The content and species of carotenoids are significantly affected by different carotenoids extraction methods. The comparison of the three methods ultrasonic assisting, grinding and HCl assisting on carotenoids extraction yield from Rhodopseudomonas faecalis PSB-B was carried out. Data ANOVA showed that ultrasound can greatly replace the conventional extraction. And then, based on ultrasonic assisting extraction method, the effect of ultrasonic time, solvent-solid ratio and ultrasonic power on the yield of carotenoids extracted from Rhodopseudomonas faecalis PSB-B was investigated using single factor and Box-Behnken experimental design. Under the extraction of temperature 20?C, N-hexane:Methanol (5:1), the optimal conditions for Ultrasonic assisted extraction of carotenoids found to be: Ultrasonic time 4.5 min, Solvent-solid ratio (mg/ml) (10:10), extraction power of 187 W. The yield of carotenoids could reach to 16.11 mg/L.
Carotenoids are colorful compounds possessing yellow, orange and red pigments. They show important biologic activities associated with antioxidant properties, such as strengthening the immune system, decreasing the risk of degenerative illnesses, reducing the risk of cardiovascular disease, and preventing macular degeneration and cataracts [
Due to their complex structure and because of the wide variety of these compounds present in vegetables, fruits, algae and bacteria, the choice of an appropriate method for extraction of intracellular bioactive compounds, including pigments is dependent on some aspects such as cell wall strength, intracellular localization, stability and the final use of compound [
A strain of photosynthetic bacterium R. faecalis PSB-B (KM272172) was separated from the sludge of the Fenhe River in Shanxi province of China.
The 1000 ml growth medium contained yeast extract 3 g, Peptone 3 g, MgSO4 0.5 g, CaCl2 0.3 g. The initial pH value was adjusted to 6.8 - 7.2. Cells were incubated at 30˚C under 60-W tungsten lamp illumination of 2000lux 3 days.
Three extraction methods for carotenoids from R. faecalis were compared. The detailed extraction process of each method was described as follows:
Ultrasonic Extraction: The method of ultrasonic extraction was measured according to the method of Gu Z [
Acid heat crushing Method: Add 5 ml 3 mol/L HCl into the centrifuge tube; soak it for 1 hour with 20˚C. After that, put it into the 100 degree celsius water to soak for 10 minutes. Then take it out and put it into the ice water to be cooled quickly. Then make the centrifugal treatment with it in the speed of 3000 r/min for 10 minutes at 4˚C. Abandon the supernatant, we get the cellular mud. Wash the cellular mud with distilled water for 3 to 5 times. Put 20 ml acetone and make the sufficient oscillation. Let it stand for 10 minutes, then make the centrifugal treatment with it in the speed of 3000 r/min for 10 minutes at 4˚C. The supernatant is the pigment crude extract liquid.
Grinding Method: Put 300 ml bacterial suspension into a centrifuge tube. Then make the centrifugal treatment with it in the speed of 3000 r/min for 10 minutes at 4˚C. Abandon the supernatant, we get the microbial precipitation. Cells were washed free of medium with distilled water three times. Thallus were freezing-dried through the vacuum freeze dryer. Put some acetone and thallus together with the ratio of liquid to solid is 40:1 (ml:g), then add some quartz sand into it. Grind it for 30 minutes, then make the centrifugal treatment with it in the speed of 3000 r/min for 10 minutes at 4˚C. The supernatant is the pigment crude extract liquid.
Determination of Carotenoids
Total carotenoids content was determined at 480 nm using a spectrophotometer (722SJinghua, China) following the recommendation of Johnson E A et al. [
Caroteniods yield (mg/L) = ADV1/0.16V2 (1)
where A is the absorbance value of diluted extraction at 480 nm, D is the dilution rate, V1 is the volume of acetone, 0.16 is the extinction coefficient of carotenoids, and V2 is the volume of fermentation liquor.
The carotenoids production were statistically evaluated by a one-way analysis of variance (ANOVA), using the SPSS 17.0. In the work described the extraction processes of carotenoids were analysed using three extraction techniques, namely ultrasound-assisted, grinding and HCl-assisted extraction, and the results are compared. Optimization of ultrasonic extraction with Box-Behnken experimental design, the single factor experiment for ultrasound assisted extraction was performed with the analysis of the effect of three factors (extraction time, solvent?solid ratio and extraction power) on extraction of carotenoids from PSB-B. The effects of three factors on carotenoid extraction were obtained. Identified low level and high level (
In the work described here the extraction processes of carotenoids were analysed using three extraction techniques, namely ultrasound-assisted, grinding and HCl-assisted extraction, and the results are compared. From the results presented in
The carotenoids yield increased with the extraction time increasing from 2 min to 8 min, but decreased when the extraction time were above 6 min, 6 min was
Factors | Symbols | Low level | High level |
---|---|---|---|
−1 | 1 | ||
Extraction time | A | 2 | 6 |
Extraction power | B | 130 | 260 |
Solvent?solid ratio | C | 0.5 | 1.5 |
NO. | A time (min) | B power (w) | C Solvent?solid ratio (mg/ml) | Carotenoids Yield (mg/L) |
---|---|---|---|---|
1 | 2 | 195 | 0.5 | 9.22 |
2 | 4 | 130 | 1.5 | 11.50 |
3 | 2 | 260 | 1 | 9.81 |
4 | 4 | 130 | 0.5 | 9.13 |
5 | 4 | 195 | 1 | 16.68 |
6 | 4 | 195 | 1 | 15.19 |
7 | 6 | 195 | 0.5 | 12.49 |
8 | 4 | 195 | 1 | 15.95 |
9 | 6 | 195 | 1.5 | 10.75 |
10 | 2 | 195 | 1.5 | 9.38 |
11 | 6 | 260 | 1 | 10.64 |
12 | 4 | 260 | 0.5 | 12.74 |
13 | 4 | 260 | 1.5 | 11.94 |
14 | 6 | 130 | 1 | 14.95 |
15 | 2 | 130 | 1 | 11.37 |
16 | 4 | 195 | 1 | 16.97 |
17 | 4 | 195 | 1 | 15.05 |
The method of broken cell wall | The yield of carotenoids (mg/L) |
---|---|
HCl assisting Grinding Ultrasound-assisted | 1.045 ± 0.052a 1.058 ± 0.049a 5.292 ± 0.061b |
Values with different letters (a, b) differ significantly (P < 0.05).
the optimal temperature at which carotenoids was extracted from Rhodopseudomonas faecalis PSB-B (
Set up six kinds of ultrasonic power: 65 W, 130 W, 195 W, 260 W, 325 W (
Index | Extraction time (min) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Carotenoids Yield (mg/L) | 2 min | 4 min | 6 min | 8 min | |||||
5.287 ± 0.060a | 5.342 ± 0.043a | 5.295 ± 0.061a | 5.364 ± 0.053a | ||||||
Extraction power (w) | |||||||||
65 W | 130 W | 195 W | 260 W | 325 W | |||||
6.237 ± 0.232a | 9.656 ± 0.138b | 9.806 ± 0.216b | 9.175 ± 0.103b | 9.064 ± 0.024b | |||||
Solvent-solid ratio (mg/ml) | |||||||||
10:1 | 10:5 | 10:10 | 10:15 | 10:20 | |||||
5.868 ± 0.231a | 8.55 ± 0.218b | 11.175 ± 0.190c | 11.138 ± 0.193c | 10.027 ± 0.141a | |||||
Values with different letters (a, b, c) differ significantly (P < 0.05).
have certain side effects on the production of carotenoids. Under the premise of ensuring the yield of carotenoids, it is more appropriate to choose 195 w for factors such as the life of the instrument [
The ratio of the optimum fermentation broth to the organic solvent determines the maximum amount of carotenoid extracted from the cells [
W stands for total carotenoids yield, A, B, C denotes the model intercept. A, B, C are the levels of extraction time, extraction power and solvent?solid ratio .By using the Design Expert version 10.0.4 (Stat-Ease, Inc.), a polynomial model describing the correlation between carotenoids yield and the three variables or conditions was obtained as follows (
W = −33.75750 + 7.21487 × A + 0.19933 × B + 30.14450 × C − 5.28846× 10−3 × A × B − 0.47500 × A × C − 0.024385 × B × C − 0.64287 × A2 − 4.03314 × 10−4 × B2 − 11.74600 × C2
with R2 = 0.86.
The correlative surface response graphs are shown in
Source | Sum of squares | Degree of freedom | Mean squares | F-value | P-value |
---|---|---|---|---|---|
Model | 100.75 | 9 | 11.19 | 4.75 | 0.0261 |
A | 10.24 | 1 | 10.24 | 4.34 | 0.0757 |
B | 0.41 | 1 | 0.41 | 0.18 | 0.4716 |
C | 1.25 × 10−5 | 1 | 1.25 × 10−5 | 5.30 × 10−6 | 0.6878 |
A2 | 27.84 | 1 | 27.84 | 11.81 | 0.0109 |
B2 | 12.23 | 1 | 12.23 | 5.18 | 0.0569 |
C2 | 36.31 | 1 | 36.31 | 15.40 | 0.0057 |
AB | 1.89 | 1 | 1.89 | 0.80 | 0.4003 |
AC | 0.90 | 1 | 0.90 | 0.38 | 0.5557 |
BC | 2.51 | 1 | 2.51 | 1.07 | 0.3363 |
Residual | 16.51 | 7 | 2.36 | ||
Lack of fit | 13.55 | 3 | 4.52 | 6.10 | 0.0565 |
Pure Error | 2.96 | 4 | 0.74 | ||
R2 | 0.86 | ||||
Adj-R2 | 0.68 | ||||
Cor Total | 117.26 | 16 |
Adequate precision = 6.039.
According to the fitted surface graphs of solvent?solid ratio and extration time (
The effect of solvent-solid ratio and extration power on carotenoids yield is shown in
Some organic solvents are not selected as the solvent to extract carotenoids due to their low boiling point. With a slight heat up during sonication the solvent will start vaporizing. Different extract solvents have significant effects on the yield of carotenoids, and the extract effects of single polar organic solvents are diversity. We chose acetone, ethanol, isopropyl alcohol, methanol and acetonitrile as extraction agent. The use of ultrasound facilitates the penetration of the isopropyl alcohol through the faecalis PSB-B cell membrane. The polar solvents help in increasing the permeability of the cell wall of the bacteria and low viscosity increases the diffusion of solvent as well as at low viscosity, acoustic cavitation takes place very easily [
Multiple sets of repetitive experiments were performed under optimal extract conditions. The test has good reproducibility, and the average extraction amount of carotenoids is 15.86 mg/L.
Extraction of carotenoids from Rhodopseudomonas faecalis PSB-B is investigated in this work. Three methods of extracting carotenoids were compared by experiments. The results showed that the ultrasonic assisted extraction method can obtain more carotenoids than others. Maximum amount of carotenoids recovered was 16.11 mg/L obtained by ultrasonic assisting using at ultrasonic time 4.5 min, solvent-solid ratio (10:10), extraction power of 187 W, N-hexane:Methanol = (5:1).
This work was financially supported by the Natural Science Foundation Project of Shanxi Province (201801D121206) and the Natural Science Foundation of Modern College of Humanities and Sciences of Shanxi Normal University (2018JCYJC01). The authors would like to thank Zhang Hong for her help in statistical analysis of data.
The authors declare no conflicts of interest regarding the publication of this paper.
Hu, Q.P. and Wang, Y. (2018) Optimization Ultrasound Assisted Extraction of Carotenoids from Rhodopseudomonas faecalis PSB-B. Open Access Library Journal, 5: e5045. https://doi.org/10.4236/oalib.1105045