Biochemical Engineering Journal 54 (2011) 111–116 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Solvent-free polyglycerol polyricinoleate synthesis mediated by lipase from Rhizopus arrhizus J.L. Gómez, J. Bastida, M.F. Máximo ∗ , M.C. Montiel, M.D. Murcia, S. Ortega Chemical Engineering Department, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain article info Article history: Received 25 February 2010 Received in revised form 26 November 2010 Accepted 4 February 2011 Available online 12 February 2011 Keywords: Polyglycerol polyricinoleate Solvent-free Lipase Immobilised enzyme Biosynthesis abstract The enzymatic biosynthesis of polyglycerol polyricinoleate (PGPR) (E-476) is described in detail for the first time. Starting from polyglycerol and polyricinoleic acid, Rhizopus arrhizus lipase was used as catalyst. The reaction, which is really a reversal of hydrolysis, takes place in the presence of a very limited amount of aqueous phase. No organic solvent is necessary to solubilise the substrates, which allows a reaction medium solely composed of the necessary substrates to be used. Immobilisation of the lipase by physical adsorption onto an anion exchange resin provided good results in terms of activity, enzyme stability and the reuse of immobilised derivative. Using this immobilised derivative, PGPR with an acid value of 16 mg KOH/g was obtained, far above the requirements of the European Commission Directive 2008/84/EC (<6 mg KOH/g). In an attempt to force the reaction equi- librium towards the synthetic pathway, polyglycerol polyricinoleate was synthesised under controlled atmosphere in a vacuum reactor with dry nitrogen intake. This equipment allowed us to synthesise PGPR with an acid value of 4.9 mg KOH/g, which complies with the European Commission Directive and the results were entirely reproducible. This investigation represents a good starting point for using the enzymatic procedure in the industrial biosynthesis of PGPR. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Polyglycerol esters have been used as food additives for many years. From the official point of view, food grade polyglycerol esters are divided in two classes: polyglycerol esters of edible fatty acids (E-number: E-475, also known as “PGFA”) and polyglycerol polyri- cinoleate (E-number: E-476, also known as “PGPR”). Polyglycerol polyricinoleate is used to maintain stable emulsions of oil and water systems with a high water content and as a viscosity modifier. In the chocolate industry, PGPR is used because it causes a notice- able reduction in the yield stress of molten chocolate. This allows chocolate to be moulded, without any air bubbles, easier coating of particulate ingredients, and the thickness of chocolate coating to be adjusted optimally. An additional property of PGPR in chocolate is its ability to limit fat bloom [1]. Known chemical methods for preparing PGPR involve autocat- alytic condensation of ricinoleic acid and alkali-catalysed reaction between the condensed ricinoleic acid and polyglycerol. These procedures have the disadvantage of requiring very long reaction times, involving high energy costs. This fact, together with the high ∗ Corresponding author. Tel.: +34 868 887367; fax: +34 868 884148. E-mail addresses: carrasco@um.es (J.L. Gómez), jbastida@um.es (J. Bastida), fmaximo@um.es (M.F. Máximo), cmontiel@um.es (M.C. Montiel), md.murcia@um.es (M.D. Murcia), dortega@um.es (S. Ortega). operating temperature can adversely affect the quality of the final product because of problems related with coloration and odours, making it unsuitable for the food industry [2]. As an alternative, we propose the biotechnological production of PGPR using lipases, which act in mild reaction conditions and produce a final product more suitable for use as a food additive. The enzymatic procedure consists of two steps. First, the ricinoleic acid is polymerised to obtain the estolide [3–5] which is then esterified with polyglycerol. Fig. 1 shows the chemical structure of all the species involved in the biosynthesis. In principle, the second step of the process can be performed in a reaction system consisting of appropriate amounts of polyglyc- erol and estolide in the presence or absence of organic non-polar solvents. The choice of solvent in this process is a key issue from a green chemistry point of view. Supercritical carbon dioxide and ionic liquids are often referred to as green solvents, but the use of a solvent-free system (SFS) is a more attractive alternative. If the elimination of solvents is technically feasible, SFS offers signifi- cant cost savings, allows higher volumetric production than organic media and facilitates the separation of products from the unreacted substrates [6]. Although great efforts have been made to develop biotransfor- mations with lipases, we have found no papers referring to the enzymatic production of PGPR. Previous works suggested that the esterification of polyglycerol with the ricinoleic acid estolide is successfully catalysed by Rhizopus arrhizus lipase [7], and so the 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.02.007