The most dominant and beneficial conjugated linoleic acids isomers (CLAs) with miscellaneous biological tasks are 9c11t-C18:2 and 10t12c-C18:2. The problem with most of the commercial CLA produced by non-optimized conventional approaches is the heterogeneity of their isomers and undesirable toxic by-products. In this study, optimization of the isomerization of the fatty acid methyl esters of the high linoleic sunflower oil was investigated through response surface methodology (RSM). The reaction temperature, the concentration of PEG400 and NaOCH 3 had positive influence on the total conjugated linoleic acid methyl esters (CLAME) production as response. However, the effect of the polyethylene glycol 400 (PEG400) concentration was more significant on response than those of other factors (p < 0.05). The reaction time and the interactions between the factors had no significant effect on response (p ≥ 0.05). The optimum point for the maximum response of 72.90% ( <i> i.e </i> ., based on the mass percentage of total fatty acid methyl esters mixture) was at 5% w/w NaOCH3, 1.06% w/w PEG400 and temperature of 140 °C.
Conjugated linoleic acid (CLA) has been found to be an extraordinary essential fatty acid with miscellaneous functional effects on the human body. The most beneficial CLA isomers participate with biological performance are 9c11t-C18:2 and 10t12c-C18:2 [
Despite CLA exists at levels of 0.3% - 0.8% (w/w) of the fat in beef and dairy products of the ruminants, this negligible level cannot provide the recommen- ded 3 - 3.4 g of CLA per day that is necessary to produce the desired physiological effects [
In order to produce commercial CLA with less unwanted isomers, Abney and Anderson (2002) applied linoleic acid methyl esters (LAMEs) as the substrate of isomerization which was found to result in the production of a high yield of conjugated linoleic acid methyl esters (CLAMEs). They proposed the isomerization of LAMEs by using negligible amounts of alkali catalyst, at a low temperature and in the presence of phase transfer catalyst (PTC) instead of solvents. Their technique resulted in increasing the degree of the isomerization from 6% without PTC to 90% [
High linoleic sunflower oil (>65% linoleic acid) fatty acid methyl esters (FAMEs) were prepared as described previously [
The first phase of the experiments was the optimization of the isomerization or conversion of LAMEs from sunflower oil into CLAMEs. The second stage involves the production of enriched CLA via four sequential steps including the saponification, hydrolysis, phase separation and purification through the two- step urea inclusion crystallization. All stages were performed using a 2 litre double-walled stainless steel pressure laboratory reactor (IKA, LR 2000 P, Germany) equipped with the pressure gauge, a mechanical stirrer, water condenser, temperature regulator, sampling outlet, and an adjustable water bath providing the desired temperatures.
The design of experiment (DOE), data analysis and optimization procedures were performed using the Minitab v.14 statistical package (Minitab Inc., 2000, State College, PA, USA). RSM was applied to determine the effect of four independent variables (i.e., reaction time, the temperature, the amounts of NaOCH3 and PEG400) or their interactions and on the mass of percentage of total CLAMEs (% w/w) as a response. Thirty isomerization treatments were designed based on a central composite design (CCD) considering five levels for each factor. The experimental matrix of isomerization is indicated in
The reaction was then terminated with the addition of 1 mL of H3PO4, during which phosphate salts were precipitated. The reactor was cooled to 80˚C, the precipitates were removed and the contents were transferred into a separatory funnel for the phase separation [
Treatment run | Time of reaction (x1, min) | Temperature (x2, ˚C) | NaOCH3 (x3, % w/w) | PEG400 (x4, % w/w) |
---|---|---|---|---|
1 | 150 | 160 | 2 | 1.5 |
2c | 210 | 140 | 3 | 1.0 |
3 | 150 | 160 | 4 | 0.5 |
4 | 150 | 120 | 2 | 0.5 |
5 | 270 | 160 | 2 | 0.5 |
6 | 270 | 120 | 2 | 1.5 |
7 | 150 | 120 | 4 | 1.5 |
8 | 270 | 120 | 4 | 0.5 |
9 | 270 | 160 | 4 | 1.5 |
10c | 210 | 140 | 3 | 1.0 |
11 | 210 | 140 | 5 | 1.0 |
12 | 210 | 140 | 1 | 1.0 |
13 | 330 | 140 | 3 | 1.0 |
14 | 90 | 140 | 3 | 1.0 |
15 | 210 | 140 | 3 | 0.0 |
16 | 210 | 180 | 3 | 1.0 |
17 | 210 | 140 | 3 | 2.0 |
18c | 210 | 140 | 3 | 1.0 |
19 | 210 | 100 | 3 | 1.0 |
20c | 210 | 140 | 3 | 1.0 |
21 | 270 | 160 | 2 | 1.5 |
22c | 210 | 140 | 3 | 1.0 |
23 | 150 | 160 | 2 | 0.5 |
24 | 150 | 120 | 4 | 0.5 |
25c | 210 | 140 | 3 | 1.0 |
26 | 270 | 160 | 4 | 0.5 |
27 | 150 | 160 | 4 | 1.5 |
28 | 270 | 120 | 2 | 0.5 |
29 | 270 | 120 | 4 | 1.5 |
30 | 150 | 120 | 2 | 1.5 |
cCenter point.
evaporator (80˚C for 1 h) and samples were stored under nitrogen at −18˚C before any further experimental procedures.
The CFAMEs (200 g, 0.679 moles) produced under the optimum condition, 200 mL water, 200 g ethanol and NaOH (38 g; 0.95 moles) were combined in the pressure lab reactor to produce sodium conjugated linoleate (CLA soap). The reactor was then sealed and nitrogen was purged )0.3 bar( to avoid the mixture from foaming under intensive stirring (300 rpm), then temperature raised and kept for 1 h at 85˚C [
The sodium salt of the conjugated fraction was transferred into the reactor. H3PO4 was added into the mixture at 80˚C and a vigorous mixing (300 rpm) was initiated to complete the conversion of CLA salts into free fatty acids until the pH of the bottom layer reached 2 - 3 [
This stage comprises a two-step urea inclusion crystallization on the free fatty acid mixture to purify the CLA. In 1st step, 1 kg of the crude CLA obtained from the previous stage was introduced into the urea-saturated methanol solution (1.5 kg/4 L) in 6 portions while mixture was stirring at 70˚C [
The composition and content of the fatty acids before and after the isomerization and purification processes were determined by GC analysis (Agilent 7890, Agilent Inc., DE, USA) equipped with flame ionization detector (FID) and autosampler injector.
The CFAMEs obtained from each isomerization treatment and the internal standard solutions were prepared following a procedure similar to what was done for the FAMEs [
where AFAMEs and AIS represent the peak areas of the individual FAMEs and the internal standard (methyl heptadecanoate), respectively. CIS and VIS are the concentration (mg/mL) and the volume of the internal standard solution (mL), and
The total conjugated dienoic acid content (w/w %) of the FAMEs which was measured by GC after the isomerization was further validated using a spectrophotometer (Ultraspec 3100 pro UV/Visible: Amersham Biosciences, Biochrom Ltd., Cambridge, England) and according to the method described in American Oil Chemists’ Society Official Method (2006) with slight modification [
where As shows absorbency at 233 nm, b and c represent the cuvette length (cm) and the concentration of the particular sample (g/L), respectively. K0 is the absorptive constant of CLAME (i.e., 0.07).
The experimental and predicted values of the total CLAMEs were calculated as the response at the points based on the experimental design. The experimental data were subjected to analysis of the variance (ANOVA) using the response surface regression procedure. Such analysis was carried out to determine the statistical significance of the isomerization parameters that affected the response, and to fit a regression relationship between the experimental data and independent variables for developing a model. A second-order polynomial equation was used to describe variations in response variables. As the response was the total CLAMEs (Y, % w/w), the generalized response surface model is given below:
where Y is the predicted response, β0 is the offset term, βi is the linear coefficient, βii and βij are the quadratic and interaction coefficients, xi and xj are the independent variables [
The numerical optimization process was performed using a response optimizer to get to the optimal point, which resulted in the desired response. Considering the mass percentage of the LAMEs is 72.90% (
The fatty acid profile of the sunflower oil obtained from GC analysis expressed
Fatty acids | Fatty acids methyl esters composition (% w/w) | ||
---|---|---|---|
Name | Formula | Transesterifieda sunflower oil | Isomerizedb sunflower oil |
Palmitic | 16:0 | 5.80 ± 0.22 | 5.60 ± 0.22 |
Palmitoleic | 16:1 | 0.20 ± 0.07 | 0.20 ± 0.01 |
Stearic | 18:0 | 3.90 ± 0.14 | 3.70 ± 0.85 |
Oleic | 18:1 | 16.70 ± 0.92 | 16.50 ± 1.02 |
Linoleic | 18:2 | 72.90 ± 0.5 | 1.50 ± 0.08 |
Linolenic | 18:3 | 0.20 ± 0.07 | NDd |
Total CLAc | 18:2 | NDd | 70.40 ± 1.50 |
c9, t11-CLA | 18:2 | NDd | 34.35 ± 1.02 |
t10, c12-CLA | 18:2 | NDd | 34.67 ± 0.84 |
Minor CLA isomers | -- | NDd | 1.60 ± 0.04 |
Minor fatty acids | -- | 0.30 ± 0.02 | 0.48 ± 0.08 |
aThe fatty acid composition of the transesterified sunflower oil (i.e., FAMEs) obtained from the optimum conditions of the transesterification (Koohi Kamali, Tan & Ling, 2012); bThe fatty acid composition of the of sunflower oil after the isomerization process (i.e., CFAMEs) resulting from the optimum conditions of the isomerization. Data represent mean ± SD (standard deviation of 3 analysis); cTotal conjugated linoleic methyl esters (w/w %) was calculated as sum of the CLA isomers; dNot detected.
in
where B is the total mass percentage of the CLAMEs in isomerized sample, and A is the mass percentage of the LAMEs in the transesterified fraction which was used as the substrate of the isomerization. The degree of the isomerization was 96.6% under the optimum condition showing that 96.6% of the LAMEs were converted into CLAMEs. In addition, the content of CLA isomers was quantified using GC (i.e., 70.40%) half of which was 9-c, 11-t and 10-t, 12-c-octadecadi- enoic acid in the sample derived from the optimum conditions.
During the 1st stage of the urea crystallization, the saturated and the monounsaturated fatty acids are trapped into urea channel shape of hexagonal crystalline
Fatty acids (% w/w) | Fatty acid profile before the purification | Fatty acid profile (1st crystallization step) | Fatty acid profile (2nd crystallization step) |
---|---|---|---|
C16:0 | 5.80 ± 0.22 | 5.00 ± 0.61 | ND |
C18:0 | 3.90 ± 0.14 | 2.80 ± 0.02 | ND |
C18:1 | 16.70 ± 0.92 | 9.040 ± 0.13 | 2.20 ± 0.01 |
C18:2 (cis-9, trans-11, CLA) | 34.35 ± 1.02 | 39.30 ± 0.14 | 45.70 ± 0.60 |
C18:2 (trans-10, cis-12, CLA) | 34.67 ± 0.84 | 41.24 ± 0.31 | 49.80 ± 0.70 |
Minor fatty acids | 4.58 ± 0.20 | 2.62 ± 0.10 | 1.30 ± 0.01 |
Data are the average of 3 analysis ± SD (standard deviation). ND; represents not detected.
complexes and can be removed from the mother liquor using filtration techniques. However, due to the smaller bulk density of saturated fatty acids than that of monoenoic, the crystallization rate of saturated fatty acids with urea is faster compared with the monoenoic fatty acids. As can be observed in
In the 2nd stage of the urea crystallization, mixtures of unsaturated fatty acids including CLA isomers and the residual amount of saturated and mono-unsa- turated fatty acids which did not form the complex in the 1st crystallization were subjected to the urea-inclusion crystallization. The CLA isomers began to form the CLA-UIC crystals and concentrated CLA was isolated from the mixture. However, alpha-linolenic acid (LNA) remained in the mother liquor and supported the earlier findings which indicated that the poly-unsaturated fatty acids with large bulk densities cannot be locked up in crystallized urea channels and remains in the liquid mixture [
The purity of the CLA isomers, which was 80.54% after at the first crystallization step increased to 95.5% by the end of second stage of the purification (
Since the conjugated double bonds reported to have a strong absorption in the region of 233 nm, formation of conjugated dienes after the alkali-isomerization was confirmed with UV spectrophotometric assay and for validating the GC results [
The mass percentages of the total CLAME for each treatment were calculated using Equation (2) and reported as the response of each isomerization treatment. The experimental data and the predicted values obtained from the software after fitting (i.e., reducing) the regression model are listed in
Treatment run | Blocks | Total conjugated linoleic acid methyl ester (Ya, % w/w) | ||
---|---|---|---|---|
Experimental value (Y0) | Predicted valueb (Yi) | Y0 - Yic | ||
1 | 3 | 64.50 | 59.56 | 4.93 |
2 | 3 | 64.20 | 63.38 | 0.81 |
3 | 3 | 63.50 | 60.95 | 2.54 |
4 | 3 | 40.71 | 43.99 | −3.29 |
5 | 3 | 50.10 | 51.03 | −0.93 |
6 | 3 | 50.25 | 52.53 | −2.28 |
7 | 3 | 63.50 | 62.46 | 1.03 |
8 | 3 | 57.20 | 53.92 | 3.27 |
9 | 3 | 62.70 | 69.49 | −6.79 |
10 | 3 | 64.10 | 63.38 | 0.71 |
11 | 2 | 69.20 | 73.08 | −3.88 |
12 | 2 | 53.00 | 53.22 | −0.22 |
13 | 2 | 68.00 | 63.15 | 4.84 |
14 | 2 | 60.40 | 63.15 | −2.75 |
15 | 2 | 35.00 | 39.25 | −4.25 |
16 | 2 | 58.10 | 58.99 | −0.89 |
17 | 2 | 61.00 | 56.33 | −4.66 |
18 | 2 | 64.00 | 63.15 | 0.84 |
19 | 2 | 42.10 | 41.07 | 1.02 |
20 | 2 | 63.80 | 63.15 | 0.64 |
21 | 1 | 57.00 | 60.18 | −3.18 |
22 | 1 | 64.20 | 64.00 | 0.19 |
23 | 1 | 55.10 | 51.64 | 3.45 |
24 | 1 | 54.00 | 54.54 | −0.54 |
25 | 1 | 63.93 | 64.00 | −0.07 |
26 | 1 | 65.70 | 61.57 | 4.12 |
27 | 1 | 67.20 | 70.11 | −2.91 |
28 | 1 | 44.10 | 44.61 | −0.51 |
29 | 1 | 65.20 | 63.08 | 2.11 |
30 | 1 | 50.50 | 53.15 | −2.65 |
aNo significant differences (p > 0.05) between experimental (Y0) and predicted values (Yi); bThe predicted values are calculated by the software and are resulting from the reduced fitted model; cY0 - Yi: residual.
The terms with non-significant effects on the response (p ≥ 0.05) were dropped from the initial model and the experimental data were re-fitted with the significant terms (p < 0.05) (
Parameter | Model term | Estimated regression coefficient | F-ratio | p-value |
---|---|---|---|---|
β0 | Intercept | −147.943 | 24.501 | 0.001 |
Linear | ||||
β1 | x1 | - | - | 0.240a |
β2 | x2 | 2.206 | 28.621 | 0.002 |
β3 | x3 | 4.965 | 50.833 | 0.003 |
β4 | x4 | 39.264 | 54.614 | 0.001 |
Quadratic | ||||
β11 | x1 * x1 | - | - | 0.800a |
β22 | x2 * x2 | −0.007 | 24.010 | 0.002 |
β33 | x3 * x3 | - | - | 0.310a |
β44 | x4 * x4 | 15.363 | 18.042 | 0.001 |
Interaction | ||||
β12 | x1 * x2 | - | - | 0.093a |
β13 | x1 * x3 | - | - | 0.351a |
β14 | x1 * x4 | - | - | 0.272a |
β23 | x2 * x3 | - | - | 0.101a |
β24 | x2 * x4 | - | - | 0.210a |
β34 | x3 * x4 | - | - | 0.282a |
R2 | - | 0.888 | - | - |
Regression (F-ratio, p-value) | - | - | 34.771 | 0.003 |
aThe non-significant term (p > 0.05) which were further eliminated from the regression fitted model. 1) Time of the reaction (min); 2) Temperature (˚C); 3) Mass percentage NaOCH3 in total reaction mixture (% w/w); 4) Mass percentage of PEG400 in total reaction mixture (% w/w).
model terms (
where Y is the response, x2 is the temperature, x3 and x4 are the concentrations of the NaOCH3 and the PEG400, respectively. The adequacy of the regression model was verified by an analysis of the model and through the measurement of the coefficient of determination (R2) [
The reaction time was one of the main parameters used in many studies for chemical synthesis of CLA [
The temperature was varied (100˚C - 180˚C) to evaluate the effectiveness of this factor on response. The analysis of the variance showed that the linear and quadratic terms of the reaction temperature significantly affected the total CLAMEs (% w/w) production (p < 0.05) (
in similarity with findings by Yang and Liu (2004), where the quadratic term of the temperature has been shown to have a negative effect on production of CLA isomers [
The NaOCH3 that was used as a preferred catalyst for a prototropic double bonds shifting in isomerization has been broadly applied in commercial organic syntheses by numerous researchers and has been found to be capable of catalyzing the reactions at low temperatures [
The PEG400 was used to improve the degree of the isomerization in a solvent free system and was chosen since it is non-toxic and commonly used in pharmaceutical and medicinal industries. In addition it can be completely removed from the mixture with strong phosphoric acid [
According to the positive sign of the corresponding coefficients (
Based on the numerical optimization, the best combination of the reaction conditions (optimum point) for the maximum production of total CLAME (72.90%) was at the reaction temperature of 140˚C, 5% (w/w) NaOCH3, and 1.06% (w/w) PEG400 based on the weight of the total mixture (
method, the sample empirically produced under the optimum conditions was then analyzed by GC and the result was compared with that of predicted by the software using t-test analysis. The value of the total CLAME (72.90%) which was predicted by the software was compared with the actual amount of the total CLAME (70.40%) that was produced under the optimum conditions (
In this research, optimization of the production of two beneficial isomers of CLA was investigated. Isomerization of the FAMEs of the sunflower oil was investigated. To optimize the isomerization condition, the effect of four variables namely reaction temperature, the concentration of PEG400 and NaOCH3 on production of total CLAME was studied. The reaction temperature, the concentration of PEG400 and NaOCH3 were found to have positive influence on the response. However, the effect of the concentration of PEG400 was found to be more significant on response than those of other factors. The quadratic effect of the temperature was negative on response. Despite the upper limit of the reaction temperature already suggested by Wenk and Haeser (2005) was 150˚C, in this study the reaction temperature of 140˚C was the optimum temperature which resulted in the best response [
I express my deep gratitude towards the almighty god for providing this opportunity to complete this study. I thank all the personal who supported me with their knowledge and experience.
Koohikamali, S. (2017) Response Surface Optimization of a Solvent Free Production of cis-9, trans-11 and trans-10, cis-12 Isomers of Conjugated Linoleic Acid Using High Linoleic Sunflower Oil. Food and Nutrition Sciences, 8, 658-677. https://doi.org/10.4236/fns.2017.86047