High-activity eggshell membrane composite enzymatic hydrolysis extraction process and extract

Abstract

The invention belongs to but not limited to the technical field of biological extraction, and discloses a high-activity eggshell membrane composite enzymatic hydrolysis extraction process and an extract, the process adopts a composite enzyme system of alkaline protease and papain for directional hydrolysis, and is combined with low-temperature ultrafiltration purification and freeze drying technologies, the total amount of amino acid in the obtained extract is greater than or equal to 79g / 100g, and the total amount of amino acid in the extract is greater than or equal to 79g / 100g. The sodium hyaluronate content is greater than or equal to 4.4 g / 100g, the collagen content is greater than or equal to 6.8%, the water solubility is greater than or equal to 95%, no skin and eye irritation is caused, the method can be widely applied to the fields of food health care, cosmetics, pet food and the like, the technical problems of loss of active ingredients and poor water solubility of a traditional process are solved, and the method is stable and controllable and suitable for industrial production. Through detection, the total amount of amino acid in the extract reaches 79.3 g / 100g, the content of sodium hyaluronate is 4.4 g / 100g, the content of collagen is 6.8%, and the extract is free of skin and eye irritation, can meet the application requirements in the fields of food health care, cosmetics and the like, is mature and controllable in process and is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of bio-extraction technology, and particularly relates to a highly active eggshell membrane complex enzymatic hydrolysis extraction process and extract. Background Technology

[0002] With the increasing demand for comprehensive utilization of biological resources and development of high-value-added raw materials, eggshell membranes, as a large amount of solid waste in the egg processing industry, are gradually attracting attention from the food, cosmetics, and pharmaceutical fields due to their high content of collagen-like proteins, glycosaminoglycans, and various bioactive substances. However, the complex fibrous network structure of eggshell membranes makes it difficult to completely separate and efficiently release their functional components using simple physical methods, thus posing a systemic technological research and development challenge.

[0003] In existing technologies, a typical approach involves enzymatic hydrolysis of eggshell membranes or the eggshell-shell membrane system to extract collagen peptides or other bioactive proteins. For example, in extracting collagen from eggshell membranes, a single enzyme is used, and orthogonal experiments are employed to optimize conditions such as pH, enzyme dosage, and material-to-liquid ratio to improve the recovery rate of specific components. This approach uses papain at room temperature, pH 5, and a material-to-water ratio of 1:100 for enzymatic hydrolysis, achieving a certain yield of collagen after a relatively long reaction time.

[0004] Another type of technical solution improves separation efficiency through compound enzymatic hydrolysis or a combination of chemical pretreatment and enzymatic hydrolysis. For example, non-starch polysaccharide enzymes are first used to hydrolyze the membrane surface fibers, and then proteases are used to further act on the structural proteins, thereby achieving the separation of the eggshell and the eggshell membrane. Although the compound enzyme system can improve the shell / membrane separation effect, this technology focuses on the separation of materials itself and does not conduct in-depth synergistic design for the systematic extraction process of high-level bioactive components such as high-purity collagen active peptides, polypeptides and other low-molecular-weight active components.

[0005] While existing technologies can achieve some of the objectives, they still have significant limitations: First, most methods focus on the extraction of single components or mechanical separation via membranes, failing to establish an optimized overall process from shell membrane separation to the release of low-molecular-weight active components. Second, the penetration and compatibility of single enzymatic hydrolysis conditions or single-enzyme systems within the complex network structure of the eggshell membrane are limited, resulting in low extraction efficiency of the target components and low water solubility and purity of the extracted products. Third, existing technologies do not fully elucidate the intrinsic synergistic mechanism between the enzymatic hydrolysis process and the extracted components, and have not constructed a process framework capable of continuous separation and purification while retaining heat-sensitive and structure-sensitive active substances. These technical shortcomings are precisely the core pain points directly addressed by this invention through an integrated design of low-temperature aqueous phase extraction, synergistic effects of compound enzymes, and molecular weight selective membrane separation. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a highly active eggshell membrane composite enzymatic extraction process.

[0007] This invention is achieved through a highly active eggshell membrane complex enzymatic hydrolysis extraction process, which includes: S1: Raw material pretreatment: Select traceable selenium-rich and antibiotic-free eggshells, remove impurities, and use a self-developed high-efficiency shell membrane separation system to separate the shell membrane under a pressure of 0.3-0.5MPa and a temperature of 10-15℃. Collect the eggshell membrane and crush it to 150-200 mesh to obtain eggshell membrane powder. S2: Low temperature extraction. Mix eggshell membrane powder with deionized water at a material-to-liquid ratio of 1:15-1:25, stir and extract at 30-35℃ for 1-2 hours, centrifuge at 8000-10000r / min for 15-20 minutes, and take the supernatant to obtain crude extract. S3: Compound enzymatic hydrolysis. Add 1.5%-2.5% of the weight of eggshell membrane powder to the crude extract, adjust the pH to 7.0-7.5, and enzymatically hydrolyze at 45-50℃ for 2.5-3.5 hours. During the enzymatic hydrolysis, stir continuously at 100-120 r / min. S4: Enzyme inactivation. Heat the enzyme hydrolysate to 85-90℃ and keep it warm for 15-20 minutes to inactivate the enzyme activity. Then cool it to room temperature. S5: Gradient purification: First, use an ultrafiltration membrane with a molecular weight cutoff of 10000 Da to purify and collect the permeate; then concentrate it by nanofiltration to a solid content of 20%-25% to obtain a purified concentrate. S6: Drying. Place the purified concentrate at -35℃ to -40℃ for freeze-drying for 18-24 hours, and then pulverize to obtain a highly active eggshell membrane extract. Furthermore, the total enzyme activity of the complex enzyme preparation in S3 is 100,000-120,000 U / g, and nitrogen gas is introduced during the enzymatic hydrolysis process to protect the active ingredients. Furthermore, in S5, the ultrafiltration operating pressure is 0.2-0.3 MPa, and the nanofiltration operating pressure is 1.0-1.2 MPa. Furthermore, the eggshell membrane powder in S1 must meet the requirements of being antibiotic-free and selenium-enriched, with a selenium content ≥0.15mg / kg. Another objective of this invention is to provide a highly active eggshell membrane extract prepared by the aforementioned process, wherein the extract has a total amino acid content ≥79g / 100g, a sodium hyaluronate content ≥4.4g / 100g, a collagen content ≥6.8%, a water solubility ≥95%, and is non-irritating to the skin and eyes. Furthermore, the highly active eggshell membrane extract is used as a raw material for food and health products, a raw material for cosmetics, a functional additive for pet food, or a nutritional fortifier in the feed industry.

[0008] 1. Raw material advantages: Relying on a fully traceable egg industry chain, we select selenium-enriched antibiotic-free eggshells, ensuring the safety and controllability of raw materials and a stable supply, thus guaranteeing product quality from the source; 2. Separation technology: A self-developed high-efficiency shell-membrane separation system is used to achieve efficient shell-membrane separation under low temperature and low pressure conditions, avoiding denaturation of active ingredients caused by high temperature; 3. Compound enzymatic hydrolysis: Alkaline protease and papain are combined to directionally hydrolyze the eggshell membrane fiber structure, precisely releasing active ingredients such as collagen and hyaluronic acid. Compared with single enzymatic hydrolysis, the extraction efficiency is increased by more than 30%. 4. Gradient purification: Gradient purification technology combining ultrafiltration and nanofiltration removes macromolecular impurities and unhydrolyzed proteins, significantly improving the product's water solubility and purity; 5. Low-temperature drying: Using freeze-drying technology, moisture is removed in a low-temperature vacuum environment to retain active ingredients to the maximum extent and ensure high product activity.

[0009] The extract obtained by this process has been tested and found to contain a total amino acid content of 79.3g / 100g, a sodium hyaluronate content of 4.4g / 100g, and a collagen content of 6.8%. It is also non-irritating to the skin and eyes and can meet the application needs of multiple fields such as food, health care, and cosmetics. The process is mature and controllable and suitable for large-scale industrial production. Attached Figure Description

[0010] Figure 1 This is a flowchart of a high-activity eggshell membrane composite enzymatic hydrolysis extraction process provided by an embodiment of the present invention; Figure 2 These are the serum cytokine concentrations of rats in the supplemented eggshell membrane and control group provided in this embodiment of the invention; Figure 3 This is a schematic diagram of improving physical fitness in 5 days provided by an embodiment of the present invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the 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 and not intended to limit the invention.

[0012] like Figure 1 As shown, this embodiment of the invention provides a highly active eggshell membrane composite enzymatic extraction process, which includes: S1: Raw material pretreatment: Select traceable selenium-rich and antibiotic-free eggshells, remove impurities, and use a self-developed high-efficiency shell membrane separation system to separate the shell membrane under a pressure of 0.3-0.5MPa and a temperature of 10-15℃. Collect the eggshell membrane and crush it to 150-200 mesh to obtain eggshell membrane powder. S2: Low temperature extraction. Mix eggshell membrane powder with deionized water at a material-to-liquid ratio of 1:15-1:25, stir and extract at 30-35℃ for 1-2 hours, centrifuge at 8000-10000r / min for 15-20 minutes, and take the supernatant to obtain crude extract. S3: Compound enzymatic hydrolysis. Add 1.5%-2.5% of the weight of eggshell membrane powder to the crude extract, adjust the pH to 7.0-7.5, and enzymatically hydrolyze at 45-50℃ for 2.5-3.5 hours. During the enzymatic hydrolysis, stir continuously at 100-120 r / min. S4: Enzyme inactivation. Heat the enzyme hydrolysate to 85-90℃ and keep it warm for 15-20 minutes to inactivate the enzyme activity. Then cool it to room temperature. S5: Gradient purification: First, use an ultrafiltration membrane with a molecular weight cutoff of 10000 Da to purify and collect the permeate; then concentrate it by nanofiltration to a solid content of 20%-25% to obtain a purified concentrate. S6: Drying. Place the purified concentrate at -35℃ to -40℃ for freeze-drying for 18-24 hours, and then pulverize to obtain a highly active eggshell membrane extract. The total enzyme activity of the compound enzyme preparation in S3 is 100,000-120,000 U / g, and nitrogen gas is introduced during the enzymatic hydrolysis process to protect the active ingredients. The ultrafiltration operating pressure in S5 is 0.2-0.3 MPa, and the nanofiltration operating pressure is 1.0-1.2 MPa. The eggshell membrane powder in S1 must meet the requirements of being antibiotic-free and selenium-enriched, with a selenium content ≥ 0.15 mg / kg. This invention provides a highly active eggshell membrane extract prepared by the aforementioned process. The extract has a total amino acid content ≥79g / 100g, a sodium hyaluronate content ≥4.4g / 100g, a collagen content ≥6.8%, a water solubility ≥95%, and is non-irritating to the skin and eyes. The highly active eggshell membrane extract is used as a raw material for food and health products, a raw material for cosmetics, a functional additive for pet food, or a nutritional fortifier in the feed industry.

[0013] Evidence related to the technical effects obtained by the embodiments of the present invention.

[0014] Evaluation of the efficacy and safety of eggshell membrane This study evaluated the efficacy and safety of oral eggshell membrane 500 mg / day for 8 weeks in 44 subjects for treating osteoarthritis-related pain and stiffness. In the overall patient assessment, over 59% of patients rated the efficacy as good or very good after 60 days of supplementation; over 75% of patients experienced moderate or significant improvement in pain and stiffness after 60 days.

[0015]

[0016]

[0017] Investigating the mechanism of action of eggshell membrane on joint pain and stiffness: After oral supplementation with eggshell membrane for 7 days, data measuring serum cytokines in rats showed a statistically significant reduction in pro-inflammatory cytokines. Preliminary evidence suggests that eggshell membrane can reduce pro-inflammatory cytokines to alleviate pain and stiffness. Figure 2 As shown.

[0018] This invention uses the 6-minute walk test (6MWT) to assess the changes in physical performance of osteoarthritis patients after 5 consecutive days of taking eggshell membrane, and compares them with a placebo-controlled cohort. Using the number of miles walked on day 0 (baseline) as the independent variable and the change in walking distance from baseline on day 5 (Δmiles) as the dependent variable, linear fitting analyses were performed on the placebo cohort and the eggshell membrane cohort, respectively. The results are shown in Figure 3.

[0019] The scatter plot and regression results show that both groups exhibited a trend of "higher baseline walking ability followed by smaller or even lower improvement in walking ability within 5 days," with negative regression slopes in both cases. However, this trend was more pronounced in the eggshell membrane cohort. Specifically, the regression slope of the eggshell membrane cohort (m² = −0.53324) was significantly greater than that of the placebo cohort (m² = −0.30506), and the correlation coefficient R was also higher (0.72959 vs 0.40622), indicating a stronger correlation between individual baseline physical fitness levels and short-term physical fitness changes after eggshell membrane intervention. This suggests that eggshell membrane intervention not only affects overall walking ability but also amplifies the modulating effect of individual differences on response amplitude, meaning that different patients showed more differentiated responses to the intervention.

[0020] As shown in Figure 3, in the group with lower baseline walking ability, the eggshell membrane cohort showed more positive Δmiles values, indicating an improvement in walking ability after 5 days. However, in the group with higher baseline walking ability, Δmiles were close to zero or slightly negative, suggesting limited room for improvement or even a slight decline. This distribution characteristic of "more pronounced benefits at low baselines and plateaus at high baselines" aligns with the general pattern of functional improvement interventions, meaning that those with initial functional limitations are more likely to observe short-term improvement effects. In contrast, the placebo cohort showed a more dispersed distribution and weaker correlation, suggesting that its changes are more likely due to individual natural fluctuations or testing errors rather than a systematic intervention effect.

[0021] The results of rat inflammatory factors shown in Figure 2 can further clarify the potential biological background of this functional improvement. Figure 2 shows that under eggshell membrane supplementation conditions of 26 mg / kg and 52 mg / kg, serum IFN-γ, IL-1β, TNF-α, IL-6, and IL-10 levels did not show significant abnormal increases and were generally similar to the control group, suggesting that short-term eggshell membrane supplementation did not induce a systemic inflammatory response. This result provides safety support for clinical performance improvement, indicating that the observed changes in walking ability are unlikely to be driven by acute inflammatory fluctuations, but are more likely related to joint function status, pain relief, or improved exercise tolerance.

[0022] In summary, the regression analysis in Figure 3 shows that the eggshell membrane cohort exhibited a stronger "baseline ability - magnitude of improvement" correlation than placebo over 5 days, particularly in osteoarthritis patients with low baseline performance, where improved walking ability was more readily observed. The cytokine results in Figure 2 suggest that this short-term intervention is well-tolerated at the immune-inflammatory level. Both findings support the conclusion that eggshell membrane may improve the performance of some osteoarthritis patients in the short term, and its effect is not accompanied by significant inflammatory activation, providing preliminary evidence for its use as a functional-improving nutritional or adjunctive intervention.

[0023] Example 1: Feasibility Verification of Basic Processes Selenium-enriched, antibiotic-free eggshells with traceable provenance were selected, cleaned to remove impurities, and then subjected to shell membrane separation at 0.4 MPa pressure and 12°C. The shell membranes were collected and pulverized to 180 mesh. The resulting powder was mixed with deionized water at a ratio of 1:20 and extracted by stirring at 32°C for 1.5 hours. Subsequently, the mixture was centrifuged at 9000 rpm for 18 minutes, and the supernatant was used as the crude extract.

[0024] Add 2% (by weight of eggshell membrane powder) of a compound enzyme preparation to the crude extract, adjust the pH to 7.2, and enzymatically hydrolyze at 48°C for 3 hours with a stirring speed of 110 rpm. After enzymatic hydrolysis, raise the temperature to 88°C and hold for 18 minutes to terminate the reaction. After cooling, perform ultrafiltration and nanofiltration concentration sequentially, and finally freeze-dry at -38°C for 20 hours to obtain a pale yellow powder extract with good water solubility and a clear solution state.

[0025] Example 2: Process for improving collagen release efficiency During the shell membrane separation stage, a pressure of 0.5 MPa and a temperature of 10°C were used for separation to enhance the integrity of the shell membrane. After being pulverized to 200 mesh, the membrane was extracted with water at a ratio of 1:25. The extraction was carried out by stirring at 30°C for 2 hours, followed by high-speed centrifugation to obtain a crude extract of the eggshell membrane.

[0026] The compound enzyme was added at a concentration of 2.5%, the pH was adjusted to 7.5, and enzymatic hydrolysis was performed at 50°C for 3.5 hours. After terminating the reaction by heat treatment, membrane separation and freeze-drying were carried out. The collagen content of the obtained product was significantly higher than that in Example 1, making it suitable for applications with high collagen supplementation requirements.

[0027] Example 3: Process for increasing the enrichment of sodium hyaluronate The shell membrane was pulverized to 150 mesh and then extracted with water at a ratio of 1:18. After extraction at 33℃ for 1.2 hours, the mixture was centrifuged. A compound enzyme was added at a rate of 1.5%, the pH was set to 7.0, and enzymatic hydrolysis was performed at 45℃ for 2.5 hours, with nitrogen gas purging throughout the process to inhibit oxidation.

[0028] The solution was then purified and concentrated using ultrafiltration at 0.2 MPa and nanofiltration at 1.0 MPa, and finally freeze-dried to obtain a light white powder with a relatively high sodium hyaluronate content and high solution viscoelasticity, making it suitable for use as a raw material for skin moisturizing.

[0029] Example 4: Mild Processes for Food-Grade Applications Membrane separation was performed at 0.3 MPa and 15°C to minimize mechanical stress on the membrane structure. The extraction stage involved a 1:15 ratio, extraction at 35°C for 1 hour, and centrifugation at 8000 rpm.

[0030] The compound enzyme was added at a ratio of 1.8%, the pH was 7.3, the enzymatic hydrolysis temperature was 47℃, and the time was 3 hours. The extract obtained after purification and freeze-drying has a mild taste and dissolves rapidly, making it suitable for use as a food fortification ingredient.

[0031] Example 5: High Concentration and High Purity Preparation Process In the membrane purification stage, the nanofiltration concentration endpoint was set at a solid content of 25% to obtain a high-concentration intermediate. After freeze-drying, a high-density powder extract was obtained, which had a high amino acid content, sufficient protein degradation, and no precipitation after water dissolution.

[0032] The product obtained by this process is more suitable for subsequent formulation processing or as a highly active intermediate raw material, which facilitates the development of downstream functional products.

[0033] Example 6: Application Process of Pet Nutritional Fortification Extracts were prepared using shell membrane raw materials with a selenium content of not less than 0.15 mg per kilogram, following the process described in Example 1, and then micronized to improve mixing uniformity.

[0034] The obtained extract was added to the pet food formula in a certain proportion to make a nutritionally fortified feed. After stability testing, no clumping, sedimentation or odor was observed under normal temperature storage conditions, showing good processing adaptability and physical stability.

[0035] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A highly active eggshell membrane complex enzymatic extraction process, characterized in that, The process includes: S1: Raw material pretreatment: Select traceable selenium-rich and antibiotic-free eggshells, remove impurities, and use a self-developed high-efficiency shell membrane separation system to separate the shell membrane under a pressure of 0.3-0.5MPa and a temperature of 10-15℃. Collect the eggshell membrane and crush it to 150-200 mesh to obtain eggshell membrane powder. S2: Low temperature extraction. Mix eggshell membrane powder with deionized water at a material-to-liquid ratio of 1:15-1:25, stir and extract at 30-35℃ for 1-2 hours, centrifuge at 8000-10000r / min for 15-20 minutes, and take the supernatant to obtain crude extract. S3: Compound enzymatic hydrolysis. Add 1.5%-2.5% of the weight of eggshell membrane powder to the crude extract, adjust the pH to 7.0-7.5, and enzymatically hydrolyze at 45-50℃ for 2.5-3.5 hours. During the enzymatic hydrolysis, stir continuously at 100-120 r / min. S4: Enzyme inactivation. Heat the enzyme hydrolysate to 85-90℃ and keep it warm for 15-20 minutes to inactivate the enzyme activity. Then cool it to room temperature. S5: Gradient purification: First, use an ultrafiltration membrane with a molecular weight cutoff of 10000 Da to purify and collect the permeate; then concentrate it by nanofiltration to a solid content of 20%-25% to obtain a purified concentrate. S6: Drying. Place the purified concentrate at -35℃ to -40℃ for freeze-drying for 18-24 hours, and then pulverize to obtain a highly active eggshell membrane extract.

2. The highly active eggshell membrane composite enzymatic extraction process according to claim 1, characterized in that, The total enzyme activity of the compound enzyme preparation in S3 is 100,000-120,000 U / g, and nitrogen gas is introduced during the enzymatic hydrolysis process to protect the active ingredients.

3. The highly active eggshell membrane composite enzymatic extraction process according to claim 1, characterized in that, The ultrafiltration operating pressure in S5 is 0.2-0.3 MPa, and the nanofiltration operating pressure is 1.0-1.2 MPa.

4. The highly active eggshell membrane composite enzymatic extraction process according to claim 1, characterized in that, The eggshell membrane powder in S1 must meet the requirements of being antibiotic-free and selenium-enriched, with a selenium content ≥ 0.15 mg / kg.

5. A highly active eggshell membrane extract prepared by the process described in any one of claims 1-4, characterized in that, The extract contains a total amino acid content ≥79g / 100g, sodium hyaluronate content ≥4.4g / 100g, collagen content ≥6.8%, water solubility ≥95%, and is non-irritating to the skin and eyes.

6. The highly active eggshell membrane extract according to claim 5, characterized in that, Used as a raw material for food and health products, a raw material for cosmetics, a functional additive for pet food, or a nutritional fortifier in the feed industry.

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