A continuous extraction method for active ingredients of microalgae

Abstract

The present invention discloses a continuous extraction method for microalgae active ingredients, comprising the following steps: after harvesting Scenedesmus, hot water extraction is carried out at a hot water temperature of 75-90 °C, the supernatant and algal sludge A are collected, the supernatant is subjected to alcohol precipitation to obtain crude polysaccharide; anhydrous methanol is added to algal sludge A, and ultrasonic extraction is carried out, the supernatant and algal sludge B are respectively collected, and the supernatant is the methanol extract; a chloroform and methanol mixed solution with a volume ratio of chloroform to methanol of 1.5-2.5:1 is added to algal sludge B, ultrasonic-assisted extraction is carried out, and the supernatant is collected to obtain crude lipid. This method can simultaneously improve the extraction rate of the methanol extract and the content of neutral lipids in the crude lipid, and the total amount of the extract can reach 39%, which can effectively reduce the remaining amount of algal residue after extraction, providing a new method for the application of microalgae biomass.

Description

Technical Field

[0001] The present invention belongs to the field of microalgae biotechnology, and particularly relates to a continuous extraction method for active components of microalgae. Background Art

[0002] The statements herein only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] As an autotrophic plant widely distributed in the natural environment, microalgae can synthesize various active substances such as polysaccharides, proteins, lipids and other high-value-added metabolites through photosynthesis, and have characteristics such as antioxidant, anti-cancer and antibacterial activities, and have application prospects in industries such as food, feed additives, pharmaceuticals, cosmetics and biodiesel production.

[0004] However, currently the main means of extracting metabolites from microalgae is to obtain a single product through solvent extraction combined with other auxiliary means such as high temperature, ultrasound, etc., such as extracting polysaccharides with hot water, and extracting oils with chloroform / methanol by ultrasound. In addition, a large amount of residual algal residue will be generated after the product is extracted. How to maximize the utilization rate of the extract and reduce the generation amount of algal residue has become a problem. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a continuous extraction method for active components of microalgae.

[0006] In order to achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] A continuous extraction method for active components of microalgae, comprising the following steps:

[0008] After harvesting Scenedesmus, extract with hot water at a hot water temperature of 75 - 90°C, collect the supernatant and algal sludge A, and obtain crude polysaccharides by alcohol precipitation of the supernatant.

[0009] Add anhydrous methanol to algal sludge A, extract by ultrasound, and collect the supernatant and algal sludge B respectively. The supernatant is the methanol extract.

[0010] Add a chloroform and methanol mixed solution to algal sludge B, with the volume ratio of chloroform to methanol being 1.5 - 2.5:1, extract with ultrasonic assistance, and collect the supernatant to obtain crude lipids.

[0011] The preservation number of the Scenedesmus is: CCTCC M 20211488.

[0012] In some embodiments, the time for hot water extraction is 2 - 6h.

[0013] In some embodiments, it further includes a step of concentrating the supernatant, and performing alcohol precipitation on the concentrated aqueous extract.

[0014] Preferably, the concentration method is rotary evaporation concentration or freeze drying.

[0015] Preferably, in the alcohol precipitation, for each alcohol precipitation, the volume ratio of the water extract to absolute ethanol is 1:3 - 5.

[0016] Preferably, the alcohol precipitation is a two-step alcohol precipitation. In the first-step alcohol precipitation, after adding absolute ethanol, it is left to stand at 0 - 10°C for 10 - 15 h, and then the supernatant is removed by centrifugation; in the second-step alcohol precipitation, after adding absolute ethanol, it is mixed evenly by ultrasonic treatment, left to stand, and then the supernatant is removed to obtain crude polysaccharide.

[0017] Low temperature reduces the solubility of polysaccharide in water, further increases the precipitation of polysaccharide, and increases the polysaccharide yield. Ultrasonic mixing enables the full mixing of water molecules and ethanol molecules, breaking the hydrogen bonds in water molecules, thereby reducing the solubility of polysaccharide in water.

[0018] In some embodiments, the frequency of ultrasonic extraction is 800 - 1000 W.

[0019] Preferably, the mass ratio of the dry weight of algal sludge A to methanol is 1:15 - 25.

[0020] In some embodiments, it further includes the step of concentrating the methanol extract by nitrogen blowing.

[0021] In some embodiments, the mass ratio of the dry weight of algal sludge B to the mixed solution of chloroform and methanol is 1:40 - 60.

[0022] Preferably, the frequency of ultrasonic-assisted extraction is 800 - 1000 W ， The treatment time is 5 - 15 min.

[0023] Further preferably, after ultrasonic-assisted extraction, an NaCl solution is added to the supernatant, shaken well, left to stand, and concentrated by nitrogen blowing to obtain crude lipid. Adding salt is beneficial for the separation of methanol and water and reduces the solubility of the extracted components in water.

[0024] Even more preferably, the mass percentage of the NaCl solution is 0.5% - 1%.

[0025] The beneficial effects obtained by one or more of the above embodiments of the present invention are as follows:

[0026] The present invention discloses a method for extracting three high-value products, namely crude polysaccharide, methanol extract, and crude lipid, from Coelastrella sp. SDEC-28. These three products have antioxidant properties, anti-cancer cell activity, and crude lipid rich in triglycerides (neutral lipid) (for biodiesel production), respectively, and have various application potentials.

[0027] Using the continuous extraction method of the present invention, since the first step is hot water extraction, the extraction process will increase the permeability of the cell membrane. Therefore, it is easier to extract intracellular substances during methanol extraction. At the same time, due to the different solvents, the methanol extract will not be extracted in the first step of water extraction. Therefore, continuous extraction can improve the extraction rate of the methanol extract.

[0028] This method can simultaneously improve the extraction rate of the methanol extract and the content of neutral lipids in the crude lipid, and the total extract can reach 39%. It can effectively reduce the remaining amount of algal residue after extraction, providing a new method for the application of microalgae biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0030] Figure 1 is a flow chart of the traditional extraction method A and the continuous extraction method B of the product of Scenedesmus SDEC-28 selected in the embodiment of the present invention;

[0031] Figure 2 is an illustration of the antioxidant properties of the crude polysaccharide of the product of Scenedesmus SDEC-28 selected in the embodiment of the present invention;

[0032] Figure 3 is an illustration of the anti-melanoma cell effect of the methanol extract of Scenedesmus SDEC-28 selected in the embodiment of the present invention;

[0033] Figure 4 is an illustration of the lipid composition of the crude lipid of Scenedesmus SDEC-28 selected in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0034] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0035] The present invention will be further described below in conjunction with embodiments.

[0036] Example 1

[0037] Cultivation of Microalgae

[0038] The crude polysaccharide, methanol extract, and crude lipid separately extracted from microalgae Coelastrella sp. SDEC-28 (deposit number: CCTCC M 20211488) have good antioxidant, anti-cancer, and biodiesel production characteristics respectively, and it is a high-quality algal strain with multiple high-value products. The microalgae were pre-cultured in BG11 medium (a common freshwater cyanobacteria and green algae medium).

[0039] SDEC-28 was inoculated into BG11 medium at an initial inoculation density of 0.5 g / L after scale-up culture. The material of the photobioreactor used for laboratory microalgae culture is plexiglass, with a total volume of 3 L. The culture system was placed in an artificial climate chamber at a temperature of 25 ± 1 °C, a light-dark cycle of 24:0, and a light intensity of 60 μmol / m 2 / s. When the algae grew to the stationary phase, centrifugation was carried out at a speed of 4000 rpm for 10 min to harvest, and algal sludge was obtained.

[0040] Continuous extraction method of microalgae products

[0041] A method for triple extraction and purification of products from high-value Scenedesmus, the process is as Figure 1 shown, and the specific operations are as follows:

[0042] 1. Extraction of crude polysaccharide

[0043] Take 10 g of ground SDEC-28 algal powder in a beaker, add 250 mL of water (1:25), mix evenly and transfer it to a round-bottom flask (1000 mL) (the volume of the added liquid should not exceed 1 / 3 of the flask volume to avoid overflow), add an appropriate amount of zeolite, and perform hot water reflux twice, each time for two hours. After each extraction, centrifuge and take the supernatant. Combine the two supernatants, and concentrate them with a rotary evaporator at 60 °C (or freeze-dry) to reduce the volume to 1 / 4 of the original. Obtain the water extract. Alcohol precipitation: Add four times the volume of absolute ethanol (80% ethanol), add it slowly, and continuously stir with a glass rod during the addition. Let it stand at 4 °C for 12 h, centrifuge and pour off the supernatant, then add 4 times 80% ethanol, sonicate for 10 min (mix evenly), let it stand for 12 h, centrifuge and remove the supernatant to obtain the crude polysaccharide, and determine the polysaccharide content by the anthrone-sulfuric acid method.

[0044] 2. Extraction of methanol extract

[0045] For the remaining algal sludge in Example 1(1), add methanol according to the ratio of dry weight 1:20, treat it with an ultrasonic crusher at an ultrasonic frequency of 80%, then centrifuge again (4000 rpm for 10 min), collect the supernatant, repeat twice, and concentrate by nitrogen blowing to obtain the methanol extract, and determine the content by the gravimetric method.

[0046] 3. Extraction of crude lipid

[0047] For the remaining algal sludge in Example 1(2), a chloroform / methanol (2:1) mixed solution was added at a ratio of 1:50 by dry weight, and it was treated with an ultrasonic crusher at an ultrasonic frequency of 80% for 10 min. Then, centrifugation was carried out again (4000 rpm for 10 min). The supernatant was poured into a separatory funnel, and this operation was repeated. 5 mL of 0.9% NaCl solution was added to the separatory funnel, shaken well, allowed to stand for 15 min, and concentrated by nitrogen blowing to obtain crude fat, and the content was obtained by the gravimetric method.

[0048] Product extraction rate

[0049] The separate extractions (the extraction methods are the same as those of the corresponding extracts in the continuous extraction) and the continuous extraction yields of crude polysaccharides, methanol extracts, and crude fat are shown in Table 1. The extraction rate of crude polysaccharides from Scenedesmus SDEC-28 by hot water extraction is 14%, and the polysaccharide content is 81%. The yield of the high-value product extracted with methanol after hot water extraction is 5% higher than that of the separate extraction. The yield of crude fat after two-step extraction is lower than that of the separate extraction. Compared with the separate extraction, the total product extraction rate of the continuous extraction can reach 39%, greatly improving the product yield and reducing the remaining algal residue.

[0050] Table 1 Extraction rates of three products by traditional extraction methods and continuous extraction methods

[0051]

[0052] Since the first step of the continuous extraction is hot water extraction, the extraction process will increase the permeability of the cell membrane. Therefore, it is easier to extract intracellular substances during methanol extraction. At the same time, due to the different solvents, the methanol extract will not be extracted in the first-step water extraction. Therefore, the continuous extraction can improve the extraction rate of the methanol extract. In the continuous extraction, since the solvent in the second step is methanol, lipids, especially polar lipids, dissolve in methanol, resulting in a lower extraction rate of crude fat compared to the single extraction.

[0053] Example 2

[0054] Determination of antioxidant properties of crude polysaccharides in microalgae Scenedesmus SDEC-28

[0055] Crude polysaccharides were added with water to prepare concentrations of 0.5, 1.0, and 2.0 mg / mL.

[0056] The ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] method, also known as the TEAC (trolox equivalent antioxidant capacity) method. After ABTS reacts with an appropriate oxidant, it will generate a blue-green ABTS cation radical (ABTS·+), which produces a characteristic absorption peak at a wavelength of 734 nm. When antioxidant substances are present, the formation of ABTS·+ will be inhibited, resulting in a decrease in absorbance. The ABTS method is simple to operate and has good result repeatability, making it very suitable for the determination of in vitro antioxidant capacity in the laboratory.

[0057] Prepare a 7 mmol / L -1 ABTS radical solution and a 140 mmol / L -1 potassium persulfate solution. Mix 5 mL of the 7 mmol / L -1 ABTS radical solution with 88 μL of the 140 mmol / L -1 potassium persulfate solution, place it in the dark for 14 h, and dilute it with water to an absorbance of about 0.7 at 734 nm before use to obtain the ABTS radical working solution. Take 3 mL of the ABTS radical working solution and mix it with 1 mL of absolute ethanol, shake for 10 s and then let it stand for 30 min as the control group; mix 1 mL of polysaccharide extracts with different concentrations with 3 mL of the ABTS radical working solution, shake for 10 s and then let it stand for 6 min, measure the absorbance at 734 nm, repeat the measurement 3 times, and take the average value; use VC as the positive control. Calculate the ABTS radical scavenging rate according to the formula:

[0058]

[0059] In the formula: A0 is the absorbance of 3 mL of the ABTS radical working solution + 1 mL of absolute ethanol;

[0060] A1 is the absorbance of 3 mL of the ABTS radical working solution + 1 mL of the polysaccharide extract.

[0061] The antioxidant properties of the SDEC-28 crude polysaccharide are as Figure 2 shown. As the concentration of the crude polysaccharide increases, the ABTS radical scavenging rate increases and the antioxidant capacity enhances. When the concentration of the SDEC-28 crude polysaccharide is 0.5 mg / mL, the ABTS radical scavenging rate can reach 92%, and the SDEC-28 crude polysaccharide has a good effect on scavenging ABTS radicals.

[0062] Example 3

[0063] Determination of the anti-melanoma cell activity of the methanol extract from Scenedesmus SDEC-28

[0064] 1) Take the mother liquor of the extract (100 mg / mL) and dilute it to 50 mg / mL, 25 mg / mL, 10 mg / mL, and 5 mg / mL with methanol respectively. Take 100 μL of the extracts at the above five concentrations and add them to 10 mL of complete DMEM medium (containing 10% fetal bovine serum) respectively, thus obtaining melanoma cell media with extract concentrations of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 100 μg / mL, and 50 μg / mL respectively.

[0065] 2) For the anti-cancer assay, melanoma A375 cells were selected and cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2. They were passaged once every 2 - 3 days, and the cells used in the experiment were in the logarithmic growth phase. Take A375 cells, wash them twice with PBS buffer, add trypsin digestion solution, adjust the cell concentration to 50000 cells / mL, inoculate them into a 96-well plate, and inoculate 200 μL of the corresponding cell suspension into each well. Incubate in an incubator at 5% CO2 and 37 °C for 4 h (the confluence reaches 40%). After the cell state tends to be stable, add extracts at different concentrations (final concentrations of 50, 100, 250, 500, 1000 μg / mL) again. At the same time, set up a blank control group (without methanol and microalgae extract) and a solvent control group (add 100 μL of methanol to 10 mL of complete medium). After continuing to incubate for 24 h, add 10 μL of cck8 solution to each well and incubate in the incubator for 2 h. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell viability:

[0066]

[0067] 3) The inhibitory effects of the methanol extracts of SDEC-28 by single extraction and continuous extraction on melanoma cells are shown in Figure 3 ., the methanol extracts under both extraction methods have inhibitory effects on melanoma cells A375 at various concentrations, and with the increase of the concentration, the inhibitory effect is further enhanced, showing an obvious dose-effect relationship. After continuous extraction, the half maximal inhibitory concentration (IC50) decreased from 148.41 to 73.69 μg / mL. It can be seen that continuous extraction not only improves the extraction rate of the methanol extract but also improves its anti-cancer biological activity.

[0068] Example 3

[0069] Lipid composition distribution of crude lipids in Scenedesmus SDEC-28

[0070] Liquid-liquid extraction separation method: Add 50 mL of each of two solvents, petroleum ether (boiling range 90°C - 120°C) and 95% water-containing methanol, to the crude fat. After shaking well, let it stand for a while. After clear stratification, remove the lower-layer methanol. Then repeat the extraction of the upper-layer solution with the same amount of methanol 2 times; add the same amount of petroleum ether to the removed methanol and repeat the extraction 2 times. Basically, the neutral lipids and polar lipids can be completely separated. The neutral lipids are in the petroleum ether, and the polar lipids are in the methanol. Then, remove the solvent by rotary evaporation, dry to constant weight, accurately weigh, and calculate the contents of the neutral lipids and polar lipids.

[0071] The lipids of microalgae are divided into two major categories: neutral lipids and polar lipids. Neutral lipids are mainly composed of triglycerides, sterol esters, and free fatty acids. Microalgae biodiesel refers to the monoalkyl fatty acid esters formed by the transesterification of microalgae oil (mainly triglycerides) and alcohols. The yield of directly extracting crude oil is 20%, but only 30% of it is neutral lipids, and the neutral lipids account for 6% of the total microalgae powder. By using continuous extraction, the yield of crude oil is 10%, and the content of neutral lipids that can be used as raw materials for biodiesel is as high as 75%, and the extraction rate of neutral lipids can reach 7.5%.

[0072] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A continuous extraction method for microalgae active ingredients, characterized in that: It includes the following steps: After harvesting Scenedesmus, perform hot water extraction at a hot water temperature of 75 - 90°C, collect the supernatant and algal sludge A. The supernatant is subjected to alcohol precipitation to obtain crude polysaccharides; Add anhydrous methanol to algal sludge A, perform ultrasonic extraction, and separately collect the supernatant and algal sludge B. The supernatant is the methanol extract; the mass ratio of the dry weight of algal sludge A to methanol is 1:15 - 25; Add a chloroform and methanol mixed solution to algal sludge B, with the volume ratio of chloroform to methanol being 1.5 - 2.5:1, perform ultrasonic - assisted extraction, and collect the supernatant to obtain crude lipids; The preservation number of the Scenedesmus is: CCTCC M 20211488; The content of neutral lipids that can be used as raw materials for biodiesel in the crude lipids reaches 75%; 2. The continuous extraction method of the microalgae active ingredient according to claim 1, characterized in that: The time for hot water extraction is 2 - 6 h.

3. The continuous extraction method of the microalgae active ingredient according to claim 2, characterized in that: It also includes the step of concentrating the supernatant, and performing alcohol precipitation on the concentrated aqueous extract.

4. The continuous extraction method of the microalgae active ingredient according to claim 3, characterized in that: The method of concentration is rotary evaporation concentration or freeze - drying.

5. The continuous extraction method of the microalgae active ingredient according to claim 1, characterized in that: In the alcohol precipitation, each time of alcohol precipitation, the volume ratio of the aqueous extract to absolute ethanol is 1:3 - 5.

6. The continuous extraction method of the microalgae active ingredient according to claim 5, characterized in that: The alcohol precipitation is two - step alcohol precipitation. In the first - step alcohol precipitation, after adding absolute ethanol, it is left to stand at 0 - 10°C for 10 - 15 h, and then the supernatant is removed by centrifugation; in the second - step alcohol precipitation, after adding absolute ethanol, it is ultrasonically mixed, left to stand, and then the supernatant is removed to obtain crude polysaccharides.

7. The continuous extraction method of the microalgae active ingredient according to claim 1, characterized in that: The frequency of ultrasonic extraction is 800 - 1000 W.

8. The continuous extraction method of the microalgae active ingredient according to claim 1, characterized in that: It also includes the step of concentrating the methanol extract by nitrogen blowing.

9. The continuous extraction method of the microalgae active ingredient according to claim 1, characterized in that: The mass ratio of the dry weight of algal sludge B to the chloroform and methanol mixed solution is 1:40 - 60.

10. The continuous extraction method of the microalgae active ingredient according to claim 9, characterized in that: The frequency of ultrasonic - assisted extraction is 800 - 1000 W, and the treatment time is 5 - 15 min.

11. The continuous extraction method of the microalgae active ingredient according to claim 10, characterized in that: After ultrasonic - assisted extraction is completed, add an NaCl solution to the supernatant, shake well, leave to stand, and after nitrogen blowing concentration, obtain crude lipids.

12. The continuous extraction method of the microalgae active ingredient according to claim 11, characterized in that: The mass percentage of the NaCl solution is 0.5% - 1%.

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