Enrichment of nervonic acid in Acer truncatum Bunge oil by combination of two-stage molecular distillation,one-stage urea complexation and five-stage solvent crystallization

Nervonic acid is the world’s first and only potent substance that can repair damaged nerve fibers and promote nerve cell regeneration with high nutritional value.The wide variety of fatty acids in plant oils and fats with similar structures makes the large-scale separation and purification of high-purity nervonic acid very difficult.A new combined process of molecular distillation,urea inclusion and solvent crystallization was established to prepare high-purity nervonic acid with the mixed fatty acids obtained after saponification and acidification of Acer truncatum Bunge oil as raw materials.First,according to the difference in the mean free path of fatty acids,molecular distillation was used to separate and remove C16 saturated fatty acid of palmitic acid and four C18-C20 fatty acids of stearic,oleic,linoleic,and linolenic acids.The content of C16-C20 fatty acids decreased from 72.92% to 19.22% after two-stage molecular distillation processes,in which the contents of saturated fatty acid of palmitic acid decreased to about 0.5%.Then,according to the difference in carbon chain length and saturation of fatty acid,the contents of C22-C24 saturated fatty acids of tetracosanoic and docosanoic acids decreased to 0.21% and 0.07% by urea inclusion with urea/free fatty acid preparation by saponification(SPOMFs)ratio as 0.6.In addition,all saturated fatty acids were basically separated.Finally,according to the difference in the solubility of fatty acids in solvents,the C18-C20 unsaturated fatty acids of oleic,linoleic,and linolenic acids and C22 unsaturated fatty acid of erucic acid were removed by solvent crystallization.The content of C18-C20 unsaturated fatty acids decreased to less than 5% with pentanol as the solvent after the first stage solvent crystallization.The content of erucic acid decreased to 3.47% with anhydrous ethanol as the solvent after the second to fifth stage solvent crystallization.The combined process of molecular distillation,urea inclusion and low temperature crystallization innovatively adopted an efficient,simple and easy-toindustrial solvent crystallization method to separate erucic and nervonic acids,obtaining nervonic acid with purity of 96.53% and final yield of 47.99%.

Diversity in seed oil content and fatty acid composition in Acer species with potential as sources of nervonic acid

Nervonic acid(NA,cis-15-tetracosenoic acid)is a very long-chain monounsaturated fatty acid that has been shown to be a core component of nerve fibers and nerve cells.It can be used to treat and prevent many neurological diseases.At present,commercially available NA is mainly derived from Acer truncatum seeds,which contain about 5%e6%NA in their seed oil.The aim of this study were to identify and analyze NA-containing Acer species that could be used as NA resource plants.For this purpose,46 Acer species seeds were collected in China and in some or all of the seed oils from these species 15 fatty acids were detected,including linoleic acid,oleic acid(C18:1D9,C18:1D11),erucic acid,palmitic acid,NA,linolenic acid(C18:3D6,9,12,C18:3D9,12,15),eicosenoic acid(C20:1D11,C20:1D13),stearic acid,behenic acid,tetracosanoic acid,arachidic acid,and docosadienoic acid.Nervonic acid was detected in all samples,but the content was highly variable among species.NA content over 9%was detected in eleven species,of which Acer elegantulum had the highest levels(13.90%).The seed oil content,seed weight,and fatty acid profiles varied among species,but the comprehensive evaluation value(W)showed that A.coriaceifolium could be a new potential NA resources plant.The results also showed that NA was significantly negatively correlated with palmitic acid,oleic acid,and eicosenoic acid,but positively correlated with eicosadienoic acid,behenic acid,erucic acid,and tetracosanoic acid,which indicate the probable pathway for NA biosynthesis in Acer plants.This study has identified Acer species that may serve as NA resources and will help guide subsequent species breeding programs.

Germplasm resources of three wood plant species enriched with nervonic acid

Nervonic acid(NA)is a very-long-chain monounsaturated fatty acid with pharmaceutical and nutraceutical functions that plays an important role in treating several neurological disorders.One major source of NA is plant seed oil.Here we report fatty acid profiles of seeds and germplasm diversity of six plant species,including three woody plants with high amounts of NA-enriched seed oil,Malania oleifera,Macaranga adenantha,and M.indica.M.oleifera had the largest seed(average 7.40 g single seed),highest oil content(58.71%),and highest NA level(42.22%).The germplasm diversity of M.oleifera is associated with its habitat but not elevation.Seeds of M.adenantha contained higher NA levels(28.41%)than M.indica(21.77%),but M.indica contained a significantly higher oil content(29.22%)and seed yield.M.adenantha germplasm varied among populations,with one population having seeds with high oil content(22.63%)and NA level(37.78%).Although M.indica grow naturally at a range of elevations,no significant differences were detected between M.indica populations.These results suggest that M.indica and M.oleifera have greater potential as a source of NA,which will contribute to constructing a germplasm resource nursery and establishing a selection and breeding program to improve the development of NA-enriched plants.

Lipid analysis of three special nervonic acid resources in China

Nervonic acid plays an important role in nutrition and function of the human body.Malania oleifera,Acer truncatum and Xanthoceras sorbifolium are China’s unique woody plant rich in nervonic acid in seed oil.This study aims to investigate the lipid composition of these 3 special resources.Their fatty acids were detected by gas chromatography coupled with flame detector(GC-FID).Triglycerides(TAGs)and phospholipids(PLs)were detected by shotgun-mass spectrometry(shotgun-MS).Results showed that M.oleifera oil presented the highest level of nervonic acid(46.20±0.22%)among the 3 oils.Seeds oil of A.truncatum and X.sorbifolium had 3.53±0.20%and 1.83±0.21%nervonic acid respectively.53 species of TAGs and 15 species of PLs were identified in M.oleifera oil,with PLs content of 499.94±22.34μg/g.In A.truncatum oil,PL and TAG species were twice more than those in M.oleifera oil,and its’content of PLs was 76.27±3.21μg/g.In X.sorbifolium oil,75 TAGs and 34 PLs were detected,with the lowest PLs at 23.84±0.17μg/g.The results demonstrated that these 3 vegetable oils have great potential to become nervonic acid supplements for human health.

Identification of KCS gene family and functional analysis of FAE-like genes from Malania oleifera

KCS(3-ketoacyl-CoA synthase)is the key enzyme catalyzing the first step of very long chain fatty acid(VLCFA)biosynthesis.Studies showed that different KCSs possessed different substrate preference.Malania oleifera are abundance of VLCFAs in its mature seeds,especially the nervonic acid,which is essential for human health.In this study,we identified and characterized 18 KCS genes in M.oleifera genome.Phylogenetic analysis showed that these KCS genes were classified into four subfamilies,including two FAE-like,six KCS-like,eight FDH-like and two CER6.We concentrated on the functional role of two FAE-like genes,Maole003085.T1 and Maole004215.T1 which encoded predicted amino acid residues of 516 and 518 in protein,respectively.Multiple sequence alignment showed that their two proteins contained the known and conserved active sites among FAE-like subfamily.Upon heterologous expression in wild type yeast(Saccharomyces cerevisiae)INVSc1,we found that Maole004215.T1 could produce four new fatty acids including C22:0/C22:1 and C24:0/C24:1,but Maole003085.T1 only produced C22:1.Besides,upon heterologous expression in mutant yeast BY4741-△elo3,we found the Maole003085.T1 could produce C24:0 and C26:0,while the Maole004215.T1 could catalyze the formation of fatty acids C24:0,C26:0 and C28:0.These results showed Maole003085.T1 and Maole004215.T1 had fatty acid elongation activity in yeast,and possessed different substrate preference in the production of different VLCFAs.Interestingly,we found Maole004215.T1 could produce nervonic acid in yeast,which provides molecular basis on the genetic improvement and genic engineering for producing nervonic acid resources by using biotechnological methods.