Method for extracting phycocyanin by using natural eutectic solvent

Abstract

The invention relates to the technical field of phycocyanin extraction, in particular to a method for extracting phycocyanin by using a natural deep-eutectic solvent, NaDES is prepared by mixing a hydrogen bond binding donor and a hydrogen bond binding receptor in a fixed proportion, the NaDES component is an ingredient allowed to be added in cosmetic regulations, and when phycocyanin is extracted, the content of the NaDES component is reduced. The phycocyanin can be directly added into cosmetic preparation without complex purification and extraction solvent removal steps such as precipitation, centrifugation, dialysis, ion exchange chromatography and the like, the method is more environment-friendly and safer, the glycerol-glucose solvent can play a role in protecting phycocyanin, so that the structure of the phycocyanin is not easily damaged by light and heat, the storage stability is improved, and the application prospect is wide. Therefore, the technical problem that in the process of extracting phycocyanin by an existing phycocyanin extraction method, the used imidazole and pyridine ionic liquid has the defects of being difficult to degrade and toxic and causes influence on the body health of workers and the environment is solved.

Description

Technical Field

[0001] The invention relates to the technical field of phycocyanin extraction, and in particular to a method for extracting phycocyanin with a natural low eutectic solvent. Background Art

[0002] Phycocyanin is a natural pigment protein with bright colors. It is a nutritious protein with a complete amino acid composition and a high content of essential amino acids. It has the effects of anti-oxidation, scavenging free radicals in the body, enhancing immunity, and anti-inflammatory, and is widely used in food, cosmetics, medicine and other fields.

[0003] Since phycocyanin is an intracellular protein, cell disruption technology is one of the key factors that directly affect the protein extraction yield. At present, the extraction methods of phycocyanin generally include ultrasound, high-pressure homogenization, repeated freeze-thaw and other methods.

[0004] Prior art N103880950A discloses a method for extracting phycocyanin using an ionic liquid aqueous two-phase system. As a new type of green solvent and functional medium material, ionic liquid has the characteristics of low vapor pressure, no volatility, strong solubility, and good chemical stability, making it a substitute for traditional organic solvents.

[0005] However, in the above-mentioned method for extracting phycocyanin, imidazole and pyridine ionic liquids are usually used as solvents during the extraction of phycocyanin. However, imidazole and pyridine ionic liquids themselves have the defects of being difficult to degrade and toxic, which may easily affect the health of workers and the environment during the extraction process. Summary of the invention

[0006] The object of the present invention is to provide a method for extracting phycocyanin using a natural low eutectic solvent, which solves the technical problem that in the process of extracting phycocyanin, imidazole and pyridine ionic liquids are usually used as solvents, and imidazole and pyridine ionic liquids themselves have the defects of being difficult to degrade and toxic, resulting in the technical problem that they are easy to affect the health of workers and the environment during the extraction process.

[0007] To achieve the above object, the present invention provides a method for extracting phycocyanin using a natural deep eutectic solvent, comprising the following steps:

[0008] Step 1: mixing a hydrogen bonding donor and a hydrogen bonding acceptor in a fixed ratio to obtain a mixture, and then heating and stirring the mixture at 80° C. until a colorless solvent is obtained;

[0009] Step 2: weighing the spirulina powder in a dry state, adding the spirulina powder to the colorless solvent, and vortexing to mix, to obtain a mixture;

[0010] Step 3: The mixture is subjected to ultrasonic extraction in an ice bath, and then centrifuged at 14800 rpm for 10 min. After diluting the mixture with water by an appropriate multiple, the supernatant is taken to detect the phycocyanin content.

[0011] Wherein, the specific method for preparing the colorless solvent in the step 1 is:

[0012] Calculate the masses of the two substances according to the molar ratio of hydrogen bond donor to hydrogen bond acceptor;

[0013] Weigh the two substances accurately according to the calculated mass and mix them in a conical flask;

[0014] The conical flask was placed on a magnetic stirrer and heated at 80°C for stirring and dissolving until a transparent and uniform liquid was generated;

[0015] After the liquid is cooled to room temperature and stabilized, 25% (w / w) water is added to the conical flask. When no crystals are precipitated after being placed away from light for more than 24 hours, a colorless solvent is obtained.

[0016] The specific method for preparing the mixture in step 2 is as follows:

[0017] Accurately weigh 0.5 g of dry spirulina powder, and add the spirulina powder to 10 ml of the colorless solvent;

[0018] Then, the spirulina powder and the colorless solvent are vortex-mixed to obtain a mixture.

[0019] Wherein, the specific method of preparing the phycocyanin in step 3 is:

[0020] The mixture is subjected to auxiliary extraction, and ultrasonic extraction is performed on ice for 2 minutes under the condition of ultrasonication for 2 seconds followed by a rest period of 2 seconds to obtain an algae slurry;

[0021] Then, the algae slurry was centrifuged at 14800 rpm for 10 min, diluted with water by an appropriate multiple, and the supernatant was taken to detect phycocyanin.

[0022] The auxiliary extraction of the mixture is achieved by using an ultrasonic cell disruptor, and the set power of the ultrasonic cell disruptor is 400W.

[0023] After the phycocyanin is prepared, the absorbance values ​​of the phycocyanin at 620 and 652 nm are measured, and the phycocyanin content is calculated according to a formula.

[0024] Wherein, the calculation formula of the phycocyanin content is:

[0025]

[0026] Where: Y pc : Phycocyanin extraction content, in milligrams per gram (mg / g);

[0027] C pc : The concentration of phycocyanin in the sample solution, in milligrams per milliliter (mg / mL);

[0028] N: dilution multiple;

[0029] V: volume of sample solution, in milliliters (mL);

[0030] M: mass of spirulina powder, in grams (g).

[0031] C pc The calculation formula is:

[0032]

[0033] Where: OD 620 : The absorbance of the sample solution at a wavelength of 620 nm;

[0034] OD 652 : The absorbance of the sample solution at a wavelength of 652 nm.

[0035] The invention discloses a method for extracting phycocyanin by using a natural low eutectic solvent. NaDES is prepared by mixing a fixed ratio of a hydrogen bond donor and a hydrogen bond acceptor. The NaDES component is an ingredient allowed to be added in cosmetic regulations. When extracting phycocyanin, it is not necessary to go through the complicated purification and extraction solvent removal steps such as precipitation, centrifugation, dialysis, ion exchange chromatography, etc., and the phycocyanin can be directly added to the preparation of cosmetics. The method is more environmentally friendly and safer. The glycerol-glucose solvent can protect the phycocyanin, making its structure less susceptible to light and heat damage, and improving the storage stability. Thus, the existing method for extracting phycocyanin solves the problem that imidazole and pyridine ionic liquids are usually used as solvents in the process of extracting phycocyanin. However, the imidazole and pyridine ionic liquids themselves have the defects of being difficult to degrade and being toxic, resulting in the technical problem that the physical health of the workers and the environment are easily affected during the extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0037] Figure 1 1 is a flowchart of a method for extracting phycocyanin using a natural deep eutectic solvent according to a first embodiment of the present invention.

[0038] Figure 21 is a diagram of the specific preparation steps of the optimized colorless solvent according to the first embodiment of the present invention.

[0039] Figure 3 1 is a diagram of specific steps for preparing the optimized mixture according to the first embodiment of the present invention.

[0040] Figure 4 1 is a diagram of the specific preparation steps of the optimized phycocyanin according to the first embodiment of the present invention.

[0041] Figure 5 FIG. 4 is a schematic diagram of the phycocyanin content of the first embodiment of the present invention.

[0042] Figure 6 Schematic diagram of phycocyanin content before and after optimization of the first embodiment of the present invention. DETAILED DESCRIPTION

[0043] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0044] First embodiment:

[0045] See also Figures 1 to 6 ,in, Figure 1 is a flow chart of the method for extracting phycocyanin using a natural deep eutectic solvent according to the first embodiment of the present invention, Figure 2 is a specific preparation step diagram of the optimized colorless solvent according to the first embodiment of the present invention, Figure 3 is a diagram of specific steps for preparing the optimized mixture according to the first embodiment of the present invention, Figure 4 is a diagram of the specific preparation steps of the optimized phycocyanin according to the first embodiment of the present invention, Figure 5 is a schematic diagram of the phycocyanin content of the first embodiment of the present invention, Figure 6 Schematic diagram of phycocyanin content before and after optimization of the first embodiment of the present invention. The present invention provides a method for extracting phycocyanin with a natural deep eutectic solvent, comprising the following steps:

[0046] S101, step 1: mixing a hydrogen bonding donor and a hydrogen bonding acceptor in a fixed ratio to obtain a mixture, and then heating and stirring the mixture at 80° C. until a colorless solvent is obtained;

[0047] S102, step 2: weighing the spirulina powder in a dry state, adding the spirulina powder to the colorless solvent, and vortexing to mix, to obtain a mixture;

[0048] S103, step 3: subjecting the mixture to ultrasonic extraction in an ice bath, and then centrifuging the mixture at 14800 rpm for 10 min, diluting the mixture by an appropriate multiple with water, taking the supernatant, and detecting the phycocyanin content.

[0049] In this embodiment, the present invention prepares NaDES by mixing a fixed ratio of hydrogen bonding donors and hydrogen bonding acceptors, and the NaDES components are ingredients allowed to be added in cosmetic regulations. When extracting phycocyanin, there is no need to go through complex purification and extraction solvent removal steps such as precipitation, centrifugation, dialysis, and ion exchange chromatography. It can be directly added to the preparation of cosmetics. This method is more environmentally friendly and safer. The glycerol-glucose solvent can protect phycocyanin, making its structure less susceptible to light and heat damage, and improving storage stability, thereby solving the existing method of extracting phycocyanin. In the process of extracting phycocyanin, imidazole and pyridine ionic liquids are usually used as solvents, and imidazole and pyridine ionic liquids themselves have the defects of being difficult to degrade and toxic, resulting in the technical problem of easily affecting the health of workers and the environment during the extraction process.

[0050] Wherein, the specific method for preparing the colorless solvent in the step 1 is:

[0051] Calculate the masses of the two substances according to the molar ratio of hydrogen bond donor to hydrogen bond acceptor;

[0052] Weigh the two substances accurately according to the calculated mass and mix them in a conical flask;

[0053] The conical flask was placed on a magnetic stirrer and heated at 80°C for stirring and dissolving until a transparent and uniform liquid was generated;

[0054] After the liquid is cooled to room temperature and stabilized, 25% (w / w) water is added to the conical flask. When no crystals are precipitated after being placed away from light for more than 24 hours, a colorless solvent is obtained. The conical flask is placed on a magnetic stirrer and heated and stirred at 80° C. for 1-2 hours to dissolve.

[0055] Secondly, the specific method of preparing the mixture in step 2 is:

[0056] Accurately weigh 0.5 g of dry spirulina powder, and add the spirulina powder to 10 ml of the colorless solvent;

[0057] Then, the spirulina powder and the colorless solvent are vortex-mixed to obtain a mixture.

[0058] Again, the specific method for preparing the phycocyanin in step three is:

[0059] The mixture is subjected to auxiliary extraction, and ultrasonic extraction is performed on ice for 2 minutes under the condition of ultrasonication for 2 seconds followed by a rest period of 2 seconds to obtain an algae slurry;

[0060] Then, the algae slurry was centrifuged at 14800 rpm for 10 min, diluted with water by an appropriate multiple, and the supernatant was taken to detect phycocyanin.

[0061] Meanwhile, the auxiliary extraction of the mixture is achieved by using an ultrasonic cell disruptor, and the set power of the ultrasonic cell disruptor is 400W.

[0062] In addition, after the phycocyanin is prepared, the absorbance values ​​of the phycocyanin at 620 and 652 nm are measured, and the phycocyanin content is calculated according to a formula. The absorbance values ​​of the phycocyanin at 620 and 652 nm are measured by an ELISA reader using a 96-well plate.

[0063] Finally, the calculation formula of the phycocyanin content is:

[0064]

[0065] Where: Y pc : Phycocyanin extraction content, in milligrams per gram (mg / g);

[0066] C pc : The concentration of phycocyanin in the sample solution, in milligrams per milliliter (mg / mL);

[0067] N: dilution multiple;

[0068] V: volume of sample solution, in milliliters (mL);

[0069] M: mass of spirulina powder, in grams (g).

[0070] C pc The calculation formula is:

[0071]

[0072] Where: OD 620 : The absorbance of the sample solution at a wavelength of 620 nm;

[0073] OD 652 : The absorbance of the sample solution at a wavelength of 652 nm.

[0074] When using a natural low eutectic solvent to extract phycocyanin in this embodiment, the masses of the corresponding two substances are calculated according to the molar ratio of the hydrogen bonding donor and the hydrogen bonding acceptor in Table 1, and the corresponding two substances are accurately weighed according to the calculated masses, and placed in a conical flask for mixing. After that, the conical flask is placed on a magnetic stirrer and heated and stirred at 80°C for dissolution. The stirring time is 1-2 hours until a transparent and uniform liquid is generated. After the liquid is cooled to room temperature and stabilized, 25% (w / w) water is added to the conical flask. When no crystals are precipitated after being placed away from light for more than 24 hours, a colorless solvent is obtained.

[0075] Table 1 Composition ratio of each component

[0076]

[0077] Accurately weigh 0.5g of spirulina powder in a dry state, and add the spirulina powder to 10ml of the colorless solvent. Then, vortex mix the spirulina powder and the colorless solvent to obtain a mixture. Then, use an ultrasonic cell disruptor to assist in extraction of the mixture. The set power of the ultrasonic cell disruptor is 400W. According to the condition of ultrasonicating for 2s and then resting for 2s, ultrasonic extraction is performed in an ice bath for 2min to obtain an algae slurry. Then, the algae slurry is centrifuged at 14800rmp for 10min, diluted with water by an appropriate multiple, and the supernatant is taken to obtain phycocyanin. When the ultrasonic cell disruptor performs ultrasonic extraction, the ultrasonic power, ultrasonication under intermittent conditions, and the temperature can be set to be consistent with the instrument. The ultrasonic process will generate heat to increase the temperature. During the ultrasonic process, the sample is placed in an ice bath so that the sample temperature does not exceed 25°C.

[0078] After the phycocyanin is prepared, the absorbance values ​​of the phycocyanin at 620 and 652 nm are measured by using a 96-well plate using an ELISA reader, and the phycocyanin content is calculated according to the following formula:

[0079]

[0080] Where: Y pc : Phycocyanin extraction content, in milligrams per gram (mg / g);

[0081] C pc : The concentration of phycocyanin in the sample solution, in milligrams per milliliter (mg / mL);

[0082] N: dilution multiple;

[0083] V: volume of sample solution, in milliliters (mL);

[0084] M: mass of spirulina powder, in grams (g).

[0085] C pc The calculation formula is:

[0086]

[0087] Where: OD 620 : The absorbance of the sample solution at a wavelength of 620 nm;

[0088] OD 652 : The absorbance of the sample solution at a wavelength of 652 nm.

[0089] By selecting four influencing factors, namely, ultrasonic time (A), ultrasonic power (B), solid-liquid ratio (C) and water content (D), a Box-Behnken response surface optimization experiment was conducted. In this optimization experiment, the phycocyanin content was used as the response value to obtain the best extraction process conditions.

[0090] After the experiment, the following experimental results were obtained:

[0091] 1. Ultrasonic assisted extraction results

[0092] The experimental results are as follows Figure 5 As shown, it can be seen that the phycocyanin content extracted by glycerol-betaine (3:1) is the highest, which is 54.34 mg / g, followed by glycerol-xylose (3:1) with a content of 49.37 mg / g and glycerol-glucose (3:1) with a content of 48.36 mg / g. Considering the costs of betaine, xylose and glucose, glycerol-glucose (3:1) was finally selected for the optimization experiment.

[0093] 2. Optimization of ultrasound-assisted extraction results by response surface methodology

[0094] The phycocyanin content before and after optimization is as follows Figure 6 As shown in the figure, the optimal extraction process conditions predicted by the response surface software are: ultrasonic time 5min, ultrasonic power 520.271W, solid-liquid ratio 1:30 and water content 30%, and the extraction content is 68.198mg / g. Subsequently, the predicted optimal parameters were verified, and at the same time, in order to meet the convenience of actual operation. Therefore, the extraction ultrasonic time of 5min, ultrasonic power 520W, solid-liquid ratio 1:30 and water content 30% were adjusted and adopted, and the experiment was repeated 3 times. Under this extraction process parameter, the phycocyanin extraction content was 67.860mg / g, which is close to the predicted value, indicating that the process condition parameters optimized by the established model are reliable and reasonable, so the model can successfully use NADES as an extraction solvent for extracting phycocyanin components.

[0095] When repeating the experiment, Figure 2As shown, the specific method for preparing the optimized colorless solvent is:

[0096] S104. Calculate the masses of the two substances according to the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor;

[0097] S105. Accurately weigh two substances according to the calculated masses, and place them in a conical flask to mix;

[0098] S106, placing the conical flask on a magnetic stirrer and heating and stirring at 80° C. to dissolve until a transparent and uniform liquid is generated;

[0099] S107. After the liquid is cooled to room temperature and stabilized, 30% (w / w) water is added to the conical flask. When no crystals are precipitated in the dark for more than 24 hours, a colorless solvent is obtained.

[0100] like Figure 3 As shown, the specific method for preparing the optimized mixture is:

[0101] S108, accurately weighing 0.5 g of dry spirulina powder, and adding the spirulina powder to 15 ml of the colorless solvent;

[0102] S109. Thereafter, the spirulina powder and the colorless solvent are vortex-mixed to obtain a mixture.

[0103] like Figure 4 As shown, the specific method for preparing the optimized phycocyanin is:

[0104] S110, performing auxiliary extraction on the mixture, using an ultrasonic cell disruptor with a set power of 520 W, ultrasonically extracting for 5 minutes in an ice bath with a 2-second interval after ultrasonication, to obtain an algae slurry;

[0105] S111. Then, the algae slurry is centrifuged at 14800 rpm for 10 min, diluted with water by an appropriate multiple, and the supernatant is taken to detect phycocyanin.

[0106] In summary, the present invention prepares NaDES by mixing a fixed ratio of hydrogen bonding donors and hydrogen bonding acceptors, and the NaDES components are ingredients allowed to be added in cosmetic regulations. When extracting phycocyanin, there is no need to go through complex purification and extraction solvent removal steps such as precipitation, centrifugation, dialysis, ion exchange chromatography, etc., and it can be directly added to the preparation of cosmetics. This method is more environmentally friendly and safer. The glycerol-glucose solvent can protect phycocyanin, making its structure less susceptible to light and heat damage, and improving storage stability. The existing method for extracting phycocyanin solves the problem that imidazole and pyridine ionic liquids are usually used as solvents in the process of extracting phycocyanin, and imidazole and pyridine ionic liquids themselves have the defects of being difficult to degrade and toxic, resulting in the technical problem that they are easy to affect the health of workers and the environment during the extraction process.

[0107] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A method for extracting phycocyanin using a natural deep eutectic solvent, characterized in that: The following steps are involved: Step 1: mixing a hydrogen bonding donor and a hydrogen bonding acceptor in a fixed ratio to obtain a mixture, and then heating and stirring the mixture at 80° C. until a colorless solvent is obtained; Step 2: weighing the spirulina powder in a dry state, adding the spirulina powder to the colorless solvent, and vortexing to mix, to obtain a mixture; Step 3: The mixture is subjected to ultrasonic extraction in an ice bath, and then centrifuged at 14800 rpm for 10 min. After diluting the mixture with water by an appropriate multiple, the supernatant is taken to detect the phycocyanin content.

2. The method for extracting phycocyanin using a natural deep eutectic solvent according to claim 1, characterized in that: The specific method for preparing the colorless solvent in step 1 is: Calculate the masses of the two substances according to the molar ratio of hydrogen bond donor to hydrogen bond acceptor; Weigh the two substances accurately according to the calculated mass and mix them in a conical flask; The conical flask was placed on a magnetic stirrer and heated at 80°C for stirring and dissolving until a transparent and uniform liquid was generated; After the liquid is cooled to room temperature and stabilized, 25% (w / w) water is added to the conical flask. When no crystals are precipitated after being placed away from light for more than 24 hours, a colorless solvent is obtained.

3. The method for extracting phycocyanin using a natural deep eutectic solvent according to claim 1, characterized in that: The specific method for preparing the mixed product in step 2 is: Accurately weigh 0.5 g of dry spirulina powder, and add the spirulina powder to 10 ml of the colorless solvent; Then, the spirulina powder and the colorless solvent are vortex-mixed to obtain a mixture.

4. The method for extracting phycocyanin using a natural deep eutectic solvent according to claim 1, characterized in that: The specific method for preparing the phycocyanin in step 3 is: The mixture is subjected to auxiliary extraction, and ultrasonic extraction is performed on ice for 2 minutes under the condition of ultrasonication for 2 seconds followed by a rest period of 2 seconds to obtain an algae slurry; Then, the algae slurry was centrifuged at 14800 rpm for 10 min, diluted with water by an appropriate multiple, and the supernatant was taken to detect phycocyanin.

5. The method for extracting phycocyanin using a natural deep eutectic solvent according to claim 4, characterized in that: The auxiliary extraction of the mixture is achieved by using an ultrasonic cell disruptor, and the set power of the ultrasonic cell disruptor is 400W.

6. The method for extracting phycocyanin using a natural deep eutectic solvent according to claim 1, characterized in that: After the phycocyanin is prepared, the absorbance values ​​of the phycocyanin at 620 nm and 652 nm are measured, and the phycocyanin content is calculated according to a formula.

7. The method for extracting phycocyanin using a natural deep eutectic solvent according to claim 6, characterized in that: The calculation formula of the phycocyanin content is: Where: Y pc : Phycocyanin extraction content, in milligrams per gram (mg / g); C pc : The concentration of phycocyanin in the sample solution, in milligrams per milliliter (mg / mL); N: dilution multiple; V: volume of sample solution, in milliliters (mL); M: mass of spirulina powder, in grams (g). C pc The calculation formula is: Where: OD 620 : The absorbance of the sample solution at a wavelength of 620 nm; OD 652 : Absorbance of the sample solution at a wavelength of 652 nm.

Images