| Probiotics has attracted wide attention around the world due to its outstanding nutritional and beneficial effects.High density,high activity,high stability are the important characteristics and production core of probiotic products,which closely related to the growth medium of strains.Limosilactobacillus reuteri(L.reuteri)has many beneficial effects,such as regulating intestinal flora,alleviating irritable bowel syndrome and enhancing immunity,and widely used to produce dietary supplements and functional foods.In recent years,whole grain foods are favored by consumers because of their "high dietary fiber,low sugar,low fat and low cholesterol",and has shown great market potential.In this study,the growth promoting effects of various cereal substrates on L.reuteri were evaluated and found that the coix seed medium had significant proliferation effect on L.reuteri.Then,transcriptomics and proteomics were used to reveal the high-density growth mechanism of L.reuteri in coix seed from gene and protein levels.Meanwhile,the main components with growth promoting effect on L.reuteri were identified in coix seed.Finally,the effects of coix seed juice fermented with L.reuteri on intestinal flora and dyslipidemia were evaluated in high fat diet-fed mice.The main conclusions were as follows:(1)The growth characteristics and fermentation activity of L.reuteri in different cereals medium(coix seed,quinoa,red rice,black rice,millet and brown rice)were analyzed.The results showed that the viable bacteria of L.reuteri in the emulsion coix seed medium was the highest(3.10?1014 CFU/m L),the number of viable bacteria in the quinoa and millet medium was 1.25?1013 and 1.53?1012 CFU/m L,respectively.The viable bacterial counts in brown rice,black rice and red rice medium were 1.18?1011,1.15?1011 and 7.10?1010 CFU/m L,respectively.The number of viable bacteria in the coix seed culture system is 100-1000 times higher than that in the optimized MRS(Man Rogosa Sharp)dense culture system reported.(2)The differentially expressed genes of L.reuteri in coix seed medium were analyzed by RNA-Seq technology during different growth cycles.The results showed that 1447 differentially expressed genes were identified in LRCS4 vs LRMS4 group(p < 0.05),including 723 significantly up-regulated genes and 724 significantly down-regulated genes;In LRCS16 vs LRMS16 group,1443 differentially expressed genes were identified(p < 0.05),including 539 significantly up-regulated genes and 604 significantly down-regulated genes;In LRCS24 vs LRMS24 group,1374 differentially expressed genes were identified(p < 0.05),including 669 significantly up-regulated genes and 705 significantly down-regulated genes.These up-regulated genes were mainly related to DNA replication,cell division,ribosome synthesis,nucleotide metabolism,amino acid metabolism,energy metabolism and transmembrane transport.(3)Label-free quantitative proteomics technology was used to interpret the differentially expressed proteins of L.reuteri in coix seed medium during different growth cycles.A total of 446 differentially expressed proteins were identified in LRCS4 vs LRMS4 group(p <0.05),including 87 up-regulated proteins and 359 down-regulated proteins;In LRCS16 vs LRMS16 group,583 differentially expressed proteins were identified(p < 0.05),including111 up-regulated proteins and 472 down-regulated proteins;In LRCS24 vs LRMS24 group,511 differentially expressed proteins were identified(p < 0.05),including 98 up-regulated proteins and 413 down-regulated proteins.Among them,the up-regulated proteins were related to DNA replication,recombination and repair,nucleotide metabolism,energy metabolism and amino acid transport.(4)Coix oil(CSO),coix polysaccharide(CPO)and coix protein(CPR)were extracted from coix seed and used as growth promoting factors to confirm the effect of growth promoting on L.reuteri.The results showed that the viable counts of L.reuteri in the medium supplemented with CSO,CPO and CPR were 5.33?1013,1.73?1012 and 9.33?1011 CFU/m L,respectively,which were significantly higher than those in MRS broth(1.47?109 CFU/m L).All the components of coix seed had growth promoting effects on L.reuteri,especially CSO.Moreover,untargeted metabolomics results indicated that 161,159 and 118 differentially metabolites were identified in the medium supplemented with CSO,CPO and CPR,including amino acids,nucleotides,sugars,vitamins and biotin.Among them,the substances related to the growth of L.reuteri were significantly reduced,such as nucleotides,amino acids and peptides.(5)The effects of coix seed juice fermented with L.reuteri on intestinal microbiota and dyslipidemia were investigated in high fat diet-fed mice.The results showed that the coix seed juice fermented with L.reuteri could regulate intestinal microbiota at different levels and reverse the imbalance of intestinal microbiota caused by high-fat diet.In particular,the relative abundance of Muribaculaceae,Lachnospiraceae,Dubosiella and Akkermansia which are negatively correlated with blood lipid levels were significantly increased,while the relative abundance of Desulfovibrionaceae,Ruminococca-ceae_UCG-014,Psychrobacter,and Staphylococcus which have positive correlation with blood lipid levels were significantly decreased. |
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