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This study was undertaken to optimize the conditions for the extraction of antibacterial activity of ^{**}), 26 h (R = −0.731^{**}) extraction time, and 50% (R = −0.075) ethanol concentration. The population of

With increasing bacterial resistance to antibiotics, there is considerable interest in investigating the antimicrobial effects of plant extracts against a range of bacteria, to develop other classes of safe and natural antimicrobials useful for infection control or for the preservation of food [

The main advantage of the evolutionary operation (EVOP) factorial design technique [

The

In the present study, therefore, the optimal conditions of extraction temperature, extraction time and ethanol concentration on the antibacterial activity of

The experimental conditions used in the first set of experiments, the corresponding antibacterial activities of cycle I and II, their differences and average values are presented in _{10}, E_{20}) were 50 °C, 14 h and 50%, respectively.

In the first set, the error limits for average, effects and changes in mean were 0.1739, 0.1235 and 0.1096, respectively. The change in mean effect was −0.2105. According to the decision-making procedure, after calculating the change in the mean effect and error limit, an examination was necessary to determine whether any change in the control (search level) experimental conditions would help to improve the objective function [

The determination of the magnitude of the change in mean effect, which is negative and large, compared to the error limit, is a requirement in order to confirm the achievement of the optimum condition. Such a situation, where some of the effects are larger in comparison to the error limit, does not ensure that the conditions in the search region (E_{10}, E_{20}) of the first set is the actual optimum and a second set of experiments is called for.

In the second set, the search level (E_{10}, E_{20}) was fixed at the best condition of Set I, at a level of E_{21}, in which the number of _{10}, E_{20}) were 65 °C, 20 h and 75%, respectively. The experimental conditions and the results of the Set II experiments are presented in _{14}. The extraction temperature, extraction time and ethanol concentration of the E_{14} point in the second set were 80 °C, 26 h and 50%, respectively. In this case, not all of the effects were smaller than the error limit, and the change in mean effect was smaller compared to the error limit even though it is positive. It has been reported that if all or any of the effects are larger than the error limits, the change in the experimental conditions may yield better results [

From the above situation, the third set of experiments was designed in which the best condition of Set II (E_{14}) was selected as the search level (E_{10}, E_{20}) for Set III. The experimental conditions and the results of Set III are shown in

In this study, it was shown that higher antibacterial activity was achieved at a higher extraction temperature of 80 °C (R = −0.800^{**}) and in a longer extraction time of 26 h (R = −0.731^{**}). However, antibacterial activity of

Although the optimal condition of

Further, a SEM study of was carried out to visualize the effects of the ethanolic extract of

The leaves of

To design an experiment for establishing proper extraction conditions, 20 g of sample was hydrolyzed and extracted in a reflux extraction apparatus under different extraction temperatures (35, 50, 65, 80 and 95 °C), extraction times (8, 14, 20, 26 and 32 h) and ethanol concentrations (25, 50, 75 and 100%) under EVOP factorial design and then freeze dried in order to establish the proper extraction conditions. The concentration of

In this study, the optimization of antibacterial activity of _{10}, E_{20}) were selected based on the results of earlier investigation on the effect of individual extraction conditions on the antibacterial activity of the ethanolic extract of _{be}) were selected with lower and higher levels of inducers compared to the initial search level (E_{b0}). Antibacterial activities of

Further, to determine the antibacterial activity of

To determine the efficacy of the extract of _{2}. Finally, the specimen was sputter-coated with gold in an ion coater for 2 min, followed by microscopic examinations (S-4300; Hitachi, Japan).

Overall, our results demonstrate that the application of EVOP-factorial design technique could serve as a potential tool to determine the optimum extraction conditions required to achieve the desired levels of antibacterial activity of natural products and their extracts for their potential utilization in the food industry to control food-borne pathogenic bacteria.

Comparison of the antibacterial activity of _{11} of Set I (extraction temperature; 35 °C, extraction time; 8 h, ethanol concentration; 25%); (

Plots of the responses against the three independent variables in EVOP. **

Scanning electron micrographs of

Experimental design for the three inducer system and results of Set I.

Experimental Conditions | E_{10} |
E_{11} |
E_{12} |
E_{13} |
E_{14} |
E_{20} |
E_{21} |
E_{22} |
E_{23} |
E_{24} |
---|---|---|---|---|---|---|---|---|---|---|

Temperature (°C) | 50(0) |
35(−) | 35(−) | 65(+) | 65(+) | 50(0) | 65(+) | 35(−) | 65(+) | 35(−) |

Time (h) | 14(0) | 8(−) | 20(+) | 8(−) | 20(+) | 14(0) | 20(+) | 8(−) | 8(−) | 20(+) |

Ethanol concentration (%) | 50(0) | 25(−) | 75(+) | 75(+) | 25(−) | 50(0) | 75(+) | 75(+) | 25(−) | 25(−) |

Antibacterial activity (cycle I) (log CFU/mL) | 6.24 | 6.75 | 6.13 | 5.70 | 5.30 | 6.05 | 5.02 | 6.70 | 5.49 | 6.50 |

Antibacterial activity (cycle II) (log CFU/mL) | 6.10 | 6.57 | 6.28 | 5.52 | 5.07 | 6.28 | 4.89 | 6.49 | 5.70 | 6.36 |

Difference (cycle I-cycle II) (log CFU/mL) | 0.14 | 0.18 | −0.15 | 0.18 | 0.23 | 0.23 | 0.13 | 0.21 | −0.21 | 0.14 |

Average activity (log CFU/mL) | 6.170 (a_{10}) |
6.660 (a_{11}) |
6.205 (a_{12}) |
5.610 (a_{13}) |
5.185 (a_{14}) |
6.165 (a_{20}) |
4.955 (a_{21}) |
6.595 (a_{22}) |
5.595 (a_{23}) |
6.430 (a_{24}) |

E_{10} to E_{24} = experiments;

Numbers in parentheses are the coded symbols of levels of the extraction conditions.

Calculation worksheet of the effects of the three-variable system and magnitude of effects and error limits of Set I.

Experimental Conditions | Calculation of Effects | |
---|---|---|

Temperature | 1/4(a_{13} + a_{14} + a_{21} + a_{23} − a_{11} − a_{12} − a_{22} − a_{24}) |
−1.1363 |

Time | 1/4(a_{12} + a_{14} + a_{21} + a_{24} − a_{11} − a_{13} − a_{22} − a_{23}) |
−0.4213 |

Ethanol concentration | 1/4(a_{12} + a_{13} + a_{21} + a_{22}− a_{11} − a_{14} − a_{23} − a_{24}) |
−0.1263 |

Temperature × time | 1/4(a_{11} + a_{14} + a_{21} + a_{22} − a_{12} − a_{13} − a_{23} − a_{24}) |
−0.1113 |

Temperature × ethanol concentration | 1/4(a_{11} + a_{13} + a_{21} + a_{24} − a_{12} − a_{14} − a_{22} − a_{23}) |
0.0188 |

Time × ethanol concentration | 1/4(a_{11} + a_{12} + a_{21} + a_{23} − a_{13} − a_{14} − a_{22} − a_{24}) |
−0.1013 |

Temperature × time × ethanol concentration | 1/4(a_{21} + a_{22} + a_{23} + a_{24} − a_{11} − a_{12} − a_{13} − a_{14}) |
−0.0213 |

Change in mean effect | 1/10(a_{11} + a_{12} + a_{13} + a_{14} + a_{21} + a_{22} + a_{23} + a_{24} − 4a_{10} − 4a_{20}) |
−0.2105 |

Standard deviation (σ) | 1/2(σ_{1} + σ_{2})=1/2(R_{1} × f_{k,n} + R_{2} × f_{k,n}) ^{(1)} |
0.1230 |

Error limits: | ||

for average | ±1.414σ (±2σ/√n) | 0.1739 |

for effects | ±1.004σ (±0.71 × 2σ/√n) | 0.1235 |

for change in mean | ±0.891σ (±0.63 × 2σ/√n) | 0.1096 |

R_{1}: (largest difference − smallest difference) in block 1; R_{2}: (largest difference − smallest difference) in block 2; f_{k,n} = constant depending on number of replications (n) and number of experiments (k) per cycle = 0.3 for n = 2 and k = 5.

Experimental design for three inducer system and results of Set II.

Experimental Conditions | E_{10} |
E_{11} |
E_{12} |
E_{13} |
E_{14} |
E_{20} |
E_{21} |
E_{22} |
E_{23} |
E_{24} |
---|---|---|---|---|---|---|---|---|---|---|

Temperature (°C) | 65(0) | 50(−) | 50(−) | 80(+) | 80(+) | 65(0) | 80(+) | 50(−) | 80(+) | 50(−) |

Time (h) | 20(0) | 14(−) | 26(+) | 14(−) | 26(+) | 20(0) | 26(+) | 14(−) | 14(−) | 26(+) |

Ethanol concentration (%) | 75(0) | 50(−) | 100(+) | 100(+) | 50(−) | 75(0) | 100(+) | 100(+) | 50(−) | 50(−) |

Antibacterial activity (cycle I) (log CFU/mL) | 4.77 | 6.19 | 4.40 | 4.80 | 4.18 | 4.87 | 4.25 | 6.11 | 4.50 | 4.51 |

Antibacterial activity (cycle II) (log CFU/mL) | 4.90 | 6.03 | 4.59 | 5.01 | 4.01 | 4.69 | 4.40 | 5.95 | 4.65 | 4.71 |

Difference (cycle I-cycle II) (log CFU/mL) | −0.13 | 0.16 | −0.19 | −0.21 | 0.17 | 0.18 | −0.15 | 0.16 | −0.15 | −0.20 |

Average activity (log CFU/mL) | 4.835 (a_{10}) |
6.110 (a_{11}) |
4.495 (a_{12}) |
4.905 (a_{13}) |
4.095 (a_{14}) |
4.780 (a_{20}) |
4.325 (a_{21}) |
6.030 (a_{22}) |
4.575 (a_{23}) |
4.610 (a_{24}) |

E_{10} to E_{24} = experiments;

Numbers in parentheses are the coded symbols of levels of the extraction conditions.

Calculation worksheet of the effects of the three-variable system and magnitude of effects and error limits of Set II.

Experimental Conditions | Calculation of Effects |
---|---|

Temperature | −0.8363 |

Time | −1.0238 |

Ethanol concentration | 0.0913 |

Temperature × time | 0.4938 |

Temperature × ethanol concentration | 0.1888 |

Time × ethanol concentration | −0.0338 |

Temperature × time × ethanol concentration | −0.0163 |

Change in mean effect | 0.0685 |

Standard deviation (σ) | 0.1140 |

Error limits: | |

for average | 0.1612 |

for effects | 0.1145 |

for change in mean | 0.1016 |

R_{1}: (largest difference − smallest difference) in block 1; R_{2}: (largest difference − smallest difference) in block 2; f_{k,n} = constant depending on number of replications (n) and number of experiments (k) per cycle = 0.3 for n = 2 and k = 5.

Experimental design for the three inducer system and results of Set III.

Experimental Conditions | E_{10} |
E_{11} |
E_{12} |
E_{13} |
E_{14} |
E_{20} |
E_{21} |
E_{22} |
E_{23} |
E_{24} |
---|---|---|---|---|---|---|---|---|---|---|

Temperature (°C) | 80(0) | 65(−) | 65(−) | 95(+) | 95(+) | 80(0) | 95(+) | 65(−) | 95(+) | 65(−) |

Time (h) | 26(0) | 20(−) | 32(+) | 20(−) | 32(+) | 26(0) | 32(+) | 20(−) | 20(−) | 32(+) |

Ethanol concentration (%) | 50(0) | 25(−) | 75(+) | 75(+) | 25(−) | 50(0) | 75(+) | 75(+) | 25(−) | 25(−) |

Antibacterial activity (cycle I) (log CFU/mL) | 4.21 | 4.38 | 4.51 | 4.41 | 4.4. | 3.96 | 4.24 | 4.65 | 4.50 | 4.39 |

Antibacterial activity (cycle II) (log CFU/mL) | 4.03 | 4.57 | 4.39 | 4.50 | 4.20 | 4.16 | 4.35 | 4.44 | 4.37 | 4.49 |

Difference (cycle I-cycle II) (log CFU/mL) | 0.18 | −0.15 | 0.12 | −0.09 | 0.20 | −0.20 | −0.11 | 0.21 | 0.13 | −0.10 |

Average activity (log CFU/mL) | 4.120 (a_{10}) |
4.475 (a_{11}) |
4.450 (a_{12}) |
4.455 (a_{13}) |
4.300 (a_{14}) |
4.060 (a_{20}) |
4.295 (a_{21}) |
4.545 (a_{22}) |
4.435 (a_{23}) |
4.440 (a_{24}) |

E_{10} to E_{24} = experiments;

Numbers in parentheses are the coded symbols of levels of the extraction conditions.

Calculation worksheet of the effects of the three-variable system and magnitude of effects and error limits of Set III.

Experimental Conditions | Calculation of Effects |
---|---|

Temperature | −0.1063 |

Time | −0.1063 |

Ethanol concentration | 0.0238 |

Temperature × time | −0.0413 |

Temperature × ethanol concentration | −0.0163 |

Time × ethanol concentration | −0.0213 |

Temperature × time × ethanol concentration | 0.0088 |

Change in mean effect | 0.2675 |

Standard deviation (σ) | 0.1140 |

Error limits: | |

for average | 0.1612 |

for effects | 0.1145 |

for change in mean | 0.1016 |

R_{1}: (largest difference − smallest difference) in block 1; R_{2}: (largest difference − smallest difference) in block 2; f_{k,n} = constant depending on number of replications (n) and number of experiments (k) per cycle = 0.3 for n = 2 and k = 5.