Method for extracting nutrients from marine processing wastewater and application thereof

Abstract

The application discloses a method for extracting nutrients from seaweed processing wastewater and application, and belongs to the technical field of extracting nutrients from seaweed processing wastewater. The method is characterized by using seaweed processing wastewater as raw material to extract nutrients from the wastewater. In the method, a gas flotation-microfiltration coupling technology is used to pretreat the wastewater, so that suspended solids and oil in the wastewater can be effectively removed, and heat-sensitive substances can be reserved. Meanwhile, a pH responsive flocculant is used to selectively remove heavy metals, so that the loss of nutrients such as protein, polysaccharide and saponin can be avoided. After the pretreatment, a combined technology of staged enzymolysis, membrane separation-adsorption, vacuum thin film evaporation and programmed cooling crystallization can be used to effectively separate and extract the protein, polysaccharide and saponin, so that the recovery rate, purity and product performance of the nutrients can be significantly improved, and technical support is provided for the reuse of the nutrients in the seaweed processing wastewater.

Description

Technical Field

[0001] This invention relates to the field of sea cucumber processing wastewater extraction technology, and more specifically to a method and application for extracting nutrients from sea cucumber processing wastewater. Background Technology

[0002] Sea cucumbers are rich in active ingredients such as protein, polysaccharides, and saponins, possessing high medicinal value. In recent years, my country's sea cucumber farming output has exceeded 204,000 tons, with a production value of approximately 60 billion yuan, indicating a vast market. As a marine product with extremely high nutritional value, current research and applications of sea cucumber processing mainly focus on sea cucumber polysaccharides, sea cucumber saponins, and sea cucumber polypeptides. After being caught, fresh sea cucumbers easily undergo autolysis due to the presence of proteolytic enzymes such as autolytic enzymes. To facilitate preservation, fresh sea cucumbers are usually processed and stored after hot water inactivation of these enzymes. However, this hot water inactivation process generates a large amount of processing wastewater, which research has found to contain abundant sugars, proteins, saponins, phospholipids, and fatty acids. In traditional processes, this wastewater is directly discharged, causing environmental pollution; furthermore, the wastewater contains a large amount of nutrients, and direct discharge would also lead to resource waste.

[0003] Currently, there are some solutions for the reuse of sea cucumber processing wastewater, such as CN 104522728A, entitled "A Method for Extracting Phospholipids and Sea Cucumber Oil from Sea Cucumber Blanching Liquid," which involves the extraction of phospholipids and sea cucumber oil from sea cucumber processing wastewater. Another example is CN 101473979 A, ​​entitled "A Method for Extracting Sea Cucumber Oil from Sea Cucumber Boiling Juice," which also involves the extraction of 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: a large molecular weight range: the wastewater contains large molecules (collagen, 10-300 kDa), medium molecules (polysaccharides, 5-50 kDa), and small molecules (saponins, <1 kDa), which traditional extraction techniques struggle to handle simultaneously. Surface activity interference: saponins generate foam (foam height >50 cm), disrupting the stability of the membrane separation process. Traditional processing techniques can lead to heat-sensitive degradation, causing over 60% of bioactive peptides to become short and denatured. They also alter the three-dimensional structure of polysaccharides (β-glucan helical unwinding >40%), resulting in decreased immunomodulatory function. These factors significantly increase the difficulty of extracting proteins, polysaccharides, and saponins from sea cucumber processing wastewater, reducing 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 and improve the recovery rate, extraction activity and purity of each nutrient is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[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 air flotation, and defoamer is added to suppress foam generation; then the wastewater is passed through an Al2O3 ceramic membrane for microfiltration to prepare pretreated wastewater.

[0009] (2) Selective precipitation of heavy metal ions: Add pH-responsive flocculant to the pretreated wastewater in step (1), adjust the pH to 5.5-6.0, stir for 20-30 min, and filter to remove heavy metal ions;

[0010] (3) Staged enzymatic hydrolysis:

[0011] First stage of enzymatic hydrolysis: Alkaline protease and cellulase are added to the wastewater after step (2), the temperature is adjusted to 44-46℃, the pH is adjusted to 8.4-8.6, and ultrasonic enzymatic hydrolysis is performed for 1.8-2.2h.

[0012] Second stage of enzymatic hydrolysis: rapidly adjust the temperature to 54-56℃, pH to 4.9-5.1, add glycosidase, and sonicate for 1.4-1.6 hours.

[0013] (4) Membrane separation-adsorption combined purification: The enzymatic hydrolysis products are sequentially purified by passing through a ceramic ultrafiltration membrane, a polyether sulfone nanofiltration membrane, and a macroporous adsorption resin.

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

[0015] Ultrafiltration synergistically combines with nanofiltration. Ultrafiltration removes large molecular impurities, reduces nanofiltration membrane fouling, and improves the membrane flux stability of nanofiltration. When nanofiltration is combined with resin adsorption, nanofiltration desalinates (Na+). + After achieving a removal rate of >90%, the resin adsorption efficiency can be improved, and the amount of saponins adsorbed can be increased.

[0016] (5) Concentration, crystallization and drying: The product purified in step (4) is concentrated to 25-30% of solid content using a vacuum thin film evaporation system; then crystals are precipitated by cooling from 40℃ to 4℃ at a rate of 0.5℃ / min, and dried to obtain the final product.

[0017] Under low-temperature vacuum conditions (absolute pressure 5-10 kPa), the boiling point of water is reduced to 30-40°C, which can effectively prevent the high-temperature degradation of heat-sensitive components (saponins, polysaccharides) and improve the 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 air flotation operation is 0.3-0.5 MPa, and the bubble density is ≥10. 5 The flotation operation time is 10-15 min; the amount of defoamer added 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 0.2-0.3μm; the crossflow velocity of the microfiltration treatment is 2.0-3.0m / s; the transmembrane pressure is 0.1-0.3MPa; the temperature of the air flotation operation and microfiltration treatment in step (1) is ≤35℃; the air flotation-microfiltration coupling technology can effectively remove suspended solids and oils in sea cucumber processing wastewater, avoid their impact on nutrient extraction, and control the operating temperature to ≤35℃, which can effectively retain heat-sensitive substances and maximize the retention of extraction activity.

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

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

[0022] In pH-responsive flocculants (chitosan / humic acid complex), chitosan (containing -NH2) and humic acid (containing -COOH / -OH) preferentially bind to Cu through coordination bonds and electrostatic adsorption under weakly acidic conditions (pH 5.5-6.0). 2+ Pb 2+ and Cd 2+ When heavy metal ions are removed, insoluble flocs are formed, while proteins (with an isoelectric point typically between pH 4 and 5) remain soluble within this pH range. Polysaccharides and saponins (stable in neutral or weakly acidic conditions) are unaffected and can be extracted subsequently. Therefore, pH-responsive flocculants can selectively remove heavy metals while avoiding the loss of nutrients.

[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: 40kHz, 200W, pulse mode 5s on / 5s off.

[0024] In the first stage of enzymatic hydrolysis: alkaline proteases hydrolyze protein-polysaccharide complexes, releasing bound polysaccharides, breaking down collagen fiber networks, and exposing cellulose and glycosidic bonds; cellulases decompose β-1,4-glycosidic bonds, degrade cell wall cellulose and chitin (the main structural components of the sea cucumber body wall), disrupt cell wall structure, increase dissolution channels, break down physical barriers, increase substrate accessibility, and make it easier for proteases / glycosidases to access target bond sites, significantly improving polysaccharide yield and avoiding the sugar chain breakage caused by traditional strong acid / alkali treatment.

[0025] As a preferred technical solution, in 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.

[0026] In the second stage of enzymatic hydrolysis: glycosidases specifically cleave sugar-protein linkages (such as O-glycosidic bonds and N-glycosidic bonds), releasing saponins and oligosaccharides, breaking down glycosidic bonds in collagen, releasing active peptides and free saponins, increasing the degree of collagen hydrolysis, and enhancing functional activity. Simultaneously, it can also remove the shielding effect of glycan chains on proteins, assisting proteases in further hydrolysis; and preventing excessive degradation of polysaccharides.

[0027] As a preferred technical solution, the ceramic ultrafiltration membrane in step (4) has a molecular weight cutoff of 10 kDa, which can effectively enrich active peptides <10 kDa; the initial flux 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 100 Da and a pore size of 0.5 nm, while oligosaccharides (molecular weight 500-3000 Da, molecular size 1.5-3 nm) can be effectively recovered; in addition, Na + The hydrated ion diameter is >0.5nm, so it can also effectively desalinate; the nanofiltration pressure of the polyethersulfone nanofiltration membrane is 0.6-0.8MPa; the macroporous resin is LX-68 resin; the sample loading flow rate is 2BV / h, and after adsorption saturation, it is eluted with a gradient of 70% ethanol, which can selectively adsorb saponin substances.

[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 operating temperature of the vacuum thin film evaporation system ≤ 45℃; the conditions of the microwave drying are as follows: 2450 MHz, 10s on / 5s off, intermittent pulse mode.

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

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

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

[0032] This invention uses sea cucumber processing wastewater as raw material to extract nutrients from the wastewater. The method employs air flotation-microfiltration coupling technology for pretreatment of the processing wastewater, which can effectively remove suspended solids and oils while retaining heat-sensitive substances. At the same time, a pH-responsive flocculant is used to selectively remove heavy metals while avoiding the loss of nutrients such as proteins, polysaccharides, and saponins. After pretreatment, a combination of staged enzymatic hydrolysis, membrane separation-adsorption, 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. This provides technical support for the reuse of nutrients in sea cucumber processing wastewater. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

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

[0036] (1) Wastewater pretreatment: Micro-nano bubbles (50μm in diameter, 0.3MPa pressure, and ≥10) are introduced into the sea cucumber processing wastewater. 5 The wastewater was subjected to air flotation (particles / ml) for 10 min, with 0.1% v / v polydimethylsiloxane added to suppress foam formation. Subsequently, the wastewater was passed through an Al2O3 ceramic membrane (pore size 0.2 μm) for microfiltration at a cross-flow velocity of 2.0 m / s and a transmembrane pressure of 0.1 MPa to prepare pretreated wastewater. The temperature of the air flotation operation and microfiltration treatment was ≤35℃.

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

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

[0039] (3) Staged enzymatic hydrolysis:

[0040] First stage enzymatic hydrolysis: Add 3% w / w alkaline protease and 1% w / w cellulase to the wastewater after step (2), adjust the temperature to 44℃, adjust the pH to 8.4, and perform enzymatic hydrolysis by ultrasound (40kHz, 200W, pulse mode 5s on / 5s off) for 1.8h.

[0041] Second stage of enzymatic hydrolysis: rapidly adjust the temperature to 54℃, pH to 4.9, add 0.5% w / w glycosidase, and enzymatically hydrolyze for 1.4h using ultrasound (40kHz, 150W, continuous mode).

[0042] (4) Membrane separation-adsorption purification: The enzymatic hydrolysis product is passed through a ceramic ultrafiltration membrane (molecular weight cutoff of 10 kDa) with an initial flux of 20 L / m 2 The sample was concentrated to a volume ratio of 1:5 and 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%). Finally, it was passed through a macroporous adsorption resin (LX-68 type) at a loading flow rate of 2 BV / h. After adsorption saturation, it was eluted with a gradient of 70% ethanol.

[0043] (5) Concentration, crystallization and drying: The product purified in step (4) was concentrated to 25% of the solid content 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. Then, the temperature was reduced from 40℃ to 4℃ at a rate of 0.5℃ / min to precipitate crystals. The product was then dried to obtain the final product.

[0044] Example 2

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

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

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

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

[0049] (3) Staged enzymatic hydrolysis:

[0050] First stage enzymatic hydrolysis: 3% w / w alkaline protease and 1% w / w cellulase were added to the wastewater after step (2), the temperature was adjusted to 46℃, the pH was adjusted to 8.6, and enzymatic hydrolysis was performed by ultrasound (40kHz, 200W, pulse mode 5s on / 5s off) for 2.2h.

[0051] Second stage of enzymatic hydrolysis: rapidly adjust the temperature to 56℃ and pH to 5.1, add 0.5% w / w glycosidase, and enzymatically hydrolyze for 1.6h using ultrasound (40kHz, 150W, continuous mode).

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

[0053] (5) Concentration, crystallization and drying: The product purified in step (4) was concentrated to 30% of the solid content 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. Then, the temperature was reduced from 40℃ to 4℃ at a rate of 0.5℃ / min to precipitate crystals. The product was then dried to obtain the final product.

[0054] Example 3

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

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

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

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

[0059] (3) Staged enzymatic hydrolysis:

[0060] First stage enzymatic hydrolysis: Add 3% w / w alkaline protease and 1% w / w cellulase to the wastewater after step (2), adjust the temperature to 45℃, adjust the pH to 8.5, and perform enzymatic hydrolysis by ultrasound (40kHz, 200W, pulse mode 5s on / 5s off) for 2.0h.

[0061] Second stage of enzymatic hydrolysis: rapidly adjust the temperature to 55℃ and pH to 5.0, add 0.5% w / w glycosidase, and enzymatically hydrolyze for 1.5h using ultrasound (40kHz, 150W, continuous mode).

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

[0063] (5) Concentration, crystallization and drying: The product purified in step (4) was concentrated to 28% of the solid content 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. Then, the temperature was reduced from 40℃ to 4℃ at a rate of 0.5℃ / min to precipitate crystals. The product was then dried to obtain the final product.

[0064] Comparative Example 1

[0065] The operation is basically the same as that in Example 3, except that step (1) does not involve air flotation.

[0066] Comparative Example 2

[0067] The operation is basically the same as in 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 in Example 3, except that the amount of alkaline protease, cellulase and glycosidase added during the staged enzymatic hydrolysis in step (3) is different, as follows:

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

[0071] Comparative Example 4

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

[0073] (I) Alcohol precipitation (primary separation)

[0074] Protein precipitation: Adjust the pH of the wastewater to 4.5 (the isoelectric point of protein), and remove the protein precipitate by centrifugation;

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

[0076] Saponin enrichment: Add ethanol to the remaining supernatant to 70%–80%, centrifuge to obtain crude saponin extract;

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

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

[0079] Saponin purification: AB-8 macroporous resin column, water → 30% → 70% ethanol gradient elution, 70% ethanol fraction for saponin enrichment;

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

[0081] Effect verification:

[0082] 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, and the experimental results are shown in Table 1.

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

[0084]

[0085] Results analysis: As shown in Table 1, Examples 1-3 were superior to Comparative Examples 1-4 in terms of the recovery rate of active peptides, polysaccharides and saponins, significantly improving the recovery rate of each nutrient.

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

[0087] Table 2 Purity of proteins, polysaccharides, and saponins by different methods

[0088]

[0089] Results analysis: As shown in Table 2, the purity of the nutrients extracted in each embodiment of the present invention reached a high level, which is better than that of comparative examples 1-4.

[0090] To verify the product performance of the sea cucumber active peptides extracted in this invention, an ACE inhibition activity assay was conducted, and the results are shown in Table 3.

[0091] Table 3. ACE inhibitory activity of bioactive peptides extracted from sea cucumbers using different methods.

[0092]

[0093] To verify the performance of the polysaccharide product extracted by this 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) of polysaccharides extracted by different methods.

[0095]

[0096] To verify the performance of the saponin product extracted in this invention, the hemolysis index (HD50) was determined, and the experimental results are shown in Table 5.

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

[0098]

[0099] The results analysis, as shown in Tables 3-5, indicate that the product performance of Examples 1-3 of the present invention is superior to that of Comparative Examples 1-4, demonstrating that the overall technical solution of the present invention can significantly improve product performance and increase its usability.

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

[0101] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded 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, Includes the following steps: (1) Wastewater pretreatment: Micro-nano bubbles are introduced into the sea cucumber processing wastewater for air flotation, and defoamer is added to suppress foam generation; then the wastewater is passed through an Al2O3 ceramic membrane for microfiltration to prepare pretreated wastewater. (2) Selective precipitation of heavy metal ions: Add pH-responsive flocculant to the pretreated wastewater in step (1), adjust the pH to 5.5-6.0, stir for 20-30 min, and filter to remove heavy metal ions; (3) Staged enzymatic hydrolysis: First stage of enzymatic hydrolysis: Add alkaline protease and cellulase to the wastewater after treatment in step (2), adjust the temperature to 44-46℃, adjust the pH to 8.4-8.6, and perform ultrasonic enzymatic hydrolysis for 1.8-2.2h; Second stage of enzymatic hydrolysis: rapidly adjust the temperature to 54-56℃, pH to 4.9-5.1, add glycosidase, and sonicate for 1.4-1.6 hours. (4) Membrane separation-adsorption combined purification: The enzymatic hydrolysis products are purified sequentially by passing through a ceramic ultrafiltration membrane, a polyethersulfone nanofiltration membrane, and a macroporous adsorption resin; (5) Concentration, crystallization and drying: The product purified in step (4) is concentrated to 25-30% of solid content using a vacuum thin film evaporation system; then crystals are precipitated by cooling from 40℃ to 4℃ at a rate of 0.5℃ / min, and microwave drying is performed to obtain the final product; The pH-responsive flocculant in step (2) is prepared by the following method: chitosan is dissolved in 1% acetic acid solution to prepare chitosan solution; sodium hydroxide solution is added to humic acid to adjust the pH to 7.0 to prepare humic acid solution; the chitosan solution and humic acid solution are mixed at a mass ratio of chitosan to humic acid of 2:1 to prepare pH-responsive flocculant. 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: 40kHz, 200W, pulse mode 5s on / 5s off. In 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: 40kHz, 150W, continuous mode.

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 for the air flotation operation is 0.3-0.5 MPa, and the bubble density is ≥10. 5 The flotation operation time is 10-15 min; the amount of defoamer added 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 crossflow 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 air flotation operation and microfiltration treatment in step (1) is ≤35℃.

4. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that, The degree of deacetylation of the chitosan is ≥85%; the amount of pH-responsive flocculant added is 0.4-0.6 g / L; and the stirring speed is 40-60 rpm.

5. The method for extracting nutrients from sea cucumber processing wastewater according to claim 1, characterized in that, The ceramic ultrafiltration membrane in step (4) has a molecular weight cutoff of 10 kDa; the initial flux of the ceramic ultrafiltration membrane is 20 L / m³. 2 The sample was concentrated to a volume ratio of 1:5 by h; 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 adsorption resin was LX-68 resin; the sample loading flow rate was 2 BV / h, and after adsorption saturation, it was eluted with a gradient of 70% ethanol.

6. 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 operating temperature of the vacuum thin-film evaporation system ≤ 45℃; the conditions of the microwave drying are as follows: 2450 MHz, 10 s on / 5 s off, intermittent pulse mode.

Images