Method for extracting organic matter components of shells

Abstract

The invention provides a method for extracting organic matter components of shells. The method comprises the following steps: soaking the shells, ultrasonically removing seawater salt and silt on the surfaces, cleaning impurities on the surfaces by sodium hypochlorite, drying, crushing, sieving, decalcifying by a composite decalcifying solution to remove calcium carbonate on the surfaces of the shells, and retaining an organic matrix; and carrying out proteolysis on the decalcified solid organic matrix, carrying out heating ultrasonic extraction by using an extraction agent for 2-3 times, merging the supernatant, and concentrating and drying to obtain the shell active substance extract. The preparation method effectively retains organic matters, can obviously improve the extraction efficiency, is easy to obtain and low in cost, and the extraction agent is safe, environment-friendly, reusable and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of high-value utilization of marine biological resources, and in particular to a method for extracting shell organic components. Background Art

[0002] With the in-depth development of global marine resources, shellfish, a major byproduct of marine shellfish farming and processing, has long been discarded in large quantities or used only as low-value-added raw materials (such as feed additives and construction materials). According to statistics, more than 10 million tons of discarded shellfish are generated globally each year, but the comprehensive utilization rate is less than 20%, resulting in not only a waste of resources but also a buildup of solid waste and a burden on the ecological environment.

[0003] Shells are primarily composed of over 95% inorganic calcium carbonate (CaCO3) and 1-5% organic matrix. For a long time, research and industrial development have focused primarily on the inorganic components of shells, with insufficient attention paid to the smaller but highly active organic components. Recent studies have shown that the organic matrix components of shells, such as conchiolin, chitin, glycosaminoglycans, and bioactive peptides, possess a variety of physiological activities, including antibacterial, anti-inflammatory, antioxidant, immunomodulatory, and bone tissue repair, showing significant application prospects in high-value-added fields such as biomedicine, tissue engineering, functional cosmetics, and biomaterials. For example, oyster shell extract has been shown to inhibit Staphylococcus aureus by over 90%, while the glycosaminoglycans abundant in scallop adductor muscle have a significant protective effect on cartilage tissue.

[0004] However, shells have a highly mineralized and dense structure, which makes the organic matrix inside them firmly wrapped between calcium carbonate crystals, making extraction difficult. Currently, common demineralization and extraction methods all have significant shortcomings: (1) Although traditional acid hydrolysis methods (such as hydrochloric acid and acetic acid treatment) can dissolve calcium carbonate, the strong acid environment easily leads to degradation of active substances, significantly weakening or even destroying their biological activity; at the same time, acid treatment produces a large amount of corrosive waste liquid, which is costly to treat and causes serious environmental pollution; (2) Although high-temperature calcination (>800℃) can remove inorganic components, the high temperature will cause the organic matter to carbonize and become ineffective, and only inorganic calcium materials can be obtained; (3) Mechanical crushing + direct extraction methods have a very low extraction rate (usually <10%) due to the dense surface of the shell, making it difficult for the solvent to penetrate into the organic layer. In addition, the method has high energy consumption and unstable process, making it difficult to meet the needs of large-scale applications.

[0005] In summary, how to achieve efficient removal of inorganic components in shells under mild conditions while fully retaining their organic active structures has become a key technical problem that restricts the high-value utilization of shells. The existing technology has not yet provided a method for extracting organic components from shells that can be efficient, environmentally friendly and suitable for industrial scale-up while ensuring biological activity. In response to the above problems, the present invention proposes a "mild decalcification-composite extraction" process path, which uses ethylenediaminetetraacetic acid (EDTA) to achieve selective chelation of calcium carbonate under a neutral pH environment, effectively retaining the structures and activities of polysaccharides, glycosaminoglycans, etc. while removing inorganic components. Further combining heating and ultrasonic synergistic extraction technology, the extraction efficiency and quality of organic active substances are greatly improved, while avoiding environmental pollution, solving the problems of high biological activity destruction rate and low extraction rate in the existing technology, and providing a practical solution for the large-scale high-value utilization of shell resources. Summary of the Invention

[0006] In view of this, the present invention proposes a method for extracting organic components from shells. By optimizing the pretreatment steps, the organic components are effectively retained, the extraction efficiency of organic active substances is greatly improved, and an innovative solution is provided for the high-value utilization of shell resources.

[0007] The technical solution of the present invention is achieved as follows: A method for extracting organic matter components from shells comprises the following steps: (1) Pretreatment: Clean, dry, and crush the shells to obtain shell powder; (2) Dynamic decalcification treatment: decalcifying the shell powder with a decalcification solution under constant temperature oscillation conditions; (3) Enzymatic hydrolysis: centrifuge the decalcified matrix, add protease to the decalcified matrix obtained after centrifugation, and inactivate the enzyme after oscillation reaction in buffer; (4) Extraction of active components: centrifuge the matrix after enzymatic hydrolysis, add the extractant to the decalcified matrix obtained after centrifugation, perform ultrasonic treatment, and combine the supernatant after ultrasonic treatment; (5) Preparation of active ingredients: The supernatant is concentrated under reduced pressure and freeze-dried to obtain shell active substances.

[0008] Furthermore, the cleaning in step (1) includes: soaking the shells to remove surface salt and sediment from the seawater, using a sodium hypochlorite solution to ultrasonically assist in cleaning to remove surface impurities, and then washing with deionized water.

[0009] Furthermore, the concentration of the sodium hypochlorite solution is 0.5%-5% (w / v), the immersion time is 10-60 minutes, the ultrasonic frequency is 20kHz-40kHz, the power is 80-300W, and the time is 25-35 minutes.

[0010] Furthermore, the drying temperature in step (1) is 55-65°C, and the particle size of the shell particles after crushing and screening is 50-200 mesh.

[0011] Furthermore, the decalcification solution in step (2) is a mixed solution of 0.4-0.6M ethylenediaminetetraacetic acid (EDTA) and 0.3-0.6M ammonium chloride (NH4Cl) prepared with 3×PBS buffer, the ratio of powder to decalcification solution is 1:10-1:30 (w / v), the reaction temperature of the decalcification process is controlled at 25-37°C, the oscillation rate is 80-100 rpm, and fresh decalcification solution is replaced every 4 hours. The decalcification treatment time is 2-8 hours.

[0012] Furthermore, the protease in step (3) includes proteinase K and papain, the buffer is Tris-HCl buffer, and after the reaction, the enzyme is inactivated by heating at 60-80°C for 8-12 minutes.

[0013] Furthermore, in the enzymatic hydrolysis treatment, the concentration of proteinase K added is 50-200 μg / mL, the concentration of papain added is 0.1-0.5 mg / mL, the reaction buffer is 50 mM Tris-HCl, the pH value is 7.5-8.0, the reaction temperature is 35-38° C., and the reaction time is 1-3 hours.

[0014] Furthermore, the extractant in step (4) is 60-95% (v / v) ethanol, and the extractant is added in an amount 10-50 times the weight of the decalcified matrix. The frequency of ultrasonic treatment is 20-60 kHz, the power is 200-500 W, the heating temperature is 55-80°C, the ultrasonic treatment time is 1-2 h, and it is repeated 1-3 times.

[0015] Furthermore, the centrifugal speed in step (3) and step (4) is 10,000-14,000 rpm, and the centrifugal time is 8-12 min.

[0016] Furthermore, the reduced pressure temperature in step (5) is 40-50° C., and after drying, the mixture is redissolved in 75-85% (v / v) methanol water.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The extraction method of shell organic matter components of the present invention can efficiently remove calcium carbonate from shells under neutral and mild conditions, avoids the structural destruction of active ingredients such as polysaccharides by a strong acid environment, and effectively retains a variety of organic active ingredients including shell chitin, glycosaminoglycans and small molecule metabolites; adopts a synergistic method of heating and ultrasound to enhance solvent penetration and cell wall breaking ability, improves the release rate and extraction efficiency of target ingredients, and maintains the integrity of active functions; the operation process is simple, the parameters are controllable, and the repeatability is strong, which is suitable for large-scale industrial promotion and the processing of various shell raw materials; the obtained extract has a high concentration of target active substances and excellent purity, is compatible with modern chromatography, mass spectrometry and other detection technologies, and can achieve subsequent efficient component separation, identification and functional verification, further promoting the refined application of shell resources in functional materials and medical research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the peak number of metabolites extracted under different extraction conditions; Figure 2 The number of metabolites identified for different pretreatment methods; Figure 3 Metabolite spectra identified by different pretreatment methods for LC-MS detection; Figure 4 The peak number of metabolites extracted with 75% ethanol by enzymatic hydrolysis and without enzymatic hydrolysis; Figure 5 The metabolite compound categories extracted by enzymatic hydrolysis with 75% ethanol; Figure 6 It is the metabolite compound category extracted by 75% ethanol without enzymatic hydrolysis. DETAILED DESCRIPTION

[0019] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0020] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0021] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0022] Example 1 The present invention provides a method for extracting organic matter components from shells, comprising the following steps: Pretreatment: Take 100g of fresh scallop shells (such as those from the Yezo scallop, Chlamys farreri, and Bay scallops) and rinse them three times with running tap water to remove surface sediment. Then soak them in deionized water to fully dissolve the seawater salt. After cleaning and removing sediment, immerse the shells in a 5% (w / v) sodium hypochlorite solution and ultrasonicate them at room temperature for 30 minutes at a frequency of 40kHz and a power of 200W, stirring twice during the process, to remove surface organic matter and microbial contamination. Remove the shells, rinse them three times with deionized water, and dry them in a 60°C oven to constant weight (approximately 12 hours). After crushing, sieve through a 200-mesh sieve, and collect the shell powder for later use.

[0023] Decalcification: Prepare a decalcification solution containing 0.5M EDTA and 0.3M NH4Cl at a pH of 7.4 using 3× PBS buffer. Add the shell powder to the solution at a ratio of 1:20 (g / mL). Place the mixture in a constant temperature shaking incubator at 37°C and 90 rpm, replacing the solution with fresh solution every 4 hours. Decalcify for 12 hours, until the shell powder becomes loose. After decalcification, transfer the mixture to a centrifuge tube and centrifuge at 12,000 rpm for 10 minutes. Discard the calcium-containing supernatant and collect the decalcified matrix.

[0024] Enzymatic hydrolysis: Add 50 μg / mL proteinase K and 0.5 mg / mL papain to the decalcified matrix and shake in Tris-HCl buffer (pH 7.0-8.0) for 1 hour. Then heat at 70°C for 10 minutes to inactivate the enzymes. Centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and collect the decalcified matrix.

[0025] (4) Organic matter extraction: Add 30 times the weight (w / w) of 75% ethanol to the decalcified matrix and thoroughly suspend. Use ultrasonic-assisted extraction at a frequency of 40 kHz, a power of 300 W, and a temperature of 65°C. Ultrasonic treatment was performed for 1 h, with stirring every 10 min. Repeat the ultrasonic extraction twice and combine the supernatants. After each sonication, centrifuge at 12,000 rpm for 30 min, collect the supernatant, and re-extract the residue using the same method.

[0026] (5) Preparation of active ingredients: The combined supernatant was transferred to a rotary evaporator and concentrated to 1 / 10 of the original volume at 45°C under reduced pressure (vacuum degree ≤ 0.08 MPa). The concentrate was divided into freeze-dried bottles, pre-frozen to -80°C for 2 h, and then freeze-dried at -56°C and vacuum degree ≤ 10 Pa for 24 h to obtain a light yellow powdered shell active substance. The obtained powder was re-dissolved in 80% methanol water.

[0027] (6) HPLC characterization: The samples were separated using a VanquishLC ultra-high performance liquid chromatography system (UHPLC) HILIC column; Column temperature 25°C; Flow rate 0.3 mL / min; Injection volume: 2 μL; Mobile phase composition: A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile; The gradient elution program is as follows: 0-1.5min, 98%B; 1.5-12 min, B changes linearly from 98% to 2%; 12-14min, B is maintained at 2%; 14-14.1 min, B changes linearly from 2% to 98%; 14.1-17min, B maintained at 98%; The samples were placed in an autosampler at 4°C during the entire analysis.

[0028] A QExactive series mass spectrometer was used to collect the primary and secondary spectra of the samples.

[0029] The samples were separated by VanquishLC ultra-high performance liquid chromatography (UHPLC) system and analyzed by mass spectrometry using a QExactive series mass spectrometer (Thermo), using electrospray ionization (ESI) positive and negative ion modes for detection.

[0030] The ESI source and mass spectrometer setting parameters were as follows: nebulizer gas auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30 psi, ion source temperature: 600 °C, spray voltage (ISVF) ±5500 V (positive and negative modes); Primary mass-to-charge ratio detection range: 80-1200Da, resolution: 60000, scanning accumulation time: 100ms, The secondary level adopted a segmented acquisition method with a scanning range of 70–1200 Da, a secondary resolution of 30,000, a scanning accumulation time of 50 ms, and a dynamic exclusion time of 4 s.

[0031] Example 2 Based on steps (1) and (2) of Example 1, the extraction conditions of step (3) were adjusted to optimize the organic matter extraction efficiency. The specific steps are as follows: Pretreatment: As in Example 1, 100 g of fresh scallop shells were taken, ultrasonically cleaned, sterilized, dried, and crushed, and then passed through a 200-mesh sieve to obtain shell powder.

[0032] As in Example 1, a decalcification solution containing 0.5 M EDTA and 0.3 M NH 4 Cl at a pH of 7.4 was used, and decalcification was performed at a ratio of 1:20 (g / mL) for 12 h. The decalcified matrix was collected by centrifugation.

[0033] As in Example 1, 50 μg / mL proteinase K and 0.5 mg / mL papain were added for enzymatic hydrolysis for 1 h. After inactivation of the enzymes, the decalcified matrix was obtained by centrifugation.

[0034] Organic matter extraction: Ultrasound-assisted extraction was performed by adding different concentrations of ethanol (60%, 75%, and 95%) and different material-liquid ratios (1:10, 1:20, 1:30, and 1:50 w / v) to the decalcified matrix. The specific experimental group settings are as follows:

[0035] Specific operations: For each experiment, the corresponding concentration of ethanol solution was added, mixed, and then sonicated for 1 hour (stirred every 10 minutes). The extraction was repeated twice, and the supernatants were combined and centrifuged at 12,000 rpm for 30 minutes.

[0036] (5) Preparation of active ingredients: As in Example 1, the combined supernatant was concentrated under reduced pressure (50°C, ≤0.08 MPa), freeze-dried (-56°C, ≤10 Pa, 24 h) to obtain shell active substances under different conditions, and re-dissolved in 80% ethanol water.

[0037] (6) HPLC characterization: Mass spectrometry conditions were the same as in Example 1 above.

[0038] result: Table 1: One-way ANOVA experimental design and results

[0039] As shown in Table 1, the extraction efficiency of metabolites reached the maximum when the ethanol concentration was 75%, the solid-liquid ratio was 1:30 (w / v), and the ultrasonic extraction was performed at 65 °C.

[0040] like Figure 1 Figure 2 shows the peak numbers of metabolites extracted under different extraction conditions. The peak number of metabolites identified was the largest when the ethanol concentration was 75%, the solid-liquid ratio was 1:30 (w / v), and the ultrasonic extraction was performed at 65°C.

[0041] Comparative Example 1 In Examples 1 and 2, it was determined that the maximum extraction efficiency of metabolites was achieved when the ethanol concentration was 75%, the solid-liquid ratio was 1:30 (w / v), and ultrasonic extraction was performed at 65°C. Therefore, these conditions were used for extraction in the following.

[0042] On the basis of Example 1, step (2) was changed, decalcification was not performed, and extraction was performed directly after pulverization. The remaining steps were the same as above.

[0043] The results are as follows Figure 2 As shown in the figure: Under the same metabolite extraction method, more metabolites were identified by mild EDTA decalcification compared with no decalcification and acetic acid decalcification, which can retain the organic components as much as possible. Figure 3 LC-MS spectra show that mild EDTA decalcification increases both the types and abundance of metabolites. The addition of NH₄Cl as a buffer maintains the pH of the EDTA decalcification solution within a stable range, promoting chelation. Furthermore, the NH₄Cl concentration in the formula provides an appropriate physiological osmotic pressure, maintaining the integrity of the organic layer and preventing the depletion of small molecule metabolites within the cells.

[0044] Comparative Example 2 In Examples 1 and 2, it was determined that the maximum extraction efficiency of metabolites was achieved when the ethanol concentration was 75%, the solid-liquid ratio was 1:30 (w / v), and ultrasonic extraction was performed at 65°C. Therefore, these conditions were used for the following extractions. Based on Example 1, step (3) was modified to omit the enzymatic hydrolysis, and the remaining steps were the same as above.

[0045] like Figure 4 、 5 As shown in Figure 6, compared with non-enzymatic hydrolysis, more compounds were identified after enzymatic hydrolysis, and more small molecules such as polysaccharides were retained. This indicates that enzymatic hydrolysis can promote the release of metabolites encapsulated in the organic matrix, thereby improving the extraction efficiency and identification of organic components.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting organic matter components from shells, characterized by: The following steps are involved: (1) Pretreatment: The shells are cleaned, dried, and crushed to obtain shell powder; (2) Dynamic decalcification: The shell powder is decalcified with a decalcification solution under constant temperature oscillation conditions; (3) Enzymatic hydrolysis: The decalcified matrix is ​​centrifuged, and protease is added to the decalcified matrix obtained after centrifugation, and the enzyme is inactivated after oscillation reaction in a buffer solution; (4) Extraction of active components: The enzymatic hydrolysis matrix is ​​centrifuged, and an extractant is added to the decalcified matrix obtained after centrifugation, and ultrasonic treatment is performed, and the supernatants after ultrasonic treatment are combined; (5) Preparation of active ingredients: The supernatant is concentrated under reduced pressure and freeze-dried to obtain shell active substances.

2. The method for extracting organic matter from shells according to claim 1, wherein: The cleaning in step (1) includes: soaking the shells to remove surface seawater salt and sediment, using sodium hypochlorite solution to ultrasonically assist in cleaning to remove surface impurities, and then washing with deionized water.

3. The method for extracting organic matter from shells according to claim 2, wherein: The concentration of the sodium hypochlorite solution is 0.5%-5% (w / v), the immersion time is 10-60 minutes, the ultrasonic frequency is 20kHz-40kHz, the power is 80-300W, and the time is 25-35 minutes.

4. The method for extracting shell organic components according to claim 1, wherein: The drying temperature in step (1) is 55-65°C, and the particle size of the shell particles after crushing and screening is 50-200 mesh.

5. The method for extracting organic matter from shells according to claim 1, wherein: The decalcification solution in step (2) is a mixed solution of 0.4-0.6M ethylenediaminetetraacetic acid and 0.3-0.6M ammonium chloride prepared with 3×PBS buffer, the ratio of powder to decalcification solution is 1:10-1:30 (w / v), the reaction temperature of the decalcification process is controlled at 25-37°C, the oscillation rate is 80-100 rpm, and fresh decalcification solution is replaced every 4 hours. The decalcification treatment time is 2-8 hours.

6. The method for extracting organic matter from shells according to claim 1, wherein: The protease in step (3) includes proteinase K and papain, and the buffer is Tris-HCl buffer. After the reaction, the enzyme is inactivated by heating at 60-80°C for 8-12 minutes.

7. The method for extracting shell organic components according to claim 6, wherein: In the enzymatic hydrolysis treatment, the concentration of proteinase K added is 50-200 μg / mL, the concentration of papain added is 0.1-0.5 mg / mL, the reaction buffer is 50 mM Tris-HCl, the pH value is 7.5-8.0, the reaction temperature is 35-38° C., and the reaction time is 1-3 hours.

8. The method for extracting organic matter from shells according to claim 1, wherein: The extractant in step (4) is 60-95% (v / v) ethanol, and the weight of the extractant is 10-50 times that of the decalcified matrix. The frequency of ultrasonic treatment is 20-60 kHz, the power is 200-500 W, the heating temperature is 55-80 ° C, the ultrasonic treatment time is 1-2 h, and it is repeated 1-3 times.

9. The method for extracting organic matter from shells according to claim 1, wherein: The centrifugal speed in step (3) and step (4) is 10000-14000 rpm, and the centrifugal time is 8-12 min.

10. The method for extracting organic matter from shells according to claim 1, wherein: The reduced pressure temperature in step (5) is 40-50° C., and after drying, the mixture is redissolved in 75-85% (v / v) methanol water.

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