Development and large-scale production of human milk fat analog by fermentation of microalgae

Background Human milk contains a complex mixture of triacylglycerols (TAG), making it challenging to recreate using common ingredients. Objective The study aimed to develop an innovative fermentation technique to produce essential human milk TAG, effectively tackling a significant hurdle in infant nutrition. Method An in-depth analysis of the literature has been conducted to identify the specific TAG to be targeted. We used a microalgal oil production platform and a two-step procedure to modify its fatty acid and TAG composition. The palmitic acid (16:0) content has been increased by classical strain improvement techniques, followed by a step involving the expression of a lysophosphatidic acid acyltransferase (LPAAT) sequence capable of esterifying 16:0 specifically at the internal position ( sn -2 palmitate) of TAG. Once the strain was stabilized, the fermentation was scaled up in a 50-L reactor to yield several kilograms of biomass. Subsequently, the oil was extracted and refined using standard oil processing conditions. Liquid chromatography-mass spectrometry was employed to monitor the TAG profile and the region specificity of 16:0 at the internal position ( sn -2 palmitate) of TAG. Results The initial strain had a 16:0 level of 25% of total fatty acids, which was increased to 30% by classical strain improvement. Simultaneously, the oleic acid level decreased from 61% to 57% of total fatty acids. Upon expression of an exogenous LPAAT gene, the level of the 16:0 esterified in the internal position of the TAG ( sn -2 palmitate) increased by a factor of 10, to reach 73% of total palmitic acid. Consequently, the concentration of oleic acid in the internal position decreased from 81% to 22% of total fatty acids, with TAG analysis confirming that the primary TAG species in the oil was 1,3-dioleoyl-2-palmitoyl-glycerol (OPO). The 50-L-scale fermentation trial confirmed the strain's ability to produce oil with a yield of >150 g of oil per liter of fermentation broth in a timeframe of 5 days, rendering the process scalable for larger-scale industrialization. Conclusion We have demonstrated the feasibility of producing a suitable TAG composition that can be effectively integrated into the formulations of infant nutrition in combination with other fats and oils to meet the infant feeding requirements.