furanthiol <![CDATA[C5H6OS]]> 69.825 28588-74-1 Bis(2-methyl-3-furyl) disulfide <![CDATA[C 10 H 10 OS2]]> 3.536 28588-75-2 2-Furylmethylthiol <![CDATA[C5H6OS]]> 10.14 35828 2-Methyl-3-methylthiopyrazine <![CDATA[C6H8N2S]]> 111.48 67952-65-2 2,5-Dimethylpyrazine <![CDATA[C6H8N2]]> 14.53 123-32-0 2,3,5-Trimethylpyrazine <![CDATA[C7H 10 N2]]> 22.89 14667-55-1 2-Acetylpyrazine <![CDATA[C6H6N2O]]> 10.27 22047-25-2 3-Mercapto-2-butanone <![CDATA[C4H8OS]]> 9.36 40789-98-8 Methylthiopropionaldehyde <![CDATA[C4H8OS]]> 5.69 3268-49-3 3-Methylbutanal <![CDATA[C5H 10 The]]> 30.81 590-86-3 2-Methylpropionaldehyde <![CDATA[C4H8O]]> 11.25 78-84-2 Phenylacetaldehyde <![CDATA[C8H8O]]> 8.29 122-78-1 p-Cresol <![CDATA[C7H8O]]> 4.32 106-44-5 2-Acetylthiazole <![CDATA[C5H5NOS]]> 37.60 24295-03-2 2-Acetyl-2-thiazoline <![CDATA[C5H7NOS]]> 32.37 29926-41-8 4-Hydroxy-2,5-dimethyl-3(2H)-furanone <![CDATA[C6H8O3]]> 4.39 3658-77-3 2-Ethyl-3,5-dimethylpyrazine <![CDATA[C8H 12 N2]]> 3.00 27043-05-6 Furfurylthiol <![CDATA[C4H4OS]]> 75.91 98-02-2 2-Methyl-3-sulfide <![CDATA[C2H6S3]]> 16.64 3658-80-8 γ-Valerolactone <![CDATA[C5H8O2]]> 41.73 108-29-2

[0052] (6) Structural identification of flavor functional peptides: The flavor peptides from soybean meal were desalted using ZipTip C18, and the flavor peptide solution was collected and freeze-dried. After drying, the powder was fully dissolved with 0.1% formic acid, centrifuged at 4°C for 20 min, and the supernatant was transferred to a sample tube. 3uL was aspirated for LC-MS / MS identification. The sample composition was analyzed by HPLC (nLC 1000) coupled with MS / MS using a chromatographic column (75 μm×150 mm, PepMap RSLC C18, 2 μm, 100 Å, USA). Mobile phase A was 0.1% formic acid, and mobile phase B was 0.1% formic acid and 80% ACN. The ion scan range was 300-1400 m / z. The database (uniprotkb_taxonomy_id_424569_2024_11_25.fasta) was searched using PEAKS software. The substance with a retention time of 22.27 min was the precursor peptide of the Maillard reaction product. Figure 2Figure 3 is the characteristic fragment ion peak of the peptide, and its sequence identification is Leucine-Glycine-Methionine-Glycine-Serine-Tyrosine-Arginine-Serine-Alanine-Leucine (LGMGSYRSAL).

[0053] (7) Flavor analysis: Soy protein peptides undergo a complex Maillard reaction to produce the characteristic flavor of grilled steak. From the perspective of core substances, thiol compounds play a key role. The active thiol (-SH) functional groups in 2-methyl-3-furanthiol and furfurylthiol molecules are generated by thermal degradation reactions of sulfur-containing amino acids such as cysteine. Their extremely low aroma perception thresholds (<1 ng / g) make them the iconic components of barbecue aroma. 2-methyl-3-furanthiol imparts a characteristic burnt aroma, while furfurylthiol adds a coffee roasting aroma. The sulfur-containing pyrazine compound 2-methyl-3-methylthiopyrazine is generated by the condensation reaction of amino acids and reducing sugars. Its methylthio group (-SCH3) structure significantly enhances the richness of the aroma of the final product.

[0054] In terms of flavor layering in the final product, the sulfide bond (-SS-) of bis(2-methyl-3-furyl) disulfide exhibits low volatility and slowly releases during roasting, continuously providing a rich, grilled steak aroma. The furan ring structure of 2-furylmethylthiol remains stable at high temperatures, continuously releasing roasted meat and coffee aromas in the middle and late stages of the reaction. Pyrazine compounds such as 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2-ethyl-3,5-dimethylpyrazine are produced through Strecker degradation reactions. Their pyrazine ring structures contribute nutty, roasted nut, and earthy notes, respectively, providing a woody foundation for the flavor profile. The thiazole compound 2-acetyl-2-thiazoline is formed through a condensation cyclization reaction between amino acids and carbonyl compounds. Its ring structure imparts a complex aroma of toasted bread and grilled meat, significantly enhancing the baked aroma dimension.

[0055] The aldehyde compounds 3-methylbutanal and 2-methylpropanal, derived from the degradation of branched-chain amino acids, offer malty and caramel notes that effectively balance the savory, meaty flavor. γ-Valerolactone, a product of fatty acid oxidation or hydroxy acid cyclization, effectively softens the sharp aroma produced by high temperatures with its low-volatility ester structure, enhancing the roundness of the flavor. The trisulfide bond structure of dimethyl trisulfide imparts a refreshing onion and cabbage note, modulating the overall aroma balance through the olfactory threshold effect. Phenylacetaldehyde, an aromatic aldehyde derived from the metabolism of phenylalanine, enhances the flavor's refinement with its honey and floral notes. The phenolic hydroxyl group structure of p-cresol, introduced through fat oxidation or smoking, simulates the characteristic smoky characteristics of charcoal grilling.

[0056] In terms of flavor enhancement, 2-acetylpyrazine and 2-acetylthiazole contribute popcorn and nutty notes through their heterocyclic structures, reinforcing the Maillard reaction characteristics of carbohydrates. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone, a caramelization product, significantly enhances caramel and tropical fruit aromas through its furanone structure. 3-Mercapto-2-butanone and methylthiopropionaldehyde, through their thiol and methylthio functional groups, complement broth and vegetable aromas.

[0057] The synergistic effect of Maillard reaction products follows a "concentration-threshold-volatility" regulation model: high concentrations of thiols dominate the core meaty aroma with their ultra-low threshold; moderate concentrations of pyrazines, aldehydes, and lactones (10-50 ng / g) build the aroma layer through moderate volatility; trace amounts of sulfide ethers, phenols, and aromatic aldehydes (<10 ng / g) contribute to the characteristic aroma through high volatility threshold effects. These compounds are generated through multiple reaction pathways, including Strecker degradation, Maillard condensation, fat oxidation, and caramelization, ultimately forming a grilled steak flavor system with thiols as the base, pyrazine-thiazole toastiness as the backbone, and aldehydes and lactone sweetness as the mellowing notes.

[0058] Example 4: A soybean protein peptide capable of generating meaty flavor through the Maillard reaction, and its preparation method and application

[0059] The method for preparing soybean protein peptides capable of generating meaty flavor through Maillard reaction comprises the following steps:

[0060] Step 1: Raw material pretreatment

[0061] drying, crushing and defatting the soybean meal to obtain defatted soybean meal powder;

[0062] Step 2: Enzymatic hydrolysis of soybean meal

[0063] Defatted soybean meal powder is added to water, and alkaline protease and flavor protease are used for tandem enzymatic hydrolysis, and soybean meal protease hydrolyzate is obtained after the enzymatic hydrolysis is completed.

[0064] Step 3: Separation and purification of soybean meal flavor peptides

[0065] The soybean meal protein hydrolysate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the 2000 Da ultrafiltration permeate was collected; the 2000 Da ultrafiltration permeate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean protein peptides;

[0066] Step 4: Mass spectrometry analysis and identification

[0067] It has been identified that the amino acid sequence of the soybean protein peptide is composed of: leucine-glycine-methionine-glycine-serine-tyrosine-arginine-serine-alanine-leucine.

[0068] A preferred embodiment is as follows: in step 1, the soybean meal is dried and then crushed, and the soybean meal powder is sieved through a 60-mesh sieve, and petroleum ether is added at a ratio of 10:1 v / w to soak the soybean meal powder, stirring once every 10 minutes, soaking for 1.5 hours, filtering to remove the solvent, and drying to obtain defatted soybean meal powder.

[0069] A preferred embodiment is as follows: in step 2, 85°C hot water is added at a ratio of 15:1 v / w to soak defatted soybean meal powder and the mixture is kept warm for 20 minutes, then the temperature is lowered to 40°C, and the pH value of the solution is adjusted to 8.8; 3000U of alkaline protease is added per gram of substrate, the mixture is kept warm for 3.5 hours, and the enzyme is inactivated by keeping warm at 85°C for 5 minutes; the pH value of the solution is adjusted to 6.3, and 650U of flavor protease is added per gram of substrate, the mixture is kept warm for 4.5 hours, and the enzyme is inactivated by keeping warm at 85°C for 5 minutes to obtain a soybean meal protein hydrolyzate.

[0070] A preferred embodiment is: in step 4, the soy protein peptide is desalted using a ZiptipC18 microchromatography column, the flavor peptide solution is collected, and freeze-dried. The dried powder is dissolved in 0.1% formic acid, centrifuged at 3°C ​​for 15 minutes, and the supernatant is transferred to a sample tube for LC-MS / MS identification; the amino acid sequence of the substance with a retention time of 22.27 minutes is: Leucine-Glycine-Methionine-Glycine-Serine-Tyrosine-Arginine-Serine-Alanine-Leucine.

[0071] The application of the soy protein peptide capable of generating a meaty flavor through the Maillard reaction is characterized in that the soy protein peptide and xylose are mixed in a weight ratio of 5:3, the mixture is dissolved in ultrapure water to prepare a solution with a concentration of 5% w / v, the pH value of the solution is adjusted to 7.5, and the solution is transferred into a reaction vessel. The reaction temperature is controlled at 105° C. and the reaction time is 25 minutes. The reaction temperature is then changed to 115° C. and the reaction time is 85 minutes to obtain a Maillard reaction product with a grilled steak flavor.

[0072] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A soybean protein peptide capable of producing a meaty flavor through the Maillard reaction, characterized in that: It is composed of the following amino acid residues: Leucine-Glycine-Methionine-Glycine-Serine-Tyrosine-Arginine-Serine-Alanine-Leucine.

2. The method for preparing a soybean protein peptide capable of generating a meaty flavor through the Maillard reaction according to claim 1, characterized in that: The following steps are involved: Step 1: Raw material pretreatment drying, crushing and defatting the soybean meal to obtain defatted soybean meal powder; Step 2: Enzymatic hydrolysis of soybean meal Defatted soybean meal powder is added to water, and alkaline protease and flavor protease are used for tandem enzymatic hydrolysis, and soybean meal protease hydrolyzate is obtained after the enzymatic hydrolysis is completed. Step 3: Separation and purification of soybean meal flavor peptides The soybean meal protein hydrolysate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the 2000 Da ultrafiltration permeate was collected; the 2000 Da ultrafiltration permeate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean protein peptides; Step 4: Mass spectrometry analysis and identification It has been identified that the amino acid sequence of the soybean protein peptide is composed of: leucine-glycine-methionine-glycine-serine-tyrosine-arginine-serine-alanine-leucine.

3. The method for preparing a soybean protein peptide capable of generating a meaty flavor through the Maillard reaction according to claim 2, characterized in that: In step 1, the soybean meal is dried and then crushed, and the soybean meal powder is obtained by passing through a 60-mesh sieve. Petroleum ether is added in a ratio of 10-15:1 v / w to soak the soybean meal powder, and the mixture is stirred once every 10-20 minutes and soaked for 1.5-3 hours. The solvent is removed by filtration, and defatted soybean meal powder is obtained after drying.

4. The method for preparing a soybean protein peptide capable of generating a meaty flavor through the Maillard reaction according to claim 2, wherein: In step 2, 85-90°C hot water is added at a ratio of 15-20:1 v / w to soak defatted soybean meal powder and the mixture is kept warm for 20-60 minutes, then cooled to 40-50°C, and the pH value of the solution is adjusted to 8.8-9.2; alkaline protease is added at a concentration of 3000-4000 U per gram of substrate, and the mixture is kept warm for enzymatic hydrolysis for 3.5-4.0 hours, and then kept warm at 85-95°C for 5-15 minutes to inactivate the enzyme; the pH value of the solution is adjusted to 6.3-6.7, and flavor protease is added at a concentration of 650-700 U per gram of substrate, and the mixture is kept warm for enzymatic hydrolysis at 45-55°C for 4.5-5.0 hours, and then kept warm at 85-95°C for 5-15 minutes to inactivate the enzyme, thereby obtaining a soybean meal protein hydrolyzate.

5. The method for preparing a soybean protein peptide capable of generating a meaty flavor through the Maillard reaction according to claim 2, characterized in that: In step 4, the soy protein peptide was desalted using a ZiptipC18 microchromatographic column, the flavor peptide solution was collected, and freeze-dried. The dried powder was dissolved in 0.1% formic acid, centrifuged at 3-5°C for 15-25 minutes, and the supernatant was transferred to a sample tube for LC-MS / MS identification. The amino acid sequence of the substance with a retention time of 22.27 minutes was: Leucine-Glycine-Methionine-Glycine-Serine-Tyrosine-Arginine-Serine-Alanine-Leucine.

6. Use of the soybean protein peptide according to claim 1 that can produce meaty flavor through Maillard reaction.

7. The use of the soybean protein peptide capable of producing meaty flavor through the Maillard reaction according to claim 1, characterized in that: Soy protein peptides and xylose are mixed in a weight ratio of 5-15:3, the mixture is dissolved in ultrapure water to prepare a solution with a concentration of 5-15% w / v, the pH value of the solution is adjusted to 7.5, and the mixture is transferred to a reaction vessel, the reaction temperature is controlled at 105-115°C, the reaction time is 25-35 minutes, and then the reaction temperature is changed to 115-125°C, the reaction time is 85-100 minutes, and a grilled steak flavor Maillard reaction product is obtained.

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