Abstract:Taking the purity and recovery of Phycocyanin from Spirulina platensis as the indexes, we investigated the effects of different wall-breaking factors in the swelling and freeze-thawing methods, and the effects of the number of mesh of activated carbon, the amount of activated carbon added and the adsorption time in the activated carbon adsorption method on the crude extraction of Phycocyanin, and optimized the parameters of the extraction process by integrating with the Response Surface Analysis (RSA) method. The optimized extraction process parameters were combined with response surface analysis to obtain the optimal conditions for wall-breaking and activated carbon adsorption.
The results showed that the optimal extraction conditions of the swelling method were 1:50 (g/mL), 24 h swelling time, and 2 times swelling, and the purity of algal blue protein was 0.47 with the yield of 2.17%; the optimal extraction conditions of the freeze-thaw method were 1:50 (g/mL), 8 h freezing time, and 2 times freeze-thawing, and the purity of algal blue protein was 0.34 with the yield of 2.19%. The optimum conditions of activated carbon adsorption were 300 mesh activated carbon, 0.7 g/20 mL of activated carbon, 10 min of adsorption time, and the purity of algal blue protein was 0.80, with a recovery rate of 73.23%. The purification by hydrophobic chromatography yielded food-grade alginate (purity P≥0.7) and pharmaceutical-grade alginate (purity P≥3.0) with a total recovery of 61.15%. Therefore, the three-step purification process of alginate by swelling-activated carbon adsorption-hydrophobic chromatography is reasonable and feasible, realizes the isolation and purification of alginate of pharmaceutical grade, reduces the cost of industrial production, and has practical value.
Phycocyanin is a kind of natural blue pigment existing in blue algae, containing open-chain tetrapyrrole structure, which has the nature of absorbing and transmitting functions, and belongs to intracellular protein, with high content in Spiroplasma[1] . Due to its high nutritional value and pharmacological effects such as anti-inflammatory, anti-fatigue, anti-tumor and immunity enhancement, algal blue protein has been widely used in food, cosmetics, health products and pharmaceuticals, etc., and has a broad application prospect[2-6] .
At present, the extraction and purification process of algal blue protein is more mature, but it is still only suitable for laboratory operation, and industrial large-scale production is still its major difficulty, and the price is expensive. The extraction and purification of algal blue protein is mainly divided into wall-breaking, crude extraction and purification of three steps, in which the cell breaking methods are lysis method[7] , ultrasonic cell-breaking method, freezing and thawing method[8] , mechanical wall-breaking method, chemical reagents method[9] , lysophospholipids method[10] , etc., the experiments are commonly used in one or two methods used in conjunction with the algal blue protein wall-breaking[11,12] .
The extraction of algal blue protein generally adopts the salt analysis method, isoelectric point precipitation method, ultrafiltration method[13-17] and adsorption method[18-21], among which the adsorption method is to use the adsorbent to adsorb the impurities or proteins in order to extract the protein, and the commonly used adsorbents are activated carbon and chitosan. Compared with the salting-out method, the reagents used in this method are inexpensive and do not cause environmental pollution, and a small amount of adsorbent can adsorb impurities without the need for a large amount of ammonium sulfate; the disadvantage of this method is the need to accurately control the dosage of the adsorbent and the adsorption time, otherwise it is easy to adsorb away from the target proteins and reduce the extraction rate. Generally speaking, this method has great potential for development, and it is expected to replace the salt analysis method to extract algal blue protein, but at present there are still few studies on this method at home and abroad.
The extraction cost of high purity algal blue protein is high and expensive, because the swelling method and freeze-thawing method have the advantages of low cost, simple operation, mild conditions and no introduction of other chemical reagents, therefore, this study is mainly on the swelling method of the material-liquid ratio, swelling time, the number of times of the swelling method, freeze-thawing method of the different solvents, material-liquid ratio, the number of times of the freeze-thawing method, and the application of the response surface modeling design experiments on the two methods of Spirulina broken wall optimization to obtain the most optimal conditions of broken wall. Response surface modeling (RSM) was applied to optimize the two wall-breaking methods of Spirulina to obtain the best wall-breaking conditions. At the same time, in order to overcome the shortcomings of activated carbon adsorption, which can easily adsorb the egg white of the mesh and reduce the recovery rate, the response surface analysis was used to optimize the method, and the effects of activated carbon mesh, the addition of activated carbon and the adsorption time were investigated to obtain the optimal activated carbon adsorption conditions, and then the food-grade and pharmaceutical-grade algal cyanine proteins were obtained by the one-step hydrophobic chromatography purification.
1 Materials and Methods
1.1 Materials
1. 1. 1 Samples and reagents
Spirulina platensis was supplied by Beihai Shengbaoda Biotechnology Co. Powdered activated carbon (100 mesh, 200 mesh, 300 mesh, 400 mesh, 500 mesh) was purchased from Beijing Meijiaoyuan Environmental Protection Engineering Co., Ltd; ButylFourose4FF hydrophobic chromatographic filler was purchased from Beijing Huideyi Science and Technology Co.
1. 1. 2 Instruments
LQ-C3001 electronic balance, purchased from Shenzhen Feiya Weighing Instrument Company Limited; GL224-1SCN electronic balance, purchased from Sedolis Scientific Instrument (Beijing) Co. UV-2600 UV-Visible Spectrophotometer, purchased from Shimadzu Instruments (Suzhou) Co., Ltd; BA2100igital digital microscope, purchased from McAuldie Industrial Group Co.
1.2 Methodology
1. 2. 1 Methods of analysis
Alginate has a maximum absorption peak at 620 nm, egg white has a maximum absorption peak at 280 nm and alginate has a maximum absorption peak at 650 nm. Based on this property, the absorbance (A) of the protein solution at 280 nm, 620 nm and 650 nm can be measured using a UV-visible spectrophotometer to calculate the purity, concentration, yield and recovery of the alginate.
(1) Algal blue protein purity .
The purity of alginate is denoted by P. Different purities of alginate have different applications, P≥0.7 for food grade, P≥3.0 for pharmaceutical grade, P≥4.0 for analytical grade, which was calculated according to the formula proposed by Yan et al[22] as follows: P≥0.7 for food grade, P≥3.0 for pharmaceutical grade, P≥4.0 for analytical grade.
P=A620/A280.
(1) ② Algal blue protein concentration .
The concentration of phycocyanin was expressed as C and calculated according to the formula proposed by Bennett et al[23] as follows.
C(mg/mL) = (A620 - 0.474 x A650)/5.34.
(2) (iii) Algal blue protein yield .
The yield of phycocyanin was expressed as Y and calculated as follows.
Y(%) = (C×V0)/(m0×1000) ×100%, (3) where C is the alginate concentration (mg/mL), V0 is the volume of crude extract (mL), and m0 is the mass of Spirulina dry powder (g).
1. 2. 2 Dissolution
Swelling method is to use pure water or low-salt solution as solvent, Spirulina absorbs water and rises up the cells, so as to flow out the effective components of the method. Specific operation is as follows: precision weighing of Spirulina powder, respectively, to investigate the material-liquid ratio (1: 10 g/mL, 1: 20 g/mL, 1: 50 g/mL, 1: 100 g/mL, 1: 200 g/mL), swelling time (6h, 12h, 18h, 24h, 36h), the number of times of swelling (1, 2, 3) on the purity of phycocyanin and the yield of the algal blue protein. The algal samples were ultrasonicated for 5 min at 4 ℃ for 12 h, and centrifuged at 4000 r/min for 15 min to obtain the supernatant, and the absorbance at 280 nm, 620 nm and 650 nm was determined respectively.
The Box Behnken design of Design-Expert software was used to optimize the wall-breaking parameters of the swelling method, and the Response Surface Analysis (RSM) was designed with the material-liquid ratio, swelling time and the number of times of swelling as the independent variables and the alginate yield as the response value. Three levels were set for each factor (Table 1), and the purity and recovery rate of alginate were used as the response variables, with a total of 17 analytical points, including five replicated centroids.
Table 1 Response surface analysis test factors and levels
Table1 Testfactorsandlevelsusedforresponsesurfaceanaly-
sis
Levels |
| Factors |
|
A:Material-liquid ratio (g/mL) A:Solid- liquid ratio (g/mL) | B: Dissolution time (h) B:Swelling time(h) | C: Number of dissolutions (times) C:Swelling times (times) | |
-1 | 1:20 | 4 | 1 |
0 | 1:50 | 8 | 2 |
1 | 1:80 | 12 | 3 |
1. 2. 3 Freeze-thaw method
Freeze-thawing method is to put the spirulina liquid under low temperature (-20℃), so that the water in the seaweed cells is rapidly frozen to form ice crystals, and then thawed quickly at room temperature, thus breaking the cells. The one-way test was conducted as follows.
4 portions of 1g Spirulina powder were weighed precisely and added with 20mL pure water, 0.01 mol/L phosphate buffer (PBS), 0.05 mol/L PBS buffer and 0.1 mol/L PBS buffer, and then ultrasonicated for 5 min, and then left to stand at -20℃ for 12 h, respectively.
② Precisely weighed 3 portions of 1 g Spirulina powder, the solvent is the best solution obtained from ① test, ultrasonic 5 min, -20 ℃ static 4 h, 8 h, 12 h, respectively; ③ The solvent is the best solution obtained from ① test, ultrasonic 5 min, -20 ℃ static 4 h, 8 h, 12 h, respectively.
(iii) Precisely weigh 5 portions of 1 g of Spirulina powder, add the optimal solvent, the solvent is the optimal solution obtained from ① test, so that the ratio of the material to liquid is 1:10 (g/mL), 1:20 (g/mL), 1:50 (g/mL), 1:100 (g/mL), 1:200 (g/mL), ultrasound for 5 min, the freezing time is the optimal conditions obtained from ②; (4) The solution is the optimal solvent obtained from the ① test.
④Precisely weigh three 1 g Spirulina powder, add the best material-liquid ratio solvent, freeze-thawing times were 1, 2 and 3 times, ultrasonic 5 min, -20 ℃ static. The above tests were thawed at room temperature, centrifuged at 4000 r/min for 15 min, 1 mL of supernatant was sucked up, diluted 10 times, and the absorbance at 280 nm, 620 nm and 650 nm were determined respectively, to analyze the effects of different solvents, freezing time, material-liquid ratio and number of freezing and thawing times on the purity and yield of algal blue protein.
The Box Behnken design of Design Expert software was used to optimize the parameters of the freeze-thaw method, and the RSM experiment was designed with the material-liquid ratio, freezing time, and the number of freezing and thawing times as the independent variables, and the algal blue protein yield as the response value. Three levels were set for each factor (Table 2), and the purity and recovery of alginate were used as the response variables to design the experiment, with a total of 17 analytical points, including five replicates of the center point.
1. 2. 4 Extraction of algal blue protein by activated carbon adsorption method
Activated carbon adsorption method is the use of activated carbon adsorption of impurities or proteins from the method of protein extraction. Single-factor test operation is as follows: ① take 5 portions of 20mL of seaweed liquid obtained from the swelling method, add 100 mesh, 200 mesh, 300 mesh, 400 mesh, 500 mesh activated carbon, static adsorption for 5 min.
② take five 20 mL of seaweed liquid obtained by swelling method, respectively, add 0.1 g, 0.3 g, 0.5 g, 0.7 g, 0.9 g of activated carbon, activated carbon mesh for the optimal mesh obtained in the previous test, static adsorption for 5 min.
③ Take 5 portions of 20 mL of seaweed liquid obtained by swelling method, add the amount of activated carbon to the optimal conditions obtained in the previous test, and leave it to be adsorbed for 5 min, 10 min, 15 min, 20 min and 25 min respectively.
The above test seaweed liquid was centrifuged at 8000 r/min for 5 min, the sediment was discarded, the supernatant was aspirated 1 mL, diluted by appropriate times, and the absorbance at 280 nm, 620 nm and 650 nm was measured respectively to analyze the effects of different activated carbon mesh, amount of activated carbon added, and adsorption time on the purity and yield of algal blue protein.
Table 2 Response surface analysis test factors and levels
Table2 Testfactorsandlevelsusedforresponsesurfaceanaly-
sis
Levels |
| Factors |
|
A:Material-liquid ratio (g/mL) A: Solid- liquid ratio (g/mL) | B:Freezing time (h) B:Freezing time(h) | C: Number of freeze-thaw cycles (times) C:Freeze- thaw times (times) | |
-1 | 1:20 | 12 | 1 |
0 | 1:50 | 18 | 2 |
1 | 1:80 | 24 | 3 |
On the basis of the one-factor test, the Box-Behnken experimental design principle of Design-Expert 10.0 software was applied to carry out the response surface test, and three factors, namely, the number of mesh of activated carbon, the amount of activated carbon added and the adsorption time were selected as the factors for the test, and three levels were set for the factors (Table 3), and the purity and recovery of phycocyanin were used as the response variables to design the test. A total of 17 analytical points with 5 replicated centers were used in the experiment.
Table 3 Response surface analysis test factors and levels
Levels |
| Factors |
|
A:Mesh of activated carbon (item) A:Number ofactive carbon mesh (mesh) | B: Activated carbon plus Intake (g/20mL) B:Amount ofactive carbon added (g/20mL) | C:Adsorption time (min) C:Adsorption time (min) | |
-1 | 200 | 0. 3 | 5 |
0 | 300 | 0. 5 | 10 |
1 | 400 | 0. 7 | 15 |
1. 2. 5 Hydrophobic chromatography purification of algal blue proteins
The algal blue protein extracted by activated carbon adsorption was concentrated to a certain concentration by ultrafiltration, and then pre-equilibrated hydrophobic chromatographic packing with 0.05 mol/L PBS phosphate buffer (containing 0.6 mol/L ammonium sulfate) was used to carry out the gradient elution, and the gradient of elution solution was 0.05 mol/L PBS (containing 0.6 mol/L ammonium sulfate), 0.05 mol/L PBS (containing 0.3 mol/L ammonium sulfate), 0.05 mol/L PBS, pure water, and the flow rate was controlled at 2 mL/min, 3 min each tube, and the 280 nm, 620 nm, and 620 nm of each tube were measured. The gradient of the eluent was 0.05 mol/L PBS (containing 0.6 mol/L ammonium sulfate), 0.05 mol/L PBS (containing 0.3 mol/L ammonium sulfate), 0.05 mol/L PBS, pure water, and the flow rate was controlled to be 2 mL/min for 3 min for each tube, and the absorbance at 280 nm, 620 nm, and 650 nm of each tube was determined to calculate the purity, and the purity and recovery were calculated by combining the protein solutions with purity 1-2, purity 2-3 and purity more than 3, respectively.
2 Results and analysis
2.1 Analysis of the results of the dissolution method
2. 1. 1 Effect of material-liquid ratio, swelling time and number of times of swelling on the purity and yield of algal blue protein
With the increase of the liquid-liquid ratio, the yield of alginate increased significantly, while the purity decreased significantly, and in the liquid-liquid ratio of 1:200 (g/mL), the yield of alginate was the highest, and the purity was the lowest [Fig. 1(a)]. With the increase of the ratio, the osmotic pressure difference between the inside and outside of Spirulina cells was bigger and bigger, and the alginate released from the broken cell wall increased, meanwhile, many other proteins might be dissolved together, so the alginate yield increased gradually and the purity decreased gradually.
Too high a material-liquid ratio will cause an increase in the actual production cost, so 1:50 (g/mL) was chosen as the optimal value for the material-liquid ratio of algal blue protein. With the prolongation of swelling time, the yield and purity of algal blue protein increased significantly [Figure 1(b)]. It means that with the extension of the swelling time, more algal blue protein is released, but the longer swelling time will cause the actual production cost increase and lower production efficiency, so we choose 24 h as the optimal value of the swelling time. With the increase of swelling times of Spiroplasma, the yield of algal blue protein increased, and the purity decreased [Figure 1(c)]. According to the needs of actual production, the optimal value was chosen to be 2 times of bloating.
2. 1. 2 Dissolution test response surface analysis optimization
The Design-Expert software was used to fit the regression of the experimental results in Table 4, and a multiple quadratic regression model was obtained for the three factors of phycocyanin yield: yield Y = 2.28 + 0.12A + 0.22B + 0.50C, and from the analysis of variance (ANOVA) of the two-response surface regression model, the correlation coefficient of the model was R2 = 0.8357, P=0.000 1<0.05, and the misfit term P=0.0970>0.05, indicating that the regression model had high fit and reliability, and could predict the optimal process more realistically. 0.05, and the misfit term P=0.0970>0.05, indicating that the fit and reliability of the regression model are high, and it can predict the optimal process in a more realistic way.
Table 4 Analysis and results of Box-Behnken test for dissolution method
test number Test number | A:Material-liquid ratio (g/mL) A: Solid- liquid ratio (g/mL) | B:Dissolution time (h) B:Swelling time(h) | C: Number of dissolutions (times) C:Swelling times (times) |
Purity | Yield (%) Yield (%) |
1 | -1 | -1 | 0 | 0. 45 | 1. 85 |
2 | 1 | -1 | 0 | 0. 38 | 2. 39 |
3 | -1 | 1 | 0 | 0. 55 | 2. 64 |
4 | 1 | 1 | 0 | 0. 42 | 2. 73 |
5 | -1 | 0 | -1 | 0. 52 | 1. 43 |
6 | 1 | 0 | -1 | 0. 33 | 1. 96 |
7 | -1 | 0 | 1 | 0. 38 | 2. 68 |
8 | 1 | 0 | 1 | 0. 39 | 2. 48 |
9 | 0 | -1 | -1 | 0. 46 | 1. 52 |
10 | 0 | 1 | -1 | 0. 47 | 1. 80 |
11 | 0 | -1 | 1 | 0. 41 | 2. 59 |
12 | 0 | 1 | 1 | 0. 44 | 2. 93 |
13 | 0 | 0 | 0 | 0. 46 | 2. 35 |
14 | 0 | 0 | 0 | 0. 42 | 2. 40 |
15 | 0 | 0 | 0 | 0. 43 | 2. 42 |
16 | 0 | 0 | 0 | 0. 41 | 2. 16 |
17 | 0 | 0 | 0 | 0. 41 | 2. 42 |
The significance test results of the coefficients of regression equation (Table 5) show that the P values of factors A and C are 0.0006, 0.000 1 (P<0.01), and the P value of BC is 0.0013 (P<0.01), respectively. The smaller the P-value and the larger the F-value of each factor, the more significant the effect of the factor on the response variable, so according to the size of the F-value in Table 5, it can be seen that the factors affecting the effect of wall-breaking by the swelling method are the number of times of swelling > the ratio of the material to liquid > the time of swelling.
Table 5 ANOVA of the regression model for the extraction rate of the dissolution method
Table5 Varianceanalysisofregression modelforextraction rateby swelling method
Source | square sum (e.g. equation of squares) Quadratic sum | Freedom Degree offreedom | mean square Mean square | F-value F value | P-value P value | significance Significance |
Model | 3. 720 | 6 | 0. 620 | 15. 51 | 0. 0002 | * * |
A | 0. 970 | 1 | 0. 970 | 24. 23 | 0. 0006 | * * |
B | 0. 097 | 1 | 0. 097 | 2. 42 | 0. 1508 |
|
C | 1. 510 | 1 | 1. 510 | 37. 64 | 0. 000 1 | * * |
AB | 0. 058 | 1 | 0. 058 | 1. 44 | 0. 2577 |
|
AC | 0. 130 | 1 | 0. 130 | 3. 15 | 0. 1062 |
|
BC | 0. 770 | 1 | 0. 770 | 19. 36 | 0. 0013 | * * |
Residualerror | 0. 400 | 10 | 0. 040 |
|
|
|
Misfitterm | 0. 290 | 6 | 0. 048 | 1. 78 | 0. 3000 |
|
Neterror | 0. 110 | 4 | 0. 027 |
|
|
|
Totaldispersion | 4. 120 | 16 |
|
|
|
|
2. 1. 3 Interaction between the factors of the dissolution method
According to the results of Box-Behnken design experiment, we made an impact curve, examined the shape of the fitted surface, and analyzed the effect of the interaction between the ratio of the material and liquid, the swelling time and the number of times of swelling on the algal blue protein yield (Fig. 2). As can be seen from Fig. 2, there is no interaction between these three factors, so according to the best combination of each factor, the optimal solution for the wall-breaking method can be obtained as the ratio of material to liquid 1:50 (g/mL), the time of solubilization is 24h, and the number of solubilization is 2 times. The predicted purity of the model was 0.47 and the predicted extraction rate was 2.16%.
2. 1. 4 Validation tests
Response surface analysis was used to optimize the optimal conditions of wall-breaking: material-liquid ratio of 1:50 (g/mL), swelling time of 24 h, the number of times of swelling 2 times, to carry out the validation test, repeated 3 times, take the average value, the measured purity of phycocyanin was 0.47, the yield of 2.17%, and the model predicted purity of 0.47 the same as the model predicts a yield rate of 2.16% close to the model predicts that the program is stable and reliable, the practical value of this program. It shows that the program is stable and reliable, and has practical value.
2. 1. 5 Before and after cell fragmentation
The microstructure of Spirulina cells before and after breakage is shown in Figure 3. After the breakage, the cylindrical spiral structure of Spirulina was broken, and a large number of cellular fragments were produced, which showed that the wall-breaking effect of the lysis method was good.
Table 6 Freeze-thaw method Box-Behnken test analysis and results
Table6 Analysisand resultsofBox-Behnken testbyfreeze-thaw method
test number Testnumber | A:Material-liquid ratio(g/mL) A:Solid-liquid ratio (g/mL) | B:Freezing time(h) B:Freezing time(h) | C: Number of freezes (times) C:Freezing times(times) | Purity | Yield (%) |
1 | -1 | -1 | 0 | 0. 42 | 1. 56 |
2 | 1 | -1 | 0 | 0. 4 | 2. 88 |
3 | -1 | 1 | 0 | 0. 47 | 1. 71 |
4 | 1 | 1 | 0 | 0. 38 | 2. 55 |
5 | -1 | 0 | -1 | 0. 46 | 1. 77 |
6 | 1 | 0 | -1 | 0. 51 | 2. 28 |
7 | -1 | 0 | 1 | 0. 46 | 2. 01 |
8 | 1 | 0 | 1 | 0. 41 | 3. 23 |
9 | 0 | -1 | -1 | 0. 45 | 2. 15 |
10 | 0 | 1 | -1 | 0. 4 | 1. 80 |
11 | 0 | -1 | 1 | 0. 38 | 1. 92 |
12 | 0 | 1 | 1 | 0. 37 | 3. 33 |
13 | 0 | 0 | 0 | 0. 37 | 2. 13 |
14 | 0 | 0 | 0 | 0. 37 | 2. 13 |
15 | 0 | 0 | 0 | 0. 39 | 2. 42 |
16 | 0 | 0 | 0 | 0. 38 | 1. 98 |
17 | 0 | 0 | 0 | 0. 38 | 2. 26 |
2. 2. 3 Interaction between different factors of the freeze-thaw method
According to the results of Box-Behnken experiment, we made the response curve diagram, examined the shape of the fitted surface, and analyzed the effects of the interactions among freezing time, material-liquid ratio, and the number of freezing and thawing times on the yield of alginate blue protein (Fig. 5). The optimal extraction conditions optimized by response surface analysis were 1∶50 (g/mL), 8 h freezing time and 3 times freezing and thawing. The predicted purity of the model was 0.38 and the predicted yield was 2.24%.
2. 2. 4 Validation tests
According to the optimal extraction conditions optimized by response surface analysis: material-liquid ratio of 1:50 (g/mL), freezing time of 8h, freeze-thaw times of 3 times, validation test, repeat 3 times, take the average value, the measured purity of algal blue protein was 0.34, the yield was 2.19%, which was close to the model prediction of purity of 0.38, the prediction of the yield of 2.24%, which indicated that the program is stable and reliable, and has practical value.
2. 3 Activated carbon adsorption method for extraction of algal blue protein
2. 3. 1 Effects of activated carbon mesh, activated carbon addition and adsorption time on the purity and yield of algal blue protein
The effects of activated carbon adsorption on the purity and yield of algal blue protein are shown in Fig. 6. When the mesh of activated carbon was 300 mesh, the purity of algal blue protein was the highest and the yield was the lowest; when the mesh of activated carbon was 400 mesh and 500 mesh, the purity tended to decrease and the yield increased [Fig. 6(a)]. Therefore, 300 mesh of activated carbon was chosen as the optimum adsorption condition. With the increase in the amount of activated carbon, the purity of algal blue protein increased significantly and then decreased, while the yield showed a significant decrease [Figure 6(b)].
When the amount of activated carbon was 0.7 g/20 mL, the purity was the highest, so it could be the optimal adsorption condition. When the adsorption time was 10 min, the purity of algal blue protein was at the maximum value; as the adsorption time was prolonged, the yield showed a decreasing trend, and the overall shape was "W"; when the adsorption time was 10-25 min, the difference of the yield was not significant [Fig. 6(c)], and therefore, 10 min was chosen as the optimal adsorption time of activated carbon.
2. 3. 2 Response surface analysis optimization of activated carbon adsorption tests
According to the results of the one-way experiments, three factors, namely, the number of activated carbon (A), the amount of activated carbon added (B) and the adsorption time (C), were selected to carry out the Box-Behnken experimental design, with a total of 17 analytical points and five replicated centroids, and the experimental design scheme and the results are shown in Table 8.
The multivariate quadratic regression simulation of the experimental data was carried out by Design-Expert10.0 software, and the regression model was alginate protein purity P=0.66-0.042A+3.750× 10-3B+6.250× 10-3C+ 0.045AB+0.030AC+0.017BC+0.063A2+0.010B2 - 4.750× 10-3C2 (R2 = 0.910 2, recovery rate RP = 71.91+ 2.61A - 4.01B - 0.90C + 1) 4.750× 10-3C2 (R2 = 0.910 2) ,alginate recovery RP = 71.91+ 2.61A - 4.01B - 0.90C + 1.78AB + 2.41AC - 0.43BC - 5.20A2 - 2.23B2 + 0.93B2 (R2 =0.9804).
The regression equations and partial regression coefficients of ANOVA for the purity and recovery of alginate are shown in Tables 9 and 10, respectively. From Table 9, we can see that the P<0.05 of the purity model indicates that the model is reliable and reaches a significant level, and the R2 of the coefficient of determination is 0.910 2, which means that the model can explain 91.02% of the variation of the response value, and the fit is more accurate; and the P>0.05 of the unfit term of the model is not significant, which means that the whole model is reasonable within the regression region, and can be used to analyze the results of alginate purity. 0.05, not significant, indicating that the whole model is reasonable within the regression region and can be used to analyze the results of alginate purity. According to the F value, it can be concluded that the factors affecting the purity of algal blue protein are the number of mesh of activated carbon > adsorption time > amount of activated carbon added.
As can be seen from Table 10, the recovery rate model with P<0.05 indicates that the model is reliable and has reached the significant level, and the coefficient of determination R2 =0.980 4 indicates that the model can explain 98.04% of the variation of response value and the fit is very accurate; the model with the misfit term P>0.05 is not significant, which indicates that the whole model is reasonable in the regression region, and it can be used to analyze the recovery rate results of the phycocyanin. It can be used to analyze the results of phycocyanin recovery. According to the F-value, it can be concluded that the factors affecting the recovery rate of algal blue protein are as follows: the amount of activated carbon added > the number of mesh of activated carbon > the adsorption time.
2. 3. 4 Interaction of different factors in activated carbon adsorption
The response surfaces of the interaction between the amount of activated carbon and adsorption time are shown in Figs. 8 and 9. The optimal conditions for the crude extraction of algal blue protein by activated carbon adsorption were 300 mesh activated carbon, 0.5 g/20 mL of activated carbon, and 10 min of adsorption time, and the predicted purity was 0.80 and the recovery was 70.56%.
2. 3. 5 Validation tests
The optimal extraction conditions obtained by response surface optimization were as follows: 300 mesh activated carbon, 0.5 g/20 mL of activated carbon, 10 min of adsorption time, experimental test, repeated for three times, and the average value was taken, and the purity of aquamarine protein was 0.80 and the recovery was 73.23%. It shows that the program is stable and reliable, and has practical value.
2.4 Hydrophobic chromatographic elution results and their spectrogram analysis
The algal blue protein was eluted sequentially, firstly, 0.05 mol/L PBS buffer (containing 0.6 mol/L PBS ammonium sulfate) was used to elute, and nearly 200 mL of yellow-green algal liquid was obtained; secondly, 0.05 mol/L PBS buffer (containing 0.3 mol/L PBS ammonium sulfate) was used to elute, and the bright blue algal liquid was obtained; again, 0.05 mol/L PBS buffer was used to elute, and the bright blue algal liquid was obtained; lastly, pure water was used to elute, and the blue-green algal liquid was obtained. PBS buffer to obtain bright blue algal liquid; finally, pure water was used to elute to obtain blue-green algal liquid. The algal solutions obtained by sequential elution were subjected to ultraviolet determination, and the algal solutions with purity 1-2, purity 2-3 and purity greater than 3 were combined respectively, and the full-wavelength scanning spectra of each algal solution are shown in Fig. 10. The substances eluted by gradient elution with 0.05 mol/L PBS buffer (containing 0.3 mol/L PBS ammonium sulfate) and 0.05 mol/L PBS buffer were mainly phycocyanin.
The results of alginate purification are shown in Table 11, after three steps of alginate purification by swelling-activated carbon adsorption-hydrophobic chromatography, the recovery rate of alginate with purity 1-2 was 14.63%, the recovery rate of alginate with purity 2-3 was 37.49%, the recovery rate of alginate with purity >3 was 9.03%, and the total recovery rate was 61.15%. The alginate with purity 2-3 can be used for subsequent purification to obtain higher purity alginate.
3 Discussion
Alginin is a kind of natural blue pigment in Spirulina, which has high nutritional and medicinal values[24] , and the total protein content is as high as 55%-65%[12] , but its source is single, the purification difficulty is large, and the production cost is high[25] . In order to further improve the wall-breaking efficiency of Spiroplasma, reduce the cost of extraction, improve the recovery and purity of the product, and isolate the pharmaceutical-grade algal blue protein, the present study was carried out to optimize the wall-breaking conditions and the extraction method of algal blue protein by the method of Response Surface Analysis (RSA).
The equipment required for the extraction of algal blue protein by swelling and freeze-thawing methods is simple and easy to operate[26,27] . In the present study, the response surface modeling experiment was carried out to test the different wall-breaking factors in the swelling and freeze-thawing methods, and the results showed that the swelling method was better than the freeze-thawing method, and the freeze-thawing method had a long time for large-scale production and was difficult to be realized. High concentration of ammonium sulfate solution (saturation greater than 40%) can precipitate phycocyanin, the disadvantage is that the introduction of a large amount of ammonium sulfate, easy to make the soil acidification and agglomeration, for the subsequent treatment of the trouble[12,26] , and activated carbon compared with ammonium sulfate has the advantages of inexpensive and easy to obtain, safe and non-polluting, large specific surface area, strong adsorption capacity and adsorption time is short and so on[7,28] .
In this study, by using activated carbon instead of ammonium sulfate used in the traditional salting out method, the heteroprotein and small molecule impurities in the crude extract of algal blue protein can be effectively removed, and it can be purified on the column without dialysis and other special treatments, which can improve the purity of algal blue protein and make the extraction process simpler and more feasible, and the nature of the extract will not be affected during the process of use, which can provide a reference for the development of the large-scale experiments in the later stage as well as the construction of the production line. However, the activated carbon adsorption method still has some problems. However, the activated carbon adsorption method is still difficult to control the adsorption conditions, and the selective mechanism of the adsorbed impurities is still unclear, which needs to be further improved.
Column chromatography method has the defects of expensive imported packing material, inconvenient cleaning, low sample volume, etc. In this study, the hydrophobic chromatographic packing produced in Beijing was used for the purification of phycocyanin, which not only reduces the cost but also obtains the pharmaceutical-grade phycocyanin, but fails to obtain the reagent-grade phycocyanin, and the subsequent test will consider the phycocyanin solution for the second time to pass through the column in order to improve the purity. At present, there is still room for improvement in the extraction and purification process of algal blue protein proposed in this study, and we will further investigate various methods comprehensively to screen out the purification process more suitable for the industrial production of algal blue protein and provide theoretical reference for the optimization of extraction process of algal blue protein.
4 Conclusion
The results of this study showed that after the optimization of the three-step purification process of alginate by swelling method, activated carbon adsorption method and hydrophobic chromatography purification, pharmaceutical-grade (P≥3.0) alginate could be isolated from Spirulina obtusususiformis, and the reagents and consumables used were inexpensive and easy to obtain, safe and non-polluting, with low extraction cost, which provided the basis for the deep processing and application in the future.
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