39. Freezing effects evaluated on complex biological systems by DSC: Baker’s yeast and microalgal cells

Abstract

When biomass is to be used in the food, feed, or pharmaceutical industry, it should be preserved and stored in order to permit transport from production to consumer. The most frequent preservation methods are freezing and drying. Differential scanning calorimetry (DSC) can be considered a powerful technique for studying thermal transitions of biological molecules related to their functionality and with biomass viability. Also, DSC has been shown to provide a convenient way to determine the chemical stability of some biomass components, such as lipids. The objective of this work was to analyze the feasibility to employ DSC as a tool to evaluate the cryo-resistance of baker’s industrial yeast ( Saccharomyces cerevisiae ) strain towards freezing and thawing and the long term conservation of intracellular lipids in Aurantiochytrium limacinum SR21 frozen microalgal cells. Yeast in stationary growth phase were exposed to two cycles of freezing (20 h and 4 h) at −20 °C after being exposed to various physical and chemical mild stresses. DSC runs were performed at 10 °C/min from 20 °C to 100 °C. Media with different C:N balances (55:1 and 6:1) were used to obtain microalgae with different PUFAs and carotenoid contents. Cells were frozen for 1, 8.5 and 30 months at −20 °C. The changes in the saturated/poly-unsaturated fatty acids (SFA/PUFAs) ratio in microalgal cells were detected by DSC under oxidative conditions (at 5 °C/min) through shifts in the onset oxidation temperature (OOT) values. Thermograms of the yeast’s systems showed two peaks that were attributed to low and high protein onset denaturation temperatures: 47–58 °C and 67–77 °C, respectively. Some pre-stress treatments (temperature, ethanol and NaCl) increased both enthalpy values and survival after freezing and thawing. DHA (C22:6, n − 3) and palmitic acid were the major PUFA and SFA, respectively produced by the SR21 cells. Microalgal growth carried out in media with 55:1 C:N ratio produced cells with higher levels of DHA and of carotenoids, when compared with those grown in media with a 6:1 C:N ratio. OOT of cells and of lipidic extracts were influenced by cellular carotenoids. Fatty acid stability in frozen microalgae could be evaluated by analyzing the shift of OOT values: the OOT values were lower for the frozen cells compared to the fresh ones and decreased as a function of frozen storage time. This behaviour could be associated with DHA degradation, in addition to carotenoid content lost. DSC onset denaturation temperatures clearly correlated with protein stability and the decrease in the enthalpy values obtained from the thermograms can provide valuable information about the corresponding effect on yeast viability. Besides, lipid stability in frozen media could be evaluated in whole microalgae cells as well as in their lipidic extracts analyzing the shift of OOT values. DSC has a high potential, as a non-chemical method, to determine stability parameters in complex biological systems and could be employed as an efficient, reliable and fast method to study proteins and fatty acids stability in whole cells and in their extracts. This work was supported by CONICET (PIP 100846, 100468), UBA (2002100100397 and I038) and ANPYCT (PICT 0928). Conflict of interest: None declared.