Oleins are obtained as a by-product in the process of the fat splitting of soapstocks, but they have low market value because they are a mixture of free fatty acids and TAG. At least 50% of it is constituted by free fatty acids and 40% by TAG, with the remaining 10% being MAG, DAG, unsaponifiable lipids and, to a lesser extent, methyl and ethyl esters.

Oleins obtained from the fat-splitting of soapstocks have various uses, including the manufacture of animal feed (fat additive) as well as other multiple technical uses by employing olein as such or via some of their derivatives obtained by chemical transformation. The most widespread use is the manufacture of biodiesel (Ribeiro et al., 2011; Narvaez Rincón et al., 2004; Pereda Marín et al., 2003), for which the olein is subjected to a process of chemical esterification with methanol catalyzed by acids (Otadi et al., 2011) and subsequently to a process of chemical transesterification catalyzed by a strong base. As a result, free fatty acids (FFA), as well as MAG, DAG and TAG can be transformed into methyl esters (FAMEs).

MAG and DAG are biodegradable compounds classified as GRAS (Generally Recognized As Safe) by the FDA (Food Drug Administration). These molecules are used as anionic surfactants, emulsifiers and stabilizers (E471) (Camino Feltes et al., 2013) for the food industry (bakery products, dairy products, and other products that may contain milk or flour) as well as for the textile industry and the plastic and biolubricant industry (formulations of oils for use in different types of machinery). They have proven to be so versatile that MAG has even shown to have antimicrobial activity (Ramesh et al., 2017). Their production, using both chemical and enzymatic reactions, can reach 90% yields when carried out in organic solvents (hexane, toluene, chloroform, acetone, etc). The disadvantage of using this type of solvents is that, in addition to denaturing the enzymes, they are toxic, carcinogenic, flammable and not biodegradable. In addition, a further purification step for the final product is required to remove these chemicals.

The glycerolysis reaction from oleins produces oils which are rich in MAG and DAG. This process can be carried out chemically or enzymatically, with the industry favoring the chemical approach (Satriana et al., 2016). Such a process is carried out with a basic catalyst (NaOH, KOH or Ca(OH)2), at high temperatures (190-250 ºC), for 3-5 h with constant agitation. The final product resulting from the reaction under these conditions usually has the following composition: MAG 30-60%, DAG 35-50% and TAG 1-20%, with certain residual acidity. Thus, in order to achieve a concentration of over 80% MAG and DAG in the final product, a further process of molecular distillation is needed (Ramesh et al., 2017; Solaesa et al., 2016). Consequently, the current industrially implemented processes of chemical glycerolysis tend to entail costly energy demands and have additional drawbacks, such as raw material degradation and darkening of the desired product due to the use of very aggressive catalysts. When a minor degradation of raw material is required, the use of Ca(OH)₂ as a catalyst is preferred. However, although smaller, the use of this catalyst cannot prevent the degradation of a significant part of the unsaturated fatty acids (Solaesa et al., 2016; Tiankui et al., 2005), with the subsequent reduction in the potential health benefits of the oil.

Enzymatic glycerolysis has several advantages over chemical glycerolysis (Fregolente et al., 2008; Csanadi et al., 2009; Kapoor et al., 2012; Camino Feltes et al., 2012). The main ones are lower working temperatures (50-80 ºC) and a reaction pH close to neutrality (pH 5.0-6.0), both having a direct impact on the quality improvement of the obtained final products. Also, energetically speaking, the process is a less costly one that can be easily scaled up and implemented at industrial scale, generating fast economic benefits and returns. However, it also has disadvantages, with the main one being the high costs associated with the enzymatic catalyst. To give some estimation of costs, increasing the enzyme load from 0.5 to 1% doubles the process costs.

Nonetheless, enzymatic catalyst is a highly viable process leading to a final product with MAG concentrations similar to or even higher than those obtained by the chemical route with the added benefit of a less degraded product (Solaesa et al., 2016; Tiankui et al., 2005).

Lipases (E.C. 3.1.1.3) belong to the family of hydrolases, since they catalyze the hydrolysis of TAG at the lipid-water interface. In addition to their physiological roles, lipases catalyze the hydrolysis or enantio- and regio-selective synthesis of a wide variety of natural substrates, as well as carrying out esterification, interesterification and transesterification reactions in non-aqueous media. This versatility explains why this enzymatic group has a great biotechnological application in the oil industry (Fariha et al., 2005; Godfrey T, 2005; Rivera-Pérez et al., 2007), and in the production of drugs, agrochemicals, etc. The main lipases of industrial interest are produced by fungi (Candida cylidracae, Aspergillus niger, Candida rugosa, Candida antarctica, Mucor miehei, Staphylococcus aureus, Rhizopus arrhizus, etc.); whereas others are extracted from bacteria. Some are non-specific enzymes, while others act specifically on a very precise fragment of the TAG. There is also another group of lipases that can work at slightly higher temperatures (60-75 °C) namely, thermostable lipases. CALB is a non-specific enzyme capable of attacking the three positions (sn-1, 2 and 3) of the TAG molecule to produce glycerol and free fatty acids (Grazia et al., 2015). Regardless of the different features of this great family of enzymes, the great advantage of the use of lipases (for low temperature fat processing processes) lies in their ability to significantly reduce the formation of trans fats, which are formed in high amounts in homologous chemical processes (Rivera-Pérez et al., 2007).

For all the reasons mentioned above, the objective of this research is to develop an enzymatic technology in a solvent-free system (Noureddini et al., 1998; Valerio et al., 2010; Vazquez et al., 2016; Wiphum et al., 2005; Zhao et al., 2013; Ziobrowski et al., 2009), with yields close to 80% (joint content of MAG and DAG).

The reaction of any Gycerolysis can be seen in Eqs. 1-3.

Finally, it is also worth mentioning that since oleins contain a high concentration of FFA (free acids concentration can represent up to 50-70% of the olein), the typical reactions of glycerolysis (Eqs. 1 to 3) compete with the reaction between glycerol and FFA (direct esterification), resulting in the glycerolysis of oleins in a set of competing reactions (Eqs. 4 to 7). A secondary reaction of direct esterification (Eq. 5) reduces the concentration of MAG but unfortunately cannot be avoided, as they happen simultaneously. Both types of syntheses lead to a balance which shows a greater shift to the right as the temperature and reaction time increase (Chemical Glycerolysis).

Thus, when the raw material has a high acidity value, such as oleins, the composition in the final product of the glycerolysis reaction is higher in TAG (up to 15-25% of total) compared to products obtained from other sources.

In this research we have studied the enzymatic glycerolysis reaction of cocoa olein in a solvent-free medium. We will study the effect of the enzyme concentration (dose), glycerol concentration, reaction time and vacuum effect in the final composition of the product (FFA and MAG, DAG and TAG contents).

The free lipase B of Candida antarctica was used in the glycerolysis of cocoa olein and produced an oil enriched in MAG and DAG in a solvent-free system. It was found that the maximum yield was reached when the reaction was carried out at low pressure (2-3 mbar) and at 65 °C, with an enzyme dose of 1% and a molar oil:glycerol ratio of 1:2. In addition, FFA enrichment of cocoa olein, used as raw material, yielded an improvement in the MAG/DAG ratio which went from MAG/DAG = 26/39% to MAG/DAG = 33/44%. Finally, we also observed a remarkable reduction in reaction time compared to the typical enzymatic processes described for this kind of reactions (Table 6).

The combination of shorter reaction times with decreasing prices of enzymes could pave the way for the scale up and industrialization of these processes for the valorization of by-products such as sunflower, soybean, olive, rapeseed and cocoa oleins from soapstocks.