Screening,identification And Omics Analysis Of Strains Degrading Limonin

Citrus is the world's largest fruit.In the world,China's citrus production is the highest.Juice and tea are by-products of citrus fruit processing.Because bitterness will appear in the citrus during processing,this bitterness affects the taste,this bitterness problem restricts the development of China's citrus processing industry.At present,enzymatic debittering is the most important method in citrus debittering.Compared with other debittering methods,it has many advantages,such as strong specificity,good effect,low cost and no damage to nutrients.Enzymatic debittering is the most promising debittering method.In this paper,the soil of perennial orange garden and the vinegar sample of Shanxi Vinegar Plant were enriched and cultured.Three strains of high-efficiency degrading limonin were screened and named 1,2 and 3 strains.The 1 strain was identified as Cellulomonas sp.by morphology and molecular biology.The 2 strain and 3 strain were identified as Ochrobactrum sp.by morphology and molecular biology.The response surface model of the 3 strain was established,the fermentation and culture conditions of the 3 strain were optimized,and the degradation mechanism of limonin in the 3strain was studied,which will provide theoretical basis for molecular transformation to improve enzyme production activity.The following are the main findings.(1)The soil of perennial orange garden and the vinegar sample of Shanxi Vinegar Plant were enriched and cultured.Through the solid plate culture method,8 strains capable of growing with limonin as the sole carbon source were screened out,and then the strains were shake-flask re-screened to obtain 3 strains with better degradability.Eight strains were selected by solid plate culture method,and they could grow with limonin as the only carbon source.The strains were rescreened in shake flasks to obtain3 strains with better degradation ability.The 1 strain was identified as Cellulomonas sp.by morphology and molecular biology.The 2 strain and 3 strain were identified as Ochrobactrum sp.by morphology and molecular biology.(2)The culture conditions of the three strains were used for single-factor experiments,and the 3 strain with better degradability was used to establish the response surface model.We obtained the best process of 3 strain limonin degradation rate:The p H is 3.5.The temperature is 30?.The nitrogen source is ammonium nitrate.The bacterial inoculation amount is 3×10~8 CFU/m L.Under this condition,the degradation rate of limonin was 78.90%,which was 2.1 times higher than that of initial culture conditions.(3)Through the Illumina Nova Seq and Pacbio Sequel platforms,the whole genome analysis of the 3 strain was carried out.The total genome length of this strain was found to be 1,250,615,224 bp,which consisted of two circular chromosomes and two circular plasmids.The Illumina Hi Seq sequencing platform was used to perform transcriptome sequencing on the 3 strain.Taking the whole genome as a reference,many differentially expressed genes were found,mainly including 2,4-dienoyl-Co A reductase(NADPH)activity,endopeptidase regulating activity,negative regulation of catalytic activity,enzyme inhibitor activity,glutamine Acid ammonia ligase activity,3-methyl-2-oxobutyrate dehydrogenase(2-methylpropionyl transfer)activity,oxidoreductase activity,pyruvate-acetyl-Co A activity,isocitrate lyase activity etc.Enriched metabolic pathways are mainly oxidative phosphorylation(ko00190),pyruvate metabolism(ko00620),propionate metabolism(ko00640),arginine and proline metabolism(ko00330),glyoxylic acid and dicarboxylic acid ester metabolism(Ko00630)etc.(4)The metabolites were analyzed by LC-MS technology.Some metabolic pathways were found,mainly including citrate cycle(TCA cycle),limonene degradation metabolism,vitamin metabolism(nicotinic acid ester and nicotinamide metabolism,pantothenic acid and Co A biosynthesis),amino acid metabolism(alanine,lysine,Metabolism of aspartic acid and glutamic acid)etc.(5)The transcriptome and metabolites were analyzed for the carbon fixation pathway of prokaryotes.It is speculated that there may be a metabolic pathway.2-Oxoglutarate is converted to isocitrate.Then isocitrate is converted to citrate,which is the end product of metabolism.It is speculated that there may be a metabolic pathway.2-Oxoglutarate is converted to isocitrate.Then isocitrate is converted to citrate,which is the end product of metabolism.