A method for extracting chlorophyll from Spirulina based on electrically induced aggregation

Abstract

The present invention discloses a method for extracting chlorophyll from spirulina based on electrical induction aggregation. The present invention extracts, concentrates and column-chromatographs the raw material spirulina powder to obtain a chlorophyll eluate having a solid content of 10-100 mg / L of ethanolic aqueous solution, selects a power supply with a voltage of 1-12 V, uses a copper rod as an electrode, and energizes for 24-48 hours to obtain a chlorophyll precipitate, centrifuges, removes the supernatant, and freeze-dries to obtain a chlorophyll freeze-dried powder. The present invention utilizes the inherent electrical conductivity of chlorophyll molecules, aggregates the chlorophyll molecules by electrical induction, and thereby precipitates the chlorophyll, thereby obtaining a chlorophyll powder that is easy to stably store, store and transport compared to a chlorophyll solution. Compared with a traditional chlorophyll preparation method, the chlorophyll extraction efficiency and chlorophyll content are improved, production costs are reduced, and operational safety is enhanced.

Description

Technical Field

[0001] The invention relates to a method for extracting chlorophyll from spirulina based on electrical induced aggregation, and belongs to the technical field of food processing. Background Art

[0002] Spirulina, a microalgae produced industrially on a large scale, is also considered a natural food. Chlorophyll, the most widely distributed natural pigment in nature, possesses numerous benefits, including anti-cancer, antibacterial, and potent antioxidant properties. Spirulina is rich in chlorophyll, with levels 2-3 times higher than in most terrestrial plants and over 10 times higher than in ordinary vegetables. Furthermore, spirulina's rapid growth and ease of cultivation ensure a reliable resource supply. Taking all these factors into consideration, spirulina's natural products, such as chlorophyll, carotenoids, and phycocyanin, are being fully utilized and transformed into high-value-added products for widespread use in industries including food, medicine, and health supplements.

[0003] The traditional method of extracting spirulina pigment mainly uses organic solvents such as methanol, ethanol, and acetone for extraction, assisted by microwave or ultrasound to obtain chlorophyll extract solution. In order to improve the purity of chlorophyll, the extracted chlorophyll solution is further subjected to column chromatography separation, impurity removal and other steps to obtain a chlorophyll solution with higher purity. The extraction and impurity removal process of chlorophyll consumes a large amount of solvent, and the steps are cumbersome. Moreover, chlorophyll products mostly exist in the form of solutions. Compared with solutions, chlorophyll powder has the advantages of better storage, wider application scenarios, and easy transportation. Chlorophyll also exists in the form of salt, which poses certain food safety issues.

[0004] Patent CN202111613032.5 obtains chlorophyll by extracting, concentrating, column chromatography, supercritical CO2 extraction and degreasing the raw material spirulina powder, and drying it. The above method requires acetone dissolution after column chromatography and then supercritical CO2 extraction and degreasing. The operation steps are relatively cumbersome and the resulting chlorophyll solution is not very suitable. If a powder is required, it needs to be further rotary evaporated for a long time and then freeze-dried.

[0005] Patent CN201410323936.8 provides a method for the fractional extraction of nutrients from spirulina. Using fresh spirulina mud as the raw material, the mud is dehydrated with high-concentration ethanol and chlorophyll and carotenoids are simultaneously extracted, resulting in a chlorophyll extract and a carotenoid extract. Patent CN200710130682.8 provides a spirulina extract and preparation method. The extract contains nutrients primarily including spirulina chlorophyll, spirulina phycocyanin, and spirulina polysaccharides, with the spirulina chlorophyll content being only approximately 8%. Both of these extraction methods present certain issues regarding the purity of the spirulina chlorophyll. High-purity chlorophyll is crucial for its targeted applications, and whether these two methods can produce sufficiently high-purity chlorophyll warrants further verification and improvement. Furthermore, the product of Patent CN201410323936.8 is in solution form, making it inconvenient to store, use, and transport.

[0006] Patent CN202111613019.X provides a preparation process for sodium copper chlorophyllin from spirulina. The raw material spirulina is subjected to extraction, concentration, fat removal, alkali addition, acidification and copper addition, impurity removal 1, salt formation, and impurity removal 2 to obtain the finished product of sodium copper chlorophyllin. The above method is not only relatively cumbersome in terms of operation steps, but the prepared sodium copper chlorophyllin contains copper. Long-term intake of this copper salt can easily lead to copper accumulation in the human body, posing a potential food safety risk. Summary of the Invention

[0007] To address these issues, the present invention provides a method for extracting chlorophyll from spirulina based on electrically induced aggregation. This method utilizes the inherent electrical conductivity of chlorophyll molecules to electrically aggregate them, thereby precipitating the chlorophyll. This method then produces a chlorophyll powder that is more stable, easier to store, and transport than a chlorophyll solution. This innovation has significant potential for producing spirulina chlorophyll powder. Compared to traditional chlorophyll preparation methods, it improves chlorophyll extraction efficiency and chlorophyll content, reduces production costs, and enhances operational safety.

[0008] The technical solution of the present invention is: a method for extracting chlorophyll from spirulina based on electrically induced aggregation, which is characterized in that the chlorophyll eluate obtained after leaching, concentrating and column chromatography of raw spirulina powder is prepared into a solution containing ethanol / water (v / v=1:2-6) with a solid content of 10-100 mg / L (which may contain other alcohol solvents used for elution, such as n-butanol, etc.), a power supply with a voltage of 1-12V, a copper rod as an electrode, and power is applied for 24-48 hours to obtain a chlorophyll precipitate, which is centrifuged, the supernatant is removed, and the solution is freeze-dried to obtain a freeze-dried chlorophyll powder for storage.

[0009] Preferably, ethanol / water (v / v=1:4) is selected, and the effect is best when the above ratio is used.

[0010] The preparation method of the raw material spirulina powder is to extract, concentrate and column chromatograph the raw material spirulina powder, which is added to a mixed solvent of anhydrous ethanol and petroleum ether (volume ratio is 1:1-3) for extraction, and then the anhydrous ethanol and residue are removed by refrigerated centrifugation, and then water is added to extract impurities, and the petroleum ether phase is concentrated, and the concentrate is chromatographed by a chromatography column filled with neutral alumina to obtain an eluent containing chlorophyll.

[0011] Preferably, the preparation method is as follows: taking spirulina powder, adding it to anhydrous ethanol and petroleum ether (the volume ratio of the two is 1:2), homogenizing, and then freezing centrifuging to obtain the supernatant, filtering the supernatant and mixing it with an equal volume of water for separation; retaining the upper petroleum ether phase, washing it with water, drying it, and concentrating it by rotary evaporation to obtain a crude chlorophyll extract; passing the crude chlorophyll extract through a chromatography column filled with neutral alumina, and eluting carotene, lutein and chlorophyll with petroleum ether-acetone (v / v=9:1), petroleum ether-acetone (v / v=7:3), and n-butanol-ethanol-water (v / v / v=3:1:1) as eluents in sequence, and collecting the chlorophyll eluate for later use.

[0012] The technical effects of the present invention are:

[0013] (1) More convenient method for preparing chlorophyll powder

[0014] Compared to chlorophyll solutions, chlorophyll powders are more stable and maintain their activity for a relatively long time. They are less susceptible to environmental factors such as light, oxygen, and temperature. Furthermore, chlorophyll powders are relatively easy to store and transport, requiring no special storage conditions. They can also be measured as needed, offering flexibility and allowing for formulation based on specific production needs.

[0015] In the present invention, due to the special effect of chlorophyll under the electric field, chlorophyll precipitation is accurately obtained and then freeze-dried to obtain chlorophyll powder. Compared with the traditional method (taking CN202111613032.5 as an example), the present invention avoids a series of impurity removal processes after column chromatography in the traditional method and quickly obtains chlorophyll powder with wider applicability, thereby simplifying the impurity removal operation, shortening the impurity removal time, and increasing the chlorophyll concentration. On this basis, the use of organic reagents is further reduced, production costs are reduced, and safety is improved.

[0016] (2) Easy to operate

[0017] Compared to existing technologies, this technique is significantly easier to use for electrically induced pigment aggregation. Simply by applying electricity, it effectively induces pigment aggregation under specific conditions. This straightforward and intuitive process not only provides a convenient and efficient means for experimenters to easily implement and control the reaction in a laboratory setting, but also holds great potential for widespread application in related research and industrial production.

[0018] (3) Increase chlorophyll content

[0019] The present invention can obtain a relatively high content of chlorophyll when extracting chlorophyll. High-content chlorophyll extracts are crucial in a wide range of applications, so the present technical solution has significant advantages in this regard.

[0020] In summary, the present invention offers advantages in safety, efficiency, cost, purity, and other aspects. By fully utilizing the electrical conductivity of materials and building on mature technologies, the present invention can more reliably extract a variety of active substances, providing a more feasible and reliable solution for applications in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the change in clarity of four natural pigment solutions (25 mg / L β-carotene, chlorophyll, lycopene, and betaine in ethanol / water (v / v = 1:4)) within 96 hours of power application.

[0022] Figure 2 The changes of chlorophyll ethanol aqueous solution after 24 hours of electricity (no electricity at room temperature (Group A), electricity at room temperature (Group B));

[0023] Figure 3 This is the flow chart for chlorophyll extraction from Spirulina;

[0024] Figure 4 The following are the characteristic spectral curves of the solution of freeze-dried chlorophyll powder; Figure A: UV absorption spectrum; Figure B: Fluorescence emission spectrum under 436nm excitation light;

[0025] Figure 5 Diagram of the plant for extracting chlorophyll using the electric induction technology;

[0026] Figure 6 Figure 1 is the spectral characteristic curve of chlorophyll ethanol aqueous solution after power is applied; Figure A: UV absorption spectrum; Figure B: Fluorescence emission spectrum under 436nm excitation light. DETAILED DESCRIPTION

[0027] The effects are illustrated below with reference to the examples and accompanying drawings. The chlorophyll used in Example 1 of the present invention was self-extracted and had a purity of 86.6%; the β-carotene was provided by Shanghai Yuanye Biotechnology Co., Ltd. and was analytically pure; the betaine was also provided by Shanghai Yuanye Biotechnology Co., Ltd. and was analytically pure; and the lycopene was provided by Shanghai Aladdin Biochemical Technology Co., Ltd. and was analytically pure.

[0028] Example 1:

[0029] This study selected four common natural pigments, including β-carotene and chlorophyll found in spirulina, as well as lycopene and betaine, to observe the macroscopic changes in these pigment solutions (25 mg / L ethanol / water (v / v = 1:4) solutions of β-carotene, chlorophyll, lycopene, and betaine) under the same electrical conditions. In the experiment, we used a power supply voltage of 1.5V and selected copper rods with a diameter of 0.5mm as electrodes. The results are as follows: Figure 1 The effect of electrochemically promoting pigment aggregation was evaluated by observing the clarity of the solution after 96 hours of electrochemical treatment. The results showed that significant precipitation was observed only after a period of electrochemical treatment in the chlorophyll solution. This provides strong experimental evidence for further exploration of the mechanism and potential applications of electrochemically promoting pigment aggregation.

[0030] Figure 2 The results show the changes in the solution of 25mL of chlorophyll ethanol / water (v / v = 1:4) under power conditions. A large amount of precipitation is clearly visible. Based on this observation, we can further extend the research and use specific metals as electrodes and connect them to a 1.5V power supply. After a specific power supply period, we successfully obtained chlorophyll aggregation and precipitation, thereby achieving efficient chlorophyll extraction. This experimental result provides an important experimental basis and data support for subsequent related research.

[0031] Example 2:

[0032] (1) Chlorophyll extraction

[0033] Take 200g of spirulina powder, add 200mL of anhydrous ethanol and 400mL of petroleum ether, and homogenize for 5 minutes. After refrigerated centrifugation for 10 minutes, remove the supernatant, filter, and mix with an equal volume of water for separation. Retain the upper petroleum ether phase, wash with water, dry, and concentrate by rotary evaporation until a residual volume of 200mL is retained to obtain a crude chlorophyll extract. Pass the crude chlorophyll extract through a chromatography column filled with neutral alumina, and elute carotene, lutein, and chlorophyll using petroleum ether-acetone (v / v = 9:1), petroleum ether-acetone (v / v = 7:3), and n-butanol-ethanol-water (v / v / v = 3:1:1) as eluents. Collect the chlorophyll eluate (chlorophyll content is approximately 8% based on dry matter) for later use.

[0034] (2) Electrofocusing

[0035] See also Figure 3 , add water to the obtained chlorophyll eluate to ethanol / water (v / v=1:4) in the solution (in addition to ethanol and water, the n-butanol contained in the solution has a certain promoting effect on the aggregation of chlorophyll molecules). At this time, the solid content of the solution is about 50 mg / L (if the concentration is too high, additional ethanol / water (v / v=1:4) can be added for dilution). Then, a power supply with a voltage of 1.5V and two copper rods with a diameter of 0.5mm and a length of 15cm are selected as electrodes. The power is turned on for 24h to obtain chlorophyll precipitation. Centrifuge at 10000g for 10min, remove the supernatant, and freeze-dry for 24h to obtain chlorophyll freeze-dried powder for storage.

[0036] The present invention dissolves 0.75 mg of freeze-dried powder in 25 mL of anhydrous ethanol and performs fluorescence spectrum and ultraviolet absorption spectrum analysis. The results are as follows: Figure 4 As shown. The UV absorption spectrum shows a series of characteristic peaks, which are consistent with the UV-visible absorption spectrum characteristics of Chl a. Specifically, in the UV spectrum, we can clearly observe some significant characteristic peaks. At 430nm, we see the typical absorption characteristics of the Soret band, which is a major absorption peak of the Chl a molecule. In the range of 490-600nm, there is a weak Q x band absorption, and at 660 nm we observed Q y The UV absorption and fluorescence spectra of the samples were analyzed. The UV absorption and fluorescence spectra showed a characteristic absorption band, a characteristic feature of the Chl a molecular spectrum. Fluorescence emission spectroscopy was also performed on the samples. Under 436 nm excitation, a fluorescence emission curve was obtained, which exhibited a distinct peak at 680 nm. In summary, the results of the UV absorption and fluorescence spectroscopy analyses fully demonstrate that the Chl a molecular structure is preserved in the freeze-dried powder, providing strong support for further research.

[0037] Chlorophyll content was determined using a UV-visible spectrophotometer at wavelengths A and645 and A 663 The absorbance value was measured and the chlorophyll content was calculated using the following formula (1):

[0038] Chl (mg / L) = 8.04*A 663 +20.29*A 645 (1)

[0039] We are at wavelength A 645 and A 663 The absorbance of the chlorophyll solution was measured at 10.67 mg / L. Further calculations revealed a chlorophyll concentration of 0.27 mg / L in the freeze-dried powder solution. The extract contained 35.56% chlorophyll, compared to approximately 8% (on a dry matter basis) in the column chromatography eluate. This significantly increases the chlorophyll content of the product through electrical aggregation.

[0040] Based on this technology, we are expected to design an efficient chlorophyll extraction device with the following structure: Figure 5 As shown, the system comprises an external power supply, an automatic lifting device, two copper electrodes, and a raw material pool. Its operating principle is as follows: a crude chlorophyll extract is placed in a larger container (i.e., the raw material pool). Two copper rods are then introduced as electrodes, and an electric current is passed through the system for a period of time. By introducing an electric current into the crude extract, we have triggered a highly efficient chlorophyll precipitation mechanism. Under the action of the electric current, the chlorophyll molecules rapidly undergo a coagulation reaction, forming precipitable solid particles on a macroscopic scale. This innovative method can produce more chlorophyll product in a shorter period of time. Compared to traditional chlorophyll purification methods or methods that rely on heating and evaporating the solution to extract chlorophyll, our method not only saves a lot of energy but also improves overall safety during the operation. Furthermore, the electric current stimulation during the operation has a relatively small impact on the environment, in contrast to the release of volatile organic compounds generated by traditional heating methods.

[0041] In summary, this electrochemical-based chlorophyll extraction technology offers an efficient, energy-efficient, and safe option. By fully leveraging the inherent properties of the chlorophyll molecule, we can achieve higher yields of chlorophyll in a shorter time, while also providing a viable solution for energy conservation and operational safety.

[0042] The UV absorption spectrum and fluorescence emission spectrum of chlorophyll ethanol / water (v / v=1:4) solution under 436nm excitation light were analyzed for 10-50 minutes. The data are as follows: Figure 6As shown in the figure, the UV absorption spectra clearly show that all five absorption curves exhibit typical chlorophyll spectral characteristics, and the five curves essentially overlap. This fully demonstrates that no new substances are generated during the electrical induction process, and the solution concentration remains unchanged. This fundamentally eliminates the possible influence of new substance formation and solution concentration changes on solution properties. The fluorescence curve shows a significant fluorescence quenching phenomenon, clearly indicating that chlorophyll aggregation occurred during the electrical induction process.

[0043] Example 3: Application of large-scale chlorophyll precipitation and extraction equipment in factories

[0044] (1) Loading into the container: Adjust the chlorophyll eluate (prepared in step (1) of Example 2) to a solution of ethanol / water (v / v = 1:4) with a solid content of about 50 mg / L and pour it into a container with a capacity of 5 to 100 L, ensuring that the container can adapt to the required volume. This container will serve as the main working area for chlorophyll extraction (i.e., Figure 5 shown in the raw material pool);

[0045] (2) Applying current: Two copper rods with a diameter of 50 cm and a length of 2 m are introduced into the container as electrodes, ensuring that they are properly immersed in the liquid. Subsequently, a direct current of 1.5 to 12 V is applied to the system to initiate the precipitation of chlorophyll.

[0046] (3) Chlorophyll precipitation: The power supply will continue for 48 hours to allow the chlorophyll to fully precipitate. After the precipitation is completed, a large amount of chlorophyll sediment can be observed at the bottom of the container. At this time, the liquid portion can be removed from the top and the sediment can be freeze-dried for 24-48 hours to obtain chlorophyll freeze-dried powder for storage.

[0047] Example 4: Development of microalgae chlorophyll cocktail

[0048] (1) Mixed alcoholic beverage: At the beginning of the cocktail preparation, pour 40 mL of 40% vol vodka, 20 mL of purified water, 50 mL of Sprite, 20 mL of creamer, and 10 g of fructose into a homogenizer. Homogenize for 3 minutes to ensure that all ingredients are thoroughly mixed.

[0049] (2) Injection of chlorophyll: The freeze-dried chlorophyll powder of Example 4 was added to the prepared cocktail and homogenized using a homogenizer for 1 min to ensure that the chlorophyll was evenly dispersed throughout the cocktail.

[0050] (3) Personalized adjustment: The preparation process of cocktails is not limited to vodka. You can also choose different types of liquor as base liquor, such as rum, gin, tequila, whiskey, brandy, etc. According to different personal tastes, you can flexibly adjust the type of base liquor used in the production.

[0051] Example 5: Natural chlorophyll as green pigment for oil painting

[0052] (1) Preparation of varnish: Grind the dammar resin into a fine powder and pour it into gauze. Take twice the amount of turpentine and place it in a sealable container. Soak the dammar resin and gauze in the turpentine and seal it tightly to prevent turpentine from evaporating. Let it stand for two to three days until the dammar resin is completely dissolved, then remove impurities.

[0053] (2) Preparation of Gum Arabic Binder: Gum Arabic resin was crushed and placed in a container. Dissolved in twice the amount of boiling water, and then filtered to remove impurities. Subsequently, 6 g / L of borax was added to the gum Arabic solution and stirred thoroughly.

[0054] (3) Preparation of the final product: Mix the prepared varnish, gum arabic binder and chlorophyll powder in an appropriate proportion.

Claims

1. A method for extracting chlorophyll from Spirulina based on electrically induced aggregation, characterized in that: The chlorophyll eluate obtained after leaching, concentrating, and column chromatography of the raw spirulina powder is prepared into an ethanol-water solution with a solid content of 10-100 mg / L and a volume ratio of ethanol to water of 1:2-6. A power supply with a voltage of 1-12 V and copper rods as electrodes is applied for 24-48 hours to obtain a chlorophyll precipitate. The solution is centrifuged, the supernatant is removed, and the solution is freeze-dried to obtain a freeze-dried chlorophyll powder. The raw material spirulina powder is subjected to extraction, concentration, and column chromatography, specifically, the following steps: taking the spirulina powder, adding it to anhydrous ethanol and petroleum ether in a volume ratio of 1:2, homogenizing, and then freezing and centrifuging to obtain a supernatant, filtering the supernatant, mixing it with an equal volume of water, and separating the liquids; retaining the upper petroleum ether phase, washing it with water, drying it, and concentrating it by rotary evaporation to obtain a crude chlorophyll extract; passing the crude chlorophyll extract through a chromatography column filled with neutral alumina, and eluting carotene, lutein, and chlorophyll with petroleum ether-acetone in a ratio of v / v=9:1, petroleum ether-acetone in a ratio of v / v=7:3, and n-butanol-ethanol-water in a ratio of v / v / v=3:1:1 in sequence as eluents, and collecting the chlorophyll eluate for later use.

2. The method according to claim 1, wherein: The volume ratio of ethanol to water in the solution containing ethanol and water is 1:

4.

3. The method according to claim 2, wherein: The solution containing ethanol and water also contains an alcohol solvent used for column chromatography elution.

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