Extraction process of umami peptide

Abstract

The present application relates to the field of umami peptide, in order to solve the problem of long time spent in the existing extraction process, provide a kind of umami peptide extraction process, comprising the following steps: S100, meat paste is dispersed in deionized water, adjust pH to 7-9, add ascorbic acid, after reaction, obtain animal source pretreatment liquid;S200, adjust the pH of animal source pretreatment liquid to 6-7, then add slow-release enzyme, and gradually add bacteria mud dispersion liquid, after reaction, after homogenization treatment process, obtain substrate;S300, substrate is obtained after enzymolysis, purification, impurity removal, drying to obtain umami peptide.The lysine residues in meat paste protein are consumed by ascorbic acid, which promotes the heterologous crosslinking between bacteria source protein and animal source protein.Slow-release enzyme with sodium ion concentration control slow-release speed, combined with the preparation method and adding method of bacteria mud dispersion liquid, while ensuring the uniformity and stability of the system crosslinking, the crosslinking time and subsequent enzymolysis time are as low as possible.

Description

Technical Field

[0001] This invention relates to the field of umami peptides, and more specifically, to an extraction process for umami peptides. Background Technology

[0002] The applicant filed a patent application on January 30, 2026, with application number 202610129636.9, entitled "A Processing Technology for Compounding Umami Peptide Extracts to Replace MSG." This technology utilizes TG enzymes to cross-link proteins from different sources, thereby improving substrate stability. However, TG enzymes can promote cross-linking not only between heterologous proteins but also between homologous proteins, ultimately requiring protein hydrolysis to obtain peptides that enhance umami flavor. This competition between homologous and heterologous proteins for cross-linking means that utilizing cross-linking between heterologous proteins to improve substrate stability requires a longer cross-linking time, which in turn requires more time for subsequent enzymatic hydrolysis. Summary of the Invention

[0003] The purpose of this invention is to provide a process for extracting umami peptides, which solves the problem of the long extraction time required by existing extraction processes.

[0004] The embodiments of the present invention are achieved through the following technical solutions:

[0005] An extraction process for umami peptides includes the following steps:

[0006] S100 and meat paste are dispersed in deionized water, and the pH value is adjusted to 7-9. Ascorbic acid is added, and after reaction, animal-derived pretreatment solution is obtained.

[0007] S200, after adjusting the pH of the animal-derived pretreatment solution to 6-7, add slow-release enzyme and gradually add bacterial sludge dispersion. After the reaction, the substrate is obtained through a homogenization process; the bacterial sludge is dispersed in a sodium salt buffer system.

[0008] S300: The substrate is subjected to enzymatic hydrolysis, purification, impurity removal, and drying to obtain umami peptides;

[0009] The method for preparing the sustained-release enzyme includes the following steps:

[0010] A100, sodium alginate solution and TG enzyme solution are mixed to obtain a mixture;

[0011] A200. The mixture is added dropwise to a continuously stirred calcium chloride solution. After the reaction, gel particles are obtained.

[0012] A300: After placing the gel particles in a chitosan solution, react them under continuous stirring, and then obtain the slow-release enzyme by washing and drying.

[0013] This invention reduces cross-linking between animal-derived proteins by consuming lysine residues in the ascorbic acid, thereby promoting heterologous cross-linking between fungal and animal-derived proteins. Furthermore, by gradually adding fungal sludge, the initial proportion of sludge in the reaction system is relatively low, resulting in greater physical distance between fungal proteins. Influenced by the higher animal protein content, the fungal proteins tend to cross-link with animal proteins rather than cross-linking with themselves.

[0014] The purpose of adjusting the pH to 6-7 is at least to: provide an environment for cross-linking of bacterial and animal proteins; and to minimize the tendency for free ascorbic acid to exert its protective effect again.

[0015] In step S200 of this invention, a slow-release enzyme is added to avoid the rapid occurrence of cross-linking reactions and excessive cross-linking caused by the simultaneous addition of a large amount of free TG enzyme. Initially, due to the presence of a large amount of unreacted substrate, the amount of TG enzyme released is relatively small, yet a large number of cross-linking sites can be found, resulting in a faster cross-linking rate. However, as the reaction progresses, the substrate gradually forms a cross-linked network, reducing the number of cross-linking sites and increasing steric hindrance. Therefore, even with an increase in TG release, the cross-linking rate will not become excessively high, thus increasing the stability of the system's cross-linking rate and facilitating the control of the degree of cross-linking.

[0016] Preparation process of meat paste: Animal raw materials are processed into minced meat. The minced meat is placed in a 55℃ constant temperature water bath and continuously stirred for 1-1.5 minutes, then cooled to below 10℃. An equal mass of deionized water is added to the cooled minced meat, and it is then mashed into a paste. Papain is added, and the mixture is stirred at 35℃ for 10-15 minutes. Vitamin C is then added, and the mixture is stirred evenly to obtain meat paste for later use. The animal raw materials consist of chicken and shrimp in a 4:3 mass ratio. The fungal raw materials consist of porcini mushrooms, shaggy ink cap mushrooms, and king oyster mushrooms in a 2:1:1 mass ratio.

[0017] Because the slow-release enzyme in this invention primarily acts on the heterologous cross-linking between animal-derived and bacterial-derived proteins, the functional loss caused by the TG enzyme's action on homologous protein cross-linking decreases, thus reducing the required total dosage and release rate per unit time of TG enzyme. Furthermore, this invention employs a gradual addition of bacterial sludge to reduce cross-linking between bacterial proteins, thereby ensuring that the TG enzyme release rate is optimally matched to the addition rate. Directly using the TG enzyme slow-release agent in patent application number 202610129636.9 is unlikely to achieve the aforementioned expectations. The optimal pH value for TG enzyme is typically 6-7, with a narrow adjustable range, and the optimal temperature is around 50°C. Without affecting TG activity, this invention provides a method for preparing the slow-release enzyme, using salt to regulate the slow-release rate to coordinate with the addition of bacterial sludge.

[0018] In this invention, the TG enzyme is initially embedded in a calcium alginate gel network, and then a polyelectrolyte complex membrane is formed by electrostatic complexation between the gel particles and chitosan. When a solution containing sodium ions is added to the reaction system containing the slow-release enzyme, the sodium ions in the solution undergo ion exchange with Ca²⁺ in the calcium alginate gel network. This exchange breaks the cross-linking bonds between Ca²⁺ and sodium alginate molecules, causing the internal gel network to swell, thereby releasing the embedded TG enzyme. An increase in sodium ion concentration promotes the release of the TG enzyme.

[0019] The solvent for the TG enzyme solution is deionized water.

[0020] Preferably, the concentration of sodium alginate solution is 1.8wt%-2.5wt%, the concentration of TG enzyme solution is 1wt%-5wt%, the volume ratio of TG enzyme solution to sodium alginate solution is 1:(4-6), the concentration of calcium chloride solution is 0.3-0.6mol / L, and the volume ratio of the mixed solution to calcium chloride solution is 1:(7-15); the concentration of chitosan solution is 1.5wt%-1.8wt%, and the mass ratio of sodium alginate to chitosan is 1:(8-10).

[0021] The concentration and amount of calcium chloride, the ratio of sodium alginate to chitosan, and reaction parameters all affect the release rate of the gel.

[0022] Appropriately increasing the amount of chitosan can not only participate in film formation, but also form an additional protective barrier on the surface of the slow-release enzyme to resist the rapid penetration of high concentrations of sodium ions, so as to better achieve slow release.

[0023] Preferably, in step A200, the reaction temperature is 25-30℃, the reaction time is 35-50 min, the stirring speed is 200-300 rpm, and the dropping speed is 30-45 drops / min.

[0024] The needle inner diameter is 0.4-0.6 mm. The reaction time is the incubation time after the dripping is complete.

[0025] Too rapid a dripping rate can lead to an uneven gel particle structure and the formation of internal cavities, potentially causing burst release during subsequent use and failing to achieve a stable, sustained-release effect. Too slow a dripping rate results in a dense gel structure, causing the TG enzyme to be released too slowly, especially initially.

[0026] Preferably, in step A300, the pH value of the chitosan solution is 5.0-5.5, the reaction temperature is 20-30℃, and the reaction time is 10-20 min.

[0027] The solvent for the chitosan solution is a 1 wt% acetic acid solution.

[0028] Preferably, in step S100, the amount of ascorbic acid added is 1wt%-3wt% of the meat paste, the reaction temperature is 25-35℃, and the reaction time is 30-60min;

[0029] In step S200, the amount of slow-release enzyme added is 1wt%-1.5wt% of the bacterial sludge, the reaction temperature is 25-35℃, and the reaction time is 12-18min.

[0030] Because the TG enzyme reduces the cross-linking of homologous proteins in this invention, the amount of slow-release enzyme added is reduced. To avoid the separation of bacterial and animal proteins, they need to be cross-linked. In this invention, by minimizing cross-linking between homologous proteins, the reaction time in step S200 can also be significantly reduced.

[0031] Preferably, the preparation method of the fungal mud dispersion includes: pulverizing fungal raw materials to obtain fungal mud; dispersing the fungal mud in deionized water at a mass ratio of 1:(4-6), adjusting the pH value to 5-6.5, adding cellulase and chitosanase, heating to 40-60℃, reacting for 1-1.5h, adjusting the pH value to 6.5-7.5, and then adding sodium carboxymethyl cellulose, sodium hydrogen phosphate and sodium dihydrogen phosphate to finally obtain the fungal mud dispersion;

[0032] Based on the volume of deionized water, the addition amount of sodium hydrogen phosphate is 20-22 g / L, the addition amount of sodium dihydrogen phosphate is 3-3.5 g / L, and the addition amount of sodium carboxymethyl cellulose is 0.1 wt%-2 wt% of the bacterial sludge.

[0033] After the bacterial sludge is hydrolyzed by cellulase and chitosanase, the bacterial proteins are exposed, making them more susceptible to cross-linking reactions. Since the bacterial sludge dispersion in this invention is added gradually, a certain degree of dispersion stability needs to be maintained; therefore, sodium carboxymethyl cellulose is added as an additional agent.

[0034] Preferably, in step S100, the mass ratio of meat paste to deionized water is 1:(2-3).

[0035] The bacterial sludge dispersion was added to the animal-derived pretreatment solution in several batches. The first addition was 1 / 5 to 1 / 4 of the total volume of the bacterial sludge dispersion. When the system viscosity reached 280-320 mPa·s, the second addition was started, with the amount added being 1 / 3 to 3 / 5 of the total volume of the bacterial sludge dispersion. When the system viscosity reached 360-440 mPa·s, the third addition was started. The remaining bacterial sludge dispersion was added after the third addition. When the system viscosity reached 500-600 mPa·s, the mixture was homogenized to obtain the substrate.

[0036] The total volume ratio of the bacterial sludge dispersion to the animal-derived pretreatment solution is 1:(1-1.8).

[0037] In this invention, the bacterial sludge is dispersed in a sodium salt buffer system. When new bacterial sludge is added, the sodium ion concentration in the reaction system increases synchronously, thereby accelerating the release of TG enzyme to meet the demand for TG enzyme after the addition of the new substrate. During the first addition, TG enzyme is released gradually, and the heterologous protein gradually cross-links. As cross-linking sites are consumed and steric hindrance increases, the reaction rate may still decrease even if TG enzyme is still being released. Therefore, by adding new bacterial sludge again to increase the number of cross-linkable sites and by increasing the sodium ion concentration to accelerate TG enzyme release, this at least compensates for the increased steric hindrance caused by cross-linking after the previous addition, which affects the contact between TG enzyme and protein.

[0038] In this invention, the amount and time of each addition are determined by the viscosity change rate of the reaction system. The next addition of new bacterial sludge begins once the viscosity change rate significantly decreases. During the experiment, viscosity is monitored using a rotational viscometer. Through the aforementioned experiments and viscosity change rate control, the obtained addition amount and time can minimize the reaction time of step S200 while ensuring substrate stability.

[0039] When the new substrate contains only bacterial sludge and no sodium salt, although the addition of bacterial sludge can increase the reaction rate, more bacterial sludge needs to be added to achieve the desired reaction rate. The cross-linking tendency between bacterial proteins increases, which is not conducive to improving the stability of the substrate and will also increase the subsequent enzymatic hydrolysis time.

[0040] The substrate is stored at low temperatures.

[0041] Preferably, the homogenization process is ultrasonic homogenization.

[0042] The ultrasonic homogenization process is performed at a temperature of 20-30℃, a power of 250-300W, a frequency of 15-20kHz, and a time of 5-8min.

[0043] Because there are fewer cross-links between homologous proteins in this invention, the degree of protein cross-linking required to maintain substrate stability is lower, making homogenization easier.

[0044] Preferably, in step S300, the enzymatic hydrolysis uses protease, and the hydrolysis time is 2.5-4 hours.

[0045] Because the cross-linking between homologous proteins is reduced in this invention, and the degree of cross-linking required to avoid the separation of bacterial and animal proteins is reduced to a certain extent, the enzymatic hydrolysis time in step S300 is significantly reduced.

[0046] The present invention has at least the following beneficial effects:

[0047] This invention reduces cross-linking between animal-derived proteins by consuming lysine residues in meat puree protein with ascorbic acid, thereby promoting heterologous cross-linking between fungal and animal-derived proteins. Furthermore, by gradually adding fungal puree, the initial proportion of the puree in the reaction system is relatively low, resulting in greater physical distance between fungal proteins. Influenced by the higher content of animal protein, the fungal proteins tend to cross-link with animal proteins rather than cross-link with themselves.

[0048] This invention prepares a slow-release enzyme whose release rate can be controlled by sodium ion concentration. Combined with the preparation method and addition method of the bacterial sludge dispersion, it minimizes the cross-linking time and subsequent enzymatic hydrolysis time while ensuring the uniformity and stability of the cross-linking of the system. Detailed Implementation

[0049] To make the objectives, methods, and advantages of the embodiments of the present invention clearer, the methods in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0050] Example 1: An extraction process for umami peptides, characterized by comprising the following steps:

[0051] Preparation of sustained-release enzymes:

[0052] A100, a 1.8 wt% sodium alginate solution, and a 1 wt% TG enzyme solution were mixed to obtain a mixture; the volume ratio of the TG enzyme solution to the sodium alginate solution was 1:4.

[0053] A200: The mixture is added dropwise to a 0.3 mol / L calcium chloride solution with constant stirring. After the reaction, gel particles are obtained. The volume ratio of the mixture to the calcium chloride solution is 1:7. The reaction temperature is 25℃, the reaction time is 35 min, the stirring speed is 200 rpm, the dropping speed is 30 drops / min, and the inner diameter of the dropping needle is 0.5 mm.

[0054] A300: Gel particles were placed in a 1.5 wt% chitosan solution and reacted with constant stirring. The resulting slow-release enzyme was obtained by washing and drying. The mass ratio of sodium alginate to chitosan was 1:8. The pH of the chitosan solution was 5.0, the reaction temperature was 20℃, and the reaction time was 10 min.

[0055] Preparation of meat paste: Process the animal raw materials into meat paste, place the meat paste in a 55℃ constant temperature water bath and stir continuously for 1 minute, then cool to 8℃; add an equal mass of deionized water to the cooled meat paste, break it into a paste, add 0.15% of the meat paste mass of papain, stir at 35℃ for 10 minutes, add 0.1% of the meat paste mass of vitamin C, stir evenly to obtain meat paste, set aside; the animal raw materials consist of chicken and shrimp (with shells) in a mass ratio of 4:3.

[0056] Preparation of fungal mud dispersion: The fungal raw material was pulverized to obtain fungal mud; the fungal raw material consisted of Boletus edulis, Coprinus comatus, and Pleurotus ostreatus; the mass ratio of Boletus edulis, Coprinus comatus, and Pleurotus ostreatus was 2:1:1. The fungal mud was dispersed in deionized water at a mass ratio of 1:4, the pH was adjusted to 5, cellulase and chitosanase were added, the temperature was raised to 40℃, and the reaction was carried out for 1 hour. The pH was adjusted to 6.5, and then sodium carboxymethyl cellulose, sodium hydrogen phosphate, and sodium dihydrogen phosphate were added to obtain the fungal mud dispersion.

[0057] Based on the volume of deionized water, the addition amount of sodium hydrogen phosphate is 20 g / L, the addition amount of sodium dihydrogen phosphate is 3 g / L, and the addition amount of sodium carboxymethyl cellulose is 1 wt% of the bacterial sludge.

[0058] S100, after dispersing meat paste in deionized water, added 0.4% of emulsifying stabilizer (composed of soy protein isolate and xanthan gum in a mass ratio of 3:1) by weight of the meat paste, adjusted the pH to 7, added ascorbic acid, and after reaction, obtained animal-derived pretreatment solution; the mass ratio of meat paste to deionized water was 1:2; the amount of ascorbic acid added was 1 wt% of the meat paste, the reaction temperature was 25℃, and the reaction time was 30 min;

[0059] After adjusting the pH of the animal-derived pretreatment solution to 6, add the slow-release enzyme, then add the bacterial sludge dispersion in portions. The first addition is 1 / 5 of the total volume of the bacterial sludge dispersion. When the system viscosity reaches 280 mPa·s, begin the second addition, with the amount being 3 / 5 of the total volume of the bacterial sludge dispersion. When the system viscosity reaches 360 mPa·s, begin the third addition, adding the remaining bacterial sludge dispersion. When the system viscosity reaches 480 mPa·s, heat to 80℃ and hold for 2 min, then homogenize using ultrasound for 5 min to obtain the substrate. The slow-release enzyme is added at 1 wt% of the bacterial sludge, the reaction temperature is 25℃, and the reaction time is 18 min.

[0060] The total volume ratio of the bacterial sludge dispersion to the animal-derived pretreatment solution is 1:1.

[0061] S300, adjust the pH of the substrate to 7.5, and enzymatically hydrolyze with a complex protease (neutral protease: alkaline protease: papain = 42:20:1) at 45℃ for 2 hours. Then, raise the temperature to 50℃, add the flavor protease, and enzymatically hydrolyze for 0.5 hours. Finally, raise the temperature to 95℃ to inactivate the enzyme, and cool to obtain the enzymatic hydrolysate. The complex protease accounts for 1 wt% of the substrate; the flavor protease accounts for 1 wt% of the substrate.

[0062] The enzymatic hydrolysate was centrifuged to obtain the supernatant. The supernatant was then microfiltered through a ceramic membrane (0.22 μm) (operating pressure controlled at 0.15 MPa and material temperature controlled at 30 °C), and the permeate was collected. The permeate was then passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da (operating pressure controlled at 0.2 MPa and material temperature controlled at 30 °C), and the permeate was collected to obtain the umami peptide solution.

[0063] Umami peptides are obtained by vacuum freeze-drying the umami peptide liquid.

[0064] Example 2: An extraction process for umami peptides, characterized by comprising the following steps:

[0065] Preparation of sustained-release enzymes:

[0066] A100, a 2.5 wt% sodium alginate solution, and a 5 wt% TG enzyme solution were mixed to obtain a mixture; the volume ratio of the TG enzyme solution to the sodium alginate solution was 1:6.

[0067] A200: The mixture is added dropwise to a 0.6 mol / L calcium chloride solution with constant stirring. After the reaction, gel particles are obtained. The volume ratio of the mixture to the calcium chloride solution is 1:15. The reaction temperature is 30℃, the reaction time is 50 min, the stirring speed is 300 rpm, and the dropping speed is 45 drops / min. The inner diameter of the dropping needle is 0.5 mm.

[0068] A300: Gel particles were placed in a 1.8 wt% chitosan solution and reacted with constant stirring. The resulting slow-release enzyme was obtained by washing and drying. The mass ratio of sodium alginate to chitosan was 1:10. The pH of the chitosan solution was 5.5, the reaction temperature was 30℃, and the reaction time was 20 min.

[0069] Preparation of meat paste: Process the animal raw materials into meat paste, place the meat paste in a 55℃ constant temperature water bath and stir continuously for 1 minute, then cool to 8℃; add an equal mass of deionized water to the cooled meat paste, break it into a paste, add 0.15% of the meat paste mass of papain, stir at 35℃ for 10 minutes, add 0.1% of the meat paste mass of vitamin C, stir evenly to obtain meat paste, set aside; the animal raw materials consist of chicken and shrimp (with shells) in a mass ratio of 4:3.

[0070] Preparation of fungal mud dispersion: The fungal raw material was pulverized to obtain fungal mud; the fungal raw material consisted of Boletus edulis, Coprinus comatus, and Pleurotus ostreatus; the mass ratio of Boletus edulis, Coprinus comatus, and Pleurotus ostreatus was 2:1:1. The fungal mud was dispersed in deionized water at a mass ratio of 1:6, the pH was adjusted to 6.5, cellulase and chitosanase were added, the temperature was raised to 60℃, and the reaction was carried out for 1.5 h. The pH was then adjusted to 7.5, and sodium carboxymethyl cellulose, sodium hydrogen phosphate, and sodium dihydrogen phosphate were added to obtain the fungal mud dispersion.

[0071] Based on the volume of deionized water, the addition amount of sodium hydrogen phosphate was 22 g / L, the addition amount of sodium dihydrogen phosphate was 3.5 g / L, and the addition amount of sodium carboxymethyl cellulose was 2 wt% of the bacterial sludge.

[0072] S100, after dispersing meat paste in deionized water, added 0.4% of emulsifying stabilizer (composed of soy protein isolate and xanthan gum in a mass ratio of 3:1) by weight of meat paste, adjusted the pH to 9, added ascorbic acid, and after reaction, obtained animal-derived pretreatment solution; the mass ratio of meat paste to deionized water was 1:3; the amount of ascorbic acid added was 3wt% of meat paste, the reaction temperature was 35℃, and the reaction time was 60min;

[0073] After adjusting the pH of the animal-derived pretreatment solution to 7, add the slow-release enzyme, then add the bacterial sludge dispersion in portions. The first addition is 1 / 4 of the total volume of the bacterial sludge dispersion. When the system viscosity reaches 320 mPa·s, begin the second addition, adding 1 / 3 of the total volume of the bacterial sludge dispersion. When the system viscosity reaches 440 mPa·s, begin the third addition, adding the remaining bacterial sludge dispersion. When the system viscosity reaches 600 mPa·s, heat to 80℃ and hold for 2 min, then homogenize using ultrasound for 8 min to obtain the substrate. The slow-release enzyme is added at 1.5 wt% of the bacterial sludge, the reaction temperature is 35℃, and the reaction time is 12 min.

[0074] The total volume ratio of the bacterial sludge dispersion to the animal-derived pretreatment solution was 1:1.8.

[0075] S300, substrate pH adjusted to 7.5, enzymatic hydrolysis with complex protease (neutral protease: alkaline protease: papain = 42:20:1) at 45℃ for 3 hours, then heated to 50℃, added flavor protease, and hydrolyzed for 1 hour. Finally, heated to 95℃ to inactivate the enzyme, and cooled to obtain the enzymatic hydrolysate; complex protease accounted for 1 wt% of the substrate; flavor protease accounted for 1 wt% of the substrate.

[0076] The enzymatic hydrolysate was centrifuged to obtain the supernatant. The supernatant was then microfiltered through a ceramic membrane (0.22 μm) (operating pressure controlled at 0.15 MPa and material temperature controlled at 30 °C), and the permeate was collected. The permeate was then passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da (operating pressure controlled at 0.2 MPa and material temperature controlled at 30 °C), and the permeate was collected to obtain the umami peptide solution.

[0077] Umami peptides are obtained by vacuum freeze-drying the umami peptide liquid.

[0078] Example 3: An extraction process for umami peptides, characterized by comprising the following steps:

[0079] Preparation of sustained-release enzymes:

[0080] A100, a 2 wt% sodium alginate solution, and a 2.5 wt% TG enzyme solution were mixed to obtain a mixture; the volume ratio of the TG enzyme solution to the sodium alginate solution was 1:5.

[0081] A200: The mixture was added dropwise to a 0.45 mol / L calcium chloride solution with constant stirring. After the reaction, gel particles were obtained. The volume ratio of the mixture to the calcium chloride solution was 1:12. The reaction temperature was 28℃, the reaction time was 40 min, the stirring speed was 250 rpm, and the dropping speed was 40 drops / min. The inner diameter of the dropping needle was 0.5 mm.

[0082] A300: Gel particles were placed in a 1.7 wt% chitosan solution and reacted with constant stirring. The resulting slow-release enzyme was obtained by washing and drying. The mass ratio of sodium alginate to chitosan was 1:9. The pH of the chitosan solution was 5.5, the reaction temperature was 25℃, and the reaction time was 15 min.

[0083] Preparation of meat paste: Process the animal raw materials into meat paste, place the meat paste in a 55℃ constant temperature water bath and stir continuously for 1 minute, then cool to 8℃; add an equal mass of deionized water to the cooled meat paste, break it into a paste, add 0.15% of the meat paste mass of papain, stir at 35℃ for 10 minutes, add 0.1% of the meat paste mass of vitamin C, stir evenly to obtain meat paste, set aside; the animal raw materials consist of chicken and shrimp (with shells) in a mass ratio of 4:3.

[0084] Preparation of fungal mud dispersion: Fungal raw materials were pulverized to obtain fungal mud; the fungal raw materials consisted of Boletus edulis, Coprinus comatus, and Pleurotus ostreatus; the mass ratio of Boletus edulis, Coprinus comatus, and Pleurotus ostreatus was 2:1:1. The fungal mud was dispersed in deionized water at a mass ratio of 1:5, the pH was adjusted to 6, cellulase and chitosanase were added, the temperature was raised to 50℃, and the reaction was carried out for 1.25 h. The pH was adjusted to 7, and then sodium carboxymethyl cellulose, sodium hydrogen phosphate, and sodium dihydrogen phosphate were added to obtain the fungal mud dispersion.

[0085] Based on the volume of deionized water, the addition amount of sodium hydrogen phosphate was 21 g / L, the addition amount of sodium dihydrogen phosphate was 3.2 g / L, and the addition amount of sodium carboxymethyl cellulose was 1.5 wt% of the bacterial sludge.

[0086] S100, after dispersing meat paste in deionized water, added 0.4% of emulsifying stabilizer (composed of soy protein isolate and xanthan gum in a mass ratio of 3:1) by weight of the meat paste, adjusted the pH to 8, added ascorbic acid, and after reaction, obtained animal-derived pretreatment solution; the mass ratio of meat paste to deionized water was 1:2.5; the amount of ascorbic acid added was 2wt% of the meat paste, the reaction temperature was 30℃, and the reaction time was 45min;

[0087] After adjusting the pH of the animal-derived pretreatment solution to 6.5, add the slow-release enzyme, then add the bacterial sludge dispersion in portions. The first addition is 23% of the total volume of the bacterial sludge dispersion. When the system viscosity reaches 300 mPa·s, begin the second addition, which is 42% of the total volume of the bacterial sludge dispersion. When the system viscosity reaches 400 mPa·s, begin the third addition, adding the remaining bacterial sludge dispersion. When the system viscosity reaches 540 mPa·s, heat to 80℃ and hold for 2 minutes, then homogenize using ultrasound for 6 minutes to obtain the substrate. The slow-release enzyme is added at 1.25 wt% of the bacterial sludge, the reaction temperature is 30℃, and the reaction time is 15 minutes.

[0088] The total volume ratio of the bacterial sludge dispersion to the animal-derived pretreatment solution was 1:1.4.

[0089] S300, adjust the pH of the substrate to 7.5, and enzymatically hydrolyze with a complex protease (neutral protease: alkaline protease: papain = 42:20:1) at 45℃ for 2 hours. Then, raise the temperature to 50℃, add the flavor protease, and enzymatically hydrolyze for 1 hour. Finally, raise the temperature to 95℃ to inactivate the enzyme, and cool to obtain the enzymatic hydrolysate. The complex protease accounts for 1 wt% of the substrate; the flavor protease accounts for 1 wt% of the substrate.

[0090] The enzymatic hydrolysate was centrifuged to obtain the supernatant. The supernatant was then microfiltered through a ceramic membrane (0.22 μm) (operating pressure controlled at 0.15 MPa and material temperature controlled at 30 °C), and the permeate was collected. The permeate was then passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da (operating pressure controlled at 0.2 MPa and material temperature controlled at 30 °C), and the permeate was collected to obtain the umami peptide solution.

[0091] Umami peptides are obtained by vacuum freeze-drying the umami peptide liquid.

[0092] Comparative Example 1: The difference from Example 3 is that the slow-release enzyme was replaced with a TG enzyme slow-release agent, which includes TG enzyme, coconut fiber modified sodium alginate, gelatin and β-cyclodextrin in a mass ratio of 1:3:2:2.

[0093] Coconut fiber modified sodium alginate was prepared by the following method: Coconut shell fibers were pulverized to 100 mesh and heated to 1200℃ for 2 hours under inert gas protection to generate biochar. After cooling to room temperature, the biochar was mixed with corn stalk nanofibers at a mass ratio of 1:2. Deionized water was added to prepare a suspension with a material-to-liquid ratio of 1:40 g / mL. The suspension was stirred at 50℃ for 3 hours (stirring rate 150 r / min). After stirring, the solid product was collected by filtration, washed, and dried to obtain coconut fiber. Coconut fiber and sodium alginate were added to deionized water at a mass ratio of 1:5 (material-to-liquid ratio 1:30 g / mL). The suspension was stirred at 60℃ for 4 hours (stirring rate 200 r / min) and then ultrasonically dispersed (ultrasonic power 250 W, frequency 25 kHz, treatment time 20 min). After ultrasonic dispersion, the suspension was freeze-dried until the moisture content was ≤5% to obtain coconut fiber modified sodium alginate.

[0094] Modified sodium alginate and gelatin from coconut fiber were mixed and added to deionized water at 40°C (material-to-liquid ratio 1:50, g / mL). After stirring and dispersing evenly, β-cyclodextrin was added and stirred evenly again. After cooling to 25°C, TG enzyme was added and stirred evenly. Then, the mixture was spray-dried (material temperature around 55°C) to obtain TG enzyme sustained-release agent.

[0095] Comparative Example 2: The difference from Example 3 is that the concentration of the calcium chloride solution is 1 mol / L.

[0096] Comparative Example 3: The difference from Example 3 is that the dripping rate is 20 drops / minute.

[0097] Comparative Example 4: The difference from Example 3 is that the dripping rate is 60 drops / minute.

[0098] Comparative Example 5: The difference from Example 3 is that the mass ratio of sodium alginate to chitosan is 1:5.

[0099] Comparative Example 6: The difference from Example 3 is that the mass ratio of sodium alginate to chitosan is 1:15.

[0100] Comparative Example 7: The difference from Example 3 is that ascorbic acid was not added in step S100.

[0101] Comparative Example 8: The difference from Example 3 is that sodium hydrogen phosphate and sodium dihydrogen phosphate were not added to the bacterial sludge dispersion.

[0102] Comparative Example 9: The difference from Example 3 is that the bacterial sludge dispersion was added to the animal-derived pretreatment solution all at once.

[0103] Comparative Example 10: The difference from Example 3 is that when the system viscosity reaches 400 mPa·s, a second addition is started, and the reaction time in step S200 is increased to 24 min.

[0104] Comparative Example 11: The difference from Example 3 is that the amount of secondary addition is 70% of the total volume of the bacterial sludge dispersion.

[0105] Experiment 1: The stability of the substrates in Examples 1-3 and Comparative Examples 1-11 was tested using the following method:

[0106] The method for detecting the stability of the substrate before enzymatic hydrolysis is as follows: Take 80 mL of the substrate of each sample before enzymatic hydrolysis and place it in an open beaker. In an environment with a temperature of 25℃ and a relative humidity of 50%, let it stand for 8.5 h, 10.5 h, and 12.5 h, and observe whether there are any abnormal phenomena such as stratification or precipitation in the test tube.

[0107] The method for detecting the stability of substrates after enzymatic hydrolysis is as follows: take equal amounts of the substrates from Examples 1-3 and Comparative Examples 1-11 in a beaker, and observe the condition of the material in the beaker after enzymatic hydrolysis according to the corresponding enzymatic hydrolysis method.

[0108] The test results are shown in Table 1. The degree of stratification, from low to high, is: no stratification, slight stratification, and obvious stratification; the degree of sedimentation, from low to high, is: no sedimentation, a small amount of sedimentation, and a large amount of sedimentation.

[0109] Table 1

[0110]

[0111] A comparison of the experimental results of Comparative Example 1 and Example 3 shows that when using the TG sustained-release agent from the patent application number 202610129636.9, the substrate is difficult to maintain stability for a long time. During enzymatic hydrolysis, precipitation and slight stratification are easily formed. The reason may be that there is insufficient cross-linking between heterologous proteins.

[0112] A comparison of the experimental results of Comparative Examples 2-6 and Example 3 shows that the parameters in the slow-release enzyme preparation process affect the stability of the substrate and the enzymatic hydrolysis system. This may be because a mismatch between the slow-release rate and the addition process of the bacterial sludge dispersion leads to uneven cross-linking reaction and an excessively low degree of cross-linking.

[0113] A comparison of the experimental results of Comparative Example 7 and Example 3 shows that when ascorbic acid is not added in step S100, the cross-linking time provided by the present invention is too short, making it difficult to maintain the stability of the substrate and the stability of the enzymatic hydrolysis system.

[0114] A comparison of the experimental results of Comparative Example 8 and Example 3 shows that when the release rate of TG enzyme is not adjusted by sodium ion concentration, the substrate and the enzymatic hydrolysis system, as in Comparative Example 1, are difficult to maintain uniformity and stability.

[0115] A comparison of the experimental results of Comparative Example 9 and Example 3 shows that after the entire bacterial sludge dispersion was added to the animal-derived pretreatment solution at once, it was difficult to maintain uniformity and stability in both the substrate and the enzymatic hydrolysis system. The applicant hypothesizes that this is because, with the increased cross-linking tendency between bacterial proteins, heterologous proteins cannot fully undergo cross-linking at a relatively short time.

[0116] A comparison of the experimental results of Comparative Example 10 and Example 3 shows that when the timing of the addition of the bacterial sludge dispersion did not match the release rate of the TG enzyme, the system viscosity was too high, the degree of cross-linking was too large, and the steric hindrance was too large during the second addition, thus increasing the overall cross-linking reaction time. The stability of the substrate and the enzymatic hydrolysis system decreased slightly. The applicant hypothesizes that this is because the uniformity of cross-linking was affected to some extent, for example, the concentration of TG enzyme near the slow-release enzyme was relatively high.

[0117] A comparison of the experimental results of Comparative Example 11 and Example 3 shows that when the amount of bacterial sludge dispersion added is not matched with the TG enzyme release rate, it is difficult to maintain uniformity and stability in both the substrate and the enzymatic hydrolysis system. The applicant hypothesizes that the reason is that adding too much bacterial sludge at once increases the cross-linking tendency between bacterial proteins.

[0118] Experiment 2: After obtaining umami peptides according to the extraction process provided in Examples 1-3, the umami peptides and maltodextrin were mixed evenly in a mixer at a mass ratio of 100:65 to obtain polypeptide umami seasoning. Then, the umami intensity, safety, and water solubility of the product and the blank example (commercially available monosodium glutamate) were tested. The test method is described in the patent application number 202610129636.9. The test results are shown in Table 2.

[0119] Table 2

[0120]

[0121] The experimental results show that, using the extraction process provided by this invention, the product still retains its good umami flavor even with a significantly shortened processing time.

[0122] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A process for extracting umami peptides, characterized in that, Includes the following steps: S100 and meat paste are dispersed in deionized water, and the pH value is adjusted to 7-9. Ascorbic acid is added, and after reaction, animal-derived pretreatment solution is obtained; the mass ratio of meat paste to deionized water is 1:(2-3). Preparation process of meat paste: The animal raw materials are processed into meat paste. The meat paste is placed in a 55℃ constant temperature water bath and stirred continuously for 1-1.5 minutes, and then cooled to below 10℃. An equal mass of deionized water is added to the cooled meat paste, and it is crushed into a paste. Papain is added, and it is stirred at a constant temperature of 35℃ for 10-15 minutes. Vitamin C is added and stirred evenly to obtain meat paste. The animal raw materials consist of chicken and shrimp in a mass ratio of 4:

3. S200. After adjusting the pH of the animal-derived pretreatment solution to 6-7, add the slow-release enzyme and gradually add the bacterial sludge dispersion. After the reaction, the substrate is obtained through a homogenization process. The bacterial sludge is dispersed in a sodium salt buffer system. The total volume ratio of the bacterial sludge dispersion to the animal-derived pretreatment solution is 1:(1-1.8). The bacterial sludge dispersion was added to the animal-derived pretreatment solution in several batches. The first addition was 1 / 5 to 1 / 4 of the total volume of the bacterial sludge dispersion. When the system viscosity reached 280-320 mPa·s, the second addition was started, with the amount added being 1 / 3 to 3 / 5 of the total volume of the bacterial sludge dispersion. When the system viscosity reached 360-440 mPa·s, the third addition was started. The remaining bacterial sludge dispersion was added after the third addition. When the system viscosity reached 480-600 mPa·s, the mixture was homogenized to obtain the substrate. The preparation method of the fungal mud dispersion includes: pulverizing fungal raw materials to obtain fungal mud; dispersing the fungal mud in deionized water at a mass ratio of 1:(4-6), adjusting the pH value to 5-6.5, adding cellulase and chitosanase, heating to 40-60℃, reacting for 1-1.5h, adjusting the pH value to 6.5-7.5, then adding sodium carboxymethyl cellulose, sodium hydrogen phosphate and sodium dihydrogen phosphate, and finally obtaining the fungal mud dispersion; the fungal raw materials are composed of *Helicobacter pylori*, *Coprinus comatus* and *Agaricus bisporus* in a mass ratio of 2:1:

1. Based on the volume of deionized water, the addition amount of sodium hydrogen phosphate is 20-22 g / L, and the addition amount of sodium dihydrogen phosphate is 3-3.5 g / L; the addition amount of sodium carboxymethyl cellulose is 1 wt%-2 wt% of the bacterial sludge. S300 and substrate were successively hydrolyzed by complex protease and flavor protease, followed by enzyme inactivation, purification, impurity removal, and drying to obtain umami peptides; the total hydrolysis time was 2.5-4 hours; the complex protease consisted of neutral protease, alkaline protease, and papain in a mass ratio of 42:20:

1. The method for preparing the sustained-release enzyme includes the following steps: A100, sodium alginate solution and TG enzyme solution are mixed to obtain a mixture; A200. The mixture is added dropwise to a continuously stirred calcium chloride solution. After the reaction, gel particles are obtained. A300: After placing the gel particles in the chitosan solution, react with constant stirring, and then obtain the slow-release enzyme by washing and drying. The concentration of sodium alginate solution is 1.8wt%-2.5wt%, and the concentration of TG enzyme solution is 1wt%-5wt%; the volume ratio of TG enzyme solution to sodium alginate solution is 1:(4-6); the concentration of calcium chloride solution is 0.3-0.6mol / L, and the volume ratio of the mixed solution to calcium chloride solution is 1:(7-15); the concentration of chitosan solution is 1.5wt%-1.8wt%, and the mass ratio of sodium alginate to chitosan is 1:(8-10). In step A200, the reaction temperature is 25-30℃, the reaction time is 35-50 min, the stirring speed is 200-300 rpm, and the dropping speed is 30-45 drops / min.

2. The extraction process according to claim 1, characterized in that, In step A300, the pH of the chitosan solution is 5.0-5.5, the reaction temperature is 20-30℃, and the reaction time is 10-20 min.

3. The extraction process according to claim 1, characterized in that, In step S100, the amount of ascorbic acid added is 1wt%-3wt% of the meat paste, the reaction temperature is 25-35℃, and the reaction time is 30-60min. In step S200, the amount of slow-release enzyme added is 1wt%-1.5wt% of the bacterial sludge, the reaction temperature is 25-35℃, and the reaction time is 12-18min.

4. The extraction process according to claim 1, characterized in that, The homogenization process is ultrasonic homogenization. The ultrasonic homogenization process is performed at a temperature of 20-30℃, a power of 250-300W, a frequency of 15-20kHz, and a time of 5-8min.

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