Process optimization of ultrasound-assisted extraction of astaxanthin

 Astaxanthin is a kind of carotenoid with strong antioxidant activity, which has been widely used in the fields of medicine, functional food, and cosmetics. In recent years, the global astaxanthin market has been growing rapidly and is expected to reach 3.4 billion U.S. dollars by 2030. Red algae is a kind of unicellular green algae, can synthesize a large amount of astaxanthin under stress culture conditions, the content can reach 5% of the dry weight of the cell, which is one of the best sources of natural astaxanthin. Because of the dense cell wall of Rhodococcus aureus, it is necessary to break the wall of Rhodococcus aureus when extracting astaxanthin. Among them, physical methods such as high-pressure homogenization and ultrasound-assisted wall-breaking are favored because of the advantages of less damage to the active ingredients and low cost[4] .

 

Currently, organic solvents such as dichloromethane, acetone, and ethyl acetate are generally used to extract astaxanthin. However, these organic solvents have certain toxicity and safety risks. The use of non-toxic green solvents to replace toxic solvents, reduce the pollution of the extraction process and improve the safety of the product has become a key issue in astaxanthin extraction. Zou et al[5] used ethyl acetate/ethanol mixture as solvent to extract astaxanthin from Rhodococcus pyrenoidus, and the highest yield of astaxanthin was achieved when the ethanol content was 50%. In recent years, new solvents such as supercritical CO2[5] , which has no solvent residue, and ionic liquids, which have low melting points and high solvency capacity, have also been used for astaxanthin extraction, and some progress has been achieved. However, the above extraction methods are based on algal powder as raw material, the drying process of algal powder needs to consume a lot of energy, which greatly increases the cost of astaxanthin.

In this study, the green and non-toxic ethanol was used as the extraction solvent, and ultrasonic-assisted method was used to extract astaxanthin from fresh red algae. The effects of different extraction times, material-liquid ratios and ultrasonic power on the extraction rate of astaxanthin were investigated, and the optimal process parameters were optimized by response surface methodology, and the kinetic process under the optimal extraction process was discussed.

1 Experiment

1.1 Materials and instruments

Fresh algae of Rhodococcus pyrenoidus, provided by the Key Laboratory of Preparation and Functional Development of Algae Active Substances in Fujian Province; astaxanthin standard (HPLC≥98%), Shanghai Yuanye Biotechnology Co.

High Performance Liquid Chromatograph (HPLC) 2695, 2489, Waters Corporation; H1650-W High Speed Centrifuge, Hunan Xiangyi Experimental Instrument Development Co.

 

1. 2 Experimental methodology

1. 2. 1 Extraction method

Fresh red algae were used as the raw material for the experiment. 1.00 g of fresh algae was weighed, and anhydrous ethanol was added in different ratios, mixed well, and placed in the ultrasonic equipment for assisted extraction. The extraction time, ultrasonic power and material-liquid ratio were varied to investigate the effects of the above factors on the extraction rate of astaxanthin.

 

1. 2. 2 One-way experiments

(1) Extraction time

The material-liquid ratio was fixed at 1∶ 10, the ultrasonic power was 65.4 W, and the extraction time was changed (10 min, 20 min, 30 min, 40 min) to analyze the extraction rate of astaxanthin.

(2) Ultrasonic power

The material-liquid ratio was fixed at 1∶ 10, the optimal extraction time was selected, and the ultrasonic power was varied (65.4 W, 168 W, 276 W) to analyze the extraction rate of astaxanthin.

(3) Material-liquid ratio

The optimal time and ultrasonic power of the above optimization were chosen, and the feed/liquid ratio was changed (2.5%, 5%, 10%, 20%) to analyze the astaxanthin extraction rate, and each experiment was repeated three times.

 

1. 2.3 Response surface experimental design

Table 1 Independent parameters and their coded levels

 

independent factor	level (of achievement etc)
	-1	0	1
Extraction time (X1  )/min	20	40	60
Ultrasonic power r(X2  )/W	20	60	100
Material-liquid ratio (X3  )/ (g/mL)	2. 5	5	7. 5

 

The three factors of extraction time (X1), ultrasonic power (X2) and material-liquid ratio (X3) were investigated, and the data were analyzed by SAS9.0 statistical analysis software with astaxanthin yield (Y) as the response value. The levels of the three factors are shown in Table 1.

 

1.3 Analysis of astaxanthin

The astaxanthin content was analyzed by high performance liquid chromatography (HPLC) as described in Liu et al [8].

 

1.4 Kinetics of astaxanthin extraction

The astaxanthin was extracted under the above optimal conditions, and the concentration of astaxanthin in the extract was measured at regular intervals until equilibrium was reached. Primary and secondary kinetic models were used to fit the astaxanthin extraction process.

 

1.5 Analysis of antioxidant activity

The scavenging ability of 1 ,1- Diphenyl- 2- trinitrophenylhydrazine radical ( 1 , 1- Diphenyl- 2 - trinitrophenylhydrazine radical , DPPH - ) and Hydroxyl radical (- OH) was determined with reference to the method of Liu, Lulu et al [9].

 

2 Results and Discussion

2.1 Optimization of extraction conditions

2. 1. 1 One-way optimization of extraction conditions

The results of one-factor optimization are shown in Fig. 1. It can be seen that the astaxanthin yield increased with the extension of extraction time, reaching 19.87 mg/g DCW at 40 min. The increase in astaxanthin yield was not obvious when the extraction time was prolonged thereafter. On the contrary, the astaxanthin yield gradually decreased with the increase of ultrasonic power. This is mainly due to the cave-in effect of ultrasound, which affects the stability of astaxanthin, and the higher the ultrasound power, the more likely to cause astaxanthin degradation. When the material-liquid ratio was increased from 1:40 to 1:5, the amount of astaxanthin extracted increased and then decreased, up to a maximum of 32.7 mg/g, at which time the material-liquid ratio was 1:20. This is related to the solubilization equilibrium of astaxanthin. At a high feed-liquid ratio, the concentration of astaxanthin in the liquid phase (ethanol) was higher and approached the equilibrium point very quickly, which resulted in a small mass transfer driving force, thus affecting the transfer process of astaxanthin from the solid phase (microalgae) to the liquid phase[10] . Therefore, an extraction time of 40 min, an ultrasonic power of 65.4 W, and a material-liquid ratio of 1:20 were selected for subsequent experiments.

 

2. 1. 2 Response surface methodology to optimize extraction conditions

Response surface methodology was used to further optimize the extraction conditions, and the response value of astaxanthin yield (Y) was used as the response value, and the experimental design and results were shown in Table 2. The data in Table 2 were fitted into a response surface regression model to obtain the following regression equations with ultrasound time (X1), ultrasound power (X2), and material-liquid ratio (X3) as independent variables.

y = 1 217. 189 + 123. 730 1x1 + 254. 904 3x2 - 172. 552 5x3 +

1. 967 742x-186. 705 9x1x2 -3. 39x1x3 -267. 938 6x+34. 055 03x2x3 -

193. 047 3X3 2

Table 2 Box-Behnken experimental design and result

 

Run	X1	X2	X3	Astaxanthin Yield/ (mg/g)
1	-1	-1	0	15. 66
2	-1	1	0	35. 88
3	1	-1	0	31. 44
4	1	1	0	35. 83
5	0	-1	-1	26. 99
6	0	-1	1	20. 81
7	0	1	-1	31. 80
8	0	1	1	29. 19
9	-1	0	-1	34. 52
10	1	0	-1	36. 38
11	-1	0	1	27. 12
12	1	0	1	29. 22
13	0	0	0	35. 16
14	0	0	0	35. 42
15	0	0	0	34. 07

 

Table 3 Analysis of variance ( ANOVA) for response

surface methodology

 

	(number of) degrees of freedom (physics)	mean square	F-value	P-value	significance
mould	9	140 466 1	6. 896 148	0. 023 4	*
X1	1	122 473	5. 411 502	0. 067 5
X2	1	519 809. 4	22. 967 91	0. 004 9	*
X3	1	238 194. 9	10. 524 7	0. 022 8	*
X1 2	1	14. 296 64	0. 000 632	0. 980 9

 

From the response surface analysis of variance (Table 3), it can be seen that the regression model was well fitted (coefficient of determination R2 = 0. 925 4) and significant (P<0. 05), which can be used for theoretical prediction of optimization of astaxanthin extraction. As shown in Table 3, ultrasonic power (X2 ) was the most significant factor in the primary term (P = 0. 004 9), followed by liquid-liquid ratio (X3 ) (P < 0. 022 8); ultrasonic power (X2 2 ) was the most significant factor in the secondary term (P < 0. 018 8); and ultrasonic time and ultrasonic power (X1 X2 ) were most significant factors in the cross-correlation (P = 0. 055). The most significant effect of intersection was on ultrasound time and ultrasound power (X1 X2 ) (P = 0. 055). The response surfaces and their contour plots in Fig. 2 also show that the contours of X1 and X3, X2 and X3 are elliptical in shape, indicating that the interaction between them is relatively large.

 

The theoretical yield of astaxanthin was maximized to 36.02 mg/g DCW by the above regression model equation, and the extraction conditions were ultrasonic time of 28 min, ultrasonic power of 87 W, and material-liquid ratio of 1∶25. In order to check the accuracy of the model prediction, the optimal conditions obtained from the above fitting were used for the extraction experiments, and the actual astaxanthin yield was obtained as (35. 93±0. 37) mg/g DCW. The experimental value of astaxanthin yield was similar to the predicted value with an error of 0.25%, which indicated that the experimental value fitted well with the predicted value of the model, and the model could be used.

 

2.2 Kinetics of astaxanthin extraction

In the present study, the first-order kinetic model and the second-order kinetic model were further used to fit the parameters under the optimal extraction conditions to investigate the effect of concentration on the extraction rate. As shown in Fig. 3, the R2 of the second-order kinetic model = 0.998 7, indicating that it can well describe the astaxanthin extraction process. On the contrary, the R2 of the first-stage kinetic fit was only 0. 882 0. Amado et al[11] found that the extraction of astaxanthin from shrimp waste also conformed to the second-stage kinetic law. This suggests that the mass transfer dynamics (i.e., Ce - Ct ) is a key factor affecting the extraction efficiency of astaxanthin, and its influence on the mass transfer rate (i.e., extraction rate) is much larger than that of the first-order kinetics. Therefore, in order to further increase the yield of astaxanthin, the use of multiple, short-time extraction can be considered to ensure a high mass transfer kinetics.

 

2.3 Analysis of antioxidant activity of astaxanthin extracts

In the present study, the antioxidant activity of astaxanthin extract prepared by the optimized process was also evaluated. The results showed that the semi-inhibitory concentrations (IC50) of astaxanthin extract against DPPH and OH radicals were (16.14 ± 1.58) μg/mL and (0.93 ± 0.18) μg/mL, respectively (Figure 4). Compared with the scavenging ability of astaxanthin extracted with DMSO by Yin et al. [12] for DPPH and OH radicals (with IC50 of 30 μg/mL and 10 μg/mL, respectively), the present method produced astaxanthin extract with lower IC50 and better antioxidant activity.

 

3 CONCLUSIONS

In this study, ethanol was used as the extraction solvent to extract astaxanthin from fresh red algae by ultrasound-assisted method, and the extraction parameters were optimized by one-way and response surface design experiments to obtain the optimal extraction conditions: ultrasound time of 28 min, material-liquid ratio of 1:25, and ultrasound power of 87 W. At this time, the yield of astaxanthin was (35.93±0.37) mg/g DCW. The effect of each factor on astaxanthin extraction content was as follows: ultrasonic power, material-liquid ratio > ultrasonic time. At the same time, the extraction kinetics of astaxanthin under the optimal conditions satisfied the secondary kinetic characteristics, i.e., the extraction rate was proportional to the square of the difference in concentration of the solution. The astaxanthin extract prepared by this method has good antioxidant activity, and its semi-inhibitory concentrations of DPPH and OH were (16.14 ± 1.58) μg/mL and (0.93 ± 0.18) μg/mL, respectively.

 

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