## 4 Discussion  
As lipids are one of the main classes of molecules considered 'building blocks' of all biological organisms, their analysis has been developed for a variety of applications. Even  
Figure 5: Carbon dioxide production during alcoholic fermentation in SGM supplemented with 0 (control) and 50 mg/L linoleic acid (linoleic acid). Mp—_M. pulcherima_; Km—_K. marxianus_; Td—_T. delbrueckii_; Sc EC—_S. cerevisiae_ EC1118. Figure 6: Percentages (top—graph) and concentrations (bottom—table, expressed in \(\mathrm{\mu}\)g/biomass) of individual lipids detected in yeasts at the end of alcoholic fermentation in SGM. Mp—_M. pulcherima_; Km—_K. marxianus_; Td—_T. delbrueckii_; Sc—_S. cerevisiae_ EC1118. Control (C)—SGM with no lipids; linoleic Acid (LA)—SGM supplemented with 50 mg/L linoleic acid. though some authors have opted for the'shotgun lipidomics' approach introduced by [PERSON] and [PERSON] [53], many researchers still prefer the more classical approach of sample preparation, then separation, followed by detection. These methods have to take into account the nature of the compounds of interest, the matrix where the lipids are present, whether they are intra- or extra-cellular, the potential interferences from the matrix, etc. These methods tend to be adapted to suit the conditions of the experiment and the instrumentation available. With an increase in accessibility to higher performance equipment, a more general method could soon replace these various (albeit similar) methods. However, this general method will have to overcome a number of issues, such as the large variation in concentrations of the compounds of interest due to the variety of matrices tested, which in themselves can be challenging. Since only what has been isolated can be analyzed, sample preparation plays a major role in the process of lipid analysis. Extracellular lipids are generally easier to isolate [26]; intracellular lipids require extensive and careful sample preparation to minimize unwanted loss of lipids [51]. One of the aspects considered relevant during the sample preparation optimization was the incorporation of pellet washes after fermentation. When, during the experiments, the media is spiked with FAS, only a certain percentage is absorbed by the yeast, resulting in residual FAS in solution [54]. Without the pellet washes, this could lead to misleading results as there is no distinction between FAS adsorbed by/absorbed on the yeast vs. residual FAS in the juice. In the literature, most fatty acid analyses are based on a methylation derivatization (the derivatives are known as FAMEs; fatty acid methyl esters), followed by GC-MS or GC-FID [26; 52; 55; 56]. This derivatization reaction is well-documented and its advantages and disadvantages have been previously discussed in the literature [52]. However, in the current study, the derivatization with BSTFA was preferred due to the stability of the resulting TMS-derivatives. As the sample preparation procedure is time consuming, especially in the case of the yeast samples, the stability of the compounds of interest (in their free or derivatized form) was paramount for the applicability of the method. Even though stability tests are not often included in method development/optimization, in this case it was considered essential. Looking at the results, the most reproducible results were for the TMS-derivatives stored at \(-20\)\({}^{\circ}\)C until analysis followed by the dried extract stored at \(-20\)\({}^{\circ}\)C, derivatization, and then immediate GC-MS analysis (Table 4). This can offer some flexibility in the practical application of the method, especially when a high number of yeast samples have to be processed. One of the issues that required particular attention was the dynamic range of the MS fitting the range of concentration of the compounds of interest. For the calibration range tested, all calibrations were quadratic, and curve fit assistant (Agilent-based software) was used to identify best calibration based on R\({}^{2}\).