Preparation Of Human Milk Fat Substitute Through Rhodococcus Opacus PD630 Fermentation

Infant formula?IF?is the main food for infants and young children,and its nutrition is directly related to the health of infants and young children.Oil and fat are the raw materials for IF.Therefore,the nutrition of oil and fat is vital concern.Currently,human milk fat substitute?HMFS?used in IF is produced by lipase-catalyzed process,which lead to the occurrence of isomers because of the acyl migration.Therefore,new approach without acyl migration is needed.There is no acyl migration occur in the synthesis of microbial oil.Rhodococcus opacus PD630 is well known for its ability to accumulate oil.The aim of the research is to prepared HMFS through R.opacus fermentation.This research involves:a)the relationship between the carbon source chain length and fatty acid composition;b)preparation of OPO and OPL through R.opacus fermentation;c)preparation of HMFS through R.opacus fermentation;d)pathways of lipids synthesis in R.opacus;e)optimization of the R.opacus fermentation process.The main conclusions are as follows:1. The morphology of R.opacus at log phase presents as a rod with a length around4?m.The fatty acid is mainly composed of palmitic acid?31.25%?and oleic acid?16.67%?when R.opacus is growing on glucose.When the chain length of carbon source is less than 12,the fatty acid composition of oil is similar to that when glucose is used as the carbon source.When the carbon source chain length is equal to or longer than 12,the carbon source chain length determines the fatty acid chain length.2. It is feasible to produce OPO and OPL by R.opacus fermentation using carbon source regulation.With regard to OPO,the best substrate oils are CIE?HOSFO:HPO1.73:1 w/w?and EO:EP=2:1?w/w?.The sum of oleic acid and palmitic acid accounts for 85-87%in the total fatty acid composition with a ratio closing to 2:1?w/w?.Palmitic acid occupied at the sn-2 position?64-74%?.The content of sn-OPO is 47%,which is a medium level comparing with other methods.For OPL,the best substate oils are CIE?HPO:SBO 21-29:79-71 w/w?and EO:EP:EL=1:1.2-1.5:1.5-2?w/w/w?.The sum of oleic acid,palmitic acid and linoleic acid accounts for more than 85%with a ratio closing to 1:1:1?w/w/w?.The sn-2 position is esterified by palmitic acid?48-82%?.?-OPL accounts for 40%in the oil of R.opacus,which is higher than the HMF,commercially available HMFS and current literature.3. It is feasible to produce HMFS similar to HMF in terms of total and sn-2 fatty acid composition and triacylglycerol by R.opacus fermentation using carbon source regulation.Six HMFSs were prepared,among which HMFS?and?were the best.The corresponding substrate oils are CIE?CNO:SBO:HSFO:AKO:FO66.1:17.5:5.4:9:2 w/w/w/w/w?and CIE?CNO:SBO:HSFO:AKO:FO57.5:26.5:4.5:9.5:2 w/w/w/w/w?.The main fatty acids of HMFS fall within the range of human milk fat with the highest similarity to human milk fat from Asia.The sn-2position was occupied by palmitic acid?43.48%-49.32%?.The most abundant triacylglycerols in the HMFS is OPL,OPO,LPL,PSO,MOL.In terms of triacylglycerols,HMFS has the highest similarity with the human milk fat from Beijing.The characteristics of HMFS meet the requirements of GB 10765-2010.4. Transcriptome analysis showed that there were complete?-oxidation,fatty acid synthesis pathway,triacylglycerols synthesis pathway,EMP pathway,ED pathway,PPP pathway in R.opacus.FAS?is the favored process in the synthesis of fatty acid.The NADPH is mainly from the malate dehydrogenase and glucose-6-phosphate dehydrogenase.Carbon flux go through EMP,TCA,PPP in the log phase,but go through ED pathway in stationary phase.The analysis found that long-chain fatty acids can enter triglyceride synthesis pathway directly,while short-chain fatty acids enter the fatty acid synthesis pathway before enter the triacylglycerol synthesis pathway.1-acyl-sn-glycerol-3-phosphate acyltransferases?AGPAT?determined the sn-2 fatty acid composition.Five AGPATs was found within the R.opacus,which have the palmitic acid preference.5. Single factor experiments showed that biomass increased linearly with glucose concentration range from 6 to 24 g/L.The optimal phosphate concentration was 0.85 to13.6 g/L.The optimal magnesium concentration was 0.24 g/L?calculated as magnesium sulfate?.The appropriate urea concentration was 0.6 g/L.The carbon-nitrogen ratio range 30-80 was conducive to the lipid accumulation.Response surface optimization showed that the optimal ethyl ester,NH₄Cl,and phosphorus contents were 23 m L/L,2.47 g/L,and 8.5 g/L,respectively.Under this condition,the biomass reached 3.3 g/L?DCW?,the oil content was 40.23%?DCW?,and the oil production was 1.33 g/L.