Screening,Genomic Characteristics And Application Of Purine Degrading Lactic Acid Bacteria

Hyperuricemia is caused by increasing uric acid content in blood due to excessive intake of purine or purine metabolism disorder.Hyperuricemia can lead to gout,hypertension,chronic kidney disease and other diseases.At present,the main way to prevent hyperuricemia is to control the intake of high purine food in the diet,which often cause the imbalance of human nutrients,and makes people unable to enjoy the pleasure of food,leading to the decline of quality of life.Lactic acid bacteria(LAB)are gram-positive cocci that can ferment sugars and produce lactic acid.Studies showed that some LAB strains have certain effects on the prevention and alleviation of hyperuricemia,which showed potential application for construction of special food for hyperuricemia population.However,due to the comong strains,the degradation characteristics and application effects are also different.In this study,LAB strains were isolated from the traditional fermented food in China to study their capability of exogenous purine degradation in food.The DNA markers for purine degrading strains were screened through genome analysis.Our study provided new LAB strains for hyperuricemia population to control purine uptake and establish special probiotic food(1)A total of 89 LAB strains were isolated from the fermented food samples by combining traditional MRS culture,DNA fingerprinting and 16S rDNA sequencing identification.The PCR amplification system was established by using(GTG)5 and ERIC-PCR primers,and the produced DNA fingerprints were used for removing.A total of 35 fingerprint patterns were obtained.According to the results of 16S rDNA sequencing,24 LAB species were identified,including L.plantarum 13 strains,Lactobacillus rhamnosus 9 strains,Lactobacillus paracasei 7 strains,Lactobacillus fermentans 7 strains,Lactococcus lactis 5 strains,Lactococcus lactis subsp.Lactis 4 strains,Lactococcus garvieae 4 strains,Lactobacillus casei 3 strains,Lactobacillus brevis 1 strains,Lactobacillus acidophilus 3 strains,Pediococcus lactis 3 strains,Enterococcus durans 3 strains,Lactobacillus taiwanensis and Lactobacillus parabuchneri 2 strains respective,Lactobacillus diolivorans 2 strains,Enterococcus faecalis 1 strains,Enterococcus faecium 1 strains,L.buchneri 1 strains,L.coryniformis subsp.Coryniformis 1 strains,L.delbrueckii 1 strains,L.delbrueckii subsp 1 strains,Pediococcus pentosus 1 strains,Enterococcus hirae 1 strains,Leuconostoc pseudomesenteroides 1 strains.The Gram staining result was consistent with that of molecular identification.Our study showed that the established isolation and identification system based on MRS plate separation-(GTG)5 and ERIC-PCR-16S rDNA sequencing is a fast and cheap method for isolation and identification of LAB strains.(2)HPLC method for purine determination and purine degradation model for LAB strains in vitro were established.A strain of L.rhamnosus(YZULr026)with high guanine degrading ability was obtained,which also showed the ability to degrade xanthine and hypoxanthine.By the purine degradation model,the degradation rates of guanine,xanthine and hypoxanthine reached to 87.85%,23.14%and 12.17%,respectively.At 37?,YZULr026 could degrade more than 85%of 20 ?g/mL guanine within 2 h.The heat inactivated L.rhamnosus YZULr026 showed the weak ability degrading 25%of 20 ?g/mL guanine was weak;the supernatant and precipitation of the cell finders of YZULr026 showed higher ability to degrade purine.The results of acid and bile salt tolerance test of YZULr026 showed that the number of viable bacteria decreased from 7.69 1g CFU/mL to 3.5 1g CFU/mL at pH 2 for 6 h and grew well at pH 3 and pH 4;the number of viable bacteria decreased from 7.69 1g CFU/mL to 4.111g CFU/mL at 0.5%bile salt concentration for 6 h,which indicated that YZULr026 has good acid and bile salt tolerance.(3)The xanthine oxidase inhibitory activity and antioxidant activity of LAB were determined.The results showed that a strain of Enterococcus faecium(named SR937)with xanthine oxidase inhibitory activity was obtained.The results showed that the metabolites and cell contents of SR937 cells had xanthine oxidase inhibitory effect,and the inhibition rates were 37.67%and 27.7%,respectively;the results of antioxidant activity test showed that the metabolites and contents of Enterococcus faecium SR937 could scavenge 42.97%,67.27%of superoxide anion free radicals,89.61%and 45.5%of DPPH free radicals.Among the tested strains,SR937 had the strongest total antioxidant capacity,which provided a new strain for further construction of multi strain probiotics for hyperuricemia population.(4)The whole genome sequences of six purine degrading LAB strains were determined.The results showed that the gene coding numbers of the six strains ranged from 1988 to 3269,with an average length of 840.39-878.53 bp.The coding region accounted for 83.36-87.15%of the total genome length.The(G+C)content of the gene region ranged from 43.29-53.4%,and the coding gene length was less than 1000 bp.Lactobacillus fermentum LBF1-1 was the most involved gene in go function annotation,accounting for 78.99%of all genes,followed by Pediococcus lactis QT-TP1-03,accounting for 78.67%of all genes;The number of genes from COG function annotation to metabolism ranged from 586 to 944;The number of genes that KEGG function annotated to metabolism ranged from 649 to 965.The results showed that there were diversity in gene sequences of Gene264(encoding ?-galactosidase),Lactobacillus plantarum LBP3-2(encoding prophage protein),Lactobacillus fermentum LBF1-1(encoding helix to helix transcription regulator),Lactobacillus brucelli 6004(encoding hypothetical protein),and Pediococcus lactis QT-TP1-03(encoding hypothetical protein).Gene264 of Lactobacillus rhamnosus YZULr026 shared 39-99%homology with other Lactobacillus rhamnosus strains.These results indicated that YZULr026 is a new strain,and Gene264 sequence can be used as a potential new nucleic acid marker.(5)The fermentation technology of cucumber yoghurt by Lactobacillus rhamnosus YZULr026 was studied.Taking sensory score,Lactobacillus colony number and pH value as evaluation indexes,the optimal fermentation parameters of cucumber yogurt by Lactobacillus rhamnosus YZULr026 were determined through single factor and response interview as follows:fermentation temperature 36?,fermentation time 9 h,sugar addition 5%,and inoculation 5%.Under these conditions,the sensory quality of cucumber yogurt met the evaluation standard.The total viable count of LAB was 1.57 × 108 CFU/mL,the pH value was 4.35,the protein content was 2.95%,the fat content was 3.38%,and the total solid content was 12.1%.The microbial indexes met the relevant standards,which laid a foundation for the further construction of special food for reducing uric acid based on YZULr026 strain.