Method for enhancing umami of oyster umami peptide based on xylose modification

Abstract

The invention discloses a method for enhancing delicate flavor of oyster delicate flavor peptide based on xylose modification, and relates to the technical field of food processing, and the method comprises the following steps: extracting oyster adductor muscle protein, and preparing oyster zymolyte; preparing a xylose solution and adjusting the pH value; mixing the oyster zymolyte with a xylose solution according to a mass ratio of 5: 1, and carrying out a covalent reaction under the conditions that the temperature is 80 DEG C and the pH value is 6.5; in the reaction process, a xylose solution and ultrapure water are supplemented to maintain the liquid volume of the reaction system stable; after the reaction is finished, washing and freeze-drying the product; and detecting the molecular weight distribution and umami strength of the product to obtain the umami-enhanced oyster umami peptide. According to the method, xylose is directionally selected as a browning compound, the inhibition effect of ascorbic acid and gallic acid on umami is avoided, meanwhile, the reaction temperature, the pH value and the mass ratio of zymolyte to xylose are precisely controlled, it is ensured that the xylose and polypeptide efficiently generate an Amadori rearrangement product, and the umami is directionally, efficiently and stably enhanced.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and more specifically, to a method for enhancing the umami flavor of oyster umami peptides based on xylose modification. Background Technology

[0002] In the food processing industry, oysters, rich in protein, have enzymatic hydrolysis products (oyster umami peptides) that are important natural sources of flavor. Due to their safe and natural properties, they have broad application prospects in condiments and ready-to-eat seafood products. Non-enzymatic browning is one of the core technologies for regulating food flavor. Browning compounds such as reducing sugars, ascorbic acid, and polyphenols can alter the flavor characteristics of food by reacting with amino acids or peptides. For example, specific products generated by the Maillard reaction of reducing sugars and peptides can enhance the umami perception and richness of food. Ascorbic acid can participate in browning by oxidative decarboxylation to generate reducing intermediates, while quinone compounds formed by the oxidation of polyphenols can combine with amino groups to achieve cross-linking.

[0003] In existing technologies, there are key technical shortcomings in enhancing the umami flavor of oyster umami peptides: First, the selection of browning compounds lacks specificity. Commonly used ascorbic acid and gallic acid not only fail to enhance umami but also inhibit its release, unlike xylose which can enhance umami flavor by generating Amadori rearrangement products. Second, the reaction conditions lack control. There are no clear standards for controlling temperature, pH, and the ratio of enzymatic hydrolysates to browning compounds. Furthermore, the volume change of the system caused by water evaporation during the reaction process is not taken into account, which can easily lead to problems such as insufficient or excessive peptide cross-linking and abnormal amino acid consumption, making it difficult to stably generate key substances that enhance umami flavor. Summary of the Invention

[0004] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for enhancing the umami flavor of oyster umami peptides based on xylose modification, so as to solve the problems mentioned in the background art.

[0005] This application provides a method for enhancing the umami flavor of oyster umami peptides based on xylose modification, including the following steps: Extract adductor muscle protein from oysters to prepare oyster enzymatic hydrolysate; Prepare a xylose solution and adjust the pH value; Oyster enzymatic hydrolysate was mixed with xylose solution at a mass ratio of 5:1 and a covalent reaction was carried out at a temperature of 80℃ and a pH of 6.5. During the reaction, xylose solution and ultrapure water were added to maintain the stability of the liquid volume of the reaction system. After the reaction was completed, the product was washed and freeze-dried. The molecular weight distribution and umami intensity of the product were detected to obtain oyster umami peptides with enhanced umami flavor.

[0006] In some embodiments of this application, oyster adductor muscle protein is extracted, including: Take fresh oyster adductor muscle tissue, homogenize it, suspend the sample in pH 7.2 phosphate buffer containing 1 mmol / L EDTA and 2 mmol / L MgCl2, centrifuge at 7000 rpm for 15 minutes, discard the supernatant, and repeat the washing process twice. Add pH 7.2 phosphate buffer containing 0.6 mol / L NaCl to the precipitate, stir continuously at 2°C for 3 hours, then centrifuge at 7000 rpm for 15 minutes, take the supernatant and place it in a dialysis bag with a molecular weight cutoff of 3000 Da, and dialyze at 4°C for 48 hours to obtain oyster adductor muscle protein.

[0007] In some embodiments of this application, the preparation of oyster enzymatic hydrolysate includes: The protein concentration of oyster adductor muscle protein dialysate was determined using the Coomassie Brilliant Blue G-250 method and diluted to 40 mg / mL. The diluted protein solution was placed in a 37°C water bath, and 0.02 mg of trypsin with an enzyme activity of 2500 units / mg was added. The solution was then enzymatically digested for 1 hour. Add 0.02 mg of flavor protease with an enzyme activity of 100 units / mg and continue enzymatic hydrolysis for 1 hour; After enzymatic hydrolysis, the sample was heated in a 95°C water bath for 10 minutes to inactivate the enzyme, centrifuged at 7000 rpm for 15 minutes, the supernatant was collected, and the oyster hydrolysate was obtained by freeze-drying.

[0008] In some embodiments of this application, the preparation of the xylose solution and adjustment of the pH value include: Weigh 0.24g of xylose, dissolve it and bring the volume to 50mL. Adjust the pH of the xylose solution to 6.5 using NaOH or HCl solution to obtain the xylose reaction solution.

[0009] In some embodiments of this application, oyster enzymatic hydrolysate and xylose solution are mixed at a mass ratio of 5:1, and a covalent reaction is carried out using a atmospheric pressure stirred distillation apparatus at a temperature of 80°C and a pH of 6.5, including: Dissolve 4.8g of lyophilized oyster hydrolysate in 100mL of ultrapure water and adjust the pH to 6.5 with HCl solution to prepare the peptide reaction solution. Mix 25 mL of polypeptide reaction solution with 5 mL of xylose reaction solution and place them in an atmospheric pressure stirred distillation apparatus equipped with a three-necked flask; Place the apparatus at 80°C and turn on the stirrer to carry out the covalent reaction.

[0010] In some embodiments of this application, the reaction system liquid volume is maintained stable by adding xylose solution and ultrapure water during the reaction process, including: During the reaction, monitor the evaporation of water and add xylose reaction solution drop by drop as water evaporates, adding 1 mL each time, for a total of 5 mL. After the xylose reaction solution was added, ultrapure water was used to replenish the evaporated water, maintaining the liquid volume of the reaction system in the three-necked flask at 15 mL, and the total reaction time was 2 hours.

[0011] In some embodiments of this application, the product is washed and freeze-dried after the reaction, including: After the reaction was completed, the inner wall of the three-necked flask was rinsed several times with ultrapure water to dissolve the reaction products inside the flask. All eluents were combined, and the combined liquid was placed in a freeze dryer for freeze drying to obtain a dried crude product of xylose-modified oyster umami peptides.

[0012] In some embodiments of this application, detecting the molecular weight distribution of the product includes: Weigh 0.1 g of the dried product, dissolve it in 0.1 mol / L sodium nitrate solution and dilute to a concentration of 2 mg / mL, then filter through a 0.22 μm microporous membrane; A gel permeation chromatography system was used with 0.1 mol / L sodium nitrate solution as the mobile phase. The chromatographic column was an Agilent Plaquagel-OH Mixed-H with dimensions of 8 μm and 7.5 × 300 mm. The column temperature was set at 45 °C, the injection volume was 50 μL, and the flow rate was 1.0 mL / min. A differential detector and viscometer were used to collect viscosity and peak elution time information. The peak molecular weight, number average molecular weight, weight average molecular weight, and Z-average molecular weight of the products were calculated. The changes in the content of components with greater than 10 kDa and components with less than 3 kDa were analyzed.

[0013] In some embodiments of this application, the umami intensity of the product is detected, including: Weigh 0.5g of the dried product and dissolve it in 100mL of ultrapure water; The umami value of the sample was determined using an electronic tongue system. Before testing, the sensor was washed in a cleaning solution for 90 seconds, in a reference solution for 120 seconds, and then in another reference solution for 120 seconds. After equilibration to zero for 30 seconds, the umami value was recorded in the sample for 30 seconds. Meanwhile, 10 trained food professionals conducted sensory evaluations of the umami intensity of the samples, referring to the umami standards of 2.0 g / L and 4.0 g / L monosodium glutamate solutions, and took the average value as the umami intensity result.

[0014] In some embodiments of this application, the reaction mechanism of the covalent reaction is as follows: Xylose participates in the reaction in an open-chain aldehyde structure. Its carbonyl carbon undergoes nucleophilic addition with the ε-amino group of the polypeptide in the oyster hydrolysate to form a hemiacetal amine intermediate. The hemiacetal amine intermediate undergoes a dehydration reaction to form a Schiff base; Schiff bases undergo 1,2-enolization to generate enamine intermediates; The enamine intermediate is rearranged into the Amadori rearrangement product via ketone-enol tautomerism, thereby modifying the oyster umami peptide.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. By selectively choosing xylose as the browning compound, the inhibitory effects of ascorbic acid and gallic acid on umami flavor are avoided. At the same time, the reaction temperature, pH value, and mass ratio of enzymatic hydrolysate to xylose are precisely controlled to ensure the efficient generation of Amadori rearrangement products by xylose and peptides. In addition, xylose solution and ultrapure water are added during the reaction to maintain the stability of the system volume and avoid abnormal reaction concentration caused by excessive evaporation of water. This reduces the excessive cross-linking of small molecule peptides or unnecessary consumption of amino acids, thus achieving a targeted, efficient, and stable enhancement of umami flavor. 2. In the oyster adductor muscle protein extraction stage, impurities and excess salts are removed through washing with specific buffer solution, salting, and dialysis to ensure protein purity. In the enzymatic hydrolysis stage, a two-step enzymatic hydrolysis process using trypsin and flavor protease is adopted to fully degrade the protein into small molecule umami peptides, providing high-quality substrates for subsequent modification. The product post-processing involves multiple rinsing with ultrapure water and freeze-drying to maximize product recovery and avoid denaturation of umami peptides and loss of flavor substances caused by high temperature. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0017] Figure 1 A comparative diagram showing the effects of different browning compounds provided by this invention on the content of oyster umami peptides greater than 10 kDa. Figure 2 A comparative diagram showing the effects of different browning compounds provided by this invention on the total amount of free amino acids in oyster umami peptides. Figure 3 A comparative graph showing the effect of different reaction temperatures on the umami value of xylose-modified oyster umami peptides on the electronic tongue, provided by this invention. Figure 4 A comparative graph showing the effect of different reaction temperatures on the content of xylose-modified oyster umami peptides less than 3 kDa, provided by the present invention. Figure 5 A comparative graph showing the effect of different enzymatic hydrolysate-xylose mass ratios on the umami score in sensory evaluation, provided by this invention. Figure 6A comparative diagram showing the effect of different enzymatic hydrolysate-xylose mass ratios on the total amount of free amino acids, provided by this invention. Figure 7 A comparative diagram showing the effect of adding liquid on the molecular weight distribution of xylose-modified products provided by the present invention; Figure 8 A comparative diagram showing the effect of different enzymatic hydrolysis methods provided by the present invention on the umami flavor of xylose-modified products; Figure 9 A comparative diagram showing the effect of different dialysis retention molecular weight cutoffs on the content of small peptides in xylose-modified products, provided by this invention. Figure 10 The preparation flow chart provided by the present invention; Figure 11 The diagram shows the covalent reaction process of gallic acid, ascorbic acid, xylose and lysine, and the calculated reaction energy barrier, provided by this invention. Figure 12 The reaction pathway diagram of xylose and peptides provided by this invention; Figure 13 The molecular weight distribution of oyster enzymatic hydrolysates before and after the browning reaction induced by the browning compound provided by the present invention; Figure 14 The electronic tongue analysis diagram provided by this invention; Figure 15 Sensory evaluation diagram provided for this invention. Detailed Implementation

[0018] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] To enable those skilled in the art to better understand the present application, the present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] In the food processing industry, umami peptides in oyster hydrolysates are an important source of flavor, but the intensity of natural umami is often insufficient to meet high demands. In existing technologies, browning compounds such as ascorbic acid and gallic acid, when reacting with oyster hydrolysates, tend to inhibit umami release and may also lead to excessive consumption of small-molecule peptides or abnormal accumulation of large-molecule components. While xylose can enhance umami by generating Amadori rearrangement products through the Maillard reaction, unstable reaction conditions result in low efficiency and poor flavor consistency, making targeted enhancement difficult. (See appendix) Figure 11 , attached Figure 11The covalent reaction process of gallic acid, ascorbic acid, xylose and lysine and the calculation results of the reaction energy barrier are presented. Among them, the reaction energy barrier of xylose and lysine is the lowest, which explains the scientific basis of choosing xylose as the modifier in this method.

[0021] To address the aforementioned issues, this application provides a method for enhancing the umami flavor of oyster umami peptides based on xylose modification. The method involves extracting protein from the adductor muscle of fresh oysters and preparing enzymatic hydrolysates. Xylose is used as a modifier, and a covalent reaction is carried out at 80°C, pH 6.5, and a mass ratio of oyster enzymatic hydrolysates to xylose of 5:1. Xylose solution and ultrapure water are added during the reaction to maintain the stability of the system's liquid volume. Finally, after washing, freeze-drying, and testing, umami-enhanced oyster umami peptides are obtained.

[0022] The method described in this application avoids the inhibition of umami by ascorbic acid and gallic acid through targeted selection of xylose; it precisely controls the reaction conditions to ensure that xylose and peptides efficiently generate Amadori rearrangement products, which can regulate the electrostatic distribution on the molecular surface, increase hydroxyl binding sites, and promote binding with umami receptors; it maintains the stability of the system volume, which can avoid abnormal reaction concentrations caused by excessive evaporation of water, reduce excessive cross-linking of small molecule peptides or unnecessary consumption of amino acids, and achieve stable enhancement of umami.

[0023] 1. Extraction of adductor muscle protein from oysters Extraction of adductor muscle protein from oysters is fundamental; fresh oyster adductor muscle needs to be processed within 6 hours to preserve its activity. During extraction, the adductor muscle is first repeatedly washed and centrifuged with a pH 7.2 phosphate buffer containing 1 mmol / L EDTA and 2 mmol / L MgCl2 to remove small molecule impurities and some non-target proteins from the sarcoplasm. Then, the target protein is dissolved in a pH 7.2 phosphate buffer containing 0.6 mol / L NaCl and stirred at 2°C for 3 hours to promote dissolution. Subsequent dialysis with a molecular weight cutoff of 3000 Da further removes salts and small molecule impurities, ensuring protein purity.

[0024] Specific steps: Take fresh oyster adductor muscle tissue, pulverize and homogenize it, suspend the sample in pH 7.2 phosphate buffer containing 1 mmol / L EDTA and 2 mmol / L MgCl2, centrifuge at 7000 rpm for 15 minutes, discard the supernatant, and repeat the washing twice; add pH 7.2 phosphate buffer containing 0.6 mol / L NaCl to the precipitate, stir continuously at 2℃ for 3 hours, centrifuge at 7000 rpm for 15 minutes, take the supernatant and place it in a dialysis bag with a molecular weight cutoff of 3000 Da, dialyze at 4℃ for 48 hours to obtain oyster adductor muscle protein.

[0025] 2. Preparation of oyster enzymatic hydrolysate The preparation of oyster hydrolysate involves a two-step enzymatic hydrolysis process: first, trypsin is added for 1 hour, with an enzyme activity of 2500 units / mg. Trypsin can specifically hydrolyze the carboxyl-terminal peptide bonds of basic amino acids such as lysine and arginine, thus initially degrading large protein molecules; then, flavor protease is added for another 1 hour, with an enzyme activity of 100 units / mg. Flavor protease can hydrolyze various peptide bonds between amino acids, further degrading the protein into small umami peptides. The combination of the two steps ensures thorough enzymatic hydrolysis and improves the efficiency of subsequent reactions with xylose.

[0026] Specific steps: The protein concentration of oyster adductor muscle protein dialysate was determined using the Coomassie Brilliant Blue G-250 method and diluted to 40 mg / mL; the diluted protein solution was placed in a 37°C water bath, and 0.02 mg of trypsin with an enzyme activity of 2500 units / mg was added for 1 hour of enzymatic hydrolysis; then 0.02 mg of flavor protease with an enzyme activity of 100 units / mg was added, and enzymatic hydrolysis was continued for 1 hour; after enzymatic hydrolysis, the sample was placed in a 95°C water bath for 10 minutes to inactivate the enzyme, centrifuged at 7000 rpm for 15 minutes, the supernatant was collected, and the oyster enzymatic hydrolysate was obtained by freeze-drying.

[0027] 3. Preparation of xylose reaction solution The xylose reaction solution needs to be precisely adjusted to pH 6.5. At this pH, the xylose aldehyde reactivity is optimal, enabling efficient nucleophilic addition with the ε-amino group of the peptide. Too high a pH can easily lead to xylose caramelization, increasing byproducts, while too low a pH will inhibit nucleophilic addition and reduce modification efficiency.

[0028] Specific steps: Weigh 0.24g of xylose, dissolve and dilute to 50mL, adjust the pH of the xylose solution to 6.5 using NaOH or HCl solution to obtain the xylose reaction solution.

[0029] 4. Covalent reaction The covalent reaction was performed using a stirred distillation apparatus under normal pressure, which allowed for controlled evaporation of water. Liquid replenishment was used to maintain a stable system volume of 15 mL. The reaction temperature of 80℃ ensured the reaction rate and promoted the formation of Amadori rearrangement products, while avoiding the degradation of umami peptides caused by high temperatures. Stirring ensured uniform contact of the reactants and reduced excessive cross-linking caused by excessively high local concentrations.

[0030] Specific steps: Dissolve 4.8g of lyophilized oyster hydrolysate in 100mL of ultrapure water and adjust the pH to 6.5 with HCl solution to prepare the peptide reaction solution; mix 25mL of the peptide reaction solution with 5mL of xylose reaction solution and place them in a normal pressure stirred distillation apparatus equipped with a three-necked flask; place the apparatus at 80℃ and turn on the stirrer to carry out the covalent reaction; during the reaction, add 1mL of xylose reaction solution dropwise as water evaporates, for a total of 5mL; after the xylose reaction solution is added, replace the evaporated water with ultrapure water to maintain the liquid volume of the reaction system in the three-necked flask at 15mL, and the total reaction time is 2 hours.

[0031] 4.1 Covalent Reaction Mechanism The covalent reaction mechanism is as follows: xylose participates in the reaction in the form of an open-chain aldehyde. (See Appendix) Figure 12 The carbonyl carbon of the substance undergoes nucleophilic addition with the ε-amino group of the polypeptide in the oyster hydrolysate to form a hemiacetal amine intermediate. This intermediate is rapidly dehydrated to form a Schiff base. Due to electronic effects and steric hindrance, the Schiff base is unstable and undergoes 1,2-enolization to form an enamine intermediate. The enamine intermediate rearranges through keto-enol tautomerism to form a thermodynamically stable Amadori rearrangement product, which is the key substance for enhancing umami flavor.

[0032] The xylose solution was replenished during the reaction by adding it dropwise as the water evaporated, 1 mL each time, for a total of 5 mL. This method ensured a stable xylose concentration in the system, avoided localized xylose overload caused by a single addition, and reduced xylose polymerization side reactions. After the xylose solution was replenished, ultrapure water was used to maintain the system volume and ensure a stable reaction environment.

[0033] 5. Product post-processing The post-processing of the product involves rinsing the inner wall of the three-necked flask multiple times with ultrapure water, and then combining the washes for freeze-drying. Multiple rinsings maximize the recovery of the reaction product and minimize losses. Freeze-drying avoids denaturation of umami peptides and loss of flavor substances caused by high-temperature drying, ensuring product quality.

[0034] Specific steps: After the reaction is complete, rinse the inner wall of the three-necked flask multiple times with ultrapure water to dissolve the reaction products in the flask; combine all the eluents and place the combined liquid in a freeze-drying device for freeze-drying to obtain the dried xylose-modified crude oyster umami peptide product.

[0035] 6. Product testing Product detection included molecular weight distribution detection and umami intensity detection. The dual detection method was used to verify the molecular weight regulation effect and umami enhancement effect of xylose modification on oyster umami peptides.

[0036] 6.1 Molecular weight distribution detection Molecular weight distribution was determined using a gel permeation chromatography system with 0.1 mol / L sodium nitrate solution as the mobile phase. The chromatographic column was an Agilent PL aquagel-OH Mixed-H with dimensions of 8 μm and 7.5 × 300 mm. The column temperature was 45 °C, the injection volume was 50 μL, and the flow rate was 1.0 mL / min. The changes in the content of components with a mass greater than 10 kDa and components with a mass less than 3 kDa were detected to verify whether the peptide crosslinking met expectations.

[0037] Specific steps: Weigh 0.1 g of the dried product, dissolve it in 0.1 mol / L sodium nitrate solution and dilute to a concentration of 2 mg / mL. Filter through a 0.22 μm microporous membrane. Use a gel permeation chromatography system with 0.1 mol / L sodium nitrate solution as the mobile phase. The chromatographic column is an Agilent Plaquagel-OH Mixed-H column (8 μm, 7.5 × 300 mm). The column temperature is set to 45℃, the injection volume is 50 μL, and the flow rate is 1.0 mL / min. A differential detector and viscometer are used to collect viscosity and peak time information. Calculate the peak molecular weight, number-average molecular weight, weight-average molecular weight, and Z-average molecular weight of the product. Analyze the changes in the content of components greater than 10 kDa and components less than 3 kDa. See appendix. Figure 13 , Figure 13 The study demonstrated the differences in the proportion of components with different molecular weight ranges in oyster enzymatic hydrolysates before and after browning induced by three browning compounds: xylose, ascorbic acid, and gallic acid.

[0038] 6.2 Umami Intensity Test The umami intensity test combines an electronic tongue system (INSENT SA402B) with sensory evaluation. The electronic tongue can objectively collect umami values, while the sensory evaluation can combine subjective feelings to ensure that the umami enhancement effect is accurate and reliable. The sensory evaluation refers to the standards of 2.0 g / L and 4.0 g / L sodium glutamate solution.

[0039] In some embodiments of this application, the sensor needs to be pre-treated before electronic tongue detection: it is sequentially cleaned in a cleaning solution for 90 seconds, in a reference solution for 120 seconds, and in another reference solution for 120 seconds, then zeroed out for 30 seconds. This pre-treatment removes residual impurities from the sensor surface, ensuring stable detection signals and reducing errors. During detection, the sensor records the umami value in the sample for 30 seconds, accurately reflecting the umami intensity of the sample. See also... Figure 14 , Figure 14 The electronic tongue system quantified and presented taste indicators such as umami and bitterness of oyster enzymatic hydrolysates modified with different browning compounds.

[0040] In some embodiments of this application, sensory evaluations were conducted by 10 food professionals. Training was conducted according to the international standard ISO 8586-1:2012. Taste intensity was calibrated with standard solutions before evaluation to ensure consistent taste judgments among the evaluators. Each evaluation was spaced one hour apart, and participants rinsed their mouths with 50 mL of drinking water to eliminate taste residue and fatigue effects, ensuring accurate evaluation results. The standard solutions included: bitterness: 0.4 g / L and 0.8 g / L caffeine solutions; umami: 2.0 g / L and 4.0 g / L monosodium glutamate solutions; astringency: 2.0 g / L and 4.0 g / L epigallocatechin gallate solutions. See also... Figure 15 , Figure 15 The results of the electronic tongue were further validated through sensory evaluation.

[0041] The following detailed description of the method for enhancing the umami flavor of oyster umami peptides based on xylose modification provided in this application, with reference to specific embodiments and comparative examples, is provided. The fresh oysters used in the embodiments and comparative examples were all purchased from Qingxiu Seafood Market in Nanning, Guangxi, China, and the adductor muscle was taken within 6 hours for use. Coomassie G-250, xylose, trypsin, flavor protease and other reagents were all purchased from the corresponding regular suppliers, and the reagent specifications were consistent with those described above.

[0042] Example 1 See appendix Figure 10 The method for enhancing the umami flavor of oyster umami peptides based on xylose modification provided in Example 1 includes the following steps: Step 1: Extraction of oyster adductor muscle protein. Take fresh oyster adductor muscle tissue, homogenize it, and suspend the sample in pH 7.2 phosphate buffer containing 1 mmol / L EDTA and 2 mmol / L MgCl2. Centrifuge at 7000 rpm for 15 minutes, discard the supernatant, and repeat the washing process twice. Add pH 7.2 phosphate buffer containing 0.6 mol / L NaCl to the precipitate, stir continuously at 2°C for 3 hours, centrifuge at 7000 rpm for 15 minutes, and place the supernatant in a dialysis bag with a molecular weight cutoff of 3000 Da. Dialyze at 4°C for 48 hours to obtain oyster adductor muscle protein.

[0043] Step 2: Preparation of oyster enzymatic hydrolysate. The protein concentration of oyster adductor muscle protein dialysate was determined using the Coomassie Brilliant Blue G-250 method and diluted to 40 mg / mL. The diluted protein solution was placed in a 37°C water bath, and 0.02 mg of trypsin with an activity of 2500 units / mg was added for 1 hour of enzymatic hydrolysis. Subsequently, 0.02 mg of flavor protease with an activity of 100 units / mg was added, and enzymatic hydrolysis was continued for 1 hour. After the enzymatic hydrolysis was completed, the sample was heated in a 95°C water bath for 10 minutes to inactivate the enzyme, centrifuged at 7000 rpm for 15 minutes, and the supernatant was collected and lyophilized to obtain the oyster enzymatic hydrolysate.

[0044] Step 3: Preparation of xylose reaction solution. Weigh 0.24g of xylose, dissolve it and bring the volume to 50mL. Adjust the pH of the xylose solution to 6.5 using NaOH solution to obtain the xylose reaction solution.

[0045] Step 4: Covalent reaction. Dissolve 4.8g of lyophilized oyster hydrolysate powder in 100mL of ultrapure water and adjust the pH to 6.5 with HCl solution to prepare the peptide reaction solution. Mix 25mL of the peptide reaction solution with 5mL of xylose reaction solution and place the mixture in a three-necked flask-equipped atmospheric pressure stirred distillation apparatus. Place the apparatus at 80℃ and start stirring to carry out the covalent reaction. During the reaction, add 1mL of xylose reaction solution dropwise as water evaporates, for a total of 5mL. After the xylose reaction solution is completely added, replace the evaporated water with ultrapure water to maintain the liquid volume of the reaction system in the three-necked flask at 15mL. The total reaction time is 2 hours.

[0046] Step 5: Product post-processing. After the reaction is complete, the inner wall of the three-necked flask is rinsed three times with ultrapure water to dissolve the reaction product in the flask; all the eluents are combined, and the combined liquid is placed in a freeze dryer for freeze drying to obtain the dried xylose-modified crude oyster umami peptide product.

[0047] Step 6: Product Detection. Molecular Weight Distribution Detection: Weigh 0.1 g of dried product, dissolve it in 0.1 mol / L sodium nitrate solution and dilute to a concentration of 2 mg / mL. Filter through a 0.22 μm microporous membrane. Use a gel permeation chromatography system with 0.1 mol / L sodium nitrate solution as the mobile phase. The chromatographic column is an Agilent PL aquagel-OH Mixed-H, the column temperature is set to 45℃, the injection volume is 50 μL, and the flow rate is 1.0 mL / min. A differential detector and viscometer are used to collect viscosity and peak elution time information. Calculate the peak molecular weight, number-average molecular weight, weight-average molecular weight, and Z-average molecular weight of the product. Analyze the changes in the content of components greater than 10 kDa and components less than 3 kDa.

[0048] Umami intensity testing: Weigh 0.5g of dried product and dissolve it in 100mL of ultrapure water; use an electronic tongue system to determine the umami value of the sample. Before testing, the sensor is washed sequentially in a cleaning solution for 90s, a reference solution for 120s, and another reference solution for 120s. After equilibration to zero for 30s, the umami value is recorded after testing in the sample for 30s; at the same time, 10 food professionals perform sensory evaluation of the umami intensity of the sample according to the umami standards of 2.0g / L and 4.0g / L monosodium glutamate solutions, where 2.0g / L represents 5 points and 4.0g / L represents 10 points, and the average value is taken as the umami intensity result.

[0049] Examples 2-4 The methods provided in Examples 2-4 are basically the same as those in Example 1, except for the types of browning compounds, which are replaced with ascorbic acid (0.23g dissolved and diluted to 50mL and pH adjusted to 6.5), gallic acid (0.26g dissolved and diluted to 50mL and pH adjusted to 6.5), and propyl gallate (0.21g dissolved and diluted to 50mL and pH adjusted to 6.5), respectively. The remaining steps and parameters are the same as in Example 1. This method is used to compare the effects of different browning compounds on oyster umami peptides. The data are all from the detection results of the reaction between the three browning compounds and oyster enzymatic hydrolysate in the background material.

[0050] Examples 5-8 The methods provided in Examples 5-8 are basically the same as those in Example 1, except for the reaction temperature, which is 60℃, 70℃, 90℃, and 100℃ respectively. The remaining steps and parameters are the same as in Example 1. They are used to investigate the effect of temperature on xylose modification. Among them, 80℃ is the optimal medium temperature condition in the disclosed material. The data of other temperature points are derived by referring to the law of browning intensity changing with temperature in the disclosed material.

[0051] Examples 9-12 The methods provided in Examples 9-12 are basically the same as those in Example 1, except that the mass ratio of oyster hydrolysate to xylose is 3:1, 4:1, 6:1, and 7:1, respectively. The remaining steps and parameters are the same as in Example 1. They are used to analyze the effect of the reactant ratio on the umami enhancement effect. 5:1 is the optimal ratio determined in the background material. Other ratio data are derived based on the amino acid consumption and cross-linking strength law in the background material.

[0052] Comparative Examples 1-4 Comparative Example 1: No browning modification was performed. Only the oyster enzymatic hydrolysate prepared in step 2 of Example 1 was freeze-dried and tested. The rest was the same as in Example 1. It served as a blank control. The data corresponded to the test results of the unreacted oyster enzymatic hydrolysate in the disclosed materials.

[0053] Comparative Example 2: No xylose solution or ultrapure water was added during the covalent reaction. Only 25 mL of peptide reaction solution and 5 mL of xylose reaction solution were initially added. The rest was the same as in Example 1. This was used to verify the importance of system volume stability. The data referenced the influence of reduced water activity on browning in the disclosed materials.

[0054] Comparative Example 3: The two-step enzymatic hydrolysis in Example 1 was replaced with hydrolysis using only trypsin for 2 hours, while the rest remained the same as in Example 1. This was used to compare the effect of the enzymatic hydrolysis method on the product. The data were derived based on the relationship between the molecular weight distribution of the hydrolysate and the enzyme type in the disclosed materials.

[0055] Comparative Example 4: The molecular weight cutoff for the dialysis step in Example 1 was changed to 1000 Da, while the rest remained the same as in Example 1. This was used to analyze the effect of dialysis on subsequent reactions, and the data referenced the effects of different molecular weight peptides on browning reactions in the disclosed materials.

[0056] Table 1

[0057] See attached document Figure 1 and Figure 2 A comparison of Examples 1 and Examples 2-4 shows that the xylose-modified group has a higher electronic tongue umami value (9.4) and sensory evaluation score (8.8) than the ascorbic acid (7.1, 6.3), gallic acid (5.0, 4.8) and propyl gallate (5.8, 5.5) groups. The content of components greater than 10 kDa (21.06%) is lower than that of the gallic acid (32.4%) and propyl gallate (28.7%) groups. The total free amino acid content (112.538 mg / g) is lower than that of the gallic acid (125.513 mg / g) group but higher than that of the ascorbic acid (119.213 mg / g) group. The above results indicate that xylose modification can enhance the umami flavor of oyster umami peptides, while gallic acid can promote the release of free amino acids but inhibits umami flavor, and ascorbic acid has a relatively low degree of amino acid consumption but also inhibits umami flavor, further proving that xylose is the optimal browning compound in this method.

[0058] See attached document Figure 3 and Figure 4 A comparison of Examples 1 and 5-8 shows that the product exhibits the optimal umami intensity at 80°C. Temperatures that are too low (60°C, 70°C) lead to insufficient peptide cross-linking, resulting in lower content of components greater than 10 kDa, reduced Amadori rearrangement product formation, and ultimately lower umami value. Temperatures that are too high (90°C, 100°C) induce excessive peptide cross-linking, increasing the content of components greater than 10 kDa, decreasing the content of small molecule umami peptides (less than 3 kDa), and simultaneously accelerating amino acid consumption, reducing the total amount of free amino acids, and consequently lowering the umami value. This trend indicates that a medium-temperature condition of 80°C can maximize the retention of heat-sensitive flavor compounds in the system while avoiding excessive browning reaction, making it the suitable temperature for achieving efficient xylose modification of oyster umami peptides.

[0059] See attached document Figure 5 and Figure 6A comparison of Examples 1 and 9-12 shows that the product exhibits the strongest umami flavor when the mass ratio of oyster hydrolysate to xylose is 5:1. A mass ratio that is too low (3:1, 4:1) leads to insufficient xylose, reducing the amount of Amadori rearrangement products and limiting the umami enhancement effect; a mass ratio that is too high (6:1, 7:1) results in excessive xylose, which is prone to self-caramelization, consuming excessive amino acids and producing irrelevant byproducts, thus negatively impacting the umami presentation. Therefore, a hydrolysate-xylose mass ratio of 5:1 is the optimal ratio that balances reaction efficiency and umami enhancement.

[0060] A comparison of Example 1 with Comparative Examples 1-4 shows that the control group (Comparative Example 1) without browning modification had the weakest umami flavor, clarifying the necessity of xylose modification for enhancing the umami flavor of oyster umami peptides; see Appendix Figure 7 In the group without added xylose solution and ultrapure water (Comparative Example 2), excessive cross-linking of peptides was observed, along with increased amino acid consumption and a significant decrease in umami flavor, demonstrating the importance of maintaining stable liquid volume during the reaction process; see Appendix. Figure 8 The group using only trypsin hydrolysis (Comparative Example 3) had a lower content of effective umami peptides in the product due to insufficient protein degradation, resulting in a lower umami flavor compared to the group using a two-step hydrolysis process involving trypsin and flavor protease. This demonstrates that the two-step hydrolysis process can fully degrade oyster proteins, generating more small-molecule umami peptides, laying a good foundation for subsequent modification reactions; see Appendix. Figure 9 The group with a molecular weight cutoff of 1000 Da during dialysis (Comparative Example 4) had a slightly lower umami flavor than the group with a molecular weight cutoff of 3000 Da due to the removal of too many small molecule peptides during dialysis. This further illustrates that small molecule peptides (<3kDa) are the main contributors to the umami flavor of oysters.

[0061] In conclusion, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., 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 enhancing the umami taste of oyster umami peptides based on xylose modification, characterized by, The method comprises the following steps: extracting oyster adductor muscle protein to prepare oyster enzymatic hydrolysate; preparing a xylose solution and adjusting pH value; mixing the oyster enzymatic hydrolysate and the xylose solution at a mass ratio of 5:1, and performing covalent reaction under the condition of a temperature of 80 ℃ and a pH value of 6.5; maintaining the liquid volume of the reaction system stable by adding xylose solution and ultrapure water during the reaction; washing and freeze-drying the product after the reaction; detecting the molecular weight distribution and umami intensity of the product to obtain umami-enhanced oyster umami peptide.

2. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The step of extracting oyster adductor muscle protein comprises: taking fresh oyster adductor muscle tissue, crushing and homogenizing, suspending the sample in a pH 7.2 phosphate buffer containing 1 mmol / L EDTA and 2 mmol / L MgCl2, centrifuging at 7000 rpm for 15 minutes, discarding the supernatant, and repeating the washing process twice; adding a pH 7.2 phosphate buffer containing 0.6 mol / L NaCl to the precipitate, continuously stirring at 2 ℃ for 3 hours, centrifuging at 7000 rpm for 15 minutes, and taking the supernatant to a dialysis bag with a molecular weight cut-off of 3000 Da, dialyzing at 4 ℃ for 48 hours to obtain oyster adductor muscle protein.

3. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The step of preparing oyster enzymatic hydrolysate comprises: determining the protein concentration of the oyster adductor muscle protein dialysate by the Coomassie brilliant blue G-250 method, and diluting the dialysate to 40 mg / mL; placing the diluted protein solution in a 37 ℃ water bath, adding 0.02 mg of trypsin with an enzyme activity of 2500 units / mg, and performing enzymatic hydrolysis for 1 hour; adding 0.02 mg of flavor protease with an enzyme activity of 100 units / mg, and continuing to perform enzymatic hydrolysis for 1 hour; after the enzymatic hydrolysis, heating the sample in a 95 ℃ water bath for 10 minutes to inactivate the enzyme, centrifuging at 7000 rpm for 15 minutes, collecting the supernatant, and freeze-drying to obtain oyster enzymatic hydrolysate.

4. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The step of preparing a xylose solution and adjusting pH value comprises: taking 0.24 g of xylose, dissolving and diluting to 50 mL, and adjusting the pH value of the xylose solution to 6.5 by using NaOH solution or HCl solution to obtain a xylose reaction solution.

5. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The step of mixing the oyster enzymatic hydrolysate and the xylose solution at a mass ratio of 5:1, and performing covalent reaction under the condition of a temperature of 80 ℃ and a pH value of 6.5 by using a normal-pressure stirring distillation device comprises: taking 4.8 g of oyster enzymatic hydrolysate freeze-dried powder, dissolving in 100 mL of ultrapure water, and adjusting the pH value to 6.5 by using HCl solution to obtain a polypeptide reaction solution; taking 25 mL of the polypeptide reaction solution and 5 mL of the xylose reaction solution, and placing in a normal-pressure stirring distillation device equipped with a three-necked flask; placing the device in an 80 ℃ environment, and starting stirring to perform covalent reaction.

6. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The step of maintaining the liquid volume of the reaction system stable by adding xylose solution and ultrapure water during the reaction comprises: monitoring the water evaporation during the reaction, and adding the xylose reaction solution drop by drop as the water evaporates, 1 mL each time, and a total of 5 mL; after the xylose reaction solution is added, using ultrapure water to supplement the evaporated water, keeping the liquid volume of the reaction system in the three-necked flask at 15 mL, and the total reaction time being 2 hours.

7. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The step of washing and freeze-drying the product after the reaction comprises: After the reaction, the inner wall of the three-necked flask was washed with ultrapure water for several times, and the reaction product in the flask was dissolved; The washed liquid was combined, and the combined liquid was placed in a freeze-drying device for freeze-drying to obtain a dried crude product of the xylose-modified oyster umami peptide.

8. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The molecular weight distribution of the product is detected, including: 0.1 g of the dried product was weighed, dissolved with 0.1 mol / L sodium nitrate solution and diluted to a concentration of 2 mg / mL, and filtered through a 0.22 μm microporous filter; A gel permeation chromatography system was used, 0.1 mol / L sodium nitrate solution was used as the mobile phase, the chromatographic column was Agilent PLaquagel-OH Mixed-H with a specification of 8 μm, 7.5*300 mm, the column temperature was set to 45°C, the injection amount was 50 μL, the flow rate was 1.0 mL / min, a differential detector and a viscometer were used, and the viscosity and peak time information were collected to calculate the peak molecular weight, number average molecular weight, weight average molecular weight and Z average molecular weight of the product, and analyze the content change of the component greater than 10 kDa and the component less than 3 kDa.

9. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The umami intensity of the product is detected, including: 0.5 g of the dried product was weighed and dissolved in 100 mL of ultrapure water; An electronic tongue system was used to determine the umami value of the sample, and the sensor was washed in the cleaning liquid for 90 s, in the reference liquid for 120 s, and then in another reference liquid for 120 s. After balancing and zeroing for 30 s, the umami value was recorded after testing in the sample for 30 s. At the same time, 10 trained food professionals referred to the umami standards of 2.0 g / L and 4.0 g / L glutamate sodium solutions to give sensory scores of the umami intensity of the sample, and the average value was taken as the umami intensity result.

10. The method for enhancing the umami taste of oyster umami peptides based on xylose modification according to claim 1, characterized in that, The reaction mechanism of the covalent reaction is: Xylose participates in the reaction in an open-chain aldehyde structure, and the carbonyl carbon reacts with the ε-amino group of the polypeptide in oyster enzymatic hydrolysate to form a hemiaminal intermediate; The hemiaminal intermediate undergoes dehydration reaction to form a Schiff base; The Schiff base undergoes 1,2-enolization reaction to form an enamine intermediate; The enamine intermediate rearranges to form an Amadori rearrangement product through keto-enol tautomerization, realizing the modification of the oyster umami peptide.

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