Enzymatic hydrolysis of egg white and preparation method thereof

Abstract

This invention discloses an enzymatic hydrolysis method for vinegar-egg liquid and its preparation, belonging to the field of bio-enzyme catalysis technology. The method uses acidic protease, cellulase and pectinase as hydrolysis catalysts, and includes the following steps: (1) Quail eggs are mixed with vinegar and acidic protease is added for simultaneous enzymatic hydrolysis to obtain quail egg hydrolysate; (2) Eggs are first soaked in vinegar to dissolve the shell, remove the membrane, and break them up to obtain crude vinegar-egg liquid; then, jujube and cordyceps extract is prepared; the crude vinegar-egg liquid is mixed with the extract, calcium ions are added, acidic protease is added, and the components are synergistically hydrolyzed to obtain a compound egg hydrolysate; (3) Chinese medicines such as wolfberry are extracted by enzymatic hydrolysis and heat extraction to obtain Chinese medicine extract; (4) Quail egg hydrolysate and compound egg hydrolysate are mixed, and pre-dissolved honey water, Chinese medicine extract and potassium sorbate are added for preparation; (5) After ultra-high temperature instantaneous sterilization, homogenization is performed. The product of this invention has a stable shelf life without stratification, good taste, short production cycle and is suitable for industrial continuous production.

Description

Technical Field

[0001] This invention belongs to the field of bio-enzyme catalysis technology, specifically relating to the preparation of vinegar-egg liquid containing small molecule active peptides and active ingredients derived from medicinal and edible plants by enzymatic hydrolysis, and the preparation method thereof. Background Technology

[0002] Enzymatic hydrolysis of natural plant and animal substrates to prepare highly active small-molecule functional components is a core research direction and mainstream technology in the field of biomanufacturing and functional foods. Among these, the use of enzymatic hydrolysis to specifically cleave protein peptide bonds, efficiently hydrolyzing large-molecule poultry egg protein into easily absorbed small-molecule active peptides, has been widely applied to the large-scale preparation of food-grade protein peptides. Cellulase and pectinase can specifically hydrolyze the cellulose and pectin skeleton structures of plant cell walls, breaking down the cell wall mass transfer barrier and significantly increasing the dissolution rate of intracellular active polysaccharides, flavonoids, saponins, and other functional components. This is a key technology for the efficient extraction of active ingredients from food-medicine homologous raw materials.

[0003] Vinegar-egg liquid is a traditional Chinese food and medicine product with a long history. It is made from poultry eggs and brewed vinegar as core ingredients, combined with medicinal and edible auxiliary ingredients such as jujubes, cordyceps, and goji berries. Its core functional components are small molecule peptides from poultry eggs, plant-derived active polysaccharides, organic acids, and flavonoids, possessing both nutritional and therapeutic value. With the rapid development of the health industry, vinegar-egg liquid products have shifted from homemade to standardized, industrialized production, becoming an important sub-category.

[0004] Currently, the mainstream industrial production process of vinegar-egg liquid beverages in the industry involves the following core steps: chicken eggs and quail eggs are soaked in brewed vinegar to dissolve their shells and then filtered to obtain egg-soaking liquid. Medicinal and edible raw materials such as jujubes, goji berries, and cordyceps are extracted and concentrated by long-term boiling under high pressure. The two are then mixed with honey and other auxiliary materials, sterilized, filtered, and bottled to obtain the finished product. This process has enabled the standardized production of vinegar-egg liquid beverages, solving the problem of unstable quality in homemade products. However, it completely lacks enzymatic hydrolysis technology, resulting in fundamental technical defects: First, relying solely on the weak acidity of acetic acid to decompose proteins leads to extremely low protein hydrolysis efficiency. The products are mainly large-molecule proteins, making it impossible to directionally prepare highly active small-molecule peptides, resulting in poor absorption and utilization by the human body. Furthermore, the incompletely decomposed egg white protein brings a strong sour and astringent taste and an eggy smell, requiring the addition of large amounts of sweeteners to mask the odor, thus destroying the product's original flavor and nutritional structure. Second, the medicinal and edible raw materials rely solely on hot extraction to release components, without effectively destroying the plant cell walls. This results in low dissolution rates of core functional components such as active polysaccharides and flavonoids, leading to significant waste of raw materials. Moreover, prolonged high-temperature boiling causes the degradation and inactivation of a large number of heat-sensitive active ingredients, significantly weakening the product's functionality.

[0005] To address the aforementioned issues, a few studies have attempted to incorporate enzymatic hydrolysis technology into the preparation process of vinegar-egg liquid. For example, existing patents disclose an enzymatic preparation method for vinegar-egg liquid, which involves soaking egg liquid in vinegar and then adding protease for enzymatic hydrolysis; other technologies disclose a composite enzymatic hydrolysis process for vinegar-egg liquid, using neutral protease and flavor enzymes to hydrolyze the egg liquid. However, these technologies still have many insurmountable drawbacks: First, existing technologies generally employ a step-by-step process of "acid hydrolysis followed by enzymatic hydrolysis," resulting in an unreasonable design of the enzymatic hydrolysis reaction system and an inability to achieve synergistic effects between acid hydrolysis and enzymatic hydrolysis. Furthermore, the enzyme catalytic reaction efficiency is low, leading to insufficient protein hydrolysis.

[0006] Secondly, the enzymatic extraction process for medicinal and edible raw materials often employs high-temperature, long-duration boiling extraction, which makes it difficult to achieve efficient release of active ingredients.

[0007] Third, the system stability of enzymatic hydrolysis products in existing technologies is poor. Small molecule peptides and active polysaccharides obtained by enzymatic hydrolysis are prone to molecular aggregation and sedimentation during storage, and the products are very prone to stratification and precipitation.

[0008] Therefore, developing a method for preparing vinegar-egg liquid beverages that is highly efficient, yields sufficient active ingredients, has good product stability, and is suitable for continuous industrial production has become a pressing technical problem to be solved in this field. Summary of the Invention

[0009] To address the aforementioned technical problems in the prior art, this invention aims to provide an enzymatically hydrolyzed vinegar-egg liquid and its preparation method that features high production efficiency, sufficient extraction of active ingredients, good product stability, and suitability for continuous industrial production.

[0010] One of the objectives of this invention is to provide a method for preparing enzymatically hydrolyzed vinegar egg liquid, wherein the preparation method uses acidic protease, cellulase and pectinase as hydrolysis catalysts, and prepares a composition containing small molecule active peptides from poultry eggs and active polysaccharides from plants through multi-step enzymatic hydrolysis. The preparation method includes the following steps: (1) Preparation of quail egg enzymatic hydrolysate: Mix quail eggs with brewed vinegar, control the pH of the system to 3.0~3.5, heat to 40~45℃, add acidic protease for simultaneous enzymatic hydrolysis, after enzymatic hydrolysis, stir, break, and filter to obtain quail egg hydrolysate, and quickly cool to below 10℃ for storage. (2) Preparation of egg enzymatic hydrolysate: a. Mix eggs with brewed vinegar, soak them to dissolve the eggshells, remove the egg membranes, and beat them to obtain coarse vinegar-egg liquid; b. Crush the jujubes and Cordyceps militaris, add water, adjust the pH to 4.5-5.5, keep the temperature at 50-55℃, add pectinase and cellulase for enzymatic hydrolysis, centrifuge and take the supernatant to obtain jujube and Cordyceps militaris extract. c. Mix the crude vinegar and egg liquid from step a with the jujube and cordyceps extract from step b, add calcium lactate to make the free calcium ion concentration in the system reach 50~60 mmol / L, adjust the pH to 3.0~3.5, the temperature to 40~45℃, add acidic protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, obtain the compound egg hydrolysate, and quickly cool it to below 10℃ for storage. (3) Preparation of compound extracts of traditional Chinese medicine: Grind and mix wolfberry, longan, mulberry and polygonatum, add water, adjust the pH to 4.5~5.5, temperature 50~55℃, add pectinase and cellulase for enzymatic hydrolysis, then heat to 90~95℃ for extraction, filter to obtain Chinese herbal compound extract; (4) Preparation: The quail egg enzymatic hydrolysate from step (1) is mixed with the compound chicken egg enzymatic hydrolysate from step (2) to obtain a mixed enzymatic hydrolysate. Dissolve honey in water and cool it to below 15°C to obtain honey water; First, add honey water to the mixed enzymatic hydrolysate and stir well. Then add the Chinese herbal compound extract and potassium sorbate from step (3) and stir well. (5) Sterilization and homogenization: The prepared liquid is subjected to ultra-high temperature instantaneous sterilization, which simultaneously deactivates the enzymes; then it is homogenized to obtain enzymatically hydrolyzed vinegar-egg liquid.

[0011] Preferably, in step (1), the mass ratio of quail eggs to brewed vinegar is 1:0.5~1.5, the final concentration of acidic protease in the reaction system is 1000~3000U / mL, and the enzymatic hydrolysis time is 4~6h.

[0012] Preferably, in step (1), during the synchronous enzymatic hydrolysis process, the mixture is stirred for 5 minutes every 30 minutes. The vinegar and protease in the system are evenly distributed by mechanical stirring, and the bubbles generated during the eggshell dissolution process are broken to prevent the bubbles from adhering to the egg membrane surface and forming an air film barrier, thus ensuring mass transfer efficiency.

[0013] Preferably, in step (2)a, the mass ratio of the egg to the brewed vinegar is 1:2~3, and the soaking time is 11~12h.

[0014] Preferably, in step (2)b, the mass ratio of jujube to Cordyceps militaris is 10:1, the amount of pectinase and cellulase added is 0.1%~0.3% of the substrate mass, the enzymatic hydrolysis time is 1~1.5h, the centrifugation conditions are 4000rpm and 10min, and the soluble solids content of the jujube and Cordyceps militaris extract is ≥3.0%. The enzyme activity of the cellulase is ≥10000U / g, and the enzyme activity of the pectinase is ≥30000U / g.

[0015] Preferably, in step (2)c, the final concentration of acidic protease in the reaction system is 1000~3000U / mL, and the enzymatic hydrolysis time is 6~8 hours.

[0016] Preferably, in step (3), the amount of pectinase and cellulase added is 0.1~0.2% of the substrate mass, the enzymatic hydrolysis time is 1h, and the heat extraction time is 1h.

[0017] Preferably, in step (5), the sterilization conditions are 132±2℃ for 4~6s and the discharge temperature is ≤60℃; The homogenization process involves first-stage and second-stage homogenization. The pressure for first-stage homogenization is 15-20 MPa, and the pressure for second-stage homogenization is 5-10 MPa. The particle size D90 of the homogenized material is ≤5 μm.

[0018] Preferably, the raw materials used in the preparation method include, by weight: 40-60 parts of eggs, 0.1-0.5 parts of quail eggs, 80-150 parts of brewed vinegar, 100-200 parts of honey, 1-3 parts of jujubes, 0.2-0.6 parts of wolfberries, 0.2-0.5 parts of longan, 0.1-0.3 parts of cordyceps militaris, 0.05-0.15 parts of mulberries, 0.05-0.15 parts of polygonatum, 0.05-0.1 parts of potassium sorbate, and 50-80 parts of water; The total acidity of the brewed vinegar is ≥5.0g / 100mL, and the pH value is 2.8~3.8.

[0019] The second objective of this invention is to provide an enzymatically hydrolyzed vinegar-egg liquid prepared by the method described above.

[0020] The beneficial effects of this invention include: (1) The simultaneous acid hydrolysis-enzymatic hydrolysis process for quail eggs achieves high efficiency and synergy. This invention addresses the structural differences between quail eggs and chicken eggs by employing a differentiated enzymatic hydrolysis strategy: quail eggs undergo simultaneous acid hydrolysis and enzymatic hydrolysis, while chicken eggs undergo a step-by-step process of first acid hydrolysis to dissolve the shell and remove the membrane, followed by a second enzymatic hydrolysis. Quail eggs have thin shells, high porosity, and loose membranes, allowing acetic acid and acidic proteases to work synergistically in the same system: acetic acid rapidly dissolves and releases calcium ions, while simultaneously causing acid denaturation and membrane rupture of the egg membrane collagen. The simultaneous process shortens the total reaction time to 4-6 hours, more than 60% shorter than the traditional step-by-step process (shell dissolution 11-12 hours + enzymatic hydrolysis 6-8 hours), achieving a degree of hydrolysis of 16%-20%, and a proportion of small molecule active peptides (<1000 Da) ≥60%.

[0021] (2) Precise activation of calcium ions and synergistic enhancement of the stability of the enzymatic hydrolysis system This invention utilizes the natural calcium source released from eggshell acid hydrolysis, combined with the synergistic supplementation of added calcium lactate, to precisely control the free calcium ion concentration at 50-60 mmol / L in an egg enzymatic hydrolysis system. At this concentration, calcium ions not only act as activators for acidic proteases, enhancing hydrolysis efficiency, but also stabilize the enzyme's spatial conformation by forming ionic bonds, preventing enzyme inactivation during the reaction and ensuring a highly efficient, stable, and controllable hydrolysis process.

[0022] (3) Jujube, Cordyceps militaris and eggs achieve multidimensional synergistic effects through synergistic enzymatic hydrolysis. This invention utilizes jujube and cordyceps militaris extracts with crude vinegar and egg liquid in the same acidic protease system for synergistic enzymatic hydrolysis, achieving the following multidimensional synergistic effects: Substrate complementarity enhances enzymatic hydrolysis efficiency: Egg protein and plant protein (jujube, cordyceps) act as "substrate partners" for each other, which helps to break the product feedback inhibition of single substrate enzymatic hydrolysis and improve enzymatic hydrolysis efficiency. Synergistic protection of calcium ions and plant polyphenols: Calcium ions undergo a complexation reaction with the natural polyphenols contained in jujube and cordyceps militaris in the system, which can prevent polyphenols from oxidizing and precipitating. At the same time, the antioxidant function of polyphenols can protect proteases and enzymatic hydrolysis products from being oxidized and inactivated. Glycopeptide complex formation: Egg protein peptides, jujube polysaccharides, and cordyceps polysaccharides form glycopeptide complexes with higher bioavailability through hydrogen bonds and hydrophobic interactions during enzymatic hydrolysis, achieving dual targeting effects of immune regulation and cell repair. Flavor modification and bitterness masking: The reducing sugars of jujube and the amino acids produced by enzymatic hydrolysis accumulate Maillard reaction precursors, which are then transformed into natural caramel aroma after ultra-high temperature instantaneous sterilization (UHT); Cordyceps militaris nucleotides provide umami flavor and mask the eggy smell; plant-derived hydrophilic amino acids and polysaccharides encapsulate bitter peptides through hydrophobic interactions, reducing the bitterness threshold by more than 60%. Synergistic effect of interfacial activity: Jujube and Cordyceps militaris extracts contain natural surfactants (such as jujube saponins and Cordyceps militaris polysaccharides), which can reduce the surface tension of the mixture of acetic acid and protease, improve its wetting and contact efficiency with egg white and egg yolk protein, make the enzymatic reaction interface more complete, and improve the uniformity of hydrolysis. Detailed Implementation

[0023] The following description includes certain specific details to provide a comprehensive understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments can be implemented without employing one or more of these specific details, but using other methods, components, materials, etc.

[0024] Unless otherwise required by the present invention, throughout the specification and the following claims, the words “comprising” and “including” shall be interpreted in an open-ended, inclusive sense, meaning “including but not limited to”.

[0025] Throughout this specification, the terms "an embodiment," "an embodiment," "a preferred embodiment," or "some embodiments" refer to including, in at least one embodiment, a specific reference element, structure, or feature associated with that embodiment. Therefore, the phrases "in an embodiment," "in a preferred embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0026] According to a first aspect of the present invention, a method for preparing enzymatically hydrolyzed vinegar egg liquid is provided, wherein the preparation method uses acidic protease, cellulase and pectinase as hydrolysis catalysts to prepare a composition containing small molecule active peptides from poultry eggs and active polysaccharides from plants through multi-step enzymatic hydrolysis; The preparation method includes the following steps: (1) Preparation of quail egg enzymatic hydrolysate: Mix quail eggs with brewed vinegar, control the pH of the system to 3.0~3.5, heat to 40~45℃, add acidic protease for simultaneous enzymatic hydrolysis, after enzymatic hydrolysis, stir, break up and filter to obtain quail egg hydrolysate, and quickly cool to below 10℃ for storage.

[0027] In this invention, the quail eggshell is only 0.12-0.15 mm thick, with loosely arranged calcium carbonate crystals and a porosity much higher than that of a chicken egg; the total thickness of the egg membrane is about 1 / 3 that of a chicken egg membrane, and the density of interwoven collagen fibers is low. Based on the above structural characteristics: Acetic acid first reacts with the calcium carbonate in the eggshell, rapidly dissolving the shell and releasing calcium ions. Subsequently, acetic acid causes acid denaturation of the egg membrane collagen, unfolding the triple helix structure and expanding the fibrous pores to the micrometer level. At the same time, acetic acid further acts on the egg white and yolk proteins, causing their spherical structures to unfold and fully expose the internal peptide bonds.

[0028] Based on this, acidic protease molecules can freely pass through the micron-sized pores formed after the egg membrane undergoes acid denaturation, and are evenly distributed in the egg white and yolk, specifically hydrolyzing and denatured avian egg proteins.

[0029] Acetic acid and acidic protease work synergistically, reducing the total reaction time to only 4-6 hours, which is more than 60% shorter than the traditional stepwise process of first acid hydrolysis with acetic acid and then enzymatic hydrolysis.

[0030] After enzymatic hydrolysis, the solution is rapidly cooled to below 10°C for storage. This low temperature inhibits the growth of microorganisms in the system and terminates the residual catalytic activity of proteases, preventing the over-hydrolysis from producing bitter peptides and ensuring the stability of the flavor and quality of the quail egg hydrolysate. The storage time is preferably no more than 24 hours to maximize the preservation of the bioactivity of small molecule active peptides.

[0031] (2) Preparation of egg enzymatic hydrolysate: a. Mix eggs with brewed vinegar, soak them to dissolve the eggshells, remove the egg membranes, and beat them to obtain coarse vinegar-egg liquid; In this invention, the eggshell is relatively thick (0.3~0.35mm), has low porosity, and a dense membrane. If a simultaneous enzymatic hydrolysis process is used, acetic acid and acidic protease will have difficulty penetrating the membrane in a short time. Therefore, a step-by-step process of "first acid hydrolysis to dissolve the shell, then secondary enzymatic hydrolysis" is adopted for the eggs: first, the eggshell is completely dissolved by soaking in acetic acid to generate calcium acetate; after removing the membrane, the egg liquid is broken up to remove obstacles for subsequent enzymatic hydrolysis.

[0032] b. Crush the jujubes and Cordyceps militaris, add water, adjust the pH to 4.5-5.5, keep the temperature at 50-55℃, add pectinase and cellulase for enzymatic hydrolysis, centrifuge and take the supernatant to obtain jujube and Cordyceps militaris extract. In this invention, the cell walls of jujube and Cordyceps militaris are mainly composed of cellulose and pectin. Pectinase and cellulase work synergistically at pH 4.5-5.5 and temperature 50-55℃ to hydrolyze the pectin backbone and cellulose microfibrils, causing the cell wall to disintegrate and fully releasing intracellular active polysaccharides and proteins.

[0033] c. Mix the crude vinegar-egg liquid from step a with the jujube and cordyceps extract from step b, add calcium lactate to make the free calcium ion concentration in the system reach 50-60 mmol / L, adjust the pH to 3.0-3.5, the temperature to 40-45℃, add acidic protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, obtain the compound egg hydrolysate, and quickly cool it to below 10℃ for storage.

[0034] In this invention, jujube and cordyceps extracts pre-extracted with pectinase and cellulase are synergistically enzymatically hydrolyzed with crude vinegar and egg liquid in the same acidic protease system. This design is not a simple mixing of raw materials, but is based on the following multi-dimensional synergistic mechanism: (i) Synergy between substrate complementarity and enzymatic hydrolysis efficiency Substrate diversity helps to overcome feedback inhibition: Egg proteins (such as ovalbumin and vitellin) and plant proteins (jujube and cordyceps militaris) differ significantly in amino acid sequence and spatial structure. In the same acidic protease system, the two types of proteins complement and synergize. When the enzymatic products of one protein accumulate to a level that may inhibit enzyme activity, the enzyme will switch to efficiently hydrolyzing the other protein, thereby maintaining the overall reaction rate. This allows the enzyme to act alternately between the two types of substrates, breaking the product feedback inhibition that easily occurs when hydrolyzing a single substrate, and improving the efficiency of mixed substrate hydrolysis.

[0035] Complementary amino acid profiles: Egg protein is rich in sulfur-containing amino acids (methionine, cysteine), while plant proteins (jujube, cordyceps) are rich in hydrophobic amino acids (leucine, phenylalanine) and hydrophilic amino acids (glycine, alanine). The peptide profiles produced during enzymatic hydrolysis can complement each other, giving the final product multiple functional activities, and the hydrophilic amino acids have a natural masking effect on the formation of subsequent bitter peptides.

[0036] (II) Synergistic protection of calcium ions and plant polyphenols Activation and stabilizing effects of calcium ions: Free calcium ions at a concentration of 50-60 mmol / L exhibit strong activation of acidic proteases. Calcium ions bind to specific structural domains of the enzyme molecule, inducing a conformational shift of the enzyme's active site towards a catalytically favorable direction, thus increasing enzymatic hydrolysis efficiency. Simultaneously, calcium ions act as structural stabilizers, enhancing internal interactions within the enzyme molecule through ionic bonding and preventing enzyme inactivation due to heat or shearing during the reaction. The calcium ions in the system originate from calcium acetate generated during eggshell acid hydrolysis and are supplemented by added calcium lactate; both work synergistically to achieve the optimal calcium ion concentration range.

[0037] Antioxidant and complexation protection of plant polyphenols: Natural polyphenols (such as jujube cyclic adenosine monophosphate and cordycepin precursors) contained in jujubes and cordyceps militaris are easily oxidized under acidic conditions. Calcium ions can undergo complexation reactions with these polyphenols to form stable, soluble complexes, preventing polyphenol oxidation and precipitation. Simultaneously, the antioxidant properties of polyphenols can scavenge free radicals that may be generated in the system, protecting proteases and already formed small peptides from oxidative inactivation, maintaining the chemical stability of the entire reaction system, and prolonging the effective reaction period of enzymatic hydrolysis.

[0038] (iii) Formation of glycopeptide complexes of animal-derived peptides and plant-derived polysaccharides In-situ complexation: During acidic protease hydrolysis, small peptides generated from egg protein combine with active polysaccharides (jujube polysaccharide and cordyceps polysaccharide) released from jujube and cordyceps militaris to form glycopeptide complexes through hydrogen bonds, hydrophobic interactions, or covalent bonds (enzymatic transglycosylation). The molecular weight of this complex falls between that of a simple peptide and a simple polysaccharide, retaining the rapid absorption characteristics of peptides while imparting the sustained-release function of polysaccharides, resulting in high bioavailability.

[0039] Synergistic Functions: Egg protein peptides primarily function in cell repair and immune regulation; jujube polysaccharides have blood-tonifying and calming effects; and cordyceps militaris polysaccharides have anti-inflammatory and anti-fatigue effects. When these three components form a glycopeptide complex, they can achieve a dual-targeting effect on both immune regulation and cell repair.

[0040] (iv) Synergistic modification of flavor and bitterness masking Accumulation of Maillard reaction precursors: The reducing sugars (glucose, fructose) in jujubes undergo a mild Maillard reaction with the amino acids (especially lysine and arginine) produced by the enzymatic hydrolysis of eggs, generating a large number of flavor precursors. During subsequent UHT sterilization (132±2℃, 4~6s), these precursors can be rapidly converted into pyrazine and furan aromatic compounds with roasted and caramel aromas, giving the product a natural and pleasant fragrance without the need for additional flavorings.

[0041] Umami Enhancement and Odor Masking: Cordyceps militaris is rich in nucleotides (cordycepin, adenosine, etc.), which are partially released during enzymatic hydrolysis, resulting in a significant umami enhancement effect. It can effectively mask the "eggy smell" and "vinegar pungent smell" of traditional vinegar-egg liquid.

[0042] Natural inclusion complex of bitter peptides: Hydrophilic amino acids (glycine, alanine, serine) and active polysaccharides produced by the enzymatic hydrolysis of jujube and cordyceps can form physical inclusion complexes with bitter peptides (rich in leucine and phenylalanine) that may be produced by excessive hydrolysis of egg protein through hydrophobic interactions and hydrogen bonds. This shields the bitter groups from contacting taste bud receptors, which helps the vinegar-egg liquid maintain a good taste.

[0043] (v) Synergistic effect of interfacial activity Jujube and Cordyceps militaris extracts contain natural surfactants (such as jujube saponins and Cordyceps militaris polysaccharides), which can reduce the surface tension of the acetic acid and protease mixture, improve its wetting and contact efficiency with egg white and egg yolk proteins, make the enzymatic hydrolysis reaction interface more complete, and improve the uniformity of hydrolysis.

[0044] (vi) Coordinated adaptation of pH and temperature The optimal pH for acidic proteases is 3.0–3.5, and the optimal temperature is 40–45℃. A mixture of crude vinegar and egg solution (pH approximately 3.2–3.8) and jujube and cordyceps extract (pH approximately 4.5–5.5), finely adjusted with calcium lactate and a pH adjuster, can be precisely controlled to a pH of 3.0–3.5. This pH range simultaneously satisfies the requirements for acidic protease activity, free calcium ion concentration, and polyphenol stability. A temperature of 40–45℃ balances enzyme activity, mass transfer rate, and microbial inhibition.

[0045] (3) Preparation of compound extracts of traditional Chinese medicine: Grind and mix wolfberry, longan, mulberry and polygonatum, add water, adjust the pH to 4.5-5.5, and set the temperature to 50-55℃. Add pectinase and cellulase for enzymatic hydrolysis, then heat to 90-95℃ for extraction, filter, and obtain the compound extract of traditional Chinese medicine.

[0046] In this invention, step (3) takes into account the characteristics of raw materials such as wolfberry, longan, mulberry and polygonatum, and adopts a process of enzymatic hydrolysis and cell wall breaking assistance + short-time mild hot extraction. First, the plant cell wall is fully broken by the compound enzymatic hydrolysis of cellulase and pectinase, and then the active ingredients are fully dissolved by short-time high-temperature extraction, which greatly shortens the hot extraction time, avoids the degradation and inactivation of heat-sensitive active ingredients, and improves the utilization rate of raw materials.

[0047] After the hot extraction is completed, the extract is filtered through an 80-mesh sieve to remove the filter residue, resulting in a clear Chinese herbal compound extract that can be directly used in subsequent preparation processes without additional concentration. This avoids further degradation of active ingredients during concentration, simplifies the process, and improves production efficiency.

[0048] (4) Preparation: The quail egg enzymatic hydrolysate from step (1) is mixed with the compound chicken egg enzymatic hydrolysate from step (2) to obtain a mixed enzymatic hydrolysate. Dissolve honey in water and cool it to below 15°C to obtain honey water; First, add honey water to the mixed enzymatic hydrolysate and stir well. Then add the Chinese herbal compound extract and potassium sorbate from step (3) and stir well.

[0049] In this invention, in step (4), the mixed enzymatic hydrolysate is stored at a low temperature below 10°C before preparation to inhibit the activity of residual acidic protease. The honey is pretreated by "heating up to dissolve and then cooling down": the amount of water used to dissolve the honey is 0.3 to 0.5 times the mass of the honey, and the honey is dissolved in water at 50 to 60°C. At this temperature, the honey has low viscosity and dissolves quickly. After dissolving, it is immediately cooled to below 15°C and then added to the mixed enzymatic hydrolysate. This operation ensures that the honey is fully dissolved and avoids the reactivation of residual enzyme activity caused by heating the main material. In this invention, the two egg-source enzymatic hydrolysates are premixed to form a homogeneous composite egg-source enzymatic hydrolysate system. Then, honey is added and stirred to dissolve. The sweetness of the honey neutralizes the slight acidity of the system, and the oligosaccharides in the honey can further improve the stability of the system.

[0050] Throughout the entire preparation process, the system temperature should be kept ≤15℃ to ensure that no enzymatic reaction occurs before UHT sterilization.

[0051] In a more preferred embodiment, the mixing process is carried out in a sealed sanitary mixing tank, and the stirring speed is controlled at 40~60 rpm to avoid the generation of a large number of bubbles by high-speed stirring, which may lead to the filling volume deviation in the subsequent filling process. After the mixing is completed, the density of the liquid is tested to be 1.10~1.25 g / mL, pH≥3.5, and there is no odor. After passing the test, it enters the subsequent sterilization and homogenization process.

[0052] (5) Sterilization and homogenization: The prepared liquid is subjected to ultra-high temperature instantaneous sterilization, which simultaneously deactivates the enzymes; then it is homogenized to obtain enzymatically hydrolyzed vinegar-egg liquid.

[0053] In this invention, Maillard reaction precursors accumulated in the early stage of ultra-high temperature instantaneous sterilization can be rapidly converted into trace amounts of aromatic compounds such as pyrazine and furan, giving the product a light caramel and roasted aroma.

[0054] In a preferred embodiment of the present invention, in step (1), the mass ratio of the quail egg to the brewed vinegar is 1:0.5~1.5, the final concentration of the acidic protease in the reaction system is 1000~3000U / mL, and the enzymatic hydrolysis time is 4~6h.

[0055] In this invention, the mass ratio of quail eggs to brewed vinegar is, for example, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5, preferably 1:1. When the mass ratio of quail eggs to brewed vinegar is lower than 1:0.5, the acetic acid concentration in the system is insufficient, which can easily lead to difficulty in fully dissolving the eggshell during the enzymatic hydrolysis cycle. It may also lead to inhibition of acidic protease activity and incomplete protein hydrolysis. When the material-liquid ratio is higher than 1:1.5, the acetic acid concentration in the system is too high, which can easily lead to acid denaturation of egg white protein, causing protein molecules to entangle and aggregate, obscuring the action sites of the protease, thus reducing the degree of protein hydrolysis, and may also make the final product too sour.

[0056] In this invention, the final concentration of the acidic protease refers to the number of acidic protease activity units (U / mL) per milliliter in the reaction system of step (1) at the beginning of the enzymatic hydrolysis reaction; the enzyme activity unit U of the acidic protease is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine in 1 minute at 40°C and pH 3.0.

[0057] In step (1), the final concentration of the acidic protease is preferably 2000 U / mL. When the enzyme activity concentration is below 1000 U / mL, it may lead to insufficient protein decomposition and poor elimination of the eggy smell; when the enzyme activity concentration is above 3000 U / mL, it may lead to an excessively fast enzymatic hydrolysis rate, which may result in over-hydrolysis, easily producing hydrophobic bitter peptides, damaging the product flavor, and increasing unnecessary raw material costs. An enzymatic hydrolysis time of 4-6 hours is beneficial for achieving a balance between the degree of hydrolysis and flavor, and the preferred enzymatic hydrolysis time is 5 hours.

[0058] In a preferred embodiment of the present invention, in step (1), the mixture is stirred for 5 minutes every 30 minutes during the synchronous enzymatic hydrolysis process, and the stirring speed is 30~50 rpm.

[0059] In this invention, during the simultaneous enzymatic hydrolysis in step (1), the mixture is stirred for 5 minutes every 30 minutes. This stirring ensures the uniform distribution of vinegar and acidic protease in the system, while simultaneously breaking up air bubbles generated during eggshell dissolution. This prevents bubbles from adhering to the egg membrane surface and forming an air film barrier, ensuring continuous and stable penetration of acetic acid and protease, improving mass transfer efficiency, and shortening the reaction cycle. A low-speed stirring mode is used, preferably 30-50 rpm, to avoid premature breakage of the quail eggshell due to high-speed stirring.

[0060] In a preferred embodiment of the present invention, in step (2)a, the mass ratio of the egg to the brewed vinegar is 1:2~3, and the soaking time is 11~12h.

[0061] In this invention, the mass ratio of eggs to brewed vinegar is, for example, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, or 1:3, preferably 1:2.5. When the material-to-liquid ratio is higher than 1:2, the acetic acid content in the system is insufficient, which easily leads to incomplete dissolution of the eggshell. Residual eggshell fragments adhere to the egg membrane, making manual membrane removal difficult and easily causing egg liquid loss. When the material-to-liquid ratio is lower than 1:3, the system may be in a state of high acetic acid concentration for a long time, which may easily cause excessive acid denaturation of the egg liquid, protein molecules aggregate and precipitate, and the protease cannot fully function during subsequent enzymatic hydrolysis, resulting in a decrease in the degree of protein hydrolysis. The soaking time is, for example, 11h, 11.2h, 11.3h, 11.4h, 11.5h, 11.6h, 11.7h, 11.8h, 11.9h, or 12h. The soaking time is preferably 11.5 hours. The soaking process is carried out under normal temperature and sealed conditions. During the soaking period, the eggs are gently turned over once every 4 hours to avoid insufficient dissolution of the eggshell at the bottom of the egg in contact with the can. During the soaking process, the floating egg membrane should be removed in time. After the eggshell is completely dissolved, the inner and outer egg membranes should be completely removed to avoid egg membrane residue encapsulating the egg liquid, which will lead to uneven enzymatic hydrolysis in the later stage.

[0062] In a preferred embodiment of the present invention, in step (2)b, the mass ratio of jujube to cordyceps militaris is 10:1, the amount of pectinase and cellulase added is 0.1~0.3% of the substrate mass, the enzymatic hydrolysis time is 1~1.5h, the centrifugation conditions are 4000rpm and 10min, the soluble solids content of the jujube and cordyceps militaris extract is ≥3.0%, the enzyme activity of the cellulase is ≥10000U / g, and the enzyme activity of the pectinase is ≥30000U / g.

[0063] In this invention, the mass ratio of jujube to cordyceps militaris is 10:1, which is determined based on the complementary nature of their active ingredients and the coordination of their flavors. Jujube is rich in jujube polysaccharides and cyclic adenosine monophosphate, while cordyceps militaris is rich in cordyceps polysaccharides and cordycepin. When the two are combined in this ratio, the active ingredients can be synergistically enhanced, while neutralizing the slight fishy smell of cordyceps militaris and improving the flavor of the product. In step (2), the mass ratio of the total mass of jujube and cordyceps militaris to water is 1:6~8. After pulverizing the jujube and cordyceps militaris, they are passed through an 80-mesh sieve. Pulverization can significantly increase the specific surface area of ​​the raw materials, allowing cellulase and pectinase to fully contact the plant cell walls, providing a basis for enzymatic hydrolysis and cell wall breaking.

[0064] In step (2), the substrate mass refers to the total mass of the jujube, Cordyceps militaris powder and water. The amount of pectinase and cellulase added is 0.1-0.3%. The pectinase and cellulase are mixed in a 1:1 ratio and can work synergistically on the plant cell wall: cellulase specifically hydrolyzes the cellulose skeleton of the cell wall, and pectinase hydrolyzes the pectin components in the middle layer of the cell wall. The two work together to break the cell wall barrier, allowing the active ingredients such as polysaccharides, flavonoids and cordycepin to be fully released. Compared with simple heat extraction, the dissolution rate of active polysaccharides is increased by more than 60%.

[0065] A hydrolysis time of 1-2 hours is beneficial for the complete disruption of cell walls, while avoiding excessive hydrolysis that could lead to excessive formation of reducing sugars and trigger the Maillard reaction, resulting in an excessively dark product color. Centrifuge at 4000 rpm for 10 minutes to remove plant residues and obtain a clear supernatant.

[0066] In a preferred embodiment of the present invention, in step (2)c, the final concentration of acidic protease in the reaction system is 1000~3000U / mL, and the enzymatic hydrolysis time is 6~8h.

[0067] In this invention, the final concentration of the acidic protease refers to the number of acidic protease activity units (U / mL) per milliliter in the reaction system of step (2)c at the beginning of the enzymatic hydrolysis reaction; the enzyme activity unit U of the acidic protease is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine in 1 minute at 40°C and pH 3.0.

[0068] Meanwhile, in this invention, the enzymatic hydrolysis time is set to 6-8 hours because a hydrolysis time of less than 6 hours can easily lead to insufficient yield of small molecule peptides; when the hydrolysis time exceeds 8 hours, the increase in degree of hydrolysis slows down, but bitterness may increase significantly at this time. 6-8 hours is the optimal process window for achieving a balance between "high degree of hydrolysis" and "low bitterness".

[0069] The final concentration of acidic protease is more preferably 2000 U / mL, and the hydrolysis time is more preferably 7 h. This combination of parameters is beneficial for controlling the degree of hydrolysis of egg protein.

[0070] After enzymatic hydrolysis, rapid cooling to below 10°C for storage can instantly inhibit residual enzyme activity, prevent excessive hydrolysis from producing bitter peptides, inhibit microbial growth, ensure the stability of intermediate products, and provide stable semi-finished products for subsequent formulation.

[0071] In a preferred embodiment of the present invention, in step (3), the amount of pectinase and cellulase added is 0.1~0.2% of the substrate mass, the enzymatic hydrolysis time is 1 hour, and the heat extraction time is 1 hour.

[0072] In this invention, the total mass ratio of wolfberry, longan, mulberry, and polygonatum to water is 1:6~8.

[0073] In step (3), wolfberry, longan, mulberry, and polygonatum are pulverized and preferably passed through a 60-mesh sieve. The substrate mass refers to the total mass of the above four raw material powders and water. The addition amount of pectinase and cellulase is preferably 0.15% of the substrate mass each. The two work synergistically to fully hydrolyze the cellulose and pectin components of plant cell walls, break the mass transfer barrier, and allow the active ingredients to dissolve quickly and fully during the subsequent hot extraction process.

[0074] The enzymatic hydrolysis temperature is controlled at 50-55℃ and the pH at 4.5-5.5, which are the optimal reaction conditions for cellulase and pectinase, ensuring full enzyme activity. A 1-hour hydrolysis time is sufficient to achieve complete cell wall disruption, eliminating the need for longer hydrolysis times and avoiding increased production costs. After hydrolysis, the temperature is raised to 90-95℃ for 1 hour, preferably 90℃. This temperature and duration ensure complete dissolution of intracellular active ingredients after cell wall disruption. Compared to the existing 3-hour atmospheric pressure boiling process, the hot extraction time is shortened by 2 / 3, significantly reducing the degradation of heat-sensitive active ingredients such as wolfberry polysaccharides and mulberry anthocyanins, and significantly improving the dissolution rate of total flavonoids and polysaccharides.

[0075] In a preferred embodiment of the present invention, in step (5), the sterilization conditions are 132±2℃ for 4~6s and the discharge temperature is ≤60℃.

[0076] In this invention, ultra-high temperature instantaneous sterilization (UHT) can completely kill microorganisms such as bacteria, mold, and yeast in the system, achieving commercial sterility requirements and ensuring product shelf-life safety. Simultaneously, the high temperature of 132℃ can completely inactivate residual acidic proteases, cellulases, and pectinases in the system within a very short time, achieving simultaneous sterilization and enzyme inactivation. This eliminates the need for a separate enzyme inactivation step, simplifying the production process and improving efficiency. Compared to the existing stepwise process of inactivating enzymes at 80-90℃ followed by sterilization, this simultaneous process significantly shortens the high-temperature heating time of the materials, avoiding problems such as degradation of small molecule peptides, destruction of active ingredients, darkening of product color, and deterioration of flavor caused by prolonged high temperatures, thus maximizing the preservation of the product's nutrition and flavor. The discharge temperature after sterilization is controlled at ≤60℃, more preferably 50-60℃. This temperature can be directly matched to the subsequent secondary homogenization process without additional cooling or heating, reducing energy consumption and preventing the viscosity of the material from increasing after cooling, which would affect the homogenization effect.

[0077] During the enzymatic hydrolysis of eggs at 40-45℃ for 6-8 hours, the reducing sugars (glucose and fructose) in jujubes and the amino acids (especially lysine and arginine) produced by egg enzymatic hydrolysis undergo a mild initial Maillard reaction, generating a large amount of Schiff bases and amadoli compounds (flavor precursors). During ultra-high temperature sterilization, these flavor precursors rapidly transform into trace amounts of pyrazines, furans, ketones, and other primary aromatic compounds, giving the product a subtle roasted and caramel aroma without any burnt or bitter taste. Simultaneously, the later stages of the Maillard reaction do not occur, resulting in a yellowish-brown product color (mainly from the natural pigments of jujubes, goji berries, and mulberries), rather than a dark brown.

[0078] In a preferred embodiment of the present invention, the homogenization process consists of a first-stage homogenization and a second-stage homogenization process performed sequentially. The first-stage homogenization pressure is 15~20MPa, and the second-stage homogenization pressure is 5~10MPa. The particle size D90 of the homogenized material is ≤5μm.

[0079] In this invention, a two-stage homogenization process is employed, which, compared to the single-stage homogenization of existing technologies, achieves more uniform and refined particle size and significantly improves system stability. The first-stage homogenization is high-pressure homogenization, whose core function is to break down large protein molecules and polysaccharide aggregates into smaller particles, breaking down inter-particle aggregation forces. The second-stage homogenization is low-pressure homogenization, whose core function is to evenly disperse the broken-down particles throughout the system, preventing particle re-aggregation and forming a stable, homogeneous system. The first-stage homogenization pressure is 18 MPa, and the second-stage homogenization pressure is 8 MPa. This parameter combination can stably control the particle size (D90) of the homogenized material to be ≤5 μm, more preferably ≤3 μm. By precisely controlling the particle size, the stratification and sedimentation problems caused by large particle settling in existing technologies can be solved. Accelerated shelf-life testing at 37°C verified that the product prepared by this process remained homogeneous after 30 days of accelerated testing, showing no stratification or significant sedimentation, far superior to products from existing technologies. At the same time, the refined particles can further improve the smoothness of the product's texture, eliminating any grainy or astringent feeling and enhancing the product's sensory quality.

[0080] In this invention, food-grade sodium bicarbonate or food-grade citric acid is used to adjust the pH.

[0081] In this invention, the acidic protease is a commercially available food-grade acidic protease derived from the deep fermentation culture of Aspergillus niger, with CAS number 9025-49-4.

[0082] The optimal pH range for this acidic protease is 2.5–4.0, with an optimal pH of 3.0; its pH stability range is 2.5–6.0. ​​The optimal temperature range is 40–55°C, exhibiting high catalytic activity and good stability at temperatures between 40 and 45°C.

[0083] The enzyme activity unit U of the acidic protease is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine in 1 minute at 40°C and pH 3.0. Commercially available products are commonly available in specifications of 50,000 to 100,000 U / g (solid) or 50,000 to 100,000 U / mL (liquid).

[0084] In a preferred embodiment of the present invention, the raw materials used in the preparation method include, by weight: 40-60 parts of eggs, 0.1-0.5 parts of quail eggs, 80-150 parts of brewed vinegar, 100-200 parts of honey, 1-3 parts of jujubes, 0.2-0.6 parts of wolfberries, 0.2-0.5 parts of longan, 0.1-0.3 parts of cordyceps militaris, 0.05-0.15 parts of mulberries, 0.05-0.15 parts of polygonatum, 0.05-0.1 parts of potassium sorbate, and 50-80 parts of water; The total acidity of the brewed vinegar is ≥5.0g / 100mL, and the pH value is 2.8~3.8.

[0085] In this invention, the brewed vinegar is preferably solid-state fermented vinegar. Solid-state fermented vinegar contains abundant organic acids and esters, which not only enable the acid hydrolysis and dissolution of eggshells but also enhance the flavor profile of the product, avoiding the sharp sourness of liquid-fermented vinegar. More preferably, the total acidity is ≥6.0g / 100mL, and the pH value is 3.0~3.5.

[0086] Potassium sorbate is a food-grade preservative, and its addition amount strictly complies with the limits of the National Food Safety Standard for the Use of Food Additives (GB 2760). In this system, it can effectively inhibit the growth of mold and yeast, extend the shelf life of the product, and will not have any negative impact on the product flavor or enzymatic hydrolysis reaction.

[0087] According to a second aspect of the present invention, an enzymatically hydrolyzed vinegar-egg liquid prepared by the method described above is provided.

[0088] In this invention, the enzymatically hydrolyzed vinegar-egg liquid obtained is a uniform, clear, yellowish-brown liquid, free of visible foreign impurities, and exhibits no stratification or sedimentation. The product contains at least 75% small-molecule active peptides with a molecular weight <1000 Da, significantly improving human absorption and utilization. The product has a pH value ≥3.5, a density of 1.10~1.25 g / mL, no obvious eggy or sour taste, a smooth and mellow mouthfeel, and a harmonious flavor. The 100ml product has a shelf life of up to 12 months, maintaining stability throughout its shelf life without stratification or significant sedimentation, demonstrating excellent marketability.

[0089] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0090] In the following embodiments, unless otherwise specified, all raw material components are commercially available products.

[0091] Example 1 The enzymatically hydrolyzed vinegar-egg liquid was prepared according to the process of this invention, with the following raw material ratio: 50 parts chicken eggs, 0.3 parts quail eggs, 125 parts brewed vinegar (total acid 6.0g / 100mL, pH 3.2), 150 parts honey, 2 parts jujubes, 0.4 parts wolfberries, 0.3 parts longan, 0.2 parts cordyceps militaris, 0.1 parts mulberry, 0.1 parts polygonatum, 0.08 parts potassium sorbate, and 70 parts water.

[0092] The steps for preparing enzymatically hydrolyzed vinegar-egg solution are as follows: (1) Preparation of quail egg enzymatic hydrolysate: Mix 0.3 parts of quail eggs with 0.3 parts of brewed vinegar (0.3 parts from 125 parts of total vinegar) at a mass ratio of 1:1, adjust the pH to 3.2, heat to 42℃, add acidic protease (final concentration 2000 U / mL) and enzymatically hydrolyze for 5 hours. Stir for 5 minutes every 30 minutes during the enzymatic hydrolysis (40 rpm). After the enzymatic hydrolysis is completed, break up the eggs, filter through a 120-mesh sieve to obtain the quail egg enzymatic hydrolysate, and quickly cool to below 10℃ for storage.

[0093] (2) Preparation of egg enzymatic hydrolysate: a. Mix 50 parts of eggs with 124.7 parts of brewed vinegar at a mass ratio of 1:2.5, soak at room temperature for 11.5 hours to dissolve the eggshells, remove the egg membranes, and beat to obtain coarse vinegar-egg liquid.

[0094] b. Crush 2 parts of jujube and 0.2 parts of Cordyceps militaris into powder and pass through an 80-mesh sieve. Add water equal to 7 times the total mass of the raw materials (i.e., 2.2 × 8 = 15.4 parts), adjust the pH to 5.0, heat to 52℃, and add pectinase (0.2% substrate mass, 30000 U / g enzyme activity) and cellulase (0.2% substrate mass, 10000 U / g enzyme activity) for enzymatic hydrolysis for 1.2 hours. After enzymatic hydrolysis, centrifuge (4000 rpm, 10 min) and collect the supernatant to obtain the jujube and Cordyceps militaris extract.

[0095] c. Mix the crude vinegar-egg liquid with the above-mentioned jujube and cordyceps extract, add calcium lactate to make the free calcium ion concentration reach 55 mmol / L, adjust the pH to 3.2, raise the temperature to 42℃, add acidic protease (final concentration 2000 U / mL) for enzymatic hydrolysis for 7 hours. After enzymatic hydrolysis, quickly cool to below 10℃ and store to obtain the compound egg enzymatic hydrolysate.

[0096] (3) Preparation of compound extract of traditional Chinese medicine: 0.4 parts of wolfberry, 0.3 parts of longan, 0.1 parts of mulberry and 0.1 parts of polygonatum are crushed and passed through a 60-mesh sieve. Add water with a total mass of 7 times that of the raw materials (i.e., 0.9×7=6.3 parts), adjust the pH to 5.0, raise the temperature to 52℃, add pectinase (substrate mass 0.15%, enzyme activity 30000U / g) and cellulase (substrate mass 0.15%, enzyme activity 10000U / g) for enzymatic hydrolysis for 1h, then raise the temperature to 92℃ for hot extraction for 1h, filter through an 80-mesh sieve to obtain compound extract of traditional Chinese medicine.

[0097] (4) Preparation: Mix the enzymatic hydrolysates from steps (1) and (2) to obtain a mixed enzymatic hydrolysate (keep the temperature ≤10℃). Take the remaining water (about 48.3 parts) and heat it to 55℃. Add 150 parts of honey and stir to dissolve. Cool to 12℃ to obtain honey water. First, add the honey water to the mixed enzymatic hydrolysate and stir evenly. Then, add the compound extract of traditional Chinese medicine and 0.08 parts of potassium sorbate. Stir for 15 minutes and keep the system temperature ≤12℃.

[0098] (5) Sterilization and homogenization: The prepared solution is subjected to ultra-high temperature instantaneous sterilization (132℃, 5s), and the discharge temperature is 58℃; then it is subjected to two-stage homogenization (first stage 18MPa, second stage 8MPa), and then filled to obtain enzymatically hydrolyzed vinegar-egg solution.

[0099] Example 2 The raw material ratio is the same as in Example 1.

[0100] In step (1), acidic protease with a final concentration of 1500 U / mL was used for enzymatic hydrolysis for 6 hours; In step (2)c, acidic protease at a final concentration of 1500 U / mL is used for enzymatic hydrolysis for 8 hours. The rest is the same as in Example 1.

[0101] Example 3 The raw material ratio is the same as in Example 1.

[0102] In step (1), acidic protease with a final concentration of 800 U / mL was used for enzymatic hydrolysis for 6 hours; In step (2)c, acidic protease at a final concentration of 800 U / mL is used for enzymatic hydrolysis for 8 hours; The rest is the same as in Example 1.

[0103] Example 4 The raw material ratio is the same as in Example 1.

[0104] In step (1), the pH is adjusted to 3.0, the temperature is raised to 45°C, and acidic protease is added for simultaneous enzymatic hydrolysis. In step (2)c, adjust the pH to 3.0, raise the temperature to 45°C, and add acidic protease for enzymatic hydrolysis; The rest is the same as in Example 1.

[0105] Example 5 The raw material ratio is the same as in Example 1.

[0106] In step (2)c, calcium lactate is added to bring the free calcium ion concentration to 50 mmol / L; The rest is the same as in Example 1.

[0107] Example 6 The raw material ratio is the same as in Example 1.

[0108] In step (2)c, calcium lactate is added to bring the free calcium ion concentration to 60 mmol / L; The rest is the same as in Example 1.

[0109] Comparative Example 1 The raw material ratio is the same as in Example 1.

[0110] Acidic protease is not used in steps (1) and (2)c, specifically: (1) Preparation of quail egg acid hydrolysate: Mix 0.3 parts quail eggs with 0.3 parts brewed vinegar (0.3 parts from a total of 125 parts vinegar) at a mass ratio of 1:1, and soak at room temperature (25℃) for 5 hours to dissolve the eggshells. During the soaking process, stir for 5 minutes every 30 minutes (40 rpm). After soaking, break the mixture apart, filter it through a 120-mesh sieve to obtain the quail egg hydrolysate, and quickly cool it to below 10℃ for storage.

[0111] (2) Preparation of egg acid hydrolysate: a. Mix 50 parts of eggs with 124.7 parts of brewed vinegar at a mass ratio of 1:2.5, soak at room temperature for 11.5 hours to dissolve the eggshells, remove the egg membranes, and beat to obtain coarse vinegar-egg liquid.

[0112] b. Crush 2 parts of jujube and 0.2 parts of Cordyceps militaris through an 80-mesh sieve, add 8 times the total mass of water (i.e., 17.6 parts), adjust the pH to 5.0, heat to 52℃, add pectinase (0.2% substrate mass, 30000 U / g enzyme activity) and cellulase (0.2% substrate mass, 10000 U / g enzyme activity) and hydrolyze for 1.2 h. After hydrolysis, centrifuge (4000 rpm, 10 min) and collect the supernatant to obtain the jujube and Cordyceps militaris extract.

[0113] c. Mix the crude vinegar-egg liquid with the above extract, add calcium lactate to bring the free calcium ion concentration to 55 mmol / L, adjust the pH to 3.2, raise the temperature to 42℃, without adding acidic protease, and stir at a constant temperature for 7 hours. After treatment, quickly cool to below 10℃ and store to obtain the compound egg acid hydrolysate.

[0114] The rest is the same as in Example 1.

[0115] Comparative Example 2 The raw material ratio is the same as in Example 1.

[0116] In step (2)c, no calcium lactate is supplemented, and the concentration of free calcium ions is approximately 42 mmol / L; The rest is the same as in Example 1.

[0117] Comparative Example 3 The raw material ratio is the same as in Example 1.

[0118] In step (2), instead of using a mixture of jujube and cordyceps militaris extract and crude vinegar and egg liquid for enzymatic hydrolysis, the crude vinegar and egg liquid and jujube and cordyceps militaris extract are enzymatically hydrolyzed separately. Step (2) is specifically as follows: Steps (2)a and (2)b are the same as in Example 1.

[0119] Step (2)c uses a separate enzymatic hydrolysis method: c1. Add calcium lactate separately to the crude vinegar egg liquid from step a to make the free calcium ion concentration reach 55 mmol / L, adjust the pH to 3.2, raise the temperature to 42℃, add acidic protease (final concentration 2000 U / mL) for enzymatic hydrolysis for 7 hours to obtain egg hydrolysate, and quickly cool it to below 10℃ for storage.

[0120] c2. Supplement the jujube and Cordyceps militaris extract from step b with calcium lactate to achieve a free calcium ion concentration of 55 mmol / L, adjust the pH to 3.2, raise the temperature to 42℃, add acidic protease (final concentration 2000 U / mL) for enzymatic hydrolysis for 7 hours to obtain plant extract hydrolysate, and quickly cool it to below 10℃ for storage.

[0121] c3. Physically mix the egg enzymatic hydrolysate from c1 with the plant extract enzymatic hydrolysate from c2, and stir until homogeneous to obtain a composite egg enzymatic hydrolysate.

[0122] The rest is the same as in Example 1.

[0123] Comparative Example 4 The raw material ratio is the same as in Example 1.

[0124] The difference from Example 1 is that in step (4), the honey is not dissolved in water beforehand, but is directly added to the mixed enzymatic hydrolysate and stirred. The water originally used to dissolve the honey is added as surplus water after the honey is added.

[0125] The rest is the same as in Example 1.

[0126] Performance testing 1. Determination of protein hydrolysis degree The amino nitrogen content before and after enzymatic hydrolysis was determined by the trinitrobenzenesulfonic acid (TNBS) method, and the total nitrogen content was determined by the Kjeldahl method. The test subject was enzymatically hydrolyzed vinegar-egg liquid. Degree of hydrolysis DH (%) = (Amino nitrogen content after hydrolysis - Amino nitrogen content before hydrolysis) / Total nitrogen content of the system × 100%; 2. Determination of the proportion of small molecule active peptides High-performance gel filtration chromatography (GPC), referring to GB / T 22729-2008 Marine fish oligopeptide powder, used a gel chromatography column with peptide molecular weight standards to determine the proportion of small molecule peptides with a molecular weight <1000 Da in the total protein. The results of the protein hydrolysis degree and the proportion of small molecule active peptides are shown in Table 1.

[0127] Table 1. Results of protein hydrolysis degree and proportion of small molecule bioactive peptides

[0128] 3. System stability testing 3.1 Accelerated Shelf Life Testing Following the general methods for accelerated testing of food and beverages, sealed samples were placed in a constant temperature and humidity incubator at 37±1℃ and 75% relative humidity. Samples were taken at 0d, 7d, 15d, 30d, and 60d to detect tissue state, particle size distribution, pH value, and sensory quality.

[0129] The test subject was 100ml of enzymatically hydrolyzed vinegar-egg liquid.

[0130] If the product shows no stratification, no obvious sedimentation, no significant change in D90 particle size, and no deterioration in sensory quality after 30 days of accelerated testing, it is considered stable; if it remains stable after 60 days of accelerated testing, it corresponds to a 12-month shelf life at room temperature.

[0131] This test can be used to evaluate the stability of a product during storage and predict its shelf life.

[0132] The results of the accelerated shelf-life test are shown in Table 2.

[0133] Table 2 Results of Accelerated Shelf Life Test

[0134] 3.2 Centrifugal stability test The initial finished products (0d) of each example and comparative example were centrifuged to quickly evaluate the suspension stability of the products. The smaller the amount of sediment, the more stable the system.

[0135] Take 50 mL of sample into a centrifuge tube, centrifuge at 4000 rpm for 15 min, discard the supernatant, and weigh the wet weight of the precipitate.

[0136] The results of the centrifugal stability test are shown in Table 3.

[0137] Table 3 Results of centrifugal stability test

[0138] 4. Sensory evaluation Referring to GB / T 16291.1 "General Guidelines for the Selection, Training and Management of Sensory Analysis Evaluators", 20 trained evaluators (half male and half female, aged 25-50) were organized to conduct blind evaluations. The product was diluted with 40℃ warm water at a ratio of 1:4 and placed in evaluation cups for evaluation. The sensory evaluation items and scoring criteria are shown in Table 4, and the sensory test results are shown in Table 5.

[0139] Table 4 Sensory Evaluation Items and Scoring Criteria

[0140] Table 5 Sensory Test Results

[0141] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing enzymatically hydrolyzed vinegar-egg liquid, wherein the preparation method uses acidic protease, cellulase and pectinase as hydrolysis catalysts to prepare a composition containing small molecule active peptides from poultry eggs and active polysaccharides from plants through enzymatic catalytic hydrolysis; The preparation method includes the following steps: (1) Preparation of quail egg enzymatic hydrolysate: Mix quail eggs with brewed vinegar, control the pH of the system to 3.0~3.5, heat to 40~45℃, add acidic protease for simultaneous enzymatic hydrolysis, after enzymatic hydrolysis, stir, break, and filter to obtain quail egg hydrolysate, and quickly cool to below 10℃ for storage. (2) Preparation of egg enzymatic hydrolysate: a. Mix eggs with brewed vinegar, soak them to dissolve the eggshells, remove the egg membranes, and beat them to obtain coarse vinegar-egg liquid; b. Crush the jujubes and Cordyceps militaris, add water, adjust the pH to 4.5-5.5, keep the temperature at 50-55℃, add pectinase and cellulase for enzymatic hydrolysis, centrifuge and take the supernatant to obtain jujube and Cordyceps militaris extract. c. Mix the crude vinegar and egg liquid from step a with the jujube and cordyceps extract from step b, add calcium lactate to make the free calcium ion concentration in the system reach 50~60 mmol / L, adjust the pH to 3.0~3.5, the temperature to 40~45℃, add acidic protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, obtain the compound egg hydrolysate, and quickly cool it to below 10℃ for storage. (3) Preparation of compound extracts of traditional Chinese medicine: Grind and mix wolfberry, longan, mulberry and polygonatum, add water, adjust the pH to 4.5~5.5, temperature 50~55℃, add pectinase and cellulase for enzymatic hydrolysis, then heat to 90~95℃ for extraction, filter to obtain Chinese herbal compound extract; (4) Allocation: The quail egg enzymatic hydrolysate from step (1) is mixed with the compound chicken egg enzymatic hydrolysate from step (2) to obtain a mixed enzymatic hydrolysate. Dissolve honey in water and cool it to below 15°C to obtain honey water; First, add honey water to the mixed enzymatic hydrolysate and stir well. Then add the Chinese herbal compound extract and potassium sorbate from step (3) and stir well. (5) Sterilization and homogenization: The prepared liquid is subjected to ultra-high temperature instantaneous sterilization, which simultaneously deactivates the enzymes; then it is homogenized to obtain enzymatically hydrolyzed vinegar-egg liquid.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of quail eggs to brewed vinegar is 1:0.5~1.5, the final concentration of acidic protease in the reaction system is 1000~3000U / mL, and the enzymatic hydrolysis time is 4~6h.

3. The preparation method according to claim 1, characterized in that, In step (1), the mixture is stirred for 5 minutes every 30 minutes during the synchronous enzymatic hydrolysis process. The vinegar and protease in the system are evenly distributed by mechanical stirring, and the bubbles generated during the eggshell dissolution process are broken to prevent the bubbles from adhering to the egg membrane surface and forming an air film barrier, thus ensuring mass transfer efficiency.

4. The preparation method according to claim 1, characterized in that, In step (2)a, the mass ratio of the eggs to the brewed vinegar is 1:2~3, and the soaking time is 11~12h.

5. The preparation method according to claim 1, characterized in that, In step (2)b, the mass ratio of jujube to cordyceps militaris is 10:1, the amount of pectinase and cellulase added is 0.1~0.3% of the substrate mass, the enzymatic hydrolysis time is 1~1.5h, the centrifugation conditions are 4000rpm and 10min, the soluble solids content of the jujube and cordyceps militaris extract is ≥3.0%, the enzyme activity of the cellulase is ≥10000U / g, and the enzyme activity of the pectinase is ≥30000U / g.

6. The preparation method according to claim 1, characterized in that, In step (2)c, the final concentration of acidic protease in the reaction system is 1000~3000U / mL, and the enzymatic hydrolysis time is 6~8h.

7. The preparation method according to claim 1, characterized in that, In step (3), the amount of pectinase and cellulase added is 0.1~0.2% of the substrate mass, the enzymatic hydrolysis time is 1h, and the heat extraction time is 1h.

8. The preparation method according to claim 1, characterized in that, In step (5), the sterilization conditions are 132±2℃ for 4~6s, and the discharge temperature is ≤60℃; The homogenization process involves first-stage and second-stage homogenization. The pressure for first-stage homogenization is 15-20 MPa, and the pressure for second-stage homogenization is 5-10 MPa. The particle size D90 of the homogenized material is ≤5 μm.

9. The preparation method according to any one of claims 1 to 8, characterized in that, The raw materials used in the preparation method, by weight, include: 40-60 parts of eggs, 0.1-0.5 parts of quail eggs, 80-150 parts of brewed vinegar, 100-200 parts of honey, 1-3 parts of jujubes, 0.2-0.6 parts of wolfberries, 0.2-0.5 parts of longan, 0.1-0.3 parts of cordyceps militaris, 0.05-0.15 parts of mulberries, 0.05-0.15 parts of polygonatum, 0.05-0.1 parts of potassium sorbate, and 50-80 parts of water; The total acidity of the brewed vinegar is ≥5.0g / 100mL, and the pH value is 2.8~3.

8.

10. An enzymatically hydrolyzed vinegar-egg liquid prepared according to any one of claims 1 to 9.

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