Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved.

Hydroperoxides (LOOH) measurement is a common technique to determine the degree of oxidation of oils and fats. Although it appears to have some limitations (time consuming, and uses of toxic solvents as chloroform), the iodometric titration is one of the most common methods. Herein, an alternative method by measuring the oxidation level using Fourier transform infrared (FTIR) with an attenuated total reflectance mode (ATR), based on the stoichiometric conversion of triphenylphosphine (TPP) into triphenylphosphine oxide (TPPO) by hydroperoxides, is proposed. The FTIR‐ATR spectroscopy allows for a simple and accurate detection of the TPPO formed by selective reaction with LOOH, with measurement of its specific adsorption band at 542 cm−1. Calibration is made with TPPO solubilized in butan‐2‐one and covers a range of peroxide values (PV) from ≈2 to ≈800 mEq kg−1. The comparison of the iodometric titration and TPP/TPPO assay is performed with standard hydroperoxides (tert‐butyl hydroperoxide and cumene hydroperoxide). The iodometric titration shows higher PV overestimations in comparison with the TPP/TPPO assay, for both hydroperoxides. An accelerated oxidation of different oils is assessed with the two methods, and the results confirm this observation since higher PV values are measured with the titration method. The TPP/TPPO assay coupled with the ATR‐FTIR spectroscopy appears as a simpler and faster assay, which may limit overestimation and use of toxic solvents, especially suitable for routine analysis of oil and fats. Practical Applications: One of the first indicator of oxidation in oils and fats is the presence of hydroperoxides, therefore, it is important to find a reliable method to assess these oxidation products. One of the most common methods to determine lipid hydroperoxide (LOOH) concentrations is iodometric titration. Although this method is simple to set up, it is labor‐intensive, time consuming, and uses a lot of solvent. TPP/TPPO assay coupled with FTIR‐ATR spectroscopy is a good alternative to the iodometric method since it allows a fast and robust hydroperoxide measurement, with minimal use of organic solvents. This rapid peroxide value determination can be adapted for routine quality control analyses, especially for oxidation in real‐time and accelerated aging assays to evaluate oxidative stability during shelf‐life, for industrial purpose or lab scale experiments. The TPP/TPPO coupled with FTIR‐ATR allows for a fast, simple, reasonably sensitive (adequate for most purposes) and reproducible alternative assay to conventional iodometric titration for peroxide value assessment.

Numerous different in vitro assays, labeled as “antioxidant assays,” are used intensively to predict the antioxidant capacity of phenolic compounds. Most of these methods give valuable information in terms of chemical reactivities but also present some weaknesses. It may be difficult to use them to predict antioxidant capacity in real conditions. Indeed, lipid oxidation is a complex reaction, with numerous paths and components, and antioxidant action can occur via a multitude of mechanisms, especially when different phases coexist in the lipid‐based formulation. Yet, correctly combining selected in vitro methods to extract complementary information with respect to antioxidant behaviors would be much better and will help reducing the gap between prediction and efficacy in the finished product. Thus, we hereby propose a methodology to evaluate the antioxidant properties of 12 selected natural polyphenolic extracts based on the appropriate combination of in vitro assays. The scores obtained with the DPPH test, the measure of the chelation capacity, the evaluation of antioxidants efficiency in emulsions (CAT and VESICAT assays), or in bulk oils were submitted to a statistical treatment. This analysis allowed a ranking on their global antioxidant capacities and the creation of clusters depending on their mechanisms of action and the type of media where the tests were performed.