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Vanillin was extracted from vanilla beans using pretreatment with cellulase to produce enzymatic hydrolysis, and response surface methodology (RSM) was applied to optimize the processing parameters of this extraction. The effects of heating time, enzyme quantity and temperature on enzymatic extraction of vanillin were evaluated. Extraction yield (mg/g) was used as the response value. The results revealed that the increase in heating time and the increase in enzyme quantity (within certain ranges) were associated with an enhancement of extraction yield, and that the optimal conditions for vanillin extraction were: Heating time 6 h, temperature 60 °C and enzyme quantity 33.5 mL. Calculated from the final polynomial functions, the optimal response of vanillin extraction yield was 7.62 mg/g. The predicted results for optimal reaction conditions were in good agreement with experimental values.
Vanilla (from
Interest in the use of naturallyoccurring flavors has increased dramatically in recent years, but since these natural flavorings belong to several different chemical classes, various extraction techniques must be used in order to obtain them [
Response surface methodology (RSM) is a collection of statistical and mathematical techniques useful for the improvement and optimization of complex processes. The main advantage of RSM is its ability to reduce the number of experimental trials needed to evaluate multiple parameters and their interactions, in order to provide sufficient information for statistically acceptable results [
Based on our preliminary experiments using this enzymatic technique, we have identified heating time, enzyme quantity and temperature to be key factors influencing extraction of vanillin. The overall objective of the present study was to investigate the effects of heating time, enzyme quantity and temperature on vanillin extraction, and to optimize the processing conditions using RSM to obtain high levels of vanillin.
The experiments were carried out in a random order. The experimental plan was determined according to the quadratic regression rotation combination design. Values obtained from the extraction process are presented in
Design program and experimental results of RSM.
Exp. number  Heating Time  Enzyme quantity  Temperature  Extraction yield 

/X_{1}(h)  /X_{2} (mL)  /X_{3}(°C)  /(mg/g)  
1  7.78  43.31  77.8  6.96 
2  7.78  43.31  42.16  6.94 
3  7.78  23.69  77.8  7.17 
4  7.78  23.69  42.16  7.25 
5  4.22  43.31  77.8  7.08 
6  4.22  43.31  42.16  7.04 
7  4.22  23.69  77.8  7.01 
8  4.22  23.69  42.16  7.19 
9  9  33.5  60  7.11 
10  3  33.5  60  7.01 
11  6  50  60  7.13 
12  6  17  60  7.31 
13  6  33.5  90  7.27 
14  6  33.5  30  7.41 
15  6  33.5  60  7.63 
16  6  33.5  60  7.53 
17  6  33.5  60  7.59 
18  6  33.5  60  7.65 
19  6  33.5  60  7.57 
20  6  33.5  60  7.67 

0.95  
1.02 
Variance analysis of extraction yield.
Parameter  DF  Estimate  Standard Error  t Value  Parameter Estimate  

From coded data  
Intercept  1  2.24726  0.668079  3.36  0.0072  7.609897 
X_{1}  1  0.904467  0.103066  8.78  <.0001  0.020774 
X_{2}  1  0.108797  0.018803  5.79  0.0002  −0.111128 
X_{3}  1  0.032427  0.010301  3.15  0.0104  −0.053873 
1  −0.067581  0.006165  −10.96  <0.0001  −0.608226  
X_{1}X_{2}  1  −0.003150  0.001506  −2.09  0.063  −0.155912 
1  −0.001647  0.000204  −8.09  <0.0001  −0.448465  
X_{1}X_{3}  1  0.000316  0.000829  0.38  0.7114  0.028413 
X_{2}X_{3}  1  0.000229  0.00015  1.52  0.1592  0.113291 
1  −0.000365  0.000061626  −5.92  0.0001  −0.328379 
Variance analysis of the secondorder regression model on extraction yield.
Regression  DF  Sum of Squares  RSquare  

Linear  3  0.75522  0.0626  4.55  0.0295 
Quadratic  3  1.036994  0.8601  62.44  <.0001 
Cross product  3  0.037809  0.0314  2.28  0.142 
Total Model  9  1.150324  0.9541  23.09  <.0001 
As shown in
The variation of extraction yield with enzyme quantity and temperature, at a constant heating time, is presented in
Threedimensional figures of interactive effects of heating time (X_{1}), enzyme quantity (X_{2}) and temperature (X_{3}) on extraction yield (Z). (
These data indicate that the linear effects of heating time (
The optimal processing parameters were determined in order to maximize the vanillin extraction yield. The extraction yield may be optimized from the figures (
The suitability of the model equation for predicting the optimum response values was tested using the recommended optimum conditions. This set of conditions was determined to be optimum by a RSM optimization approach, which was also used to experimentally validate and predict the value of the responses using model equations. The experimental values were found to be in agreement with the predicted ones. The optimal extraction yield of vanillin in this experiment was less than 8.06 mg/g, as has been described previously [
Vanilla beans (from cured vanilla pods of
Vanilla beans were ground in a centrifugal grinding mill (type ZM1) fitted with a 1 mm stainless steel sieve (Retsch, Haan, Germany). 10 g vanilla powder was mixed with 50 mL acetic acid buffer (pH 4.8) and cellulase in a 250 mL Erlenmeyer flask. The flask was sealed and placed in a water bath, and the process of enzymatic hydrolysis was allowed to proceed with stirring using a magnetic stirrer (IKA Works Inc., Wilmington, NC, USA). Heating time, cellulase quantity and water bath temperature were varied to allow their effects on vanillin liberation to be studied. After hydrolysis, the reaction was stopped by 10 min immersion in boiling water, and the mixture was allowed to cool. 200 mL ethanol was then added, and the mixture stirred at 50 rpm for 12 h at 35 °C. Following this, the mixture was centrifuged with a Z36 HK Super Speed Centrifuge (Labnet, USA) at 10,000 × g for 30 min at 20 °C, to separate the solid and liquid phases. 1 mL of liquid phase was removed for vanillin quantification using HPLC. The yield of vanillin extraction was expressed using Equation 2.
Vanillin levels were quantified using a slight modification of methods described previously [
According to our previous experimental findings, the most influential factors affecting extraction yield were heating time, enzyme quantity and temperature. In order to evaluate the effects and interactions of these three factors, RSM was used in designing the experiment. SAS software (version 9.0, SAS Institute Inc., USA) was used to generate the quadratic regression rotation combination design. The independent variables were heating time (X_{1}), enzyme quantity (X_{2}) and temperature (X_{3}). Each independent variable had coded levels of −1.682, −1, 0, 1 and +1.682. This design was constructed based on a threefactor and a fivelevel Box–Wilson central composite design (CCD), leading to 20 sets of experiments, allowing each experimental response to be optimized. The experimental designs with regard to the coded (X) and actual heating times, enzyme quantities and temperature levels are shown in
where Z is the response value calculated by the model; X_{1}, X_{2} and X_{3} are coded variables, corresponding to heating time, enzyme quantity and temperature, respectively. a_{1}, a_{2} and a_{3} are the linear; a_{11}, a_{22} and a_{33} are the quadratic; and a_{12}, a_{13} and a_{23} are the crossproduct effects of the X_{1}, X_{2} and X_{3} factors on the response.
Analysis of variance (ANOVA) was performed, ANOVA tables generated, and the effects and regression coefficients of individual linear, quadratic and interaction terms were determined. The statistical significance of the regression coefficients was determined using a Ttest, and the applicability of the model was assessed using coefficient of determination (
Experimental design for the optimization of vanillin extraction.
Code levels  Variables  

X_{1}/Heating time (h)  X_{2}/Enzyme quantity (mL)  X_{3}/Temperature (°C)  
r = +1.682  9  50  90 
1  7.78  43.31  77.8 
0  6  33.5  60 
−1  4.22  23.69  42.2 
−r = −1.682  3  17  30 
△j  1.78  9.81  17.8 
Response surface methodology is a useful tool to investigate the optimal conditions with regard to heating time, enzyme quantity and temperature, for extracting vanillin from vanilla beans. The coefficient of determination (
This study was funded by the Chinese Central PublicInterest Scientific Institution Basal Research Fund (1630052012001) and The National Key Technology R&D Program of China (2012BAD36B03).