Microseparation, TAG Distribution And Anti-inflammatory Function Of BCFA In Milk Fat

Intestinal inflammatory diseases of the children such as neonatal necrotizing enterocolitis?NEC?and inflammatory bowel disease?IBD?are common but sometimes seriously affect or even threat children's health.The machanisim of intestinal inflammation diseases is very complicated,which leads to the difficulty of treatment.This kind of disease is still one of the tough questions in the field of pediatric research.It was reported that cell cytokine of inflammation plays a vital role in intestinal inflammation.Premature infant feeding on human milk has lower morbidity rate of intestinal inflammation diseases than that of infant formula.Inspired by the fact that premature infant lacks vernix,Pro Brenna from Cornell University used neonatal rat to do experiments and found that mixture with branched chain fatty acids?BCFA?can reduce NEC morbidity rate by 50%,which first time connected BCFA with NEC and brought new hope for NEC treatment.However,the mechanism and effect of this finding still need to be explored and verified.The relationship between BCFA and inflammation reaction and the interaction between BCFA and human intestinal cells are still unknown.BCFA composition and structure distribution of natural lipids expecially human milk fat are unclear.These are all hash questions needing to be solved at this stage.Therefore,our research is to set up combined urea-thin layer chromatography and silver nitrate-thin layer chromatography for micro separation and determination of hard-to-detect BCFA,which was then applied to the detection of BCFA profile of human milk fat,animal milk fat and fish oil.The property and structure of BCFA were also comprehensively investigated.On this basis,particular BCFA were chosen for the reseach of anti-inflamation function and mechanism.And BCFA positional distributions in Triacylglyceride?TAG?human milk fat,animal milk fat and fish oil were first analyzed and the effects of positional distribution on cell uptake and anti-inflammatory function were investigated.The research in present work is designed to provide basis for developing functional structure lipids product with BCFA.First of all,novel method for BCFA micro-enrichment in common food fats by combining urea-thin layer chromatography?urea-TLC?with silver nitrate-thin layer chromatography?AgNO₃-TLC?was estabilished.Total BCFA composition in human milk fat,animal milk fat and fish oil were analyzed using GC-MS after the pre-enrichment.Urea dipping coating and dynamic AgNO₃ coating methods were designed to make urea-TLC plate and silver-TLC plate,and then separation parameters such as sample application method,plate dipping time,and sample amount were optimized to obtain the best seperation condition.The result showed that the optimized condition is: urea/methanol predissolving sample application method,1 min dipping time,and 200 mg sample amount.After combined urea thin layer chromatography and sliver thin layer chromatography separation,BCFA in common food fats could be better separated and condensed,which enables trace amount BCFA to be detected.Compared with traditional urea solvent separation method,the present method need only 200 mg lipids rather than more than 50 g lipids and the purity of BCFA from cow milk fat could be improved to 79.74% comprared to 11%.The analysis results of total BCFA showed that monomethyl BCFA dominated the BCFA in cow milk fat,but BCFA in human milk fat and goat milk fat were more complicated.And there were more multi-BCFA detected in fish oil.And then,BCFA in neutral lipids?NL?and polar lipids?PL?of these common food fats were aslo separated and determined.Sn-2 fatty acids profile and distribution in different food fats were analyzed.In animal milk fat,there were more BCFA in NL than in PL.In fish oil,there were more BCFA in PL than NL.In human milk,there were more BCFA in NL,among which anteiso-BCFA ratio was bigger than iso-BCFA,which is opposite in fish oil.The anteiso-BCFA ratio in animal milk falls in between.Triacylglycerol?TAG?in human milk fat,cow milk fat and goat milk fat and fish oil was first separated and extracted using thin layer chromatography.Pancreatic lipase was used to hydrolyze sn-1/3 position fatty acids to get sn-2 fatty acids.The results showed that BCFA in human milk fat preferred to locate in the sn-2 position of triacylglycerol?⁶8%?.BCFA in animal milk fat were randomly distributed in three position of TAG?³3%?.However,in fish oil,BCFA preferred to locate in sn-1/3 position instead of sn-2 position?less than 10%?.BCFA distribution ratio in sn-2 position of TAG decreased with fatty acids carbon chain length increase.From the perspect of the sn-2 distribution in TAG of food fats,BCFA in human milk was affected by diet,however,its special TAG structure distribution indicated that BCFA would be resynthesized in the body and transformed into new lipids containing BCFA which has structures and components beneficial to infants.Next,to investigate the interaction between BCFA and human intestinal cell and its function of anti-inflammation,7 kinds of BCFA were used as research objects.LPS?lipopolysaccharide?was used to induce the inflammation in cells.Single BCFA with different chain length and branch position were substrates,and DHA,EPA were references.The cell viability and gene expression of pro-inflammatory cytokine IL-8?interleukin-8?,NF-?B?nuclear factor-kappa B?and TLR 4?toll like receptor 4?were analyzed by q-PCR.The results showed that BCFA treatments could protect human intestinal cell from LPS stimulation and maintained better cell viability?P<0.05?.IL-8,NF-?B and TLR 4 expression was decreased after BCFA treatments compared to inflammatory cell without any fatty acids treatment?P<0.05?.BCFA have anti-inflammatory function,the possible mechanism of which was that BCFA triggerred TLR 4 expression suppression to inhibit LPS activation of NF-?B pathway.The anti-inflammatory function of anteiso-BCFA is better than iso-BCFA.The shorter the carbon chain is,the greater the anti-inflammatory function will be.Lastly,the effect of BCFA TAG structure on anti-inflammatory function and cell uptake were investigated.LPS induced human intestinal cells were treated with vernix sn-2 MAG and FFA,which was prepared by vernix containing higher amount of BCFA.The cell viability and gene expression of pro-inflammatory cytokine IL-8,NF-?B and TLR 4 were analyzed by q-PCR.The results showed that compared to FFA form,sn-2 MAG worked better in maintaining good cell viability of human intestinal cells and suppressing pro-inflammation cytokine IL-8 and NF-?B expression?P<0.05?.Compared to inflammated cells,vernix sn-2 MAG and FFA treatment could maintain good cell viability?P<0.05?and protect cells from LPS stimulation.Shorter chain BCFA in vernix lipid treated cells increased significantly?P<0.05?.Longer chain BCFA didn't show a significant difference.The result showed that total BCFA uptake ratio of sn-2 MAG form in human intestinal cell was more than that of free fatty acids form.Compared to animal milk fats and fish oil,BCFA in human milk fat have higher uptake rate due to sn-2 distribution preference in TAG,which indicates that BCFA from human milk fat incorporate more into intestinal cell than animal milk fat and fish oil.MAG form can enhance the anti-inflammatory function of BCFA,which indicates the positional distribution of BCFA in human milk is important to its anti-inflammatory function and cell uptake.This is the first time that BCFA-MAG was recognized as a new and prospective nutraceutical compound of medicinal interest in modulating the inflammation of patients with intestinal inflammation.To sum up,the researches of the composition and positional distribution of BCFA in milk fat and fish oil,and its uptake and anti-inflammatory function in human intestinal cell show that BCFA are a group of important and beneficial food component.