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