Selectively increasing of polyunsaturated (18:2) and monounsaturated (18:1) fatty acids in Jatropha curcas seed oil by crystallization using D-optimal design
- Jumat Salimon^{1}Email author,
- Bashar Mudhaffar Abdullah^{1} and
- Nadia Salih^{1}
https://doi.org/10.1186/1752-153X-6-65
© Salimon et al.; licensee Chemistry Central Ltd. 2012
Received: 9 December 2011
Accepted: 10 February 2012
Published: 2 July 2012
Abstract
Background
This study was done to obtain concentrated polyunsaturated fatty acid (PUFA) linoleic acid (LA; 18:2) and monounsaturated fatty acid (MUFA) oleic acid (OA; 18:1) from Jatropha curcas seed oil by urea complexation. Urea complexation is a method used by researchers to separate fatty acids (FAs) based on their molecular structure. Effects the ratio of urea-to-FAs, crystallization temperature and crystallization time on the final products of urea complexation were examined. D-optimal Design was employed to study the significance of these factors and the optimum conditions for the technique were predicted and verified.
Results
Optimum conditions of the experiment to obtain maximum concentration of LA were predicted at urea-to-FAs ratio (w/w) of 5:1, crystallization temperature of −10°C and 24 h of crystallization time. Under these conditions, the final non-urea complex fraction (NUCF) was predicted to contain 92.81% of LA with the NUCF yield of 7.8%. The highest percentage of OA (56.01%) was observed for samples treated with 3:1 urea-to-FAs ratio (w/w) at 10°C for 16 h. The lowest percentage of LA (8.13%) was incorporated into urea complex fraction (UCF) with 1:1 urea-to-FAs ratio (w/w) at 10°C for 8 h.
Conclusions
The separation of PUFA (LA) and MUFA (OA) described here. Experimental variables should be carefully controlled in order to recover a maximum content of PUFA and MUFA of interest with reasonable yield% with a desirable purity of fatty acid of interest.
Keywords
D-optimal design Optimization Polyunsaturated Monounsaturated Linoleic acid Oleic AcidBackground
Linoleic acid (LA) [also called cis,cis,-9,12-octadecadienoic acid] is an example of a polyunsaturated fatty acid (PUFA), due to the presence of two carbon double bonds. The high content of LA makes Jatropha curcas seed oil very important for industry use. LA can be used in protective coatings, plastics, surfactant, dispersants, biolubricant, and a variety of synthetic and in the preparations of other long chain compounds. The high content of LA in seed oil of J. curcas is very important to the production of oleo-chemicals [1]. Oleic acid (OA) [also called (9z)- octadec-9-enoic acid] is an example of a monounsaturated fatty acid (MUFA). A small amount of OA is used in the pharmaceutical industry, as an emulsifying agent in aerosol products [2].
There are several methods which can be used to obtain polyunsaturated fatty acids (PUFA) including freezing crystallization, urea complexation, molecular distillation, supercritical fluid extraction, silver ion complexation and lipase concentration [3] as well as high-performance liquid chromatography [4]. The most economic and most efficient technique to obtain LA in the form of fatty acids (FAs) is urea complex fractionation. This is a well-established technique for the elimination of saturated fatty acids (SFAs) and MUFA [5].
Urea complexation has the advantage that the complex crystals are extremely stable, and filtration is not carried out at low temperatures which is required for solvent crystallization of FAs. This method is preferred by many researchers because complexation depends upon configuration of the FAs moieties due to the presence of multiple double bonds, rather than pure physical properties such as melting point or solubility [5, 6]. The SFAs and MUFA easily form complexes with urea and crystallize out at cooling during urea complex fraction (UCF). These complexes can subsequently be removed by filtration. The liquid or non-urea complex fraction (NUCF) is enriched with PUFA and the crystals formed or UCF consists of SFAs and MUFA.
In this study, urea complex fractionation of a mixture of FAs of Malaysian J. curcas seed oil was carried out to obtain concentrated PUFA. The effects of urea-to-FAs ratio, crystallization temperature and crystallization time to the yield% of NUCF (Y_{ 1 }), yield% of UCF (Y_{ 5 }), percentage MUFA (OA) (Y_{ 3 } and Y_{ 7 }) and percentage PUFA (LA) (Y_{ 4 } and Y_{ 5 }) in NUCF and UCF were systematically studied.
Results and discussion
Non-urea complex fraction (NUCF)
The original fatty acids (FAs) mixture was composed of 13.19% palmitic (16:0), 6.37% stearic (18:0), 43.33% oleic (18:1) and 36.71% linoleic (18:2) acid. Average molecular weight of the FAs was 203.36 as obtained from saponification test of the original oil. The results compared well with those of [7]. The PUFA (LA) concentrate was prepared by urea complex fractionation following the technique of [8], using the FAs that was previously obtained. The purpose of this procedure was to obtain a PUFA concentrate enriched in LA and simultaneously, maintain the highest yield% of LA. The crystallization process with urea preferentially selects SFAs and MUFA, and the tendency of FAs to combine with urea decreases with increasing chain lengths [9].
D-optimal design arrangement and responses for non-urea-complexed fraction (NUCF) of Jatropha curcas seed oil
Variables levels | Responses, Y | ||||||||
---|---|---|---|---|---|---|---|---|---|
Run no. | Urea-FAs^{a}(X_{ 1 }) | Temp.^{b}(X_{ 2 }) | Time^{c}(X_{ 3 }) | Y_{ 1 }, Yield (%) | C16:0 (%) | C18:0 (%) | Y_{ 2 }, SFAs (C16:0+C18:0) (%) | Y_{ 3 }, MUFA (C18:1) (%) | Y_{ 4 }, LA (C18:2) (%) |
1 | 1 | 10 | 8 | 49 | 1.44 | - | 1.44 | 36.50 | 58.23 |
2 | 3 | 0 | 24 | 22.2 | 0.56 | 0.26 | 0.82 | 13.25 | 85.16 |
3 | 2 | 0 | 16 | 34.9 | 0.44 | - | 0.44 | 28.87 | 69.41 |
4 | 3 | -10 | 8 | 32.3 | 0.49 | - | 0.49 | 20.90 | 77.74 |
5 | 5 | 10 | 24 | 7.7 | 0.43 | - | 0.43 | 9.37 | 88.60 |
6 | 5 | -10 | 24 | 7.8 | 0.33 | - | 0.33 | 5.73 | 92.81 |
7 | 1 | 10 | 24 | 50.6 | 3.25 | 0.30 | 3.55 | 39.67 | 54.91 |
8 | 1 | -10 | 24 | 34.1 | 0.58 | - | 0.58 | 34.64 | 61.46 |
9 | 5 | 10 | 8 | 8.8 | 1.23 | - | 1.23 | 9.94 | 87.82 |
10 | 5 | 0 | 16 | 6.2 | 0.97 | - | 0.97 | 8.95 | 89.19 |
11 | 1 | -10 | 16 | 48.1 | 1.17 | - | 1.17 | 35.55 | 59.85 |
12 | 1 | 0 | 8 | 31.3 | 2.79 | - | 2.79 | 39.58 | 54.63 |
13 | 5 | 10 | 24 | 4.1 | 0.94 | 0.28 | 1.23 | 6.31 | 92.14 |
14 | 3 | 10 | 16 | 31.6 | 0.19 | - | 0.19 | 20.09 | 78.42 |
15 | 1 | 10 | 8 | 45.6 | 3.65 | 0.33 | 3.99 | 40.49 | 52.53 |
16 | 5 | -10 | 8 | 6.6 | 0.89 | - | 0.89 | 9.10 | 88.92 |
17 | 5 | 0 | 8 | 20.5 | 0.34 | - | 0.34 | 10.30 | 88.12 |
18 | 1 | 10 | 16 | 49.7 | 1.17 | - | 1.17 | 41.30 | 54.87 |
The LA% derived from the NUCF phase was relatively high, and some even greater than 90% under certain experimental conditions (Table 1). This showed that the experimental conditions were suitable for the preparation of high purity LA. However, it is difficult to completely remove all the SFAs and MUFA to obtain 100% purity of PUFA in the concentrate. [11] reported that complete removal of SFAs and MUFA by urea complexation may be impossible since some of the SFAs do not bind with urea during crystallization.
Regression coefficients of the predicted quadratic polynomial model for response variables (yield% of NUCF) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), yield % of NUCF (Y_{ 1 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 27.97 | 11.71 | 0.0010 | *** |
Linear | ||||
X _{ 1 } | -16.60 | 73.24 | 0.0001 | *** |
X _{ 2 } | 1.85 | 0.88 | 0.3767 | |
X _{ 3 } | -1.60 | 0.66 | 0.4405 | |
Quadratic | ||||
X _{ 11 } | -2.39 | 0.31 | 0.5935 | |
X _{ 22 } | 3.88 | 1.09 | 0.3263 | |
X _{ 33 } | -3.43 | 0.84 | 0.3849 | |
Interaction | ||||
X _{ 12 } | -2.44 | 1.28 | 0.2904 | |
X _{ 13 } | -2.13 | 1.02 | 0.3428 | |
X _{ 23 } | 0.69 | 0.093 | 0.7682 | |
R ^{ 2 } | 0.92 |
Regression coefficients of the predicted quadratic polynomial model for response variables (SFAs%) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), SFAs% (Y_{ 2 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 0.087 | 1.84 | 0.2005 | |
Linear | ||||
X _{ 1 } | -0.69 | 6.65 | 0.0327 | ** |
X _{ 2 } | 0.40 | 2.12 | 0.1831 | |
X _{ 3 } | -0.12 | 0.21 | 0.6600 | |
Quadratic | ||||
X _{ 11 } | 0.92 | 2.44 | 0.1572 | |
X _{ 22 } | -0.31 | 0.36 | 0.5672 | |
X _{ 33 } | 0.72 | 1.98 | 0.1972 | |
Interaction | ||||
X _{ 12 } | -0.24 | 0.68 | 0.4342 | |
X _{ 13 } | 0.041 | 0.020 | 0.8914 | |
X _{ 23 } | 0.26 | 0.67 | 0.4370 | |
R ^{ 2 } | 0.67 |
Regression coefficients of the predicted quadratic polynomial model for response variables (MUFA (OA%)) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), MUFA (OA%) (Y_{ 3 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 18.77 | 81.33 | 0.0001 | *** |
Linear | ||||
X _{ 1 } | -14.76 | 577.20 | 0.0001 | *** |
X _{ 2 } | 1.04 | 2.80 | 0.1330 | |
X _{ 3 } | -1.59 | 6.53 | 0.0339 | ** |
Quadratic | ||||
X _{ 11 } | 5.19 | 14.55 | 0.0051 | *** |
X _{ 22 } | 0.29 | 0.061 | 0.8115 | |
X _{ 33 } | -1.32 | 1.25 | 0.2951 | |
Interaction | ||||
X _{ 12 } | -0.49 | 0.52 | 0.4906 | |
X _{ 13 } | -0.39 | 0.34 | 0.5741 | |
X _{ 23 } | 1.08 | 2.26 | 0.1714 | |
R ^{ 2 } | 0.98 |
Regression coefficients of the predicted quadratic polynomial model for response variables (PUFA (LA%)) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), PUFA (LA%) (Y_{ 4 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 80.30 | 91.62 | 0.0001 | *** |
Linear | ||||
X _{ 1 } | 16.37 | 643.86 | 0.0001 | *** |
X _{ 2 } | -1.30 | 3.95 | 0.0820 | |
X _{ 3 } | 1.91 | 8.49 | 0.0195 | ** |
Quadratic | ||||
X _{ 11 } | -7.14 | 24.89 | 0.0011 | *** |
X _{ 22 } | -0.22 | 0.032 | 0.8622 | |
X _{ 33 } | 0.65 | 0.28 | 0.6133 | |
Interaction | ||||
X _{ 12 } | 0.57 | 0.64 | 0.4466 | |
X _{ 13 } | 0.18 | 0.065 | 0.8056 | |
X _{ 23 } | -1.10 | 2.13 | 0.1827 | |
R ^{ 2 } | 0.99 |
Examination of these coefficients with a T-test shows that the percentage yield of NUCF (Y_{ 1 }), percentage of MUFA (OA) (Y_{ 3 }), percentage of PUFA (LA) (Y_{ 4 }), the linear term of urea-to-FAs ratio (X_{ 1 }) and quadratic term of urea-to-FAs were highly significant (p < 0.01), while the percentage of SFAs (Y_{ 2 }), the linear term was significant at p < 0.05. Lastly, linear term of crystallization time (X_{ 3 }) for the percentage of PUFA (LA) (Y_{ 4 }) and percentage of MUFA (LA) (Y_{ 3 }) in the concentrate were significant at p < 0.05.
The results suggest that the linear effect of urea-to-FAs ratio and crystallization time are the primary determining factors for FAs separation by urea complexation. [10] concluded that these two variables significantly influenced the results of their urea complexation study. Crystallization time was found to be the insignificant factor (P > 0.05). This finding is in agreement with the results reported by other researchers [5, 6, 10].
Analysis of variance (ANOVA) for all the responses of NUCF
Source | Df | Sum of squares | Mean square | F value | P | ||
---|---|---|---|---|---|---|---|
Y _{ 1 } | Model | 3 | 4484.17 | 1494.72 | 37.83 | < 0.0001 | Significant |
Residual | 14 | 553.23 | 39.52 | ||||
lack-of-fit | 12 | 540.97 | 45.08 | 7.35 | 0.1258 | Not significant | |
Pure error | 2 | 12.26 | 6.13 | ||||
Y _{ 2 } | Model | 3 | 8.44 | 2.81 | 3.20 | 0.0560 | Not significant |
Residual | 14 | 12.29 | 0.88 | ||||
lack-of-fit | 12 | 8.73 | 0.73 | 0.41 | 0.8720 | Not significant | |
Pure error | 2 | 3.57 | 1.78 | ||||
Y _{ 3 } | Model | 9 | 3259.79 | 362.20 | 81.33 | < 0.0001 | Significant |
Residual | 8 | 35.63 | 4.45 | ||||
lack-of-fit | 6 | 23.03 | 3.84 | 0.61 | 0.7298 | Not significant | |
Pure error | 2 | 12.59 | 6.30 | ||||
Y _{ 4 } | Model | 9 | 4051.33 | 450.15 | 91.62 | < 0.0001 | Significant |
Residual | 8 | 39.31 | 4.91 | ||||
lack-of-fit | 6 | 16.80 | 2.80 | 0.25 | 0.9219 | Not significant | |
Pure error | 2 | 22.50 | 11.25 |
Figures 1, 2, 3 and 4 show increasing amount of urea and decreasing crystallization temperature which led to reduction of percentage of SFAs and MUFA (OA) in liquid NUCF. The content of PUFA (LA) in the liquid fraction would also be enriched under these conditions (Figure 4). The relationships between the parameters and FAs percentages were linear or almost linear. High concentration of PUFA (LA) could be obtained by using high ratio of urea-to-FAs at low temperatures. However, this could also reduce the yield% of liquid NUCF in the final product as more LA would be lost into urea adducts. Experimental variables should therefore be carefully controlled in order to recover a maximum content of PUFA (LA) of interest with reasonable yield% [5].
Straight-chained molecules such as SFAs readily formed stable adduct with urea. SFAs formed complexes more readily than MUFA. MUFA formed more readily inclusion compounds than PUFA (LA). Similar complexation tendency patterns were also obtained by [12]. The addition of more urea could reduce the SFAs percentage in NUCF to a minimum level; it however results in indiscriminate FAs complexation and thus reducing the amount of MUFA (OA) and PUFA (LA). A lower urea-to-FAs ratio prevented indiscriminate FAs complexation. Lower crystallization temperature can facilitate formation of more stable urea adducts, that would reduce SFAs in NUCF. Longer periods of crystallization time would allow the crystals to further stabilize. However the parameters must be set at a level to achieve an acceptable yield% of product with high purity. Higher purity of PUFA (LA) will always give lower yield of NUCF.
Urea complex fraction (UCF)
D-optimal design arrangement and responses for urea-complexed fraction (UCF) of Jatropha curcas seed oil
Variables levels | Responses, Y | ||||||||
---|---|---|---|---|---|---|---|---|---|
Run no. | Urea-FAs^{a}(X_{ 1 }) | Temp.^{b}(X_{ 2 }) | Time^{c}(X_{ 3 }) | Y_{ 5 }, Yield (%) | C16:0 (%) | C18:0 (%) | Y_{ 6 }, SFAs (C16:0+C18:0) (%) | Y_{ 7 }, MUFA (C18:1) (%) | Y_{ 8 }, LA (C18:2) (%) |
1 | 1 | 10 | 8 | 50.7 | 25.42 | 13.97 | 39.39 | 48.10 | 8.13 |
2 | 3 | 0 | 24 | 78.6 | 17.09 | 9.27 | 26.36 | 53.15 | 19.94 |
3 | 2 | 0 | 16 | 64.9 | 22.62 | 21.50 | 44.13 | 44.89 | 10.23 |
4 | 3 | −10 | 8 | 67.4 | 19.34 | 10.28 | 29.62 | 54.11 | 15.47 |
5 | 5 | 10 | 24 | 92.1 | 14.14 | 7.36 | 21.50 | 45.07 | 29.34 |
6 | 5 | −10 | 24 | 92.0 | 20.59 | 12.43 | 33.02 | 55.41 | 8.37 |
7 | 1 | 10 | 24 | 48.7 | 25.06 | 14.57 | 39.64 | 45.69 | 11.50 |
8 | 1 | −10 | 24 | 65.7 | 23.06 | 12.24 | 35.31 | 45.63 | 15.74 |
9 | 5 | 10 | 8 | 91.0 | 13.52 | 8.11 | 21.63 | 42.61 | 31.45 |
10 | 5 | 0 | 16 | 93.5 | 14.47 | 8.91 | 23.39 | 44.44 | 28.68 |
11 | 1 | −10 | 16 | 51.3 | 19.76 | 10.39 | 30.16 | 45.28 | 20.83 |
12 | 1 | 0 | 8 | 68.8 | 20.37 | 11.84 | 32.21 | 43.89 | 20.10 |
13 | 5 | 10 | 24 | 95.7 | 13.40 | 6.52 | 19.92 | 42.38 | 35.81 |
14 | 3 | 10 | 16 | 68.3 | 20.37 | 11.08 | 31.46 | 56.01 | 12.10 |
15 | 1 | 10 | 8 | 54.3 | 23.34 | 13.49 | 36.84 | 45.28 | 14.61 |
16 | 5 | −10 | 8 | 93.2 | 13.68 | 8.33 | 22.01 | 42.89 | 30.77 |
17 | 5 | 0 | 8 | 79.2 | 16.61 | 8.94 | 25.56 | 53.00 | 20.87 |
18 | 1 | 10 | 16 | 49.8 | 28.38 | 15.89 | 44.27 | 43.04 | 9.46 |
The highest percentage of MUFA (OA) (56.01%) was observed for samples treated with 3:1 urea-to-FAs ratio (w/w) at 10°C for 16 h, while the lowest percentage of PUFA (LA) (8.13%) was incorporated into the urea complex with 1:1 urea-to-FAs ratio (w/w) at 10°C for 8 h. Inclusion of more PUFA (LA) into UCF reduced the percentage of SFAs and MUFA (OA) in the samples. The process may be not suitable for industrial uses because this method cannot employ high purity SFAs (palmitic and stearic acids) and MUFA (OA).
Regression coefficients of the predicted quadratic polynomial model for response variables (yield% of UCF) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), yield % of UCF (Y_{ 5 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 72.14 | 11.29 | 0.0012 | *** |
Linear | ||||
X _{ 1 } | 16.64 | 70.27 | 0.0001 | *** |
X _{ 2 } | -1.83 | 0.82 | 0.3911 | |
X _{ 3 } | 1.65 | 0.67 | 0.4370 | |
Quadratic | ||||
X _{ 11 } | 1.97 | 0.20 | 0.6662 | |
X _{ 22 } | -4.18 | 1.21 | 0.3038 | |
X _{ 33 } | 3.74 | 0.96 | 0.3568 | |
Interaction | ||||
X _{ 12 } | 2.46 | 1.25 | 0.2965 | |
X _{ 13 } | 2.21 | 1.04 | 0.3374 | |
X _{ 23 } | -0.81 | 0.12 | 0.7362 | |
R ^{ 2 } | 0.92 |
Regression coefficients of the predicted quadratic polynomial model for response variables (SFAs%) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), SFA% (Y_{ 6 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 33.80 | 4.51 | 0.0226 | ** |
Linear | ||||
X _{ 1 } | -5.43 | 16.17 | 0.0038 | *** |
X _{ 2 } | 0.38 | 0.076 | 0.7898 | |
X _{ 3 } | 1.49 | 1.17 | 0.3101 | |
Quadratic | ||||
X _{ 11 } | -1.22 | 0.17 | 0.6943 | |
X _{ 22 } | -0.053 | 4.140E-004 | 0.9843 | |
X _{ 33 } | -3.06 | 1.39 | 0.2719 | |
Interaction | ||||
X _{ 12 } | -3.96 | 6.99 | 0.0295 | ** |
X _{ 13 } | -0.41 | 0.078 | 0.7868 | |
X _{ 23 } | -1.78 | 1.28 | 0.2913 | |
R ^{ 2 } | 0.83 |
Regression coefficients of the predicted quadratic polynomial model for response variables (MUFA (OA%)) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), MUFA (OA%) (Y_{ 7 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 52.35 | 1.43 | 0.3118 | |
Linear | ||||
X _{ 1 } | 1.40 | 1.26 | 0.2940 | |
X _{ 2 } | -1.07 | 0.72 | 0.4218 | |
X _{ 3 } | 0.78 | 0.38 | 0.5571 | |
Quadratic | ||||
X _{ 11 } | -8.33 | 9.08 | 0.0167 | ** |
X _{ 22 } | 0.71 | 0.089 | 0.7728 | |
X _{ 33 } | 1.58 | 0.43 | 0.5281 | |
Interaction | ||||
X _{ 12 } | -1.68 | 1.47 | 0.2604 | |
X _{ 13 } | 0.39 | 0.082 | 0.7821 | |
X _{ 23 } | -1.89 | 1.68 | 0.2315 | |
R ^{ 2 } | 0.61 |
Regression coefficients of the predicted quadratic polynomial model for response variables (PUFA (LA%)) in urea inclusion fractionation experiment of J. curcas seed oil
Variables | Coefficients (ß), PUFA (LA%) (Y_{ 8 }) | T | P | Notability |
---|---|---|---|---|
Intercept | 13.73 | 3.77 | 0.0375 | ** |
Linear | ||||
X _{ 1 } | 4.16 | 6.42 | 0.0350 | ** |
X _{ 2 } | 0.83 | 0.25 | 0.6321 | |
X _{ 3 } | -2.07 | 1.54 | 0.2500 | |
Quadratic | ||||
X _{ 11 } | 6.88 | 3.59 | 0.0949 | |
X _{ 22 } | -1.44 | 0.21 | 0.6592 | |
X _{ 33 } | 1.55 | 0.24 | 0.6360 | |
Interaction | ||||
X _{ 12 } | 5.61 | 9.49 | 0.0151 | ** |
X _{ 13 } | -0.15 | 6.701E-003 | 0.9368 | |
X _{ 23 } | 3.74 | 3.79 | 0.0874 | |
R ^{ 2 } | 0.80 |
Linear term of urea-to-FAs ratio was highly significant (p < 0.01) for the yield% of UCF (Y_{ 5 }) and percentage SFAs (Y_{ 6 }), while the linear term of urea-to-FAs was significant (P < 0.05) for the percentage PUFA (LA) (Y_{ 8 }). The interaction between urea-to-FAs ratio and crystallization temperature were significant (p < 0.05) for the percentage PUFA (LA) (Y_{ 8 }) and the percentage SFAs (Y_{ 6 }). Quadratic term of urea-to-FAs ratio was also significant (p < 0.05) for the percentage MUFA (OA) (Y_{ 7 }).
The coefficients of the independent variables (urea-to-FAs ratio; X_{ 1 }, crystallization temperature; X_{ 2 } and crystallization time; X_{ 3 }) determined for the quadratic polynomial models are lists in Tables 8, 9, 10 and 11 respectively. Table 8 lists the yield% of solid UCF (Y_{ 5 }), Table 9 lists the percentage SFAs (palmitic and stearic acids) (Y_{ 6 }), Table 10 lists the percentage MUFA (OA) (Y_{ 7 }) and Table 11 lists the percentage PUFA (LA) (Y_{ 8 }).
Analysis of variance (ANOVA) for all the responses of UCF
Source | Df | Sum of squares | Mean square | F value | P | ||
---|---|---|---|---|---|---|---|
Y _{ 5 } | Model | 3 | 4510.49 | 1503.50 | 35.98 | < 0.0001 | Significant |
Residual | 14 | 484.99 | 41.79 | ||||
lack-of-fit | 12 | 572.03 | 47.67 | 7.36 | 0.1258 | Not significant | |
Pure error | 2 | 12.96 | 6.48 | ||||
Y _{ 6 } | Model | 3 | 671.30 | 223.77 | 8.39 | 0.0019 | Significant |
Residual | 14 | 373.55 | 26.68 | ||||
lack-of-fit | 12 | 369.04 | 30.75 | 13.65 | 0.0702 | Not significant | |
Pure error | 2 | 4.50 | 2.25 | ||||
Y _{ 7 } | Model | 9 | 236.43 | 26.27 | 1.43 | 0.3118 | Not significant |
Residual | 8 | 146.77 | 18.35 | ||||
lack-of-fit | 6 | 139.17 | 23.19 | 6.10 | 0.1475 | Not significant | |
Pure error | 2 | 7.60 | 3.80 | ||||
Y _{ 8 } | Model | 6 | 940.28 | 156.71 | 4.42 | 0.0163 | Significant |
Residual | 11 | 390.43 | 35.49 | ||||
lack-of-fit | 9 | 348.48 | 38.72 | 1.85 | 0.4004 | Not significant | |
Pure error | 2 | 41.95 | 20.98 |
Conclusion
Optimum conditions of the experiment to obtain maximum concentration of PUFA (LA), were predicted at urea-to-FAs ratio (w/w) of 5:1, crystallization temperature of −10°C and 24 h of crystallization time. The final NUCF At this condition was predicted to contain 92.81% of PUFA (LA) with a NUCF yield of 7.8%. The highest percentage MUFA (OA) (56.01%) was observed for sample treated with a urea-to-FAs ratio (w/w) of 3:1 at 10°C for 16 h. The lowest percentage PUFA (LA) (8.13%) was incorporated into the UCF with a urea-to-FAs ratio (w/w) of 1:1 at 10°C for 8 h. All of the above mentioned factors have to be controlled to yield a reasonable amount of product with a desirable purity of FAs.
Experimental and Methods
Experimental and Methods
Independent variables and their levels for D-optimal design of the fatty acids separation
Independent variables | Variable levels | |||
---|---|---|---|---|
−1 | 0 | +1 | ||
The urea-to-FAs ratio (w/w) (g/g) | X _{ 1 } | 1 | 3 | 5 |
Crystallization temperature (Â°C) | X _{ 2 } | −10 | 0 | 10 |
Crystallization time (h) | X _{ 3 } | 8 | 16 | 24 |
Experimental design and statistical analysis
A three-factor D-optimal design was employed to study the responses, after urea inclusion fractionation. The yield of NUCF Y_{ 1 } in % by wt], SFAs (palmitic and stearic acids) Y_{ 2 } in %], MUFA (OA) Y_{ 3 } in %] and PUFA (LA) Y_{ 4 } in %] are shown in equations 1, 2, 3 and 4 respectively. The yield of UCF Y_{ 5 } in %], SFAs (palmitic and stearic acids) Y_{ 6 } in %], MUFA (OA) Y_{ 7 } in %] and PUFA (LA) Y_{ 8 } in %] after urea inclusion fractionation are shown in equations 5, 6, 7 and 8 respectively. An initial screening step was carried out using the technique describe by [5] to select the major response factors and their values.
Where B_{ 0 }; Bi; Bii and Bij are constant, linear, square and interaction regression coefficient terms, respectively, and xi and xj are independent variables. The goodness of fit of the model was evaluated by the coefficient of determination R^{ 2 } and the analysis of variance (ANOVA).
Declarations
Acknowledgment
We thank UKM and the Ministry of Science and Technology for research grant UKM-GUP-NBT-08-27-113, OUP-2012-139 and UKM-OUP-MI-2011.
Authors’ Affiliations
References
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