Dairy phospholipids: analysis, enrichment and biological application

Dairy phospholipids (PLs) are complex polar lipids with a unique amphiphilic nature underpinning their structural integrity as a membrane surrounding milk fat globules- the milk fat globule membrane (MFGM). The techno-functional capabilities, nutritional benefits and putative health associations of dairy PLs have garnered considerable interest over the last number of decades and thus significant research has focused on the analysis, isolation and application of these bioactive and functional compounds. Development of a robust and reliable analytical method for efficient recovery and accurate quantitative determination of dairy PLs was achieved in the course of comparing different lipid extraction protocols and optimisation of high performance liquid chromatography (HPLC) procedures. Reappraisal of current lipid extraction procedures based on PL recovery determined Folch as the most effective method for PL analysis with a 1.9-fold and 2.5-fold increase in PL recovery compared with Röse Gottlieb and a modified Folch, respectively. Concomitantly, optimisation of a HPLC method coupled to a charged aerosol detection system (CAD) yielded a two-fold increase in PL recovery compared to the original method. Alteration of the HPLC elution program maximised PL peak separation, and pH adjustment of the buffer reduced co-elution of PL species detected by the CAD with the result that the combined protocol yielded greater PL recoveries than that previously published, 2.30 ± 0.03 % total PL in milk. Efficiency and improved recoveries was also reflected in PL values obtained from different dairy streams, 35.32 ± 0.01 % total PL and 46.09 ± 0.01% total PL in buttermilk and butter serum, respectively. Most notably recoveries of the more acidic PLs, phosphatidylinositol (PI) and phosphatidylserine (PS) increased by 2 % and 7 % across all dairy streams analysed compared to previous studies. Generation of a novel dairy ingredient enriched in dairy PLs was achieved through development of a process at laboratory scale that combined enzymatic digestion and ultrafiltration (UF). Buttermilk with its higher residue of PL arising from buttermaking was prepared by reconstitution of buttermilk powder (BMP) substrate and subjected to extensive hydrolysis of its inherent milk protein complement followed by ultrafiltration to permeate the resulting smaller molecular weight peptide material. Screening of a number of digestive enzymes based on their proteolytic activity identified Alcalase® as the most proteolytic enzyme that generated a hydrolysate with 89.84 % of the peptides < 50 kDa in size. Comparison of 100 kDa and 50 kDa molecular weight cut off (MWCO) membranes (Sartorius Vivaflow™ 200 PES crossflow cassette) resulted in no significant difference in the peptide profiles of the obtained retentates. A 7.8-fold increase in PL (based on 6.16 ± 0.02 % and 0.79 ± 0.01 % total PL in the freeze-dried retentate and BMP starting material, respectively) was achieved based on degree of hydrolysis (DH) of 19 % and 50 kDa MWCO UF membrane filtration. This combined approach increased the lipid material 6.3-fold and reduced residual protein content 2-fold in the retentate compared to the starting BMP with no evidence of PLs detectable in the permeate, 0.00 ± 0.01 % total PL. Successful scalability of this combined process was achieved at pilot scale with an even higher 8.5-fold increase in PL material in the retentate compared to the starting material, 11.05 ± 0.02 % and 1.30 ± 0.00 % total PL, respectively. A total lipid increase of 8.7-fold and total residual protein decrease of 2.9-fold during scale-up in the pilot plant exceeded laboratory scale performance. Subsequent application of supercritical fluid extraction (SFE) treatment to the obtained PL enriched spray dried retentate yielded an enriched PL extract, 56.24 ± 0.07 % total PL (dry matter) depleted of protein and lactose, thus exceeding the dairy PL enrichment values achieved by previous researchers. Application of the highly-enriched PL extract in vitro determined a neurotrophic bioactivity. The PL extract induced a stimulatory effect on the outgrowth of cortical neuron cells. Much of the research published to date has explored the biological effects of long-chain polyunsaturated fatty acids (LC-PUFAs) strongly associated with PLs, e.g. docosahexaenoic acid (DHA). Investigation of the effect of the PL extract at different dosages determined that at a concentration of 150 µg mL-1 PL extract a 43 % increase in cortical neuron stimulation was observed compared to the control, 0 % stimulation. However, at a concentration of 300 µg mL-1 PL extract the observed stimulation decreased to 12 % suggesting a neurotoxic effect at higher dosage levels. This increase is greater than that previously published and the effective concentration, 150 µg mL-1, is in agreement with the PL levels found in human milk. This study demonstrated that dairy PLs promote the development and outgrowth of cortical neurons and open up an avenue to explore further the putative health benefits associated with dairy PLs