Synthesis of plant sterol esters catalyzed by heteropolyacid in a ...

Eur. J. Lipid Sci. Technol. 108 (2006) 13–18 Xianghe Menga Peilong Suna Qiuyue Panb Zhongping Shic Kai Yanga Rongjun Hea a

College of Biological & Environmental Engineering, Zhejiang University of Technology, Hangzhou, P. R. China b Zhejiang Economic & Trade Polytechnic, Hangzhou, P. R. China c School of Biotechnology, Southern Yangtze University, Wuxi, P. R. China

DOI 10.1002/ejlt.200500265

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Synthesis of plant sterol esters catalyzed by heteropolyacid in a solvent-free system The synthesis of phytosteryl esters is of importance due to their recent recognition and application as cholesterol-lowering agents in the food and nutraceutical industries. In this study, a synthetic route potentially useful for the large-scale production of foodgrade phytosteryl esters with high yield and purity in a solvent-free system was investigated. To examine the feasibility of replacing sodium methylate by heteropolyacid, four heteropolyacids, tungstosilicic acid, tungstophosphoric acid, molybdosilicic acid and molybdophosphoric acid, were evaluated to determine the best catalyst and the optimum conditions for the esterification reaction between various fatty acids and phytosterols. The results suggested that tungstosilicic acid was more selective towards butyric acid and caprylic acid than towards lauric acid, palmitic acid, and oleic acid. However, there was no significant discrimination in terms of the tungstosilicic acid catalyst’s selectivity to stearic acid, oleic acid, linoleic acid and alpha-linolenic acid, all with C18 chains, in the esterification reaction. The yield of phytosteryl ester was higher than 90% when the esterification reaction was carried out at 150 7C, with phytosterols and fatty acids in a molar ratio of 1 : 1.5, and catalyzed by 0.2% tungstosilicic acid in silica gel. The catalysts recovery experiments suggested that the immobilized tungstosilicic acid did not significantly lose its activity in six operation runs. As a result, the immobilized tungstosilicic acid would be a promising catalyst for replacing sodium methylate, to synthesize phytosteryl esters with fatty acids and phytosterols as the starting materials in a commercial production.

1 Introduction Dietary phytosterols including phytostanols have been widely shown in the literature to efficiently lower cholesterol absorption. Typically, 0.5–3.0 g/day of plant sterols produce a 30–80% lowering of the cholesterol absorption and a corresponding 10–15% lowering of the LDL cholesterol [1–3]. Since phytosterols are nontoxic natural products and inexpensive byproducts of food processing, the use of plant sterols as therapeutical dietary options before resorting to drug treatment to lower plasma cholesterol concentration was encouraged [4]. Despite their potential attractiveness, the usefulness of phytosterols has been limited by their higher melting point, lower solubility in both water and oil phases, and their chalky taste. As a food additive to be used broadly, phytosterols should also be conveniently incorporated into food, without the adverse organoleptic effects. For

Correspondence: Xianghe Meng, College of Biological & Environmental Engineering, Zhejiang University of Technology, Zhaohui xincun, Hangzhou, Zhejiang, 310014, P. R. China. Phone: 186–571–88320345, Fax: 186–571–88320345, e-mail: [email protected], [email protected]

this reason, several ways have been proposed to increase the solubility or bioavailability of phytosterols [5, 6]. Based on previous studies and the finding that phytosteryl esters are much more soluble than the free phytosterols in the oil phase, it has been proposed to use phytosteryl esters in oil to lower cholesterol absorption [7]. U.S. Patent No. 5,502,045 described the use of sitostanol ester in oil for the treatment of hypercholesterolemia in humans [8]. It was suggested that the cholesterol-lowering effect of sitostanol may be increased when it is ingested in a soluble form (phytosteryl ester) [9]. Methods of phytosteryl ester production are various. For example, in the 1970s and 1980s, Saroja et al. reported the synthesis of sterol fatty acid ester catalyzed by chlorine [10], bromine [11], thionyl chloride [12] or anhydride derivatives of fatty acids. However, the catalysts and reagents were not accepted for the production of food or food additives. Other researchers reported lipase-catalyzed production of phytosteryl esters, by esterification and/or transesterification methods, in organic solvent or solvent-free systems [13–18]. Enzymatic reactions have many advantages, such as high specificity, mild reaction conditions and environmental friendliness. However, the broad application of enzymatic methods was still limited

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Research Paper

Keywords: Phytosteryl ester, esterification, heteropolyacid, fatty acid, immobilization.

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by high costs of lipases, lower productivity, and longer reaction time (as long as 24 h). In addition, enzymatic synthesis of phytosteryl esters in supercritical carbon dioxide was studied by Jerry et al. [19], but it is still far from meeting industrial demands. Up to now, chemical synthesis is still the main method for commercial production of phytosteryl esters. The typical operation is a transesterification reaction of phytosterols and edible oil methyl esters, catalyzed by sodium methylate. However, the acyl profile of phytosteryl esters is difficult to control since the profile is dependent on the array of fatty acids presented in the oil employed in the reaction. In addition, the need to dispose of the strongly corrosive sodium methylate could potentially pollute the environment. Heteropolyacid is a strong solid acid with high catalytical activity and lower corrosiveness; it is often used as catalyst for esterification reactions in the food and chemical industry [20]. To develop a method for large-scale production of phytosteryl esters from phytosterols and fatty acids at high yield, low cost and low pollution, the feasibility of replacing sodium methylate by heteropolyacid salt was investigated in the present study.

Eur. J. Lipid Sci. Technol. 108 (2006) 13–18 then, a predetermined amount of support was added to the above solution, and the mixture was thoroughly stirred for 2 h. The support, impregnated with heteropolyacid, was air-dried for 1 h, and thereafter dried for 5 h at 150 7C. The prepared catalyst was cooled down to ambient temperature in a desiccator. The amount of the supported heteropolyacid catalyst can be simply calculated by subtracting the weight of the original support.

2.3 Esterification method A phytosteryl ester mixture was prepared in a 50-mL three-neck glass vessel. Before use in the esterification reaction, phytosterols were dried in an oven at 607 overnight and fatty acids were dried by a 3A molecular sieve. The esterification was carried out as follows: A mixture of phytosterol and fatty acid (unless otherwise stated, oleic acid was used in the esterification reaction) in a certain molar ratio was added to a three-neck vessel, then heated to the desired temperature under nitrogen. Drying continued for 1 h and then the catalyst was added. The reaction was continued for approximately 5 h and the conversion rate of the reaction was monitored with thinlayer chromatography (TLC).

2 Materials and methods 2.1 Materials The reagent fatty acids (butyric acid, caprylic acid, lauric acid, palmitic acid, and oleic acid, linoleic acid, alphalinolenic acid), tungstosilicic acid (H4SiW12O40 ?6 H2O), tungstophosphoric acid (H3PW12O40 ?6 H2O), molybdosilicic acid (H3SiMo12O40 ?6 H2O), molybdophosphoric acid (H3PMo12O40 ?6 H2O), silica gel, zeolite, diatomite, and activated carbon were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, P. R. China). Soy sterol mixtures (purity 95%, sitosterol 42.49%, stigmasterol 27.26%, campesterol 24.50%, brassicasterol 3.58%, others 2.17%) were obtained from Spring Fruit Biological Products Co., Ltd. (Taizhou, Jiangsu, China). All sterol standards were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Preparation of heteropolyacid catalyst support

2.4 Repeated use of catalyst When the reaction was finished, the immobilized catalyst was separated by filtration and reused in a next batch for esterification. Ten cycles of this reaction were conducted. The conversion rate of the reaction was measured after each run.

2.5 TLC [21] Samples were taken from the reaction mixtures, immobilized catalyst was separated by centrifugation, and the conversion rate was monitored by TLC on a 0.25-mm layer of silica gel G254 (Merck), using n-hexane/diethyl ether/formic acid (90 : 10 : 0.1, vol/vol/vol). Spots were located by iodine staining.

2.6 HPLC analytical procedure [22]

The absorption of all of the four heteropolyacids (tungstosilicic acid, tungstophosphoric acid, molybdosilicic acid, and molybdophosphoric acid) to a support followed the same procedure, as described below. A certain amount of heteropolyacid was solved in 100 mL distilled water until a saturated solution was formed. The support to be used was dried for 4 h at 110 7C in a hot-air dryer;

The final esterification yield was determined using the HP1050 HPLC system (Dionex Inc., Salt Lake City, UT, USA), with an Alltech 2000 evaporative light scattering detector (ELSD; Alltech Co., France). The ELSD was operated at 40 7C with N2 as a nebulizing gas at a pressure of 2.2 bar. A Shimadzu-Pack HRC-SiL column (5 mm, 25064.6 mm; Shimadzu, Tokyo) was used to separate the products and reactants. The area (%) of the produced


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phytosteryl ester was calculated as esterification rate. The mobile phase was a mixture of n-hexane, isopropanol and acetic acid (90 : 10 : 0.1, vol/vol/vol), and the flow rate was 0.5 mL/min.

Tab. 2. Synthesis ratio of phytosteryl oleate catalyzed by tungstosilicic acid immobilized on various supports in 5 h. Support

Tungstosilicic acid load [wt-%]

Phytosteryl oleate after 5 h of reaction [%]

3 Results and discussion

Silica gel Activated carbon Diatomite Zeolite

25 35 28 42

78.6 76.5 68.4 57.4

3.1 Immobilization and screening of heteropolyacid Four heteropolyacid candidates, tungstosilicic acid, tungstophosphoric acid, molybdosilicic acid and molybdophosphoric acid, were initially screened as the reaction catalysts, using the batch method. The results are shown in Tab. 1. Tab. 1. Synthesis ratio of phytosteryl oleate catalyzed by various heteropolyacids in 3 h. Heteropolyacid

Phytosteryl oleate after 3 h of reaction [%]

Tungstosilicic acid Tungstophosphoric acid Molybdosilicic acid Molybdophosphoric acid

69.8 60.4 42.7 53.6

Conditions: Heteropolyacid, 0.1%; reaction temperature, 150 7C; substrate molar ratio (oleic acid vs. phytosterol), 1 : 1; nitrogen flow rate, 1.5 mL/min; moderate agitation. Unless otherwise stated, these reaction conditions were used for the rest of the reactions. Tungstosilicic acid was found to be the best catalyst, based on high overall yields for the ester formed from oleic acid and phytosterol. This catalyst was then used in all the following esterification reactions. However, an immobilized catalyst is more desirable in an industrial process. Although silica gel, zeolite, diatomite, activated carbon, MgO, Al2O3, etc. are all commonly used, only the former four supports were examined because of the strong acidity of tungstosilicic acid. The results of the tungstosilicic acid loads on various supports and the relevant catalyst activities are listed in Tab. 2.

Note: The amount of immobilized catalyst added was calculated as a tungstosilicic acid mass in the reaction mixture of 0.2 wt-%. catalyst. If the reaction was continuously performed for a long period of time, the catalyst could be crashed into powder. It was also verified that the silica gel support was more stable; the catalyst was also advantageous over the activated carbon support in terms of a high yield of phytosteryl oleate esters. The stability of these catalysts will be discussed below in Section 3.5.

3.2 Effect of temperature The effect of the reaction temperature on the production of the phytosteryl oleate ester is shown in Fig. 1. Over the range of 90–210 7C, the catalyst activity increased with the reaction temperature. The highest ester yield of approximate 83.4% was observed at 210 7C. However, sterol loss and deterioration of free phytosterols were more severe at high temperature [23], as was observed for polyunsaturated fatty acids. Hence, unless otherwise specifically stated, a temperature of 150 7C was used in our experiments.

The results showed that silica gel and activated carbon were appropriate supports, with an ester synthesis ratio of 78.6% and 76.5%, respectively. As shown in the table, an extremely strong adsorption occurred when using heteropolyacid as an acid catalyst and activated carbon as support. Therefore, even when the catalyst is used in a liquid reaction, tungstosilicic acid or its salt could not be dissolved out. On the other hand, activated carbon has a lower heat resistance and strength as a support for the

Fig. 1. Effect of temperature on phytosteryl oleate yields.


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3.3 Molar ratio of fatty acid to phytosterol Similarly, Fig. 2 shows the effect of the molar ratio of oleic acid to phytosterols on the yield of phytosteryl oleate. The results show that when oleic acid was in excess, the conversion rate of phytosterols was greatly higher than when the substrates were supplied at stoichiometric levels. The phenomenon abides by the reaction equilibrium theory. In addition, when the oleic acid was excessively used, it could function as a solvent, which was beneficial for solubilizing the phytosterols, reducing the viscosity of the substrate and in turn improving the mass transfer in the esterification reaction system. However, the excessive use of oleic acid could not improve the efficiency of the esterification reaction. An excess of non-reacted oleic acid would lead to a huge cost increase in the downstream isolation process. The optimum substrate molar ratio should ensure that the esterification reaction is performed with high yield and efficiency. Hence, a molar ratio (oleic acid vs. phytosterol) of 1.5 was the optimum under the conditions of a temperature of 150 7C and an immobilized tungstosilicic acid catalyst load of 0.2 wt-%.

3.4 Effect of varying the fatty acid chain length The synthesis of alternative esters formed between various fatty acids and phytosterol was also evaluated. The results from the preliminary esterification reactions conducted in the batch mode were then applied to study the reaction of phytosterols and fatty acids with varying chain lengths. A reaction temperature of 150 7C and a molar ratio of 1.5 : 1 (fatty acids vs. phytosterols) were used for these reactions. An immobilized catalyst load of 0.2 wt-% was used for all of the reported synthesis reactions. The results are summarized in Fig. 3 for the even-carbon number fatty acids from C8 to C18.

Fig. 3. Tungstosilicic acid-catalyzed esterification of phytosterols with various saturated and unsaturated fatty acids.

Yields of over 90% were attained for all of the esters formed, regardless of fatty acid chain length or identity of the sterol/stanol reactant. As shown in Fig. 3, the yields of the phytosteryl esters varied slightly as the chain length of the fatty acid increased; however, even for the C18 esters, a yield of 90% was achieved with phytosterols. Reaction yields in excess of 95% were achieved in the case of the formation of the C4, C8 esters. Similar results were attained for the formation of the phytosteryl esters with C12, C16 and C18 fatty acids. It should be noted that there was no significant discrimination in terms of the tungstosilicic acid catalyst’s selectivity to stearic acid, oleic acid, linoleic acid and alpha-linolenic acid, all with a C18 chain, in the esterification reaction. The reaction yield was above 90%.

3.5 Operational stability of immobilized tungstosilicic acid

Fig. 2. Effect of substrate molar ratio on the esterification of oleic acid with phytosterols.

The stability of immobilized tungstosilicic acid during high-temperature esterification was examined (Fig. 4). At the end of each cycle, the catalyst was separated and reused in the next batch of reaction. The synthesis ratio was then measured and plotted in the figure. Fig. 4 indicates that the phytosteryl ester synthesis ratio after six cycles (30 h) still remained above the high level of 80%, suggesting that the catalyst did not significantly lose its activity after repeated use for six cycles. The half-life of the catalyst was 110 h, calculated by fitting the data with an exponential model.


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References [1] F. H. Mattson, S. M. Grundy, J. R. Crouse: Optimizing the effect of plant sterols on cholesterol absorption in man. Am J Clin Nutr. 1982, 35, 697–700. [2] T. A. Miettinen, T. E. Strandberg, H. Gylling: Noncholesterol sterols and cholesterol lowering by long-term simvastatin treatment in coronary patients: Relation to basal serum cholestanol. Arterioscler Thromb Vasc Biol. 2000, 20, 1340– 1346. [3] A. F. Vuorio, H. Gylling, H. Turtola, K. Kontula, P. Ketonen, T. A. Miettinen: Stanol ester margarine alone and with simvastatin lowers serum cholesterol in families with familial hypercholesterolemia caused by the FH-North Karelia mutation. Arterioscler Thromb Vasc Biol. 2000, 20, 500–506.

Fig. 4. Stability of tungstosilicic acid/silica gel catalyst for repeated use.

[4] Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults: Executive summary of the third report of the National Cholesterol Education, Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. J Am Med Assoc. 2001, 285, 2486–2497. [5] A. Akashe, M. Miller: U.S. Patent 6,267,963 (2001). [6] A. Akashe, M. Miller: U.S. Patent 6,274,574 (2001).

4 Conclusions In summary, a batch method was developed for testing the feasibility of conducting esterification reactions catalyzed by heteropolyacid, with phytosterols and fatty acids as the starting substrates. Various heteropolyacids were also evaluated with respect to their efficacy to catalyze the above reaction in a solvent-free system. Reaction yields were optimized with respect to reaction temperature and molar ratio of fatty acids to phytosterols. The synthesis of alternative esters formed by various fatty acids and phytosterols was also evaluated, and high yield was achieved accordingly. The present study demonstrated that the immobilized tungstosilicic acid could be a promising catalyst for replacing sodium methylate in the process of commercially synthesizing phytosteryl esters from fatty acids and phytosterols. It should be noted that a relatively higher reaction temperature and a longer reaction time of the heteropolyacidcatalyzed esterification of phytosterols would be required, compared with the high-temperature-assisted enzymatic method; however, from the viewpoints of industrial economics and environmental friendliness, it might be more practical for large-scale industrial application.

Acknowledgment We greatly appreciate Dr. Fuming Zhang of the Department of Chemistry, Biology and Chemical and Biological Engineering, Resselaer Polytechnic Institute, USA, for his help in editing our manuscript.


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[19] J. W. King, J. M. Snyder, H. Frykman, A. Neese: Sterol ester production using lipase-catalyzed reactions in supercritical carbon dioxide. Eur Food Res Technol. 2001, 212, 566–569. [20] A. Fujita, E. Kadowaki, T. Higashi, H. Uchida: U.S. Patent 20 040 152 915 (2004). [21] E. W. Hammond: Thin layer chromatography. In: Chromatography for the Analysis of Lipids. CRC Press, Boca Raton, FL (USA) 1993, pp. 21–24.


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