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Optimizing conditions for the purification of crude octacosanol extract from rice bran wax by molecular distillation analyzed using response surface methodology
Journal of Food Engineering 70 (2005) 47–53 www.elsevier.com/locate/jfoodeng
Optimizing conditions for the purification of crude octacosanol extract from rice bran wax by molecular distillation analyzed using response surface methodology Fang Chen, Tongyi Cai, Guanghua Zhao, Xiaojun Liao, Linyu Guo, Xiaosong Hu
*
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Received 11 March 2004; accepted 13 September 2004 Available online 2 November 2004
Abstract The purification of crude octacosanol extract from transesterified rice bran wax was carried out by molecular distillation (MD). A central composite rotate design (CCRD) was used in order to optimize the experimental parameters: distilling temperature and vacuum degree. Through the response surface methodology (RSM), the optimal MD conditions were determined and the quadratic response surfaces were drawn from the mathematical models. The results suggest that the distillng temperature and vacuum degree significantly affected both octacosanol content and yield of purified products, while the interaction of distilling temperature and vacuum degree was not significant in the two models. The optimum conditions of the purification of crude octacosanol extract were: distilling temperature 176.1 C and vacuum degree 1.29 Torr. Optimal values predicted by RSM for the octacosanol content and yield of purified product were 25.93% and 51.09%, respectively. Close agreement between experimental and predicted values was obtained. 2004 Elsevier Ltd. All rights reserved. Keywords: Molecular distillation; Purification; Octacosanol; Rice bran wax; Response surface methodology
1. Introduction Octacosanol is one kind of higher aliphatic alcohol. It is a water-insoluble and stable compound. Its health benefits like blood lipid or cholesterol lowering (Arruzazabala et al., 1994; Kato et al., 1995), and athletic performance improving effects (Rapport, 2000; Shimura, Hasegawa, Takano, & Suzuki, 1987) have been proven with no side effects. Thus the high stability and encouragingly biological activities of octacosanol make it become a potential candidate for a supplement in foods, medicine and cosmetics. Octacosanol has been widely found in many plants, e.g. in leaf, bark and stem waxes of rye grass, apple peel, *
Corresponding author. Fax: +86 10 62343553. E-mail address: [email protected] (X. Hu).
0260-8774/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.09.011
and wheat germ (Kazuko, Kumlko, & Yohel, 1991). Recently, rice bran wax (RBW) has been reported to be one of the best sources containing this natural compound (Tolloch, 1976). Chemically, the RBW is composed of esters with 46 up to 60 carbon atoms, in which the most of aliphatic alcohols and aliphatic acids have 26–30 carbon atoms (Wang, Zhou, & Ni, 1998). To extract and purify octacosanol from RBW, column chromatography and recrystallization are popular methods (Steve, George, Ezra, Kirkpatrick, & Darwin, 1963). However, a serious pollution and low safety due to a lot of organic liquid used in these purified methods has become the largest obstacle for their application in food and pharmaceutical industries. Unlike these conventional methods, molecular distillation (MD) can avoid using any organic solvents in the purification, even if it can remove any harmful solvent
48
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
residues in the products, resulting in the generation of a much smaller waste and higher safety (Christensen & Reineccius, 1995; Wu & Zhang, 2000). MD is a special case of short-path distillation in which the distance between evaporating and condensing surfaces is less than the mean free path of the molecules involved in high vacuum (Feng, Yang, & Yu, 2002; Ridway, 1956). This technology is considered as one of the best methods separating and purifying natural product, especially for substances with high-molecular mass, high viscosity and high melting point (Armando, Paolo, & Carlo, 1994; Ooi, Choo, Yap, Basiron, & Ong, 1994). On the other hand, response surface methodology (RSM) is effective for responses that are influenced by many factors and their interactions, which was originally described by Box and Wilson (1951). Many studies indicated that it is useful for developing, improving and optimizing processes (Atkinson & Donev, 1992; San Martin, Jaime, & Martinez, 2003). In the present study, MD is used as a main method for the purification of crude octacosanol extract from transesterified RBW, and its working conditions such as distilling temperature and vacuum degree is optimized by RSM in order to obtain the highest octacosanol content, at the same time, to maintain the yield in the purified product as large as possible. The detailed process and the effect of MD conditions on the purification are described through mathematical model for the first time, although a few of references have referred to purification of crude octacosanol extract by MD (Feng et al., 2002).
Fig. 1. Schematic diagram of molecular distillation apparatus of falling film.
Table 1 Central composite design for the prediction and optimization of purification of octacosanol crude extracts by MD
2. Materials and methods 2.1. Materials RBW was obtained from a local rice bran oil processor. All solvents/chemicals used were of analytical grade or GC grade. Ethanol, n-butanol, cyclohexane, octacosanol, and 1,3,5-triphenylbenzene (TPB) were purchased from Sigma Chemical Co. (Beijing, PR China). The MD apparatus (2000 ml) (Pope Scientific Co, USA) was used for purification and its schematic diagram is shown in Fig. 1. 2.2. Experimental design
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Independent variables
Response variablesa
Temperature (C) (X1)
Vacuum degree (Torr) (X2)
Octacosanol content (%) (Y1)
Purification yield (%) (Y2)
200 (1)b 200 (1) 180 (1) 180 (1) 204.14 (1.414) 175.86 (1.414) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0)
1.5 (1) 0.5 (1) 1.5 (1) 0.5 (1) 1.0 (0) 1.0 (0) 1.707 (1.414) 0.293 (1.414) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0)
17.28 16.02 21.19 17.55 15.3 28.15 23.37 15.82 22.95 22.65 22.93 23.17 22.52 22.84 23.25 22.41
62.9 80.07 50.68 61.05 83.82 40.51 44.36 82.69 56.33 50.27 51.42 51.93 56.46 57.35 49.08 52.97
a
RSM was applied to determine the working conditions of MD for the purification of crude octacosanol extract from RBW. CCRD was used to investigate effects of two independent variables (purification conditions), distilling temperature (X1) and vacuum degree (X2), on dependant variables (Yn) of the purification. Coded and original levels for process or independent
Mean values of triplicate determinations. Numbers in parentheses are coded symbols for levels of independent parameters as per the text. b
variables are shown in Table 1. The correspondence between the coded and actual values can be obtained using the following formula:
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
Z ¼ ðX X 0 Þ=DX ;
ð1Þ
where Z is the coded value, X is the corresponding actual value, X0 is the actual value in the center of the domain, and DX is the increment of X corresponding to 1 unit of Z. In our experiment the Z1 (coded value of distilling temperatures) and Z2 (coded value of vacuum degrees) were given by Eqs. (2) and (3): Z 1 ¼ ðX 1 190Þ=10;
ð2Þ
Z 2 ¼ ðX 2 190Þ=10:
ð3Þ
The complete design consisted of 16 experimental points including eight replications of the center points, and the triplicate purifications were performed at all design points in randomized order. 2.3. Extraction of crude octacosanol extract from transesteried RBW The transesterification reaction of the RBW was carried out in a round-bottomed flask equipped with a temperature controller and a stainless-steel double-arm blender. At first, the RBW was added to the flask, and heated until it completely melted. A n-butanol solution containing 0.2% KOH was added to the melted RBW with continuous stirring. The above solution was refluxed for 8 h. The reaction mixtures were cooled to 0 C, and subsequently washed with distilled water to neutral. The resulting dried solid was extracted with ethanol at 70 C to obtain the crude octacosanol extract (containing 12.12% octacosanol). 2.4. Purification of crude octacosanol extract by MD The MD was used to further purify crude octacosanol extract obtained from transesterified RBW. Our preliminary studies (data not shown) indicated that the distilling temperature and vacuum degree were two major factors responsible for the further purification of crude octacosanol extract, while other factors had little effects on it. Therefore, the flow rate of feed, temperature of condensing surface, and rotate rate of scraper were not included as CCRD factors, and they were set at 3 ml/min, 90 C, and 50 rpm respectively. The feed was heated to melt and admitted into still. After setting the parameters, feeding valve was turned on, and the degassed feed liquid was immediately forced quickly down the evaporating surface and spread to a very thin film by scraper. Heated walls and high vacuum drive the more volatile components (lighter components) to the closely positioned internal condensing surface (distillates) as the less volatile components (heavier components) continue down the cylinder (residues). The resulting fractions, thus separated, exit through indivi-
49
dual discharge outlets. The distillates were collected to calculate the yield and determine octacosanol content. 2.5. Determination of octacosanol content by GLC On the basis of the methods of Kazuko et al. (1991) and Gonzalez, Magraner, and Acosta (1996), 0.200 g TPB was dissolved in 100.00 ml cyclohexane. The TPB concentration in this solution was 2.000 mg/ml. Calibration curve were obtained by injecting standard solutions with concentrations of octacosanol ranging from 100 lg/ml to 900 lg/ml. 0.025 g of sample, which was obtained by transesterification in conjunction with MD from RBW, was dissolved in 3.0 ml cyclohexane under the help of ultrasonic wave at 40 C. Then 1.5 ml of 2.000 mg/ml TPB solution was added to the above solution as internal standard. The resulting solution was made up to 5.0 ml with cyclohexane before being subjected to GLC analysis. GLC was performed with a Hewlett Packard 6890 A and a HP-5 (column 30 m · 320 lm · 0.25 lm). The gas flow rates for N2, H2 and air were 45, 40, 450 ml/min, respectively. The operating temperatures were set as follows: injector, 320 C; detector, 330 C; initial oven temperature 230 C, keeping 6 min, with a ramp rate 10 C/min to 280 C, keeping 20 min, then with a ramp rate 20 C/min to 300 C. 2.6. Statistical analysis The corresponding purified products were subjected to analyze for dependent variables (responses), such octacosanol content (Y1) and purification yield (Y2). Mean value of triplicate determines was analyzed to fit the following second-order polynomial models to all dependent Y variables. The model proposed for each response of Y is Y ¼ A0 þ A1 X 1 þ A2 X 2 þ A11 X 21 þ A12 X 1 X 2 þ A22 X 22
ð4Þ
where X1 and X2 correspond to independent variables, namely, distilling temperature and vacuum degree, respectively. The An values represent corresponding regression coefficients. The data were analyzed by RSM using Statistica software version 6.0 (Statsoft Inc., Tulsa, OK, USA). The significance of the model was tested using variance analysis (F-test). The effects of the variables were displayed in surface graphs. Response surface and contour plots were developed using fitter polynomial equations. When the results showed a saddle point in response surfaces, optimal conditions were determined using analysis of ridge. Then subsequent confirmatory experiments were carried out to validate their equations.
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F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
3. Results and discussion Octacosanol content (Y1) and purification yield (Y2) for each set of variable combinations during MD were showed in Table 1. Multiple regression coefficients were calculated by employing the least square technique to predict quadratic polynomial models for Y1 and Y2. The regression analysis for the two responses indicated that results were highly significant (p < 0.01) under the models, respectively, therefore they could be used for explanation of the responses observed. 3.1. Octacosanol content
(Z1Z2) and quadratic term of distilling temperature ðZ 22 Þ was not significant; nevertheless, also they had been kept in the equation in order to diminish the errors. Table 3 showed the variance analysis of the secondorder regression model on the octacosanol content of purified product. The complete quadratic model showed an excellent fit; furthermore, it appeared to reasonably represent the data. Thus, the responses were sufficiently explained by the regression equation. In order to make it more directly express the effect of the distilling temperature and vacuum degree, we take Eqs. (2) and (3) into Eq. (5) and get Eq. (6) as follows: Y 1 ¼ 398:54 þ 4:8X 1 þ 44:68X 2 0:01X 21
To examine conditions that might affect the octacosanol content (Y1) of purified product, its regression model was predicted by Eq. (5) as follows: Z ¼ 22:84 1:27Z 1 þ 1:95Z 2 0:60Z 1 Z 2 þ 3:01Z 21 1:69Z 22
ð5Þ
The analysis of variance for this model was given in Table 2. There was a high significant regression (a < 0.01) relationship between the dependent variable (Y1), which responds to the octacosanol content of purified product, and the two responding variables, Z1 and Z2, which represent distilling temperature and vacuum degree, respectively. According to this model, linear terms of distilling temperature (Z1, a < 0.01) and vacuum degree (Z2, a < 0.01), and quadratic term of vacuum degree ðZ 22 ; a < 0:01Þ reached high significant. The result suggested that the change of distilling temperature and vacuum degree had a significant effect on the octacosanol content of purified product. In contrast, the interaction of distilling temperature and vacuum degree
0:12X 1 X 2 9:09X 22
ð6Þ
The response surface of octacosanol content (Y1), distilling temperature (X1), and vacuum degree (X2) was shown in Fig. 2. From this three-dimensional plot, the region of high content can be easily identified and the effect of distilling temperature and vacuum degree on the octacosanol content of purified product were reflected: the octacosanol content reduced as the distilling temperature increased, possibly because higher distilling temperature reduced the effective mean free path of the molecules more sharply while causing more collisions in the gap between the evaporating and condensing surfaces (Wang & Xu, 2002), resulting in more amount of components can be condensed. The vacuum degree had different effects on the octacosanol content. Under less vacuum degree the octacosanol content increased by increasing the vacuum degree, in contrast, the octacosanol content decreased by increasing the vacuum degree under greater vacuum degree. This was related to the changes of effective mean free path of the molecules under different vacuum.
Table 2 Estimated regression model of relationship between response variables (octacosanol content) and independent variables (X1, X2) Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
A1 A2 A1A2 A11 A22
1 1 1 1 1
69.69 30.33 1.42 11.91 41.77
69.69 30.33 1.42 11.91 41.77
17.81 7.75 3.04 0.36 10.68
4.96 4.96 4.96 4.96 4.96
10.04 10.04 10.04 10.04 10.04
** ** NS NS **
Notability:
**(a
< 0.01); NS: no significance.
Table 3 Variance analysis of the second-order regression model on octacosanol content of purified product Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
Regress Lack of fit Residual error Total error
5 3 10 15
155.10 30.89 39.12 194.22
31.02 10.30 3.91
7.93 3.28
3.33 3.71
5.64 6.55
** **
Notability:
**(a
< 0.01).
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
51
Fig. 3. Response surface for the effect of temperature and vacuum degree on purification yield by MD. Fig. 2. Response surface for the effect of temperature and vacuum degree on octacosanol content of purified product by MD.
3.2. Purification yield of crude octacosanol extract The regression model of purification yield of crude octacosanol extract was predicted by Eq. (7) as follows: Y 2 ¼ 53:73 þ 11:56Z 1 10:22Z 2 þ 4:43Z 21 1:70Z 1 Z 2 þ 5:11Z 22
ð7Þ
The result of variance analysis in Table 4 indicated that there was a high statistically significant multiple regression relationship (p < 0.01) between the whole independent variables and the response variable, as using the regression equation to describe the relation-
ship between those experimental factors and the response value. None of the whole model was shown to have lack of fit in Table 5. In this model, the values of response variable Y2 were affected significantly by the linear terms of distilling temperature (Z1, a < 0.01), vacuum degree (Z2, a < 0.01), quadratic terms of distilling temperature ðZ 21 ; a < 0:01Þ and vacuum degree (Z2, a < 0.01), while the interaction of distilling temperature and vacuum degree (Z1Z2) had no significant effect on the purification yield. In like manner, we take the Eqs. (2) and (3) into Eq. (7) and get Eq. (8) as follows: Y ¼ 1404:94 15:30X 1 þ 3:83X 2 þ 0:04X 21 0:34X 1 X 2 þ 20:17X 22
ð8Þ
Table 4 Estimated regression model of relationship between response variables (purification yield) and independent variables (X1, X2) Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
A1 A2 A1A2 A11 A22
1 1 1 1 1
1069.1 835.15 11.56 194.19 251.5
1069.1 835.15 11.56 194.19 251.5
39.41 30.79 0.57 6.45 8.46
4.96 4.96 4.96 4.96 4.96
10.04 10.04 10.04 10.04 10.04
** ** NS ** **
Notability: (**a < 0.01); NS: no significance.
Table 5 Variance analysis of the second-order regression model on purification yield Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
Regress Lack of fit Residual error Total error
5 3 10 15
2361.50 203.22 260.45 2621.95
472.3 67.74 26.04 174.8
18.14 2.60
3.33 3.71
5.64 6.55
** **
Notability: (**a < 0.01).
52
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
Table 6 Predicted and experimental values of the response at optimum conditions Response
Optimum conditions Distilling temperature (C)
Distilling vacuum (Torr)
Octacosanol content (%) Yield (%)
176.1 176.1
1.29 1.29
The influences of the distilling temperature and vacuum degree on the purification yield were shown in Fig. 3. There was a negative correlation between the yield and octacosanol content (Figs. 2 and 3). In other words, under certain conditions of MD the octacosanol content increased at the cost of decrease of purification yield. 3.3. Optimization According to the canonical analysis described by Khuri and Cornell (1987), the stationary points were located for the corresponding responses. In our study the search criteria were to obtain the highest octacosanol content while to maintain the yield of the purified product as large as possible. Therefore, the optimum MD conditions were obtained as 176.1 C and 1.29 Torr from ridge analysis, which correspond to the distilling temperature and the vacuum degree, respectively. The adequacy of the model equation for predicting the optimum response values was tested in the experiments using the above MD conditions. Predicted and experimental values were given in Table 6. The experimental values (25.93% for the octacosanol content and 51.09% for the yield) were in perfect agreement to the predicted values (25.36% for the octacosanol content and 51.53% for the yield), a result confirming the adequacy of the predicted models (Table 6).
4. Conclusion RSM was effective for estimating the effect of the distilling temperature and vacuum degree of MD, as well as determining the optimal conditions. Response surface were drawn from the regression models to visualize the robustness of the purification method. Moreover, MD was a valuable alternative technique for the purification of crude octacosanol extract from RBW. The optimal parameters in the process of the purification were determined as the distilling temperature 176.1 C and vacuum degree 1.29 Torr, respectively.
Acknowledgement The project was supported by the China Agricultural Science and Education Funds (No. 2001-01-E02), China
Predicted value
Experimental value
25.36 51.53
25.93 51.09
High-Tech (863) Project (No. 2002AA248011) and Beijing Natural Science Foundation (No. 6041003).
References Armando, L., Paolo, B., & Carlo, M. (1994). A new short path distillation system applied to the reduction of cholesterol in butter and lard. Journal of American Oil Chemists’ Society, 71(6), 609–614. Arruzazabala, M. L., Carbajal, D., Mas, R., Molina, V., Valdes, S., & Laguna, A. (1994). Cholesterol-lowering effects of policosanol in rabbits. Biological Research, 27, 205–208. Atkinson, A. C., & Donev, A. N. (1992). Optimum experimental designs. Oxford: Oxford University Press (pp. 132–189). Box, G. E. P., & Wilson, K. G. (1951). On the experimental attainment of optimum conditions. Journal of the Royal Statistical Society, 13, 1–45. Christensen, K. R., & Reineccius, G. A. (1995). Aroma extract dilution analysis of aged Cheddar cheese. Journal of Food Science, 60(2), 212–220. Feng, W., Yang, C., & Yu, H. (2002). Molecular distillation technology and daily-use chemical industry: (I) The principle and characteristics of molecular distillation. Chemical Industry and Engineering Progress, 32(5), 74–77 (in Chinese). Gonzalez, B. L. J., Magraner, H. Z. L., & Acosta, G. P. C. (1996). Analytical procedure for the determination of 1-octacosanol in plasma by solvent extraction and capillary gas chromatography. Journal of Chromatography B, 682, 359–363. Kato, S., Karino, K., Hasegawa, J., Nagasaki, A., Eguchi, M., & Ichinose, T. (1995). Octacosanol affects lipid metabolism in rats fed on a high fat diet. British Journal of Nutrition, 73, 433–442. Kazuko, K., Kumlko, A., & Yohel, H. (1991). Free primary alcohols in oils and waxes from germs, kernels and other components of nuts, seeds, fruits and cereals. Journal of the American Oil Chemists’ Society, 68(11), 869–872. Khuri, A. A., & Cornell, J. A. (1987). Response surface: design and analyses. Marcel Dekker Inc. (pp. 1–17, 254). Ooi, C. K., Choo, Y. M., Yap, S. C., Basiron, Y., & Ong, A. S. H. (1994). Recovery of carotenoids from palm oil. Journal of American Oil Chemists’ Society, 71(4), 423–426. Rapport, L. J. (2000). Nutraceuticals (3): octacosanol. Pharmaceutical Journal, 265, 170–171. Ridway, W. (1956). Molecular distillation. London: Chapman and Hall Press (pp. 1–63). San Martin, M. E., Jaime, F. M. R., & Martinez, B. F. (2003). Selective nixtamalization of fractions of maize grain (Zea mays L.) and their use in the preparation of instant tortilla flours analyzed using response surface methodology. Cereal Chemistry, 80(1), 13–19. Shimura, S., Hasegawa, T., Takano, S., & Suzuki, T. (1987). Studies on the effect of octacosanol on motor endurance in mice. Nutritional Reproduction International, 36, 1029–1038. Steve, V. D., George, W., Ezra, L., Kirkpatrick, C. V., & Darwin, M. J. (1963). Pharmaceutical compositions comprising high molecular weight fatty alcohols and derivatives. UK 923047.
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53 Tolloch, A. P. (1976). Chemistry and biochemistry of natural waxes. Amsterdam: Elsevier (pp. 50–86). Wang, J., & Xu, S. (2002). Application status of molecular distillation technology. Chemical Industry and Engineering Progress, 21(7), 4–7 (in Chinese).
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Wang, Y., Zhou, H., & Ni, P. (1998). Analysis of components of rice bran wax. China Lipids, 23(6), 93–94 (in Chinese). Wu, P., Zhang, D., & Zhang Q. (2000). Principle and industrial application of short path distillation. Chemical Industry and Engineering Progress, (1), 4951 (in Chinese).
Optimizing conditions for the purification of crude octacosanol extract from rice bran wax by molecular distillation analyzed using response surface methodology Fang Chen, Tongyi Cai, Guanghua Zhao, Xiaojun Liao, Linyu Guo, Xiaosong Hu
*
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Received 11 March 2004; accepted 13 September 2004 Available online 2 November 2004
Abstract The purification of crude octacosanol extract from transesterified rice bran wax was carried out by molecular distillation (MD). A central composite rotate design (CCRD) was used in order to optimize the experimental parameters: distilling temperature and vacuum degree. Through the response surface methodology (RSM), the optimal MD conditions were determined and the quadratic response surfaces were drawn from the mathematical models. The results suggest that the distillng temperature and vacuum degree significantly affected both octacosanol content and yield of purified products, while the interaction of distilling temperature and vacuum degree was not significant in the two models. The optimum conditions of the purification of crude octacosanol extract were: distilling temperature 176.1 C and vacuum degree 1.29 Torr. Optimal values predicted by RSM for the octacosanol content and yield of purified product were 25.93% and 51.09%, respectively. Close agreement between experimental and predicted values was obtained. 2004 Elsevier Ltd. All rights reserved. Keywords: Molecular distillation; Purification; Octacosanol; Rice bran wax; Response surface methodology
1. Introduction Octacosanol is one kind of higher aliphatic alcohol. It is a water-insoluble and stable compound. Its health benefits like blood lipid or cholesterol lowering (Arruzazabala et al., 1994; Kato et al., 1995), and athletic performance improving effects (Rapport, 2000; Shimura, Hasegawa, Takano, & Suzuki, 1987) have been proven with no side effects. Thus the high stability and encouragingly biological activities of octacosanol make it become a potential candidate for a supplement in foods, medicine and cosmetics. Octacosanol has been widely found in many plants, e.g. in leaf, bark and stem waxes of rye grass, apple peel, *
Corresponding author. Fax: +86 10 62343553. E-mail address: [email protected] (X. Hu).
0260-8774/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.09.011
and wheat germ (Kazuko, Kumlko, & Yohel, 1991). Recently, rice bran wax (RBW) has been reported to be one of the best sources containing this natural compound (Tolloch, 1976). Chemically, the RBW is composed of esters with 46 up to 60 carbon atoms, in which the most of aliphatic alcohols and aliphatic acids have 26–30 carbon atoms (Wang, Zhou, & Ni, 1998). To extract and purify octacosanol from RBW, column chromatography and recrystallization are popular methods (Steve, George, Ezra, Kirkpatrick, & Darwin, 1963). However, a serious pollution and low safety due to a lot of organic liquid used in these purified methods has become the largest obstacle for their application in food and pharmaceutical industries. Unlike these conventional methods, molecular distillation (MD) can avoid using any organic solvents in the purification, even if it can remove any harmful solvent
48
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
residues in the products, resulting in the generation of a much smaller waste and higher safety (Christensen & Reineccius, 1995; Wu & Zhang, 2000). MD is a special case of short-path distillation in which the distance between evaporating and condensing surfaces is less than the mean free path of the molecules involved in high vacuum (Feng, Yang, & Yu, 2002; Ridway, 1956). This technology is considered as one of the best methods separating and purifying natural product, especially for substances with high-molecular mass, high viscosity and high melting point (Armando, Paolo, & Carlo, 1994; Ooi, Choo, Yap, Basiron, & Ong, 1994). On the other hand, response surface methodology (RSM) is effective for responses that are influenced by many factors and their interactions, which was originally described by Box and Wilson (1951). Many studies indicated that it is useful for developing, improving and optimizing processes (Atkinson & Donev, 1992; San Martin, Jaime, & Martinez, 2003). In the present study, MD is used as a main method for the purification of crude octacosanol extract from transesterified RBW, and its working conditions such as distilling temperature and vacuum degree is optimized by RSM in order to obtain the highest octacosanol content, at the same time, to maintain the yield in the purified product as large as possible. The detailed process and the effect of MD conditions on the purification are described through mathematical model for the first time, although a few of references have referred to purification of crude octacosanol extract by MD (Feng et al., 2002).
Fig. 1. Schematic diagram of molecular distillation apparatus of falling film.
Table 1 Central composite design for the prediction and optimization of purification of octacosanol crude extracts by MD
2. Materials and methods 2.1. Materials RBW was obtained from a local rice bran oil processor. All solvents/chemicals used were of analytical grade or GC grade. Ethanol, n-butanol, cyclohexane, octacosanol, and 1,3,5-triphenylbenzene (TPB) were purchased from Sigma Chemical Co. (Beijing, PR China). The MD apparatus (2000 ml) (Pope Scientific Co, USA) was used for purification and its schematic diagram is shown in Fig. 1. 2.2. Experimental design
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Independent variables
Response variablesa
Temperature (C) (X1)
Vacuum degree (Torr) (X2)
Octacosanol content (%) (Y1)
Purification yield (%) (Y2)
200 (1)b 200 (1) 180 (1) 180 (1) 204.14 (1.414) 175.86 (1.414) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0) 190 (0)
1.5 (1) 0.5 (1) 1.5 (1) 0.5 (1) 1.0 (0) 1.0 (0) 1.707 (1.414) 0.293 (1.414) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0) 1.0 (0)
17.28 16.02 21.19 17.55 15.3 28.15 23.37 15.82 22.95 22.65 22.93 23.17 22.52 22.84 23.25 22.41
62.9 80.07 50.68 61.05 83.82 40.51 44.36 82.69 56.33 50.27 51.42 51.93 56.46 57.35 49.08 52.97
a
RSM was applied to determine the working conditions of MD for the purification of crude octacosanol extract from RBW. CCRD was used to investigate effects of two independent variables (purification conditions), distilling temperature (X1) and vacuum degree (X2), on dependant variables (Yn) of the purification. Coded and original levels for process or independent
Mean values of triplicate determinations. Numbers in parentheses are coded symbols for levels of independent parameters as per the text. b
variables are shown in Table 1. The correspondence between the coded and actual values can be obtained using the following formula:
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
Z ¼ ðX X 0 Þ=DX ;
ð1Þ
where Z is the coded value, X is the corresponding actual value, X0 is the actual value in the center of the domain, and DX is the increment of X corresponding to 1 unit of Z. In our experiment the Z1 (coded value of distilling temperatures) and Z2 (coded value of vacuum degrees) were given by Eqs. (2) and (3): Z 1 ¼ ðX 1 190Þ=10;
ð2Þ
Z 2 ¼ ðX 2 190Þ=10:
ð3Þ
The complete design consisted of 16 experimental points including eight replications of the center points, and the triplicate purifications were performed at all design points in randomized order. 2.3. Extraction of crude octacosanol extract from transesteried RBW The transesterification reaction of the RBW was carried out in a round-bottomed flask equipped with a temperature controller and a stainless-steel double-arm blender. At first, the RBW was added to the flask, and heated until it completely melted. A n-butanol solution containing 0.2% KOH was added to the melted RBW with continuous stirring. The above solution was refluxed for 8 h. The reaction mixtures were cooled to 0 C, and subsequently washed with distilled water to neutral. The resulting dried solid was extracted with ethanol at 70 C to obtain the crude octacosanol extract (containing 12.12% octacosanol). 2.4. Purification of crude octacosanol extract by MD The MD was used to further purify crude octacosanol extract obtained from transesterified RBW. Our preliminary studies (data not shown) indicated that the distilling temperature and vacuum degree were two major factors responsible for the further purification of crude octacosanol extract, while other factors had little effects on it. Therefore, the flow rate of feed, temperature of condensing surface, and rotate rate of scraper were not included as CCRD factors, and they were set at 3 ml/min, 90 C, and 50 rpm respectively. The feed was heated to melt and admitted into still. After setting the parameters, feeding valve was turned on, and the degassed feed liquid was immediately forced quickly down the evaporating surface and spread to a very thin film by scraper. Heated walls and high vacuum drive the more volatile components (lighter components) to the closely positioned internal condensing surface (distillates) as the less volatile components (heavier components) continue down the cylinder (residues). The resulting fractions, thus separated, exit through indivi-
49
dual discharge outlets. The distillates were collected to calculate the yield and determine octacosanol content. 2.5. Determination of octacosanol content by GLC On the basis of the methods of Kazuko et al. (1991) and Gonzalez, Magraner, and Acosta (1996), 0.200 g TPB was dissolved in 100.00 ml cyclohexane. The TPB concentration in this solution was 2.000 mg/ml. Calibration curve were obtained by injecting standard solutions with concentrations of octacosanol ranging from 100 lg/ml to 900 lg/ml. 0.025 g of sample, which was obtained by transesterification in conjunction with MD from RBW, was dissolved in 3.0 ml cyclohexane under the help of ultrasonic wave at 40 C. Then 1.5 ml of 2.000 mg/ml TPB solution was added to the above solution as internal standard. The resulting solution was made up to 5.0 ml with cyclohexane before being subjected to GLC analysis. GLC was performed with a Hewlett Packard 6890 A and a HP-5 (column 30 m · 320 lm · 0.25 lm). The gas flow rates for N2, H2 and air were 45, 40, 450 ml/min, respectively. The operating temperatures were set as follows: injector, 320 C; detector, 330 C; initial oven temperature 230 C, keeping 6 min, with a ramp rate 10 C/min to 280 C, keeping 20 min, then with a ramp rate 20 C/min to 300 C. 2.6. Statistical analysis The corresponding purified products were subjected to analyze for dependent variables (responses), such octacosanol content (Y1) and purification yield (Y2). Mean value of triplicate determines was analyzed to fit the following second-order polynomial models to all dependent Y variables. The model proposed for each response of Y is Y ¼ A0 þ A1 X 1 þ A2 X 2 þ A11 X 21 þ A12 X 1 X 2 þ A22 X 22
ð4Þ
where X1 and X2 correspond to independent variables, namely, distilling temperature and vacuum degree, respectively. The An values represent corresponding regression coefficients. The data were analyzed by RSM using Statistica software version 6.0 (Statsoft Inc., Tulsa, OK, USA). The significance of the model was tested using variance analysis (F-test). The effects of the variables were displayed in surface graphs. Response surface and contour plots were developed using fitter polynomial equations. When the results showed a saddle point in response surfaces, optimal conditions were determined using analysis of ridge. Then subsequent confirmatory experiments were carried out to validate their equations.
50
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
3. Results and discussion Octacosanol content (Y1) and purification yield (Y2) for each set of variable combinations during MD were showed in Table 1. Multiple regression coefficients were calculated by employing the least square technique to predict quadratic polynomial models for Y1 and Y2. The regression analysis for the two responses indicated that results were highly significant (p < 0.01) under the models, respectively, therefore they could be used for explanation of the responses observed. 3.1. Octacosanol content
(Z1Z2) and quadratic term of distilling temperature ðZ 22 Þ was not significant; nevertheless, also they had been kept in the equation in order to diminish the errors. Table 3 showed the variance analysis of the secondorder regression model on the octacosanol content of purified product. The complete quadratic model showed an excellent fit; furthermore, it appeared to reasonably represent the data. Thus, the responses were sufficiently explained by the regression equation. In order to make it more directly express the effect of the distilling temperature and vacuum degree, we take Eqs. (2) and (3) into Eq. (5) and get Eq. (6) as follows: Y 1 ¼ 398:54 þ 4:8X 1 þ 44:68X 2 0:01X 21
To examine conditions that might affect the octacosanol content (Y1) of purified product, its regression model was predicted by Eq. (5) as follows: Z ¼ 22:84 1:27Z 1 þ 1:95Z 2 0:60Z 1 Z 2 þ 3:01Z 21 1:69Z 22
ð5Þ
The analysis of variance for this model was given in Table 2. There was a high significant regression (a < 0.01) relationship between the dependent variable (Y1), which responds to the octacosanol content of purified product, and the two responding variables, Z1 and Z2, which represent distilling temperature and vacuum degree, respectively. According to this model, linear terms of distilling temperature (Z1, a < 0.01) and vacuum degree (Z2, a < 0.01), and quadratic term of vacuum degree ðZ 22 ; a < 0:01Þ reached high significant. The result suggested that the change of distilling temperature and vacuum degree had a significant effect on the octacosanol content of purified product. In contrast, the interaction of distilling temperature and vacuum degree
0:12X 1 X 2 9:09X 22
ð6Þ
The response surface of octacosanol content (Y1), distilling temperature (X1), and vacuum degree (X2) was shown in Fig. 2. From this three-dimensional plot, the region of high content can be easily identified and the effect of distilling temperature and vacuum degree on the octacosanol content of purified product were reflected: the octacosanol content reduced as the distilling temperature increased, possibly because higher distilling temperature reduced the effective mean free path of the molecules more sharply while causing more collisions in the gap between the evaporating and condensing surfaces (Wang & Xu, 2002), resulting in more amount of components can be condensed. The vacuum degree had different effects on the octacosanol content. Under less vacuum degree the octacosanol content increased by increasing the vacuum degree, in contrast, the octacosanol content decreased by increasing the vacuum degree under greater vacuum degree. This was related to the changes of effective mean free path of the molecules under different vacuum.
Table 2 Estimated regression model of relationship between response variables (octacosanol content) and independent variables (X1, X2) Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
A1 A2 A1A2 A11 A22
1 1 1 1 1
69.69 30.33 1.42 11.91 41.77
69.69 30.33 1.42 11.91 41.77
17.81 7.75 3.04 0.36 10.68
4.96 4.96 4.96 4.96 4.96
10.04 10.04 10.04 10.04 10.04
** ** NS NS **
Notability:
**(a
< 0.01); NS: no significance.
Table 3 Variance analysis of the second-order regression model on octacosanol content of purified product Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
Regress Lack of fit Residual error Total error
5 3 10 15
155.10 30.89 39.12 194.22
31.02 10.30 3.91
7.93 3.28
3.33 3.71
5.64 6.55
** **
Notability:
**(a
< 0.01).
F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
51
Fig. 3. Response surface for the effect of temperature and vacuum degree on purification yield by MD. Fig. 2. Response surface for the effect of temperature and vacuum degree on octacosanol content of purified product by MD.
3.2. Purification yield of crude octacosanol extract The regression model of purification yield of crude octacosanol extract was predicted by Eq. (7) as follows: Y 2 ¼ 53:73 þ 11:56Z 1 10:22Z 2 þ 4:43Z 21 1:70Z 1 Z 2 þ 5:11Z 22
ð7Þ
The result of variance analysis in Table 4 indicated that there was a high statistically significant multiple regression relationship (p < 0.01) between the whole independent variables and the response variable, as using the regression equation to describe the relation-
ship between those experimental factors and the response value. None of the whole model was shown to have lack of fit in Table 5. In this model, the values of response variable Y2 were affected significantly by the linear terms of distilling temperature (Z1, a < 0.01), vacuum degree (Z2, a < 0.01), quadratic terms of distilling temperature ðZ 21 ; a < 0:01Þ and vacuum degree (Z2, a < 0.01), while the interaction of distilling temperature and vacuum degree (Z1Z2) had no significant effect on the purification yield. In like manner, we take the Eqs. (2) and (3) into Eq. (7) and get Eq. (8) as follows: Y ¼ 1404:94 15:30X 1 þ 3:83X 2 þ 0:04X 21 0:34X 1 X 2 þ 20:17X 22
ð8Þ
Table 4 Estimated regression model of relationship between response variables (purification yield) and independent variables (X1, X2) Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
A1 A2 A1A2 A11 A22
1 1 1 1 1
1069.1 835.15 11.56 194.19 251.5
1069.1 835.15 11.56 194.19 251.5
39.41 30.79 0.57 6.45 8.46
4.96 4.96 4.96 4.96 4.96
10.04 10.04 10.04 10.04 10.04
** ** NS ** **
Notability: (**a < 0.01); NS: no significance.
Table 5 Variance analysis of the second-order regression model on purification yield Variance source
DF
SS
MS
F-value
F(0.05)
F(0.01)
Notability
Regress Lack of fit Residual error Total error
5 3 10 15
2361.50 203.22 260.45 2621.95
472.3 67.74 26.04 174.8
18.14 2.60
3.33 3.71
5.64 6.55
** **
Notability: (**a < 0.01).
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F. Chen et al. / Journal of Food Engineering 70 (2005) 47–53
Table 6 Predicted and experimental values of the response at optimum conditions Response
Optimum conditions Distilling temperature (C)
Distilling vacuum (Torr)
Octacosanol content (%) Yield (%)
176.1 176.1
1.29 1.29
The influences of the distilling temperature and vacuum degree on the purification yield were shown in Fig. 3. There was a negative correlation between the yield and octacosanol content (Figs. 2 and 3). In other words, under certain conditions of MD the octacosanol content increased at the cost of decrease of purification yield. 3.3. Optimization According to the canonical analysis described by Khuri and Cornell (1987), the stationary points were located for the corresponding responses. In our study the search criteria were to obtain the highest octacosanol content while to maintain the yield of the purified product as large as possible. Therefore, the optimum MD conditions were obtained as 176.1 C and 1.29 Torr from ridge analysis, which correspond to the distilling temperature and the vacuum degree, respectively. The adequacy of the model equation for predicting the optimum response values was tested in the experiments using the above MD conditions. Predicted and experimental values were given in Table 6. The experimental values (25.93% for the octacosanol content and 51.09% for the yield) were in perfect agreement to the predicted values (25.36% for the octacosanol content and 51.53% for the yield), a result confirming the adequacy of the predicted models (Table 6).
4. Conclusion RSM was effective for estimating the effect of the distilling temperature and vacuum degree of MD, as well as determining the optimal conditions. Response surface were drawn from the regression models to visualize the robustness of the purification method. Moreover, MD was a valuable alternative technique for the purification of crude octacosanol extract from RBW. The optimal parameters in the process of the purification were determined as the distilling temperature 176.1 C and vacuum degree 1.29 Torr, respectively.
Acknowledgement The project was supported by the China Agricultural Science and Education Funds (No. 2001-01-E02), China
Predicted value
Experimental value
25.36 51.53
25.93 51.09
High-Tech (863) Project (No. 2002AA248011) and Beijing Natural Science Foundation (No. 6041003).
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