Complete Polar Lipid Profile of Kefir Beverage by Hydrophilic Interaction Liquid Chromatography with HRMS and Tandem Mass Spectrometry

Abstract

Kefir, a fermented milk product produced using kefir grains, is a symbiotic consortium of bacteria and yeasts responsible for driving the fermentation process. In this study, an in-depth analysis of kefir's lipid profile was conducted, with a focus on its phospholipid (PL) content, employing liquid chromatography with high-resolution mass spectrometry (LC-HRMS). Nearly 300 distinct polar lipids were identified through hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform orbital-trap MS and linear ion-trap tandem MS/MS. The identified lipids included phosphatidylcholines (PCs), lyso-phosphatidylcholines (LPCs), phosphatidylethanolamines (PEs) and lyso-phosphatidylethanolamines (LPEs), phosphatidylserines (PSs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The presence of lysyl-phosphatidylglycerols (LyPGs) was identified as a key finding, marking a lipid class characteristic of Gram-positive bacterial membranes. This discovery highlights the role of viable bacteria in kefir and underscores its probiotic potential. The structural details of minor glycolipids (GLs) and glycosphingolipids (GSLs) were further elucidated, enriching the understanding of kefir's lipid complexity. Fatty acyl (FA) composition was characterized using reversed-phase LC coupled with tandem MS. A mild epoxidation reaction with meta-chloroperoxybenzoic acid (m-CPBA) was performed to pinpoint double-bond positions in FAs. The dominant fatty acids were identified as C18:3, C18:2, C18:1, C18:0 (stearic acid), C16:0 (palmitic acid), and significant levels of C14:0 (myristic acid). Additionally, two isomers of FA 18:1 were distinguished: ∆9-cis (oleic acid) and ∆11-trans (vaccenic acid). These isomers were identified using diagnostic ion pairs, retention times, and accurate m/z values. This study provides an unprecedented level of detail on the lipid profile of kefir, shedding light on its complex composition and potential nutritional benefits.

Keywords: LC-ESI-tandem MS; fatty acyl chains; food; kefir; phospholipids.

Figure 1

Representative total ion current (TIC) chromatograms by HILIC-ESI-HR-FTMS of a lipid extract from kefir sample 1, analyzed in both positive (A) and negative (B) ion modes. The abbreviations indicate the individual lipid classes separated during the analysis: Hex₂Cer (Dihexosylcerebroside), PG (Phosphatidylglycerol), PI (Phosphatidylinositol), PE (Phosphatidylethanolamine), PS (Phosphatidylserine), PC (Phosphatidylcholine), and SM (Sphingomyelin).

Figure 2

Phospholipid class distributions, as evaluated by HILIC-ESI-MS considering class-related peak areas, of kefir samples 1 and 2 (A,B), respectively, and milk (C).

Figure 3

Tandem MS spectra for key lipid species identified in kefir samples: (A) HexCer d18:1/22:0, observed as a chlorinated adduct with an m/z value of 818.6; (B) Hex₂Cer d18:1/24:0, detected as a deprotonated molecule at m/z 972.7, and (C) DGDG 34:1 as a deprotonated species at m/z 917.7.

Figure 4

(A) Tandem MS spectrum at m/z 875.6 of lysyl-PG 34:1. MS³ spectra of ions at m/z 747.5 (B) and m/z 673.5 (C) isolated from precursor at m/z 875.6. All species were detected as deprotonated molecules. PG and PA stand for phosphatidylglycerols and phosphatidic acids, respectively.

Scheme 1

Proposed fragmentation pathway of lysyl-phosphatidylglycerols. The ions specifically generated from the fragmentation of LyPG 18:1/16:0 at m/z 875.6 of Figure 4 are highlighted in blue.

Figure 5

Total ion current chromatograms by RPLC-ESI(-)-HR-FTMS of a kefir lipid extract (sample 2) analyzed in two steps: (A) following hydrolysis of the extracted lipids and (B) after subsequent epoxidation of the same sample.

Figure 6

MS/MS spectrum of deprotonated-epoxidated (A) FA 18:1, ∆9 and (B) FA 18:1, ∆11, which elute at 30.0 and 29.4 min, respectively (see Figure 5).