ORIGINAL PAPER

Optimization of Phytosterols Recovery from Soybean OilDeodorizer Distillate

Feng Yan • Haojun Yang • Jianxin Li • Huiling Wang

Received: 4 October 2011 / Revised: 19 January 2012 / Accepted: 19 January 2012 / Published online: 8 February 2012

� AOCS 2012

Abstract A large amount of phytosterols in the bound

form remains in the waste residue during the traditional

process of recovering tocopherols and sterols from soybean

oil deodorizer distillate (SODD). In order to avoid the loss

of natural resources, we developed a process to recover the

maximum amount of phytosterols from SODD. The pro-

cess includes saponification, methyl esterification, and

crystallization. The purpose of saponification and methyl

esterification were to decompose the bound phytosterols

and to esterify the fatty acids, respectively. The yield of

sterols was dependent on saponification and solvent crys-

tallization. The conditions of saponification and solvent

crystallization were optimized by single-factor tests and

response surface methodology, respectively. The sterol

yield obtained under the optimized conditions was 6.64%.

This value is much greater than 4.43% obtained by the

traditional industrial process. The purity of the recovered

sterols was 94.7%.

Keywords Soybean oil deodorizer distillate (SODD) �Phytosterols � Steryl esters � Crystallization � Response

surface methodology

Introduction

Phytosterols (plant sterols) structurally resemble choles-

terol, but with different side-chain configurations. Phytos-

terols are integral natural components of cell membranes in

vegetable oils, nuts, seeds, and grains and are additives in

functional foods (e.g. margarines). Phytosterols have a

wide spectrum of biological effects including anti-inflam-

matory, anti-oxidative, and anti-carcinogenic activities [1].

Their cholesterol-lowering capacities have been exten-

sively researched. Several studies have shown that plant

sterols inhibit intestinal absorption of cholesterol, thereby

lowering total plasma cholesterol [2].

Deodorizer distillates from edible oils constitute a major

industrial source of phytosterols [3]. Deodorization is the last

step in the refining of edible oils and involves the removal of

some undesirable components of natural fats and oils. The

treatment is conducted at 200 �C in vacuo for several hours.

The by-product thus obtained is called ‘‘deodorizer distillate’’

and consists of a complex mixture of various compound

families including fatty acids, sterols, tocopherols, sterol

esters, glycerides, hydrocarbons and others. Soybean oil

deodorizer distillate (SODD) is especially interesting due to

the large amount of useful products it contains, and is an

important source of natural tocopherols and phytosterols [4].

Preparing high-purity concentrates of phytosterols and

tocopherols involves a series of physical and chemical

treatment steps. Several studies have been conducted to

recover phytosterols and tocopherols from deodorization

distillate, such as crystallization [5, 18], solvent extraction

F. Yan (&) � H. Wang

State Key Laboratory of Hollow Fiber Membrane Materials

and Processes, School of Environmental and Chemical

Engineering, Tianjin Polytechnic University,

Tianjin 300160, People’s Republic of China

e-mail: yanfeng.yxm@hotmail.com

H. Yang

E-Tech Energy Technology Development Corporation,

Tianjin, People’s Republic of China

J. Li (&)

Laboratory for Membrane Materials and Separation Technology,

Sustainable Technology Research Center, Shanghai Advanced

Research Institute, Chinese Academy of Sciences,

Shanghai 201203, People’s Republic of China

e-mail: jxli0288@yahoo.com.cn

123

J Am Oil Chem Soc (2012) 89:1363–1370

DOI 10.1007/s11746-012-2023-0

[6], supercritical fluids extraction [7, 8], supercritical

extraction with a multistage countercurrent column [9],

transesterification to form methyl esters followed by frac-

tional distillation [10], neutralization and washing [11],

membrane separation [12], enzymatic esterification [13,

14], and batch adsorption [15]. Phytosterols cannot be

removed by molecular distillation because their molecular

weights and volatilities are similar to those of tocopherols

[16]. It is difficult to recover concentrates of tocopherols

and phytosterols in good yield and high quality. Since

phytosterols are insoluble in some cold solvents, they could

be obtained by crystallization [13].

In industry production, a comprehensive process is

widely adopted. The process includes methyl esterification

by using sulfuric acid as a catalyst, transesterification by

using an alkaline catalyst, crystallization and filtration to

separate sterols, and molecular distillation to produce

concentrated tocopherols, fatty acid methyl esters (FAME)

and waste residue; however, there are two key problems in

the process. One is the low content of tocopherols (about

50%) in the concentrated fractions. The other is that a large

amount of bound sterols (namely sterol esters) is lost in the

waste residue [17]. The percentages of sterols (in the bound

form) are about 30, 17, and 20% of waste residue from

CGOG TECH. Bioengineering Co. Ltd. (Tianjin, China);

Heilongjiang Jiusan Oil & Fat Co. Ltd. (Heilongjiang

Province, China,); and Jiangsu Spring Fruit Biological

Products Co. Ltd. (Jiangsu Province, China), respectively.

The purpose of the present study was to develop a suitable

process to recover phytosterols not only in free state, but

also in the bound form from SODD.

Materials and Methods

Materials

Soybean oil deodorizer distillate and a standard sample of

mixed phytosterols (95.30% phytosterols) were supplied by

CGOG TECH. Bioengineering Co. Ltd. (Tianjin, China).

Cholesterols ([97% purity) were purchased from Tianjin

Guangfu Fine Chemical Research Institute (Tianjin,

China). Other chemicals, such as acetic anhydride, pyri-

dine, methanol, ethanol, n-propanol, petroleum ether, ace-

tone, butanone, cyclohexanone, benzene, toluene, were all

of analytical grade and were used as received.

Reaction Procedure

Saponification of SODD

Soybean oil deodorizer distillate (30 g) was dissolved in

30 mL ethanol in a 250-mL three-neck flask equipped

with a reflux condenser, a dropping funnel, and a nitrogen

duct. After the mixture was heated to reflux, NaOH

solution (30 mL) was added dropwise through the drop-

ping funnel. The mixture reacted at reflux temperature and

under N2 for 2 h after adding NaOH. After saponification,

excess acid was added to the mixture. The acidified

mixture was then transferred to a separatory funnel, and

the organic phase was washed with water three times. The

residue water in the organic phase was evaporated under

vacuum. The fatty acid steryl esters in SODD were

transformed into free sterols and FFA, and the glycerides

were transformed into FFA and glycerol. In order to

decompose the steryl esters completely, the effects of

NaOH dosage on the free sterol content in the organic

phase after saponification and acidify was investigated by

using single-factor tests. Two replicates for each dosage

of NaOH were done to obtain mean sterol contents in the

organic phases.

Methyl Esterification

The organic phase obtained after saponification was

esterified with methanol catalyzed by using sulfuric acid.

After esterification, the mixture was repeatedly washed

with water until pH 7 was achieved. During the process, the

FFA were then transformed into FAME. The organic phase

was then evaporated under vacuum.

Phytosterol Recovery by Crystallization

Phytosterols were recovered from the feed solution

obtained after saponification and methyl esterification by

using solvent crystallization. The feedstock was dissolved

in different solvents at 60 �C. Then the solution was

cooled to a ripening temperature and was kept at this

temperature for a ripening time. Phytosterol crystals were

finally obtained by vacuum filtration. The solvent selec-

tion, ripening temperature, and ripening time were varied

to optimize the crystallization conditions by using a

central-composite design (CCD). The independent factors

A, B and C represented proportion of feed solution to

solvent, ripening temperature (�C), and ripening time (h),

respectively. Five levels (-1.68, -1, 0, ?1 and ?1.68)

CCD was performed to determine the best combination

effect of the three crystallization parameters. The layout

of the CCD and the experimental response (sterols yield,

%) obtained with each run are shown in Table 1 and all

experiments were randomized. Two replicates were done

for each run to obtain the mean yield of sterols. The

fitting degree of the model was evaluated by the coef-

ficient of determination (R2) and the analysis of variance

(ANOVA).

1364 J Am Oil Chem Soc (2012) 89:1363–1370

123

Analytical Methods

Determination of Sterol Yield

The yield of sterols was calculated by using Eq. 1.

Sterol yield

¼ the mass of phytosterols recovered from SODD=

the mass of SODD ð1Þ

Determination of Sterol Content

A GC (Agilent 6890N, USA) with an HP-5 capillary col-

umn was used to analyze the content of sterols in the feed

solution and in the sterol products. All samples were

derivatized with acetic anhydride before testing. Choles-

terol was used as an internal standard. Analytical condi-

tions were: 280 �C column temperature; 300 �C FID

detector; N2 carrier gas; and temperature program of 1 min

at 100 �C, followed by heating to 250 �C at 35 �C/min, and

then to 280 �C at 30 �C/min (where it was held for

30 min).

The purity of sterols was calculated by using Eq. 2 [17]

Y ¼ FAsample � minternal

Ainternal � msample

� 100% ð2Þ

where Y is the content of sterols (%), F is the correction

factor, Asample is the total peak area of sterols in a sample,

Ainternal is the total peak area of internal standard, minternal is

the mass of the internal standard (mg), and msample is the

mass of a sample (mg). The correction factor F was

obtained from the standard sample (95.30% purity) from

CGOG TECH. Bioengineering Co., Ltd.

Results and Discussion

The free sterol content in SODD was 4.36% and increased

to 9.30% in the feed solution after SODD was saponified

and to 9.12% in the feed solution after methyl esterifica-

tion. More than one-half of the sterols was in the bound

form in SODD. Furthermore, the free sterol content was

6.80% in the feed solution after methyl esterification and

transesterification in industrial production from CGOG

TECH. Bioengineering Co. Ltd. The yield of sterols was

only 4.43% in the industrial production. The results indi-

cate that the present process including saponification can

transfer the bound sterols into free sterols more efficiently,

and the process is promising for sterol recovery from

SODD.

Optimization of Saponification

The dosage of NaOH for the saponification was explored

as shown in Fig. 1. NaOH dosage influenced the

Table 1 The setting of central

composite design and the

observed response values

Run Code parameter Actual parameter values Yield of sterols

Y (%)A B C A (g/ml) B (�C) C (h)

1 -1.00 -1.00 -1.00 2.00 0.00 16.00 5.64

2 0.00 0.00 0.00 3.00 5.00 24.00 6.58

3 0.00 0.00 0.00 3.00 5.00 24.00 6.60

4 0.00 -1.68 0.00 3.00 -3.41 24.00 6.04

5 0.00 0.00 1.68 3.00 5.00 37.45 6.26

6 -1.00 -1.00 1.00 2.00 0.00 32.00 5.64

7 -1.00 1.00 1.00 2.00 10.00 32.00 5.75

8 0.00 0.00 -1.68 3.00 5.00 10.55 6.72

9 0.00 0.00 0.00 3.00 5.00 24.00 6.58

10 1.00 -1.00 -1.00 4.00 0.00 16.00 6.16

11 1.00 1.00 1.00 4.00 10.00 32.00 6.24

12 0.00 0.00 0.00 3.00 5.00 24.00 6.64

13 0.00 0.00 0.00 3.00 5.00 24.00 6.68

14 1.00 -1.00 1.00 4.00 0.00 32.00 6.46

15 -1.68 0.00 0.00 1.32 5.00 24.00 5.72

16 0.00 1.68 0.00 3.00 13.41 24.00 5.84

17 1.68 0.00 0.00 4.68 5.00 24.00 6.37

18 1.00 1.00 -1.00 4.00 10.00 16.00 5.52

19 -1.00 1.00 -1.00 2.00 10.00 16.00 5.44

20 0.00 0.00 0.00 3.00 5.00 24.00 6.68

J Am Oil Chem Soc (2012) 89:1363–1370 1365

123

decomposition of bound sterols. The free sterol content in

the feed solution after saponification, C (%), increased

from 6.64 to 9.30% as the NaOH/SODD mass ratio

increased from 0.17/1 to 0.33/1. The free sterol content did

not increase further with an increased NaOH/SODD ratio.

Therefore, the optimized dosage of NaOH for the saponi-

fication was 0.33/1 NaOH/SODD mass ratio.

In order to confirm whether there was unreacted sterol

ester left in the organic phase, column chromatography was

used to separate free sterols from the organic phase when

the NaOH/SODD mass ratio was 0.33:1. The FTIR spec-

trum of decomposed SODD after separating free sterols is

presented in Fig. 2, where the C=O and C–O–C stretching

bands of the ester group were 1,744 and 1,168 cm-1,

respectively (curve A) and the two wave peaks disappeared

when SODD was saponified (curve B). This result indi-

cated that there was no/little unreacted sterol esters left in

the organic phase after saponification.

Phytosterol Recovery by Crystallization After Methyl

Esterification

Selection of Solvents for Crystallization

In the present work, a variety of solvents including various

alcohols, alkanes, ketones, esters, and aromatics were used

in the crystallization of phytosterols from feed solution

after methyl esterification (Table 2). Methanol gave the

best crystallization efficiency with high yield (4.48%) and

high purity (95.33%). When alcohols or ketones were used

as crystallization solvents, the yield of phytosterols

decreased as the length of hydrocarbon chains increased.

No phytosterols crystals precipitated in n-propanol and

butanone, probably due to increased solubility of sterols as

the hydrocarbon chain length of alcohols and ketones

increased. Furthermore, the sterols crystallized only at low

temperature (-8 �C) when benzene or toluene was used as

solvent. Methanol, petroleum ether and ethyl acetate gave

higher yields of sterols.

As discussed above, low productivity of sterols was

obtained when a single solvent was used for the crystalli-

zation. The yield of phytosterols in a solvent–water system

was much higher than that in a single solvent (Fig. 3). The

yield of phytosterols increased from 3.35 to 6.55% as the

water/petroleum ether ratio (V/V) increased from 0 to 0.16.

This may be because sterols tend to form hydrates with

water, which leads to their decreased solubility. Accord-

ingly, the yield of phytosterols extracted from a water-

bearing feed solution would be much higher than from an

anhydrous feed [18]. As shown, further increases in the

water content in petroleum ether had little effect on phy-

tosterol yield. By contract, the phytosterol yield increased

5

6

7

8

9

10

NaOH/SODD mass ratio (w/w)

C (

%)

0.17:1 0.20:1 0.25:1 0.33:1 0.50:1

Fig. 1 Effect of NaOH dosage on the content of free sterols in feed

solution after saponification

4000 3500 3000 2500 2000 1500 1000 500

2675

2853

2924

723

939

1096

1168

1242

1461

1744

1710

Wavenumber cm-1

Tra

nsm

ittan

ce

A

B

Fig. 2 The FT-IR spectra of SODD oil (A) and decomposed SODD

oil (B)

Table 2 The effects of different solvents on crystallization of

phytosterols

Run Solvents TRa (�C) Yield of sterols (%) Purityb (%)

1 Methanol 6 4.48 95.33

2 Ethanol 6 1.17 93.74

3 n-Propanol 6 No crystal

4 Acetone 6 2.57 91.82

5 Butanone 6 No crystal

6 Benzene -8 2.18 82.86

7 Toluene -8 1.36 91.85

8 Petroleum ether 6 3.35 93.12

9 Ethyl acetate 6 3.20 93.68

a Ripening temperatureb Purity of sterol sample

1366 J Am Oil Chem Soc (2012) 89:1363–1370

123

from 4.48 to 5.68% as water/methanol ratio increased from

0 to 0.12. In the same way, the yield of phytosterols

increased to 5.43% as the water/ethyl acetate ratio was

increased to 0.16. The purities of sterol samples obtained

from the solvent–water systems were all [94%. A petro-

leum ether–water co-solvent system could give higher yield

of sterols. The optimal yield of phytosterols was 6.64% with

95.88% purity at 0.24 water/petroleum ether ratio (V/V).

Optimization of Crystallization Conditions

by Single-Factor Tests

The yield of recovered sterols increased from 4.40% to a

maximum of 6.70% as the feed solution to solvent ratio

increased from 1:2 to 1:0.33 (w/V) (Fig. 4a). The yield of

recovered sterols increased as solvent dosage decreased.

Some sterols remain dissolved in the solvent and do not

precipitate. However, it showed a negative influence on

sterols precipitation with further decrease of solvent dos-

age. As shown in Fig. 4a, at a range from 1:0.33 to 1:0.2,

the yield of recovered sterols decreased as a result of

insufficient phytosterol dissolution. Thus, the optimum

feed solution to solvent ratio was 1:0.33. The sterol yield

decreased as ripening temperature increased, while it

increased when ripening time increased (Fig. 4b, c).

Therefore, the optimum crystallization conditions to obtain

the highest sterol yield were ripening at 5 �C for 24 h.

RSM Model Fitting

RSM was applied to model and optimize the crystallization

conditions of phytosterols recovered from SODD after

saponification and methyl esterification. The significant

quadratic models and the corresponding significant model

term for all responses are tabulated in Table 3.

The best-fitting models were determined through mul-

tiple linear regressions with backward elimination. The

accuracy of the models was evaluated by a coefficient of

determination (R2). The determination coefficient (R2 =

0.8192) indicated that 81.92% of the variation in sterol

yield was attributed to the independent variables (Table 3).

On the other hand, a lower coefficient of variation

(CV = 3.45%) indicated better precision and reliability of

the experiments carried out [19]. The CV as the ratio of the

standard error of estimate to the mean value of the

observed response (as a percentage) was a measure of the

reproducibility of the model and as a general rule a model

is considered to be reasonably reproducible if the CV is not

[10% [20]. By applying diagnostic plots, including normal

probability plotting of residuals, and plotting residuals

versus predicted, assumptions of normality, independence

0.0 0.1 0.2 0.3 0.42

3

4

5

6

7

8

ethyl acetate

petroleum ether

Yie

ld (

%)

Ratio of water/solvent (V/V)

methanol

Fig. 3 Effect of water as a co-solvent on yield of phytosterols by

crystallization

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Proportion of feed solution to solvent (w/V)

Yie

ld (

%)

1:2 1:1 1:0.5 1:0.33 1:0.25 1:0.2

0 5 10 15 204.0

4.5

5.0

5.5

6.0

6.5

7.0

Yie

ld (

%)

Ripening temperature ( oC)

8 16 24 324.0

4.5

5.0

5.5

6.0

6.5

7.0

Yie

ld (

%)

Ripening time (h)

(a)

(b)

(c)

Fig. 4 Effect of crystallization condition single factors on the yield

of phytosterols a ratio of feed solution to solvent, b ripening

temperature, c ripening time

J Am Oil Chem Soc (2012) 89:1363–1370 1367

123

and randomness of the residual were satisfied. The fitted

model for phytosterol yield was accepted. The adequate

precision value was a measure of the ‘‘signal-to-noise

ratio’’ for the responses. A ratio [4 was considered to be

adequate model discrimination [21]. The 6.280 ratio indi-

cated an adequate signal to noise ratio (Table 3). The

predictive model could be used to navigate the space

defined by CCD.

The P value was used as the tool to check the signifi-

cance of each coefficient, which also indicated the inter-

action strength of each parameter. The smaller the P value

was, the greater the significance of the regression coeffi-

cient. In other words, the higher the F value was for the

model, the lower probability that the value was for the

model, which also indicated the greater significance of

the fitted model. In the present study, the model F value of

5.04 indicated the model was significant. There was only a

0.94% chance that a ‘‘Model F-Value’’ this large could

occur due to noise. Values of \0.0500 ‘‘Prob [ F’’ indi-

cated model terms were significant. As shown in Table 3,

the liner term of feed solution to solvent ratio (A) had a

large effect on the sterol yield and was significant due to

the high F value. The quadratic term of feed solution to

solvent ratio, ripening time and ripening temperature were

all significant; however, the effect of interaction between

any two variables did not affect the sterols yield.

Analyzing the contour plots for yield of sterols was the

best way to evaluate the relationships between responses,

variables and interactions. The dimensional response sur-

face was plotted (Fig. 5) as a function of the interaction of

feed solution to solvent ratio (A) and ripening temperature

(B) at a medium ripening time 24 h. With each variable

increasing, the sterol yield initially increased and then

slightly decreased. The responses obtained were convex

Table 3 ANOVA for the

regression model and respective

model terms

Source Sum of

squares

Degree

of freedom

Mean of

square

F value P value

Prob [ FRemarks

Model 2.01 9 0.23 5.04 0.0094 Significant

A 0.49 1 0.49 10.62 0.0082 Significant

B 0.020 1 0.020 0.43 0.5271 Not significant

C 0.20 1 0.20 4.26 0.0660 Not significant

AB 5.000E-003 1 5.000E-003 0.11 0.7492 Not significant

AC 8.450E-003 1 8.450E-003 0.18 0.6782 Not significant

BC 7.200E-003 1 7.200E-003 0.16 0.7016 Not significant

A2 0.72 1 0.72 15.45 0.0028 Significant

B2 0.46 1 0.46 9.93 0.0103 Significant

C2 0.46 1 0.46 9.93 0.0103 Significant

Residual 0.46 10 0.046

Lack of fit 0.45 5 0.091 45.52 0.0004 Significant

Pure error 9.950E-003 5 9.950E-003

R2: 0.8192 CV%: 3.45 Adequate precision: 6.280

10.00

7.50

5.00

2.50

0.002.00 2.50 3.00 3.50 4.00

B: Ripening temperature A

Ratio of feed solution to solvent

Fig. 5 Response surface curve and contour plot showing predicted

response surface of phytosterol yield as a function of feed solution to

solvent ratio and ripening temperature

1368 J Am Oil Chem Soc (2012) 89:1363–1370

123

which suggested well-defined optimum operating condi-

tions. The dimensional response surfaces of interactions

between feed solution to solvent ratio (A) and ripening time

(C), and between ripening temperature (B) and ripening

time (C) were similar to that between feed solution to

solvent ratio (A) and ripening temperature (B).

Optimization of Phytosterol Crystallization Conditions

For crystallization conditions, the RSM clearly indicated

that optimum phytosterol recovery (6.69%) was produced

with a feed solution to solvent ratio (A), ripening temper-

ature (B) and ripening time (C) at 3.41:1 (g/ml), 4.48 �C,

and 26.47 h, respectively. In order to confirm the predicted

results of the optimized model, experiments were carried

out with the crystallization conditions at these conditions.

The sterol yield obtained at the optimized conditions was

6.64%, which suggested the model was reliable. The purity

of recovered phytosterols under the optimized conditions

was 94.7%.

Purity and Structure Analysis of Recovered

Phytosterols

The results from GC analysis of recovered phytosterols

showed that cholesterol (internal standard) and four kinds

of sterols eluted with the relative retention time (RT) of

17.019, 17.875, 19.687, 20.631 and 22.545 min, respec-

tively. According to the peak areas of sterols and Eq. 1, the

purity of the recovered phytosterols sample was 96.95%.

GC–MS spectrometry was further utilized to determine

the exact kind of sterol corresponding to the relative

retention time. Brassicasterols (RT: 17.88 min), campes-

terols (RT: 19.68 min), stigmasterols (RT: 20.63 min) and

b-sitosterols (RT: 22.55 min) were present, which was in

agreement with our previous work [17]. Furthermore, the

brassicasterol, campesterol, stigmasterol, and beta-sitos-

terol contents of the recovered phytosterols were 0.81,

27.72, 23.69 and 44.72 wt%, respectively. In addition, GC–

MS showed that there were impurities in the sterol prod-

ucts; the impurities were sterol derivatives by oxidation/

reduction, such as hydrogenation of sterols.

Conclusions

We developed a saponification, methyl esterification and

solvent crystallization process for phytosterol recovery

from SODD. The conditions of saponification and solvent

crystallization were optimized. Petroleum ether with water

as cosolvent could generate desirable crystallization. The

predicted yield of the recovered sterols (6.69%) was con-

sistent with the experimental result 6.64% (94.7% purity)

under optimum crystallization conditions (3.41:1 g/ml feed

solution to solvent ratio, 26.5 h ripening time, 4.5 �C rip-

ening temperature). Our process is a promising route to

recover phytosterols from SODD.

Acknowledgements The authors gratefully acknowledge the

financial support of the National High Technology Research and

Development Program of China (‘‘863’’ Program, Grant

No.2009AA03Z223), National Science and Technology Major Pro-

ject (2011ZX05011) and Tianjin ‘‘Double Five’’ Science Foundation

(Grant No. SWPY 20080004). The authors also thank Ms. Ming Huo

and Mr. Daogeng Wu for their contributions.

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