Using Caenorhabditis Elegans Model To Assess The Effect Of Streptococcus Salivarius F286 And S.parasanguinis F278 Isolated From Human Breast Milk On Host

Streptococcus salivarius and S.parasanguinis are bacterial species prevalent in human breast milk.In the previous research in our lab,we found Streptococcus salivarius F286 and S.parasanguinis F278 strains in the milk had great potential to colonize infant gut by using human breast milk microbiota-associated mouse model.However,few studies investigated the effects of milk S.salivarius and S.parasanguinis on host health.C.elegans is a convenient model to assess the probiotic potential of bacteria.To study the effects of S.salivarius F286 and S.parasanguinis F278 on the lifespan,progeny and growth of C.elegans.From L4 stage,C.elegans were fed S.salivarius F286,S.parasanguinis F278 and E.coli OP50(standard food for C.elegans),respectively,and the lifespan,body size and progeny were compared among them.Bleached nematodes eggs were placed on the lawn of S.salivarius F286,S.parasanguinis F278,and E.coli OP50,respectively,and the survival rate and body size of 3-day-old worms were compared.Compared with E.coli OP50 feeding,S.salivarius F286 and S.parasanguinis F278 feeding from L4 stage significantly prolonged the mean lifespan of C.elegans to similar extent.Nematodes fed S.parasanguinis F278 from L4 stage,but not those fed S.salivarius F286,showed significantly decreased body sizes compared to counterparts fed OP50.Both Streptococcus strains significantly reduced the brood size of the worms.Three-day-old nematodes hatching on the F286 and F278 lawns had significantly smaller body size than those hatching on OP50,but F286nematodes were significantly bigger in size than F278 worms.The survival rate of 3-day-old nematodes hatching on F286 and F278 lawn was 100%and 97%,respectively.S.salivarius F286 and S.parasanguinis F278 showed similar extent of anti-aging effects in C.elegans,but restricted the growth and progeny of worms,indicating the two breast milk Streptococcus strains extend longevity at least partially via dietary restriction.S.salivarius F286restricted the growth of C.elegans to significantly less extent than S.parasanguinis F278,indicating F286 may have probiotic mechanisms other than dietary restriction for the anti-aging effect.To screen for beneficial bacteria with anti-inflammatory property,the culture supernatant or heat-killed cells of the test bacterial strains are co-cultured with human colon adenocarcinoma HT29 cells stimulated with TNF-?,and assayed is whether the inflammation factors secreted by HT29 cells are reudecd.One premise of this test is that the culture supernatant or heat-killed cells of the bacteria strains kill less than 10%HT29 cells.In our previous work,Streptococcus salivarius F286 and S.parasanguinis F278 were isolated from the human breast milk.Prior to investigating the anti-inflammatory ability of the two strains,we used MTT assay to test the cytotoxicity of the culture supernatants and heat-killed cells of S.salivarius F286 and S.parasanguinis F278 at different concentrations to HT29 cells.Both undiluted and diluted culture supernatants of the two strains had no cytotoxicity on HT29 cells;the cytotoxicity of heat-killed cells of F286 at concentrations of 5×10~5~7.5×10~6 cfu/mL and of heat-killed cells of F278with concentration of 5×10~5~2.5×10~6 cfu/mL on HT29 cells was lower than10%,while 1×10~8 cfu/mL heat-killed cells of F286 and F278 killed 23±5.3%and 22±5.3%of HT29 cells,respectively.Consequently,it is feasible to conduct anti-inflammation assay with undiluted culture supernatants of S.salivarius F286 and S.parasanguinis F278,and 5×10~5~7.5×10~6 cfu/mL heat~killed F286 cells and 5×10~5~2.5×10~6 cfu/mL heat-killed F278 cells in HT29 cell model.