A method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction

Abstract

The present invention belongs to the technical field of extraction of natural biopolymers, and particularly relates to a method for extracting Spirulina platensis protein assisted by deep eutectic solvents and ethanol. The method of the present invention includes: mixing a hydrogen bond donor and a hydrogen bond acceptor, heating and stirring evenly in an oil bath, adding water and stirring evenly, and cooling to room temperature to form a colorless and transparent liquid to obtain a deep eutectic solvent; mixing Spirulina powder with the deep eutectic solvent, oscillating and extracting in a hot water bath to obtain an extract; centrifuging the extract at high speed, collecting the supernatant to obtain a Spirulina platensis protein extract; mixing the Spirulina platensis protein extract with ethanol, extracting and centrifuging at low temperature to obtain a precipitate; redissolving the precipitate in water, dialyzing to desalt, and drying to obtain Spirulina platensis protein. The method of the present invention does not require adjusting the pH value, is relatively economical and environmentally friendly; the method of the present invention can significantly improve the purity and yield of Spirulina platensis protein, and can provide a reference for the extraction of high-quality proteins from natural raw materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction of natural biopolymers, and particularly relates to a method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction. Background Art

[0002] Spirulina platensis is a microalgae plant growing in tropical and subtropical alkaline lakes, with extremely high nutritional value. Its protein content is as high as 60% - 70%. In addition, it also contains nutrients such as polysaccharides, chlorophyll, β-carotene, vitamins, and essential fatty acids, and has been widely used in the fields of food, medicine, and cosmetics. A large number of experiments have shown that Spirulina platensis protein has multiple effects such as anti-tumor, antioxidant, immunomodulatory, hypoglycemic, and promoting the regeneration of animal blood cells. How to produce Spirulina platensis protein in a green and efficient manner has become a research hotspot in the field of food processing.

[0003] Currently, the conventional methods for plant protein extraction mainly include enzymatic hydrolysis, organic solvent extraction, salting out, alkali dissolution and acid precipitation, etc. However, these methods still have the following defects: (1) The operation cost of enzymatic hydrolysis is relatively high, and the obtained protein purity is relatively low; (2) The process of organic solvent extraction is relatively complex, and the residual solvent is likely to cause environmental pollution; (3) The salting-out method takes a long time and has a low extraction rate; (4) The alkali dissolution and acid precipitation operation is complex and requires adjusting the pH value, which is not conducive to large-scale industrial application. At the same time, high pH conditions may decompose the structures of lysine and cysteine in proteins, reducing the overall quality and acceptability of proteins.

[0004] Therefore, it is of great practical significance to establish a simple, efficient, pollution-free and low-cost method for plant protein extraction. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction. This method uses a recyclable deep eutectic solvent as the extraction solvent, without the need to adjust the pH value, which is relatively economical and environmentally friendly; the deep eutectic solvent-assisted ethanol extraction can significantly improve the purity and yield of Spirulina platensis protein, and can provide a reference for the extraction of high-quality proteins from natural raw materials.

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

[0007] In the first aspect, the present invention provides a method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction, and the method includes the following steps:

[0008] Step S1, preparation of deep eutectic solvent:

[0009] Mix the hydrogen bond acceptor and the hydrogen bond donor, heat and stir evenly in an oil bath to form a stable mixed system, add water and stir evenly, and cool to room temperature to form a colorless transparent liquid to obtain a deep eutectic solvent.

[0010] In the present invention, the water addition amount in adding water and stirring evenly is 10% - 50% of the volume percentage of the mixed system. For example, the volume percentage can be 10%, 11%, 12%, 13%, 15%, 16%, 20%, 22%, 23%, 25%, 27%, 28%, 30%, 32%, 35%, 37%, 40%, 41%, 43%, 46%, 48% or 50%.

[0011] Step S2, extraction of Spirulina platensis protein:

[0012] Mix Spirulina platensis powder with the deep eutectic solvent, extract by shaking in a hot water bath to obtain an extract;

[0013] In the present invention, the Spirulina platensis powder is Spirulina platensis powder after ultrafine grinding. In the extraction by shaking in a hot water bath, the mass ratio of Spirulina platensis powder to the deep eutectic solvent is 1:20 - 1:60 g / mL, the extraction temperature by shaking in a hot water bath is 35 - 55 °C, and the extraction time is 0.5 - 2.5 h.

[0014] Centrifuge the extract at high speed, collect the supernatant to obtain a Spirulina platensis protein extract;

[0015] In the present invention, centrifuging the extract at high speed means cooling the extract to room temperature, and then centrifuging at 0 - 4 °C and 6000 - 8000 rpm for 10 - 20 min.

[0016] Mix the Spirulina platensis protein extract with ethanol, extract and centrifuge at low temperature to obtain a precipitate;

[0017] In the present invention, the extraction and centrifugation at low temperature include standing at 0 - 4 °C for 12 - 24 h and centrifuging at 6000 - 8000 rpm for 10 - 20 min.

[0018] Redissolve the precipitate in water, dialyze to desalt, and dry to obtain Spirulina platensis protein.

[0019] In the present invention, the dialysis desalting means that after redissolving the precipitate with an appropriate amount of distilled water, transfer it to a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, dialyze at 0 - 4 °C for 24 - 48 h, and change the distilled water every 3 - 6 h.

[0020] In the present invention, the drying is freeze-drying.

[0021] Preferably,

[0022] The hydrogen bond acceptor is choline chloride;

[0023] The hydrogen bond donor is selected from one of urea, acetic acid, lactic acid, oxalic acid, glycerol and ethylene glycol;

[0024] The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 1:3. For example, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor can be 1:1, 1:2 or 1:3.

[0025] Preferably,

[0026] The temperature of the oil bath heating is 80 - 110 °C; for example, the temperature of the oil bath heating can be 80 °C, 81 °C, 82 °C, 83 °C, 85 °C, 87 °C, 90 °C, 92 °C, 95 °C, 98 °C, 100 °C, 101 °C, 103 °C, 105 °C, 107 °C or 110 °C.

[0027] The time of the oil bath heating is 1 - 3 h; for example, the time of the oil bath heating can be 1 h, 1.2 h, 1.5 h, 2 h, 2.2 h, 2.5 h or 3 h.

[0028] Preferably,

[0029] The mass ratio of the Spirulina platensis powder to the deep eutectic solvent is 1:20 - 1:60 g / mL; for example, the mass ratio of the Spirulina platensis powder to the deep eutectic solvent is 1:20 g / mL, 1:30 g / mL, 1:40 g / mL, 1:50 g / mL or 1:60 g / mL.

[0030] The temperature of the hot water bath oscillating extraction is 35 - 55 °C; for example, the temperature of the hot water bath oscillating extraction is 35 °C, 36 °C, 37 °C, 38 °C, 40 °C, 41 °C, 43 °C, 45 °C, 48 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C or 55 °C.

[0031] The time of the hot water bath oscillating extraction is 0.5 - 2.5 h; for example, the time of the hot water bath oscillating extraction is 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 1.2 h, 1.3 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, 2.2 h, 2.3 h or 2.5 h.

[0032] Preferably,

[0033] The volume ratio of the Spirulina platensis protein extract to the ethanol is 1:1 to 1:5; for example, the volume ratio of the Spirulina platensis protein extract to the ethanol is 1:1, 1:2, 1:3, 1:4 or 1:5.

[0034] In a second aspect, a Spirulina platensis protein is provided, and the Spirulina platensis protein is prepared by the method of the present invention.

[0035] In a third aspect, a health food is provided, which comprises a safe and effective amount of the Spirulina platensis protein as described in the present invention.

[0036] In a fourth aspect, the application of the Spirulina platensis protein prepared by the method described in the present invention in the preparation of health foods is provided.

[0037] In a fifth aspect, the application of the Spirulina platensis protein prepared by the method described in the present invention in the preparation of pharmaceuticals is provided.

[0038] In a sixth aspect, the application of the Spirulina platensis protein prepared by the method described in the present invention in the preparation of cosmetics is provided.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] Deep eutectic solvents (DESs) are novel green extraction solvents, which are mainly composed of a hydrogen bond donor and a hydrogen bond acceptor, and have excellent properties such as low cost, easy synthesis, low volatility, non-toxicity, and biodegradability. DESs can be recovered by rotary evaporation and reused without further purification. The deep eutectic solvent-assisted ethanol extraction system of the present invention mixes the deep eutectic solvent containing the target extract with ethanol. After reaching a certain ratio, the target product aggregates in one phase, while other impurities are concentrated in the other phase, thereby achieving the purpose of extracting and separating the Spirulina platensis protein. The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor, the mass ratio of the Spirulina platensis powder to the deep eutectic solvent, the parameters of oil bath heating, the parameters of hot water bath shaking extraction, the volume ratio of the Spirulina platensis protein extract to ethanol, the amount of water added and its volume percentage, the parameters of high-speed centrifugation of the leaching solution, the parameters of low-temperature extraction centrifugation, and the process of dialysis desalination in the deep eutectic solvent-assisted ethanol extraction system of the present invention are key steps, which play an important role in the enrichment of the target product, the separation of impurities, and the extraction amount and extraction purity of the target product.

[0041] Specifically,

[0042] (1) Compared with the traditional water extraction method, the method of the present invention does not require additional adjustment of the pH value, avoiding the influence of a high pH environment on the functional properties of proteins.

[0043] (2) Compared with the ordinary organic solvent method, the deep eutectic solvent used in the present invention is easy to synthesize, non-toxic, low-volatile, biodegradable, and recyclable, more economical and safe, and is an efficient green extraction solvent.

[0044] (3) The purity, extraction amount, and total phycobiliprotein content of the Spirulina platensis protein obtained in the present invention are relatively high, significantly superior to the traditional water extraction technology. The emulsifying activity index and solubility of the obtained Spirulina platensis protein can reach 438.59m 2 / g and 20.04%, and the purity of phycocyanin is 0.4.

[0045] (4) The method of the present invention has mild conditions, simple operation, and is suitable for large-scale industrial production applications. Description of the Drawings

[0046] Figure 1 Appearance diagrams of deep eutectic solvents with different molar ratios and water addition amounts;

[0047] Figure 2 Effect of different deep eutectic solvents on the extraction amount of Spirulina platensis protein;

[0048] Figure 3 Effect of deep eutectic solvents with different water addition amounts on the extraction amount of Spirulina platensis protein;

[0049] Figure 4 Effect of different extraction conditions on the extraction amount of Spirulina platensis protein;

[0050] Figure 5 Extraction amount and purity of Spirulina platensis protein obtained by deep eutectic solvent-assisted ethanol extraction. Detailed Embodiments

[0051] The present invention will be specifically described below in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0052] Example 1

[0053] This example provides a method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction, and the method includes the following steps:

[0054] Step S1, preparation of deep eutectic solvent:

[0055] Mix choline chloride and urea at a molar ratio of 1:2, heat and stir in an 80 °C oil bath for 2 h, then add 10% distilled water, stir evenly, and cool to room temperature to obtain a colorless and transparent liquid, thus obtaining a deep eutectic solvent;

[0056] Step S2, extraction of Spirulina platensis protein:

[0057] Mix 1 g of ultra-finely ground Spirulina platensis powder with the deep eutectic solvent at a mass ratio of 1:20 g / mL, stir and extract at 45 °C for 2.0 h to obtain an extraction solution;

[0058] Centrifuge the extraction solution at 4 °C and 7000 rpm for 15 min, collect the supernatant to obtain a Spirulina platensis protein extraction solution;

[0059] Add absolute ethanol to the extract of Spirulina platensis in a volume ratio of 1:2. After mixing evenly, let it stand at 4 °C for 12 h to precipitate Spirulina platensis protein. Then centrifuge at 7000 rpm for 15 min to collect the precipitate of Spirulina platensis protein.

[0060] Dissolve the precipitate in an appropriate amount of distilled water, put it into a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, and dialyze at 4 °C for 24 h, changing the distilled water every 4 h. Collect the dialysate and freeze-dry it to obtain Spirulina platensis protein.

[0061] The protein content in the supernatant was detected by Coomassie brilliant blue colorimetry, and the extraction yield of Spirulina platensis protein was found to be 47.88 ± 2.16 mg / g.

[0062] In this invention, the extraction yield of protein from Spirulina platensis was also detected by changing the type of hydrogen bond donor, the molar ratio of hydrogen bond acceptor to hydrogen bond donor, and the amount of water added to the system in Example 1. See Figure 2 and Table 1 for details.

[0063] Table 1 Extraction Yields of Spirulina platensis Protein Obtained with Different Deep Eutectic Solvents

[0064]

[0065]

[0066] Note: CC-UE is choline chloride-urea; CC-AA is choline chloride-acetic acid; CC-LA is choline chloride-lactic acid; CC-OA is choline chloride-oxalic acid; CC-GY is choline chloride-glycerol; CC-EG is choline chloride-ethylene glycol.

[0067] Combined with Figure 2 and Table 1, it can be seen that the extraction yields of Spirulina platensis protein obtained with different deep eutectic solvents are different, and the extraction yields of Spirulina platensis protein obtained with deep eutectic solvents containing 10% distilled water are all higher than those without distilled water. This is mainly because the hydrogen bond donors and hydrogen bond acceptors that make up the deep eutectic solvents are different, which leads to differences in the viscosity, polarity, and number of hydrogen bonds of different solvents, thus affecting the extraction yield of Spirulina platensis protein. Among all the solvents, the Spirulina platensis protein content obtained with the deep eutectic solvent composed of choline chloride-glycerol at a molar ratio of 1:3 and containing 10% distilled water is the highest, reaching 58.49 ± 3.53 mg / g.

[0068] Example 2

[0069] This example provides a method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction. The method includes the following steps:

[0070] Step S1, preparation of deep eutectic solvent:

[0071] Mix choline chloride and glycerol in a molar ratio of 1:3, heat and stir in an 80 °C oil bath for 2 h, then add 30% distilled water, stir evenly, and cool to room temperature to form a colorless and transparent liquid to obtain a deep eutectic solvent;

[0072] Step S2, extraction of Spirulina platensis protein:

[0073] Mix 1 g of ultrafinely ground Spirulina platensis powder with the deep eutectic solvent at a mass ratio of 1:20 g / mL, stir and extract at 45 °C for 2.0 h to obtain an extract;

[0074] Centrifuge the extract at 4 °C and 8000 rpm for 15 min, collect the supernatant to obtain a Spirulina platensis protein extract;

[0075] Add absolute ethanol to the Spirulina platensis protein extract at a volume ratio of 1:2, mix evenly, then let it stand at 4 °C for 12 h to precipitate Spirulina platensis protein, and then centrifuge at 8000 rpm for 10 min to collect the Spirulina platensis protein precipitate;

[0076] Dissolve the precipitate in an appropriate amount of distilled water, load it into a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, dialyze at 4 °C for 36 h, and change the distilled water every 5 h during this period; collect the dialysate, and after freeze-drying, Spirulina platensis protein is obtained.

[0077] The protein content in the supernatant was detected by the Coomassie brilliant blue colorimetric method, and the extraction amount of Spirulina platensis protein was found to be 125.99 ± 4.88 mg / g.

[0078] The present invention also detected the extraction amount of Spirulina platensis protein by changing the water addition amount of the deep eutectic solvent in Example 2, as specifically shown in Figure 3 . From Figure 3 It can be seen that when the water addition amount in the system increases from 0% to 30%, the extraction amount of Spirulina platensis protein gradually increases, and when the water addition amount is 30%, the maximum extraction amount reaches 125.99 ± 4.88 mg / g; when the water addition amount exceeds 30%, as the water addition amount continues to increase, the extraction amount of Spirulina platensis protein begins to decrease. This may be because the introduction of distilled water reduces the viscosity of the deep eutectic solvent, making it more conducive to the dissolution of protein, thereby leading to an increase in the extraction amount. However, too high a water addition amount will cause the hydrogen bond network structure to be damaged, weakening the interaction between the solvent and the protein, and ultimately resulting in a decrease in the protein extraction amount.

[0079] Example 3

[0080] This example provides a method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction, and the method includes the following steps:

[0081] Step S1, Preparation of deep eutectic solvent:

[0082] Mix choline chloride and glycerol at a molar ratio of 1:3, heat and stir in an 80 °C oil bath for 2 h, then add 30% distilled water, stir evenly, and cool to room temperature to form a colorless transparent liquid to obtain the deep eutectic solvent;

[0083] Step S2, Extraction of Spirulina platensis protein:

[0084] Mix 1 g of ultrafinely ground Spirulina platensis powder with the deep eutectic solvent at a mass ratio of 1:30 g / mL, stir and extract at 40 °C for 2.0 h to obtain an extract;

[0085] Centrifuge the extract at 4 °C and 7500 rpm for 15 min, collect the supernatant to obtain the Spirulina platensis protein extract;

[0086] Add absolute ethanol to the Spirulina platensis protein extract at a volume ratio of 1:2, mix evenly, then let it stand at 4 °C for 36 h to precipitate the Spirulina platensis protein, and then centrifuge at 8000 rpm for 15 min to collect the Spirulina platensis protein precipitate;

[0087] Dissolve the precipitate in an appropriate amount of distilled water, load it into a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, dialyze at 4 °C for 36 h, and change the distilled water every 4 h during this period; collect the dialysate, and after freeze-drying, the Spirulina platensis protein is obtained.

[0088] The Coomassie brilliant blue colorimetric method was used to detect the protein content in the supernatant, and the extraction amount of Spirulina platensis protein was found to be 180.87 ± 3.64 mg / g.

[0089] The present invention also used choline chloride - glycerol (molar ratio 1:3, water addition 30%) as the extractant to study the effects of the solid - liquid ratio (mass ratio) ( Figure 4 in A), extraction time ( Figure 4 in B), and extraction temperature ( Figure 4 in C) in the extraction process on the extraction amount of Spirulina platensis protein. For details, see Figure 4 . From Figure 4 it can be seen that under the conditions of a fixed extraction time of 2.0 h and an extraction temperature of 45 °C, the extraction amount of Spirulina platensis protein first increases and then decreases with the increase of the mass ratio, and reaches a maximum value of 164.14 ± 5.35 mg / g at a mass ratio of 1:30 g / mL. This is mainly because when the mass ratio is too small, the viscosity of the system is large, resulting in the obstruction of the dissolution of Spirulina platensis protein; as the amount of solvent added increases, the concentration difference between the material and the solvent gradually increases, which is more conducive to mass transfer, and the dissolution of Spirulina platensis protein is more complete. However, too high a mass ratio may lead to a slower temperature rise in the system within the same time, thereby affecting the extraction amount of Spirulina platensis protein.

[0090] Similarly, under the conditions of a fixed mass ratio of 1:30 g / mL and an extraction temperature of 45 °C, the extraction yield of Spirulina platensis protein first increased and then decreased with the prolongation of the extraction time, and reached the maximum value of 154.91±5.02 mg / g when the extraction time was 2.0 h. This may be because the appropriate prolongation of the extraction time is more conducive to the full dissolution of Spirulina platensis protein. However, if the extraction time is too long, more impurities will dissolve in the system, which will affect the extraction yield of Spirulina platensis protein.

[0091] Similarly, under the conditions of a fixed mass ratio of 1:30 g / mL and an extraction time of 2.0 h, the extraction yield of Spirulina platensis protein first increased and then decreased with the increase of the extraction temperature, and reached the maximum value of 180.87±3.64 mg / g when the temperature was 40 °C. Appropriate increase in the extraction temperature has a certain promoting effect on the infiltration of plant tissues and is conducive to the dissolution of proteins. However, too high an extraction temperature may lead to the dissolution of other substances or cause protein denaturation, thereby reducing the extraction yield of proteins.

[0092] Comparative Example 1

[0093] Under the same conditions as in Example 3, using distilled water as the extraction agent, Spirulina platensis protein was extracted by the traditional water extraction method. The obtained protein extraction yield was 170.49 mg / g, while the protein extraction yield obtained by deep eutectic solvent-assisted ethanol extraction was 180.87 mg / g, which was 6.1% higher than the former; for details, see Figure 5 . The protein content in Spirulina platensis protein obtained by different methods was determined by the Kjeldahl method, and the protein purity was calculated according to formula (1).

[0094]

[0095] It can be seen from Figure 5 that the purity of Spirulina platensis protein obtained by choline chloride-glycerol deep eutectic solvent-assisted ethanol extraction was 92.8%, which was 20.5% higher than that of the traditional water extraction method.

[0096] Experiment 1 Analysis of the main protein components in Spirulina platensis protein

[0097] Using the choline chloride-glycerol deep eutectic solvent (molar ratio 1:3, system water addition 30%) in Example 3 as the extraction solvent to extract Spirulina platensis protein, and taking the Spirulina platensis protein obtained by the traditional water extraction method as the control, the main component composition of the obtained Spirulina platensis protein was analyzed.

[0098] [PC]=A 620 -0.474A 652 ] / 5.34 (2)

[0099] [APC]=[A652 -0.208 A 620 / 5.09 (3)

[0100] [PE] = [A 562 -2.41(PC) - 0.849(APC)] / 9.62 (4)

[0101] Total phycobiliprotein content mg(g) = [(PC) + (APC) + (PE)] × V × 100 / m (5)

[0102] Use a spectrophotometer to measure the absorbance of the sample solution, and calculate the main protein components in Spirulina platensis protein through formulas (2), (3), (4) and (5):

[0103] Among them, [PC], [APC] and [PE] are the contents (mg / g) of phycocyanin, allophycocyanin and phycoerythrin respectively, V is the system of the sample solution, and m is the sample mass.

[0104] Table 2 Main protein components of Spirulina platensis protein

[0105]

[0106] As can be seen from Table 2, the contents of phycocyanin, allophycocyanin and phycoerythrin in Spirulina platensis protein extracted by choline chloride - glycerol deep eutectic solvent - assisted ethanol extraction are 92.96 mg / g, 94.71 mg / g and 59.74 mg / g respectively, and the total phycobiliprotein content reaches 247.41 mg / g. Compared with the Spirulina platensis protein obtained by the traditional water extraction method, except that the phycocyanin content is slightly lower, the contents of allophycocyanin, phycoerythrin and total phycobiliprotein are increased by 59.6%, 191.2% and 38.9% respectively.

[0107] Experiment 2 Analysis of the functional properties of Spirulina platensis protein

[0108] Use the choline chloride - glycerol deep eutectic solvent (molar ratio 1:3, water addition in the system 30%) in Example 3 as the extraction solvent to extract Spirulina platensis protein, and analyze the representative functional properties of the obtained Spirulina platensis protein. See Table 3 for details.

[0109] Determination of emulsifying activity: Disperse 35 mL of the sample dispersion (20 mg / mL) with 15 mL of perilla oil, and homogenize it at 12600 rpm for 2 min by a T18 high - speed disperser. Then suck 50 μL of the freshly prepared emulsion from the bottom of the beaker, vortex - mix it with 5 mL of SDS solution (0.1%, w / v), and measure the absorbance of this mixed system at 500 nm. After storing the emulsion system at room temperature for 10 min, follow the same

[0110]

[0111] Measure the absorbance of the sample again using the same method. Calculate the emulsifying activity index and emulsifying stability index of the sample according to formulas (6) and (7) respectively.

[0112] In the formula, DF is the dilution factor. C and θ represent the oil phase volume fraction, protein concentration, and optical path respectively. A0 is the absorbance of the initial sample, and A 10 is the absorbance of the sample after standing for 10 min. ΔA is the difference between A0 and A 10 .

[0113] Solubility determination: Disperse 250 mg of sample powder in 25 mL of deionized water and continuously

[0114]

[0115] stir for 2 h at room temperature. After centrifuging at 6000 rpm for 15 min, collect the supernatant and determine the protein content in it by the Coomassie Brilliant Blue G250 colorimetric method. Calculate the solubility of the protein according to formula (8).

[0116] In the formula, m1 and m represent the protein content in the supernatant and the dispersion respectively.

[0117] Table 3 Functional properties of Spirulina platensis protein

[0118]

[0119] As can be seen from Table 3, the emulsifying activity index and solubility of the Spirulina platensis protein obtained by choline chloride-glycerol deep eutectic solvent-assisted ethanol extraction are 438.59 m 2 / g and 20.04% respectively. This indicates that Spirulina platensis protein has relatively good functional properties and is expected to be applied in the fields of health food, medicine, or cosmetics development.

[0120] Experiment 3 Appearance analysis of deep eutectic solvents

[0121] Figure 1 are the appearance diagrams of deep eutectic solvents with different molar ratios and water addition amounts. From Figure 1It can be seen that when no distilled water is added, the choline chloride-lactic acid (CC-LA) and choline chloride-oxalic acid (CC-OA) with a molar ratio of 1:1, the CC-LA with a molar ratio of 1:2, choline chloride-glycerol (CC-GY), choline chloride-acetic acid (CC-AA), choline chloride-ethylene glycol (CC-EG), and the CC-LA, CC-GY, CC-AA, and CC-EG mixed systems with a molar ratio of 1:3 all appear as colorless transparent liquids after being dissolved by oil bath heating and cooled, indicating the formation of deep eutectic solvents. After adding 10% distilled water, the appearance of the choline chloride-urea (CC-UE) with a molar ratio of 1:2 and the CC-GY with a molar ratio of 1:1 mixed system changes from white waxy solid crystals to colorless transparent liquids, indicating that during the preparation of deep eutectic solvents, the molar ratio of hydrogen bond donors to hydrogen bond acceptors and the water content in the system have an important influence on their formation. In addition, among the 6 mixed systems of hydrogen bond donors and hydrogen bond acceptors, the CC-LA mixed systems with different molar ratios all appear as colorless transparent liquids with or without the introduction of distilled water, indicating that it is easier for them to form deep eutectic solvents.

[0122] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0123] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also encompassed by the appended claims.

Claims

1. A method for extracting Spirulina platensis protein by deep eutectic solvent-assisted ethanol extraction, characterized in that, The method includes the following steps: Step S1, preparation of deep eutectic solvent: Mix a hydrogen bond acceptor and a hydrogen bond donor, heat and stir evenly in an oil bath, add 30% distilled water and stir evenly, and cool to room temperature to form a colorless transparent liquid to obtain the deep eutectic solvent; Step S2, extraction of Spirulina platensis protein: Mix Spirulina platensis powder with the deep eutectic solvent, extract by shaking in a water bath, and obtain an extract; Centrifuge the extract at high speed, collect the supernatant to obtain a Spirulina platensis protein extract; Mix the Spirulina platensis protein extract with ethanol, extract and centrifuge at low temperature to obtain a precipitate; the low-temperature extraction and centrifugation includes standing at 0-4 °C for 12-24 h and centrifuging at 6000-8000 rpm for 10-20 min; Redissolve the precipitate in water, dialyze to desalt, and dry to obtain Spirulina platensis protein; the dialysis desalting is to redissolve the precipitate with an appropriate amount of distilled water, transfer it to a dialysis bag with a molecular weight cut-off of 8-14 kDa, dialyze at 0-4 °C for 24-48 h, and change the distilled water every 3-6 h; The hydrogen bond acceptor is choline chloride; The hydrogen bond donor is glycerol; The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:3; The temperature of the oil bath heating is 80-110 °C; The time of the oil bath heating is 1-3 h; The mass ratio of the Spirulina platensis powder to the deep eutectic solvent is 1:20-1:60 g / mL; The temperature of the extraction by shaking in the water bath is 35-55 °C; The time of the extraction by shaking in the water bath is 0.5-2.5 h; The volume ratio of the Spirulina platensis protein extract to the ethanol is 1:1-1:5.

Images