Chicken bone enzymatic hydrolysate rich in taste peptides, and preparation method and application in chicken soup products thereof

Abstract

The application discloses a chicken bone enzymatic hydrolysate rich in taste peptides and a preparation method and application in chicken soup products thereof, and relates to the field of food biotechnology. The application comprises the following steps: S100, fresh chicken bones are cooked under high temperature and high pressure, and chicken bone extract and bone residues are obtained through solid-liquid separation; S200, the bone residues are mixed with water, and two types of compound enzymes are added in sequence to perform directional enzymolysis through a step-by-step temperature control enzymolysis process; S300, after the enzymolysis liquid is inactivated, the chicken bone extract obtained in the step S100 is combined, three-stage membrane filtration is adopted, and the cut-off liquid with a molecular weight of 300-1500 Da is collected; and S400, after the cut-off liquid is concentrated and dried, the target chicken bone enzymatic hydrolysate is obtained. The application realizes efficient enrichment of 300-1500 Da taste peptides, the taste peptides in the combined liquid of the enzymatic hydrolysate and the extract account for >72% of the total peptide content, the taste activity of small molecule peptides is retained, and the product has rich chicken meat aroma and long-lasting thick feeling.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, specifically to a chicken bone enzymatic hydrolysate rich in flavor peptides, its preparation method, and its application in chicken soup products. Background Technology

[0002] Every year, a large amount of chicken carcasses and bones are generated as processing byproducts. These chicken bones are rich in nutrients such as protein, collagen, and minerals, but currently they are mainly used to produce feed or low-value-added products, resulting in low resource utilization. With the development of the food industry, how to efficiently utilize chicken bone resources to prepare high-value-added flavor bases has become a research hotspot.

[0003] Enzymatic hydrolysis is a process that uses proteases to break down proteins into small peptides and amino acids. It is widely used in the deep processing of animal and plant proteins. The small peptides produced from chicken bones after enzymatic hydrolysis are not only easily absorbed but also have flavor-enhancing properties, which can improve the umami, richness, and aftertaste of food.

[0004] However, existing technologies mainly focus on the total peptide content or specific functions (such as antioxidant and salty flavor) of enzymatic hydrolysates, with insufficient research on the targeted generation and molecular weight distribution control of flavor peptides. In particular, the dense structure of chicken bone protein makes efficient release and targeted cleavage difficult with single or simple stepwise enzymatic hydrolysis, resulting in low yields of flavor peptides and insufficient flavor intensity in the products. Furthermore, current processes often employ direct concentration and drying of the hydrolysate, failing to selectively enrich flavor peptides. This leads to higher levels of off-flavor peptides and non-flavor components in the products, limiting their application in high-end chicken soup products. Summary of the Invention

[0005] The purpose of this invention is to provide a chicken bone enzymatic hydrolysate rich in flavor peptides, its preparation method, and its application in chicken soup products. The enzymatic hydrolysate has a specific molecular weight distribution and flavor characteristics. The preparation method achieves efficient utilization of chicken bone protein and targeted enrichment of flavor peptides, which can improve the flavor quality of chicken soup products.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A method for preparing chicken bone enzymatic hydrolysate rich in flavor peptides includes the following steps:

[0008] Step S100: Fresh chicken bones are steamed under high temperature and pressure, and chicken bone extract and bone residue are obtained by solid-liquid separation;

[0009] Step S200: Add water to the above bone residue and mix it. Use a stepwise temperature-controlled enzymatic hydrolysis process to add two types of compound enzyme systems for targeted enzymatic hydrolysis.

[0010] Step S300: After inactivating the enzyme in the above enzymatic hydrolysate, combine it with the chicken bone extract obtained in step S100, and use a three-stage membrane filtration to collect the retentate with a molecular weight of 300~1500 Da.

[0011] S400. After concentrating and drying the above retentate, the target chicken bone enzymatic hydrolysate is obtained.

[0012] This invention proposes for the first time a synergistic technical solution for the entire process of gradient-directed enzymatic hydrolysis, rapid temperature-locked enzyme inactivation, and three-stage membrane filtration. In the first stage, a three-enzyme complex system of papain, bromelain, and alkaline protease is used to fully leverage the synergistic cleavage of different peptide bonds by these three endonucleases, efficiently opening the dense structure of collagen. In the second stage, a combination of flavor protease and trypsin is used, along with a dynamic pH control strategy, to achieve the targeted release of flavor peptides. This two-stage enzymatic hydrolysis design, which first opens the backbone and then releases the flavor, combined with rapid temperature-locked enzyme inactivation to protect the target peptides, and three-stage membrane filtration for precise enrichment of the 300-1500 Da molecular weight range, forms a complete technical closed loop. The chicken bone hydrolysate obtained by this invention contains over 72% flavor peptides with a molecular weight of 300-1500 Da, a figure far exceeding the 40%-50% of conventional enzymatic hydrolysis processes. The product is rich in characteristic flavor peptide sequences such as Ala-Glu-Asp, giving it a rich chicken aroma and a lasting aftertaste.

[0013] The various steps in this invention exhibit close synergy, collectively ensuring the high quality of the final product. Step S100, high-pressure cooking, not only extracts soluble flavor precursors from chicken bones but, more importantly, moderately denatures and loosens the collagen in the bone fragments, exposing numerous enzyme cleavage sites for subsequent enzymatic hydrolysis. Step S200, gradient enzymatic hydrolysis, builds upon this foundation. In the first stage, a combination of three endonucleases works synergistically to efficiently degrade large protein molecules into medium-length peptides. In the second stage, a combination of flavor protease and trypsin, combined with dynamic pH control, directionally releases flavor peptides from the ends of these peptides. This two-stage, continuous process, with enzymes remaining inactive in between, maximizes the synergistic effect of the enzymes. Step S300, rapid temperature-locked enzyme inactivation, precisely controls the heating rate, isothermal time, and cooling rate to ensure complete enzyme inactivation while maximizing the preservation of the generated flavor peptides. Subsequently, the extract is combined with the chicken bone extract and filtered through a three-stage membrane system, achieving precise enrichment of the flavor peptides. Step S400, low-temperature vacuum concentration and spray drying, further preserve the product's flavor and activity.

[0014] Further, in step S100, fresh chicken bones are washed and crushed, water is added, and the mixture is steamed at 95~125℃ and 0.1~0.2MPa for 1~3 hours to obtain chicken bone extract and bone residue through solid-liquid separation; the mass ratio of chicken bones to water is 1:1~3; in step S200, the mass ratio of bone residue to water is 1:2~4.

[0015] Step S100, through the coordinated control of temperature, material-liquid ratio, and time, not only obtained chicken bone extract, but also prepared high-quality bone residue for the gradient enzymatic hydrolysis in step S200. Through precise cooking conditions, the collagen structure in the bone residue was appropriately opened and the enzyme cleavage sites were fully exposed, thereby creating the best substrate state for the subsequent synergistic effect of papain, bromelain, and alkaline protease.

[0016] Furthermore, the stepwise temperature-controlled enzymatic hydrolysis process includes the following:

[0017] First stage: Adjust the pH to 6.5~7.5, raise the temperature to 50~60℃, add complex protease I composed of endopeptide protease, and enzymatically hydrolyze for 1~2 hours;

[0018] Second stage: Adjust the pH to 6.0~7.0, cool to 45~55℃, add complex protease II composed of flavor protease and trypsin, and continue enzymatic hydrolysis for 2~4 hours;

[0019] In the second stage, the pH was maintained at 6.8-7.0 for the first hour, and then gradually decreased to 6.2-6.5.

[0020] Conventional stepwise enzymatic hydrolysis often only considers the optimal temperature or pH differences of different enzymes, adding them sequentially in chronological order, essentially still a superposition of independent reactions. This invention sets the first stage as the skeleton-opening stage, employing a three-enzyme complex system of papain, bromelain, and alkaline protease. These three endonucleases act on different peptide bond sites in collagen, synergistically disrupting its dense helical structure and exposing numerous cleavage sites. The purpose of the first stage is not to achieve the depth of hydrolysis, but rather to create the optimal substrate state for the targeted release in the second stage.

[0021] The second stage employs a combination system of flavor protease and trypsin. Flavor protease has both endopeptidation and exopeptidation activities, and can release flavor amino acids from the peptide chain ends, while trypsin specifically cleaves specific peptide bonds. The synergistic effect of the two can directionally enrich small molecule flavor peptides.

[0022] In the second stage, the present invention introduces dynamic pH control. The pH is maintained at 6.8-7.0 for the first hour to maintain the high activity of trypsin, and then gradually reduced to 6.2-6.5 to stimulate the optimal function of flavor protease. This pH change strategy of fast first and slow later, and cleavage first and release later, allows the two enzymes to perform their respective functions and take turns to act at different stages, so as to achieve the precise release of flavor peptides.

[0023] Furthermore, the complex protease I is composed of papain, bromelain, and alkaline protease in a mass ratio of 1~2:1~2:1; the mass ratio of flavor protease to trypsin in the complex protease II is 2~4:1.

[0024] Further, in step S300, the enzyme inactivation method includes the following: rapidly heating to 90~100℃ at a heating rate of ≥5℃ / min, maintaining the temperature at the enzyme inactivation temperature for 5~15 minutes, and then rapidly cooling to ≤40℃ at a cooling rate of ≥8℃ / min.

[0025] Rapid heating (≥5℃ / min) is to shorten the residence time of the enzyme solution in the optimal temperature range of the protease, so as to avoid excessive degradation of the generated flavor peptides; precise isothermal control (5~15 minutes) is to shorten the heat treatment time as much as possible while ensuring complete denaturation and inactivation of the enzyme protein; rapid cooling (≥8℃ / min) is to quickly pass through the temperature-sensitive zone and "lock in" the molecular structure and flavor activity of the flavor peptides.

[0026] Furthermore, the tertiary membrane filtration includes the following:

[0027] The first stage uses a ceramic membrane with a pore size of 0.1~0.2μm for microfiltration and collects the permeate; the second stage passes the permeate from the first stage through a nanofiltration membrane with a molecular weight cutoff of 1500Da and collects the permeate.

[0028] The third stage involves passing the second-stage permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da, collecting the 300-1500 Da cutoff fraction as the target product.

[0029] This invention is the first to construct a three-stage membrane system consisting of ceramic microfiltration, 1500Da nanofiltration permeation, and 300Da nanofiltration retention, achieving an excellent effect of target peptide proportion ≥70%. The first-stage ceramic membrane (0.1~0.2μm) removes large colloid particles and unhydrolyzed proteins, creating a clean environment for subsequent fine separation; the second-stage 1500Da nanofiltration membrane collects the permeate, actively discarding large molecular peptides (>1500Da), which often have low flavor contribution or even bitterness; the third-stage 300Da nanofiltration membrane retains the 300~1500Da component as the target product, while allowing free amino acids and small molecular peptides <300Da to permeate.

[0030] Furthermore, the concentration is a vacuum concentration, wherein the vacuum concentration temperature is 50~55℃, the vacuum degree is 0.08~0.09MPa, and the final solid content is 35~40%.

[0031] This invention employs a vacuum concentration temperature of 50-55℃, which significantly reduces the degradation risk of heat-sensitive flavor peptides compared to the high temperatures required for atmospheric pressure evaporation (typically 80-100℃). Flavor peptides are mostly small molecule peptides, and their spatial conformation and flavor activity are highly sensitive to temperature. Excessively high concentration temperatures may cause peptide chains to coil, aggregate, or even undergo Maillard reactions with reducing sugars, thereby weakening their umami and rich flavor perception. The vacuum environment reduces the system pressure to 0.08-0.09 MPa, allowing water to vaporize rapidly at low temperatures, shortening the concentration time and avoiding the cumulative damage to flavor peptides caused by prolonged heating.

[0032] The drying process is spray drying, with an inlet air temperature of 120~140℃, an outlet air temperature of 80~90℃, and a feed rate of 50~70mL / min.

[0033] This invention employs a parameter combination of inlet air temperature of 120-140℃ and outlet air temperature of 80-90℃ to achieve instantaneous drying of materials. The concentrated liquid is dispersed into tiny droplets by an atomizer, resulting in a dramatic increase in specific surface area. Moisture evaporation occurs instantly upon contact with hot air, and the droplet temperature is maintained at the wet-bulb temperature (approximately 45-55℃), far lower than the inlet air temperature, thus avoiding thermal damage to flavor peptides. Precise control of the outlet air temperature at 80-90℃ ensures that the final product moisture content is below 4%, inhibiting microbial growth and preventing peptide molecular structure collapse due to over-drying. The matching of the feed rate of 50-70 mL / min with the inlet and outlet air temperatures allows the droplets sufficient residence time within the drying tower to complete dehydration, while preventing wall adhesion or overheating.

[0034] In the final powdered product, the flavor peptides are locked in an amorphous solid matrix with extremely low molecular mobility, effectively preventing aggregation and inactivation during storage. At the same time, the powder has excellent solubility and can be quickly reconstituted in water, restoring the original peptide molecular distribution and flavor activity of the concentrate.

[0035] A chicken bone enzymatic hydrolysate rich in flavor peptides prepared by the preparation method described above, wherein flavor peptides with a molecular weight of 300-1500 Da account for >72% of the total peptide content.

[0036] The application of the chicken bone hydrolysate rich in flavor peptides in the preparation of chicken soup products.

[0037] Furthermore, the chicken soup products include concentrated chicken soup, ready-to-eat chicken soup, chicken soup flavored compound seasonings, or chicken essence.

[0038] Compared with the prior art, the beneficial effects of the present invention are:

[0039] This invention utilizes a synergistic process involving gradient-directed enzymatic hydrolysis, rapid temperature-locked enzyme inactivation, and three-stage membrane fractionation to achieve highly efficient enrichment of flavor peptides ranging from 300 to 1500 Da. In the combined hydrolysate and extract, flavor peptides with a molecular weight of 300-1500 Da account for >72% of the total peptide content, preserving the flavor activity of small-molecule peptides while avoiding the formation of bitter peptides due to excessive hydrolysis. The product possesses a rich chicken aroma and a lingering aftertaste. Furthermore, the chicken bone hydrolysate prepared by this invention also exhibits antioxidant bioactivity, with its DPPH free radical scavenging IC50 value reduced to 0.32 mg / mL. Detailed Implementation

[0040] The present invention will now be further described.

[0041] Example 1

[0042] A method for preparing chicken bone enzymatic hydrolysate rich in flavor peptides includes the following steps:

[0043] Step S100: After washing and crushing the fresh chicken bones, add water and cook at 110℃ and 0.15MPa for 2 hours. Separate the solid and liquid to obtain chicken bone extract and bone residue; the mass ratio of chicken bones to water is 1:2.

[0044] Step S200: Add water to the above bone residue and mix it with water at a mass ratio of 1:3. Use a stepwise temperature-controlled enzymatic hydrolysis process and add two types of compound enzyme systems for targeted enzymatic hydrolysis.

[0045] The stepwise temperature-controlled enzymatic hydrolysis process includes the following:

[0046] First stage: Adjust pH to 7, raise temperature to 55℃, add complex protease I composed of endopeptides, and enzymatically hydrolyze for 1.5 hours; the complex protease I is composed of papain, bromelain and alkaline protease in a mass ratio of 1.5:1.5:1.

[0047] Second stage: Cool down to 50℃, add complex protease II composed of flavor protease and trypsin, and continue enzymatic hydrolysis for 3 hours. Keep the pH at 6.9 for the first hour, and then gradually decrease the pH to 6.3. The mass ratio of flavor protease to trypsin in the complex protease II is 3:1.

[0048] Step S300: After inactivating the enzyme in the above enzymatic hydrolysate, combine it with the chicken bone extract obtained in step S100, and use a three-stage membrane filtration to collect the retentate with a molecular weight of 300~1500 Da.

[0049] The enzyme inactivation method includes the following steps: rapidly heating to 95°C at a heating rate of 6°C / min, maintaining the temperature at this inactivation temperature for 10 minutes, and then rapidly cooling to 38°C at a cooling rate of 9°C / min.

[0050] The three-stage membrane filtration includes the following:

[0051] The first stage uses a ceramic membrane with a pore size of 0.12 μm for microfiltration and collects the permeate; the second stage passes the permeate from the first stage through a nanofiltration membrane with a molecular weight cutoff of 1500 Da and collects the permeate.

[0052] The third stage involves passing the second-stage permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da, collecting the 300-1500 Da cutoff fraction as the target product.

[0053] S400. After concentrating and drying the above retentate, the target chicken bone enzymatic hydrolysate is obtained.

[0054] The concentration is a vacuum concentration, wherein the vacuum concentration temperature is 53°C, the vacuum degree is 0.085 MPa, and the final solid content is 38%.

[0055] The drying process is spray drying, with an inlet air temperature of 130°C, an outlet air temperature of 85°C, and a feed rate of 60 mL / min.

[0056] Example 2

[0057] A method for preparing chicken bone enzymatic hydrolysate rich in flavor peptides includes the following steps:

[0058] Step S100: After washing and crushing the fresh chicken bones, add water and cook at 95℃ and 0.1MPa for 1 hour. Separate the solid and liquid to obtain chicken bone extract and bone residue; the mass ratio of chicken bones to water is 1:1.

[0059] Step S200: Add water to the above bone residue and mix it with water at a mass ratio of 1:2. Use a stepwise temperature-controlled enzymatic hydrolysis process and add two types of compound enzyme systems for targeted enzymatic hydrolysis.

[0060] The stepwise temperature-controlled enzymatic hydrolysis process includes the following:

[0061] First stage: Adjust the pH to 6.5, raise the temperature to 50°C, add complex protease I composed of endopeptides, and enzymatically hydrolyze for 1 hour; the complex protease I is composed of papain, bromelain and alkaline protease in a mass ratio of 1:1:1.

[0062] Second stage: Cool down to 45℃, add complex protease II composed of flavor protease and trypsin, and continue enzymatic hydrolysis for 2 hours. Keep the pH at 6.8 for the first hour, and then gradually decrease the pH to 6.2. The mass ratio of flavor protease to trypsin in the complex protease II is 2:1.

[0063] Step S300: After inactivating the enzyme in the above enzymatic hydrolysate, combine it with the chicken bone extract obtained in step S100, and use a three-stage membrane filtration to collect the retentate with a molecular weight of 300~1500 Da.

[0064] The enzyme inactivation method includes the following steps: rapidly heating to 90°C at a heating rate of 5°C / min, maintaining the temperature at this inactivation temperature for 5 minutes, and then rapidly cooling to 40°C at a cooling rate of 8°C / min.

[0065] The three-stage membrane filtration includes the following:

[0066] The first stage uses a ceramic membrane with a pore size of 0.1 μm for microfiltration and collects the permeate; the second stage passes the permeate from the first stage through a nanofiltration membrane with a molecular weight cutoff of 1500 Da and collects the permeate.

[0067] The third stage involves passing the second-stage permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da, collecting the 300-1500 Da cutoff fraction as the target product.

[0068] S400. After concentrating and drying the above retentate, the target chicken bone enzymatic hydrolysate is obtained.

[0069] The concentration is a vacuum concentration, wherein the vacuum concentration temperature is 50°C, the vacuum degree is 0.08 MPa, and the final solid content is 35%.

[0070] The drying process is spray drying, with an inlet air temperature of 120°C, an outlet air temperature of 80°C, and a feed rate of 50 mL / min.

[0071] Example 3

[0072] A method for preparing chicken bone enzymatic hydrolysate rich in flavor peptides includes the following steps:

[0073] Step S100: After washing and crushing the fresh chicken bones, add water and cook at 125℃ and 0.2MPa for 3 hours. Separate the solid and liquid to obtain chicken bone extract and bone residue; the mass ratio of chicken bones to water is 1:1~3.

[0074] Step S200: Add water to the above bone residue and mix it with water at a mass ratio of 1:4. Use a stepwise temperature-controlled enzymatic hydrolysis process and add two types of compound enzyme systems for targeted enzymatic hydrolysis.

[0075] The stepwise temperature-controlled enzymatic hydrolysis process includes the following:

[0076] First stage: Adjust the pH to 7.5, raise the temperature to 60℃, add complex protease I composed of endopeptides, and enzymatically hydrolyze for 2 hours; the complex protease I is composed of papain, bromelain and alkaline protease in a mass ratio of 2:2:1.

[0077] Second stage: Cool down to 55℃, add complex protease II composed of flavor protease and trypsin, and continue enzymatic hydrolysis for 4 hours. Keep the pH at 7.0 for the first hour, and then gradually decrease the pH to 6.5. The mass ratio of flavor protease to trypsin in the complex protease II is 4:1.

[0078] Step S300: After inactivating the enzyme in the above enzymatic hydrolysate, combine it with the chicken bone extract obtained in step S100, and use a three-stage membrane filtration to collect the retentate with a molecular weight of 300~1500 Da.

[0079] The enzyme inactivation method includes the following steps: rapidly heating to 100°C at a heating rate of 7°C / min, maintaining the temperature at the enzyme inactivation temperature for 15 minutes, and then rapidly cooling to 35°C at a cooling rate of 10°C / min.

[0080] The three-stage membrane filtration includes the following:

[0081] The first stage uses a ceramic membrane with a pore size of 0.2 μm for microfiltration and collects the permeate; the second stage passes the permeate from the first stage through a nanofiltration membrane with a molecular weight cutoff of 1500 Da and collects the permeate.

[0082] The third stage involves passing the second-stage permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da, collecting the 300-1500 Da cutoff fraction as the target product.

[0083] S400. After concentrating and drying the above retentate, the target chicken bone enzymatic hydrolysate is obtained.

[0084] The concentration is a vacuum concentration, wherein the vacuum concentration temperature is 55°C, the vacuum degree is 0.09 MPa, and the final solid content is 40%.

[0085] The drying process is spray drying, with an inlet air temperature of 140°C, an outlet air temperature of 90°C, and a feed rate of 70 mL / min.

[0086] Comparative Example 1

[0087] In step S200, complex protein I and complex protein II are added simultaneously and enzymatically hydrolyzed at pH 7.0 and 55°C for 4.5 hours. The remaining parameters and steps are the same as in Example 1.

[0088] Comparative Example 2

[0089] In step S300, enzyme inactivation was performed using a conventional water bath heating method, with the temperature increased to 95°C at a rate of approximately 2°C / min, held at that temperature for 10 minutes, and then allowed to cool naturally. The remaining parameters and steps were the same as in Example 1.

[0090] Comparative Example 3

[0091] In step S300, tertiary membrane filtration is not used. The material combined with the chicken bone extract after enzyme inactivation is directly passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate is collected as the target product. The remaining parameters and steps are the same as in Example 1.

[0092] Comparative Example 4

[0093] In step S200, the pH was kept constant at 7.0 throughout the second stage. All other parameters and steps were the same as in Example 1.

[0094] Comparative Example 5

[0095] In step S200, complex protease I is a mixture of neutral protease and papain in a 1:1 ratio, and complex protease II is a flavor protease added alone. The enzymatic hydrolysis time, temperature and other conditions, and the remaining steps are the same as in Example 1.

[0096] Comparative Example 6

[0097] A method for preparing chicken bone enzymatic hydrolysate includes the following steps:

[0098] 1) Clean and crush the chicken carcass, steam it with water vapor, add an aqueous solution containing 0.7% citric acid, soak it at 50℃ for 8 hours, and filter to separate the leachate and residue.

[0099] 2) Take the extract and add 2 kg of papain, 2 kg of trypsin, and 1 kg of aminopeptidase to every 1000 kg of chicken bones at 50℃ for 3 hours of enzymatic hydrolysis; then add 5.5 kg of flavor protease and continue enzymatic hydrolysis for 6 hours.

[0100] 3) After completion, quickly raise the temperature to 85℃ and hold for 15 minutes to inactivate the enzyme;

[0101] 4) Cool to room temperature, adjust pH to 7.0, inoculate with Bifidobacterium at a 5% inoculum, and ferment anaerobically for 5 hours;

[0102] 5) Sterilize by heating, centrifuge to remove bacteria and other impurities, concentrate and dry the supernatant to obtain chicken protein peptides.

[0103] Comparative Example 7

[0104] In step S200, the complex protease I is a mixture of papain, bromelain, and alkaline protease in a mass ratio of 0.5:1.5:1. The remaining parameters and steps are the same as in Example 1.

[0105] Comparative Example 8

[0106] In step S200, the complex protease I is a mixture of papain, bromelain, and alkaline protease in a mass ratio of 1.5:0.5:1. The remaining parameters and steps are the same as in Example 1.

[0107] Comparative Example 9

[0108] In step S200, the complex protease I is composed of papain, bromelain, and alkaline protease in a mass ratio of 1.5:1.5:1.8. The remaining parameters and steps are the same as in Example 1.

[0109] Comparative Example 10 (originally 11)

[0110] In step S200, the complex protease II is a mixture of flavor protease and trypsin in a 1:1 mass ratio. The remaining parameters and steps are the same as in Example 1.

[0111] Comparative Example 11 (originally 13)

[0112] In step S200, the complex protease II is a mixture of flavor protease and trypsin at a mass ratio of 5:1. The remaining parameters and steps are the same as in Example 1.

[0113] Comparative Example 12 (originally 14)

[0114] In step S200, complex protease I is a mixture of papain, bromelain, and alkaline protease in a mass ratio of 0.8:2.2:0.8; complex protease II is a mixture of flavor protease and trypsin in a mass ratio of 1.5:1. The remaining parameters and steps are the same as in Example 1.

[0115] The properties of the chicken bone enzymatic hydrolysates prepared by the methods in Examples 1-3 and Comparative Examples 1-12 are shown in Table 1.

[0116] Table 1. Performance of chicken bone enzymatic hydrolysates prepared by the methods of Examples 1-3 and Comparative Examples 1-12

[0117]

[0118] Note: The standard formula for calculating protein recovery rate (or nitrogen recovery rate) is: Protein recovery rate (%) = (Protein content in enzymatic hydrolysis supernatant / Total protein content in raw material) × 100%, which is determined by the Kjeldahl method.

[0119] The target peptide percentage refers to the percentage of peptides with a molecular weight between 300 and 1500 Da in the combined solution of the enzymatic hydrolysate and chicken bone extract before entering the three-stage membrane filtration system, relative to the total peptide mass in the combined solution.

[0120] The Ala-Glu-Asp content refers to the mass (mg) of Ala-Glu-Asp contained in the combined solution of the enzymatic hydrolysate and chicken bone extract in step S200 (after enzymatic hydrolysis and before tertiary membrane filtration in step S300).

[0121] As shown in Table 1, Example 1 achieved the following optimal results: target peptide percentage of 78.5%, Ala-Glu-Asp content of 2.8 mg / g, DPPH IC50 of 0.32 mg / mL, degree of hydrolysis of 14.2%, and protein recovery rate of 92.6%. These indicators demonstrate the synergistic effect of the present invention.

[0122] In Example 2 (lower limit process), the target peptide ratio was 72.3%, and in Example 3 (upper limit process), the target peptide ratio was 75.8%. Both maintained excellent performance within the process range, demonstrating the robustness and broad applicability of the technical solution of the present invention.

[0123] In Comparative Example 1, the target peptide accounted for 61.2%, significantly lower than in Example 1. Simultaneous enzymatic hydrolysis caused interference between the first-stage endonuclease and the second-stage flavor enzymes and trypsin in the same system. The flavor enzymes prematurely cleaved the already generated peptides, disrupting the phased design of backbone opening and flavor release, resulting in a significant decrease in the yield of the target peptide.

[0124] In Comparative Example 2, the target peptide accounted for 69.3%, lower than in Example 1. Existing technologies generally consider enzyme inactivation only requires reaching a certain temperature, with little attention paid to the heating and cooling rates. This invention, for the first time, quantifies the enzyme inactivation process as a rapid temperature-locking process with a heating rate ≥5℃ / min and a cooling rate ≥8℃ / min. By shortening the enzyme's residence time in the active zone, the retention rate of flavor peptides is effectively improved.

[0125] Comparative Example 3 showed the highest protein recovery rate (94.3%), but the lowest proportion of the target peptide (48.6%). Example 1 showed a protein recovery rate of 92.6% and a target peptide proportion of 78.5%. This demonstrates that the three-stage membrane filtration process accurately enriches the target peptide while actively discarding some non-target components (>1500Da macromolecular peptides and <300Da small molecule peptides), achieving a balance between quality and yield.

[0126] The higher the proportion of the target peptide, the lower the DPPH IC50 value and the stronger the antioxidant activity. Example 1 IC50 = 0.32 mg / mL, Comparative Example 3 IC50 = 0.73 mg / mL. This is because the 300-1500 Da peptides are rich in aromatic amino acids (tyrosine, tryptophan, phenylalanine) with antioxidant activity; precise enrichment enhances antioxidant function.

[0127] In Comparative Example 4, the pH was kept constant at 7.0 throughout the second stage. The activity of flavor proteases was limited under alkaline conditions, and their ability to release flavor amino acids from the peptide chain ends was weakened. Although trypsin had high activity at pH 7.0, it lacked the synergistic effect of flavor proteases, resulting in insufficient overall flavor release.

[0128] In Comparative Example 5, the target peptide accounted for 63.8%, significantly lower than in Example 1. The first stage used a combination of neutral protease and papain, lacking bromelain. Bromelain has a unique ability to open the helical structure of collagen; its absence resulted in insufficient loosening of the dense collagen in the bone residue, leading to incomplete exposure of the cleavage sites. The second stage used only flavor protease, lacking the specific cleavage effect of trypsin, resulting in insufficient flavor release.

[0129] In Comparative Example 6, the target peptide accounted for 60.7%, and the protein recovery rate was the lowest at 80.2%. Although this process employs stepwise enzymatic hydrolysis, it suffers from the following problems: citric acid soaking pretreatment may alter the isoelectric point of the protein, affecting hydrolysis efficiency; the enzyme combination lacks bromelain and alkaline protease, resulting in insufficient scaffold opening; and multiple treatment steps lead to protein loss, resulting in a recovery rate of only 80.2%. The overall process is complex, yet the yield of the target peptide is low, validating the superiority of the precise enzymatic hydrolysis-rapid enzyme inactivation-membrane enrichment route of this invention.

[0130] In Comparative Example 7, the target peptide accounted for 68.3%. The low proportion of papain resulted in insufficient opening of the backbone in the first stage, insufficient cleavage of peptide bonds containing phenylalanine and tyrosine, incomplete exposure of enzyme cleavage sites, and subsequent inhibition of flavor release. The proportion of the target peptide was lower than that in Example 1.

[0131] In Comparative Example 8, the target peptide accounted for 67.5%. The low proportion of bromelain weakened the ability to open the collagen helical structure, and the dense collagen in the bone residue was not fully loosened, resulting in incomplete exposure of the enzyme cleavage sites and a decrease in the yield of flavor peptides.

[0132] In Comparative Example 9, the target peptide accounted for 67.5%. The proportion of alkaline protease was higher than the upper limit, resulting in excessive endopeptidation in the first stage, producing a large number of medium-length peptides. These peptides were further degraded into small peptides and free amino acids of <300 Da in the second stage, while the proportion of the target 300~1500 Da peptides decreased.

[0133] In Comparative Example 10, the target peptide accounted for 70.5%, and the low proportion of flavor protease resulted in insufficient exonuclease activity, weakening the ability to release flavor amino acids from the peptide chain ends. The content of the characteristic flavor peptide Ala-Glu-Asp was only 2.0 mg / g, which was lower than 2.8 mg / g in Example 1.

[0134] In Comparative Example 11, the target peptide accounted for 72.8%. Although the high proportion of flavor protease resulted in sufficient release of flavor amino acids, the specific cleavage effect of trypsin was relatively weakened, and the generation of some flavor peptide sequences that require trypsin cleavage to be exposed was hindered.

[0135] In Comparative Example 12, the target peptide accounted for 63.5%. Multiple deviations combined to result in insufficient opening of the backbone and incomplete release of flavor. The synergistic effect of each step was destroyed, and the overall effect was significantly lower than that of Example 1, which verified the overall synergistic effect of the enzyme ratio combination of the present invention.

[0136] The following examples demonstrate the effect of the chicken bone hydrolysate rich in flavor peptides of the present invention in chicken soup products.

[0137] Example 4

[0138] Preparation of concentrated chicken broth: Ingredients: 400g fresh chicken carcass, cleaned and chopped; 150g chicken breast, chopped; 30g chicken bone hydrolysate, prepared in Example 1; 12g salt; 5g ginger; 5g scallion; 800g water.

[0139] Preparation method:

[0140] 1. Clean the chicken carcass and chicken breast thoroughly. Chop the chicken carcass into pieces and cut the chicken breast into pieces.

[0141] 2. Place the chicken carcass and chicken breast into a pot, add 8 parts water, bring to a boil over high heat, and skim off any foam.

[0142] 3. Add ginger and scallions, and simmer over low heat for 2 hours;

[0143] 4. Filter through a 100-mesh sieve to obtain approximately 650g of chicken broth;

[0144] 5. While still hot, add the chicken bone hydrolysate and salt of this invention, and stir until dissolved and homogeneous;

[0145] 6. Vacuum concentrate until the solid content is 35% (55℃, 0.08MPa), yielding approximately 280g of concentrated chicken broth;

[0146] 7. Fill and sterilize at 121℃ for 20 minutes to obtain the finished concentrated chicken soup.

[0147] Comparative Example 4: Prepared according to the same formula and steps, but without adding the chicken bone hydrolysate of the present invention, and replacing it with an equal amount of water.

[0148] Sensory evaluation: The concentrated chicken broth was diluted at a ratio of 1:5 (concentrated broth: water) and then blind-tested by 20 professional sensory evaluators. A 10-point scale (comprehensive evaluation of umami intensity, body, and aftertaste persistence) was used, and the results are shown in Table 2.

[0149] Table 2 Sensory Evaluation Results

[0150]

[0151] The concentrated chicken broth with the added chicken bone hydrolysate of this invention showed a 1.9-point increase in sensory score compared to control example 1, representing a relative improvement of 26%. This indicates that the hydrolysate of this invention has a significant flavor-enhancing effect on concentrated chicken broth.

[0152] Example 5

[0153] Preparation of ready-to-eat chicken soup:

[0154] Ingredients: 300g fresh chicken carcass, cleaned and chopped; 200g chicken breast, cut into pieces; 20g chicken bone hydrolysate, prepared in Example 1; 8g salt; 5g ginger; 10g red dates; 5g goji berries; 800g water.

[0155] Preparation method:

[0156] 1. Clean the chicken carcass and chicken thigh meat thoroughly. Chop the chicken carcass into pieces and cut the chicken thigh meat into pieces.

[0157] 2. Place the chicken carcass and chicken thigh meat into a pot, add 800g of water, bring to a boil over high heat, and skim off the foam.

[0158] 3. Add ginger, red dates, and goji berries, and simmer over low heat for 1.5 hours;

[0159] 4. Filter through a 100-mesh sieve to obtain approximately 700g of chicken broth;

[0160] 5. Add the chicken bone hydrolysate and salt of this invention, and stir until dissolved and homogeneous;

[0161] 6. Fill and sterilize at 121℃ for 20 minutes to obtain ready-to-eat chicken soup.

[0162] Compare with Example 2

[0163] Prepared according to the same formula and steps, but without adding the chicken bone hydrolysate of the present invention, and replacing it with an equal amount of water.

[0164] Sensory evaluation: The ready-to-eat chicken soup was directly heated and then blind-tested by 20 professional sensory evaluators. A 10-point scale (comprehensive evaluation of umami intensity, body, and aftertaste persistence) was used, and the results are shown in Table 2.

[0165] Table 3 Sensory evaluation results

[0166]

[0167] As shown in Table 3, the sensory score of the ready-to-eat chicken soup with the added chicken bone hydrolysate of the present invention was 1.5 points higher than that of Control Example 2, representing a relative increase of 20%. The chicken soup of Example 5, when consumed at room temperature, had no obvious greasiness and a rich flavor, superior to traditionally boiled chicken soup. This indicates that the enzymatic hydrolysate of the present invention also plays a significant flavor-enhancing role in ready-to-eat chicken soup.

[0168] Example 6

[0169] Preparation of chicken broth flavored compound seasoning:

[0170] Chicken bone enzymatic hydrolysate, 5g, prepared according to the method in Example 1; salt 45g; white sugar 15g; monosodium glutamate 20g; disodium inosinate 1g; onion powder 3g; garlic powder 2g; white pepper powder 1g; starch 8g.

[0171] Preparation method:

[0172] 1. Pass all raw materials through a 60-mesh sieve to ensure uniform fineness;

[0173] 2. Place in a 3D mixer and mix for 20 minutes;

[0174] 3. After passing inspection, the product is packaged to obtain chicken soup flavored compound seasoning.

[0175] Compared with Example 3, the main ingredients of commercially available chicken soup seasoning are: salt, monosodium glutamate, white sugar, starch, chicken powder, and spices.

[0176] Comparative Example 4 was prepared according to the same formula, but without adding the chicken bone hydrolysate of the present invention, and instead using an equal amount of starch.

[0177] Application testing: 1g of each seasoning sample was dissolved in 100mL of boiling water, and then blind-tested by 20 professional sensory evaluators. The results are shown in Table 4.

[0178] Table 4 Sensory Evaluation Results

[0179]

[0180] As shown in Table 4, the compound seasoning with added chicken bone hydrolysate of the present invention has a significantly better sensory score than commercially available products and the control example without added hydrolysate. The addition of flavor peptides to the compound seasoning of the present invention enhances the richness of the base flavor, and the seasoning of the present invention, at the same dosage, increases the chicken soup flavor intensity by 17% compared to commercially available products.

[0181] Example 7

[0182] Preparation of chicken bouillon seasoning:

[0183] 8g of chicken bone enzymatic hydrolysate, prepared according to the method in Example 1; 42g of salt; 12g of white sugar; 30g of monosodium glutamate; 0.5g of disodium inosinate; 5.5g of corn starch; 0.5g of turmeric powder for coloring; and an appropriate amount of water for granulation.

[0184] Preparation method:

[0185] 1. Mix all powdered ingredients thoroughly;

[0186] 2. Add an appropriate amount of water to make a soft material, and granulate it by passing it through a 12-mesh sieve;

[0187] 3. Dry at 60℃ until moisture content ≤3%;

[0188] 4. Granulate, sieve, and package to obtain the finished chicken essence.

[0189] Example 5 is a commercially available chicken essence: a well-known brand of commercially available chicken essence.

[0190] Compare with Example 6

[0191] Chicken essence was prepared using the same formula as the chicken bone enzymatic hydrolysate (48.6% target peptide) prepared by Comparative Example 3 (single-stage ultrafiltration).

[0192] Compare with Example 7

[0193] Chicken bone hydrolysate prepared using the same method as Comparative Example 9 was used to prepare chicken essence according to the same formula.

[0194] Application testing: 2g of each chicken essence sample was dissolved in 200mL of boiling water, and then blind-tested by 20 professional sensory evaluators. The results are shown in Table 5.

[0195] Table 5 Sensory Evaluation Results

[0196]

[0197] As shown in Table 5, the chicken essence containing the chicken bone hydrolysate of the present invention had a significantly better sensory score than Control Example 5 and Control Example 6.

[0198] Compared with the chicken bone hydrolysate prepared by the method of Example 9 in Comparative Example 7, the proportion of alkaline protease in Comparative Example 9 was too high, resulting in excessive hydrolysis. Although the degree of hydrolysis was improved, more bitter peptides and peptides with too small a molecular weight were produced. The proportion of the target 300~1500 Da peptides decreased, and the sensory quality deteriorated.

[0199] At the same addition amount, the chicken broth flavor intensity of the chicken essence of the present invention is 13% higher than that of the enzymatic hydrolysate prepared by the method of Comparative Example 3, and the aftertaste is more lasting.

[0200] Sensory evaluation method: A double-blind method was used to score the samples. The scores were comprehensively evaluated from three dimensions: umami intensity, body, and aftertaste persistence. Each sample was evaluated three times, and the average value was taken.

Claims

1. A method for preparing chicken bone enzymatic hydrolysate rich in flavor peptides, characterized in that, Includes the following steps: S100. Fresh chicken bones are steamed under high temperature and pressure, and chicken bone extract and bone residue are obtained by solid-liquid separation. S200. Add water to the above bone residue and mix it. Use a stepwise temperature-controlled enzymatic hydrolysis process to add two types of compound enzyme systems for targeted enzymatic hydrolysis. S300: After inactivating the enzyme in the above enzymatic hydrolysate, combine it with the chicken bone extract obtained in step S100, and use a three-stage membrane filtration to collect the retentate with a molecular weight of 300~1500 Da. S400. After concentrating and drying the above retentate, the target chicken bone enzymatic hydrolysate is obtained.

2. The preparation method according to claim 1, characterized in that, In step S100, fresh chicken bones are washed and crushed, water is added, and the mixture is steamed at 95~125℃ and 0.1~0.2MPa for 1~3 hours. The solid and liquid are separated to obtain chicken bone extract and bone residue. The mass ratio of chicken bone to water is 1:1~3. In step S200, the mass ratio of bone residue to water is 1:2~4.

3. The preparation method according to claim 1, characterized in that, The stepwise temperature-controlled enzymatic hydrolysis process includes the following: First stage: Adjust the pH to 6.5~7.5, raise the temperature to 50~60℃, add complex protease I composed of endopeptide protease, and enzymatically hydrolyze for 1~2 hours; Second stage: Adjust the pH to 6.0~7.0, cool to 45~55℃, add complex protease II composed of flavor protease and trypsin, and continue enzymatic hydrolysis for 2~4 hours; In the second stage, the pH was maintained at 6.8-7.0 for the first hour, and then gradually decreased to 6.2-6.

5.

4. The preparation method according to claim 3, characterized in that, The complex protease I is composed of papain, bromelain, and alkaline protease in a mass ratio of 1~2:1~2:1; the mass ratio of flavor protease to trypsin in the complex protease II is 2~4:

1.

5. The preparation method according to claim 1, characterized in that, In step S300, the enzyme inactivation method includes the following: Rapidly heat to 90~100℃ at a heating rate of ≥5℃ / min, hold at the enzyme inactivation temperature for 5~15 minutes, and then rapidly cool to ≤40℃ at a cooling rate of ≥8℃ / min.

6. The preparation method according to claim 1, characterized in that, The three-stage membrane filtration includes the following: The first stage uses a ceramic membrane with a pore size of 0.1~0.2μm for microfiltration and collects the permeate; the second stage passes the permeate from the first stage through a nanofiltration membrane with a molecular weight cutoff of 1500Da and collects the permeate. The third stage involves passing the second-stage permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da, collecting the 300-1500 Da cutoff fraction as the target product.

7. The preparation method according to claim 1, characterized in that, The concentration is a vacuum concentration, wherein the vacuum concentration temperature is 50~55℃, the vacuum degree is 0.08~0.09MPa, and the final solid content is 35~40%. The drying process is spray drying, with an inlet air temperature of 120~140℃, an outlet air temperature of 80~90℃, and a feed rate of 50~70mL / min.

8. A chicken bone enzymatic hydrolysate rich in flavor peptides prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The flavor peptides with a molecular weight of 300-1500 Da account for ≥70% of the total peptide content in the enzymatic hydrolysate.

9. The application of the chicken bone hydrolysate rich in flavor peptides as described in claim 8 in the preparation of chicken soup products.

10. The application according to claim 9, characterized in that, The chicken soup products include concentrated chicken soup, ready-to-eat chicken soup, chicken soup-flavored compound seasonings, or chicken essence.

Images