Human Milk Structural Lipids’ Preparation,Nutritional Properties,and Their Effects On Cognitive Behavior

Human milk fat contributes to more than 50% of the infant’s energy requirements and regulates various metabolic activities.Human milk fat is composed of complex natural lipids,including triglycerides(TAGs),glycerophospholipids(GPs),and sterol lipids.Among these,TAGs account for 98% of total fat and show a unique fatty acid composition and positional distribution.Palmitic acid(PA),the most abundant longchain saturated fatty acid in human milk fat,constitutes 20-25% of total fatty acids,with >70% of it occupying the sn-2 position.This forms the characteristic structural lipid known as sn-2 palmitate,distinguishing human milk fat from vegetable oils and animal milk fats.In recent years,an increasing body of evidence shows that formula supplemented with sn-2 palmitate has a significant role for infant health in different directions,such as fat digestion and absorption,calcium absorption,bone development,fecal consistency,sleep duration,intestinal microbiota,and neurodevelopment in infants,with the majority of these outcomes being established on 1,3-dioleoyl-2-palmitoyl-glycerol(OPO).In fact,the lipid composition of human milk is not constant and is influenced by geography,ethnicity,and diet.For example,1-oleoyl-2-palmitoyl-3-linoleoylglycerol(OPL)is the most abundant TAG in Chinese human milk compared with foreign human milk.However,studies on OPL are still limited;therefore,this study systematically investigated the influence of OPO and OPL feeding on digestion and absorption,lipid metabolism,intestinal microbiota,and brain function to clarify their nutritional differences,and finally provided the mechanisms by which dietary human milk structural lipids(HMSL)influenced cognitive function.The main results were as follows:1.The chemoenzymatic preparation process of high-purity OPOThe low-purity OPO and OPL produced by enzymatic synthesis significantly interfere with the health benefits of the research subjects;therefore,OPO and OPL with purity >80% were used as the research subjects in this study.OPL(≥ 80%)can be obtained from reagent companies,and this study investigated the chemoenzymatic preparation process of high-purity OPO,which includes reversible acylation with oleic acid(OA)as the acyl donor and irreversible acylation with vinyl oleate(OV)as the acyl donor.Among them,the yield(57.11%)and purity(98.23%)of 1,3-diolein(1,3-DO)produced by irreversible acylation were significantly higher than that of 1,3-DO obtained from reversible acylation(35.25% of yield and 93.23% of purity),when the reaction parameters of the irreversible acylation reaction were as follows: the substrate ratio was 2:1(OV: glycerol;mol/mol),the enzyme dosage was 8% Novozym 435,the reaction time was 6 h,and the reaction temperature was 8 °C.Correspondingly,the purity of OPO products obtained from the irreversible acylation method(97.17%)was higher than that produced by the reversible acylation method(91.62%).2.Digestion and absorption properties of OPO and OPLIn response to the lack of clarity regarding the differences in digestion and absorption characteristics between OPO and OPL,the present study investigated the differences in digestion and absorption processes between OPO and OPL using C57BL/6J mice.Results showed that the gastric emptying rates of OPO and OPL were similar.The digestion curves of OPO and OPL in the small intestine showed a bimodal pattern.Of which,the OPO content peaked at 0.5 h and 3 h,while the OPL concentration peaked at 0.5 h and 4 h.The content of OPL at 4 h(0.46 mg/g)was higher than that of OPO at 3 h(0.26 mg/g).Meanwhile,the area under the curve(AUC)of OPO(1.472)concentration in the intestinal contents was lower than that of OPL(1.749)during the 5 h monitoring time,indicating that the digestion rate of OPO was higher than that of OPL.Moreover,OPL group mice plasma contained a higher concentration of TG(AUC: 12.33)when compared with the OPO group(AUC: 11.38)during the 5 h monitoring time,indicating that OPL was more easily absorbed into the blood than OPO.The postprandial plasma LDL-C concentration and LDL-C/HDL-C ratio in the OPL group were higher than in the OPO group.3.OPO was more beneficial to improving the liver metabolism than OPL-fedThe OPO and OPL absorbed into the circulation system could affect mice’s growth and liver lipid metabolism.Results showed that structural lipids feeding decreased mice body weight,liver TG,TC,LDL-C concentrations,and LDL/HDL ratio when compared with the Control(Con)group,which was attributed to the significant elevated liver ACOX1 m RNA expression in the structural lipids feeding groups.These indicators were lower in the mice of OPO group except for liver TC when compared with the OPL group.According to the serum lipidomics results,compared with the Con group,OPO and OPL feeding increased the chain length and the number of double bonds of lipids in the serum.Of which,dietary OPO significantly increased the abundance of very long-chain unsaturated fatty acids(VLCPUFAs),which might play an important role in the development of the brain in early life.Additionally,dietary OPL increased serum PC levels,while OPO feeding elevated PC and PE levels.The differential lipid analysis results showed that,OPO increased the abundance of OArich TAGs(18:1 TAGs)in serum and decreased the abundance of Ac Cas(acylcarnitine)and Car Es(carnitine esters)compared to the Con group,while OPL elevated 5 PCs levels and reduced 5 TAGs levels.Of note,Ac Cas and Car Es were the differential lipids between the OPO and OPL groups,and their abundance were significantly higher in the OPL group than in the OPO group,indicating that dietary OPL reduced the liver fatty acid β oxidation efficiency compared with the OPO feeding.This was consistent with the lower expression levels of PPAR α and ACOX1 in the liver of the OPL group.These results suggested that dietary OPO was more beneficial to improving the liver metabolism than OPL-fed,such as reducing lipid accumulation,increasing VLCPUFAs biosynthesis,elevating glycerophospholipid metabolism,and enhancing fatty acid βoxidation.4.OPL was more beneficial to improving the intestinal bacterial community compared with OPOBased on the digestion and absorption processes of OPO and OPL,it was found that some structural lipids were not absorbed in the small intestine and entered the large intestine for microbial fermentation,thereby affecting the gut microbiota.This study investigated the effects of OPO and OPL on the microbial community using in vitro and in vivo models.The results of β-diversity showed that the effects of OPO and OPL on the gut bacterial composition were similar.Analysis of the differential genera revealed that both OPO and OPL significantly increased the relative abundance of Coriobacteriaceae＿UCG-002 and [Clostridium]＿innocuum＿group compared with the Con group.Of which,Coriobacteriaceae＿UCG-002 was strongly related to brain development in early life.Moreover,OPO specifically elevated the relative abundance of Enterococcus,Candidatus＿Stoquefichus and Sphingobacterium,while OPL specifically increased the relative abundance of Parabacterioides,Alistipes,Bacteroides,Alloprevotella,and Parasutterella.These results indicated that OPL was more favorable for the growth of probiotic bacteria in the mice’s gut than OPO,such as Parabacterioides,Alistipes,Bacteroides,and Alloprevotella.In the in vitro fermentation,OPO and OPL significantly increased the relative abundance of the probiotic Veillonella and significantly decreased the relative abundance of the pathogenic Vibrio compared with the Con group;moreover,OPO specifically elevated the abundance of Lactobacillus,while OPL specifically reduced the relative abundance of pathogenic bacteria such as Solibacillus,Pseudomonas,Brachybacterium,Staphylococcus,Paracoccus,and UCG-005.Similarly,in the fermentation broth,OPL treatment was more favorable for improving the growth of probiotics and inhibiting the multiplication of pathogenic bacteria when compared with the OPO interventions.In terms of short-chain fatty acids(SCFAs),both OPO and OPL increased the concentrations of acetate,propionate,and butyrate in the mice’s gut and fermentation broth.Of these,the concentrations of acetate,propionate,and total SCFAs in the OPL group were higher than in the OPO group.These results suggested that OPL was more beneficial to improving the intestinal bacterial community compared with OPO,such as enhancing the multiplication of beneficial bacteria(in vivo: Parabacterioides,Alistipes,and Bacteroides,etc.;in vitro: Veillonella,etc.)and inhibiting the growth of pathogenic bacteria(in vitro: Pseudomonas,Brachybacterium,and Staphylococcus,etc.).5.HMSL regulated brain functions by influencing hepatic lipid metabolismThe above results showed that dietary HMSL increased the levels of VLCPUFAs in the blood and the abundance of beneficial bacteria in the gut,which might play an important role in infant neurodevelopment.Therefore,this study further investigated the effects of OPO and OPL on mice’s cognitive behavior and clarified the underlying mechanisms using metabolomics and lipidomics.Results showed that,OPO and OPL feeding enhanced the mice’s working memory and recognition memory,elevated the level of brain-derived neurotrophic factor(BDNF),and reduced neuronal damage compared with the Con group.Brain metabolomics results showed that dietary OPO and OPL influenced the mice’s brain function by enhancing the brain tricarboxylic acid(TCA)cycle and energy metabolism and increasing levels of VLCPUFAs(such as DGLA,DPA,AA,and DHA),as well as the abundance of LPC and LPE(LPC/E)in the brain.The VLCPUFAs cannot be synthesized de novo within the brain;therefore,we further analyzed the expression of MFSD2 A and the hepatic lipid composition.Briefly,HMSL feeding promoted the elongation and desaturation of FA(18:3)and FA(20:5)in the liver to produce VLCPUFAs and increased the incorporation rate of VLCPUFAs in the LPC/E.This increased the transport of VLCPUFAs to the brain via MFSD2 A,which elevated the levels of VLCPUFAs in the brain,regulating the mice’s brain function.Correlation analysis results showed that intestinal Coriobacteriaceae＿UCG-002 abundance and acetate concentration were strongly positively associated with hepatic VLCPUFAs concentration and the levels of brain metabolites involved in the TCA cycle,suggesting that the increase of acetate concentration in the intestine might promote the elongation and desaturation of fatty acids in the liver and enhance the TCA cycle in the brain.In summary,this study investigated the effects of OPO and OPL on digestion and absorption,lipid metabolism,gut bacterial composition,and cognitive behavior to clarify their nutritional differences.Of which,OPO was more beneficial to the body metabolism than OPL,such as decreasing hepatic lipid accumulation,increasing the biosynthesis of VLCPUFAs,elevating glycerophospholipid metabolism,and enhancing fatty acid β oxidation.While OPL was more favorable to intestinal microecology than OPO,such as increasing SCFAs production,increasing the abundance of probiotics,and inhibiting the growth of pathogenic bacteria.Additionally,this study provided the mechanism by which dietary HMSL influenced brain function.Briefly,HMSL feeding regulated brain function by increasing hepatic LPC/E-VLCPUFA biosynthesis and enhancing VLCPUFAs transport to the brain via MFSD2 A.These findings provide a theoretical basis for clarifying the physiological functions of HMSL and are important for guiding the commercialization of structural lipids.