Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Optimization of Chemical Synthesis of
Phytosterol Esters with Polyunsaturated
Fatty Acids by Response Surface
Methodology
Optimization of Chemical Synthesis of
Phytosterol Esters with Polyunsaturated
Fatty Acids by Response Surface
Methodology
Fenghong Huang, Qianchun Deng, Qingde Huang, Xing Liao, Changsheng Liu, Pin Zhang, Guangyuan NiOil Crops Research InstituteChinese Academy of Agricultural SciencesWuhan, 430062China
Staffs in the institute for Oilseeds processing and nutritional research
3 research fellows5 associate research fellows12 assistant research fellows8 Students
Staffs in the Oil Crops Research Institute:about 240
Major Research fields
Oil seeds processing technologyBiomass energy (Bio-diesel, bio-oil, biogas, etc.) Oil chemical productsMeal FeedOil and protein sciences Healthy and functional foods
Lipid lowering function foods: Kangxinling soft capsule Product serial number:卫食健字(2003)第0022号
1. IntroductionCVD disease
TOP ONE of disease leading to death in 2004: heart diseaseCVD disease: 1710 ten thousand in 2004, 2340 ten thousand in 2030Plasma LDL cholesterol concentration and risk for CHD: Positive correlation
Mechanism of action which phytosterols decrease cholesterol absorptionOrigin: Christopher P.F.,2006
The cholesterol-lowering action of phytosterol / phytostanols
Advantages• Cholesterol-lowering ability demonstrated in both humans and animals • National Cholesterol Education Program (NCEP) : encouraged consumption • A daily intake of 2–2.5 g : an average reduction in LDL cholesterol of up to 14%
Disadvantages• Not benefit for the absorbing of lipid-soluble vitamin• Very low solubility in edible oil • Insoluble in the micellar phase in a digestive organs• Presented in appropriate physical form
Prescription omega-3 acid ethyl esters (P-OM3) and lipid levels insubjects with hypertriglyceridemia (≥500 mg/dL) Origin: Harris et al, 2008
The cholesterol-lowering action of Polyunsaturated fatty acids (PUFAs)
Advantages• Known to reduce plasma total and low density lipoprotein (LDL)
cholesterol concentrations relative to triglycerides containing predominantly saturated fatty acids.
DisadvantagesPoor oxidative stabilityResult in oxidative damage to the bodyVery sensitive to heat, light etc.
The cholesterol-lowering action of phytosterol / phytostanols ester
Promoting efficacy by Esterification of sitostanol or sitosterol with fatty acids
• Enhance solubility in mayonnaise and margarines: • Solubility of ester is 10 times of sterol• Enhance dispersion in the intestine• PUFA connected with ester contribute to the efficacy
Generally Recognised as Safe Status (GRAS) by FDA in the USAFOSHU in JapanSeveral EU countries
Schematic diagram of the effects of plant stanol ester consumption on cholesterol metabolism in humans.
Origin: JOGCHUM PLAT AND RONALD P. MENSINK, 2002
Enzymatic methods:• Moderate reaction conditions and little by-products• Requiring organic solvents • Long reaction time • High costs and stay on the stage of laboratory research
Chemical methods• Shorter reaction time and simple reaction condition• Requiring higher temperature • Undesired by-products: dehydrated, oxysterols or oxidation products• Harmful catalysts • For industrial use
Synthesis Technology of phytosterol / phytostanols ester
To provide a food grade process: direct esterification reactionResponse surface methodology (RSM)
• Needs less number of experiments• More efficient and easier to arrange and to interpret in comparison to
othersFour reaction parameters were considered
• Temperature• Reaction time• Substrate mass ratio• Catalyst amount
Objective
2. MethodsMaterials
PUFA: linolenic acid 80%, linoleic acid 15%, oleic acid 5% Phytosterols: β-Sitosterol 77%, campesterol 17%, stigmasterol 5%
Direct esterification of phytosterols esters with PUFAWell-mixed by air table at 280 rpm Heated and stirred under the vacuum of the 0.03-0.04MPaStopped by cold water Distilled water was added to remove the catalyst Dried by anhydrous sodium sulfate
Optimum reaction conditions: Box–Behnken designPurification: Silica gel column chromatography Structure analysis: TLC, GC analysis, FT-IR analysisPhysicochemical properties analysis
Peroxide value (POV) and conjugated diene value (CD)
Direct esterification of phytosterols esters with PUFAWell-mixed by air table at 280 rpm Heated and stirred under the vacuum of the 0.03-0.04MPaStopped by cold water Distilled water was added to remove the catalyst Dried by anhydrous sodium sulfate
Optimum reaction conditions: Box–Behnken designPurification: Column chromatographyStructure analysis: TLC, GC analysis, FT-IR analysisPhysicochemical properties analysis
peroxide value (POV) and conjugated diene value (CD)Statistical analysis: the “Design Expert” software (Version 6.0.4.0, Stat-Ease Inc., Minneapolis, USA) statistical package
Gas chromatogram of mixture after reaction: (1)solvent: chloroform; (2)PUFA; (3) campesterol; (4) stigmasterol; (5) β-Sitosterol; (6-8)phytosterol esters of PUFA
Degree of esterification (DE, %)= 100BB A
×+
where A = peak area of total phytosterols (i.e., campesterol+ stigmasterol+β-Sitosterol); B = peak area of total phytosterol esters of PUFA.
Calculation of degree of esterification
3. Results
Sodium bisulfate was selected for the following experiments
Catalyst screening
Table 1 Effect of different catalysts on the DE
Catalyst Sodium
Methoxide
Sodium
Acetate
Sodium
Benzoate Citric Acid
Sodium
Bisulfate
Anhydrous
Sodium
Sulfite
Potassium
Chloride
Calcium
Hydroxide
Sodium
Hydroxide
DE(%) 54.61±2.80 54.88±1.93 53.22±0.43 59.43±1.03 88.36±0.35 74.02±1.95 58.78±0.96 54.09±0.11 49.15±1.90
Single factor experiments
Temperature 120℃, time 8 h, substrate mass ratio 4:1
Temperature 120℃, time 8 h, Catalyst 2%
Figure 1 Effects of single factor on Degree of esterification
The optimum range of reaction parameters: Catalyst 0.5-3.5%, time 6-10h, Temperature 110-150℃, substrate mass ratio 4:1
Temperature 120℃, substrate mass ratio 4:1, Catalyst 2%
substrate mass ratio 4:1, Catalyst 2%, time 8 h
Figure 1 Effects of single factor on Degree of esterification
94.110(130)0(8)0(2)15
87.670(130)0(8)0(2)14
94.040(140)0(9)0(2)13
91.581(150)1(10)0(2)12
94.661(150)-1(6)0(2)11
59.12-1(110)1(10)0(2)10
51.69-1(110)-1(6)0(2)9
81.251(150)0(8)1(3.5)8
89.941(150)0(8)-1(0.5)7
79.51-1(110)0(8)1(3.5)6
20.44-1(110)0(8)-1(0.5)5
93.460(130)1(10)1(3.5)4
72.680(130)1(10)-1(0.5)3
87.780(130)-1(6)1(3.5)2
40.660(130)-1(6)-1(0.5)1
Temperature (℃)
Reaction time(h)
Catalyst amount(%,w/w)
Y(DE,%)X3X2X1
Run no.Table 2 Box–Behnken design matrix of three variables and the experimentally observed response
Box-Benhnken Design
Y=β0+∑βiXi+∑βiiXi2+∑βijXiXj
ANOVA for quadratic model
Source SSa DFb MSc F-valueProb. (P) >
F
Model 7073.73 9 785.97 18.99 0.0024
Residual
(error) 206.90 5 41.38
Lack of fit 179.59 3 59.86 4.38 0.1913
Pure error 27.31 2 13.65
Total 7280.63 14
R2 = 0.9716; CV = 8.47; Adj. R2 = 0.9204. a SS, sum of squares. b DF, degrees of freedom. c MS, mean square.
Table 3
A good agreement between experimental and predicted values and indicates that the mathematical model is very reliable
Model fitting
Effects of
parameters
Model coefficient estimated by multiple linear regression
Model termParameter
estimate Standard error Computed t-value P-value
Intercept 91.94 3.71 24.782 0.0000
X1 14.78 2.27 6.511 0.0013
X2 5.26 2.27 2.317 0.0688
X3 18.33 2.27 8.075 0.0005
X1X2 -6.58 3.22 -2.043 0.0960
X1X3 -16.94 3.22 -5.261 0.0033
X2X3 -2.63 3.22 -0.817 0.4511
X12 -12.38 3.35 -3.696 0.0140
X22 -5.91 3.35 -1.764 0.1379
X32 -11.77 3.35 -3.513 0.0170
Table 4
Order of effect is as follows: X1X3>X1X2 > X2X3.
Figure 2 Contour plots of the combined effects of catalyst amount,reaction time and temperature on phytosterols ester with PUFA
Attaining optimum condition and model verification
Y=91.94+14.78X1+5.26X2+18.33X3-6.58X1X2-16.94X1X3-2.63X2X3-12.38X1
2-5.91X22-11.77X3
2
• the maximum point derived from the model:– Reaction condition: 2% of sodium bisulfate, 8.5h of reaction time,
145℃ of temperature– Predicted value of DE: 99.53%.
The second-order polynomial Equation:
Validation of the models• DE: 98.86 ± 0.0692%
• In good agreement with the predicted value
6.11±0.0083.3083±0.7380(140)0(9)0(2)15
6.13±0.0013.2293±0.9650(130)0(8)0(2)14
6.22±0.0073.5282±0.3250(140)0(9)0(2)13
10.01±0.0038.7082±0.3091(150)1(10)0(2)12
7.43±0.0045.7781±0.7231(150)-1(6)0(2)11
4.73±0.0062.2893±0.416-1(110)1(10)0(2)10
4.04±0.0042.0098±1.296-1(110)-1(6)0(2)9
8.49±0.0028.0480±0.7351(150)0(8)1(3.5)8
12.13±0.0098.7134±0.0231(150)0(8)-1(0.5)7
4.95±0.0063.3971±0.484-1(110)0(8)1(3.5)6
5.17±0.0053.8770±0.289-1(110)0(8)-1(0.5)5
6.28±0.0035.2960±1.4260(130)1(10)1(3.5)4
8.11±0.0066.3083±0.5370(130)1(10)-1(0.5)3
5.14±0.0023.5160±0.3330(130)-1(6)1(3.5)2
8.13±0.0027.2293±0.3000(130)-1(6)-1(0.5)1
Temperature (℃)Reaction time (h)Catalyst amount(%,w/w)
CD(mmol/kg)
POV(meq/kg)
X3X2X1Run no.
Table 5 Results of POV and CD values for the Box-Benhnken design
The POV value and CD value of the central point experiment were The POV value and CD value of the central point experiment were 3.36 3.36 meqmeq / kg and 6.15 / kg and 6.15 mmolmmol/kg oil/kg oil
Physiochemical properties
Food grade processReaction conditions
• vacuum 0.03-0.04Mpa• mass ratio of PUFA: phytosterols=4:1• catalyst amount: 3%• reaction temperature: 130℃• reaction time: 8h
DE: 96.27 ± 0.0256%POV: 3.05 meq / kgCD value: 5.75 mmol / kg
Phytosterols
Figure 3 The TLC graph of phytosterols ester with PUFA
TLC (Thin layer chromatography) analysis
Phytosterols esters
PUFA
GC (Gas chromatography) analysis
Figure 4 Gas chromatogram of phytosterols ester with PUFA
3420cm-1: -OH1710cm-1: C=O1176cm-1: C-O-C3088cm-1: unsaturated double bond
FT-IR (Fourier transform infrared spectroscopy) analysis
Figure 5 FT-IR spectrum of phytosterols ester with PUFA
4. Conclusion
Food grade processCatalyst: safe, easy to removeSolvent: PUFAwithout the use of watertrapping agentsGender condition
High DE: above 96%The products exhibits a good physiochemical properties andpossess a high food safety
Thank you foryour attention