Method for extracting nutrient substances from sea cucumber processing wastewater and application

Abstract

The invention discloses a method for extracting nutrient substances from sea cucumber processing wastewater and application, and belongs to the technical field of sea cucumber processing wastewater extraction. According to the method, the sea cucumber processing wastewater is used as a raw material, nutrient substances in the wastewater are extracted, the processing wastewater is pretreated by adopting an air floatation-microfiltration coupling technology, suspended solids and grease in the wastewater can be effectively removed, and heat-sensitive substances are reserved; meanwhile, a pH response type flocculating agent is adopted for removing heavy metal in a high-selectivity mode, meanwhile, loss of nutrient substances including protein, polysaccharide and saponin is avoided, after pretreatment is completed, a staged enzymolysis technology, a membrane separation-adsorption combined technology, a vacuum thin film evaporation technology and a programmed cooling crystallization technology are cooperated, protein, polysaccharide and saponin can be effectively separated and extracted, and the quality of the product is improved. The recovery rate, the purity and the product performance of each nutrient substance are remarkably improved, and technical support is provided for reutilization of nutrient components in sea cucumber processing wastewater.

Description

Technical Field

[0001] The present invention relates to the technical field of extracting sea cucumber processing wastewater, and more particularly to a method and application of extracting nutrients from sea cucumber processing wastewater. Background Art

[0002] Sea cucumbers are rich in active ingredients such as proteins, polysaccharides, and saponins, and possess high medicinal value. In recent years, my country's sea cucumber aquaculture has exceeded 204,000 tons, with an output value of approximately 60 billion yuan, representing a vast market. As a highly nutritious marine product, sea cucumber processing and application research currently focuses primarily on polysaccharides, saponins, and peptides. Fresh sea cucumbers are susceptible to peeling and autolysis after harvesting due to the presence of proteolytic enzymes such as autolytic enzymes. To facilitate preservation, fresh sea cucumbers are typically de-enzymed in hot water before processing and storage. This de-enzyme process generates significant amounts of processing wastewater, and research has found that this wastewater contains significant amounts of nutrients such as sugars, proteins, saponins, phospholipids, and fatty acids. Traditionally, this wastewater is directly discharged, causing environmental pollution. Furthermore, this wastewater contains numerous nutrients, and direct discharge also results in a waste of resources.

[0003] Currently, there are some proposals for reusing sea cucumber processing wastewater, such as publication number CN 104522728A, entitled "A Method for Extracting Phospholipids and Sea Cucumber Oil from Sea Cucumber Blanching Solution," which relates to extracting phospholipids and sea cucumber oil from sea cucumber processing wastewater. Another example is publication number CN 101473979 A, ​​entitled "A Method for Extracting Sea Cucumber Oil from Sea Cucumber Boiled Juice," which also relates to extracting sea cucumber oil from sea cucumber processing wastewater. However, there is currently little research on the simultaneous extraction of proteins, polysaccharides, and saponins from sea cucumber processing wastewater. This may be due to the wide molecular weight range: the wastewater contains large molecules (collagen, 10-300 kDa), medium molecules (polysaccharides, 5-50 kDa), and small molecules (saponins, <1 kDa), making it difficult to address both with traditional extraction techniques. Surfactant interference: Saponins generate foam (bubble height >50 cm), disrupting the stability of the membrane separation process. Traditional processing techniques can lead to heat-sensitive degradation, resulting in the denaturation of over 60% of active peptides. They also alter the three-dimensional structure of polysaccharides (β-glucan helical structure unwinding >40%), leading to a decrease in immunomodulatory function. These factors significantly increase the difficulty of extracting proteins, polysaccharides, and saponins from sea cucumber processing wastewater, reducing both the extraction rate and the activity of the extracted products.

[0004] Therefore, how to provide a method for extracting nutrients from sea cucumber processing wastewater to improve the recovery rate, extraction activity and purity of each nutrient is a technical problem that technicians in this field urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a method and application for extracting nutrients from sea cucumber industrial wastewater, which can successfully extract proteins (active peptides), polysaccharides and saponins from sea cucumber industrial wastewater, and to a certain extent improve the extraction rate and purity of the products, and also improve the performance of each product.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for extracting nutrients from sea cucumber processing wastewater, characterized by comprising the following steps:

[0008] (1) Wastewater pretreatment: Micro-nano bubbles are introduced into the sea cucumber processing wastewater for flotation operation, and a defoaming agent is added to suppress foaming; the wastewater is then passed through an Al2O3 ceramic membrane for microfiltration treatment to prepare pretreated wastewater;

[0009] (2) Selectively precipitating heavy metal ions: adding a pH-responsive flocculant to the wastewater pretreated in step (1), adjusting the pH to 5.5-6.0, stirring for 20-30 minutes, and filtering to remove heavy metal ions;

[0010] (3) Staged enzymatic hydrolysis:

[0011] The first stage of enzymatic hydrolysis: add alkaline protease and cellulase to the wastewater treated in step (2), adjust the temperature to 44-46°C, adjust the pH to 8.4-8.6, and perform ultrasonic enzymatic hydrolysis for 1.8-2.2 hours;

[0012] The second stage of enzymatic hydrolysis: quickly adjust the temperature to 54-56 ° C, pH to 4.9-5.1, add glycosidase, and perform ultrasonic enzymatic hydrolysis for 1.4-1.6 hours;

[0013] (4) Membrane separation-adsorption combined purification: The enzymatic hydrolysis product is purified by passing through a ceramic ultrafiltration membrane, a polyethersulfone nanofiltration membrane, and a macroporous adsorption resin in sequence;

[0014] When membrane separation and adsorption are combined, the following synergistic effects are achieved:

[0015] Ultrafiltration cooperates with nanofiltration, ultrafiltration removes large molecular impurities, reduces nanofiltration membrane pollution, and improves the membrane flux stability of nanofiltration membrane; when nanofiltration cooperates with resin adsorption, nanofiltration desalination (Na + After the removal rate is greater than 90%), the resin adsorption efficiency can be improved and the saponin adsorption capacity can be increased.

[0016] (5) Concentration, crystallization, and drying: The purified product from step (4) was concentrated to a solid content of 25-30% using a vacuum thin film evaporation system; the temperature was then reduced from 40°C to 4°C at a rate of 0.5°C / min to precipitate crystals, which were then dried to obtain the final product.

[0017] In a low-temperature vacuum environment (absolute pressure 5-10kPa), the boiling point of water drops to 30-40°C, which can effectively avoid high-temperature degradation of heat-sensitive ingredients (saponins, polysaccharides) and improve activity retention rate.

[0018] As a preferred technical solution, the diameter of the micro-nano bubbles in step (1) is 50-100 μm; the air pressure of the flotation operation is 0.3-0.5 MPa, and the bubble density is ≥10 5 The flotation operation time is 10-15 minutes; the addition amount of the defoaming agent is 0.1-0.2% v / v.

[0019] As a preferred technical solution, the pore size of the Al2O3 ceramic membrane in step (1) is 0.2-0.3 μm; the cross-flow velocity of the microfiltration treatment is 2.0-3.0 m / s; the transmembrane pressure is 0.1-0.3 MPa; the temperature of the flotation operation and microfiltration treatment in step (1) is ≤35°C; the flotation-microfiltration coupling technology can effectively remove suspended matter and grease in sea cucumber processing wastewater, avoiding their influence on nutrient extraction, and controlling the operating temperature to ≤35°C can effectively retain heat-sensitive substances and maximize the extraction activity.

[0020] As a preferred technical solution, the pH-responsive flocculant in step (2) is prepared by the following method: dissolving chitosan in 1% acetic acid solution to prepare a chitosan solution; adding sodium hydroxide solution to humic acid and adjusting the pH to 7.0 to prepare a humic acid solution; mixing the chitosan solution and the humic acid solution in a mass ratio of chitosan to humic acid of 2:1 to prepare a pH-responsive flocculant.

[0021] As a preferred technical solution, the deacetylation degree of the chitosan is ≥85%; the addition amount of the pH-responsive flocculant is 0.4-0.6 g / L; and the rotation speed of the stirring treatment is 40-60 rpm.

[0022] Chitosan (with -NH2) and humic acid (with -COOH / -OH) in pH-responsive flocculants (chitosan / humic acid complexes) can preferentially bind to Cu by coordination bonds and electrostatic adsorption under weakly acidic conditions (pH 5.5-6.0). 2+ , Pb 2+ and Cd 2+ Heavy metal ions such as ions are removed by pH responsive flocculants, forming insoluble flocs. Proteins (usually with an isoelectric point of pH 4-5) remain dissolved within this pH range, while polysaccharides and saponins (stable at neutral or weakly acidic conditions) remain unaffected and can be subsequently extracted. Therefore, pH-responsive flocculants can remove heavy metals with high selectivity while avoiding nutrient loss.

[0023] As a preferred technical solution, in step (3), the amount of alkaline protease added in the first stage of enzymatic hydrolysis is 3% w / w, and the amount of cellulase added is 1% w / w; the ultrasonic enzymatic hydrolysis conditions are as follows: 40 kHz, 200 W, pulse mode 5 s on / 5 s off;

[0024] In the first stage of enzymatic hydrolysis: alkaline protease can hydrolyze protein-polysaccharide complexes, release bound polysaccharides, disintegrate the collagen fiber network, and expose cellulose and glycosidic bonds; cellulase decomposes β-1,4-glycosidic bonds, degrades cell wall cellulose and chitin (the main structural components of the sea cucumber body wall), destroys the cell wall structure, increases dissolution channels, breaks down physical barriers, increases substrate accessibility, makes it easier for proteases / glycosidases to access target bonds, significantly improves polysaccharide yield, and avoids the sugar chain breakage caused by traditional strong acid / alkali treatment.

[0025] As a preferred technical solution, the amount of glycosidase added in the second stage enzymatic hydrolysis in step (3) is 0.5% w / w; the ultrasonic enzymatic hydrolysis conditions are as follows: 40 kHz, 150 W, continuous mode.

[0026] In the second stage of enzymatic hydrolysis, glycosidases specifically cleave sugar-protein bonds (such as O-glycosidic bonds and N-glycosidic bonds), releasing saponins and oligosaccharides. These decompose the glycosidic bonds in collagen, releasing active peptides and free saponins, increasing the degree of collagen hydrolysis and enhancing its functional activity. This also removes the shielding effect of sugar chains on proteins, assisting proteases in further hydrolysis and preventing excessive degradation of polysaccharides.

[0027] As a preferred technical solution, the molecular weight cut-off of the ceramic ultrafiltration membrane in step (4) is 10 kDa, which can effectively enrich the active peptide segment of <10 kDa; the initial flux of ultrafiltration of the ceramic ultrafiltration membrane is 20 L / m 2 h, concentrated to a volume ratio of 1:5; the polyethersulfone nanofiltration membrane has a molecular weight cutoff of 100Da and a pore size of 0.5nm, and oligosaccharides (molecular weight 500-3000Da, molecular size 1.5-3nm), so oligosaccharides can be effectively recovered; in addition, Na + The diameter of the hydrated ions is greater than 0.5 nm, so it can also effectively desalinate; the nanofiltration pressure of the polyethersulfone nanofiltration membrane is 0.6-0.8 MPa; the macroporous resin is LX-68 resin; the sample flow rate is 2BV / h, and after adsorption saturation, it is gradient eluted with 70% ethanol, which can selectively adsorb saponins.

[0028] As a preferred technical solution, the conditions of the vacuum thin film evaporation system in step (5) are as follows: absolute pressure 8 kPa, scraper speed 200 rpm, feed rate 5 L / min, and vacuum thin film evaporation system operating temperature ≤ 45°C; the conditions of the microwave drying are as follows: 2450 MHz, 10 s on / 5 s off, intermittent pulse mode.

[0029] Another object of the present invention is to provide: application of the above method in extracting nutrients from sea cucumber processing wastewater; the nutrients include proteins, polysaccharides and saponins.

[0030] Another object of the present invention is to provide: nutrients extracted by the above method.

[0031] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention uses sea cucumber processing wastewater as raw material to extract nutrients from the wastewater. The method adopts flotation-microfiltration coupling technology to pretreat the processed wastewater, which can effectively remove suspended matter and grease in the sewage and retain heat-sensitive substances; at the same time, a pH-responsive flocculant is used to highly selectively remove heavy metals while avoiding the loss of nutrients such as protein, polysaccharide and saponin. After the pretreatment, the coordinated staged enzymatic hydrolysis technology, membrane separation-adsorption combined technology, vacuum thin film evaporation and programmed cooling crystallization technology can effectively separate and extract proteins, polysaccharides and saponins, significantly improving the recovery rate, purity and product performance of each nutrient, and providing technical support for the reuse of nutrients in sea cucumber processing wastewater. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for extracting nutrients from sea cucumber processing wastewater comprises the following steps:

[0036] (1) Wastewater pretreatment: Micro-nano bubbles (diameter 50 μm, air pressure 0.3 MPa, bubble density ≥ 10) are introduced into the sea cucumber processing wastewater. 5 The wastewater was subjected to flotation treatment with 0.1% v / v polydimethylsiloxane (PDMS) for 10 minutes, and 0.1% v / v polydimethylsiloxane was added to suppress foaming. The wastewater was then passed through an Al2O3 ceramic membrane (pore size 0.2 μm) and microfiltered at a cross-flow velocity of 2.0 m / s and a transmembrane pressure of 0.1 MPa to prepare pretreated wastewater. The temperature for the flotation and microfiltration treatments was ≤35°C.

[0037] (2) Selective precipitation of heavy metal ions: adding a pH-responsive flocculant at a dosage of 0.4 g / L to the pretreated wastewater in step (1), adjusting the pH to 5.5, stirring at 40 rpm for 20 min, and filtering to remove heavy metal ions;

[0038] The pH-responsive flocculant was prepared by the following method: chitosan (deacetylation degree ≥85%) was dissolved in 1% acetic acid solution to prepare a chitosan solution; sodium hydroxide solution was added to humic acid to adjust the pH to 7.0, and then mixed with the chitosan solution in a mass ratio of chitosan to humic acid of 2:1 to prepare a pH-responsive flocculant.

[0039] (3) Staged enzymatic hydrolysis:

[0040] The first stage of enzymatic hydrolysis: 3% w / w alkaline protease and 1% w / w cellulase were added to the wastewater treated in step (2), the temperature was adjusted to 44°C, the pH was adjusted to 8.4, and ultrasonic hydrolysis (40kHz, 200W, pulse mode 5s on / 5s off) was carried out for 1.8h.

[0041] The second stage of enzymatic hydrolysis: the temperature was quickly adjusted to 54°C and the pH to 4.9, 0.5% w / w glycosidase was added, and ultrasonic enzymatic hydrolysis (40 kHz, 150 W, continuous mode) was performed for 1.4 h;

[0042] (4) Membrane separation-adsorption purification: The enzymatic hydrolysis product was passed through a ceramic ultrafiltration membrane (molecular weight cut-off of 10 kDa) with an initial flux of 20 L / m 2 h, concentrated to a volume ratio of 1:5, then passed through a polyethersulfone nanofiltration membrane (molecular weight cutoff of 100 Da, nanofiltration membrane pore size of 0.5 nm, nanofiltration pressure of 0.6 MPa, recovery rate set at 70%), and finally passed through a macroporous adsorption resin (LX-68 type) at a flow rate of 2 BV / h. After adsorption saturation, it was eluted with a 70% ethanol gradient;

[0043] (5) Concentration, crystallization and drying: The purified product of step (4) was concentrated to a solid content of 25% using a vacuum thin film evaporation system at an absolute pressure of 8 kPa, a scraper speed of 200 rpm, and a feed rate of 5 L / min. The temperature was then reduced from 40°C to 4°C at a rate of 0.5°C / min to precipitate crystals, and dried to obtain the final product.

[0044] Example 2

[0045] A method for extracting nutrients from sea cucumber processing wastewater comprises the following steps:

[0046] (1) Wastewater pretreatment: Micro-nano bubbles (diameter 100 μm, air pressure 0.5 MPa, bubble density ≥ 10) are introduced into the sea cucumber processing wastewater. 5The wastewater was subjected to flotation treatment with 0.2% v / v polydimethylsiloxane (PDMS) for 15 minutes, and 0.2% v / v polydimethylsiloxane was added to suppress foaming. The wastewater was then passed through an Al2O3 ceramic membrane (pore size 0.3 μm) and microfiltered at a cross-flow velocity of 3.0 m / s and a transmembrane pressure of 0.3 MPa to prepare pretreated wastewater. The temperature for the flotation and microfiltration treatments was ≤35°C.

[0047] (2) Selective precipitation of heavy metal ions: adding a pH-responsive flocculant at a dosage of 0.6 g / L to the pretreated wastewater in step (1), adjusting the pH to 6.0, stirring at 60 rpm for 30 min, and filtering to remove heavy metal ions;

[0048] The pH-responsive flocculant was prepared by the following method: chitosan (deacetylation degree ≥85%) was dissolved in 1% acetic acid solution to prepare a chitosan solution; sodium hydroxide solution was added to humic acid to adjust the pH to 7.0, and then mixed with the chitosan solution in a mass ratio of chitosan to humic acid of 2:1 to prepare a pH-responsive flocculant.

[0049] (3) Staged enzymatic hydrolysis:

[0050] The first stage of enzymatic hydrolysis: 3% w / w alkaline protease and 1% w / w cellulase were added to the wastewater treated in step (2), the temperature was adjusted to 46°C, the pH was adjusted to 8.6, and ultrasonic (40kHz, 200W, pulse mode 5s on / 5s off) enzymatic hydrolysis was carried out for 2.2h.

[0051] The second stage of enzymatic hydrolysis: the temperature was quickly adjusted to 56°C and the pH to 5.1, 0.5% w / w glycosidase was added, and ultrasonic enzymatic hydrolysis (40 kHz, 150 W, continuous mode) was performed for 1.6 h;

[0052] (4) Membrane separation-adsorption purification: The enzymatic hydrolysis product was passed through a ceramic ultrafiltration membrane (molecular weight cut-off of 10 kDa) with an initial flux of 20 L / m 2 h, concentrated to a volume ratio of 1:5, then passed through a polyethersulfone nanofiltration membrane (molecular weight cut-off of 100 Da, nanofiltration membrane pore size of 0.5 nm, nanofiltration pressure of 0.8 MPa, recovery rate set at 70%), and finally passed through a macroporous adsorption resin (LX-68 type) at a flow rate of 2 BV / h. After adsorption saturation, it was eluted with a 70% ethanol gradient;

[0053] (5) Concentration, crystallization and drying: The purified product of step (4) was concentrated to a solid content of 30% using a vacuum thin film evaporation system at an absolute pressure of 8 kPa, a scraper speed of 200 rpm, and a feed rate of 5 L / min. The temperature was then reduced from 40°C to 4°C at a rate of 0.5°C / min to precipitate crystals, and dried to obtain the final product.

[0054] Example 3

[0055] A method for extracting nutrients from sea cucumber processing wastewater comprises the following steps:

[0056] (1) Wastewater pretreatment: Micro-nano bubbles (diameter 80 μm, air pressure 0.4 MPa, bubble density ≥ 10) were introduced into the sea cucumber processing wastewater. 5 The wastewater was subjected to flotation treatment with 0.2% v / v polydimethylsiloxane (PDMS) for 15 minutes, and 0.2% v / v polydimethylsiloxane was added to suppress foaming. The wastewater was then passed through an Al2O3 ceramic membrane (pore size 0.3 μm) and microfiltered at a cross-flow velocity of 2.0 m / s and a transmembrane pressure of 0.2 MPa to prepare pretreated wastewater. The temperature for the flotation and microfiltration treatments was ≤35°C.

[0057] (2) Selective precipitation of heavy metal ions: adding a pH-responsive flocculant at a dosage of 0.5 g / L to the pretreated wastewater in step (1), adjusting the pH to 5.8, stirring at 50 rpm for 25 min, and filtering to remove heavy metal ions;

[0058] The pH-responsive flocculant was prepared by the following method: chitosan (deacetylation degree ≥85%) was dissolved in 1% acetic acid solution to prepare a chitosan solution; sodium hydroxide solution was added to humic acid to adjust the pH to 7.0, and then mixed with the chitosan solution in a mass ratio of chitosan to humic acid of 2:1 to prepare a pH-responsive flocculant.

[0059] (3) Staged enzymatic hydrolysis:

[0060] The first stage of enzymatic hydrolysis: 3% w / w alkaline protease and 1% w / w cellulase were added to the wastewater treated in step (2), the temperature was adjusted to 45°C, the pH was adjusted to 8.5, and ultrasonic (40kHz, 200W, pulse mode 5s on / 5s off) enzymatic hydrolysis was carried out for 2.0h

[0061] The second stage of enzymatic hydrolysis: the temperature was quickly adjusted to 55°C and the pH to 5.0, 0.5% w / w glycosidase was added, and ultrasonic enzymatic hydrolysis (40 kHz, 150 W, continuous mode) was performed for 1.5 h;

[0062] (4) Membrane separation-adsorption purification: The enzymatic hydrolysis product was passed through a ceramic ultrafiltration membrane (molecular weight cut-off of 10 kDa) with an initial flux of 20 L / m 2 h, concentrated to a volume ratio of 1:5, then passed through a polyethersulfone nanofiltration membrane (molecular weight cut-off of 100 Da, nanofiltration membrane pore size of 0.5 nm, nanofiltration pressure of 0.7 MPa, recovery rate set at 70%), and finally passed through a macroporous adsorption resin (LX-68 type) at a flow rate of 2 BV / h. After adsorption saturation, it was eluted with a 70% ethanol gradient;

[0063] (5) Concentration, crystallization and drying: The purified product of step (4) was concentrated to a solid content of 28% using a vacuum thin film evaporation system at an absolute pressure of 8 kPa, a scraper speed of 200 rpm, and a feed rate of 5 L / min. The temperature was then reduced from 40°C to 4°C at a rate of 0.5°C / min to precipitate crystals, and dried to obtain the final product.

[0064] Comparative Example 1

[0065] The operation is basically the same as that of Example 3, except that step (1) is not subjected to flotation treatment.

[0066] Comparative Example 2

[0067] The operation is basically the same as that of Example 3, except that the mass ratio of chitosan to humic acid in step (2) is 1:1.

[0068] Comparative Example 3

[0069] The operation is basically the same as that of Example 3, except that the addition amounts of alkaline protease, cellulase and glycosidase in the staged enzymatic hydrolysis process in step (3) are different, as follows:

[0070] The addition amount of alkaline protease was 3% w / w, the addition amount of cellulase was 0.5% w / w, and the addition amount of glycosidase was 1% w / w.

[0071] Comparative Example 4

[0072] The operation is basically the same as that of Example 3, except that step (4) adopts the traditional alcohol precipitation-column chromatography method instead of the membrane separation-adsorption purification operation of Example 3. The specific process of the traditional alcohol precipitation-column chromatography method is as follows:

[0073] (1) Alcohol precipitation method (primary separation)

[0074] Protein precipitation: adjust the wastewater pH to 4.5 (protein isoelectric point) and centrifuge to remove protein precipitates;

[0075] Polysaccharide precipitation: add ethanol to the supernatant to a concentration of 30% to 40%, let it stand at 4°C for 12 hours, and centrifuge to obtain crude polysaccharide;

[0076] Saponin enrichment: add ethanol to the remaining supernatant to 70% to 80% and centrifuge to obtain a crude saponin extract;

[0077] (2) Column chromatography (fine purification)

[0078] Polysaccharide purification: DEAE ion exchange column, 0-0.5M NaCl gradient elution, collecting the 0.2-0.3M elution fraction;

[0079] Saponin purification: AB-8 macroporous resin column, water → 30% → 70% ethanol gradient elution, 70% ethanol section to enrich saponins;

[0080] Protein purification: gel filtration column (Sephadex G-50) to remove small molecule impurities.

[0081] Effect verification:

[0082] In order to verify the extraction effects of Examples 1-3 and Comparative Examples 1-4 of the present invention, the recovery rates of active peptides, polysaccharides and saponins in different groups were measured respectively. The experimental results are shown in Table 1.

[0083] Table 1 Recovery rates of proteins, polysaccharides and saponins by different methods

[0084]

[0085] Result analysis: From the data in Table 1, it can be seen that Examples 1-3 are superior to Comparative Examples 1-4 in terms of the recovery rates of active peptides, polysaccharides and saponins, and the recovery rates of various nutrients are significantly improved.

[0086] The purity of the recovered products of each nutrient in different groups was further determined, and the experimental results are shown in Table 2.

[0087] Table 2 Purity of proteins, polysaccharides and saponins in different groups

[0088]

[0089] Result analysis: From the content in Table 2, the purity of the nutrients extracted from each embodiment of the present invention reached a high level and was better than that of comparative examples 1-4.

[0090] In order to verify the product performance of the sea cucumber active peptide extracted by the present invention, an ACE inhibitory activity determination experiment was carried out, and the experimental results are shown in Table 3.

[0091] Table 3 ACE inhibitory activity of active peptides extracted from sea cucumbers by different methods

[0092]

[0093] In order to verify the performance of the polysaccharide product extracted by the present invention, experiments were conducted to determine the sulfate content and molecular weight distribution CV value. The experimental results are shown in Table 4.

[0094] Table 4 Sulfate content and molecular weight distribution CV values ​​of polysaccharides extracted by different methods

[0095]

[0096] In order to verify the performance of the saponin product extracted by the present invention, the hemolytic index (HD50) was determined, and the experimental results are shown in Table 5.

[0097] Table 5 Hemolytic index (HD50) of saponins extracted by different methods

[0098]

[0099] Results analysis shows that the product performance of Examples 1-3 of the present invention is better than that of Comparative Examples 1-4, which indicates that the overall technical solution of the present invention can significantly improve product performance and increase its usable value.

[0100] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0101] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting nutrients from sea cucumber processing wastewater, characterized in that: The steps include: (1) Wastewater pretreatment: Micro-nano bubbles are introduced into the sea cucumber processing wastewater for flotation operation, and a defoaming agent is added to suppress foaming; the wastewater is then passed through an Al2O3 ceramic membrane for microfiltration treatment to prepare pretreated wastewater; (2) Selectively precipitating heavy metal ions: adding a pH-responsive flocculant to the wastewater pretreated in step (1), adjusting the pH to 5.5-6.0, stirring for 20-30 minutes, and filtering to remove heavy metal ions; (3) Staged enzymatic hydrolysis: The first stage of enzymatic hydrolysis: add alkaline protease and cellulase to the wastewater treated in step (2), adjust the temperature to 44-46°C, adjust the pH to 8.4-8.6, and perform ultrasonic enzymatic hydrolysis for 1.8-2.2 hours; The second stage of enzymatic hydrolysis: quickly adjust the temperature to 54-56 ° C, pH to 4.9-5.1, add glycosidase, and perform ultrasonic enzymatic hydrolysis for 1.4-1.6 hours; (4) Membrane separation-adsorption combined purification: The enzymatic hydrolysis product is purified by passing through a ceramic ultrafiltration membrane, a polyethersulfone nanofiltration membrane, and a macroporous adsorption resin in sequence; (5) Concentration, crystallization, and drying: The purified product from step (4) was concentrated to a solid content of 25-30% using a vacuum thin film evaporation system; the temperature was then reduced from 40°C to 4°C at a rate of 0.5°C / min to precipitate crystals, which were then microwave-dried to obtain the final product.

2. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that: The diameter of the micro-nano bubbles in step (1) is 50-100 μm; the air pressure of the flotation operation is 0.3-0.5 MPa, and the bubble density is ≥10 5 The flotation operation time is 10-15 minutes; the addition amount of the defoaming agent is 0.1-0.2% v / v.

3. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that: The pore size of the Al2O3 ceramic membrane in step (1) is 0.2-0.3 μm; the cross-flow velocity of the microfiltration treatment is 2.0-3.0 m / s; the transmembrane pressure is 0.1-0.3 MPa; and the temperature of the flotation operation and microfiltration treatment in step (1) is ≤35°C.

4. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that: The pH-responsive flocculant in step (2) is prepared by the following method: dissolving chitosan in 1% acetic acid solution to prepare a chitosan solution; adding sodium hydroxide solution to humic acid and adjusting the pH to 7.0 to prepare a humic acid solution; mixing the chitosan solution and the humic acid solution in a mass ratio of chitosan to humic acid of 2:1 to prepare a pH-responsive flocculant.

5. The method for extracting nutrients from sea cucumber processing wastewater according to claim 4, characterized in that: The deacetylation degree of the chitosan is ≥85%; the addition amount of the pH-responsive flocculant is 0.4-0.6 g / L; and the rotation speed of the stirring treatment is 40-60 rpm.

6. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that: In step (3), the amount of alkaline protease added in the first stage of enzymatic hydrolysis was 3% w / w, and the amount of cellulase added was 1% w / w; the ultrasonic enzymatic hydrolysis conditions were as follows: 40 kHz, 200 W, pulse mode 5 s on / 5 s off; Step (3) The amount of glycosidase added in the second stage of enzymatic hydrolysis is 0.5% w / w; the ultrasonic enzymatic hydrolysis conditions are as follows: 40 kHz, 150 W, continuous mode.

7. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that: The molecular weight cut-off of the ceramic ultrafiltration membrane in step (4) is 10 kDa; the initial flux of ultrafiltration of the ceramic ultrafiltration membrane is 20 L / m 2 h, concentrated to a volume ratio of 1:5; the polyethersulfone nanofiltration membrane had a molecular weight cutoff of 100 Da and a pore size of 0.5 nm; the nanofiltration pressure of the polyethersulfone nanofiltration membrane was 0.6-0.8 MPa; the macroporous resin was LX-68 resin; the sample loading rate was 2 BV / h, and after adsorption saturation, gradient elution was performed using 70% ethanol.

8. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that: The conditions of the vacuum thin film evaporation system in step (5) are as follows: absolute pressure 8 kPa, scraper speed 200 rpm, feed rate 5 L / min, and vacuum thin film evaporation system operating temperature ≤ 45°C; the conditions of the microwave drying are as follows: 2450 MHz, 10 s on / 5 s off, intermittent pulse mode.

9. Use of the method according to any one of claims 1 to 8 in extracting nutrients from sea cucumber processing wastewater, wherein the nutrients include proteins, polysaccharides and saponins.

10. Nutrients extracted by the method according to any one of claims 1 to 8.

Images