Ascorbic acid-based oyster umami peptide browning targeted inhibition and nutritional ingredient synergistic preservation method

Abstract

The invention discloses an ascorbic acid-based oyster umami peptide browning targeted inhibition and nutritional ingredient synergistic preservation method, and relates to the technical field of aquatic product processing, and the method comprises the following steps: selecting fresh crassostrea gigas adductor muscle, washing with sterile water, carrying out standing pretreatment, and crushing and pulping by adopting a grinding technology to obtain oyster primary pulp; adding an ascorbic acid solution into the oyster primary pulp, stirring and mixing under a dark condition, and balancing for a preset time, so as to construct a browning substrate targeted binding system, and the like. According to the invention, through targeted combination of ascorbic acid and a browning substrate, phenolic substances, free amino acids and other components which are easy to cause browning are locked in advance; an ascorbic acid-composite antioxidant synergistic effect mechanism is formed by combining with a composite antioxidant composed of a green tea extract and citric acid, the problems that a traditional single anti-browning means is limited in effect and prone to being affected by the environment and losing efficacy are effectively solved, the browning risk of the whole processing flow of the oyster flavor peptide is reduced, and the natural color and luster of the product are reserved to the maximum extent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquatic product processing, in particular to a method for inhibiting the browning of oyster umami peptides and synergistically retaining nutritional components based on ascorbic acid. BACKGROUND

[0002] In the field of food processing, oysters become an important raw material for preparing umami peptides due to their high-quality protein, umami amino acids, and various active ingredients. Oyster umami peptides not only provide rich umami flavor to food, but also have certain nutritional value, and have broad application prospects in the fields of condiments and health foods. However, during the processing of oysters, the phenolic substances and free amino acids contained therein are prone to non-enzymatic browning reactions with oxygen in the air or under the action of polyphenol oxidase and peroxidase, resulting in dark and dull color of the product and affecting the sensory quality. At the same time, the browning reaction may also be accompanied by the loss of umami amino acids and the destruction of umami peptide structure, further reducing the flavor and nutritional value of the product.

[0003] In the existing processing of oyster umami peptides, the technology for inhibiting browning has obvious limitations. On the one hand, traditional anti-browning methods mostly use single antioxidants, such as adding ascorbic acid or citric acid alone. Such methods can only block the browning reaction from a single link and cannot precisely target the core browning substrates such as phenolic substances and free amino acids, resulting in unstable anti-browning effect and being easily affected by environmental factors such as processing temperature, oxygen concentration, and pH value, thus failing to cover the browning risks in the whole processing flow of oyster slurry preparation, enzymatic hydrolysis, and drying. On the other hand, due to the lack of a targeting binding mechanism for browning substrates, traditional technology cannot reduce the reactants of the browning reaction from the source, and even if a large amount of antioxidant is added, it is also difficult to completely prevent browning from occurring, and on the contrary, the excessive antioxidant may affect the flavor of the product. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a method for inhibiting the browning of oyster umami peptides based on ascorbic acid and synergistically retaining nutritional components, which solves the problems mentioned in the background art.

[0005] To achieve the above-mentioned purposes, the present application is implemented by the following technical scheme: a method for inhibiting the browning of oyster umami peptides based on ascorbic acid and synergistically retaining nutritional components, comprising the following steps: Fresh long oyster adductor muscle is selected, washed with sterile water, and then subjected to standing pretreatment. The oyster is broken into slurry by grinding technology to obtain oyster slurry; An ascorbic acid solution is added to the oyster slurry, and the mixture is stirred and balanced under light shielding conditions for a preset time to construct a browning substrate targeting binding system; The complex protease is added to the browning substrate target binding system in a preset ratio, the complex protease is composed of trypsin and flavor protease in a specific enzyme activity ratio, the browning substrate target binding system is adjusted to a preset pH and temperature, and a segmented enzymatic hydrolysis reaction is performed; During the enzymatic hydrolysis reaction, intermittent ultrasonic assisted treatment is adopted, and the activity changes of polyphenol oxidase and peroxidase in the browning substrate target binding system are monitored in real time, and the ultrasonic parameters are dynamically adjusted according to the enzyme activity curve; After the enzymatic hydrolysis reaction is completed, a composite antioxidant composed of green tea extract and citric acid is added to construct an ascorbic acid-composite antioxidant synergistic color protection system, and vacuum drying treatment is performed after stirring and mixing.

[0006] Preferably, fresh long oyster adductor muscle is selected, washed with sterile water, and then subjected to standing pretreatment, broken by grinding technology to obtain oyster original slurry, including: Select fresh long oysters, separate the adductor muscle after opening the shell, remove the fascia and impurities, and select adductor muscle with complete shape and no damage; The surface of the adductor muscle is washed with sterile water, and the water temperature is controlled in the range of 0-4℃ during the washing process to avoid premature browning of the adductor muscle caused by temperature fluctuations; The washed adductor muscle is placed in a 0-4℃ environment for a predetermined time to stabilize the adductor muscle tissue; A grinding device equipped with a low-temperature cooling device is used to break and grind the slurry, and the slurry temperature is monitored in real time during the grinding process to ensure that the slurry temperature is always maintained in the range of 0-4℃, and the oyster original slurry is obtained.

[0007] Preferably, ascorbic acid solution is added to the oyster original slurry, stirred and mixed under light-proof conditions for a predetermined time to construct a browning substrate target binding system, including: Dissolve ascorbic acid in sterile ultrapure water to prepare ascorbic acid solution with a specific concentration, and use light-proof containers during the dissolution process to avoid oxidation of ascorbic acid caused by light; The ascorbic acid solution is slowly added to the low-temperature oyster original slurry at a preset volume ratio, and a magnetic stirrer is used for stirring during the addition process, with the stirring rate controlled within a predetermined range to avoid the generation of a large amount of bubbles caused by excessive stirring; The mixed system is placed in a light-proof environment and balanced for a predetermined time under low-temperature conditions to ensure that the ascorbic acid is fully contacted and combined with the browning substrate in the original slurry, and a browning substrate target binding system is constructed; During the balancing process, the redox potential of the system is measured every predetermined time to ensure that the system is in a stable reducing state.

[0008] Preferably, the complex protease is added to the browning substrate target binding system in a preset proportion, the complex protease is composed of trypsin and flavor protease in a specific enzyme activity ratio, the browning substrate target binding system is adjusted to a preset pH and temperature, and a segmented enzymatic hydrolysis reaction is carried out, including: According to the protein content of the low-temperature oyster plasma, the total amount of complex protease is determined; According to the specific enzyme activity ratio of trypsin and flavor protease, the corresponding mass of the two proteases is weighed and mixed uniformly to obtain the complex protease; The complex protease is slowly added to the browning substrate target binding system, and stirring is performed to uniformly disperse the protease; The pH of the system is slowly adjusted to the preset range using an edible acid-base regulator, and the pH change of the system is monitored in real time during the adjustment process to avoid sudden increases and decreases in pH; The adjusted system is transferred to a constant temperature water bath device, the water bath temperature is controlled to a first preset temperature, and the first stage of enzymatic hydrolysis reaction is carried out; After the first stage of enzymatic hydrolysis reaction is completed, the water bath temperature is adjusted to a second preset temperature, the second stage of enzymatic hydrolysis reaction is carried out, and the segmented enzymatic hydrolysis reaction is completed.

[0009] Preferably, according to the protein content of the low-temperature oyster plasma, the total amount of complex protease is determined, including: The protein concentration of the low-temperature oyster plasma is determined by the Coomassie brilliant blue G-250 method; The total protein mass in the plasma is calculated according to the protein concentration; According to the enzyme activity efficiency of the complex protease and the enzymatic hydrolysis reaction time, the proteinase addition amount corresponding to each unit mass of protein is set; According to the total protein mass and the proteinase addition amount corresponding to each unit mass of protein, the total amount of complex protease is calculated; During the calculation process, a preset proportion of the remaining amount is reserved to ensure that the protease can fully degrade the protein in the plasma to generate sufficient umami peptides.

[0010] Preferably, an intermittent ultrasonic auxiliary treatment is used to monitor the activity changes of polyphenol oxidase and peroxidase in the system in real time, and the ultrasonic parameters are dynamically adjusted according to the enzyme activity curve, including: The enzymatic hydrolysis system is placed in an ultrasonic device, the working time and intermittent time of ultrasonic treatment are set, and an intermittent ultrasonic treatment mode is formed; During ultrasonic treatment, the activity of polyphenol oxidase and peroxidase in the system is monitored in real time using an enzyme activity determination kit; According to the measured enzyme activity data, an enzyme activity change curve is drawn to analyze the influence law of ultrasonic treatment on enzyme activity; When the enzyme activity is higher than the preset threshold, the working time ratio of ultrasonic treatment is increased or the ultrasonic power is adjusted to the preset range. When the enzyme activity is lower than the preset threshold, the working time proportion of ultrasonic treatment is reduced or the ultrasonic power is adjusted to another preset range, so as to realize dynamic adjustment of the ultrasonic parameters; During the dynamic adjustment process, the temperature change of the system is monitored synchronously to avoid the system temperature exceeding the suitable range for enzymolysis due to heat production of ultrasonic treatment.

[0011] Preferably, the activity of polyphenol oxidase and peroxidase in the system is monitored in real time by using an enzyme activity assay kit, which includes: According to the instructions of the enzyme activity assay kit, the reagents and reaction system required for enzyme activity assay are prepared; During the intermittent stage of ultrasonic treatment, a preset volume of sample is taken from the enzymolysis system and quickly placed in a low-temperature environment to terminate the enzymatic reaction; The sample is pretreated according to the requirements of the kit to remove impurities and interfering substances; The pretreated sample is mixed with the reagents in the kit and reacted at a specific temperature for a preset time; The absorbance value of the reaction system at a specific wavelength is measured by using a spectrophotometer, and the activity of polyphenol oxidase and peroxidase in the sample is calculated according to the absorbance value and the standard curve of enzyme activity.

[0012] Preferably, after the enzymolysis reaction is completed, a composite antioxidant composed of green tea extract and citric acid is added to construct an ascorbic acid-composite antioxidant synergistic color protection system, and vacuum drying treatment is performed after stirring and mixing, including: The green tea extract is purified to remove impurities and bitter components, obtaining high-purity green tea extract; The high-purity green tea extract and citric acid are mixed in a preset mass ratio, dissolved in sterile ultrapure water to obtain a composite antioxidant solution; After the enzymolysis reaction is completed, the composite antioxidant solution is added to the enzymolysis system in a preset proportion, and a stirring device is used to stir and mix to uniformly disperse the composite antioxidant in the enzymolysis system; After stirring is completed, the enzymolysis system is placed in a vacuum drying device, the drying temperature is set to a temperature range of 40-45℃, and the vacuum degree is controlled within a preset range; During the drying process, the moisture content of the system is monitored in real time, and the drying time and vacuum degree are adjusted according to the change of the moisture content, and when the moisture content of the system decreases to a preset target value, the drying process is stopped; After drying is completed, the product is cooled to room temperature to obtain the dried oyster umami peptide product.

[0013] Preferably, it further includes the steps of quality detection and analysis of the dried oyster umami peptide product, including: Take a dry product sample, use an amino acid automatic analyzer to determine the composition and content of free amino acids in the sample, and analyze the content of umami amino acids; The molecular weight distribution of the sample is determined by a gel permeation chromatography system, and the proportion of small molecule umami peptides is analyzed. The absorbance of the sample at a specific wavelength is determined by a UV-visible spectrometer to evaluate the degree of browning of the sample. The electronic tongue system is used in combination with sensory evaluation to evaluate the umami intensity, bitterness intensity and other taste characteristics of the sample. According to the quality detection results, the key parameters in the method are optimized to improve the quality and nutrient retention effect of oyster umami peptides.

[0014] After obtaining the dried oyster umami peptide product, the storage stability guarantee step based on browning and crosslinking monitoring is also included: The dried product sample is taken out from the storage environment at a predetermined period, and the content ratio of macromolecular components and small molecule umami peptides in the sample is determined by a gel permeation chromatography system to determine whether excessive crosslinking has occurred. The Lab value of the sample is determined by a color difference meter to evaluate the degree of browning during storage. If the proportion of macromolecular components exceeds the predetermined range or the degree of browning reaches the warning threshold, adjust the temperature and humidity of the storage environment to reduce temperature and humidity fluctuations to ensure that the umami characteristics and nutritional ingredients of the dried product remain stable during the storage period.

[0015] The present application provides a method for targeted inhibition of oyster umami peptide browning based on ascorbic acid and synergistic retention of nutritional ingredients. It has the following beneficial effects: (1) Through the targeted combination of ascorbic acid and browning substrates, the components such as phenolic substances and free amino acids that are prone to cause browning are locked in advance; combined with the composite antioxidant composed of green tea extract and citric acid, an ascorbic acid-composite antioxidant synergistic mechanism is formed, effectively solving the problem of limited effect of traditional single anti-browning method and easy failure due to environmental influence, reducing the browning risk of the whole process of oyster umami peptide processing, and maximizing the retention of the natural color of the product; (2) According to the difference in the optimal reaction conditions of trypsin and flavor protease, the enzyme is designed in sections, first degrading macromolecular proteins, and then deeply converting them into small molecule umami peptides; at the same time, by monitoring the activity of browning-related enzymes in real time, the ultrasonic parameters are dynamically adjusted to improve the efficiency of enzyme hydrolysis, increase the proportion of small molecule umami peptides, and inhibit the activity of polyphenol oxidase and peroxidase, avoiding the intensification of browning and the destruction of umami peptide activity during the enzyme hydrolysis process, breaking through the bottleneck of traditional enzyme hydrolysis process that efficiency and quality are difficult to be considered; (3) From the low-temperature grinding of oyster raw pulp, to the low-temperature equilibrium of browning inhibition, to the constant temperature control of enzymatic reaction, and finally to the vacuum gentle drying, the temperature is controlled throughout the whole process to avoid protein denaturation, amino acid loss and umami peptide degradation caused by high temperature; the vacuum environment further reduces the oxidation reaction, compared with the traditional high-temperature drying process, the retention rate of free amino acids, especially umami amino acids, is improved, and the nutritional and flavor quality of the product is guaranteed; (4) Through multi-dimensional quality detection, covering amino acid composition, molecular weight distribution, browning degree and taste characteristics, the process short board is identified and the key parameters are optimized to continuously improve the product quality; at the same time, a regular monitoring and environment adjustment mechanism is established to real-time control the cross-linking of umami peptide and the browning degree, dynamically adjust the storage temperature and humidity, solve the problem of product deterioration and quality decline in traditional storage process, and prolong the shelf life of the product. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The flowchart provided by the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0018] First of all, it should be pointed out that in the processing of oyster umami peptide, the traditional process generally faces the problems of difficult control of browning, easy loss of nutritional ingredients, and easy destruction of umami peptide activity. The conventional anti-browning means is mostly single addition of antioxidant, which cannot realize the targeted combination of browning substrate; the enzymatic process lacks dynamic regulation mechanism, resulting in unstable protease activity; the high temperature in the drying link easily intensifies browning and nutrient loss. These problems superimposed make it difficult to balance the color, nutrition and flavor quality of oyster umami peptide product, and cannot meet the needs of high-quality food processing.

[0019] The present application constructs an oyster umami peptide browning targeted inhibition and nutritional ingredient synergistic retention method based on ascorbic acid. Through oyster raw pulp preparation, browning substrate targeted combination system construction, segmented enzymolysis, intermittent ultrasonic assistance, synergistic color protection and vacuum gentle drying, precise inhibition of browning and synergistic retention of nutrition and umami are realized.

[0020] Referring to the accompanying Figure 1 , the specific implementation of each step will be described in detail in combination with the embodiments.

[0021] I. Oyster raw pulp preparation Oyster original pulp preparation is through the selection, washing, standing and low temperature grinding treatment of fresh long oyster adductor muscle to obtain oyster original pulp with uniform texture and no premature browning, which lays a foundation for subsequent browning inhibition and enzymatic reaction.

[0022] It should be noted that the oyster adductor muscle tissue is fragile, and temperature fluctuation can easily cause the activity of polyphenol oxidase and peroxidase to increase, leading to premature browning; impurities and residual fascia can affect the subsequent enzymatic efficiency and product quality, therefore the temperature and impurity removal during the preparation process need to be strictly controlled.

[0023] Fresh and live long oysters are selected, shelled with special tools, and the adductor muscle is separated. The fascia, damaged tissue and other impurities are removed by manual screening, and the adductor muscle with complete shape and bright color is retained. The adductor muscle is washed with sterile water, and the water temperature is controlled in the range of 0-4℃ by a constant temperature water tank during the washing process. The water flow speed is adjusted to a slow rinsing state to avoid tissue damage caused by water impact and prevent temperature fluctuation from activating browning related enzymes.

[0024] The washed adductor muscle is transferred to a sterile preservation box and placed in a 0-4℃ refrigerated environment for a predetermined time. The standing time is adjusted according to the freshness of the adductor muscle. The adductor muscle with high freshness can be placed for 1 hour, and the adductor muscle with slightly lower freshness can be placed for 2 hours, so that the adductor muscle tissue returns to a stable state and reduces the degree of cell rupture during subsequent grinding.

[0025] Colloid grinding equipment equipped with low temperature cooling device is used for crushing and pulping. The device cavity is pre-cooled to 0-4℃ before grinding. The standing adductor muscle is put into the grinding cavity in batches, and the input amount of each batch should not exceed 80% of the rated capacity of the device to avoid overloading and insufficient grinding. The temperature of the pulp is monitored in real time by the temperature sensor during the grinding process. When the temperature exceeds 4℃, the low temperature cooling device is automatically started to ensure that the temperature of the pulp is always maintained in the range of 0-4℃. The grinding speed is set to 3000 rpm, and the grinding time is 5 minutes. The oyster original pulp with uniform texture is obtained. The original pulp needs to be immediately transferred to a low temperature container for sealing and preservation, waiting for subsequent processing.

[0026] It should be noted that the oyster original pulp preparation process needs to be carried out in a sterile environment, and the operating tools and containers need to be sterilized at high temperature. If the temperature of the pulp continues to rise during the grinding process and the cooling device cannot control it, the grinding should be stopped immediately, the pulp should be transferred to a 0-4℃ refrigerated environment for cooling, and the grinding should be continued after the temperature meets the standard.

[0027] II. Construction of browning substrate targeted binding system The browning substrate targeted binding system was constructed by adding ascorbic acid solution to oyster pulp and stirring it at low temperature and in the dark to allow the ascorbic acid to fully bind with the browning substrate in the pulp, thereby achieving targeted inhibition of browning while maintaining a stable reducing state of the system.

[0028] It should be noted that ascorbic acid is easily oxidized by light and high temperature, losing its anti-browning activity; improper stirring rate can easily generate bubbles, introducing oxygen and aggravating oxidation. Therefore, it is necessary to strictly control the dissolution environment, stirring rate and equilibrium conditions.

[0029] Ascorbic acid powder was placed in a brown, light-proof container, and sterile ultrapure water was added. The mixture was then stirred at low speed using a magnetic stirrer at a speed of 200 rpm until the ascorbic acid was completely dissolved. This prepared an ascorbic acid solution of a specific concentration. The concentration was adjusted according to the browning risk of the oyster pulp. The concentration of pulp with a high browning risk was set at 0.2 mol / L, and the concentration of pulp with a low browning risk was set at 0.1 mol / L.

[0030] Ascorbic acid solution was slowly added to low-temperature oyster pulp at a preset volume ratio of 1:10. During the addition process, the magnetic stirrer was kept running continuously at a stirring speed of 300 rpm to avoid generating a large number of bubbles due to excessive stirring, while ensuring that the ascorbic acid solution and oyster pulp were mixed evenly. The mixed system was then transferred to a light-proof reactor, and the reactor temperature was controlled at 0-4°C by a constant temperature jacket. The equilibration time was set to 2 hours to allow the ascorbic acid to fully contact and bind with the browning substrates (such as phenolic substances and free amino acids) in the pulp, thus constructing a browning substrate targeted binding system.

[0031] During the equilibration process, samples were taken every 30 minutes, and the redox potential of the system was measured using a redox potentiometer. The potential value under normal reduction conditions should be maintained between -100 and -50 mV. If the potential value is higher than -50 mV, it indicates that the ascorbic acid is oxidized too quickly, and an appropriate amount of ascorbic acid solution needs to be added. If the potential value is lower than -100 mV, it indicates that there is an excess of ascorbic acid, and the subsequent equilibration time can be appropriately shortened to ensure that the system is always in a stable reduction state.

[0032] It should be noted that the preparation and addition of ascorbic acid solution must be carried out in the dark, and the reaction vessel must be made of opaque material. If frequent fluctuations in the redox potential of the system are found during sampling and testing, the sealing of the reaction vessel must be checked to prevent oxygen from leaking into the system.

[0033] III. Segmented Enzymatic Hydrolysis Reaction The staged enzymatic hydrolysis reaction involves determining the amount of compound protease to be added based on the protein content of the oyster pulp, adjusting the system to a suitable pH and temperature, and carrying out enzymatic hydrolysis in stages to achieve efficient degradation of proteins into umami peptides while avoiding accelerated browning during the enzymatic hydrolysis process.

[0034] It should be noted that the optimum temperature and pH of different proteases are different, and a single enzyme stage cannot accommodate the activity of two proteases; improper addition of protease can lead to insufficient protein degradation or excessive enzyme hydrolysis, affecting the yield and quality of umami peptides, so a segmented enzyme hydrolysis mode and precise control of enzyme addition are required.

[0035] 3.1 Determination of complex protease addition The protein concentration of the low-temperature oyster homogenate was determined by the Coomassie Brilliant Blue G-250 method. The specific operation is as follows: take 1 mL of oyster homogenate sample, add 5 mL of Coomassie Brilliant Blue G-250 reagent, mix evenly, and stand for 5 minutes. The absorbance value was determined at 595 nm wavelength by spectrophotometer. The protein concentration of the homogenate was calculated according to the protein standard curve; according to the protein concentration and the total volume of the homogenate, the total protein mass in the homogenate was calculated; referring to the enzyme activity efficiency of complex protease and the enzyme hydrolysis reaction time, the proteinase addition amount corresponding to 1 g of protein was set to 0.01 g, and the proteinase with high enzyme activity efficiency could be appropriately reduced to 0.008 g, and the enzyme hydrolysis reaction time was set to 2 hours; according to the total protein mass and the proteinase addition amount corresponding to unit mass of protein, the total addition amount of complex protease was calculated; during the calculation process, 10% of the amount was reserved to ensure that the proteinase could fully degrade the protein in the homogenate to generate sufficient umami peptides.

[0036] 3.2 Segmented enzyme hydrolysis operation According to the enzyme activity ratio of 1:1 of trypsin and flavor protease, the corresponding mass of the two proteases was weighed, the trypsin enzyme activity was 2500 units / mg, and the flavor protease enzyme activity was 100 units / mg. The two proteases were placed in a sterile container and gently mixed to obtain a complex protease; the complex protease was slowly added to the browning substrate target binding system, and the stirrer was stirred at a stirring rate of 400 revolutions per minute for 10 minutes to make the protease evenly dispersed in the system; the pH of the system was slowly adjusted to 7.2 using edible hydrochloric acid and sodium hydroxide solution, and the pH value change of the system was monitored in real time by pH meter during the adjustment process. After each drop of acid-base solution, stir for 30 seconds before measuring the pH value to avoid sudden rise and fall of the pH value.

[0037] The adjusted system was transferred to a constant temperature water bath device, the water bath temperature was controlled to 37℃, and the first stage enzyme hydrolysis reaction was carried out. The first stage reaction time was 1 hour, and the trypsin activity was mainly used to degrade large molecular proteins; after the first stage enzyme hydrolysis reaction, the water bath temperature was adjusted to 50℃, and the second stage enzyme hydrolysis reaction was carried out. The second stage reaction time was 1 hour, and the flavor protease activity was mainly used to further degrade small molecular peptides into umami peptides, and the segmented enzyme hydrolysis reaction was completed.

[0038] It should be noted that the enzymatic hydrolysis process needs to be sampled every 20 minutes, and the absorbance value is measured at 280 nm wavelength by UV spectrophotometer to monitor the progress of protein degradation; if the enzymatic efficiency is found to be lower than expected, the temperature and pH value need to be checked to see if they are in the appropriate range, and if necessary, a small amount of complex protease is added.

[0039] Four, intermittent ultrasonic assisted treatment Intermittent ultrasonic assisted treatment is to use intermittent ultrasonic mode to process the system during the enzymatic hydrolysis reaction process, monitor the browning-related enzyme activity in real time, and dynamically adjust the ultrasonic parameters to improve the enzymatic efficiency while inhibiting the browning enzyme activity.

[0040] It should be noted that continuous ultrasonic treatment can easily lead to an increase in system temperature, affecting the activity of protease; improper ultrasonic parameters may activate browning enzymes, so intermittent mode should be used and parameters should be adjusted dynamically according to enzyme activity.

[0041] The enzymatic hydrolysis system is transferred to an ultrasonic treatment container with temperature control, the working time of ultrasonic treatment is set to 30 seconds, the intermittent time is 1 minute, forming an intermittent ultrasonic treatment mode; the initial ultrasonic power is set to 200 W, which is adjusted according to the subsequent enzyme activity monitoring results; during the ultrasonic treatment process, the activities of polyphenol oxidase and peroxidase in the system are monitored in real time every 15 minutes using the polyphenol oxidase and peroxidase activity assay kit.

[0042] The specific determination operation is as follows: according to the kit instructions, prepare the reagents such as substrate solution, color reagent and reaction system; during the intermittent stage of ultrasonic treatment, 0.5 mL of sample is taken from the enzymatic hydrolysis system and quickly placed in a 0-4℃ environment to terminate the enzymatic reaction; centrifuge the sample according to the kit requirements, the centrifugal speed is 6000 rpm, the centrifugal time is 10 minutes, and the impurities and interfering substances are removed; take 0.1 mL of supernatant after centrifugation, mix with reagents in the kit, and react in a 37℃ constant temperature water bath for 15 minutes; the absorbance value of the reaction system is measured at 420 nm wavelength by spectrophotometer, and the activities of polyphenol oxidase and peroxidase in the sample are calculated according to the absorbance value and enzyme activity standard curve.

[0043] According to the determined enzyme activity data, the enzyme activity change curve is drawn to analyze the influence law of ultrasonic treatment on enzyme activity; when the enzyme activity is higher than the preset threshold (polyphenol oxidase activity > 50 U / mL, peroxidase activity > 80 U / mL), the working time proportion of ultrasonic treatment is increased, the working time is adjusted to 40 seconds, the intermittent time is kept for 1 minute, or the ultrasonic power is adjusted to 250 W; when the enzyme activity is lower than the preset threshold (polyphenol oxidase activity < 30 U / mL, peroxidase activity < 50 U / mL), the working time proportion of ultrasonic treatment is reduced, the working time is adjusted to 20 seconds, the intermittent time is kept for 1 minute, or the ultrasonic power is adjusted to 150 W, so as to realize the dynamic adjustment of ultrasonic parameters.

[0044] During the dynamic adjustment process, the temperature sensor synchronously monitors the temperature change of the system, and when the temperature exceeds the suitable range of enzymolysis (exceeding 37°C in the first stage and exceeding 50°C in the second stage), the cooling system of the ultrasonic container is started to ensure that the temperature of the system is maintained in the suitable range.

[0045] It should be noted that the ultrasonic treatment container should be made of ultrasonic-resistant material to avoid releasing impurities during ultrasonic treatment; if the enzyme activity monitoring result shows that the activities of the two enzymes continue to increase, it is necessary to check whether the ultrasonic equipment is working normally, and if necessary, replace the ultrasonic treatment mode.

[0046] Five, synergistic color protection and vacuum drying treatment The synergistic color protection and vacuum drying treatment is to add a composite antioxidant to construct a synergistic color protection system after the enzymatic reaction is completed, and then to remove water by vacuum gentle drying, so as to inhibit browning and maximize the retention of nutritional ingredients and umami peptide activity.

[0047] It should be noted that the system still has the risk of browning after the enzymatic reaction is completed, and the effect of single ascorbic acid color protection is limited; high-temperature drying is easy to cause denaturation of umami peptides and loss of nutrients, so it is necessary to construct a synergistic color protection system and use a gentle drying method.

[0048] The green tea extract is placed in a purification device, and macroporous resin adsorption method is used to remove impurities and bitter components therein. The specific operation is as follows: the green tea extract solution is passed through a macroporous resin column, the flow rate is controlled to be 1 mL / min, after adsorption is completed, 70% ethanol solution is used for elution, the eluate is collected and concentrated under reduced pressure, and high-purity green tea extract is obtained; the high-purity green tea extract and citric acid are mixed according to a mass ratio of 3:1, dissolved in sterile ultrapure water, the stirring rate is set to 300 revolutions / minute, and stirring is continued until complete dissolution, and a composite antioxidant solution is obtained.

[0049] After the enzymatic reaction is completed, the complex antioxidant solution is added to the enzymatic system at a volume ratio of 1:20, and the stirring device is used to stir and mix at a stirring rate of 400 revolutions per minute for 15 minutes, so that the complex antioxidant is uniformly dispersed in the enzymatic system to construct an ascorbic acid-complex antioxidant synergistic color protection system; after stirring is completed, the enzymatic system is transferred to a vacuum drying device, and a mild temperature range of 40-45°C is set for the drying temperature, and the vacuum degree is controlled in the range of 0.08-0.1 MPa.

[0050] During the drying process, the moisture content of the system is monitored in real time by a moisture meter, and the moisture content is measured every 30 minutes; when the moisture content is higher than 15%, the drying temperature and the vacuum degree are kept unchanged; when the moisture content is reduced to 10%-15%, the drying temperature is appropriately reduced to 38-40°C; when the moisture content is reduced to below 5%, the drying process is stopped; after the drying is completed, the product is transferred to a sterile cooling room and cooled to room temperature to obtain the dried oyster umami peptide product.

[0051] It should be noted that the complex antioxidant solution needs to be prepared and used immediately to avoid the decrease in activity caused by long-term storage; the vacuum drying equipment needs to be tested for vacuum degree before use to ensure good sealing of the equipment and prevent air from being introduced during the drying process.

[0052] Six, quality detection and analysis Quality detection and analysis is to detect the amino acid composition, molecular weight distribution, browning degree and taste characteristics of the dried oyster umami peptide product, and to optimize the process parameters and improve the product quality according to the detection results.

[0053] It should be noted that the quality detection results directly reflect the effectiveness of the process, and only by comprehensively mastering the various indicators of the product can the key parameters be accurately adjusted, so a multi-dimensional detection method needs to be used.

[0054] Take 0.1 g of the dried product sample and use an amino acid automatic analyzer to determine the composition and content of free amino acids in the sample. The chromatographic conditions are as follows: buffer flow rate is 20 mL / h, reaction liquid flow rate is 10 mL / h, separation column is Na type cation exchange resin column, ultraviolet detection wavelength is 570 nm and 440 nm, column temperature is programmed to 55°C, 65°C and 77°C in turn, sample injection volume is 50 μL, and the content of umami amino acids (glutamic acid, aspartic acid, etc.) is analyzed according to the determination results; take 0.1 g of the dried product sample, dissolve it with 0.1 mol / L sodium nitrate solution and dilute it to 2 mg / mL, filter it through a 0.22 μm microporous filter membrane, and then use a gel permeation chromatography system to determine the molecular weight distribution of the umami peptides in the sample. The chromatographic column is Agilent PL aquagel-OH Mixed-H, the column temperature is set to 45°C, the sample injection amount is 50 μL, the mobile phase is 0.1 mol / L sodium nitrate solution, and the flow rate is 1.0 mL / min. The proportion of small molecule umami peptides (<3 kDa) is analyzed.

[0055] Take 0.01 g of the dried product sample, dilute it 50 times with ultrapure water, filter it through a 0.22 μm water filter membrane, and then use a ultraviolet-visible spectrometer to determine the absorbance of the sample at a wavelength of 420 nm. The lower the absorbance value, the lighter the degree of browning, and the degree of browning of the sample is evaluated. Take 0.5 g of the dried product sample, dissolve it in 100 mL of ultrapure water, and use an electronic tongue system to determine the umami intensity and bitterness intensity of the sample. At the same time, 10 trained food professionals are selected for sensory evaluation. The evaluators need to retain the sample solution in their mouths for 10 seconds before scoring. The evaluation interval is 1 hour, and 50 mL of drinking water is consumed for mouth cleaning. The average score is taken as the sensory evaluation result.

[0056] According to the quality detection results, the key parameters in the method are optimized. If the content of umami amino acids is low, the enzyme activity ratio or enzyme hydrolysis time of the compound protease can be adjusted. If the degree of browning is high, the concentration of ascorbic acid or the amount of compound antioxidant can be increased. If the proportion of small molecule umami peptides is low, the ultrasonic parameters or enzyme hydrolysis temperature can be optimized, thereby improving the quality and nutrient retention effect of oyster umami peptides.

[0057] Seven, storage stability guarantee After obtaining the dried oyster umami peptide product, the crosslinking and browning degree during storage are regularly monitored, and the storage environment parameters are adjusted to ensure that the product remains stable during the storage period.

[0058] It should be noted that temperature and humidity fluctuations during storage can easily lead to excessive crosslinking and browning of umami peptides, affecting product quality, so a regular monitoring and environmental adjustment mechanism needs to be established.

[0059] The dried oyster umami peptide product is sealed and packaged, and then placed in a storage environment with a temperature of 25°C and a relative humidity of 50%; every 7 days, a sample of the dried product is taken from the storage environment, 0.1 g of the sample is dissolved in 0.1 mol / L sodium nitrate solution, and then the content ratio of macromolecular components (>10 kDa) and small-molecule umami peptides (<3 kDa) in the sample is determined by using a gel permeation chromatography system to determine whether excessive cross-linking has occurred; at the same time, 0.1 g of the sample is dissolved in ultrapure water, and then the Lab value of the sample is determined by using a color difference meter; the lower the L value and the higher the a value and b value, the more serious the browning degree, and the browning degree during storage is evaluated.

[0060] If the content ratio of macromolecular components exceeds the preset range of 15% or the browning degree reaches the warning threshold (L value <60, a value >10, b value >50), the temperature of the storage environment is adjusted to below 20°C, and the relative humidity is controlled to be below 40%; the temperature is reduced and the humidity fluctuation is reduced to ensure that the umami characteristics and nutritional ingredients of the dried product remain stable during the storage period; after each adjustment of the environmental parameters, the monitoring period is shortened to 3 days until the product state returns to stable, and then the regular monitoring period of 7 days is restored.

[0061] It should be noted that the storage container should be made of a sealed moisture-proof material to prevent external moisture and oxygen from entering; if the product quality continues to decline, it is necessary to check whether the packaging is damaged, and if necessary, the product should be repackaged or used in advance.

[0062] In summary, the present application establishes a whole-process temperature control and mild drying system to achieve efficient retention of nutritional ingredients; proposes a process combining segmented enzymatic hydrolysis and intermittent ultrasonic dynamic regulation to achieve efficient generation and activity protection of umami peptides; constructs a browning substrate targeted combination and synergistic color protection double system to achieve precise inhibition of browning from the source; and designs a quality detection and storage stability guarantee closed-loop mechanism to ensure the stable quality of the product throughout its life cycle.

[0063] Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application.

Claims

1. A method for targeted inhibition of browning of oyster umami peptides and synergistic retention of nutrients based on ascorbic acid, characterized in that: Includes the following steps: Fresh long oyster adductor muscles were selected, rinsed with sterile water, and then subjected to static pretreatment. The adductor muscles were then crushed and pulped using grinding technology to obtain oyster pulp. Ascorbic acid solution was added to the oyster pulp, and the mixture was stirred and equilibrated for a preset time under light-protected conditions to construct a browning substrate targeted binding system. A complex protease is added to the browning substrate targeted binding system in a preset ratio. The complex protease is composed of trypsin and flavor protease in a specific enzyme activity ratio. The browning substrate targeted binding system is adjusted to a preset pH and temperature to carry out a segmented enzymatic hydrolysis reaction. During the enzymatic hydrolysis process, intermittent ultrasound-assisted treatment was used to monitor the activity changes of polyphenol oxidase and peroxidase in the browning substrate targeted binding system in real time, and the ultrasound parameters were dynamically adjusted according to the enzyme activity curve. After the enzymatic hydrolysis reaction is completed, a complex antioxidant composed of green tea extract and citric acid is added to construct an ascorbic acid-complex antioxidant synergistic color-protecting system. After stirring and mixing, the mixture is vacuum dried.

2. The method according to claim 1, characterized in that, Fresh adductor muscles of oysters were selected, rinsed with sterile water, and then pretreated by static settling. The resulting oyster pulp was obtained by grinding and crushing the pulp, comprising: Fresh oysters were selected, and after opening the shells, the adductor muscles were separated, the fascia and impurities were removed, and the adductor muscles with intact shape and no damage were selected. Rinse the surface of the adductor muscle with sterile water, keeping the water temperature between 0 and 4°C during the rinsing process. After rinsing, place the adductor muscle in an environment of 0-4℃ and let it stand for a preset time. The grinding equipment equipped with a low-temperature cooling device is used for crushing and pulping. The pulp temperature is monitored in real time during the grinding process to ensure that the pulp temperature is always maintained in the range of 0 to 4℃, so as to obtain oyster pulp.

3. The method according to claim 1, characterized in that, Ascorbic acid solution was added to the oyster pulp, and the mixture was stirred and equilibrated for a preset time under light-protected conditions to construct a browning substrate targeted binding system, including: Ascorbic acid was dissolved in sterile ultrapure water to prepare an ascorbic acid solution of a specific concentration. The dissolution process was carried out in a light-proof container. The ascorbic acid solution was slowly added to the low-temperature oyster paste at a preset volume ratio, and the mixture was stirred with a magnetic stirrer while being added, with the stirring rate controlled within a preset range. The mixed system was placed in a dark environment and equilibrated for a preset time under low temperature conditions to allow ascorbic acid to fully contact and bind with the browning substrate in the original pulp, thereby constructing a browning substrate targeted binding system. During the equilibration process, the redox potential of the system is measured at preset intervals to ensure that the system is in a stable reducing state.

4. The method according to claim 1, characterized in that, A complex protease, composed of trypsin and flavor protease in a specific enzyme activity ratio, is added to the browning substrate targeting binding system according to a preset ratio. The browning substrate targeting binding system is adjusted to a preset pH and temperature to perform a segmented enzymatic hydrolysis reaction, including: The total amount of compound protease added is determined based on the protein content of the low-temperature oyster paste. Weigh out the corresponding mass of the two proteases according to the specific enzyme activity ratio of trypsin and flavor protease, and mix them evenly to obtain a compound protease. The complex protease was slowly added to the browning substrate targeting binding system, and the mixture was stirred to ensure uniform dispersion of the protease. The pH of the system is slowly adjusted to the preset range using food-grade pH adjusters. During the adjustment process, the pH value of the system is monitored in real time to avoid sudden increases or decreases in pH value. The adjusted system was transferred to a constant temperature water bath device, and the water bath temperature was controlled to the first preset temperature to carry out the first stage of enzymatic hydrolysis reaction. After the first stage of enzymatic hydrolysis is completed, the water bath temperature is adjusted to the second preset temperature to carry out the second stage of enzymatic hydrolysis, thus completing the segmented enzymatic hydrolysis reaction.

5. The method according to claim 4, characterized in that, Based on the protein content of the aforementioned low-temperature oyster paste, the total amount of compound protease to be added is determined, including: The protein concentration in low-temperature oyster slurry was determined using the Coomassie Brilliant Blue G-250 method. Calculate the total protein content in the original pulp based on the protein concentration; Based on the enzyme activity efficiency and enzymatic hydrolysis time of the complex protease, the amount of protease to be added per unit mass of protein was determined. The total amount of complex protease added is calculated based on the total protein mass and the amount of protease added per unit mass of protein. During the calculation process, a margin is reserved according to a preset ratio to ensure that the protease can fully degrade the protein in the original pulp and generate sufficient umami peptides.

6. The method according to claim 1, characterized in that, During the enzymatic hydrolysis process, intermittent ultrasound-assisted treatment is employed to monitor the activity changes of polyphenol oxidase and peroxidase in the system in real time. Ultrasound parameters are dynamically adjusted based on the enzyme activity curves, including: The enzymatic hydrolysis system is placed in an ultrasonic device, and the working time and interval of ultrasonic treatment are set to form an intermittent ultrasonic treatment mode. During the ultrasonic treatment process, the activities of polyphenol oxidase and peroxidase in the system were monitored in real time using an enzyme activity assay kit. Based on the measured enzyme activity data, an enzyme activity change curve was plotted to analyze the influence of ultrasonic treatment on enzyme activity. When enzyme activity is higher than a preset threshold, increase the proportion of ultrasonic treatment time or adjust the ultrasonic power to a preset range. When the enzyme activity is below a preset threshold, the proportion of working time for ultrasonic treatment is reduced or the ultrasonic power is adjusted to another preset range to achieve dynamic adjustment of ultrasonic parameters. During the dynamic adjustment process, the temperature change of the system is monitored simultaneously to avoid the system temperature from exceeding the suitable range for enzymatic hydrolysis due to heat generated by ultrasonic treatment.

7. The method according to claim 6, characterized in that, During the ultrasonic treatment process, the activity of polyphenol oxidase and peroxidase in the system is monitored in real time using an enzyme activity assay kit, including: Prepare the reagents and reaction system required for enzyme activity assay according to the instructions of the enzyme activity assay kit; During the intervals of ultrasonic treatment, a sample of a predetermined volume is removed from the enzymatic hydrolysis system and placed in a low-temperature environment to terminate the enzymatic reaction. Pre-treat the samples according to the kit requirements to remove impurities and interfering substances; Mix the pretreated sample with the reagents in the kit and react at a specific temperature for a preset time. The absorbance of the reaction system at a specific wavelength was measured using a spectrophotometer. Based on the absorbance value and the enzyme activity standard curve, the activities of polyphenol oxidase and peroxidase in the sample were calculated.

8. The method according to claim 1, characterized in that, After the enzymatic hydrolysis reaction is completed, a composite antioxidant consisting of green tea extract and citric acid is added to construct an ascorbic acid-composite antioxidant synergistic color-protecting system. After stirring and mixing, the system undergoes vacuum drying, including: The green tea extract is purified to remove impurities and bitter components, resulting in a high-purity green tea extract. The high-purity green tea extract and citric acid are mixed at a preset mass ratio and dissolved in sterile ultrapure water to obtain a composite antioxidant solution. After the enzymatic hydrolysis reaction is completed, the composite antioxidant solution is added to the enzymatic hydrolysis system in a preset ratio, and the mixture is stirred using a stirring device to ensure that the composite antioxidant is evenly dispersed in the enzymatic hydrolysis system. After stirring, place the enzymatic hydrolysis system in a vacuum drying device, set the drying temperature to a range of 40-45℃, and control the vacuum degree within the preset range. During the drying process, the moisture content of the system is monitored in real time, and the drying time and vacuum degree are adjusted according to the moisture content changes. When the moisture content of the system drops to the preset target value, the drying process is stopped. After drying, the product was cooled to room temperature to obtain the dried oyster umami peptide product.

9. The method according to claim 1, characterized in that, The method also includes a step of quality detection and analysis of the dried oyster umami peptide product, including: Take dried product samples and use an automatic amino acid analyzer to determine the composition and content of free amino acids in the samples, and analyze the content of umami amino acids. The molecular weight distribution of umami peptides in the samples was determined using a gel permeation chromatography system, and the proportion of small molecule umami peptides was analyzed. The degree of browning of the sample was assessed by measuring the absorbance of the sample at a specific wavelength using a UV-Vis spectrometer. The umami intensity, bitterness intensity, and other taste characteristics of the samples were evaluated by combining an electronic tongue system with sensory evaluation. Based on the quality test results, key parameters in the method were optimized to improve the quality and nutrient retention of oyster umami peptides.

10. The method according to claim 1, characterized in that, After obtaining the dried oyster umami peptide product, the process also includes a storage stability assurance step based on browning and cross-linking monitoring: The dried product samples were taken out from the storage environment according to the preset cycle, and the content ratio of macromolecular components and small umami peptides in the samples was determined by gel permeation chromatography system to determine whether excessive cross-linking had occurred. Meanwhile, the Lab value of the samples was measured using a colorimeter to assess the degree of browning during storage; If the proportion of macromolecular components exceeds the preset range or the degree of browning reaches the warning threshold, adjust the temperature and humidity of the storage environment, reduce temperature and reduce humidity fluctuations, and ensure that the umami characteristics and nutritional components of the dried product remain stable during the storage period.

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