Chinese Bulletin of Botany ›› 2021, Vol. 56 ›› Issue (6): 722-731.DOI: 10.11983/CBB21064
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Qing Miao, Pa-he-er-ding Sabiha, Siyu Zeng, Qifang Pan()
Received:
2021-04-17
Accepted:
2021-09-17
Online:
2021-11-01
Published:
2021-11-12
Contact:
Qifang Pan
Qing Miao, Pa-he-er-ding Sabiha, Siyu Zeng, Qifang Pan. Green Extraction Method and Optimization of Steviosides from Stevia rebaudiana by Natural Deep Eutectic Solvent[J]. Chinese Bulletin of Botany, 2021, 56(6): 722-731.
No. | Component 1 | Component 2 | Component 3 | Mole ratio (v/v) |
---|---|---|---|---|
NADES-1 | 1,2-propanediol | Glycerol | Water | 8:1:1 |
NADES-2 | 1,2-propanediol | Glycerol | Water | 1:1:1 |
NADES-3 | 1,2-propanediol | Glucose | Lactic acid | 3:1:5 |
NADES-4 | 1,2-propanediol | Water | 2:1 | |
NADES-5 | 1,2-propanediol | Glycerol | 1:2 | |
NADES-6 | Glycerol | Water | 9:1 |
Table 1 Different combinations of natural deep eutectic solvents
No. | Component 1 | Component 2 | Component 3 | Mole ratio (v/v) |
---|---|---|---|---|
NADES-1 | 1,2-propanediol | Glycerol | Water | 8:1:1 |
NADES-2 | 1,2-propanediol | Glycerol | Water | 1:1:1 |
NADES-3 | 1,2-propanediol | Glucose | Lactic acid | 3:1:5 |
NADES-4 | 1,2-propanediol | Water | 2:1 | |
NADES-5 | 1,2-propanediol | Glycerol | 1:2 | |
NADES-6 | Glycerol | Water | 9:1 |
No. | 1 | 2 | 3 | 4 | 5 | 6 |
---|---|---|---|---|---|---|
Stevioside (mg∙mL-1) | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 |
Rebaudioside A (mg∙mL-1) | 3 | 1.5 | 0.75 | 0.375 | 0.1875 | 0.09375 |
Table 2 Standard solution concentration
No. | 1 | 2 | 3 | 4 | 5 | 6 |
---|---|---|---|---|---|---|
Stevioside (mg∙mL-1) | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 |
Rebaudioside A (mg∙mL-1) | 3 | 1.5 | 0.75 | 0.375 | 0.1875 | 0.09375 |
Level | Factor | ||
---|---|---|---|
A: Temperature (°C) | B: Power (J∙s-1) | C: Time (min) | |
-1 | 60 | 50 | 30 |
0 | 70 | 65 | 60 |
1 | 80 | 80 | 90 |
Table 3 Factors and levels of response surface methodology
Level | Factor | ||
---|---|---|---|
A: Temperature (°C) | B: Power (J∙s-1) | C: Time (min) | |
-1 | 60 | 50 | 30 |
0 | 70 | 65 | 60 |
1 | 80 | 80 | 90 |
Time (min) | Mobile phase C (%) | Mobile phase B (%) |
---|---|---|
0 | 80 | 20 |
3 | 80 | 20 |
15 | 30 | 70 |
20 | 30 | 70 |
20.1 | 80 | 20 |
27 | 80 | 20 |
Table 4 Gradient elution procedure
Time (min) | Mobile phase C (%) | Mobile phase B (%) |
---|---|---|
0 | 80 | 20 |
3 | 80 | 20 |
15 | 30 | 70 |
20 | 30 | 70 |
20.1 | 80 | 20 |
27 | 80 | 20 |
Figure 3 The effects of ultrasonic power on the concentration of stevioside (A) and rebaudioside A (B) Different lowercase letters indicate significant differences (P< 0.05).
Figure 4 The effects of extraction temperature on the concentration of stevioside (A) and rebaudioside A (B) Different lowercase letters indicate significant differences (P< 0.05).
Figure 5 The effects of extraction time on the concentration of stevioside (A) and rebaudioside A (B) Different lowercase letters indicate significant differences (P< 0.05).
Run units | A: Tempe- rature (°C) | B: Power (J∙s-1) | C: Time (min) | Stevioside concentration (mg∙mL-1) | Rebaudioside A concentration (mg∙mL-1) |
---|---|---|---|---|---|
1 | 70 | 50 | 30 | 2.30 | 1.11 |
2 | 80 | 65 | 90 | 2.08 | 0.90 |
3 | 70 | 80 | 90 | 2.77 | 1.03 |
4 | 70 | 50 | 90 | 0.11 | 0.09 |
5 | 70 | 80 | 30 | 1.71 | 0.70 |
6 | 60 | 65 | 90 | 2.57 | 1.18 |
7 | 60 | 65 | 30 | 2.59 | 1.06 |
8 | 80 | 80 | 60 | 2.00 | 0.83 |
9 | 60 | 80 | 60 | 2.52 | 1.02 |
10 | 70 | 65 | 60 | 1.52 | 0.64 |
11 | 70 | 65 | 60 | 1.52 | 0.64 |
12 | 70 | 65 | 60 | 1.52 | 0.64 |
13 | 80 | 50 | 60 | 0.06 | 0.02 |
14 | 70 | 65 | 60 | 1.52 | 0.64 |
15 | 70 | 65 | 60 | 1.52 | 0.64 |
16 | 80 | 65 | 30 | 1.31 | 0.65 |
17 | 60 | 50 | 60 | 0.49 | 0.35 |
Table 5 Response surface experimental results
Run units | A: Tempe- rature (°C) | B: Power (J∙s-1) | C: Time (min) | Stevioside concentration (mg∙mL-1) | Rebaudioside A concentration (mg∙mL-1) |
---|---|---|---|---|---|
1 | 70 | 50 | 30 | 2.30 | 1.11 |
2 | 80 | 65 | 90 | 2.08 | 0.90 |
3 | 70 | 80 | 90 | 2.77 | 1.03 |
4 | 70 | 50 | 90 | 0.11 | 0.09 |
5 | 70 | 80 | 30 | 1.71 | 0.70 |
6 | 60 | 65 | 90 | 2.57 | 1.18 |
7 | 60 | 65 | 30 | 2.59 | 1.06 |
8 | 80 | 80 | 60 | 2.00 | 0.83 |
9 | 60 | 80 | 60 | 2.52 | 1.02 |
10 | 70 | 65 | 60 | 1.52 | 0.64 |
11 | 70 | 65 | 60 | 1.52 | 0.64 |
12 | 70 | 65 | 60 | 1.52 | 0.64 |
13 | 80 | 50 | 60 | 0.06 | 0.02 |
14 | 70 | 65 | 60 | 1.52 | 0.64 |
15 | 70 | 65 | 60 | 1.52 | 0.64 |
16 | 80 | 65 | 30 | 1.31 | 0.65 |
17 | 60 | 50 | 60 | 0.49 | 0.35 |
Source | Sum of squares | Degree of freedom | Mean square | F value | P-value |
---|---|---|---|---|---|
Model | 9.98 | 9 | 1.11 | 8.00 | 0.0060** |
A: Temperature | 0.9351 | 1 | 0.9351 | 6.75 | 0.0355* |
B: Power | 4.58 | 1 | 4.58 | 33.08 | 0.0007** |
C: Time | 0.0188 | 1 | 0.0188 | 0.1355 | 0.7237 |
AB | 0.0018 | 1 | 0.0018 | 0.0128 | 0.9130 |
AC | 0.1520 | 1 | 0.1520 | 1.10 | 0.3297 |
BC | 2.64 | 1 | 2.64 | 19.09 | 0.0033** |
A² | 0.0265 | 1 | 0.0265 | 0.1910 | 0.6752 |
B² | 0.4768 | 1 | 0.4768 | 3.44 | 0.1059 |
C² | 1.21 | 1 | 1.21 | 8.71 | 0.0214* |
Residual | 0.9697 | 7 | 0.1385 | ||
Lack of fit | 0.9697 | 3 | 0.3232 | ||
Pure error | 0.0000 | 4 | 0.0000 | ||
Cor total | 10.95 | 16 | |||
R2 | 0.9114 | ||||
Radj2 | 0.7975 | ||||
Rpred2 | -0.4173 | ||||
Adeq precision | 10.9996 |
Table 6 Analysis of the model variances of stevioside concentration
Source | Sum of squares | Degree of freedom | Mean square | F value | P-value |
---|---|---|---|---|---|
Model | 9.98 | 9 | 1.11 | 8.00 | 0.0060** |
A: Temperature | 0.9351 | 1 | 0.9351 | 6.75 | 0.0355* |
B: Power | 4.58 | 1 | 4.58 | 33.08 | 0.0007** |
C: Time | 0.0188 | 1 | 0.0188 | 0.1355 | 0.7237 |
AB | 0.0018 | 1 | 0.0018 | 0.0128 | 0.9130 |
AC | 0.1520 | 1 | 0.1520 | 1.10 | 0.3297 |
BC | 2.64 | 1 | 2.64 | 19.09 | 0.0033** |
A² | 0.0265 | 1 | 0.0265 | 0.1910 | 0.6752 |
B² | 0.4768 | 1 | 0.4768 | 3.44 | 0.1059 |
C² | 1.21 | 1 | 1.21 | 8.71 | 0.0214* |
Residual | 0.9697 | 7 | 0.1385 | ||
Lack of fit | 0.9697 | 3 | 0.3232 | ||
Pure error | 0.0000 | 4 | 0.0000 | ||
Cor total | 10.95 | 16 | |||
R2 | 0.9114 | ||||
Radj2 | 0.7975 | ||||
Rpred2 | -0.4173 | ||||
Adeq precision | 10.9996 |
Source | Sum of squares | Degree of freedom | Mean square | F value | P-value |
---|---|---|---|---|---|
Model | 1.51 | 9 | 0.1682 | 4.53 | 0.0294* |
A: Temperature | 0.1834 | 1 | 0.1834 | 4.94 | 0.0616 |
B: Power | 0.4988 | 1 | 0.4988 | 13.45 | 0.0080** |
C: Time | 0.0126 | 1 | 0.0126 | 0.3396 | 0.5783 |
AB | 0.0044 | 1 | 0.0044 | 0.1185 | 0.7408 |
AC | 0.0040 | 1 | 0.0040 | 0.1083 | 0.7517 |
BC | 0.4567 | 1 | 0.4567 | 12.31 | 0.0099** |
A² | 0.0173 | 1 | 0.0173 | 0.4660 | 0.5168 |
B² | 0.0951 | 1 | 0.0951 | 2.56 | 0.1534 |
C² | 0.2538 | 1 | 0.2538 | 6.84 | 0.0346* |
Residual | 0.2597 | 7 | 0.0371 | ||
Lack of fit | 0.2597 | 3 | 0.0866 | ||
Pure error | 0.0000 | 4 | 0.0000 | ||
Cor total | 1.77 | 16 | |||
R2 | 0.8536 | ||||
Radj2 | 0.6653 | ||||
Rpred2 | -1.3429 | ||||
Adeq precision | 7.9551 |
Table 7 Analysis of the model variances of rebaudioside A concentration
Source | Sum of squares | Degree of freedom | Mean square | F value | P-value |
---|---|---|---|---|---|
Model | 1.51 | 9 | 0.1682 | 4.53 | 0.0294* |
A: Temperature | 0.1834 | 1 | 0.1834 | 4.94 | 0.0616 |
B: Power | 0.4988 | 1 | 0.4988 | 13.45 | 0.0080** |
C: Time | 0.0126 | 1 | 0.0126 | 0.3396 | 0.5783 |
AB | 0.0044 | 1 | 0.0044 | 0.1185 | 0.7408 |
AC | 0.0040 | 1 | 0.0040 | 0.1083 | 0.7517 |
BC | 0.4567 | 1 | 0.4567 | 12.31 | 0.0099** |
A² | 0.0173 | 1 | 0.0173 | 0.4660 | 0.5168 |
B² | 0.0951 | 1 | 0.0951 | 2.56 | 0.1534 |
C² | 0.2538 | 1 | 0.2538 | 6.84 | 0.0346* |
Residual | 0.2597 | 7 | 0.0371 | ||
Lack of fit | 0.2597 | 3 | 0.0866 | ||
Pure error | 0.0000 | 4 | 0.0000 | ||
Cor total | 1.77 | 16 | |||
R2 | 0.8536 | ||||
Radj2 | 0.6653 | ||||
Rpred2 | -1.3429 | ||||
Adeq precision | 7.9551 |
Figure 6 Response surface analysis of extraction temperature, ultrasonic power and extraction time for stevioside and rebaudioside A (A) Response surface (3D) showing effect of temperature and power on stevioside; (B) Response surface (3D) showing effect of temperature and time on stevioside; (C) Response surface (3D) showing effect of power and time on stevioside; (D) Response surface (3D) showing effect of temperature and power on rebaudioside A; (E) Response surface (3D) showing effect of temperature and time on rebaudioside A; (F) Response surface (3D) showing effect of power and time on rebaudioside A
Temperature (°C) | Power (J∙s-1) | Time (min) | Stevioside concentration (mg∙mL-1) | Rebaudioside A concentration (mg∙mL-1) | |
---|---|---|---|---|---|
Initial condition | 60 | 65 | 30 | 2.59±0.04 | 1.06±0.08 |
Optimal condition | 60 | 80 | 90 | 3.48±0.36* | 1.42±0.18* |
Table 8 Extraction concentration of stevioside and rebaudioside A by different extraction conditions
Temperature (°C) | Power (J∙s-1) | Time (min) | Stevioside concentration (mg∙mL-1) | Rebaudioside A concentration (mg∙mL-1) | |
---|---|---|---|---|---|
Initial condition | 60 | 65 | 30 | 2.59±0.04 | 1.06±0.08 |
Optimal condition | 60 | 80 | 90 | 3.48±0.36* | 1.42±0.18* |
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