In-Depth Lipidomic Analysis of Molecular Species of Triacylglycerides, Diacylglycerides, Glycerophospholipids, and Sphingolipids of Buttermilk by GC-MS/FID, HPLC-ELSD, and UPLC-QToF-MS.

Buttermilk, a byproduct of butter manufacturing, has gained considerable attention due to its high concentration of polar lipids as phospho- and sphingolipids from the milk fat globule membrane (MFGM). These polar lipids (PLs) are essential components of all cellular membranes and exert a variety of indispensable metabolic, neurological, and intracellular signaling processes. Despite its importance, there are few research studies that report a comprehensive characterization of the lipid molecular species of MFGM that could contribute to a better understanding of their putative healthful activities. In this study, procedures such as pressurized liquid extraction of polar and nonpolar lipids and their fractionation by flash chromatography have been carried out. The obtained fractions were submitted to an exhaustive characterization from a lipidomic point of view. The characterization includes new data about the identification and quantification of triacylglycerides (TAG), diacylglycerides (DAG), and phospho- and sphingolipids using different chromatographic techniques. The fatty acid profile was comparable to that of the milk fat but with a highly diverse composition of fatty acids. Molecular species have also been determined by using ultra-high performance liquid chromatography/quadruple-time-of-flight mass spectrometry (UPLC/QToF-MS). The TAG (16:0/16:0/6:0) and TAG (16:0/16:0/8:0) were the predominant saturated TAG species and TAG (14:0/18:1/16:0) and TAG (16:0/16:0/18:1) presented the highest content of monounsaturated TAG species. Furthermore; over 30 molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) could be identified within PL, with PC (16:0/18:1) being the most abundant species. Whereas C16:0 was found to be the preferred FA in TAGs, it was C18:1 in PLs. Several ganglioside species have also been characterized with d18:1 ceramide moiety and secondary acyl chains ranging from C20:0 to C26:1. This approach could broaden the applications of high-resolution mass spectrometry for a better understanding of the role of MFGM and its functionality.

Appraisal of the suitability of two-stage extraction process by combining compressed fluid technologies of polar lipid fractions from chia seed.

Although triacylglycerols (TAG) are the major constituents of chia oil, it also contains minor lipid fractions that include phospholipids (PL) among other desirable components. Its amphiphilic character and excellent biocompatibility make PL appropriate for numerous applications with technological and nutritional significanceand potential health benefits. Given the difficulties entailed by the PL isolation, the efficiency for extracting such compounds using two environmental friendly techniques, pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) was evaluated. By using PLE with food-grade ethanol (EtOH), an oil recovery close to 100% was achieved in just 10 min. This oil extract was particularly rich in α-linolenic acid (ALA; 70%) as compared to the oil extracted by SFE (56%). In the case of SFE, the oil recovery was only 87% but increased to 99% when ethanol was added to CO2. However the use of co-solvent did not affect the fatty acid profile of the supercritical extracts or their TAG composition, where the high molecular weight TAG species were the predominant in all cases. With the exception of SFE without co-solvent, all methods applied were capable of extracting the PL fraction, although the content and distribution of the individual components present in this fraction differed markedly depending on the extraction conditions used. In this context, the use of a sequential extraction process, combining SFE and PLE was particularly interesting. The re-extraction by PLE of the chia cake, previously defatted by SFE, allowed to obtain an oil extract highly enriched in PLs, whose content exceeded 16% and with a higher PL species than the rest of the oil extracts.

Production of omega 3-rich oils from underutilized chia seeds. Comparison between supercritical fluid and pressurized liquid extraction methods.

Chia seeds constitute a promising source of α-linolenic acid (ALA). In the present work, an underutilized and cheaper set of chia seeds, which were discarded after the harvest according to quality criteria - named in this work as low oil content seeds (LOCS) - have been evaluated as a potential source for obtaining PUFA-enriched oils against the commonly studied high-quality chia seeds denoted as high oil content seeds (HOCS) in this study. Two efficient and environmental friendly techniques, supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), were evaluated to optimize the extraction process of chia oil. At 60 °C, by using pressurized food-grade ethanol, recoveries close to 100% were achieved from both sets of seeds in a short extraction time (10 min). By using SFE, the greatest oil extraction yield (>95%) was attained at the highest pressure and temperature conditions (45 MPa and 60 °C) after 240 min. At the early stage of SFE extraction, both LOCS and HOCS exhibited a similar kinetic behavior, reaching oil extraction rates of 0.59 g oil/min and 0.64 g oil/min, respectively. No differences were found between the fatty acid profile of the oils extracted from LOCS and HOCS both by PLE and SFE. ALA and linoleic acid (LA) concentrations ranged between 65-68% and 17-23% respectively, and a predominance of high molecular weight triglycerides (≥ CN50), was found in all extracted oils. In conclusion, LOCS might constitute a new suitable raw material for the production of ALA-enriched oils. Concerning the extraction methods assayed, the oil was almost entirely recovered by both PLE and SFE at the used conditions.

Buttermilk and Krill Oil Phospholipids Improve Hippocampal Insulin Resistance and Synaptic Signaling in Aged Rats.

Impaired glucose metabolism and mitochondrial decay greatly increase with age, when cognitive decline becomes rampant. No pharmacological or dietary intervention has proven effective, but proper diet and lifestyle do postpone the onset of neurodegeneration and some nutrients are being investigated. We studied insulin signaling, mitochondrial activity and biogenesis, and synaptic signaling in the hippocampus and cortex following dietary supplementation with bioactive phospholipid concentrates of krill oil (KOC), buttermilk fat globule membranes (BMFC), and a combination of both in aged rats. After 3 months of supplementation, although all groups of animals showed clear signs of peripheral insulin resistance, the combination of KOC and BMFC was able to improve peripheral insulin sensitivity. We also explored brain energy balance. Interestingly, the hippocampus of supplemented rats-mainly when supplemented with BMFC or the combination of KOC and BMFC-showed an increase in intracellular adenosine triphosphate (ATP) levels, whereas no difference was observed in the cerebral cortex. Moreover, we found a significant increase of brain-derived neurotrophic factor (BDNF) in the hippocampus of BMFC+KO animals. In summary, dietary supplementation with KOC and/or BMFC improves peripheral and central insulin resistance, suggesting that their administration could delay the onset of these phenomena. Moreover, n-3 fatty acids (FAs) ingested as phospholipids increase BDNF levels favoring an improvement in energy state within neurons and facilitating both mitochondrial and protein synthesis, which are necessary for synaptic plasticity. Thus, dietary supplementation with n-3 FAs could protect local protein synthesis and energy balance within dendrites, favoring neuronal health and delaying cognitive decline associated to age-related disrepair.

Antiproliferative activity of buttermilk lipid fractions isolated using food grade and non-food grade solvents on human cancer cell lines.

Buttermilk is a dairy by-product with a high content of milk fat globule membranes (MFGMs), whose protein constituents are reported to be antiproliferative. Lipids represent about half of the composition of MFGM. The aim of this study was to isolate buttermilk lipid fractions and evaluate their potential antiproliferative effect. Selective extraction with food grade or non-food grade solvents was performed. Antiproliferative effectiveness of lipid extracts and their neutral and polar fractions was evaluated on nine human cancer cell lines. Fractions obtained using food grade ethanol gave a higher yield than those obtained using non-food grade solvents, and they effectively inhibited cell viability of the cancer cell lines investigated. These fractions, rich in phospho- and sphingolipids, were strongly antiproliferative against human ovary and colon cancer cells. This observation allowed us to hypothesize further analyses aimed at promoting the use of buttermilk polar lipid fractions as functional food additives.

A high-performance direct transmethylation method for total fatty acids assessment in biological and foodstuff samples.

Isolation is the main bottleneck in the analysis of fatty acids in biological samples and foods. In the last few decades some methods described direct derivatization procedures bypassing these steps. They involve the utilization of methanolic HCL or BF3 as catalysts, but several evidences from previous works suggest these reagents are unstable, lead to the formation of artifacts and alter the distribution of specific compounds as hydroxy fatty acids or CLA. However, the main issue is that they are excellent esterification reagents but poor in transterification, being not suitable for the analysis of all lipid classes and leading to erroneous composition quantitations. The present research work is a comprehensive comparison of six general methylation protocols using base, acid or base/acid catalysts plus a proposed method in the analysis of total fatty acids in lipid standards mixtures, foodstuff and biological samples. The addition of aprotic solvents to the reaction mixture to avoid alterations was also tested. Results confirmed that procedures solely involving acid catalyst resulted in incomplete derivatizations and alteration of the fatty acid profile, partially corrected by addition of the aprotic solvent. The proposed method combining sodium methoxyde and sulfuric acid showed absence of alteration of the FAME profile and the best values for response factors (short chain fatty acids to PUFA), accuracy in the determination of total cholesterol and derivatization performance, thus showing a high reliability in the determination of the total fatty acid composition in biological samples and foods.