Method for extracting bioactive substances of microalgae in high-salt and alkali environment and application thereof

Abstract

This disclosure provides a method for extracting bioactive substances from microalgae in high-salt-alkali environments, specifically including low-temperature plasma pretreatment; mixed enzymatic hydrolysis to break down cell walls, the mixed enzymes including cellulase and pectinase; ultrasonic extraction with a salt-tolerant extract; and purification by aqueous two-phase extraction followed by hydrophobic chromatography. The technical solution disclosed in this invention solves the problems of low extraction rate, high cost, and salt-alkali interference in the existing extraction of bioactive substances from high-salt-alkali microalgae, achieving (1) increasing the extraction rate of bioactive substances from high-salt-alkali microalgae to ≥90%; (2) shortening the purification process to ≤24 hours, with a target product purity of ≥95%; and (3) reducing the interference of the salt-alkali environment on the extraction process and reducing the amount of organic solvent used by more than 30%.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a highly efficient method for the extraction and purification of bioactive substances (such as β-carotene, phycocyanin, and polysaccharides) from microalgae (such as Dunaliella salina and Chlorella) growing in high saline-alkali environments (such as salt lakes in Xinjiang), which is suitable for the industrial development of microalgae resources under extreme environments. Background Technology

[0002] Microalgae generally refers to a collective term for microorganisms containing chlorophyll a and capable of photosynthesis, belonging to a type of protist organism. Microalgae cultivated or produced in large quantities using biotechnology belong to four phyla: Chlorophyta, Cyanophyta, Chrysophyta, and Rhodophyta. Chlorophyta includes Dunaliella salina and Chlorella vulgaris, among others. Dunaliella salina is extremely salt-tolerant, surviving in near-freshwater to saturated saline environments (salinity > 5 mol / L NaCl), and is commonly found in high-salinity environments such as salt pans and salt lakes. Chlorella vulgaris is a highly adaptable green algae that can rapidly reproduce in environments with a wide salinity range (freshwater to brackish water), temperatures of 10-36℃ (optimal 24-28℃), and light intensities of approximately 150 μmol / m² / s.

[0003] Microalgae have a wide range of applications in many fields, such as functional foods, health products, and the pharmaceutical industry. Microalgae cells contain high-value nutrients and chemical raw materials, including proteins, lipids, algal polysaccharides, β-carotene, and various inorganic elements (such as Cu, Fe, Se, Mn, Zn, etc.). For example, Chlorella has a protein content as high as 50%-57%, is rich in fats, carbohydrates, and vitamins (A, C, E), and has a chlorophyll content of 3000-7000 mg / 100g. Dunaliella salina contains high concentrations of β-carotene (13% of dry weight), α-carotene, and chlorophyll, with α-carotene having an antioxidant capacity more than 10 times that of β-carotene; it is also rich in glycerol (50%), protein (40%), vitamins (such as vitamins A and E), minerals (more than 70 kinds), and polysaccharides, with elemental ratios highly similar to those in human body fluids.

[0004] However, existing technologies for extracting these nutrients have the following problems: (1) Low extraction efficiency: Traditional organic solvents (such as acetone and ethanol) have poor penetration into the cell walls of high-salt-alkali microalgae, and the extraction rate of β-carotene is only 50%-60% (Li et al., 2021); Patent CN112233456A uses traditional ultrasound-assisted extraction of β-carotene from Dunaliella salina, which takes 6 hours and has an extraction rate of 65%; impurity ions (such as Ca²⁺ and Mg²⁺) in the crude extract of salt lake microalgae cause column blockage and require frequent regeneration (Wang et al., 2022). (2) High purification cost: Chromatography requires multiple steps (such as ion exchange + gel filtration), takes more than 48 hours, and has a target product loss rate of more than 30%. (3) Salt and alkali interference: High concentrations of Na⁺ / Cl⁻ inhibit enzymatic hydrolysis or membrane separation efficiency, resulting in polysaccharide purity of less than 80%. Summary of the Invention

[0005] The present invention aims to solve the problems of low extraction rate, high cost and salt and alkali interference in the existing extraction of active substances from microalgae in high salt and alkali environments.

[0006] The primary objective of this invention is to provide a method for extracting bioactive substances from microalgae in high-salt-alkaline environments.

[0007] A second objective of this invention is to provide bioactive substances prepared by the above method.

[0008] A third objective of this invention is to provide an application of the above-described method.

[0009] First, this invention provides a method for extracting bioactive substances from microalgae in high-salt-alkaline environments, the specific steps of which are as follows: Step S1, low-temperature plasma pretreatment; Step S2, mixed enzymatic hydrolysis to break the cell wall, the mixed enzymes include cellulase and pectinase, the mass concentration of cellulase is 0.5%-2% (w / v) and the mass concentration of pectinase is 0.3%-1% (w / v), the mass concentration ratio of the two is about 1.5:1-2:1; Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 2:1-4:1 and a solid-liquid ratio of 1:10-1:20 (g / mL).

[0010] Step S4: Aqueous two-phase extraction followed by hydrophobic chromatography purification. The extract uses PEG and ammonium sulfate, with the PEG 4000-8000 having a mass concentration of 8%-12% and the ammonium sulfate having a mass concentration of 12%-18%.

[0011] Preferably, in step S1, the low-temperature plasma parameters are a power of 150-250 W and a time of 3-10 minutes.

[0012] Preferably, the concentration of cellulase in step S2 is 1.2%, and the concentration of pectinase is 0.8%; the ratio of the two is preferably 1:0.6.

[0013] Preferably, the enzymatic hydrolysis in step S2 is performed at a pH of 5-6 and a temperature of 50-60°C.

[0014] Preferably, in step S3, the ultrasonic extraction parameters are 40-55 kHz, the temperature is 50-60℃, and the time is 24-30 minutes.

[0015] Preferably, in step S3, the molar ratio of isopropanol / choline chloride eutectic solvent is 3:1, and the material-to-liquid ratio is 1:15 (g / mL). Preferably, the hydrophobic chromatography medium in step S4 is phenyl / butyl agarose gel.

[0016] Furthermore, the present invention provides microalgal bioactive substances prepared by the above method.

[0017] Furthermore, the present invention provides the application of microalgae prepared by the above method in the preparation of functional foods, health products and pharmaceutical raw materials by the above method. Beneficial effects

[0018] This invention provides a method for extracting bioactive substances from microalgae in high-salt-alkali environments. The composite cell wall disruption technology disclosed in this invention, combined with low-temperature plasma pretreatment and mixed enzymatic hydrolysis, can directionally disrupt the cell walls of high-salt microalgae. The salt-tolerant extraction solvent disclosed in this invention uses isopropanol / choline chloride eutectic solvent, which is suitable for high-salt environments and removes metal ions simultaneously. The integrated purification process disclosed in this invention uses aqueous two-phase extraction followed by hydrophobic chromatography to remove pigments and salts in one step. The technical solution disclosed in this invention solves the problems of low extraction rate, high cost, and salt-alkali interference in the extraction of bioactive substances from high-salt-alkali microalgae, and achieves (1) increasing the extraction rate of bioactive substances from high-salt-alkali microalgae to ≥90%; (2) shortening the purification process to ≤24 hours, with a target product purity of ≥95%; and (3) reducing the interference of the salt-alkali environment on the extraction process and reducing the amount of organic solvent used by more than 30%. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure.

[0020] In this invention, low-temperature plasma (LTP) is an ionized gas composed of active particles such as high-energy electrons, ions, and free radicals, with an overall temperature close to room temperature (20-40℃). Its characteristics include: (1) High activity: It generates ·OH, O2⁻, H2O2, O3 (ozone), NO (nitric oxide), O (atomic oxygen), etc. through electric field excitation. The specific types depend on the active substances such as plasma gas (e.g., air, oxygen, nitrogen / oxygen mixture). The bond energy of biomass chemical bonds (e.g., CC bond ~3.6 eV, CH bond ~4.3 eV) is significantly lower than the energy of high-energy electrons in LTP (1-10 eV). Therefore, electrons can directly break chemical bonds or indirectly oxidize / reduce through active species. (2) Non-thermal effect-dominated: The inactivation process mainly relies on chemical action rather than thermal effect, so it is safer for host cells (e.g., microalgae, cells, bacteria) or heat-sensitive materials (e.g., proteins, polymers).

[0021] Example 1: Extraction of β-carotene from Dunaliella salina

[0022] Step S0: Take 10 g of Dunaliella salina powder cultured in a high salinity environment (salinity 10%). Step S1, low-temperature plasma pretreatment, 200 W, 5 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 0.5% and the concentration of pectinase is 0.3%. The reaction is carried out at pH 5.0 and 50°C for 2 hours. Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 2:1 and a solid-liquid ratio of 1:10. Ultrasonic extraction is performed at 50 kHz and 50 °C for 30 minutes.

[0023] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 8% and the concentration of ammonium sulfate is 12%.

[0024] HPLC analysis showed that the yield of β-carotene was 91.6%, which was significantly different from the 58.7% yield of the control group extracted with acetone. The residual salt content after aqueous two-phase extraction was <0.5%, and the hydrophobic chromatography purity was 97.9%.

[0025] Example 2: Extraction of β-carotene from Dunaliella salina

[0026] Step S0: Take 10 g of Dunaliella salina powder cultured in a high salinity environment (salinity 10%). Step S1, low-temperature plasma pretreatment, 220 W, 5 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 1.2% and the concentration of pectinase is 0.8%. The reaction is carried out at pH 5.0 and 50°C for 2 hours. Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 3:1 and a solid-liquid ratio of 1:15. Ultrasonic extraction is performed at 50 kHz and 50 °C for 30 minutes.

[0027] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 10% and the concentration of ammonium sulfate is 15%.

[0028] HPLC analysis showed that the yield of β-carotene was 95.1%, which was significantly different from the 58.7% yield of the control group extracted with traditional acetone. The residual salt content after aqueous two-phase extraction was <0.5%, and the hydrophobic chromatography purity was 98.5%.

[0029] Example 3: Extraction of β-carotene from Dunaliella salina

[0030] Step S0: Take 10 g of Dunaliella salina powder cultured in a high salinity environment (salinity 10%). Step S1, low-temperature plasma pretreatment, 220 W, 7 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 1.5% and the concentration of pectinase is 0.4%. The reaction is carried out at pH 5.5 and 55°C for 2 hours. Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 3:1 and a solid-liquid ratio of 1:15. Ultrasonic extraction is performed at 50 kHz and 50 °C for 30 minutes.

[0031] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 10% and the concentration of ammonium sulfate is 15%.

[0032] HPLC analysis showed that the yield of β-carotene was 93.1%, which was significantly different from the 58.7% yield of the control group extracted with acetone. The residual salt content after aqueous two-phase extraction was <0.5%, and the hydrophobic chromatography purity was 98.0%.

[0033] Example 4: Extraction of β-carotene from Dunaliella salina

[0034] Step S0: Take 10 g of Dunaliella salina powder cultured in a high salinity environment (salinity 10%). Step S1, low-temperature plasma pretreatment, 220 W, 7 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 1%, the concentration of pectinase is 0.5%, the pH is 5.5, and the reaction is carried out at 55°C for 2 hours. Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 4:1 and a solid-liquid ratio of 1:10. Ultrasonic extraction is performed at 40 kHz and 50 °C for 30 minutes.

[0035] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 10% and the concentration of ammonium sulfate is 15%.

[0036] HPLC analysis showed that the yield of β-carotene was 92.6%, which was significantly different from the 58.7% yield of the control group extracted with acetone. The residual salt content after aqueous two-phase extraction was <0.5%, and the hydrophobic chromatography purity was 98.1%.

[0037] Example 5: Extraction of β-carotene from Dunaliella salina

[0038] Step S0: Take 10 g of Dunaliella salina powder cultured in a high salinity environment (salinity 10%). Step S1, low-temperature plasma pretreatment, 250 W, 10 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 2% and the concentration of pectinase is 1%. The reaction is carried out at pH 5.5 and 55°C for 2 hours. Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 4:1 and a solid-liquid ratio of 1:25. Ultrasonic extraction is performed at 50 kHz and 60 °C for 25 minutes.

[0039] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 10% and the concentration of ammonium sulfate is 15%.

[0040] HPLC analysis showed that the yield of β-carotene was 92.6%, which was significantly different from the 58.7% yield of the control group extracted with traditional acetone. The residual salt content after aqueous two-phase extraction was <0.5%, and the hydrophobic chromatography purity was 98.4%.

[0041] Example 6: Extraction of polysaccharides from Chlorella vulgaris in salt lakes

[0042] Step S0: Take 10 g of Chlorella vulgaris from the salt lake, pulverize it to 40 mesh, and defatt it by reflux with ethanol; Step S1, low-temperature plasma pretreatment, 220 W, 8 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 1.2% and the concentration of pectinase is 0.72%. The reaction is carried out at pH 5.0 and 50°C for 3 hours. Step S3: Ultrasonic extraction with a eutectic solvent. Isopropanol and choline chloride are mixed to form a eutectic solvent. The ratio of isopropanol to choline chloride in the eutectic solvent is 1:20. Ultrasonic extraction is performed at 50 kHz and 50 °C for 30 minutes.

[0043] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 12% and the concentration of ammonium sulfate is 16%.

[0044] Step S5: Remove protein with chloroform-n-butanol, decolorize with H2O2, precipitate with ethanol, and dry at 60℃.

[0045] This method achieves a polysaccharide recovery rate of 80.3% and a protein impurity removal rate of 60%. Compared with the traditional ultrafiltration + ion exchange method, the time is shortened by 16% (24 hours → 20 hours), and the polysaccharide purity is increased from 82% to 86.3%.

[0046] Example 7 Extraction of polysaccharides from Chlorella vulgaris in salt lakes

[0047] Step S0: Take 10 g of Chlorella vulgaris from the salt lake, pulverize it to 40 mesh, and defatt it by reflux with ethanol; Step S1: Low-temperature plasma pretreatment, 230 W, 10 minutes; Step S2: Mixed enzymatic hydrolysis to break the cell wall. The mixed enzymes include cellulase and pectinase. The concentration of cellulase is 1.2% and the concentration of pectinase is 0.72%. The reaction is carried out at pH 5.0 and 50°C for 3 hours. Step S3: Ultrasonic extraction with a eutectic solvent. Isopropanol and choline chloride are mixed to form a eutectic solvent. The ratio of isopropanol to choline chloride in the eutectic solvent is 1:20. Ultrasonic extraction is performed at 50 kHz and 55°C for 35 minutes.

[0048] Step S4: Aqueous two-phase extraction with tandem hydrophobic chromatography of phenyl / butyl agarose gel, using PEG and ammonium sulfate as the extractant, wherein the concentration of PEG 4000-8000 is 12% and the concentration of ammonium sulfate is 16%.

[0049] Step S5: Remove protein with chloroform-n-butanol, decolorize with H2O2, precipitate with ethanol, and dry at 65°C.

[0050] This method achieves a polysaccharide recovery rate of 81.3% and a protein impurity removal rate of 60%. Compared with the traditional ultrafiltration + ion exchange method, the time is shortened by 25% (24 hours → 18 hours), and the polysaccharide purity is increased from 82% to 89.8%.

[0051] Although this disclosure has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are all within the scope of protection claimed by this disclosure.

Claims

1. A method for extracting bioactive substances from microalgae in high-salt-alkaline environments, the specific steps of which are as follows: Step S1, low-temperature plasma pretreatment; Step S2, mixed enzymatic hydrolysis to break the cell wall, the mixed enzymes include cellulase and pectinase, the mass concentration of cellulase is 0.5%-2% (w / v) and the mass concentration of pectinase is 0.3%-1% (w / v), the mass concentration ratio of the two is about 1.5:1-2:1; Step S3: Ultrasonic extraction with salt-tolerant extractant. The extractant includes isopropanol and choline chloride eutectic solvent. The isopropanol / choline chloride eutectic solvent has a molar ratio of 2:1-4:1 and a solid-liquid ratio of 1:10-1:20 (g / mL). Step S4: Aqueous two-phase extraction followed by hydrophobic chromatography purification. The extract uses PEG and ammonium sulfate, with the concentration of PEG 4000-8000 being 8%-12% and the volume concentration of ammonium sulfate being 12%-18%.

2. The method according to claim 1, characterized in that, In step S1, the low-temperature plasma pretreatment parameters are power 150-250 W and time 3-10 minutes.

3. The method according to claim 1, characterized in that, In step S2, the concentration of cellulase is 1.2% and the concentration of pectinase is 0.8%; the preferred ratio of the two is 1:0.

6.

4. The method according to claim 1, characterized in that, In step S2, the enzymatic hydrolysis to break the cell wall occurs at a pH of 5-6 and a temperature of 50-60℃.

5. The method according to claim 1, characterized in that, The ultrasonic extraction parameters in step S3 are 40-55 kHz, temperature 50-60℃, and time 24-30 minutes.

6. The method according to claim 1, characterized in that, The isopropanol / choline chloride eutectic solvent molar ratio in step S3 is 3:1, and the material-to-liquid ratio is 1:15 (g / mL).

7. The method according to claim 1, characterized in that, The medium for hydrophobic chromatography in step S4 is phenyl / butyl agarose gel.

8. Microalgae bioactive substances prepared by the method according to any one of claims 1 to 7.

9. The application of the microalgae bioactive substances prepared by the method of any one of claims 1 to 7, and the method of claim 8, in the preparation of raw materials and excipients for functional foods, health products, and pharmaceutical products.