Elsevier

Food Chemistry

Volume 190, 1 January 2016, Pages 960-967
Food Chemistry

Production and concentration of monoacylglycerols rich in omega-3 polyunsaturated fatty acids by enzymatic glycerolysis and molecular distillation

https://doi.org/10.1016/j.foodchem.2015.06.061Get rights and content

Highlights

  • MAGs rich in ω-PUFAs is produced by combining enzymatic glycerolysis with SPD process.

  • SPD enables a distillate stream of MAGs with 91% purity and 94% overall recovery.

  • The capability of SPD to directly concentrate n-3 PUFAs in MAG form is demonstrated.

  • A processing diagram for scalable production of n-3 PUFAs enriched MAG is mapped out.

Abstract

Production of monoacylglycerols (MAGs) rich in ω-3 polyunsaturated fatty acids (n-3 PUFAs) was conducted through short path distillation (SPD) of an acylglycerol mixture (containing 67% MAGs) produced by enzymatic glycerolysis of sardine oil with glycerol. A stepwise SPD process in a UIC KDL 5 system (vacuum 10−3 mbar, feeding flow 1.0 mL/min) was proceeded: the first distillation performed at evaporator temperature (TE) of 110 °C to remove glycerol completely and most of FFAs; and the second distillation at optimized TE 155 °C; resulting in a stream distillate with 91% purity and 94% overall recovery of MAGs. This work also demonstrated that SPD is able to concentrate n-3 PUFAs in MAG form by distilling at proper TE e.g. 125 °C, where n-3 PUFAs are concentrated in the residues. Moreover, this work mapped out a complete processing diagram for scalable production of n-3 PUFAs enriched MAGs as potential food emulsifier and ingredient.

Introduction

Fish oil is rich in ω-3 (n-3) polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The n-3 PUFAs play important roles in human health, and epidemiological and clinical studies have shown that EPA and DHA reduce the risk of coronary heart disease and help in brain, neural, and retinal developments (De Deckere et al., 1998, Komprda, 2012, Kris-Etherton et al., 2002, Nichols et al., 2014).

There are several reasons for producing concentrates of n-3 PUFAs, but their chemical form has to be taken into account, as n-3 PUFAs in free fatty acids (FFAs), ethyl esters (EE), acylglycerols (AG), or phospholipids have different bioavailabilities. Studies have shown that human absorption of n-3 PUFAs in EE form is poor, primarily because EEs are poor substrates for pancreatic lipase. Although PUFAs in FFA form are absorbed more efficiently than PUFAs in EE or TAG form, they could present irritant effects and are very prone to autooxidation (Hernandez, 2014, Lawson and Hughes, 1988). Some studies have reported that the oxidative stability of PUFAs in TAG form is higher than PUFAs in EE form. Taking these considerations into account, MAG and DAG containing n-3 PUFAs are expected to have good application potentials, e.g. in functional foods, dietary supplements, or pharmaceuticals. Furthermore, these molecules, especially MAG, have excellent emulsifying properties, and MAG represent about 70% of the synthetic emulsifiers currently produced. For some applications, at least 90% pure MAG is required (Fregolente, Batistella, Maciel Filho, & Wolf Maciel, 2006). MAGs are usually produced by chemical glycerolysis employing inorganic alkaline catalysts at high temperatures. These processes are not suitable for heat-sensitive oils such as fish oils. Therefore, the enzymatic process has become an attractive alternative approach for polyunsaturated MAG production because the reaction can be carried out under mild conditions (Feltes, de Oliveira, Block, & Ninow, 2013).

The most widely used immobilized lipases used in enzymatic glycerolysis reactions are from Candida antarctica (lipase B, Novozym® 435), Rhizomucor miehei (Lipozyme RM IM), Pseudomonas fluorescens (IM-AK), and Thermomyces lanuginose (Lipozyme TL IM) (Feltes et al., 2013). Several researchers use Novozym® 435 as biocatalyst to mediate glycerolysis reactions due to its high MAG yield (Damstrup et al., 2006, Voll et al., 2011, Yang et al., 2005). This lipase is not water-dependent, and works successfully in glycerolysis systems, even in the presence of solvents (Feltes et al., 2013). This is important, as solvents are usually required in glycerolysis reactions due to the immiscibility of the reactants (glycerol and oil or free fatty acids). Damstrup et al. (2005) evaluated various solvents and concluded that the highest MAG yield by glycerolysis was obtained with tertiary alcohols, tert-butanol (TB) and tert-pentanol (TP). The relatively low log P value of these tertiary alcohols (0.35 and 0.89, respectively) indicates both hydrophilic and hydrophobic characteristics, ensuring miscibility of the reactants. Moreover, C. antarctica lipase B does not act on tertiary alcohols due to steric hindrance (Yang et al., 2005). The liquid–liquid equilibria (LLE) of sardine oil + glycerol + tert-alcohols have been determined previously (Solaesa, Bucio, Sanz, Beltrán, & Rebolleda, 2013). LLE data help to minimize the amount of solvent needed to create a homogeneous reaction system, which leads to an improvement in mass transfer, obtaining high reactions yields in short reaction time.

For commercial interests, several methods have been reported for concentrating n-3 PUFA derivatives. Generally, a combination of techniques is used, such as an enzymatic reaction (hydrolysis, ethanolysis or glycerolysis) followed by molecular distillation, also known as short path distillation (SPD), low-temperature crystallization, urea complexation or supercritical fluid fractionation (Gámez-Meza et al., 2003, Lin et al., 2006, Xu, 2003). Crystallization at low temperature has some technical limitations, such as molecular association and mixed-crystal formation and use of large quantities of organic solvents (Brown & Kolb, 1955). On the other hand, urea complexation presents some technical advantages in fractionation of PUFAs such as high efficiency and better selectivity. However, for concentrating PUFAs for human consumption the use of urea should be avoided, where formation of ethyl carbamate, an animal carcinogen is reported (Canas & Yurawecz, 1999). In case of supercritical fluid fractionation, most studies are directed towards fractionating the FFAs or their esters, which are more soluble in supercritical fluids. To design an efficient process, more knowledge on the solubility and phase equilibria of other compounds in the supercritical fluid is still needed (Farvin & Surendraraj, 2012). Whereas, in SPD, compounds with different boiling points are separated under vacuum, which decreases the evaporation temperature and minimize the residence time, which enable heat-sensitive compounds to be separated with minimal thermal degradation. Therefore, this technology has been widely used in lipid areas. Most of the studies about the separation of MAG from DAG and TAG mixtures by SPD are focused on purification of saturated and monounsaturated compounds such as monoolein by glycerolysis from coconut oil (Zha et al., 2014), or high oleic sunflower oil (Zhu, Li, Wang, Yang, & Ma, 2011). Concentration of MAG and DAG by SPD from glycerolysis of palm olein (Yeoh et al., 2014), soybean oil (Fregolente, Pinto, Wolf-Maciel, & Filho, 2010) or camellia oil (Zheng et al., 2014) have been also reported. Processing of marine oils by SPD to remove pollutants and oxidation products has been reported (Oliveira and Miller, 2014, Oterhals and Berntssen, 2010); however, the studies focused on PUFA purification mainly obtained the PUFA concentrate in FFA or EE form (Breivik et al., 1997, Kahveci and Xu, 2011, Rossi et al., 2012, Zhang et al., 2013). Other studies have used SPD to remove ethyl esters subsequently to ethanolysis in order to obtain an acylglycerol product (mainly DAG) rich in DHA and EPA (Lyberg and Adlercreutz, 2008, Valverde et al., 2013, Valverde et al., 2012). However, the preparation of a product with a high concentration of MAG rich in n-3 PUFA has not been well documented yet.

In this work, acylglycerols rich in n-3 PUFA were prepared by enzymatic glycerolysis of sardine oil using immobilized C. antarctica Lipase B (Lipozyme 435) in TP. The MAG fraction was purified using SPD. Different evaporation temperatures were evaluated to maximize the MAG recovery at high purity in the distillate. The main purpose of this study was to develop an effective distillation process for concentration of n-3 PUFA in MAG form.

Section snippets

Materials

Refined sardine oil was kindly provided by Industrias Afines S.L. (Pontevedra, Spain). The fatty acid (FA) composition of sardine oil and the mole percentage of FAs at the sn-2 position of TAG is presented in Table 1 (Solaesa, Bucio, Sanz, Beltrán, & Rebolleda, 2014). More than 80% of DHA was found at sn-2 position.

Lipase B from C. antarctica (Lipozyme 435, immobilized lipase) was generously supplied by Novozymes A/S (Bagsvaerd, Denmark). The water content of Lipozyme 435 was 3.5 ± 0.3% as

Effect of substrate molar ratio and enzyme concentration in the production of MAG

To determine an optimum substrate mole ratio, the effect of glycerol/oil mole ratio on the reaction rate was studied at three levels (1:1, 3:1 and 5:1) at 50 °C with 10 wt% of lipase. The results are shown in Fig. 1A. The substrate mole ratio can influence the reaction in different ways. An increase in glycerol amount will increase the theoretical equilibrium conversion, and shift the equilibrium towards MAG production. On the other hand, an excess of glycerol will affect the polarity as well as

Conclusion

This work reported the optimization of an SPD process for separating a mixture of acylglycerols, free fatty acids, and glycerol, obtained from an enzymatic glycerolysis of sardine oil, which produced high concentration of MAG (67%). The products (TAG, DAG, MAG, and FFA) contain n-3 PUFAs, and SPD is a suitable process to preserve their stability. A two-step distillation was applied to remove firstly at 110 °C as TE the glycerol and part of FFA (63.5%), and then a second distillation at 155 °C

Acknowledgements

The authors acknowledge to the Spanish Government through MINECO and the European Regional Development Fund (ERDF) for financial support to the project CTQ2012-39131-C02-01. AGS acknowledges University of Burgos for a pre-doctoral fellowship.

References (40)

  • H. Breivik et al.

    Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid

    Journal of the American Oil Chemists’ Society

    (1997)
  • J.B. Brown et al.

    Applications of low temperature crystallization in the separation of the fatty acids and their compounds

    Progress in the Chemistry of Fats and Other Lipids

    (1955)
  • B.J. Canas et al.

    Ethyl carbamate formation during urea complexation for fractionation of fatty acids

    Journal of the American Oil Chemists’ Society

    (1999)
  • M.L. Damstrup et al.

    Solvent optimization for efficient enzymatic monoacylglycerol production based on a glycerolysis reaction

    Journal of the American Oil Chemists’ Society

    (2005)
  • M.L. Damstrup et al.

    Production of heat-sensitive monoacylglycerols by enzymatic glycerolysis in tert-pentanol: Process optimization by response surface methodology

    Journal of the American Oil Chemists’ Society

    (2006)
  • E.A.M. De Deckere et al.

    Health aspects of fish and n-3 polyunsaturated fatty acids from plant and marine origin

    European Journal of Clinical Nutrition

    (1998)
  • K.H.S. Farvin et al.

    Utilization of marine lipids for the production of PUFA, squalene, wax esters and biodiesel for commercial application

    Fish Oil: Production, Consumption and Health Benefits

    (2012)
  • M.M.C. Feltes et al.

    The production, benefits, and applications of monoacylglycerols and diacylglycerols of nutritional interest

    Food and Bioprocess Technology

    (2013)
  • L.V. Fregolente et al.

    Optimization of distilled monoglycerides production

    Applied Biochemistry and Biotechnology

    (2006)
  • P.B.L. Fregolente et al.

    Monoglyceride and diglyceride production through lipase-catalyzed glycerolysis and molecular distillation

    Applied Biochemistry and Biotechnology

    (2010)
  • Cited by (93)

    • Enzymatic preparation of mono- and diacylglycerols: A review

      2023, Grain and Oil Science and Technology
    • A systematic review of mathematical modeling for molecular distillation technologies

      2023, Chemical Engineering and Processing - Process Intensification
    View all citing articles on Scopus
    View full text