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Response surface methodology (RSM) along with central composite design (CCD) was applied to optimize the freeze drying conditions for purified pectinase from mango (

In terms of production and consumer acceptance, mango is universally a very important and popular tropical fruit and tops the super fruit list [

^{2}^{2} values. As presented in the table, the significant (^{2} and adjusted ^{2} value varied from 0.944–0.997 and 0.972–0.989, respectively and were obtained for all response variables studied. In addition, no significant (_{1}), Arabic gum (X_{2}) and maltodextrin (X_{3}) on each of the response variables. The significance of the

The pectinase activity was significantly (

The results (

One of the desirable outcomes of freeze drying optimization is the higher stability of the enzyme during storage. The results indicate that stability of pectinase is significantly increased in the presence of coating agents. Based on the results (

The target of optimization is to achieve optimum levels of independent variables that result in desirable response goals so the individual and overall optimization procedures were performed to achieve this target. To determine the optimum region, numerical and graphical optimization procedures were employed. Graphical optimization (using 3D surface plot) was used to determine the optimum region and numerical optimization was employed to determine the exact optimum points. In addition, a comparison was made between the experimental data and predicted value due to investigate the adequacy of the response surface equations [

The adequacy of the response surface equations is indicated by a comparison between the experimental value and the predicted data. The comparison is done by generating a fitted-line plot (with experimental values on X-axis and predicted values on Y-axis) for the results obtained, showing how close it is to or how far it deviates from the fitted line. ^{2} (>0.95) for fitted line plots and overall closeness of these variables, thus indicating that the response surface model is adequate for predicting the varied enzymatic properties as functions of the conditions in freeze drying.

Mango fruits (

One hundred and five kilograms of mango were washed with distilled water and then were peeled with stainless steel knife. Subsequently, mango peel (five hundred grams) was cut into small pieces (3 mm × 3 mm × 1 mm) and blended with a commercial laboratory blender 32BL79 (Dynamic Corporation of America, Torrington, CT, USA) in 50 mL of sodium hydrogen phosphate (pH 4.3) at 4 °C for 4 min. Following this, the homogenate sample was filtered using cheesecloth to remove plant tissue.

Aqueous two-phase system (ATPS) based polyethylene glycol (PEG) and salt was used to purify pectinase from mango peel. The highest purification factor (13.2) and yield (96.7%) of pectinase was achieved in concentration of 14% (w/w) PEG 4000 and 14% (w/w) potassium phosphate at pH 7.0 with addition of 3% (w/v) of NaCl to 25% (w/w) of feedstock [

Different concentrations of Arabic gum (−1.21, 10.21%, w/v) and maltodextrin (0.73, 7.26%, w/v) were prepared and added to (−2.66, 62.66 mg/mL) of purified pectinase. Subsequently, the mixtures were frozen at −40 °C for 24 h before the freeze drying. Then, frozen samples were lyophilised at −40 °C for 24 h on a VIRTIS Genesis freeze dryer.

The pectinase activity was determined according to the procedure of Miller [

Yield of pectinase was determined by dividing pectinase activity after freeze drying (E) to activity of enzyme before encapsulation (F) and multiplying of result to 100 [

The storage of purified pectinase was for a predetermined period of one week at a temperature of 4 °C. Storage efficiency, which is the ratio of enzyme activity after storage to its initial activity multiplied 100, determined the storage stability (

The response surface methodology (RSM) has been identified as an important method in the statistical design. Collection of mathematical and statistical method useful for experimental design, optimization procedure and analysis of data is RSM [_{1}), yield of enzyme (Y_{2}) and storage stability (Y_{3}) of pectinase from mango peel. It should be noted, the corresponding experimental design and the concentration of coating agents range were considered based on preliminary studies. Twenty purified enzymes were encapsulated based on central composite design, including eight factorial points, six axial points (±α) and six center points (

where, Y is the response variable, β_{0}, β_{1}, β_{2}, β_{3}, β_{11}, β_{22}, β_{33}, β_{12}, β_{13} and β_{23} are the regression coefficient for constant, linear, quadratic and interaction effect, respectively. The adequacy of polynomial equations was investigated by determination of coefficient (^{2}) [^{2} of at least 0.80 is proof of a good model fit while larger values of absolute

In this study, response surface methodology was used to investigate the main and interaction effects of important independent variables on activity and stability of pectinase from mango peel. Main and interaction effect of pectinase content, Arabic gum and maltodextrin showed a significant (

_{3}-alginate composite gel

Response surface plots showing the significant (

Fitted line plot indicating the closeness between predicted values (Y1) and experimental value (Y0) for pectinase activity (

Regression coefficients ^{2}, adjusted ^{2} and probability values of the response surface models.

Regression Coefficient | Pectinase Activity (Y_{1}, U/mL) |
Yield (Y_{1}, %) |
Storage Stability (Y_{3}, %) |
---|---|---|---|

b_{0} |
87.13 | 11.12 | 83.68 |

b_{1} |
8.50 | 1.75 | 3.50 |

b_{2} |
6.00 | 0.25 | 6.00 |

b_{3} |
6.44 | 0.49 | 6.44 |

b_{1}^{2} |
5.68 | 1.68 | 6.67 |

b_{2}^{2} |
26.40 | 1.69 | 24.99 |

b_{3}^{2} |
26.88 | 3.60 | 20.98 |

b_{12} |
6.00 | 0.37 | 6.00 |

b_{13} |
1.50 | 1.00 | 1.50 |

b_{23} |
9.00 | 0.25 | 14.00 |

^{2} |
0.994 | 0.997 | 0.991 |

^{2} ( |
0.981 | 0.989 | 0.972 |

Regression ( |
0.000 |
0.000 |
0.000 |

Lack of fit ( |
0.106 |
0.322 |
0.262 |

b_{i}: The estimated regression coefficient for the main linear effects; b_{i}^{2}: The estimated regression coefficient for quadratic effects; b_{ij}: The estimated regression coefficient for the interaction effects; 1: Pectinase content; 2: Arabic gum; 3: Maltodextrin;

Significant (

No significant (

F-ratio and

Variables | Main Effects | Quadratic Effects | Interaction Effects | |||||||
---|---|---|---|---|---|---|---|---|---|---|

| ||||||||||

X_{1} |
X_{2} |
X_{3} |
X_{1}^{2} |
X_{2}^{2} |
X_{3}^{2} |
X_{1}X_{2} |
X_{1}X_{3} |
X_{2}X_{3} | ||

Pectinase | 0.001 |
0.000 |
0.011 |
0.003 |
0.004 |
0.003 |
0.002 |
0.372 | 0.000 | |

Activity (Y_{1}, U/mL) |
F-ratio | 30.80 | 147.86 | 15.36 | 29.5 | 34.56 | 19.83 | 274.56 | 0.963 | 27.87 |

| ||||||||||

Yield (Y_{2}, %) |
0.000 |
0.001 |
0.000 |
0.033 |
0.019 |
0.002 |
0.178 | 0.009 |
0.000 | |

F-ratio | 528.54 | 69.88 | 104.24 | 10.24 | 14.28 | 54.02 | 2.65 | 22.84 | 564.06 | |

| ||||||||||

Storage Stability (Y_{3}, %) |
0.004 |
0.000 |
0.000 |
0.200 | 0.010 |
0.042 |
0.016 |
0.410 |
0.000 | |

F-ratio | 24.80 | 108.16 | 139.94 | 2.18 | 16.72 | 4.36 | 12.88 | 0.79 | 70.05 |

X_{1}, X_{2} and X_{3}: The main effect of pectinase content, Arabic gum and maltodextrin, respectively; X_{1}^{2}, X_{2}^{2} and X_{3}^{2}: The quadratic effect of pectinase content, Arabic gum and maltodextrin, respectively; X_{1}X_{2}: The interaction effect of pectinase content and Arabic gum; X_{1}X_{3}: The interaction effect of pectinase content and maltodextrin; X_{2}X_{3}: The interaction effect of Arabic gum and maltodextrin.

Significant (

Level of independent variables established according to central composite design (CCD).

Independent Variable Levels | Independent Variables Levels | ||||
---|---|---|---|---|---|

| |||||

Axial (−α) | Low | Center | High | Axial (+α) | |

Pectinase content (mg/mL) | −2.66 | 10 | 30 | 50 | 62.66 |

Arabic gum (%, w/v) | −1.21 | 1 | 4.5 | 8 | 10.21 |

Maltodextrin(%, w/v) | 0.73 | 2 | 4 | 6 | 7.26 |

Matrix of central composite design (CCD).

Treatment | Block | Pectinase Content (mg/mL) | Arabic Gum (%, w/v) | Maltodextrin (%, w/v) |
---|---|---|---|---|

1 |
1 | 30.00 | 4.50 | 4.00 |

2 |
1 | 30.00 | 4.50 | 4.00 |

3 | 1 | 10.00 | 8.00 | 6.00 |

4 | 1 | 10.00 | 1.00 | 2.00 |

5 | 1 | 50.00 | 1.00 | 6.00 |

6 | 1 | 50.00 | 8.00 | 2.00 |

7 | 2 | 30.00 | 4.50 | 0.73 |

8 | 2 | 30.00 | 4.50 | 7.26 |

9 | 2 | 30.00 | 10.21 | 4.00 |

10 | 2 | 62.66 | 4.50 | 4.00 |

11 |
2 | 30.00 | 4.50 | 4.00 |

12 |
2 | 30.00 | 4.50 | 4.00 |

13 | 3 | 30.00 | −1.21 | 4.00 |

14 | 3 | −2.66 | 4.50 | 4.00 |

15 |
3 | 30.00 | 4.50 | 4.00 |

16 | 3 | 10.00 | 1.00 | 6.00 |

17 | 3 | 10.00 | 8.00 | 2.00 |

18 | 3 | 50.00 | 1.00 | 2.00 |

19 |
3 | 30.00 | 4.50 | 4.00 |

20 | 3 | 50.00 | 8.00 | 6.00 |

centre point.