Extraction method and optimization method of red-skin algae polysaccharide

Abstract

The invention relates to an extraction method of a red-skin algae polysaccharide, which comprises the following steps: mixing red-skin algae powder with a deep eutectic solvent, extracting polysaccharide in the red-skin algae under the assistance of ultrasonic waves to obtain a leaching solution, centrifuging, taking supernate, then adding absolute ethyl alcohol for alcohol precipitation, centrifuging, collecting precipitate, and freeze-drying to obtain the red-skin algae polysaccharide, wherein the eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor is one or more of urea, ethylene glycol, glycerol, malonic acid and oxalic acid, the hydrogen bond acceptor is choline chloride, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 2: 1. According to the method, different hydrogen bond donors and choline chloride are prepared into the eutectic solvent, then the eutectic solvent is organically combined with ultrasonic-assisted extraction to extract the red-skin algae polysaccharide, compared with a traditional hot water extraction method, the extraction rate of the red-skin algae polysaccharide and the activity of resisting oxidation and reducing blood fat are remarkably improved, and the method has the advantages of being short in consumed time, low in energy consumption, environmentally friendly and the like.

Description

Technical Field

[0001] The invention relates to the technical field of plant polysaccharide extraction, and in particular to a method for extracting dulse algae polysaccharide and an optimization method thereof. Background Art

[0002] Palmate red algae belongs to the Rhodophyta. Its dry algae are rich in polysaccharides, proteins, vitamins, unsaturated fatty acids and other nutrients. Among them, red algae polysaccharides can play the role of anti-toxin, anti-cancer, immune regulation, anti-aging, and lowering blood sugar. Therefore, the research on the extraction process of palmate red algae polysaccharides is of great significance to the development and utilization of red algae and the study of active natural drugs in its algae. At present, polysaccharides are mainly extracted and separated by hot water, acid, alkali and different organic solvents, but these traditional extraction methods often have problems such as time-consuming, inefficient, and not environmentally friendly.

[0003] As a new generation of environmentally friendly green solvents, deep eutectic solvents (DESs) have the advantages of high extraction rate, complete structure, and certain selective extraction compared with traditional solvents, and have great potential in the extraction of natural products. However, there are no reports on the application of deep eutectic solvents in the extraction of polysaccharides from dulse.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for extracting dulse polysaccharide and an optimization method thereof.

[0006] The technical solution adopted by the present invention is:

[0007] On one hand, the present invention provides a method for extracting dulse polysaccharide, comprising the following steps: mixing dulse powder with a low eutectic solvent, extracting polysaccharide in the dulse under the assistance of ultrasound to obtain an extract, centrifuging, taking the supernatant, then adding anhydrous ethanol for alcohol precipitation, centrifuging, collecting the precipitate, and freeze-drying to obtain the dulse polysaccharide;

[0008] The low eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor is one or more of urea, ethylene glycol, glycerol, malonic acid and oxalic acid, the hydrogen bond acceptor is choline chloride, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 2:1.

[0009] The present invention proposes a method for extracting polysaccharides from dulse seaweed, which comprises preparing a low eutectic solvent with different hydrogen bond donors and choline chloride, and then organically combining the low eutectic solvent with ultrasonic-assisted extraction to extract the polysaccharides from the dulse seaweed. Compared with the traditional hot water extraction method, the method significantly improves the extraction rate of dulse seaweed polysaccharides and the antioxidant and lipid-lowering activities, and has the characteristics of short time, low energy consumption, and green environmental protection, and can provide a theoretical basis and technical reference for the high-value development and utilization of dulse seaweed resources.

[0010] Preferably, the deep eutectic solvent uses ethylene glycol as the hydrogen bond donor, and the water content of the deep eutectic solvent is 10-50% v / v.

[0011] More preferably, the water content of the deep eutectic solvent is 20% v / v.

[0012] Preferably, the liquid-to-solid ratio of the low eutectic solvent to the dulse powder is 20-60:1 mL / g.

[0013] More preferably, the liquid-to-solid ratio of the low eutectic solvent to the dulse powder is 40:1 mL / g.

[0014] Preferably, the extraction conditions are: extraction temperature is 30-90°C, and extraction time is 20-60min.

[0015] More preferably, the extraction conditions are: extraction temperature is 75° C., and extraction time is 40 min.

[0016] Another aspect of the present invention provides a method for optimizing the process conditions of the extraction method of dulse polysaccharide as described in any of the above technical solutions, comprising the following steps:

[0017] S1. Five factors affecting the extraction rate of dulse polysaccharide were screened out: the type of deep eutectic solvent, the water content of the deep eutectic solvent, the liquid-to-solid ratio of the deep eutectic solvent to dulse powder, the extraction time, and the extraction temperature. The extraction rate of dulse polysaccharide was used as an indicator to conduct a single factor experiment.

[0018] S2. According to the single factor experimental results of step S1, three factors that have a significant impact on the extraction rate of dulse polysaccharides are selected: the water content of the deep eutectic solvent, the liquid-to-solid ratio of the deep eutectic solvent to dulse powder, and the extraction time. A three-factor three-level response surface experiment is performed using the Box-Behnken design principle;

[0019] S3. Response surface software was used for analysis, and a quadratic polynomial regression equation model was established to determine the optimal extraction conditions of dulse polysaccharides.

[0020] The invention optimizes the process parameters of extracting dulse algae polysaccharides by using an ultrasound-assisted low eutectic solvent through single factor experiments and a response surface analysis method, further improves the polysaccharide extraction rate, and shortens the extraction time.

[0021] Preferably, the quadratic polynomial regression equation model:

[0022] Y=+9.00+1.58A+0.1999B-0.0065C-0.0515AB+0.0388AC-0.1162BC-4.07A2-0.3224B2-0.0901C2, wherein A is the water content of the low eutectic solvent, B is the liquid-to-solid ratio of the low eutectic solvent to the red algae powder, C is the extraction time, and Y is the red algae polysaccharide extraction rate.

[0023] Preferably, the optimal extraction conditions of the dulse polysaccharide are: the water content of the low eutectic solvent is 21.9% v / v, the liquid-to-solid ratio of the low eutectic solvent to the dulse powder is 42.9:1 mL / g, and the extraction time is 38.8 min.

[0024] The extraction rate of dulse polysaccharide extracted by the ultrasonic-assisted low eutectic solvent method under the optimal process conditions can reach 9.138%, which is 168.43% higher than that of the traditional hot water extraction method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of the type of deep eutectic solvent on the extraction rate of dulse polysaccharide in Example 1;

[0026] Figure 2 The effect of the water content of the deep eutectic solvent on the extraction rate of dulse polysaccharide in Example 1;

[0027] Figure 3 The effect of the liquid-to-solid ratio of the deep eutectic solvent to the dulse algae powder on the extraction rate of dulse algae polysaccharide in Example 1;

[0028] Figure 4 The effect of extraction time on the extraction rate of dulse polysaccharide in Example 1;

[0029] Figure 5 The effect of extraction temperature on the extraction rate of dulse polysaccharide in Example 1;

[0030] Figure 6 The response surface diagram and contour diagram of the effect of the interaction of various factors on the extraction rate of dulse polysaccharide in Example 1;

[0031] Figure 7 This is a purity test chart of dulse polysaccharide obtained under the best extraction process in Example 1;

[0032] Figure 8The infrared spectrum of dulse polysaccharide obtained under the best extraction process in Example 1

[0033] Fig. 9 The DPPH free radical scavenging ability of the dulse algae polysaccharide obtained under the optimal extraction process in Example 1;

[0034] Fig.10 is the ABTS free radical scavenging ability of the dulse algae polysaccharide obtained under the optimal extraction process in Example 1;

[0035] Fig.11 is the scavenging ability of dulse polysaccharide obtained under the optimal extraction process in Example 1 on hydroxyl radicals;

[0036] Fig.12 is the superoxide anion free radical scavenging ability of the dulse algae polysaccharide obtained under the optimal extraction process in Example 1;

[0037] Fig.13 is the binding capacity of the dulse polysaccharide obtained under the optimal extraction process in Example 1 to anhydrous sodium cholate;

[0038] Fig.14 is the binding capacity of dulse polysaccharide obtained under the best extraction process in Example 1 to sodium taurocholate;

[0039] Fig.15 is the binding capacity of dulse polysaccharide obtained under the best extraction process in Example 1 to sodium glycocholate;

[0040] Fig.16 It is the binding capacity of dulse polysaccharide obtained under the optimal extraction process in Example 1 to sodium deoxycholate. DETAILED DESCRIPTION

[0041] The technical scheme in the specific embodiment of the present invention is described in detail and completely below. Obviously, the described embodiment is only a part of the specific implementation of the overall technical scheme of the present invention, rather than all implementations. Based on the overall concept of the present invention, all other embodiments obtained by ordinary technicians in this field fall within the scope of protection of the present invention.

[0042] Example 1

[0043] A method for extracting dulse algae polysaccharide comprises the following steps: mixing a low eutectic solvent with dulse algae powder at a liquid-to-solid ratio of 20-60:1 mL / g, extracting polysaccharides in the dulse algae with the assistance of ultrasound with a power of 180 W, the extraction temperature being 30-90° C., the extraction time being 20-60 min, obtaining an extract, centrifuging at a speed of 5000 r / min for 20 min, taking a supernatant, then adding anhydrous ethanol 4 times the volume of the supernatant for alcohol precipitation for 12 h, centrifuging at a speed of 5000 r / min for 20 min, collecting the precipitate, and freeze-drying for 48 h to obtain dulse algae polysaccharide;

[0044] The low eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor is one or more of urea, ethylene glycol, glycerol, malonic acid and oxalic acid, the hydrogen bond acceptor is choline chloride, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 2:1;

[0045] The water content of the deep eutectic solvent is 10-50% v / v.

[0046] The process conditions are optimized by the following method, including the following steps:

[0047] S1. Five factors affecting the extraction rate of dulse polysaccharides were screened out: the type of deep eutectic solvent, the water content of the deep eutectic solvent, the liquid-to-solid ratio of the deep eutectic solvent to dulse powder, the extraction time, and the extraction temperature. A single factor experiment was conducted with the extraction rate of dulse polysaccharides as an indicator.

[0048] S11. The single factor experiment of the type of deep eutectic solvent is as follows:

[0049] According to the above-mentioned extraction method of dulse polysaccharide, under the conditions of fixed liquid-to-solid ratio of low eutectic solvent to dulse powder at 40:1 mL / g, extraction temperature at 70°C, and extraction time at 30 min, the extraction rate of dulse polysaccharide was determined when the low eutectic solvents were choline chloride-urea, choline chloride-ethylene glycol, choline chloride-glycerol, choline chloride-malonic acid, and choline chloride-oxalic acid. The polysaccharide content was first determined according to the phenol-sulfuric acid method, and then the extraction rate of dulse polysaccharide was calculated. The results were recorded in Figure 1 .

[0050] Depend on Figure 1 It can be seen that the extraction rate of dulse polysaccharide composed of choline chloride and ethylene glycol is the highest, and is much higher than that of organic acid-based deep eutectic solvents. This may be attributed to the different physical and chemical properties between different types of deep eutectic solvents and dulse polysaccharide, such as viscosity and solubility. Therefore, different types of deep eutectic solvents have different extraction abilities for dulse polysaccharide. Therefore, the present invention selects choline chloride and ethylene glycol as the best deep eutectic solvent combination for subsequent optimization experiments.

[0051] S12. The single factor experiment of water content of deep eutectic solvent is as follows:

[0052] According to the above-mentioned extraction method of dulse polysaccharide, under the conditions of fixed low eutectic solvent as choline chloride-ethylene glycol, liquid-to-solid ratio of low eutectic solvent to dulse powder as 40:1mL / g, extraction temperature as 70°C, and extraction time as 30min, the extraction rate of dulse polysaccharide was determined when the water content of the low eutectic solvent was 10%, 20%, 30%, 40%, and 50% (v / v). The results are recorded in Figure 2 .

[0053] Depend on Figure 2 It can be seen that when the water content is 10%-20%, the extraction rate of dulse algae polysaccharide increases with the increase of water content. When the water content is 20%, the extraction rate of dulse algae polysaccharide is as high as 7.942%; when the water content exceeds 20%, that is, when the water content is 30%-50%, the extraction rate of dulse algae polysaccharide decreases with the increase of water content. At the same time, we can find that when the water content is 40%-50%, the extraction rate of dulse algae polysaccharide almost no longer decreases. This may be because when the water content increases to 40%, the intermolecular force of the low eutectic solvent has been reduced to the minimum, making it impossible to crack the polysaccharides in the dulse algae, so the decrease in the extraction rate at 50% is not very obvious. Therefore, the present invention selects the water content of the low eutectic solvent to be 20% as the best, so as to carry out subsequent optimization experiments.

[0054] S13. The single factor experiment of the liquid-to-solid ratio of the deep eutectic solvent and the dulse powder is as follows:

[0055] According to the above-mentioned extraction method of dulse polysaccharide, under the conditions of fixed low eutectic solvent as choline chloride-ethylene glycol (water content of 20%), extraction temperature of 70°C, and extraction time of 30min, the extraction rate of dulse polysaccharide was determined when the liquid-to-solid ratio of the low eutectic solvent to dulse powder was 20:1mL / g, 30:1mL / g, 40:1mL / g, 50:1mL / g, and 60:1mL / g. The results are recorded in Figure 3 .

[0056] Depend on Figure 3 It can be seen that when the liquid-to-material ratio is 20-40mL / g, as the amount of the low eutectic solvent increases, the extraction rate of the dulse polysaccharide gradually increases. When the liquid-to-material ratio is 40mL / g, the dulse polysaccharide extraction rate can reach 7.787%. This is because, in this interval, the concentration difference between the dulse algae cells and the external low eutectic solvent makes the dulse algae cells more easily broken, so the dulse polysaccharide extraction rate rises, but when the liquid-to-material ratio reaches 50-60mL / g, due to too much low eutectic solvent, the interaction force between the dulse algae cells and the low eutectic solvent is weakened, which is not conducive to extraction, so the dulse polysaccharide extraction rate decreases rapidly. Therefore, the present invention selects a liquid-to-material ratio of 40:1mL / g for the dulse algae powder to carry out subsequent optimization experiments.

[0057] S14. The single factor experiment of extraction time is as follows:

[0058] According to the above-mentioned extraction method of dulse polysaccharide, under the conditions of fixed low eutectic solvent as choline chloride-ethylene glycol (water content of 20%), liquid-to-solid ratio of low eutectic solvent to dulse powder of 40:1mL / g, extraction temperature of 70°C, the extraction rate of dulse polysaccharide was measured when the extraction time was 20min, 30min, 40min, 50min, and 60min respectively. The results are recorded in Figure 4 .

[0059] Depend on Figure 4 It can be seen that when the extraction time is 20min-40min, the extraction rate of dulse polysaccharide increases with the increase of extraction time, and reaches the maximum at 40min, which is 8.793%; when the extraction time is 50min-60min, the extraction rate decreases with the increase of time. This may be attributed to the fact that after the extraction time reaches 40min, further extension of the extraction time may cause the structure of dulse polysaccharide to be destroyed and degraded, thus resulting in a decrease in the extraction rate. Therefore, the present invention selects an extraction time of 40min as the best, so as to carry out subsequent optimization experiments.

[0060] S15. The single factor experiment of extraction temperature is as follows:

[0061] According to the above-mentioned extraction method of dulse polysaccharide, under the conditions of fixed low eutectic solvent as choline chloride-ethylene glycol (water content of 20%), liquid-to-solid ratio of low eutectic solvent to dulse powder of 40:1mL / g, and extraction time of 40min, the extraction rate of dulse polysaccharide was measured at extraction temperatures of 30℃, 45℃, 60℃, 75℃, and 90℃. The results are recorded in Figure 5 .

[0062] Depend on Figure 5 It can be seen that when the extraction temperature is lower than 75°C, the extraction rate of dulse polysaccharide increases with the increase of temperature, reaching a maximum of 8.010% at 75°C; when the temperature reaches 75°C-90°C, the polysaccharide extraction rate begins to decrease. Analysis shows that the excessively high temperature destroys and degrades the structure of dulse polysaccharide, resulting in a decrease in the extraction rate. Therefore, the present invention selects an extraction temperature of 75°C as the best for subsequent optimization experiments.

[0063] S2. According to the single factor experimental results of step S1, under the conditions of fixing the deep eutectic solvent as choline chloride-ethylene glycol and the extraction temperature as 75°C, three factors with a more significant effect on the extraction rate of dulse polysaccharides were selected: the water content of the deep eutectic solvent, the liquid-to-solid ratio of the deep eutectic solvent to dulse powder, and the extraction time. The Box-Behnken design principle was used to conduct a three-factor three-level response surface experiment. The experimental design and results are shown in Table 1.

[0064] Table 1 Response surface experimental design and results

[0065]

[0066] S3. Response surface software was used for analysis, and a quadratic polynomial regression equation model was established to determine the optimal extraction conditions of dulse polysaccharides.

[0068] Specifically, the Design Expert 11.0.1 software was used to perform multivariate regression fitting on the data in Table 1, and the quadratic polynomial regression equation model was obtained:

[0069] Y=+9.00+1.58A+0.1999B-0.0065C-0.0515AB+0.0388AC-0.1162BC-4.07A2-0.3224B2-0.0901C2, wherein A is the water content of the low eutectic solvent, B is the liquid-to-solid ratio of the low eutectic solvent to the red algae powder, C is the extraction time, and Y is the red algae polysaccharide extraction rate.

[0070] The difference analysis of the regression equation is shown in Table 2.

[0071] Table 2 Analysis of variance of regression equation

[0072]

[0073] As shown in Table 2, the influence of A and A2 in the model constructed by the present invention is extremely significant (P<0.01). The model is extremely significant (P<0.01), and the lack of fit term is not significant (P>0.05), indicating that the model is established. The correlation coefficient R 2 =0.9923, indicating that the experimental value of the model is highly consistent with the predicted value; the correction correlation coefficient R 2 =0.9825, indicating that the model can explain 98.25% of the response value variation. 2 =0.9923 and AdjustedR 2 =0.9825 are both close to 1, so the accuracy and versatility of this model are high; since the coefficient of variation CV=4.6232%<10%, the credibility and precision of this response surface experiment are high. Therefore, this model can be used to optimize the parameters of the red algae polysaccharide extraction process.

[0074] The data in Table 2 were fitted with a quadratic multiple regression using Design Expert 11.0.1 software. The response surface and contour plots of this model are shown in Figure 6 The contour map can be used to see whether the interaction between the variables is significant. A circle indicates that the interaction between the two is not significant, while an ellipse indicates that it is significant. Figure 6(C) and (D) show that the contour lines of the interaction between the water content of the deep eutectic solvent and the extraction time are elliptical, indicating that the interaction between the two has a very significant effect on the extraction rate of dulse algae polysaccharide, and with the increase of the water content of the deep eutectic solvent and the extraction time, the extracted dulse algae polysaccharide first increases and then decreases. Figure 6 (A) and (B) show that the contour lines of the interaction between the water content of the low eutectic solvent and the liquid-to-solid ratio are elliptical, indicating that the interaction between the two is extremely significant, and with the increase of the water content of the low eutectic solvent and the liquid-to-solid ratio, the extracted dulse algae polysaccharide first increases and then decreases. Figure 6 (E) and (F) show that the contour lines of the interaction between the liquid-to-solid ratio and the extraction time are almost circular, indicating that the interaction between the two is not significant, which is consistent with the insignificant C in the variance analysis. The response surface curve is relatively smooth, indicating that the liquid-to-solid ratio and the extraction time have little effect on the extraction rate of red algae polysaccharides. With the increase of the liquid-to-solid ratio and the extraction time, the extracted red algae polysaccharides first increase slightly and then slowly decrease.

[0075] Based on the response surface methodology, the optimal extraction conditions of dulse polysaccharide were analyzed as follows: the water content of the low eutectic solvent was 21.9% v / v, the liquid-to-solid ratio of the low eutectic solvent to dulse powder was 42.9:1 mL / g, and the extraction time was 38.8 min. Under these process conditions, the extraction rate of dulse polysaccharide was predicted to be 9.262%. The error of the response surface results was verified by three parallel experiments, and the final extraction rate of dulse polysaccharide was 9.138% (relative error was 1.34%), indicating that the model can accurately predict the extraction rate of dulse polysaccharide and the conditions for optimizing the process are very reliable. The extraction rate increased by 168.43% compared with the traditional hot water extraction method, which can be promoted for industrial application.

[0076] Structural characterization and performance testing

[0077] The dulse polysaccharide obtained under the optimal extraction conditions in Example 1 was purified by deproteinization using the Sevage method to obtain refined dulse polysaccharide, and structural characterization and performance testing were performed with reference to existing testing methods.

[0078] Structural characterization

[0079] (1) Purity test of dulse polysaccharide (UV-visible spectroscopy)

[0080] Depend on Figure 7 It can be seen from the analysis that the dulse polysaccharide solution has no ultraviolet absorption at 260nm and 280nm, which indicates that the protein and nucleic acid in the dulse polysaccharide prepared in the embodiment of the present invention are removed relatively cleanly, and the purity of the dulse polysaccharide is relatively high.

[0081] (2) Fourier infrared spectroscopy detection of red algae polysaccharides

[0082] from Figure 8 It can be seen that the red algae polysaccharide is at 3394.97, 2936.71, 1374.88, 1154.04, 1024.52, 771.48 and 528.57 cm -1 The absorption band is at 3200-3600cm -1 It is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars. Specific analysis shows that the following functional groups and compounds may exist in red algae polysaccharide: 3394.97cm -1 It is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars; at 2936.71cm -1 The absorption peak at 1374.88cm is attributed to CH stretching vibration, indicating that the red algae polysaccharide contains methyl or methylene. -1 The absorption peak at 1024.62cm usually corresponds to the bending vibration of the CH bond, indicating that there are some organic compounds containing carbonyl or carboxyl groups in the red algae polysaccharide, such as fatty acids, esters, ketones or aldehydes. -1 The absorption peak at 1154.04cm belongs to the stretching vibration of CO. This absorption peak is commonly found in polysaccharides, cellulose, hemicellulose, pectin, esters and other compounds containing ester bonds. -1 The absorption peak at 771.48 cm is usually related to the stretching vibration of COC or COH, indicating that polysaccharides or other compounds containing ether bonds or alcohol hydroxyl groups exist in the red algae polysaccharide. -1 The absorption peak at 528.57 cm is usually related to the out-of-plane bending vibration of the CH bond in the aromatic compound, indicating that the red algae polysaccharide contains aromatic compounds; -1 There is an absorption peak at , indicating that the red algae polysaccharide contains compounds that bind to metal ions.

[0083] (3) Determination of molecular weight distribution of dulse polysaccharide (HPLC gel permeation chromatography)

[0084] After measurement, the number average molecular weight (Mn) of the dulse algae polysaccharide of the present invention is 41.41KDa, the peak molecular weight (Mp) is 17.85KDa, the weight average molecular weight (Mw) is 109.90KDa, and the z-average molecular weight (Mz) is 254.82KDa. The peak molecular weight is much smaller than the weight average molecular weight and the z-average molecular weight, indicating that although there are a large number of medium-sized molecules in the dulse algae polysaccharide, there are still a considerable number of larger molecules and smaller molecules. The molecular weight distribution broadband index (Mw / Mn) is 2.65, indicating that the molecular weight distribution is relatively wide, and there is a wide range of molecular weights in the dulse algae polysaccharide. These conclusions show that the molecular weight distribution of dulse algae polysaccharide presents a certain polymorphism, from smaller molecules to larger molecules.

[0085] Performance Testing

[0086] (1) Antioxidant activity test (free radical scavenging method)

[0087] (1.1) DPPH free radical scavenging experiment

[0088] like Fig. 9 As shown, the scavenging effect of dulse polysaccharide on DPPH free radicals increases with the increase of its mass concentration. When its mass concentration is 20 mg / mL, the average scavenging rate of dulse polysaccharide on DPPH free radicals is 73.13%.

[0089] (1.2) ABTS free radical scavenging experiment

[0090] like Fig.10 As shown, the scavenging effect of dulse polysaccharide on ABTS free radicals increases with the increase of its mass concentration. When its mass concentration is 10 mg / mL, the average scavenging rate of dulse polysaccharide on ABTS free radicals is 42.37%.

[0091] (1.3) Hydroxyl radical scavenging experiment

[0092] like Fig.11 As shown, the scavenging effect of dulse polysaccharide on hydroxyl free radicals increases with the increase of its mass concentration. When its mass concentration is 50 mg / mL, the average scavenging rate of dulse polysaccharide on hydroxyl free radicals is 59.49%.

[0093] (1.4) Superoxide anion free radical scavenging experiment

[0094] like Fig.12 As shown, the scavenging effect of dulse algae polysaccharide on superoxide anion free radicals first increases and then decreases with the increase of its mass concentration. When its mass concentration is 30 mg / mL, the average scavenging rate of dulse algae polysaccharide on superoxide anion free radicals is the maximum of 72.09%.

[0095] (2) Lipid-lowering activity test (in vitro simulated digestion method combined with UV-spectrophotometry)

[0096] Specifically, it includes experiments on the combination of dulse polysaccharide with anhydrous sodium cholate, sodium taurocholate, sodium glycocholate, and sodium deoxycholate. The results are recorded in Figure 13-16 .

[0097] from Figure 13-16It can be seen that dulse polysaccharide has a certain binding ability to four kinds of bile salts, namely, anhydrous sodium cholate, sodium taurocholate, sodium glycocholate and sodium deoxycholate, and the binding ability of dulse polysaccharide to the above bile salts increases with the increase of its mass concentration. When its mass concentration is 50 mg / mL, the average binding rates of dulse polysaccharide to anhydrous sodium cholate, sodium taurocholate, sodium glycocholate and sodium deoxycholate are 53.30%, 82.38%, 71.37% and 74.27%, respectively.

[0098] In summary, the present invention has the following advantages:

[0099] (1) The ultrasound-assisted choline chloride-ethylene glycol deep eutectic solvent extraction method of the present invention has the advantages of being green, efficient, having high extraction rate and biological activity compared with the traditional hot water extraction method, and promotes the high-value development and utilization of red algae resources;

[0100] (2) The present invention uses single factor experiments and response surface experiments to optimize the process parameters of ultrasound-assisted deep eutectic solvent extraction of dulse algae polysaccharides, further improving the extraction rate and efficiency of dulse algae polysaccharides and reducing energy consumption, which has broad application prospects;

[0101] (3) The present invention uses ultrasound-assisted low eutectic solvent to extract red algae polysaccharides, which have strong antioxidant activity and lipid-lowering activity, and can provide a theoretical basis and technical support for the research and development of red algae polysaccharides as functional foods, medicines, etc.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting dulse polysaccharide, comprising the following steps: The dulse powder is mixed with a low eutectic solvent, and polysaccharides in the dulse are extracted with the assistance of ultrasound to obtain an extract, centrifuge, take the supernatant, then add anhydrous ethanol for alcohol precipitation, centrifuge, collect the precipitate, and freeze-dry to obtain the dulse polysaccharide; The low eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor is one or more of urea, ethylene glycol, glycerol, malonic acid and oxalic acid, the hydrogen bond acceptor is choline chloride, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 2:

1.

2. The method for extracting dulse polysaccharide according to claim 1, characterized in that: The deep eutectic solvent uses ethylene glycol as the hydrogen bond donor, and the water content of the deep eutectic solvent is 10-50% v / v.

3. The method for extracting dulse polysaccharide according to claim 2, characterized in that: The water content of the deep eutectic solvent is 20% v / v.

4. The method for extracting dulse polysaccharide according to claim 1, characterized in that: The liquid-to-solid ratio of the low eutectic solvent to the dulse powder is 20-60:1 mL / g.

5. The method for extracting dulse polysaccharide according to claim 4, characterized in that: The liquid-to-solid ratio of the low eutectic solvent to the red algae powder is 40:1 mL / g;.

6. The method for extracting dulse polysaccharide according to claim 1, characterized in that: The extraction conditions are: extraction temperature is 30-90° C., and extraction time is 20-60 min.

7. The method for extracting dulse polysaccharide according to claim 6, characterized in that: The extraction conditions are as follows: extraction temperature is 75° C., and extraction time is 40 min.

8. A method for optimizing the process conditions of the extraction method of dulse polysaccharide according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Five factors affecting the extraction rate of dulse polysaccharide were screened out: the type of deep eutectic solvent, the water content of the deep eutectic solvent, the liquid-to-solid ratio of the deep eutectic solvent to dulse powder, the extraction time, and the extraction temperature. The extraction rate of dulse polysaccharide was used as an indicator to conduct a single factor experiment. S2. According to the single factor experimental results of step S1, three factors that have a significant impact on the extraction rate of dulse polysaccharides are selected: the water content of the deep eutectic solvent, the liquid-to-solid ratio of the deep eutectic solvent to dulse powder, and the extraction time, and a three-factor three-level response surface experiment is performed using the Box-Behnken design principle; S3. Response surface software was used for analysis, and a quadratic polynomial regression equation model was established to determine the optimal extraction conditions of dulse polysaccharides.

9. The process conditions optimization method for extracting dulse polysaccharide according to claim 8, characterized in that: The quadratic polynomial regression equation model: Y=+9.00+1.58A+0.1999B-0.0065C-0.0515AB+0.0388AC-0.1162BC-4.07A2-0.3224B2-0.0901C2, wherein A is the water content of the low eutectic solvent, B is the liquid-to-solid ratio of the low eutectic solvent to the red algae powder, C is the extraction time, and Y is the red algae polysaccharide extraction rate.

10. The process condition optimization method for extracting dulse polysaccharide according to claim 8, characterized in that: The optimal extraction conditions of the dulse polysaccharide are as follows: the water content of the low eutectic solvent is 21.9% v / v, the liquid-to-solid ratio of the low eutectic solvent to the dulse powder is 42.9:1 mL / g, and the extraction time is 38.8 min.

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