Astaxanthin, one of the main xanthophyll carotenoid pigments, possesses 500-fold and 38-fold times stronger free radical antioxidant activity of vitamin E and
As one of the potent organisms for production of astaxanthin,
In this study, four different methods, hydrochloric acid pretreatment followed by acetone extraction (HCl-ACE), hexane/isopropanol (6 : 4, v/v) mixture solvents extraction (HEX-IPA), methanol extraction followed by acetone extraction (MET-ACE, 2-step extraction), and soy-oil extraction, were employed to extract astaxanthin from
Astaxanthin extraction by HCl-ACE method was modified according to the procedures reported by Sarada et al. [
HEX-IPA binary solvents extraction method consists of transferring 10 mg of the lyophilized organisms into 2 mL of hexane/isopropanol (6 : 4, v/v) binary organic solvents for 20 min in an ice-water bath temperature and ultrasonically assistant extraction. The mixture of cell biomass, extract, and solvent was separated by means of centrifugation at 3500 rpm at 4°C for 5 min, followed by concentration under vacuum. The extraction yield was calculated in dry basis and expressed in % (w/w-dry basis). HPLC estimation was employed for analysis of astaxanthin content. All the steps were carried out in light protection and filled with nitrogen.
In this procedure, ten milligrams biomass was weighed into a 15 mL screw top amber glass vial and ultrasonically extracted in an ice-water bath with 1 mL methanol and acetone for 5 min in sequential order [
The oil-soy extraction method was performed in triplicate according to the procedure presented by Sachindra and Mahendrakar [
Oil consisting of astaxanthin extraction yield by different methods was calculated with
To evaluate the extraction efficiency of different methods, the morphology of
The extracts were subjected to high performance liquid chromatography (HPLC) (LC-20AT; Shimadzu, Beijing, China) equipped with ZORBOX 300-SB C18 column for astaxanthin content determination. The conditions were as follows: eluants were (A) acetone and (B) methanol: H2O (9 : 1 v/v) with the flow rate of 0.8 mL/min and column temperature was 40°C. A gradient concentration program was employed as follows: B was run at 80 to 20% for 25 min, 20% for 10 min, and 20 to 80% for 5 min. The detection wavelength was monitored at 460 nm.
The fatty acid profiles in lipid extracts were quantitatively analyzed by 1H-NMR method, which is based on the fact that the amplitude of 1H-NMR signal is proportional to the number of hydrogen nuclei contained in the molecule [
1H-NMR spectral peak assignment*.
Signal | Chemical shift (ppm) | Functional group |
---|---|---|
1 | 0.82–0.94 | –CH3 (terminal methyl protons (saturated, oleic and linoleic)) |
2 | 0.94–1.03a | –CH3 (terminal methyl protons (linolenic)) |
3 | 1.20–1.43 | –(CH2)n–(methylene protons (saturated)) |
4 | 1.55–1.69 | –OCO–CH2–CH2–(_–methylene protons (carbonyl)) |
5 | 1.93–2.13 | –CH2–CH CH–(allyl methylene protons) |
6 | 2.25–2.36 | –OCO–CH2–(_–methylene protons) |
7 | 2.73–2.87 | HC–CH2–CH (divinyl methylene protons) |
8 | 4.10–4.35 | –CH2OCOR (methylene protons (glyceryl)) |
9 | 5.23–5.29 | CHOCOR (proton on carbon atom 2 of glyceryl group) |
10 | 5.29–5.43 | –CH CH–(olefinic protons) |
aThis chemical shift range was changed to 0.94–0.99 ppm in all subsequent peak integration measurements to exclude signal contribution from unassigned peak at 1.01 ppm.
The antioxidant activity of the extracts was measured on the basis of the scavenging activity of the stable DPPH free radical [
Each sample of 1 mL was added to 2.5 mL of 0.2 mol/L phosphate buffers (pH 6.6) and 1 mL 1% (w/v) potassium ferricyanide. The mixture was incubated at 50°C for 20 min and cooled rapidly. Then 2.5 mL of 10% (w/v) trichloroacetic acid was added to the mixture, which was then centrifuged at 3500 rpm for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% (w/v) ferric chloride in a test tube. After a 10 min reaction, the absorbance of the resulting solution was measured at 700 nm by a UV756CRT spectrophotometer [
All reported data were collected in triplicate, and the statistical analysis was performed using SAS 9.0 software (SAS Institute, Inc., Cary, NC, USA). Analytical data were expressed as mean ± SE (standard error of the mean).
The extraction yield and total astaxanthin content (TAC) values obtained by four different extraction techniques are presented in Table
Effect of different extraction methods on oil yield and total astaxanthin content (TAC) of extracts from
Extraction techniquea | Solvent/raw material ratio (mL/g) | Extraction time (min) | Extraction oil yield (%, w/w) | TAC (mg/g-cell) |
---|---|---|---|---|
HCl-ACE | 200 | 20 |
|
|
HEX-IPA | 100 | 20 |
|
|
MET-ACE | 400 | 20 |
|
|
Oil soy | 8 | 120 |
|
|
The morphologies of
Photoes and SEM analysis of
1H-NMR spectroscopy experiments were carried out in order to gather information about the quantitative fatty acid (FA) composition in the total lipid extract by four different extraction methods and the results are deposited in Figure
1H-NMR spectroscopy results for FA compositions profiles*.
Fatty acid compositions (%) | HCl-ACE | HEX-IPA | MET-ACE | Oil-soy |
---|---|---|---|---|
Linolenic acid |
25.93 | 20.75 | 20.63 | 30.8 |
Linoleic acid |
42.81 | 17.89 | 36.67 | 34.9 |
Oleic acid (monounsaturated FA) | 13.93 | 34.54 | 14.75 | 14.0 |
Saturated FA | 17.33 | 26.82 | 27.94 | 21.1 |
|
1.65 | 0.86 | 1.78 | 1.13 |
Total monounsaturated FA/total Saturated FA | 0.80 | 1.29 | 0.53 | 0.66 |
Total PUFA/total Saturated FA | 3.97 | 1.44 | 2.05 | 3.11 |
NMR determination for fatty acids profiles of extracts obtained by four different extraction techniques from
To evaluate the effect of four different extraction methods on the quality of total lipid extracts from
DPPH radical scavenging activities (a) and reducing powers (b) of astaxantin products from
The total oil yield and TCA extraction from
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was financially supported by the Natural Science Foundation of China (31270858, 31070709).