CN117924301A

CN117924301A - Method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability

Info

Publication number: CN117924301A
Application number: CN202410087536.5A
Authority: CN
Inventors: 李方巍; 王淑玉; 曾名湧
Original assignee: Sanya Institute Of Oceanography Ocean University Of China
Current assignee: Sanya Institute Of Oceanography Ocean University Of China
Priority date: 2024-01-22
Filing date: 2024-01-22
Publication date: 2024-04-26

Abstract

The invention discloses a method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability. Uniformly mixing HBD and HBA, adding deionized water, and magnetically stirring at 70-80 ℃ until colorless transparent clear liquid is formed, thus obtaining NADES extract; mixing the NADES extract with spirulina powder, breaking wall by a homogenizer, magnetically stirring and extracting at 30-70 ℃, freezing and centrifuging after extraction is completed, and taking supernatant to obtain the spirulina chlorophyll NADES extract. Experiments prove that the spirulina chlorophyll NADES extract prepared by the method obviously improves the photostability and the heat stability of chlorophyll. The invention applies the cheap and easily available environment-friendly NADES to the extraction of the spirulina chlorophyll for the first time, solves the problems of environmental pollution and human health hazard of the traditional extraction method, and can also improve the stability of the chlorophyll.

Description

Method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability

Technical Field

The invention belongs to the technical field of food processing, and particularly relates to a method for extracting spirulina chlorophyll and improving chlorophyll stability in a green way.

Background

Spirulina is a green food with rich nutrition and comprehensive functions, and is listed in the development plan of human food resources in the 21 st century by the United nations grain and agriculture organization (FAO) and is known as the most ideal food in the 21 st century. The spirulina is rich in a plurality of active substances such as polysaccharide, polyphenol, chlorophyll, carotenoid, unsaturated fatty acid and the like, has a plurality of health care effects such as antivirus, antitumor, antifatigue, improving organism immunity and the like, and is known as a miniature green functional nutrition treasury. The fat-soluble pigment chlorophyll in the spirulina is used as a natural pigment, can replace artificial pigment to develop color, and has various good biological activities such as antioxidation, anti-tumor, bacteriostasis, anti-inflammation, anti-mutagenesis and the like. Therefore, chlorophyll has wide application in food, nutrition, medicine, cosmetics and other aspects, and has higher commercial value.

At present, the method for extracting chlorophyll mainly adopts enzymolysis or ultrasonic-assisted organic solvent extraction, and a large amount of organic reagents such as methanol, acetone, petroleum ether and the like are used in the extraction process, so that the environment is polluted, the human health is endangered, and certain potential safety hazard exists. CN201910838715.7 discloses that organic reagents such as ethanol, acetone, etc. are used to extract chlorophyll and aromatic oil from the pepper leaves at the same time, and the extraction process reduces the addition amount of organic solvents. CN201710650214.7 relates to a method for extracting chlorophyll by adding enzyme, which mainly comprises the steps of adding cellulase to the pretreated enteromorpha for breaking the wall, and extracting the chlorophyll by using organic solvents such as methanol, acetone, isopropanol and the like. CN201610176075.4 shows a method for extracting chlorophyll from green plant leaves, which mainly comprises the steps of raw material preparation, leaching, filtering, concentrating, eluting, drying and the like, wherein in the extraction process, acetone solution is mainly used for extracting chlorophyll in the raw material. CN201710169066.7 discloses an extraction process of active substances in spirulina, mainly using ethanol soxhlet extraction method to obtain sodium zinc chlorophyllin, wherein hydrochloric acid, acetone and petroleum ether are inevitably used to complete the extraction process.

The deep eutectic solvent (Deep eutectic solvents, DES) is a green solvent with simple and easily available raw materials, high thermal stability, low volatility and easy synthesis, and has been widely used in various fields such as biology, medicine and green chemistry since the first proposal in 2001, and is considered to be one of the most important findings in the 21 st century. Deep eutectic solvents are typically binary or ternary systems consisting of a Hydrogen Bond Donor (HBD) and a Hydrogen Bond Acceptor (HBA), with polyols, urea, carboxylic acids, etc. often acting as Hydrogen Bond Donors (HBD), choline chloride, amino acids, etc. acting as Hydrogen Bond Acceptors (HBA). DES can form hydrogen bonds with the target compound, so many compounds exhibit high solubility in DES. Based on this property, DES is widely used for extraction of bioactive substances, and DES whose main component is primary metabolites of sugar, amino acid, organic acid, etc., is called natural deep eutectic solvent (Natural deep eutectic solvents, NADES). The natural source of NADES is wide, easy to synthesize, biodegradable, and green and non-toxic, and its application in the food industry exhibits higher safety compared to organic agents. Compared with the traditional chlorophyll extraction method which still has the problems of organic solvent residue, environmental pollution, safety in the extraction process and the like, the NADES has great potential in the green chemistry field.

CN202011060450.1 uses choline chloride, glycerol and citric acid (the molar ratio is 1:2:1), and a certain volume of ultrapure water is added to prepare NADES, and the NADES is combined with an ultrasonic extraction technology to optimize the isoflavone extraction process of the red clover, so that the component extraction process is more green, efficient and environment-friendly. CN202110812322.6 uses NADES (including choline chloride, citric acid, malic acid, D-glucose and D-fructose) with different components to assist in ultrasonic extraction of perilla leaf essential oil, and compared with the traditional method, the obtained perilla leaf essential oil has higher yield in ultrasonic-assisted NADES extraction, has stronger oxidation resistance and better antibacterial effect, and is considered that the ultrasonic-assisted NADES extraction can be applied to the field of natural perfume raw material extraction as a novel extraction method. CN202010034722.4 uses NADES (hydrogen bond acceptor is one of choline chloride and DL-menthol, and hydrogen bond donor is one of gluconic acid, tartaric acid and lactic acid) to extract carotenoid from apricot residue, tomato residue and carrot residue, and the method is efficient, environment-friendly, simple and rapid.

As is clear from the above patents, the extraction of bioactive substances using NADES is cleaner and more efficient than the conventional extraction methods, but no report on the use of NADES for chlorophyll extraction is currently found. The existing extraction method of chlorophyll is to assist the extraction of organic solvent by ultrasonic and enzymolysis methods to improve the extraction efficiency, and the restriction of the organic solvent can not be thoroughly removed although the consumption of the organic solvent in the extraction process is reduced. Based on the property that NADES itself is easy to form hydrogen bond with target compound, the extraction of bioactive substances by using NADES has been widely applied to the field of natural product extraction, so that NADES can be tried to be applied to the extraction of spirulina chlorophyll, and the extraction process is more environment-friendly.

Disclosure of Invention

In view of the above problems, the present invention provides a green method for extracting spirulina chlorophyll and improving chlorophyll stability. According to the invention, the natural deep eutectic solvent is used for replacing the organic solvent to extract chlorophyll in the spirulina, so that the use of toxic and harmful organic reagents can be avoided, and the problems of environmental pollution and human health hazard of the traditional extraction method are solved; meanwhile, the stability of chlorophyll can be improved, and a new idea is provided for widening chlorophyll application channels and prolonging the shelf life of products.

The technical scheme of the invention is as follows: a green method for extracting spirulina chlorophyll and improving chlorophyll stability is characterized in that,

(1) Preparation of NADES extract

Uniformly mixing HBD (hydrogen bond donor) and HBA (hydrogen bond acceptor), adding deionized water, and magnetically stirring at 70-80 ℃ to form colorless transparent clear liquid, thus obtaining NADES extract;

(2) Preparation of spirulina chlorophyll

Mixing the NADES extract with spirulina powder, breaking wall by a homogenizer, magnetically stirring and extracting at 30-70 ℃, freezing and centrifuging after extraction is completed, and taking supernatant to obtain the spirulina chlorophyll NADES extract.

Wherein HBD is betaine; the HBA is one of triethylene glycol, glycerol and xylitol, preferably xylitol. When the HBD is betaine and the HBA is xylitol, the specific steps are preferably as follows:

(1) Preparation of NADES extract

According to the ratio (molar ratio) of the amounts of the substances 1:2, uniformly mixing betaine and xylitol, adding a proper amount of deionized water, and magnetically stirring at 70-80 ℃ until colorless transparent clear liquid is formed, so that the water content is 30-35%;

(2) Preparation of spirulina chlorophyll

Uniformly mixing the prepared NADES extract with spirulina powder (feed-liquid ratio is 1:100-150), breaking the walls by using a homogenizer, extracting under the auxiliary magnetic stirring of water bath at 40-60 ℃, magnetically stirring for 90-150 min to finish the extraction process, removing algae residues by freezing and centrifuging at 4 ℃, and taking supernatant to obtain the spirulina chlorophyll NADES extract for later use.

Repeatedly extracting the algae residues according to the step (2) to improve the extraction rate of the spirulina chlorophyll.

Experiments prove that the spirulina chlorophyll NADES extract prepared by the method obviously improves the photostability and the heat stability of chlorophyll.

The invention also discloses application of the spirulina chlorophyll NADES extract prepared by the method in preparation of functional jelly, functional beverage and antioxidant toner.

The invention has the technical effects that:

1. According to the invention, the cheap and easily available environment-friendly NADES is applied to the extraction of the spirulina chlorophyll for the first time, and the NADES is used for successfully extracting the chlorophyll, so that the extraction rate is up to 65%, and the three advantages of economic benefit, high-efficiency extraction and environment protection are considered;

2. The prior art focuses on how to extract chlorophyll, and avoids the key problem of stability of the extracted chlorophyll, which affects the application of the chlorophyll. The invention successfully improves the light stability and the heat stability of chlorophyll on the premise of efficiently extracting the chlorophyll, solves the key problem, prolongs the shelf life of natural pigment products, and is beneficial to the further application of the chlorophyll products;

3. The reagents used in the invention are biodegradable and green nontoxic NADES, the innovation meets the requirements of green chemistry, and the use of toxic and harmful organic reagents is avoided, so that the safety of the extraction process and the product is greatly improved.

Drawings

FIG. 1 is a graph showing comparison of the results of extraction of spirulina chlorophyll by 8 NADES, wherein: 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 figure, the left side is prepared NADES, and the right side is corresponding algae powder extract; f, sequentially extracting NADES, algae powder extract and 5 times diluted algae powder extract from left to right;

FIG. 2 is an ultraviolet spectrum of chlorophyll extracts of 8 NADES spirulina; wherein: a: bet-Teg; b: chCl-prop; c: bet-Lev; d: chCl-MA; e: bet-Gly; f: chCl-Gly; g: bet-Xyl; h: chCl-Xyl;

FIG. 3 is a graph comparing the stability variation of each group with illumination for 0 and 6 hours; A. b, C, D, E are respectively set of Bet-Teg, bet-Gly, bet-Xyl, chlorophyll ethanol/water (v/v=1:4) solution, chlorophyll ethanol solution;

FIG. 4 is a graph showing the change of ultraviolet spectrum of each group in the illumination range of 0 to 6 hours, wherein A, B, C, D is respectively a Bet-Teg group, a Bet-Gly group, a Bet-Xyl group and a chlorophyll control group;

FIG. 5 shows the change in degradation rate of Chl in each group after 6 hours of continuous illumination, A, B, C, D, E being respectively a Bet-Teg group, a Bet-Gly group, a Bet-Xyl group, a chlorophyll ethanol/water (v/v=1:4) solution, a chlorophyll ethanol solution;

FIG. 6 is a graph of ultraviolet absorption spectrum (A graph) and chlorophyll concentration change (B graph) of Bet-Xyl extracts with different deionized water contents;

FIG. 7 shows ultraviolet absorption spectra (A graph) and chlorophyll concentration change (B graph) of Bet-Xyl algae powder extract at different extraction temperatures;

FIG. 8 shows ultraviolet absorption spectra (A graph) and chlorophyll concentration change (B graph) of Bet-Xyl algae powder extract at different extraction times;

FIG. 9 shows ultraviolet absorption spectra (A graph) and chlorophyll concentration change conditions (B graph) of Bet-Xyl algae powder extract solutions with different feed liquid ratios;

FIG. 10 is an ultraviolet absorption spectrum of the Bet-Xyl algae meal extract;

FIG. 11 is a fluorescence excitation spectrum (A plot) of a Bet-Xyl algae powder extract with an emission wavelength of 676nm and a fluorescence emission spectrum (B plot) of a Bet-Xyl algae powder extract with an excitation wavelength of 400 nm;

FIG. 12 is a thermogravimetric analysis of Bet-Xyl-Chl with Chl, wherein A: TG curve; b: DTG curve.

Detailed Description

The effects thereof are described below with reference to the embodiments and drawings.

Preparation of NADES extract

The preparation method of the NADES extract comprises the following steps: HBD (component 1) and HBA (component 2) are magnetically stirred to a transparent and clear state in a water bath at 70-80 ℃ according to a certain mass ratio. To achieve a good solution state for some fluidity of the NADES, a certain amount of deionized water was added to each of the eight NADES. The specific formulation of NADES is shown in Table 1.

Table 1 eight formulations of NADES extract

2. Screening of different NADES extract for spirulina chlorophyll extraction effect

The main experimental method is as follows: 10ml of each of the eight prepared NADES extracts is added with 0.2g of spirulina powder, homogenized for 10min, and magnetically stirred for 90min at 40 ℃ to finish the extraction. And (3) freezing and centrifuging at 4 ℃ and taking supernatant to obtain NADES algae powder extract, and analyzing substances contained in the extract through ultraviolet absorption spectrum.

As shown in FIG. 1, it was observed that A, B, E, F, G groups of possible pigment substances in spirulina were extracted. The above eight extracts of NADES were subjected to uv spectroscopy and the results are shown in figure 2. The ultraviolet absorption spectrum of the extract liquid of three NADES including Bet-Teg, bet-Gly and Bet-Xyl can be observed to be consistent with the absorption peaks of chlorophyll and phycocyanin, and two substances of chlorophyll and phycocyanin are considered to be extracted. However, the extraction method of phycocyanin is relatively mature at present, so chlorophyll is selected as a main research target.

3. Determination of chlorophyll degradation Rate

The extraction finds that three NADES of Bet-Teg, bet-Gly and Bet-Xyl extract chlorophyll and phycocyanin, and in order to further screen out NADES which is most suitable for extracting chlorophyll, three groups of extracting solutions of Bet-Teg, bet-Gly and Bet-Xyl are subjected to determination of chlorophyll degradation rate. The extract was diluted to a chlorophyll concentration of 6mg/L using the corresponding NADES, and chlorophyll ethanol/water (v/v=1:4) solution (concentration 6 mg/L) and chlorophyll ethanol solution (concentration 6 mg/L) were prepared using chlorophyll extracted by a conventional organic solvent extraction method as controls. The mixture is continuously illuminated for 6 hours at the illumination incubator of 4 ℃ and 60% illumination intensity (namely 19200 Lux), and the content change of chlorophyll in the system is observed.

The main method for extracting chlorophyll by using the traditional organic solvent is as follows: 200g of spirulina powder is taken, 200mL of absolute ethyl alcohol and 400mL of petroleum ether are added, and the mixture is homogenized for 5 minutes. And (5) freezing and centrifuging for 10 minutes, taking supernatant, and mixing with the equal volume of water for liquid separation after suction filtration. And (3) reserving the petroleum ether phase at the upper layer, washing with water, drying, and concentrating by rotary evaporation until 200mL remains to obtain the chlorophyll crude extract. And (3) allowing the chlorophyll crude extract to pass through a chromatographic column with neutral alumina as a filler, eluting carotene, lutein and chlorophyll by sequentially using petroleum ether-acetone (v/v=9:1), petroleum ether-acetone (v/v=7:3) and n-butanol-ethanol-water (v/v=3:1:1) as eluents, and collecting chlorophyll eluent (the chlorophyll content is about 8% in terms of dry matter) for later use.

The Bet-Teg group, bet-Gly group, bet-Xyl group, chlorophyll ethanol/water (v/v=1:4) solution, chlorophyll ethanol solution 0h and 6h pairs such as shown in fig. 3, can be seen from fig. 3: after illumination for 6 hours, chlorophyll in three groups of NADES extracting solutions including Bet-Teg, bet-Gly and Bet-Xyl has good stability (color fading is not obvious), chlorophyll ethanol/water (v/v=1:4) solution and chlorophyll ethanol solution has poor stability and obvious color fading after continuous illumination for 6 hours.

As shown in FIG. 4, after the ultraviolet spectrum is changed from 0 to 6 hours, the chlorophyll degradation rates of three groups of NADES extract solutions, including Bet-Teg, bet-Gly and Bet-Xyl, are lower than those of ethanol/water (v/v=1:4) and chlorophyll in an ethanol system after the ultraviolet spectrum is continuously illuminated for 6 hours under the illumination intensity of 19200 Lux. The method has the advantages that compared with the traditional organic solvent method, the method for extracting chlorophyll from spirulina by using NADES can effectively improve the illumination stability of chlorophyll and improve the defect that chlorophyll is easy to degrade under illumination.

The results of the change in the degradation rate of Chl in each group after 6 hours of continuous illumination are shown in fig. 5, wherein the chlorophyll degradation rate of the Bet-Xyl group is significantly lower than that of the chlorophyll ethanol/water (v/v=1:4) solution, the chlorophyll ethanol solution and the other two groups of NADES extract, and the chlorophyll illumination stability is considered to be superior to that of the other groups. Therefore, bet-Xyl is considered to be the most suitable NADES for subsequent extraction of spirulina chlorophyll.

Condition optimization of chlorophyll extraction by nades

And respectively taking different NADES water contents, extraction temperatures, extraction time, feed-liquid ratios and the like as factors to examine the influence of each factor on the chlorophyll extraction concentration.

4.1 Effect of NADES moisture content on chlorophyll content

The addition of a certain amount of water to the NADES can act to increase solubility, adjust pH and viscosity. Meanwhile, the preparation time and the preparation temperature can be shortened. Therefore, the proper moisture content in NADES is critical for extraction of spirulina chlorophyll. Experiments show that the Bet-Xyl is difficult to form transparent and clear liquid under the condition that the water content is lower than 30%, which is unfavorable for subsequent application. Therefore, the Bet-Xyl with the water content of 30-50% is prepared in sequence, and then chlorophyll is extracted. Homogenizing, extracting, centrifuging to obtain Bet-Xyl algae powder extractive solutions (10 ml each, adding 0.1g spirulina powder, homogenizing for 10min to break wall, magnetically stirring at 40deg.C for 90min to extract, centrifuging at 4deg.C, collecting supernatant to obtain NADES algae powder extractive solution), and measuring chlorophyll concentration.

As shown in FIG. 6, the chlorophyll concentration in the Bet-Xyl extract gradually decreased as the water content of the Bet-Xyl increased. The chlorophyll concentration of the 30% moisture content extract was not significantly different from that of the 35% moisture content extract, but the 30% solution was more viscous than the 35% solution, and was unsuitable for food production. Therefore, the water content of the Bet-Xyl is considered to be 30-35% as the optimal water content for extracting spirulina chlorophyll from the Bet-Xyl.

4.2 Effect of extraction temperature on chlorophyll content

Equal amount of Bet-Xyl (water content 35%) was used for extraction of spirulina chlorophyll. 10ml of each of the above materials is taken, 0.1g of spirulina powder is added, the wall is broken after homogenizing for 10min, and the magnetic stirring is carried out for 90min at 30-70 ℃ to finish the extraction. Freezing and centrifuging at 4deg.C, collecting supernatant to obtain NADES algae powder extractive solution, and measuring chlorophyll concentration.

As shown in FIG. 7, the ultraviolet absorption spectrum and chlorophyll concentration change of the Bet-Xyl algae powder extract at different extraction temperatures show a trend that the chlorophyll concentration in the Bet-Xyl extract increases after decreasing with increasing extraction temperature. Therefore, the extraction temperature of 40-60 ℃ is considered to be the optimal extraction temperature for extracting spirulina chlorophyll from Bet-Xyl.

4.3 Effect of extraction time on chlorophyll content

Equal amount of Bet-Xyl (water content 35%) was used for extraction of spirulina chlorophyll. 10ml of each of the above materials is taken, 0.1g of spirulina powder is added, the wall is broken after homogenizing for 10min, and the magnetic stirring is carried out for 30-150 min at 50 ℃ to finish the extraction. Freezing and centrifuging at 4deg.C, collecting supernatant to obtain NADES algae powder extractive solution, and measuring chlorophyll concentration.

As shown in FIG. 8, the ultraviolet absorption spectrum and chlorophyll concentration change of the Bet-Xyl algae powder extract at different extraction times show a trend that the chlorophyll concentration in the Bet-Xyl extract generally increases and then decreases with increasing extraction time. Therefore, the extraction time of 90 to 150min is considered to be the optimal extraction time for extracting spirulina chlorophyll from Bet-Xyl.

4.4 Effect of feed to chlorophyll content

In the process of extracting spirulina chlorophyll by using NADES, the feed-liquid ratio is also an important index for influencing the extraction effect. Considering that the water content of the NADES selected in the experiment is low (35%), the viscosity is high, so that the extracting solution is excessively viscous or even pasty when the liquid is low in the extracting process, and the full operation of the extracting process is affected. Taking an equal amount of algae powder, and when the feed liquid ratio is lower than 1:100, the extract is pasty and cannot be subjected to subsequent operations, so that the following steps are respectively carried out according to 1: extracting spirulina chlorophyll with a feed liquid ratio of 100-1:300. Homogenizing for 10min to break wall, and magnetically stirring at 50deg.C for 120min to extract. And (3) freezing and centrifuging at 4 ℃ and taking supernatant to obtain the Bet-Xyl algae powder extract, and determining the chlorophyll concentration.

The ultraviolet absorption spectrum and chlorophyll concentration change conditions of the Bet-Xyl algae powder extract with different feed liquid ratios are shown in fig. 9, and the concentration of the Bet-Xyl algae powder extract is continuously reduced along with the reduction of the feed liquid ratio, and the total chlorophyll content in the extract is further calculated by taking the volume difference of the extract into consideration. The ultraviolet spectrum of the extracting solution is observed to find that the ratio of chlorophyll to phycocyanin in the system is continuously reduced along with the reduction of the feed liquid ratio, so that the extracting of spirulina chlorophyll is most favorable when the feed liquid ratio is 1:100-1:150.

In summary, under the optimal process conditions, namely, the water content of the Bet-Xyl is 30-35%, the extraction temperature is 40-60 ℃, the extraction time is 1-2 h, and the feed-liquid ratio is 1:100-1:150.

Characterization of 5NADES spirulina chlorophyll extract

Under the optimal process conditions, namely, the water content of the Bet-Xyl is 35%, the extraction temperature is 50 ℃, the extraction time is 2 hours, the feed-liquid ratio is 1:100, and the NADES spirulina chlorophyll extract is prepared and characterized.

5.1 Ultraviolet Spectrum

The ultraviolet absorption spectrum of the Bet-Xyl algae powder extract prepared according to the optimal process is shown in figure 10, and the observation shows that the Bet-Xyl algae powder extract has a strong absorption peak between 650 and 700nm and accords with the characteristic absorption peak of Chl, and the calculation shows that 10ml NADES is used for extracting 0.1g algae powder for one time, and the concentration of the extracted chlorophyll is 17.75mg/L. I.e. 0.1775mg extracted from 0.1g algae powder.

5.2 Fluorescence Spectrum

And measuring the excitation spectrum of the Bet-Xyl algae powder extract between 350 and 500nm by taking 676nm as an emission wavelength. In addition, excitation at 400nm, fluorescence emission spectra between 600 and 750nm were measured. All measurements were performed using 96-well plates at room temperature.

FIG. 11 (A) shows the excitation spectrum of the extract of Bet-Xyl algae powder with emission peak at 676nm, which shows strong absorption peak between 380 and 450nm, and which is consistent with the characteristic absorption of Chl. FIG. 11 (B) is a graph showing fluorescence emission spectra at an excitation wavelength of 400nm, 400nm corresponding to an absorption wavelength of Chl a, with emission peak at 676nm, which is the fluorescence emission of Chl a.

5.3 Determination of chlorophyll extraction Rate of Spirulina

Accurately weighing 0.1g of algae powder, measuring the total chlorophyll content in the algae powder, and simultaneously using Bet-Xyl to fully extract chlorophyll therein, and calculating the extraction rate. The specific method comprises the following steps:

Determination of total chlorophyll content of algae powder: firstly, extracting phycocyanin in spirulina powder by using deionized water. Homogenizing the algae powder and deionized water for 5min to break wall, centrifuging at 4deg.C under centrifugal force of 8000 Xg for 10min, and removing supernatant. Dissolving the precipitate with deionized water, homogenizing, and centrifuging until the supernatant is colorless. Dissolving the precipitate with ethanol, breaking wall with a homogenizer for 15min, centrifuging at 4deg.C under centrifugal force 8000 Xg for 10min, and collecting supernatant. Dissolving the precipitate with ethanol, breaking the wall of the precipitate for 10 to 15 minutes by using a homogenizer again, and centrifuging under the same condition until the supernatant is colorless after centrifuging. Collecting all supernatant, suction filtering, and measuring chlorophyll content.

The Chl extraction method using Bet-Xyl is as follows: mixing Bet-Xyl and algae powder uniformly (feed-liquid ratio 1:100), breaking wall by using a homogenizer for 5min, performing magnetic stirring extraction for 2h in water bath with the assistance of 50 ℃, centrifuging for 10min under the condition of 4 ℃ and centrifugal force 10000 Xg, and collecting supernatant. Dissolving the precipitate with Bet-Xyl, breaking wall again with homogenizer for 5min, extracting for 2 hr, and centrifuging under the same conditions until the supernatant is colorless and transparent. All supernatants were collected and chlorophyll content was measured.

The extraction rate is calculated as follows:

According to the method, chlorophyll content in 0.1g spirulina powder is about 0.5344mg, and 0.3466mg can be extracted by repeatedly extracting with Bet-Xyl for several times. The calculation shows that the extraction rate of the spirulina chlorophyll is about 64.85% by using the Bet-Xyl.

Determination of the thermal stability of chlorophyll extract of spirulina NADES

Thermogravimetric analysis (TG-DTA) can reflect the temperature at which the architecture collapses. To evaluate the thermal stability of Chl in the Bet-Xyl system, the thermogravimetric curve of Bet-Xyl-Chl was measured under nitrogen atmosphere using a relaxation-resistant STA 449F3 instrument.

The thermogravimetric analysis curves of Bet-Xyl-Chl and Chl are shown in FIG. 12 (A: TG curve; B: DTG curve), and the temperature rising range of TG is 30-400 ℃. Among them, the mass loss of Bet-Xyl-Chl is divided into two stages, and at the initial stage Bet-Xyl-Chl shows a mass loss of about 30%, which may be caused by the decomposition of water. The later quality loss is mainly caused by the joint decomposition of betaine, xylitol and chlorophyll components. The initial decomposition temperature is the highest temperature at which the NADES remains liquid without decomposing. As can be seen from FIG. 12, the initial decomposition temperature of the Chl sample was around 165℃and the initial decomposition temperature of the Bet-Xyl-Chl was around 210℃as compared with the Chl sample, indicating that the thermal stability of Chl was improved in the Bet-Xyl system. Meanwhile, by observing the DTG curves of the two, the maximum decomposition rate temperature of Chl is about 224.7 ℃ and the maximum decomposition rate temperature of Bet-Xyl-Chl is about 286.6 ℃. This is consistent with the results obtained from the TGA profile. Therefore, the thermal stability of Chl in the NADES system is considered to be superior to Chl extracted by the conventional organic solvent method.

In summary, the invention discovers a NADES, namely Bet-Xyl, which can effectively extract chlorophyll from spirulina, and the extraction rate is as high as 65%. After the spirulina chlorophyll extracted by the NADES method is continuously illuminated for 6 hours under the illumination intensity of 19200Lux, the chlorophyll degradation rate is lower than that of the chlorophyll extracted by the traditional organic solvent method, and the NADES extraction method is proved to be successful in improving the photostability of the chlorophyll. The thermogravimetric analysis shows that the initial decomposition temperature and the maximum decomposition rate temperature of chlorophyll extracted by using the NADES are higher than those of chlorophyll extracted by the traditional organic solvent method, and the NADES extraction method is proved to successfully improve the thermal stability of chlorophyll.

Example 2: preparation of spirulina NADES extract functional jelly

(1) NADES preparation: the ratio of the mass is 1:2, respectively taking Bet (betaine) and Xyl (xylitol), uniformly mixing, adding a proper amount of deionized water, and magnetically stirring at 70-80 ℃ until colorless transparent clear liquid is formed, so that the water content is 35%;

(2) Preparation of spirulina chlorophyll: extracting chlorophyll from spirulina powder by using prepared NADES (Bet-Xyl), uniformly mixing Bet-Xyl and spirulina powder (feed liquid ratio 1:100), breaking wall for 5min by using a homogenizer, performing magnetic stirring under water bath auxiliary magnetic stirring at 40 ℃ for 90min to complete the extraction process, freezing and centrifuging at 4 ℃ to remove algae residues, and collecting supernatant to obtain spirulina chlorophyll NADES extract for later use;

(3) Preparing functional jelly: taking 6mL of water, 1mL of spirulina chlorophyll NADES extract, 1g of konjak powder, 0.5g of carrageenan and 0.05g of xanthan gum and 0.3g of citric acid, uniformly mixing, heating to 90 ℃, decocting for 30min, and then injecting the jelly glue solution into a mold for cooling and molding.

Example 3: preparation of spirulina NADES extract functional beverage

(1) The ratio of the mass is 1:2, respectively taking Bet (betaine) and Xyl (xylitol), uniformly mixing, adding a proper amount of deionized water, and magnetically stirring at 70-80 ℃ until colorless transparent clear liquid is formed, so that the water content is 35%;

(2) Preparation of spirulina chlorophyll: extracting chlorophyll from spirulina powder by using prepared NADES (Bet-Xyl), uniformly mixing Bet-Xyl and spirulina powder (feed liquid ratio 1:100), breaking wall for 5min by using a homogenizer, performing magnetic stirring under water bath auxiliary magnetic stirring at 40 ℃ for 150min to complete the extraction process, freezing and centrifuging at 4 ℃ to remove algae residues, and collecting supernatant to obtain spirulina chlorophyll NADES extract for later use;

(3) Functional beverage preparation: the functional beverage is prepared by adding 10 parts of spirulina chlorophyll NADES extract, 2 parts of mint leaves and 100 parts of purified water into the functional beverage according to weight parts, uniformly mixing to obtain beverage primary juice, adding a buffering agent into the beverage primary juice to adjust the pH of the beverage primary juice to 7-7.5, adding a sweetener, and mixing.

Example 4: antioxidant toner containing spirulina NADES extract

(2) Preparation of spirulina chlorophyll: extracting chlorophyll from spirulina powder by using prepared NADES (Bet-Xyl), uniformly mixing Bet-Xyl and spirulina powder (feed liquid ratio 1:100), breaking wall for 5min by using a homogenizer, performing magnetic stirring under water bath auxiliary magnetic stirring at 40 ℃ for 120min to complete the extraction process, freezing and centrifuging at 4 ℃ to remove algae residues, and taking supernatant to obtain spirulina chlorophyll NADES extract for later use;

(3) Preparation of antioxidant toner: and uniformly mixing 4 parts of glycerin, 1 part of sodium hyaluronate, 5 parts of spirulina NADES extract, 89 parts of water and 1 part of ethanol at the temperature of 70-80 ℃ to obtain the antioxidant toner.

Claims

1. A green method for extracting spirulina chlorophyll and improving chlorophyll stability is characterized in that,

The improvement of chlorophyll stability is to improve the light stability and the heat stability of chlorophyll;

The method comprises the following steps:

(1) Preparation of NADES extract

Uniformly mixing HBD and HBA, adding deionized water, and magnetically stirring at 70-80 ℃ until colorless transparent clear liquid is formed, thus obtaining NADES extract;

(2) Preparation of spirulina chlorophyll

2. The method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability according to claim 1, wherein said HBD is betaine; the HBA is one of triethylene glycol, glycerol and xylitol.

3. A method of green extraction of spirulina chlorophyll and improvement of chlorophyll stability according to claim 2, wherein said HBD is betaine; HBA is xylitol.

4. A method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability according to claim 3, wherein,

(1) Preparation of NADES extract

According to the mass ratio 1:2, uniformly mixing betaine and xylitol, adding a proper amount of deionized water, and magnetically stirring at 70-80 ℃ until colorless transparent clear liquid is formed, so that the water content is 30-35%;

(2) Preparation of spirulina chlorophyll

Mixing the prepared NADES extract with spirulina powder uniformly, breaking the wall by using a homogenizer, extracting under the auxiliary magnetic stirring of water bath at 40-60 ℃, magnetically stirring for 90-150 min to finish the extraction process, freezing and centrifuging at 4 ℃ to remove algae residues, and taking supernatant to obtain the spirulina chlorophyll NADES extract for later use.

5. The method for green extraction of spirulina chlorophyll and improvement of chlorophyll stability according to claim 4, wherein the mass-to-volume ratio of spirulina powder to prepared NADES extract is 1:50-150.

6. The method for green extraction of chlorophyll from spirulina and improvement of chlorophyll stability as in claim 4, wherein said step (2) is repeated for extraction of said spirulina residues to increase the extraction yield of chlorophyll from spirulina.

7. A spirulina chlorophyll NADES extract prepared by the method of any one of claims 4-6.

8. The use of spirulina chlorophyll NADES extract as claimed in claim 7 in the preparation of functional jelly, functional beverage and antioxidant toner.