A green method for extracting chlorophyll from spirulina and improving stability of chlorophyll

Abstract

The application discloses a kind of green extraction spirulina chlorophyll and improve the method of chlorophyll stability.The application is mixed uniformly with HBD and HBA, deionized water is added, and is stirred by magnetic force under 70~80 DEG C to form colorless transparent clear liquid, obtain NADES extraction fluid;NADES extraction fluid and spirulina powder are mixed, and homogenizer is broken wall, and is extracted by magnetic stirring under 30~70 DEG C, after extraction, freeze centrifugation, and take supernatant, spirulina chlorophyll NADES extraction fluid is obtained.The test proves that spirulina chlorophyll NADES extraction fluid prepared by the above method, the light stability and thermal stability of chlorophyll are obviously improved.The application is first applied to spirulina chlorophyll extraction with cheap and easy to obtain, green and environmental protection NADES, solves the problem that traditional extraction method pollutes environment and harms human health, and also can improve the stability of chlorophyll.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability. Background Technology

[0002] Spirulina is a nutrient-rich and functionally comprehensive green food. The Food and Agriculture Organization of the United Nations (FAO) has included it in its 21st-century human food resource development plan and hailed it as "the most ideal food of the 21st century." Spirulina is rich in polysaccharides, polyphenols, chlorophyll, carotenoids, unsaturated fatty acids, and other active substances, possessing various health benefits such as antiviral, antitumor, anti-fatigue, and immune-boosting effects, earning it the reputation of a "miniature green functional nutrient treasure trove." The fat-soluble pigment chlorophyll in spirulina, as a natural pigment, can not only replace artificial pigments for coloring but also possesses various beneficial biological activities such as antioxidant, antitumor, antibacterial, anti-inflammatory, and antimutagenic properties. Therefore, chlorophyll has wide applications in food, nutrition, medicine, and cosmetics, and has high commercial value.

[0003] Currently, the main methods for extracting chlorophyll are enzymatic hydrolysis or ultrasound-assisted organic solvent extraction. These processes utilize large amounts of organic reagents such as methanol, acetone, and petroleum ether, polluting the environment, harming human health, and posing certain safety hazards. CN201910838715.7 discloses the simultaneous extraction of chlorophyll and aromatic oil from Sichuan pepper leaves using organic reagents such as ethanol and acetone, reducing the amount of organic solvent added. CN201710650214.7 relates to a method for extracting chlorophyll using enzymes, primarily involving adding cellulase to pretreated *Ulva prolifera* to disrupt its cell wall, and then using organic solvents such as methanol, acetone, and isopropanol to extract the chlorophyll. CN201610176075.4 demonstrates a method for extracting chlorophyll from green plant leaves, mainly including steps such as raw material preparation, extraction, filtration, concentration, elution, and drying. In this extraction process, acetone solution is primarily used to extract chlorophyll from the raw materials. CN201710169066.7 discloses an extraction process for active substances in Spirulina, which mainly uses ethanol Soxhlet extraction to obtain sodium zinc chlorophyll, inevitably using hydrochloric acid, acetone, and petroleum ether to complete the extraction process.

[0004] Deep eutectic solvents (DES) are green solvents that are simple to obtain, have high thermal stability, low volatility, and are easy to synthesize. Since their initial proposal in 2001, they have been widely used in various fields such as biology, medicine, and green chemistry, and are considered one of the most important discoveries of the 21st century. Deep eutectic solvents are generally binary or ternary systems composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs). Polyols, urea, and carboxylic acids often act as hydrogen bond donors (HBDs), while choline chloride and amino acids act as hydrogen bond acceptors (HBAs). DES can form hydrogen bonds with target compounds, so many compounds exhibit high solubility in DES. Based on this property, DES is widely used in the extraction of bioactive substances. DES whose main components are primary metabolites such as sugars, amino acids, and organic acids are called natural deep eutectic solvents (NADES). NADES has abundant natural sources, is easy to synthesize, is biodegradable, and is green and non-toxic. Compared with organic reagents, it exhibits higher safety in the food industry. Traditional chlorophyll extraction methods still suffer from problems such as organic solvent residues, environmental pollution, and safety concerns during the extraction process. In contrast, NADES has shown great potential in the field of green chemistry.

[0005] CN202011060450.1 describes the preparation of NADES using choline chloride, glycerol, and citric acid (molar ratio 1:2:1) with the addition of a certain volume of ultrapure water. The extraction process of isoflavones from red clover is optimized by combining NADES with ultrasonic extraction technology, resulting in a greener, more efficient, and environmentally friendly extraction process. CN202110812322.6 describes the use of different NADES components (including choline chloride, citric acid, malic acid, D-glucose, and D-fructose) to assist in the ultrasonic extraction of perilla leaf essential oil. It was found that compared to traditional methods, ultrasonic-assisted NADES extraction yielded a higher rate, and the resulting perilla leaf essential oil exhibited stronger antioxidant properties and better antibacterial effects. Therefore, ultrasonic-assisted NADES extraction is considered a novel extraction method applicable in the field of natural fragrance raw material extraction. CN202010034722.4 describes the use of NADES (with hydrogen bond acceptors being choline chloride or DL-menthol, and hydrogen bond donors being gluconic acid, tartaric acid, or lactic acid) to extract carotenoids from apricot pomace, tomato pomace, and carrot pomace. This method is efficient, environmentally friendly, simple, and rapid.

[0006] As can be seen from the above patents, using NADES to extract bioactive substances is cleaner and more efficient than traditional extraction methods. However, no reports have been found regarding the use of NADES for chlorophyll extraction. Existing chlorophyll extraction methods mostly use methods such as ultrasound and enzymatic hydrolysis to assist organic solvent extraction to improve extraction efficiency. Although this reduces the amount of organic solvent used in the extraction process, it still cannot completely eliminate the constraints of organic solvents. Based on the property that NADES readily forms hydrogen bonds with target compounds, its use in extracting bioactive substances is already widely applied in the field of natural product extraction. Therefore, it is worth exploring the application of NADES in the extraction of spirulina chlorophyll, which would make the extraction process more environmentally friendly. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a green method for extracting chlorophyll from Spirulina and improving its stability. This invention uses a natural deep eutectic solvent instead of organic solvents to extract chlorophyll from Spirulina. This not only avoids the use of toxic and harmful organic reagents, solving the problems of environmental pollution and harm to human health associated with traditional extraction methods, but also improves the stability of chlorophyll, providing a new approach to broadening the application channels of chlorophyll and extending product shelf life.

[0008] The technical solution of this invention is: a method for green extraction of chlorophyll from Spirulina and improvement of chlorophyll stability, characterized in that...

[0009] (1) Preparation of NADES extract

[0010] Mix HBD (hydrogen bond donor) and HBA (hydrogen bond acceptor) evenly, add deionized water, and stir magnetically at 70-80°C until a colorless and transparent clear liquid is formed, which is the NADES extract.

[0011] (2) Preparation of Spirulina chlorophyll

[0012] Mix the NADES extract with spirulina powder, homogenize the mixture, and extract with magnetic stirring at 30-70°C. After extraction, freeze and centrifuge, and collect the supernatant to obtain the spirulina chlorophyll NADES extract.

[0013] HBD is betaine; HBA is one of triethylene glycol, glycerol, and xylitol, preferably xylitol. When HBD is betaine and HBA is xylitol, the preferred steps are:

[0014] (1) Preparation of NADES extract

[0015] Betaine and xylitol were mixed evenly according to a molar ratio of 1:2. An appropriate amount of deionized water was added, and the mixture was magnetically stirred at 70-80°C until a colorless and transparent clear liquid was formed, with a water content of 30-35%.

[0016] (2) Preparation of Spirulina chlorophyll

[0017] The prepared NADES extract was mixed evenly with spirulina powder (material-to-liquid ratio 1:100-150), and the cell walls were broken using a homogenizer. Extraction was carried out under magnetic stirring in a water bath at 40-60℃ for 90-150 minutes to complete the extraction process. The algal residue was removed by centrifugation at 4℃, and the supernatant was collected to obtain the spirulina chlorophyll NADES extract for later use.

[0018] The algal residue was extracted repeatedly in step (2) to improve the extraction rate of spirulina chlorophyll.

[0019] Experiments have shown that the Spirulina chlorophyll NADES extract prepared by the above method significantly improves the photostability and thermostability of chlorophyll.

[0020] The present invention also discloses the application of the Spirulina chlorophyll NADES extract prepared by the above method in the preparation of functional jellies, functional beverages and antioxidant toners.

[0021] The technical effects of this invention are:

[0022] 1. This invention is the first to apply inexpensive, readily available, and environmentally friendly NADES to the extraction of chlorophyll from Spirulina. Using NADES, chlorophyll was successfully extracted with an extraction rate of up to 65%, taking into account the advantages of economic benefits, high-efficiency extraction, and environmental friendliness.

[0023] 2. Most existing technologies focus on how to extract chlorophyll, while avoiding the key issue of the stability of the extracted chlorophyll, which affects its application. This invention, while efficiently extracting chlorophyll, successfully improves the photostability and thermostability of chlorophyll, solving this key problem, extending the shelf life of natural pigment products, and facilitating the further application of chlorophyll products.

[0024] 3. All reagents used in this invention are biodegradable and green and non-toxic NADES. This innovation meets the requirements of green chemistry, avoids the use of toxic and harmful organic reagents, and greatly improves the safety of the extraction process and the product. Attached Figure Description

[0025] Figure 1The image shows a comparison of the results of extracting chlorophyll from Spirulina using eight different NADES formulations: A: Bet-Teg; B: ChCl-prop; C: Bet-Lev; D: ChCl-MA; E: Bet-Gly; F: ChCl-Gly; G: Bet-Xyl; H: ChCl-Xyl. In the image, the left side shows the prepared NADES, and the right side shows the corresponding algal powder extract. Image F, from left to right, shows NADES, algal powder extract, and algal powder extract diluted 5 times.

[0026] Figure 2 The UV spectra of eight NADES-containing Spirulina chlorophyll extracts are shown below; where: A: Bet-Teg; B: ChCl-prop; C: Bet-Lev; D: ChCl-MA; E: Bet-Gly; F: ChCl-Gly; G: Bet-Xyl; H: ChCl-Xyl.

[0027] Figure 3 The graph shows the stability changes of each group after 0 and 6 hours of light exposure; A, B, C, D, and E are the Bet-Teg group, the Bet-Gly group, the Bet-Xyl group, the chlorophyll ethanol / water (v / v = 1:4) solution, and the chlorophyll ethanol solution, respectively.

[0028] Figure 4 The image shows the changes in ultraviolet spectra of each group from 0 to 6 hours of light exposure. A, B, C, and D are the Bet-Teg group, Bet-Gly group, Bet-Xyl group, and chlorophyll control group, respectively.

[0029] Figure 5 To show the changes in the degradation rate of Chl in each group after 6 hours of continuous light exposure, A, B, C, D, and E are the Bet-Teg group, Bet-Gly group, Bet-Xyl group, chlorophyll ethanol / water (v / v = 1:4) solution, and chlorophyll ethanol solution, respectively.

[0030] Figure 6 The ultraviolet absorption spectra of Bet-Xyl extracts with different deionized water contents (Figure A) and the changes in chlorophyll concentration (Figure B) are shown.

[0031] Figure 7 The ultraviolet absorption spectra (Figure A) and chlorophyll concentration changes (Figure B) of Bet-Xyl algal powder extracts at different extraction temperatures are shown.

[0032] Figure 8 The ultraviolet absorption spectra (Figure A) and chlorophyll concentration changes (Figure B) of Bet-Xyl algal powder extracts at different extraction times are shown.

[0033] Figure 9The ultraviolet absorption spectra (Figure A) and chlorophyll concentration changes (Figure B) of Bet-Xyl algal powder extracts with different material-to-liquid ratios;

[0034] Figure 10 The image shows the ultraviolet absorption spectrum of Bet-Xyl algal powder extract.

[0035] Figure 11 Figure A shows the fluorescence excitation spectrum of Bet-Xyl algal powder extract with an emission wavelength of 676 nm and the fluorescence emission spectrum of Bet-Xyl algal powder extract with an excitation wavelength of 400 nm.

[0036] Figure 12 The thermogravimetric analysis curves of Bet-Xyl-Chl and Chl are shown, where A: TG curve; B: DTG curve. Detailed Implementation

[0037] The effects are illustrated below with reference to the embodiments and accompanying drawings.

[0038] 1. Preparation of NADES extract

[0039] The preparation method of NADES extract is as follows: HBD (component 1) and HBA (component 2) are mixed in a certain molar ratio and stirred magnetically in a water bath at 70-80℃ until a transparent and clear state is achieved. To ensure that NADES has a certain fluidity and achieves a good solution state, a certain amount of deionized water is added to each of the eight NADES types. The specific formulation of NADES is shown in Table 1.

[0040] Table 1. Formulations of eight NADES extracts

[0041]

[0042]

[0043] 2. Screening of the effects of different NADES extraction solutions on the extraction of chlorophyll from Spirulina

[0044] The main experimental method was as follows: 10 ml of each of the eight prepared NADES extracts was added, along with 0.2 g of Spirulina powder. The mixture was homogenized for 10 min and then magnetically stirred at 40 °C for 90 min to complete the extraction. After centrifugation at 4 °C, the supernatant was collected to obtain the NADES powder extract. The substances contained in the extract were analyzed by ultraviolet absorption spectroscopy.

[0045] NADES extraction results, for example Figure 1 As shown, pigments from Spirulina may have been extracted from groups A, B, E, F, and G. Ultraviolet spectroscopy was performed on the extracts of the above eight NADES compounds, and the results are as follows. Figure 2Observation of their ultraviolet absorption spectra revealed that the ultraviolet spectra of the three NADES extracts—Bet-Teg, Bet-Gly, and Bet-Xyl—matched the absorption peaks of chlorophyll and phycocyanin, indicating that both chlorophyll and phycocyanin were extracted. However, since the extraction method for phycocyanin is relatively mature, chlorophyll was chosen as the primary research target.

[0046] 3. Determination of chlorophyll degradation rate

[0047] Extraction revealed that chlorophyll and phycocyanin were extracted from three NADES groups: Bet-Teg, Bet-Gly, and Bet-Xyl. To further screen for the most suitable NADES for chlorophyll extraction, the chlorophyll degradation rate of the three extracts was determined. The extracts were diluted to a chlorophyll concentration of 6 mg / L using the corresponding NADES. Chlorophyll ethanol / water (v / v = 1:4) solution (6 mg / L) and chlorophyll ethanol solution (6 mg / L) were prepared as controls using traditional organic solvent extraction methods. The systems were continuously irradiated at 4°C and 60% light intensity (19200 Lux) for 6 hours, and the changes in chlorophyll content were observed.

[0048] The main method for extracting chlorophyll using traditional organic solvents is as follows: Take 200g of spirulina powder, add 200mL of anhydrous ethanol and 400mL of petroleum ether, and homogenize for 5 minutes. After centrifugation at freeze for 10 minutes, collect the supernatant, filter it, and mix it with an equal volume of water for separation. Retain the upper petroleum ether phase, wash it with water, dry it, and concentrate it by rotary evaporation to the remaining 200mL to obtain a crude chlorophyll extract. Pass the crude chlorophyll extract through a chromatography column packed with neutral alumina, and elute carotene, lutein, and chlorophyll sequentially 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. Collect the chlorophyll eluent (chlorophyll content is approximately 8% on a dry matter basis) for later use.

[0049] Comparison of Bet-Teg group, Bet-Gly group, Bet-Xyl group, chlorophyll ethanol / water (v / v = 1:4) solution, and chlorophyll ethanol solution at 0h and 6h: Figure 3 As shown, from Figure 3 It can be seen that after 6 hours of light exposure, the chlorophyll in the three NADES extracts (Bet-Teg, Bet-Gly, and Bet-Xyl) showed good stability (no obvious fading), while the chlorophyll ethanol / water (v / v = 1:4) solution and the chlorophyll ethanol solution showed poor stability and obvious fading after 6 hours of continuous light exposure.

[0050] Changes in UV spectrum from 0 to 6 hours are as follows Figure 4As shown, observation of their ultraviolet spectra revealed that after 6 hours of continuous illumination at a light intensity of 19200 Lux, the chlorophyll degradation rates in the Bet-Teg, Bet-Gly, and Bet-Xyl NADES extracts were all lower than those in the ethanol / water (v / v = 1:4) and ethanol systems. This indicates that using NADES to extract chlorophyll from Spirulina can effectively improve the light stability of chlorophyll compared to traditional organic solvent extraction methods, mitigating its tendency to degrade easily under light.

[0051] The degradation rate of Chl in each group after 6 hours of continuous light exposure is as follows: Figure 5 As shown, the chlorophyll degradation rate of the Bet-Xyl group was significantly lower than that of the chlorophyll ethanol / water (v / v = 1:4) solution, the chlorophyll ethanol solution, and the other two NADES extraction solutions, indicating that its chlorophyll photostability was superior to the other groups. Therefore, Bet-Xyl is considered the most suitable NADES for subsequent extraction of spirulina chlorophyll.

[0052] 4. Optimization of conditions for chlorophyll extraction using NADES

[0053] The effects of different NADES moisture content, extraction temperature, extraction time and material-liquid ratio on the chlorophyll extraction concentration were investigated.

[0054] 4.1 Effect of NADES water content on chlorophyll content

[0055] Adding a certain amount of water to NADES can increase solubility and adjust pH and viscosity. It can also shorten preparation time and lower preparation temperature. Therefore, the appropriate water content in NADES is crucial for the extraction of spirulina chlorophyll. Experiments showed that Bet-Xyl is difficult to form a clear liquid when the water content is below 30%, which is not conducive to subsequent applications. Therefore, Bet-Xyl with water contents of 30-50% was prepared sequentially, followed by chlorophyll extraction. The Bet-Xyl algal powder extract was obtained by homogenization, extraction, and centrifugation (10 ml of each extract was added to 0.1 g of spirulina algal powder, homogenized for 10 min to break the cell wall, and then magnetically stirred at 40℃ for 90 min to complete the extraction. After centrifugation at 4℃, the supernatant was collected, which is the NADES algal powder extract). The chlorophyll concentration was then measured.

[0056] like Figure 6As shown, the chlorophyll concentration in the Bet-Xyl extract gradually decreased with increasing water content. There was no significant difference in chlorophyll concentration between the extract with 30% and 35% water content; however, the 30% solution was more viscous than the 35% solution, making it unsuitable for food production. Therefore, a water content of 30–35% is considered the optimal water content for extracting chlorophyll from Spirulina.

[0057] 4.2 Effect of extraction temperature on chlorophyll content

[0058] Equal amounts of Bet-Xyl (35% water content) were used for the extraction of chlorophyll from Spirulina. 10 ml of each sample was added to 0.1 g of Spirulina powder, homogenized for 10 min to break the cell wall, and then magnetically stirred at 30–70 °C for 90 min to complete the extraction. After centrifugation at 4 °C, the supernatant was collected to obtain the NADES algal powder extract, and the chlorophyll concentration was determined.

[0059] The UV absorption spectra and chlorophyll concentration changes of Bet-Xyl algal powder extracts at different extraction temperatures are as follows: Figure 7 As shown, with increasing extraction temperature, the chlorophyll concentration in the Bet-Xyl extract generally exhibits a trend of first increasing, then decreasing, and then increasing again. Therefore, it is considered that an extraction temperature of 40–60℃ is the optimal extraction temperature for Bet-Xyl to extract chlorophyll from Spirulina.

[0060] 4.3 Effect of extraction time on chlorophyll content

[0061] Equal amounts of Bet-Xyl (35% water content) were used for the extraction of chlorophyll from Spirulina. 10 ml of each sample was added to 0.1 g of Spirulina powder, homogenized for 10 min to break the cell wall, and then magnetically stirred at 50℃ for 30–150 min to complete the extraction. After centrifugation at 4℃, the supernatant was collected to obtain the NADES algal powder extract, and the chlorophyll concentration was determined.

[0062] The UV absorption spectra and chlorophyll concentration changes of Bet-Xyl algal powder extracts at different extraction times are as follows: Figure 8 As shown, the chlorophyll concentration in the Bet-Xyl extract generally showed a trend of first increasing and then decreasing with increasing extraction time. Therefore, it is considered that an extraction time of 90–150 min is the optimal extraction time for spirulina chlorophyll from Bet-Xyl.

[0063] 4.4 Effect of substrate-to-liquid ratio on chlorophyll content

[0064] In the extraction of spirulina chlorophyll using NADES, the solid-liquid ratio is a crucial factor affecting the extraction efficiency. Considering the low water content (35%) and high viscosity of the NADES used in the experiment, a low solid-liquid ratio would result in an excessively viscous extract, even a paste-like consistency, hindering the extraction process. When equal amounts of algal powder were used, the extract was paste-like at a solid-liquid ratio below 1:100, making subsequent operations impossible. Therefore, spirulina chlorophyll extraction was performed at solid-liquid ratios ranging from 1:100 to 1:300. After homogenization for 10 minutes to break the cell walls, extraction was completed by magnetic stirring at 50℃ for 120 minutes. The supernatant was collected after centrifugation at 4℃ to obtain the Bet-Xyl algal powder extract, and the chlorophyll concentration was determined.

[0065] The UV absorption spectra and chlorophyll concentration changes of Bet-Xyl algal powder extracts with different material-to-liquid ratios are as follows: Figure 9 As shown, the concentration of the Bet-Xyl algal powder extract continuously decreases with the decrease of the solid-liquid ratio. Considering the volume difference of the extract, the total chlorophyll content in the extract was further calculated. Observation of the ultraviolet spectrum of the extract revealed that the ratio of chlorophyll to phycocyanin in the system continuously decreased with the decrease of the solid-liquid ratio. Therefore, it is considered that a solid-liquid ratio of 1:100 to 1:150 is most favorable for the extraction of spirulina chlorophyll.

[0066] In summary, the optimal process conditions obtained are: Bet-Xyl moisture content 30-35%, extraction temperature 40-60℃, extraction time 1-2h, and material-to-liquid ratio 1:100-1:150.

[0067] Characterization of 5NADES Spirulina chlorophyll extract

[0068] Under the optimal process conditions obtained, namely, Bet-Xyl water content of 35%, extraction temperature of 50℃, extraction time of 2h, and material-liquid ratio of 1:100, NADES Spirulina chlorophyll extract was prepared and characterized.

[0069] 5.1 Ultraviolet Spectrum

[0070] The ultraviolet absorption spectrum of the Bet-Xyl algal powder extract prepared according to the optimal process is as follows: Figure 10 As shown, observations revealed a strong absorption peak in the Bet-Xyl algal powder extract between 650 and 700 nm, consistent with the characteristic absorption peak of Chl. Calculations indicate that a single extraction using 10 ml of NADES from 0.1 g of algal powder yielded a chlorophyll concentration of 17.75 mg / L. This means that 0.1775 mg of chlorophyll was extracted from 0.1 g of algal powder.

[0071] 5.2 Fluorescence spectrum

[0072] The excitation spectrum of Bet-Xyl algal powder extract was measured between 350 and 500 nm using an emission wavelength of 676 nm. Additionally, fluorescence emission spectra were measured between 600 and 750 nm using excitation at 400 nm. All measurements were performed using 96-well plates at room temperature.

[0073] Figure 11 Figure A shows the excitation spectrum of Bet-Xyl algal powder extract with an emission peak at 676 nm. It exhibits a strong absorption peak between 380 and 450 nm, which is consistent with the characteristic absorption of Chl. Figure 11 (Figure B) shows the fluorescence emission spectrum at an excitation wavelength of 400 nm, which corresponds to the absorption wavelength of Chl a. The emission peak in the figure is located at 676 nm, which is the fluorescence emission of Chl a.

[0074] 5.3 Determination of Spirulina chlorophyll extraction rate

[0075] Accurately weigh 0.1g of algal powder, measure the total chlorophyll content in the algal powder, and simultaneously use Bet-Xyl to fully extract the chlorophyll, calculating the extraction rate. The specific method is as follows:

[0076] Determination of total chlorophyll content in spirulina powder: First, phycocyanin was extracted from spirulina powder using deionized water. The powder and deionized water were homogenized for 5 min to break the cell walls, then centrifuged at 8000×g for 10 min at 4℃, and the supernatant was removed. The precipitate was dissolved in deionized water, and the homogenization and centrifugation were repeated until the supernatant was colorless. The precipitate was then dissolved in ethanol, homogenized for 15 min, and centrifuged at 8000×g for 10 min at 4℃, and the supernatant was collected. The precipitate was dissolved in ethanol again, homogenized for another 10–15 min, and centrifuged under the same conditions until the supernatant was colorless. All supernatants were collected, filtered, and the chlorophyll content was measured.

[0077] The method for extracting Chl using Bet-Xyl is as follows: Mix Bet-Xyl with algal powder (material-to-liquid ratio 1:100), homogenize for 5 min using a homogenizer, and extract with magnetic stirring in a 50℃ water bath for 2 h. Centrifuge at 4℃ and 10000×g for 10 min, and collect the supernatant. Dissolve the precipitate with Bet-Xyl, homogenize again for 5 min, and extract for 2 h. Centrifuge under the same conditions until the supernatant is colorless and transparent. Collect all supernatants and measure their chlorophyll content.

[0078] The extraction rate is calculated as follows:

[0079]

[0080] Following the above method, the chlorophyll content in 0.1g of Spirulina powder is approximately 0.5344mg. Using Bet-Xyl for repeated extractions, a maximum of 0.3466mg can be extracted. Calculations show that the extraction rate of chlorophyll from Spirulina using Bet-Xyl is approximately 64.85%.

[0081] 6. Determination of the thermal stability of Spirulina chlorophyll extract by NADES

[0082] Thermogravimetric analysis (TG-DTA) can reflect the temperature at which the crystal structure of a system collapses. To evaluate the thermal stability of Chl in the Bet-Xyl system, the thermogravimetric curves of Bet-Xyl-Chl and Chl were determined using a Netzsch STA 449F3 instrument under a nitrogen atmosphere.

[0083] Thermogravimetric analysis curves of Bet-Xyl-Chl and Chl are as follows: Figure 12 (A: TG curve; B: DTG curve) As shown, the TG heating range is 30℃~400℃. The mass loss of Bet-Xyl-Chl occurs in two stages. In the initial stage, Bet-Xyl-Chl shows approximately 30% mass loss, likely due to water decomposition. The later mass loss is mainly due to the combined decomposition of betaine, xylitol, and chlorophyll components. The initial decomposition temperature is the highest temperature at which NADES remains liquid without decomposing. Figure 12 The initial decomposition temperature of the Chl sample was found to be around 165℃, while that of Bet-Xyl-Chl was around 210℃, shifting towards higher temperatures compared to the Chl sample. This indicates improved thermal stability of Chl within the Bet-Xyl system. Furthermore, the DTG curves of both samples showed that the maximum decomposition rate temperature of Chl was approximately 224.7℃, while that of Bet-Xyl-Chl was approximately 286.6℃. This is consistent with the results obtained from the TGA curves. Therefore, it is concluded that the thermal stability of Chl in the NADES system is superior to that of Chl extracted using traditional organic solvent methods.

[0084] In summary, this invention has discovered a NADES, namely Bet-Xyl, that can effectively extract chlorophyll from Spirulina, achieving an extraction rate as high as 65%. The chlorophyll extracted using the NADES method, after continuous illumination at 19200 Lux for 6 hours, exhibited a lower degradation rate than chlorophyll extracted using traditional organic solvent methods, demonstrating that the NADES extraction method successfully improves the photostability of chlorophyll. Thermogravimetric analysis revealed that the initial decomposition temperature and maximum decomposition rate temperature of the chlorophyll extracted using NADES were both higher than those of chlorophyll extracted using traditional organic solvent methods, proving that the NADES extraction method successfully improves the thermal stability of chlorophyll.

[0085] Example 2: Preparation of functional jelly made from Spirulina NADES extract

[0086] (1) Preparation of NADES: Take Bet (betaine) and Xyl (xylitol) in a molar ratio of 1:2 and mix them evenly. Add an appropriate amount of deionized water and stir magnetically at 70-80℃ until a colorless and transparent clear liquid is formed with a water content of 35%.

[0087] (2) Preparation of Spirulina chlorophyll: Chlorophyll was extracted from Spirulina powder using the prepared NADES (Bet-Xyl). Bet-Xyl was mixed evenly with the algal powder (material-liquid ratio 1:100), and the cell walls were broken by homogenizing for 5 min. The extraction process was carried out under magnetic stirring in a water bath at 40℃ for 90 min to complete the extraction process. The algal residue was removed by centrifugation at 4℃, and the supernatant was taken to obtain the Spirulina chlorophyll NADES extract for later use.

[0088] (3) Preparation of functional jelly: Take 6 mL of water, 1 mL of Spirulina chlorophyll NADES extract, 1 g of konjac powder, 0.5 g of carrageenan, 0.05 g of xanthan gum, and 0.3 g of citric acid, mix them evenly, heat to 90℃ and cook for 30 min, then pour the jelly liquid into the mold and cool to form.

[0089] Example 3: Preparation of functional beverage from Spirulina NADES extract

[0090] (1) Take Bet (betaine) and Xyl (xylitol) in a molar ratio of 1:2 and mix them evenly. Add an appropriate amount of deionized water and stir magnetically at 70-80℃ until a colorless and transparent clear liquid is formed, with a water content of 35%.

[0091] (2) Preparation of Spirulina chlorophyll: Chlorophyll was extracted from Spirulina powder using the prepared NADES (Bet-Xyl). Bet-Xyl was mixed evenly with the algal powder (material-liquid ratio 1:100), and the cell walls were broken by homogenizing for 5 min. The extraction process was carried out under magnetic stirring in a water bath at 40℃ for 150 min to complete the extraction process. The algal residue was removed by centrifugation at 4℃, and the supernatant was taken to obtain the Spirulina chlorophyll NADES extract for later use.

[0092] (3) Preparation of functional beverage: This functional beverage is prepared by adding 10 parts of spirulina chlorophyll NADES extract, 2 parts of mint leaves, and 100 parts of purified water by weight. The mixture is mixed evenly to obtain the initial juice of the beverage. A buffer is added to adjust the pH to 7-7.5, and then a sweetener is added and mixed to obtain the final product.

[0093] Example 4: Preparation of an antioxidant toner containing spirulina NADES extract

[0094] (1) Take Bet (betaine) and Xyl (xylitol) in a molar ratio of 1:2 and mix them evenly. Add an appropriate amount of deionized water and stir magnetically at 70-80℃ until a colorless and transparent clear liquid is formed, with a water content of 35%.

[0095] (2) Preparation of Spirulina chlorophyll: Chlorophyll was extracted from Spirulina powder using the prepared NADES (Bet-Xyl). Bet-Xyl was mixed evenly with the algal powder (material-liquid ratio 1:100), and the cell walls were broken by homogenizing for 5 min. The extraction process was carried out under magnetic stirring in a water bath at 40℃ for 120 min to complete the extraction process. The algal residue was removed by centrifugation at 4℃, and the supernatant was taken to obtain the Spirulina chlorophyll NADES extract for later use.

[0096] (3) Preparation of antioxidant toner: At 70-80℃, 4 parts of glycerin, 1 part of sodium hyaluronate, 5 parts of spirulina NADES extract, 89 parts of water and 1 part of ethanol are mixed evenly to obtain the antioxidant toner.

Claims

1. A method for green extraction of chlorophyll from Spirulina and improvement of chlorophyll stability, characterized in that, The improvement of chlorophyll stability refers to improving the light stability and thermal stability of chlorophyll. The method includes the following steps: (1) Preparation of NADES extract Mix HBD and HBA thoroughly, add deionized water, and stir magnetically at 70-80°C until a colorless and transparent clear liquid is formed, which is the NADES extract; HBD is betaine; HBA is xylitol. (2) Preparation of Spirulina chlorophyll Mix the NADES extract with spirulina powder, homogenize the mixture, and extract with magnetic stirring at 30-70°C. After extraction, freeze and centrifuge, and collect the supernatant to obtain the spirulina chlorophyll NADES extract.

2. The method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability as described in claim 1, characterized in that, (1) Preparation of NADES extract Mix betaine and xylitol in a molar ratio of 1:2 until homogeneous, add an appropriate amount of deionized water, and stir magnetically at 70-80℃ until a colorless, transparent, clear liquid is formed, with a water content of 30-35%. (2) Preparation of Spirulina chlorophyll The prepared NADES extract was mixed evenly with spirulina powder, and the cell walls were broken using a homogenizer. Extraction was carried out under magnetic stirring in a water bath at 40-60 ℃ for 90-150 min to complete the extraction process. The algal residue was removed by centrifugation at 4 ℃, and the supernatant was collected to obtain the spirulina chlorophyll NADES extract for later use.

3. The method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability as described in claim 2, characterized in that, The mass-to-volume ratio of the spirulina powder to the prepared NADES extract is 1:50~150.

4. The method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability as described in claim 2, characterized in that, The algal residue was extracted repeatedly in step (2) to improve the extraction rate of spirulina chlorophyll.

5. Spirulina chlorophyll NADES extract prepared by the method according to any one of claims 2-4.

6. The application of the Spirulina chlorophyll NADES extract according to claim 5 in the preparation of functional jellies, functional beverages, and antioxidant toners.

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