Eggshell membrane extract containing chondroitin sulfate and oligopeptides and methods of making same

Abstract

The present application provides a chitosan sulfate and oligopeptide-containing eggshell membrane extract and a preparation method thereof. The preparation method of the chitosan sulfate and oligopeptide-containing eggshell membrane extract comprises the following steps: adding water with a pH value of 7.5±0.2 into an eggshell membrane, then adding a mixed preparation containing alkaline protease, cellulase, pectinase and sodium sulfide, performing enzymolysis, filtering a crude product, and drying a filtrate, and the chitosan sulfate and oligopeptide-containing eggshell membrane extract is obtained. The chitosan sulfate and oligopeptide-containing eggshell membrane extract can be used as a low-cost high-protein raw material additive, can replace high-priced protein raw materials, and can improve the immune capacity of a body.

Description

Technical Field

[0001] This invention relates to the field of eggshell waste application technology, specifically to an eggshell membrane extract containing chondroitin sulfate and oligopeptides and its preparation method. Background Technology

[0002] my country generates approximately 4 million tons of waste eggshells annually, resulting in a serious waste of resources. How to effectively reuse these waste eggshells is of significant research value.

[0003] The eggshell membrane, also known as the "eggshell coat," is a fibrous membrane located between the eggshell and the egg white. It has a mesh-like structure and is a double-layered, water-insoluble membrane consisting of an inner and an outer membrane. The outer membrane is 50 μm to 70 μm thick, while the inner membrane is 15 μm to 30 μm thick. The polysaccharides and proteins contained in the eggshell membrane are relatively complex. How to effectively and simultaneously obtain multiple functional components from the eggshell membrane and conduct applied research is a pressing problem that needs to be solved. Summary of the Invention

[0004] Therefore, it is necessary to provide an eggshell membrane extract containing chondroitin sulfate and oligopeptides, and its preparation method.

[0005] The present invention adopts the following technical solution:

[0006] This invention provides a method for preparing an eggshell membrane extract containing chondroitin sulfate and oligopeptides, comprising the following steps: obtaining an eggshell membrane; adding water with a pH of 7.5±0.2 to the eggshell membrane, then adding a mixed preparation containing alkaline protease, cellulase, pectinase, and sodium sulfide, and performing enzymatic hydrolysis to obtain a crude enzymatic hydrolysis product; filtering the crude enzymatic hydrolysis product, collecting the filtrate containing chondroitin sulfate and oligopeptides, and drying it to obtain the final product.

[0007] In some embodiments, the preferred ratio of eggshell membrane to water is 1 g: (3~5) mL.

[0008] In some embodiments, the preferred composition of the mixture is: starch as a carrier, 10-25 wt% alkaline protease (50,000 U / g), 10-25 wt% cellulase (2,000 U / g), 10-25 wt% pectinase (10,000 U / g), and 7-9 wt% sodium sulfide. Preferably, the amount of the mixture added is 1 wt% of the eggshell membrane input.

[0009] In some embodiments, the enzymatic hydrolysis process conditions are: temperature 45~55℃, time 6~8h.

[0010] In some embodiments, the drying temperature is 50~100°C, preferably 55~65°C.

[0011] In the eggshell membrane extract containing chondroitin sulfate and oligopeptides obtained by the above preparation method, the content of the oligopeptides is not less than 50%, and the content of the chondroitin sulfate is not less than 15%; more preferably, the content of the oligopeptides is not less than 55%, and the content of the chondroitin sulfate is not less than 25%. Furthermore, in the oligopeptides, peptide components with a molecular weight ≤2000 Da account for more than 90%.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] This invention utilizes eggshell membrane to prepare an eggshell membrane extract containing chondroitin sulfate and oligopeptides. In the feed industry, it can be used as a high-protein raw material additive, and in the pharmaceutical and food industries, it can be used as an excipient with effects such as enhancing human physique, fighting bacteria, beautifying the skin, and anti-aging. It has great application potential. Attached Figure Description

[0014] Figure 1 The images show the state diagrams of the eggshell membrane enzymatic hydrolysis products obtained by different preparation processes.

[0015] Figure 2 Images showing the dried state of eggshell membrane extracts obtained through different preparation processes. Detailed Implementation

[0016] Patent document CN115382877A discloses "a method for separating eggshell and eggshell membrane, comprising the following steps: (1) washing the eggshell with membrane, placing it in a container, and adding water to the container; (2) adding a non-starch polysaccharide enzyme to the container for a first enzymatic hydrolysis; (3) adding a protease to the first enzymatic hydrolysis product for a second enzymatic hydrolysis to obtain an enzymatic hydrolysate containing eggshell and eggshell membrane; (4) inactivating the enzyme in the enzymatic hydrolysate and drying it to obtain the separated eggshell and eggshell membrane." Stepwise enzymatic hydrolysis reduces the interaction force between the eggshell and eggshell membrane, achieving the separation of the eggshell and eggshell membrane. However, this patent document does not substantially study how to effectively and simultaneously obtain multiple functional components in the eggshell membrane for synergistic application.

[0017] The technical concept of this invention lies in making full use of eggshell waste to prepare an eggshell membrane extract containing both chondroitin sulfate and oligopeptides (molecular weight ≤2000Da) from the eggshell membrane.

[0018] Chondroitin sulfate has a protective effect on corneal collagen fibers, promotes the growth of fibers in the matrix, thereby promoting the healing of corneal wounds and improving symptoms of dry eyes. It can also be used as a drug for treating joint diseases in arthritis. It is often used in combination with glucosamine and has the effects of relieving pain and promoting cartilage regeneration. It has a certain effect on improving age-related degenerative arthritis and rheumatoid arthritis. It is a commonly used joint protection and health care product.

[0019] Oligopeptides can improve the permeability of capillary walls and gastrointestinal mucosa, accelerate nutrient digestion and absorption, prevent pathogen invasion, enhance the body's immunity and resistance, and help eliminate toxins. Clinically, oligopeptides primarily inhibit cholesterol rise, promote insulin secretion, regulate blood sugar, improve diabetes symptoms, and inhibit angiotensin-converting enzyme activity, thus lowering blood pressure. They also have a regulatory effect on hyperlipidemia, hyperglycemia, and hypertension. Furthermore, oligopeptides can enhance immunity by stimulating lymphocyte proliferation, enhancing macrophage phagocytic capacity, improving the body's ability to resist external pathogens, and reducing morbidity.

[0020] By obtaining eggshell membrane extracts containing both chondroitin sulfate and oligopeptides (molecular weight ≤2000Da), it can not only be used as a functional additive for high-protein raw materials, but also as a functional excipient in the pharmaceutical and food fields with effects such as enhancing physical fitness, antibacterial properties, beauty enhancement, and anti-aging.

[0021] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0022] Key experimental material sources:

[0023] Eggshell membrane: Eggshell membrane prepared from discarded and broken eggs collected from breeding farms, referring to the method steps and process conditions of Example 3 in patent document CN115382877A.

[0024] The mixed formulation consists of: 15wt% alkaline protease 50000 U / g, 15wt% cellulase 2000 U / g, 15wt% pectinase 10000 U / g, sodium sulfide 8%, and starch as the carrier.

[0025] Experimental Example 1

[0026] This experimental example provides a method for preparing eggshell membrane extract, including the following steps:

[0027] S1, Obtain the eggshell membrane.

[0028] S2, Extraction:

[0029] At a material-to-liquid mass-to-volume ratio of 1g:4mL, water with a pH of 7.5±0.2 was added to the eggshell membrane as an extraction solvent for extraction at a temperature of 50±5℃ for 6~8h to obtain the crude product.

[0030] S3, Impurity Removal and Drying:

[0031] The crude product was filtered through a 60-mesh sieve, and the filtrate was collected and dried at 60±5℃ to obtain the eggshell membrane extract, designated as Sample 1. Further pulverization yielded the final product.

[0032] Experimental Example 2

[0033] This experimental example provides a method for preparing eggshell membrane extract, including the following steps:

[0034] S1, Obtain the eggshell membrane.

[0035] S2, Extraction:

[0036] Add water with a pH of 7.5±0.2 to the eggshell membrane at a mass-to-volume ratio of 1g:4mL, then add sodium sulfide at a feed amount of 1wt% of the eggshell membrane feed amount as an extractant and perform extraction at a temperature of 50±5℃ for 6~8h to obtain crude product.

[0037] S3, Impurity Removal and Drying:

[0038] The crude product was filtered through a 60-mesh filter, the filtrate was collected, and dried in an environment at 60±5℃ to obtain the eggshell membrane extract, which was designated as sample 2.

[0039] Experimental Example 3

[0040] This experimental example provides a method for preparing eggshell membrane extract, including the following steps:

[0041] S1, Obtain the eggshell membrane.

[0042] S2, Extraction:

[0043] Add water with a pH of 7.5±0.2 to the eggshell membrane at a mass-to-volume ratio of 1g:4mL, then add cellulase (2000 U / g) at a rate of 1wt% of the eggshell membrane as the extraction agent. Extract at 50±5℃ for 6~8h to obtain the crude product.

[0044] S3, Impurity Removal and Drying:

[0045] The crude product was filtered through a 60-mesh filter, the filtrate was collected, and dried in an environment at 60±5℃ to obtain the eggshell membrane extract, which was designated as sample 3.

[0046] Test Example 4

[0047] This experimental example provides a method for preparing eggshell membrane extract, including the following steps:

[0048] S1, Obtain the eggshell membrane.

[0049] S2, Extraction:

[0050] Add water with a pH of 7.5±0.2 to the eggshell membrane at a mass-to-volume ratio of 1g:4mL, then add pectinase (10000 U / g) at a rate of 1wt% of the eggshell membrane as the extraction agent, and perform extraction at a temperature of 50±5℃ for 6~8h to obtain the crude product.

[0051] S3, Impurity Removal and Drying:

[0052] The crude product was filtered through a 60-mesh filter, the filtrate was collected, and dried in an environment at 60±5℃ to obtain the eggshell membrane extract, which was designated as sample 4.

[0053] Experimental Example 5

[0054] This experimental example provides a method for preparing eggshell membrane extract, including the following steps:

[0055] S1, Obtain the eggshell membrane.

[0056] S2, Extraction:

[0057] According to the material-liquid mass-volume ratio of 1g:4mL, water with a pH of 7.5±0.2 was added to the eggshell membrane, and then alkaline protease (50000 U / g) was added at a rate of 1wt% of the eggshell membrane as the extraction agent. Extraction was carried out at a temperature of 50±5℃ for 6~8h to obtain crude product.

[0058] S3, Impurity Removal and Drying:

[0059] The crude product was filtered through a 60-mesh filter, the filtrate was collected, and dried in an environment at 60±5℃ to obtain the eggshell membrane extract, which was designated as sample 5.

[0060] Experimental Example 6

[0061] This experimental example provides a method for preparing an eggshell membrane chondroitin sulfate-oligopeptide extract, comprising the following steps:

[0062] S1, Obtain the eggshell membrane.

[0063] S2, Extraction:

[0064] According to the material-liquid mass-volume ratio of 1g:4mL, water with a pH of 7.5±0.2 was added to the eggshell membrane, followed by a mixed preparation containing alkaline protease (50000 U / g), cellulase (2000 U / g), pectinase (10000 U / g), and sodium sulfide. The amount of the mixture added was 1wt% of the eggshell membrane as the extraction agent, and enzymatic hydrolysis was carried out at a temperature of 50±5℃ for 6~8h to obtain the crude enzymatic hydrolysis product.

[0065] S3, Impurity Removal and Drying:

[0066] The crude enzymatic hydrolysis product was filtered through a 60-mesh filter, the filtrate was collected, and dried in an environment at 60±5℃ to obtain chondroitin sulfate-oligopeptide eggshell membrane extract, which was designated as sample 6.

[0067] Experimental Example 7

[0068] This experimental example provides a method for preparing eggshell membrane extract. The raw materials and extraction process parameters used are the same as those in Experiment 6, except that the drying temperature is adjusted to 90±5℃.

[0069] The eggshell membrane extract prepared in this experiment is designated as Sample 7.

[0070] Experimental Example 8

[0071] This experimental example provides a method for preparing eggshell membrane extract, the process parameters and steps of which are the same as in Example 6, the only difference being that the extractant does not contain pectinase, but only a mixture of alkaline protease (50000 U / g), cellulase (2000 U / g), and sodium sulfide is used. The eggshell membrane extract prepared in this experimental example is designated as Sample 8.

[0072] Experimental Example 9

[0073] This experimental example provides a method for preparing an eggshell membrane extract. The process parameters and steps are the same as in Example 6, except that the extractant does not contain sodium sulfide, but is a mixture of pectinase (10000 U / g), alkaline protease (50000 U / g), and cellulase (2000 U / g). The eggshell membrane extract prepared in this experimental example is designated as Sample 9.

[0074] The states of the eggshell membrane reaction products extracted during the preparation process and the dried products in Examples 1 to 9 were tracked and compared. Statistical charts are shown below. Figure 1 and 2 .

[0075] The following tests were performed on the eggshell membrane extracts prepared in the above experimental examples:

[0076] 1) The test method for chondroitin sulfate is to be performed in accordance with the provisions of GB / T 20365-2006.

[0077] 2) The test method for protein content is as follows: the protein content shall be tested in accordance with the provisions of GB5009.5-2016.

[0078] Acid-soluble proteins: Determination of acid-soluble protein content according to GB / T22492-2008.

[0079] Free amino acids: The amino acid content was determined according to the method in GB / T 18246-2019.

[0080] Peptide content = acid-soluble protein - free amino acids.

[0081] 3) The test method for the molecular weight distribution range of oligopeptides is as follows: refer to GB / T22492-2008 Method for Determination of Relative Molecular Mass Distribution of Peptides.

[0082] Statistical analysis of the content of eggshell membrane extracts obtained by different preparation processes

[0083]

[0084] Statistical analysis of protein molecular weight distribution in eggshell membrane extracts obtained by different preparation processes

[0085]

[0086] Statistical table of the content composition of eggshell membrane extract in sample 6

[0087]

[0088] Furthermore, it is worth noting that, through extensive research, this application has discovered that, when using eggshell membrane as raw material, only by employing a mixed preparation containing a specific ratio of alkaline protease, cellulase, pectinase, and sodium sulfide, combined with specific enzymatic hydrolysis and drying process conditions, can an eggshell membrane extract be obtained with an overall content of not less than 55% oligopeptides, not less than 25% chondroitin sulfate, and a peptide component ratio of ≤2000 Da of more than 90%.

[0089] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an eggshell membrane extract containing chondroitin sulfate and oligopeptides, characterized in that, Includes the following steps: Obtain the eggshell membrane; Add water with a pH of 7.5±0.2 to the eggshell membrane, then add a mixed preparation containing alkaline protease, cellulase, pectinase and sodium sulfide, and carry out enzymatic hydrolysis to obtain crude enzymatic hydrolysis product; The crude enzymatic hydrolysis product is filtered, and the filtrate containing chondroitin sulfate and oligopeptides is collected, dried, and then obtained. The mixed preparation consists of: starch as a carrier, 10-25 wt% alkaline protease 50000 U / g, 10-25 wt% cellulase 2000 U / g, 10-25 wt% pectinase 10000 U / g, and 7-9 wt% sodium sulfide. The enzymatic hydrolysis process conditions are: temperature 45~55℃, time 6~8h; The drying temperature is 55~65℃.

2. The method for preparing the eggshell membrane extract containing chondroitin sulfate and oligopeptides according to claim 1, characterized in that, The ratio of eggshell membrane to water is 1g:(3~5)mL.

3. The method for preparing the eggshell membrane extract containing chondroitin sulfate and oligopeptides according to claim 1, characterized in that, The amount of the mixed preparation added is 1 wt% of the eggshell membrane input.

4. The method for preparing the eggshell membrane extract containing chondroitin sulfate and oligopeptides according to any one of claims 1 to 3, characterized in that, Of the oligopeptides, peptide components with a molecular weight ≤2000 Da account for more than 90%.

5. The method for preparing the eggshell membrane extract containing chondroitin sulfate and oligopeptides according to any one of claims 1 to 3, characterized in that, The extract contains at least 50% oligopeptides and at least 15% chondroitin sulfate.

6. Eggshell membrane chondroitin sulfate-oligopeptide extract prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the eggshell membrane extract containing chondroitin sulfate and oligopeptides as described in claim 6 in the preparation of pharmaceutical excipients.

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