Cleavage process of softening enzyme of small molecule peptide material of bird's nest based on gradient temperature control

Abstract

The present application relates to the technical field of food processing, and discloses a softening enzymolysis process of bird's nest small molecule peptide material based on gradient temperature control, which comprises the following steps: firstly, gradient temperature rising physical softening is performed on bird's nest slurry to gently relax the compact structure thereof; then, three-stage sequential enzymolysis is performed in sequence, that is, preliminary degradation is performed by endoprotease, further cutting is performed by exoprotease, and finally, depth and directional enzymolysis is performed by glycoside hydrolase and specific endopeptidase in cooperation; and the product is obtained after inactivation, separation, refinement and drying. Through the gradient temperature control physical softening, three-stage sequential enzymolysis and integration of the front and rear processes, the present application solves the problems existing in the prior art, such as poor substrate accessibility, low enzymolysis efficiency and specificity, wide product distribution, non-outstanding functional ingredients and loose process flow.

Description

Technical Field

[0001] This invention patent relates to the field of food processing technology, specifically to a gradient temperature-controlled enzymatic hydrolysis process for softening small molecule peptides from bird's nest. Background Technology

[0002] Bird's nest, a traditional and prized food, is primarily composed of bird's nest protein (glycoprotein). Traditionally, it is consumed mainly through stewing, but this method has low extraction efficiency, and the large protein molecules are not easily absorbed directly by the human body, resulting in limited bioavailability. To improve the processing depth and added value of bird's nest, enzymatic hydrolysis technology has emerged as a method for processing it.

[0003] A typical enzymatic hydrolysis process for bird's nest involves cleaning, soaking, and pulping the bird's nest. The mixture is then directly adjusted to a single, suitable temperature, and one or more proteases are added for one-time or staged enzymatic hydrolysis. The resulting product is then inactivated, filtered, concentrated, and dried. These methods aim to degrade bird's nest proteins into small peptides and amino acids.

[0004] However, existing enzymatic hydrolysis processes for bird's nest still have several significant drawbacks: Bird's nest raw materials have a special physical structure with dense and highly cross-linked properties. When directly enzymatically hydrolyzed, the contact area between the enzyme and the substrate is limited, resulting in problems such as insufficient enzymatic hydrolysis, long reaction time, and large amount of enzyme used. This leads to the residue of some large molecular fragments and low yield of target small molecule peptides. Secondly, most processes employ a single temperature or a simple two-stage temperature approach for enzymatic hydrolysis, failing to fully consider the differences in optimal operating temperatures of different types of proteases (such as endopeptidases, exopeptidases, and glycosidases), as well as the dynamic changes in the physical properties of materials at different temperatures. Constant temperature or simple variable temperature conditions cannot synergistically optimize material accessibility and enzyme activity efficiency, thus affecting the overall effectiveness of multi-enzyme synergy. Furthermore, the selection and addition sequence of enzymes in existing enzymatic hydrolysis processes are rather crude. They are typically simple combinations and simultaneous additions of multiple enzymes, or the order of addition lacks fine design. This crude approach makes it difficult to achieve directional and orderly degradation of peptide chains, easily leading to problems such as uncontrollable enzymatic hydrolysis, excessively wide molecular weight distribution of product peptides, and low content of characteristic short peptides with specific functional potential. Finally, traditional processes do not pay enough attention to the pretreatment of materials before enzymatic hydrolysis (such as physical softening), which is often disconnected from the enzymatic hydrolysis process and fails to form a coherent and synergistic technical chain from the loosening of physical structure to the precise cutting by biological enzymatic hydrolysis.

[0005] In view of this, we propose a gradient temperature control-based enzymatic hydrolysis process for softening small molecule peptides in bird's nest. Summary of the Invention

[0006] The purpose of this invention is to provide a gradient temperature-controlled enzymatic hydrolysis process for softening small molecule peptides in bird's nest, in order to solve the problems existing in the background technology.

[0007] To achieve the above objectives, this invention provides the following technical solution: The gradient temperature-controlled enzymatic hydrolysis process for softening small molecule peptides in bird's nest includes: S1. Select bird's nest raw materials, clean them to remove visible impurities, and then use hydraulic shearing to perform preliminary crushing to obtain bird's nest fragment slurry; S2. The bird's nest fragment slurry is placed in a programmable temperature-controlled reaction vessel, first kept warm and soaked in the first temperature range, and then heated to the second temperature range at a constant heating rate and kept warm. The second temperature range is higher than the first temperature range. During this process, gentle stirring is continuously carried out to relax and initially swell the network structure of the bird's nest fragments. S3. After completing the gradient heating physical softening, adjust and maintain the temperature of the slurry in the third temperature range suitable for the action of endopeptidase, add endopeptidase to the slurry, and carry out the first stage of enzymatic hydrolysis under constant temperature conditions. The endopeptidase is used to randomly break the peptide bonds inside the bird's nest protein and initially degrade the macromolecular protein into polypeptide fragments. S4. After completing the first-stage enzymatic hydrolysis, without inactivation treatment, exonuclease is directly added to the reaction system, and the slurry temperature is adjusted and maintained in the fourth temperature range suitable for the action of exonuclease to carry out the second-stage enzymatic hydrolysis reaction. The exonuclease cuts from the end of the polypeptide fragment to further generate peptides with smaller molecular weight. S5. After completing the secondary enzymatic hydrolysis, maintain the system temperature or adjust it to the fifth temperature range, and simultaneously add glycoside hydrolase and specific telopeptidase to the reaction system to carry out the third stage of synergistic enzymatic hydrolysis. The glycoside hydrolase is used to weaken or cut the glycoside bonds that connect peptide chains in bird's nest, and the specific telopeptidase is used to specifically recognize and cut the peptide bonds at the ends of specific amino acid sequences exposed after being acted upon by the endopeptidase and / or exopeptidase. S6. After completing the three-stage synergistic enzymatic hydrolysis, the reaction system is rapidly heated to the inactivation temperature that completely inactivates all enzymes and maintained for a period of time. Then, the inactivated enzymatic hydrolysate is centrifuged and the supernatant is collected to obtain a primary solution containing bird's nest small molecule peptides. S7. The primary solution is filtered and separated sequentially through a microfiltration membrane and an ultrafiltration membrane system, and the permeate within the target molecular weight range is collected. Then, the permeate is concentrated under low temperature vacuum to obtain bird's nest small molecule peptide concentrate. S8. Spray-dry or vacuum freeze-dry the concentrated bird's nest small molecule peptide solution to obtain a powdered bird's nest small molecule peptide product.

[0008] Preferably, in step S2, the first temperature range is a cold water immersion range, the second temperature range is a sub-high temperature softening range that is higher than room temperature but lower than the range where proteins undergo significant denaturation, and the heating rate is a uniform and slow heating rate.

[0009] Preferably, in step S3, the endopeptide is one or a combination of alkaline protease, neutral protease, or complex protease.

[0010] Preferably, in step S4, the exonuclease is one or a combination of two of aminopeptidase and carboxypeptidase.

[0011] Preferably, in step S5, the glycoside hydrolase is an enzyme that can act on the glycan chains in the characteristic glycoproteins of bird's nest, and the specific telopeptidase is one of proline-specific endopeptidase and glutamate-specific endopeptidase.

[0012] Preferably, the enzymatic hydrolysis reactions in steps S3, S4, and S5 are all carried out under constant pH conditions, and the pH of the reaction system is maintained by automatically adding acid or alkali solution.

[0013] Preferably, the enzymatic hydrolysis reaction times of steps S3, S4, and S5 are independently controlled, and the time of the subsequent enzymatic hydrolysis reaction covers or is longer than the time of the previous enzymatic hydrolysis reaction.

[0014] Preferably, the centrifugation in step S6 is high-speed centrifugation, and the pore size of the microfiltration membrane in step S7 is larger than the molecular weight cutoff of the ultrafiltration membrane, wherein the molecular weight cutoff of the ultrafiltration membrane corresponds to the upper limit of the molecular weight of the target small molecule peptide.

[0015] Preferably, the temperature of the low-temperature vacuum concentration in step S7 is in the low-temperature range, and the vacuum degree is under medium vacuum conditions.

[0016] A bird's nest small molecule peptide product prepared by any of the processes described herein, characterized in that its molecular weight is mainly distributed in the target small molecular weight range and is rich in characteristic short peptide sequences generated by the action of specific telopeptidases.

[0017] By employing the above technical solution, this invention patent provides a gradient temperature-controlled enzymatic hydrolysis process for softening small molecule peptides in bird's nest. It possesses at least the following beneficial effects: This invention patent adopts a "gradient heating physical softening" step, first soaking at low temperature and then heating up to sub-high temperature for heat preservation, which achieves a gentle and gradual loosening of the dense bird's nest material structure, solves the problem of "limited contact area between enzyme and substrate and insufficient enzymatic hydrolysis", creates extremely favorable physical conditions for subsequent enzymatic hydrolysis reaction, and achieves the effect of greatly improving substrate accessibility without using violent chemical or physical means. By coupling "gradient temperature control" with "three-stage sequential enzymatic hydrolysis", the temperature and action stage are precisely matched according to the characteristics of different enzymes. This solves the problem that "using a single temperature or a simple two-stage temperature cannot synergistically optimize material accessibility and the activity efficiency of multiple enzymes". It maximizes the synergy between thermal energy utilization and biocatalytic efficiency, thereby speeding up the overall reaction rate and relatively reducing the amount of enzyme used. By constructing an ordered and controllable directional degradation logic through the addition sequence and action flow of "endopeptidase → exopeptidase → (glycoside hydrolase + specific telopeptidase)", an orderly and controllable directional degradation logic was constructed. This solved the problem of "coarse enzyme addition sequence leading to uncontrollable enzymatic hydrolysis process and excessively wide molecular weight distribution of products". It achieved precise guidance of the degradation pathway of bird's nest protein, making the molecular weight distribution of products more concentrated and improving the selectivity and yield of target small molecule peptides. By integrating "gradient heating physical softening" with "multi-stage sequential enzymatic hydrolysis" into a continuous and smooth process, physical pretreatment and biological enzymatic hydrolysis are no longer two isolated links. This solves the problem of "disconnection between material pretreatment and enzymatic hydrolysis" and achieves full-process optimization from macroscopic structural loosening to microscopic molecular cutting. The process has strong continuity, high overall efficiency, and good controllability. By adopting a tandem approach (especially from the first to the second stage) that allows products to directly enter the next enzymatic hydrolysis stage without inactivation, the operation steps are simplified, avoiding efficiency losses and potential contamination or side reactions caused by frequent temperature adjustments, inactivation, pH adjustments, etc. The product of the previous stage is directly used as the substrate of the next stage, the reaction system is continuous, and energy and material utilization is more economical. By employing the steps of "membrane separation purification" and "low-temperature vacuum concentration / drying", the purity and quality of the final product are ensured. Microfiltration removes residual insoluble particles, and ultrafiltration precisely retains components with molecular weights above the target value, thereby strictly controlling the molecular weight range of the finished small molecule peptides. Low-temperature concentration and drying maximize the protection of the activity and structural integrity of the heat-sensitive small molecule peptides.

[0018] In summary, this invention solves the problems of poor substrate accessibility, low enzymatic efficiency and specificity, wide product distribution, lack of prominent functional components, and loose process flow in existing bird's nest enzymatic hydrolysis processes by using gradient temperature-controlled physical softening, three-stage sequential enzymatic hydrolysis (especially the introduction of specific enzymes at the end for synergistic effect), and integration of upstream and downstream processes. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of the invention, form part of this application: Figure 1 This is a schematic diagram of the overall process of this invention patent. Detailed Implementation

[0020] The technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] Please see Figure 1 The present invention describes a gradient temperature-controlled enzymatic hydrolysis process for softening small molecule peptides in bird's nest, comprising: S1. Select bird's nest raw materials, clean them to remove visible impurities, and then use hydraulic shearing to perform preliminary crushing to obtain bird's nest fragment slurry; It should be noted that hydraulic shearing crushing uses the intense shearing force generated by high-speed water flow to break up bird's nest materials. Compared with traditional mechanical grinding or cutting, hydraulic shearing can reduce localized overheating caused by frictional heat, thus better protecting the natural conformation of bird's nest proteins and avoiding unnecessary denaturation during the pretreatment stage. At the same time, it can form a slurry with more uniform particle size and better flowability, laying the foundation for subsequent uniform heating and enzymatic hydrolysis reactions.

[0022] S2. The bird's nest fragment slurry is placed in a programmable temperature-controlled reaction vessel, first kept warm and soaked in the first temperature range, and then heated to the second temperature range at a constant heating rate and kept warm. The second temperature range is higher than the first temperature range. During this process, gentle stirring is continuously carried out to relax and initially swell the network structure of the bird's nest fragments. It should be noted that the prolonged immersion in cold water in the "first temperature range" aims to allow water molecules to fully penetrate the dense protein fiber network of the bird's nest. The "constant heating rate" programmed heating to the "second temperature range" (sub-high temperature softening range) is a gentle heat treatment process. Slow heating avoids sudden temperature changes that could cause rapid denaturation and coagulation of the protein surface, forming a dense layer that hinders internal hydration and heat transfer. During this process, the heat gradually weakens the non-covalent forces, such as hydrogen bonds, that maintain the bird's nest network structure. Combined with the mechanical action of gentle stirring, this causes the bird's nest's three-dimensional structure to gradually loosen and become fluffy, significantly swelling in volume, thereby exposing more potential enzyme activity sites.

[0023] S3. After completing the gradient heating physical softening, adjust and maintain the temperature of the slurry in the third temperature range suitable for the action of endopeptidase, add endopeptidase to the slurry, and carry out the first stage of enzymatic hydrolysis under constant temperature conditions. The endopeptidase is used to randomly break the peptide bonds inside the bird's nest protein and initially degrade the macromolecular protein into polypeptide fragments. It should be noted that after physical softening, the specific surface area and accessibility of bird's nest material are greatly improved, making enzymatic hydrolysis more efficient. The role of endopeptides (such as alkaline proteases) is similar to "scissors," randomly cutting at specific peptide bonds inside protein molecules, "pulverizing" the complete large protein molecules of bird's nest into a series of smaller polypeptide fragments. This stage is the beginning and foundation of enzymatic hydrolysis, determining the initial size and distribution of the subsequent enzymatic hydrolysis substrate (polypeptide fragments).

[0024] S4. After completing the first-stage enzymatic hydrolysis, without inactivation treatment, exonuclease is directly added to the reaction system, and the slurry temperature is adjusted and maintained in the fourth temperature range suitable for the action of exonuclease to carry out the second-stage enzymatic hydrolysis reaction. The exonuclease cuts from the end of the polypeptide fragment to further generate peptides with smaller molecular weight. It should be noted that the reaction system of the endopeptidase is directly used as the substrate system of the exopeptidase, realizing a "tandem" reaction of enzymes. Exopeptides (such as aminopeptidase and carboxypeptidase) hydrolyze amino acid residues one by one from the N-terminus or C-terminus of the polypeptide chain, which can effectively and rapidly shorten the peptide chain length and is a key step in obtaining smaller peptides with lower molecular weights. This design avoids the tedious operation of intermediate inactivation and subsequent temperature and pH adjustment, improving efficiency and continuity.

[0025] S5. After completing the secondary enzymatic hydrolysis, maintain the system temperature or adjust it to the fifth temperature range, and simultaneously add glycoside hydrolase and specific telopeptidase to the reaction system to carry out the third stage of synergistic enzymatic hydrolysis. The glycoside hydrolase is used to weaken or cut the glycoside bonds that connect peptide chains in bird's nest, and the specific telopeptidase is used to specifically recognize and cut the peptide bonds at the ends of specific amino acid sequences exposed after being acted upon by the endopeptidase and / or exopeptidase. It's important to note that bird's nest protein is a glycoprotein, with glycan chains linked to peptide chains via glycosidic bonds, which may shield some enzyme cleavage sites. Glycoside hydrolases can hydrolyze these glycosidic bonds, removing steric hindrance and exposing more peptide segments or terminals protected by the glycan chains, creating new sites for peptidases. Specific terminal peptidases (such as proline-specific endopeptidases) can precisely cleave peptide bonds at the sites of specific amino acid residues such as proline exposed in the first two stages, generating short peptides with specific sequences. The synergistic effect of these two enzymes achieves a more thorough deconstruction of the complex structure of bird's nest and may enrich peptide segments with specific physiological activities.

[0026] S6. After completing the three-stage synergistic enzymatic hydrolysis, the reaction system is rapidly heated to the inactivation temperature that completely inactivates all enzymes and maintained for a period of time. Then, the inactivated enzymatic hydrolysate is centrifuged and the supernatant is collected to obtain a primary solution containing bird's nest small molecule peptides. It should be noted that after the enzymatic hydrolysis reaction is completed, all enzyme proteins must be denatured and inactivated by heating (e.g., 85-95℃) to terminate the reaction and prevent the enzymatic hydrolysis from continuing during subsequent processing or storage, which would lead to product instability. The purpose of centrifugation is to remove the enzyme proteins that have coagulated and precipitated due to heat inactivation, insoluble particulate matter that has not been completely hydrolyzed, and other solid impurities, thereby obtaining a clear enzymatic hydrolysate.

[0027] S7. The primary solution is filtered and separated sequentially through a microfiltration membrane and an ultrafiltration membrane system, and the permeate within the target molecular weight range is collected. Then, the permeate is concentrated under low temperature vacuum to obtain bird's nest small molecule peptide concentrate. It should be noted that the process involves first microfiltration (MF) to remove any small particles, colloids, or bacteria that may remain after centrifugation, followed by ultrafiltration (UF). Depending on the molecular weight range of the target small peptide (e.g., setting a cutoff of 3000 Daltons or 1000 Daltons), peptides larger than this value and residual large proteins are retained, allowing the target small peptide to pass through, thus achieving molecular weight fractionation and purification of the product. Low-temperature vacuum concentration involves evaporating water at lower temperatures (e.g., <60°C) and under reduced pressure, aiming to avoid the damage of heat-sensitive small peptides to high temperatures and obtain a high-concentration peptide solution.

[0028] S8. Spray dry or vacuum freeze dry the concentrated bird's nest small molecule peptide solution to obtain a powdered bird's nest small molecule peptide product. It should be noted that spray drying is highly efficient and suitable for industrial production, but the inlet air temperature needs to be controlled to avoid overheating. Vacuum freeze drying removes moisture through sublimation at low temperatures, which maximizes the protection of peptide activity and structure, resulting in excellent product solubility, but at a higher cost. The choice can be made based on different requirements for product quality and production costs.

[0029] In step S2, the first temperature range is the cold water immersion range, the second temperature range is the sub-high temperature softening range which is higher than room temperature but lower than the significant protein denaturation range, and the heating rate is a uniform and slow heating rate. It is worth noting that the cold water immersion range typically refers to 4-25℃, and the sub-high temperature softening range typically refers to 40-55℃. The rate of uniform and slow heating should be controlled at 0.1-0.5℃ per minute. This parameter design ensures that the relaxation of the bird's nest network structure is a gentle and controllable physical process, rather than a violent chemical denaturation, thus preserving a good substrate state for subsequent enzymatic hydrolysis.

[0030] In step S3, the endopeptidase is one or a combination of alkaline protease, neutral protease, or complex protease. It is worth noting that alkaline proteases (such as Alcalase) exhibit high activity and a wide range of cleavage sites under slightly alkaline conditions; neutral proteases function under mild conditions; and complex proteases are mixtures of various endopeptides with an even broader spectrum of activity. The specific choice should be based on a comprehensive consideration of factors such as the peptide profile of the target product, the pH conditions of the process, and cost.

[0031] In step S4, the exonuclease is one or a combination of two of aminopeptidase and carboxypeptidase; It is worth noting that aminopeptidase hydrolyzes peptide chains from the N-terminus (amino end), while carboxypeptidase hydrolyzes from the C-terminus (carboxyl end). Using both in combination can simultaneously "trim" both ends of the peptide chain, reducing chain length more rapidly and increasing the yield of small peptides.

[0032] In step S5, the glycoside hydrolase is an enzyme that can act on the glycan chains in the characteristic glycoprotein of bird's nest, and the specific telopeptidase is one of proline-specific endopeptidase and glutamate-specific endopeptidase. It is worth noting that the glycan chains in bird's nest glycoproteins are mostly O-linked or N-linked, and corresponding O-glycosidases or N-glycosidases can be selected. Proline-specific endonucleases (such as Protease P) can specifically cleave the peptide bonds formed at the carboxyl terminus of proline residues, and the resulting proline-terminated short peptides often have better stability (resistance to gastrointestinal protease hydrolysis) and potential biological activity.

[0033] The enzymatic hydrolysis reactions in steps S3, S4, and S5 are all carried out under constant pH conditions, and the pH of the reaction system is maintained by automatically adding acid or alkali solution. It is worth noting that pH is one of the most critical factors affecting enzyme activity and stability. Each enzyme has its optimal pH range. Maintaining a constant pH in the reaction system through automatic pH monitoring and a dropping system ensures that each stage of the enzymatic hydrolysis reaction proceeds under conditions of peak enzyme activity, thereby guaranteeing the stability and reproducibility of reaction rate and efficiency.

[0034] The enzymatic hydrolysis reaction times of steps S3, S4, and S5 are controlled independently, and the time of the subsequent enzymatic hydrolysis reaction is equal to or longer than the time of the previous enzymatic hydrolysis reaction. It is worth noting that independently controlling the time facilitates the optimization of the reaction extent at each stage. By specifying that the reaction time of the subsequent stage covers or exceeds that of the previous stage, it ensures that the substrate produced by the previous stage of enzymatic hydrolysis has sufficient time to be fully utilized by the enzyme in the subsequent stage. For example, after the second stage (exonuclease) reaction begins, the first stage (endonuclease) reaction may still be in progress. This time overlap design conforms to the kinetics of enzymatic hydrolysis and is conducive to the completeness of the reaction.

[0035] The centrifugation separation described in step S6 is high-speed centrifugation, and the pore size of the microfiltration membrane in step S7 is larger than the molecular weight cutoff of the ultrafiltration membrane. The molecular weight cutoff of the ultrafiltration membrane corresponds to the upper limit of the molecular weight of the target small molecule peptide. It is worth noting that high-speed centrifugation (e.g., 10,000-15,000 g) can effectively separate solid particles larger than micrometers. Microfiltration membranes typically have pore sizes of 0.1-0.2 micrometers and are used to remove submicrometer particles; the molecular weight cutoff (MWCO) of ultrafiltration membranes is selected based on the product design goals. For example, if the target is small peptides with a molecular weight below 1000 Daltons, then an ultrafiltration membrane with an MWCO of 1000 is selected. The order of microfiltration followed by ultrafiltration conforms to the conventional logic of membrane filtration, removing large particles first to protect the subsequent ultrafiltration membrane.

[0036] The temperature of the low-temperature vacuum concentration in step S7 is in the low-temperature range, and the vacuum degree is under medium vacuum conditions. It is worth noting that the low-temperature range generally refers to temperatures below 60℃, and moderate vacuum conditions typically refer to absolute pressures in the range of 10-100 kPa. Under these conditions, the boiling point of water decreases, allowing for rapid concentration at lower temperatures. This effectively prevents the destruction of heat-sensitive small molecule peptides by high temperatures, ensuring the bioactivity of the final product.

[0037] A bird's nest small molecule peptide product prepared by any of the processes described herein, characterized in that its molecular weight is mainly distributed in the target small molecular weight range and is rich in characteristic short peptide sequences generated by the action of specific telopeptidases. It is worth noting that through precise quantitative separation using ultrafiltration membranes, the molecular weight of the vast majority of peptides in the final product can be concentrated within a preset target range (e.g., <1000 Daltons or <500 Daltons), resulting in a more concentrated distribution. Simultaneously, due to the addition of specific telopeptidases such as proline-specific endopeptidases during the process, the final product will be rich in corresponding characteristic short peptide sequences (e.g., short peptides containing terminal proline). These characteristic peptides may be related to specific physiological functions of the product, constituting a distinctive feature that differentiates this product from ordinary enzymatic hydrolysates of bird's nest.

[0038] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0039] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A gradient temperature-controlled enzymatic hydrolysis process for softening small molecule peptides in bird's nest, characterized in that, The method includes: S1. Select bird's nest raw materials, clean them to remove visible impurities, and then use hydraulic shearing to perform preliminary crushing to obtain bird's nest fragment slurry; S2. The bird's nest fragment slurry is placed in a programmable temperature-controlled reaction vessel, first kept warm and soaked in the first temperature range, and then heated to the second temperature range at a constant heating rate and kept warm. The second temperature range is higher than the first temperature range. During this process, gentle stirring is continuously carried out to relax and initially swell the network structure of the bird's nest fragments. S3. After completing the gradient heating physical softening, adjust and maintain the temperature of the slurry in the third temperature range suitable for the action of endopeptidase, add endopeptidase to the slurry, and carry out the first stage of enzymatic hydrolysis under constant temperature conditions. The endopeptidase is used to randomly break the peptide bonds inside the bird's nest protein and initially degrade the macromolecular protein into polypeptide fragments. S4. After completing the first-stage enzymatic hydrolysis, without inactivation treatment, exonuclease is directly added to the reaction system, and the slurry temperature is adjusted and maintained in the fourth temperature range suitable for the action of exonuclease to carry out the second-stage enzymatic hydrolysis reaction. The exonuclease cuts from the end of the polypeptide fragment to further generate peptides with smaller molecular weight. S5. After completing the secondary enzymatic hydrolysis, maintain the system temperature or adjust it to the fifth temperature range, and simultaneously add glycoside hydrolase and specific telopeptidase to the reaction system to carry out the third stage of synergistic enzymatic hydrolysis. The glycoside hydrolase is used to weaken or cut the glycoside bonds that connect peptide chains in bird's nest, and the specific telopeptidase is used to specifically recognize and cut the peptide bonds at the ends of specific amino acid sequences exposed after being acted upon by the endopeptidase and / or exopeptidase. S6. After completing the three-stage synergistic enzymatic hydrolysis, the reaction system is rapidly heated to the inactivation temperature that completely inactivates all enzymes and maintained for a period of time. Then, the inactivated enzymatic hydrolysate is centrifuged and the supernatant is collected to obtain a primary solution containing bird's nest small molecule peptides. S7. The primary solution is filtered and separated sequentially through a microfiltration membrane and an ultrafiltration membrane system, and the permeate within the target molecular weight range is collected. Then, the permeate is concentrated under low temperature vacuum to obtain bird's nest small molecule peptide concentrate. S8. Spray-dry or vacuum freeze-dry the concentrated bird's nest small molecule peptide solution to obtain a powdered bird's nest small molecule peptide product.

2. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, In step S2, the first temperature range is the cold water immersion range, the second temperature range is the sub-high temperature softening range which is above room temperature but below the level of significant protein denaturation, and the heating rate is a uniform and slow heating rate.

3. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, In step S3, the endopeptide is one or a combination of alkaline protease, neutral protease, or complex protease.

4. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, In step S4, the exonuclease is one or a combination of two of aminopeptidase and carboxypeptidase.

5. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, In step S5, the glycoside hydrolase is an enzyme that can act on the glycan chains in the characteristic glycoproteins of bird's nest, and the specific telopeptidase is one of proline-specific endopeptidase and glutamate-specific endopeptidase.

6. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, The enzymatic hydrolysis reactions in steps S3, S4, and S5 are all carried out under constant pH conditions, and the pH of the reaction system is maintained by automatically adding acid or alkali solutions.

7. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, The enzymatic hydrolysis reaction times of steps S3, S4, and S5 are controlled independently, and the time of the subsequent enzymatic hydrolysis reaction is equal to or longer than the time of the previous enzymatic hydrolysis reaction.

8. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, The centrifugation separation in step S6 is high-speed centrifugation. The pore size of the microfiltration membrane in step S7 is larger than the molecular weight cutoff of the ultrafiltration membrane. The molecular weight cutoff of the ultrafiltration membrane corresponds to the upper limit of the molecular weight of the target small molecule peptide.

9. The enzymatic hydrolysis process for softening small molecule peptides in bird's nest based on gradient temperature control according to claim 1, characterized in that, The temperature of the low-temperature vacuum concentration in step S7 is in the low-temperature range, and the vacuum degree is under medium vacuum conditions.

10. A bird's nest small molecule peptide product prepared by the process described in any one of claims 1 to 9, characterized in that, Its molecular weight is mainly distributed in the target low molecular weight range and is rich in characteristic short peptide sequences produced by specific telopeptidases.

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