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

Abstract

The invention discloses soybean protein small peptide capable of generating beef bone soup meat flavor through Maillard reaction and a preparation method and application thereof. The preparation method comprises the following specific steps: drying, crushing, sieving, degreasing and drying fresh 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 according to a mass spectrometry result, and the sequence of the functional peptide is as follows: alanine-arginine-phenylalanine-glutamic acid-histidine-lysine-alanine-valine-tyrosine-serine-tyrosine-proline-tyrosine (ARFEHKAVYSYPY); then preparing the beef bone soup flavor substance through 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, the Maillard reaction product bovine bone soup is rich in meat flavor, and meat flavor functional substances 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 capable of generating beef bone soup meat flavor through Maillard reaction, and a preparation method and application thereof. Background Art

[0002] Soybeans are one of China's important food crops, rich in oil and protein. Soybean meal, produced after soybean de-oiling or oil extraction, is high in protein. Enzymatic hydrolysis of these proteins produces 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 producing a beef bone broth flavor through the Maillard reaction. The functional peptide has the following sequence: Alanine-Arginine-Phenylalanine-Glutamic Acid-Histidine-Lysine-Alanine-Valine-Tyrosine-Serine-Tyrosine-Proline-Tyrosine (ARFEHKAVYSYPY).

[0004] To achieve the above-mentioned and other related purposes, the technical solution provided by the present invention is: a soybean protein peptide that can produce the meaty aroma of beef bone soup through the Maillard reaction, which is composed of the following amino acid residues: alanine-arginine-phenylalanine-glutamic acid-histidine-lysine-alanine-valine-tyrosine-serine-tyrosine-proline-tyrosine.

[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 beef bone broth flavor through the Maillard reaction comprises the following steps:

[0006] Step 1: Raw material pretreatment

[0007] The soybean meal is dried, crushed, and sieved, and the soybean meal powder is soaked with petroleum ether to prepare defatted soybean meal;

[0008] Step 2: Enzymatic hydrolysis of soybean meal

[0009] Defatted soybean meal is added to water, and alkaline protease and flavor protease are used for tandem enzymatic hydrolysis, and soybean meal protease hydrolysate 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 3000 Da, and the 3000 Da ultrafiltration permeate was collected; the 3000 Da ultrafiltration permeate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2000 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: alanine-arginine-phenylalanine-glutamic acid-histidine-lysine-alanine-valine-tyrosine-serine-tyrosine-proline-tyrosine.

[0014] The preferred technical solution is: in step 1, the soybean cake is dried and then crushed, and the soybean cake powder is passed through an 80-mesh sieve to obtain the soybean cake powder, petroleum ether is added at a ratio of 10-15:1 v / w to soak the soybean cake powder, the mixture is stirred once every 10-20 minutes, the mixture is soaked for 1.5-3 hours, the solvent is removed by filtration, and the defatted soybean cake powder is obtained after drying.

[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 20-40 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 microchromatography column, the flavor peptide solution is collected, and the solution is freeze-dried. After drying, the 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 39.90 minutes is: alanine-arginine-phenylalanine-glutamic acid-histidine-lysine-alanine-valine-tyrosine-serine-tyrosine-proline-tyrosine.

[0017] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the use of the soybean protein peptide that can produce the meaty aroma of beef bone soup 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 the roasted beef bone soup meat 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 present invention has a clear amino acid sequence structure, the Maillard reaction product, the beef bone soup, has a strong meat flavor, and meat flavor functional substances are analyzed; and the process of the present invention can be put into industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 beef bone soup 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 an 80-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 for 2 hours, filter out the solvent, and dry to obtain defatted soybean meal powder;

[0030] (3) Enzymatic hydrolysis of soybean meal: 50 g defatted soybean meal powder 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 3000 Da, and the permeate was collected; the 3000 Da ultrafiltration permeate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean meal flavor functional peptides;

[0032] (5) Preparation of beef bone soup flavor: 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, and the reaction time was 90 min to obtain beef bone soup flavor.

[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: 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 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 product in Example 1

[0035] Detected flavors Molecular formula Content (ng / g) CAS number 2-Methyl-3-furanthiol <![CDATA[C5H6OS]]> 15.67 2489-45-6 Furfurylthiol <![CDATA[C4H4OS]]> 38.21 98-02-2 1-Octen-3-ol <![CDATA[C8H 16 The]]> 28.9 3391-86-4 2-Thiophenethiol <![CDATA[C4H4S2]]> 49.12 7774-74-5 Bis(2-methyl-3-furyl) disulfide <![CDATA[C 10 H 10 O2S2]]> 8.9 32538-18-4 Methylfurfuryl disulfide <![CDATA[C6H6O2S2]]> 28.34 1438-91-1 2,5-Dimethylpyrazine <![CDATA[C6H8N2]]> 58.43 123-32-0 2,3-Dimethylpyrazine <![CDATA[C6H8N2]]> 11.23 5910-89-4 2,3,5-Trimethylpyrazine <![CDATA[C7H 10 N2]]> 32.14 14667-55-1 2-Ethyl-3,5-dimethylpyrazine <![CDATA[C8H 12 N2]]> 36.78 15707-23-4 2-Acetylpyrazine <![CDATA[C6H6N2O]]> 19.87 22047-25-2 Hexanal <![CDATA[C6H 12 The]]> 45.32 66-25-1 Nonanal <![CDATA[C9H 18 The]]> 9.87 124-19-6 Benzaldehyde <![CDATA[C7H6O]]> 33.56 100-52-7 trans-2-hexenal <![CDATA[C6H 10 The]]> 25.43 6728-26-3 3-Methylbutanal <![CDATA[C5H 10 The]]> 28.11 590-86-3 Furfural <![CDATA[C5H4O2]]> 42.34 98-01-1 2-Acetylfuran <![CDATA[C6H6O2]]> 13.45 1192-62-7 4-Hydroxy-2,5-dimethyl-3(2H)-furanone <![CDATA[C6H8O3]]> 39.21 3658-77-3 2-Acetylthiazole <![CDATA[C5H5NOS]]> 17.89 24295-03-2 Benzothiazole <![CDATA[C7H5NS]]> 46.54 95-16-9 phenol <![CDATA[C6H6O]]> 11.56 108-95-2 Pyrrolidine <![CDATA[C4H9N]]> 35.67 123-75-1 Pyridine <![CDATA[C5H5N]]> 52.12 110-86-1

[0036] Example 2: A soybean protein peptide capable of generating beef bone soup 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 an 80-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 for 2 hours, filter out the solvent, and dry to obtain defatted soybean meal powder;

[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 3000 Da, and the permeate was collected; the 3000 Da ultrafiltration permeate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, and the intercepted liquid was collected and freeze-dried to obtain soybean meal flavor functional peptides;

[0041] (5) Preparation of beef bone soup flavor: 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 beef bone soup 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 it through an 80-mesh sieve to obtain soybean meal powder, add petroleum ether at a ratio of 10:1 (v / w) to soak the soybean meal powder, stir it every 15 minutes, soak it for 2 hours, filter it to remove the solvent, and dry it to obtain defatted soybean meal powder;

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

[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 3000 Da, and the 3000 Da ultrafiltration permeate was collected; the 3000 Da ultrafiltration permeate was filtered using an ultrafiltration membrane with a molecular weight cutoff of 2000 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 the beef bone soup flavor Maillard reaction product.

[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 MRPs analyzed by GC-MS. 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]]> 2.34 2489-45-6 Furfurylthiol <![CDATA[C4H4OS]]> 45.67 98-02-2 1-octen-3-ol <![CDATA[C8H 16 The]]> 23.45 3391-86-4 2-Thiophenethiol <![CDATA[C4H4S2]]> 56.78 7774-74-5 Bis(2-methyl-3-furyl) disulfide <![CDATA[C 10 H 10 O2S2]]> 6.78 32538-18-4 Methylfurfuryl disulfide <![CDATA[C6H6O2S2]]> 34.56 1438-91-1 2,5-Dimethylpyrazine <![CDATA[C6H8N2]]> 67.89 123-32-0 2,3-Dimethylpyrazine <![CDATA[C6H8N2]]> 9.01 5910-89-4 2,3,5-Trimethylpyrazine <![CDATA[C7H 10 N2]]> 25.61 14667-55-1 2-Ethyl-3,5-dimethylpyrazine <![CDATA[C8H 12 N2]]> 48.48 15707-23-4 2-Acetylpyrazine <![CDATA[C6H6N2O]]> 15.15 22047-25-2 Hexanal <![CDATA[C6H 12 The]]> 59.59 66-25-1 Nonanal <![CDATA[C9H 18 The]]> 7.77 124-19-6 Benzaldehyde <![CDATA[C7H6O]]> 41.41 100-52-7 trans-2-hexenal <![CDATA[C6H 10 The]]> 33.33 6728-26-3 3-Methylbutanal <![CDATA[C5H 10 The]]> 22.22 590-86-3 Furfural <![CDATA[C5H4O2]]> 55.55 98-01-1 2-Acetylfuran <![CDATA[C6H6O2]]> 10.1 1192-62-7 4-Hydroxy-2,5-dimethyl-3(2H)-furanone <![CDATA[C6H8O3]]> 50.5 3658-77-3 2-Acetylthiazole <![CDATA[C5H5NOS]]> 14.14 24295-03-2 Benzothiazole <![CDATA[C7H5NS]]> 60.6 95-16-9 phenol <![CDATA[C6H6O]]> 8.88 108-95-2 Pyrrolidine <![CDATA[C4H9N]]> 44.44 123-75-1 Pyridine <![CDATA[C5H5N]]> 66.66 110-86-1

[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 39.90 min was the precursor peptide of the Maillard reaction product. Figure 2The characteristic fragment ion peak of the peptide is shown in Figure 1, and its sequence is identified as Alanine-Arginine-Phenylalanine-Glutamic Acid-Histidine-Lysine-Alanine-Valine-Tyrosine-Serine-Tyrosine-Proline-Tyrosine (ARFEHKAVYSYPY).

[0053] (7) Flavor analysis: Soy protein peptides generate multiple flavor volatiles through the Maillard reaction, Strecker degradation, and sugar dehydration cyclization, forming 24 major volatile flavor compounds. Among the sulfur-containing compounds, 2-methyl-3-furanthiol is the core of the beef bone soup aroma and meat aroma due to its extremely low threshold (0.0002 ng / g). Furfurylthiol and 2-thiophenethiol are generated by the decarboxylation and desulfurization reaction of the cysteine ​​thiol group and the furan ring degraded from xylose, respectively, which provide roasted nut aroma, coffee aroma, and spicy sulfur aroma; bis(2-methyl-3-furanyl) disulfide and methylfurfuryl disulfide are formed by thiol oxidation or condensation, and enhance the richness and persistence of the aroma with a low threshold (1-10 ng / g). Among nitrogen-containing pyrazines, 2,5-dimethylpyrazine, the primary contributor to toasted and nutty aromas, is formed through the condensation and cyclization of aldehydes and ammonia generated by Strecker degradation of alanine and lysine, along with α-dicarbonyl compounds from sugar cleavage. 2,3-Dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2-ethyl-3,5-dimethylpyrazine enhance toasty and caramel aromas through methyl and ethyl substituents, related to reactions involving phenylalanine and tyrosine. 2-Acetylpyrazine, produced by acetylation of the pyrazine ring, contributes smoky and sweet nutty notes. Among aldehydes, hexanal and trans-2-hexenal, produced by Strecker degradation of lysine and isomerization of sugar-amino acid condensates, respectively, contribute grassy and leafy notes. 3-Methylbutyraldehyde, produced by valine degradation, imparts a sweet malty aroma. Benzaldehyde and furfural, formed by decarboxylation of phenylalanine and dehydration cyclization of xylose, respectively, contribute sweet almond and toasted caramel aromas. Among furans, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, produced through the 1,2-enolization of xylose, is a key component in the caramel aroma. 2-Acetylfuran (10.1 / 13.45 ng / g), produced through the acetylation of the furan ring, enhances the sweet, roasted fruit aroma. Among heterocyclic compounds, benzothiazole, formed through the condensation and cyclization of cysteine ​​and phenylalanine, is stable at high temperatures and imparts a roasted nutty aroma with a slight bitterness. Pyridine and pyrrolidine (formed through amino acid decarboxylation and Strecker degradation, respectively) contribute roasted grain aromas and sweet amine notes, enhancing flavor complexity.

[0054] In summary, sulfur-containing compounds dominate the characteristic aroma through the cysteine ​​thiol reaction, pyrazines rely on amino acid-sugar condensation to build the ox bone aroma skeleton, furans and aldehydes are degraded by sugar to improve the sweetness and freshness, and together form a synergistic complex flavor of ox bone soup, roasted aroma and caramel flavor.

[0055] 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 beef bone broth flavor through the Maillard reaction, characterized in that: It is composed of the following amino acid residues: Alanine-Arginine-Phenylalanine-Glutamic Acid-Histidine-Lysine-Alanine-Valine-Tyrosine-Serine-Tyrosine-Proline-Tyrosine.

2. The method for preparing a soybean protein peptide capable of generating beef bone broth aroma through the Maillard reaction according to claim 1, characterized in that: The following steps are involved: Step 1: Raw material pretreatment The soybean meal is dried, crushed, and sieved, and the soybean meal powder is soaked with petroleum ether to prepare defatted soybean meal; Step 2: Enzymatic hydrolysis of soybean meal Defatted soybean meal is added to water, and alkaline protease and flavor protease are used for serial enzymatic hydrolysis, and soybean meal protease hydrolysate 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 3000 Da, and the 3000 Da ultrafiltration permeate was collected; the 3000 Da ultrafiltration permeate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2000 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: alanine-arginine-phenylalanine-glutamic acid-histidine-lysine-alanine-valine-tyrosine-serine-tyrosine-proline-tyrosine.

3. The method for preparing a soybean protein peptide capable of generating beef bone broth aroma 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 an 80-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 beef bone broth aroma through the Maillard reaction according to claim 2, characterized in that: 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 20-40 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 beef bone broth aroma 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 with 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 39.90 minutes was: alanine-arginine-phenylalanine-glutamic acid-histidine-lysine-alanine-valine-tyrosine-serine-tyrosine-proline-tyrosine.

6. Use of the soybean protein peptide according to claim 1 that can generate the aroma of beef bone soup through the Maillard reaction.

7. The use of the soybean protein peptide capable of generating the aroma of beef bone soup 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 the beef bone broth meat-flavored Maillard reaction product is obtained.

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