Genomic,physiological And Probiotic Properties Of Streptococcus Thermophilus KLDS 3.1003 For Industrial And Therapeutic Applications

Lactic acid bacteria(LAB)are an important group of microorganisms with multiple applications in the food and agricultural sectors of the world.With the development of omics technology,the molecular basis for which LAB exert beneficial health properties are being explored.Considered as the second most important LAB strain in the world,Streptococcus thermophilus has been conferred with the ‘Generally Recognized As Safe(GRAS)' status in the United States and the ‘Qualified Presumption of Safety QPS)' status in the European Union because its use has not been associated with any known health risks in the food industry over the years.Several strains of Streptococcus thermophilus have also been shown to have a number of in vitro and in vivo probiotic properties ranging from production of a range of short chain fatty acids(SCFAs)to moderately high survivability levels under simulated gastrointestinal tract conditions.However,many of these properties are strainspecific and there is not only the need to study specific strains of interest but to further elucidate,using the latest molecular tools available,the molecular bases for its probiotic propensities.The prevalence of illnesses and mortalities caused by foodborne and vaginal pathogens are a global burden that has warranted research interests.The aim of this doctoral research is to understudy the genomic,physiologic and probiotic properties both in vitro and in vivo of the strain,Streptococcus thermophilus KLDS 3.1003 in order to explore its possible applications in the food and allied industries.The first phase of this research entailed the DNA extraction and bioinformatics of the S.thermophilus KLDS 3.1003 genome.After successful sequencing and deposition of its genome at the National Centre for Biotechnology Information(NCBI),a range of analyses were carried out – evolutionary relationship,circular genome map construction,MG-RAST annotation,comparison with the genome of other notable S.thermophilus genomes,EPS-gene cluster construction,amino-acid coding gene elucidation and predicted structure of some of its gene-coding enzymes involved in carbohydrate fermentation using the SWISS-MODEL analyser.The genome of S.thermophilus KLDS 3.1003 has a 1,899,956 bp chromosome with a G+C content of 38.92%,1,995 genes,and 6 r RNAs.With the exception of S.thermophilus M17TZA496,S.thermophilus KLDS 3.1003 has more t RNAs(amino acid coding genes)compared to all S.thermophilus strains available on the NCBI database.MG-RAST annotation showed that this strain has 317 subsystems with most genes associated with amino acid and carbohydrate metabolism.This strain also has a unique EPS gene cluster containing 23 genes,and may be a mixed dairy starter culture.In depth amino acid coding gene analysis shows that S.thermophilus KLDS 3.1003 can synthesize all twenty(20)essential amino acids required for proper body functioning as well as animal feed formulations.In the second phase,we investigated the antimicrobial properties in vitro of selected strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus on food-borne pathogens Staphylococcus aureus ATCC 25923,Escherichia coli ATCC 25922 and vaginal pathogen Gardnerella vaginalis ATCC 14018.The oxford cup method was used to evaluate the antimicrobial activities of the bacteria and their respective cell-free supernatants in vitro.This method was also used to estimate the effects of catalase,proteinase K,papain and p H on antimicrobial potentials of cell-free supernatants.Lactic and acetic acid production levels of each strain using three different carbon sources(1% glucose,1% sucrose and 1% FOS)were determined by HPLC.Bile and acid tolerance assays were also evaluated.Results show that S.thermophilus LMD-9 and L.bulgaricus KLDS 1.0207 had the highest antimicrobial activities and that catalase treatment had the greatest inhibitory effect on antagonistic capabilities of these strains.There was no significant difference(P<0.05)between the activities of S.thermophilus LMD-9 and S.thermophilus KLDS 3.1003.At p H 3 and after 3 h of incubation,L.bulgaricus KLDS 1.0207 and S.thermophilus SMQ-301 had the highest and lowest tolerance levels respectively L.bulgaricus KLDS 1.0207 and S.thermophilus SMQ-301 produced the highest levels of lactic and acetic acids respectively using the FOS-supplemented media.Over all,we showed in vitro that S.thermophilus KLDS 3.1003 possessed comparably desirable properties as S.thermophilus LMD-9 even though it is not a bacteriocin-producing strain.Finally,the third phase evaluated in vivo the suppressive and immune-modulatory effects of S.thermophilus KLDS 3.1003 and L.bulgaricus KLDS 1.0207 strains against E.coli ATCC 25922 and S.aureus ATCC 25923 pathogens.A total of 40 BALB/c mice were purchased and divided into 10 treatment groups(control and 9 treatments).A concentration of 1 X 108 cfug-1 each of S.thermophilus KLDS 3.1003 and L.bulgaricus KLDS 1.0207 were used in this phase.Animals were given one week to acclimatize and then fed treatment diets for 14 days.Weekly weights were measured and all data collected were analysed using the SPSS v22.0 statistical package.Afterwards,animals were fasted for 16 h,blood samples collected,and various organs harvested for serum/biochemical and histospathological and necropsy analyses respectively.Our results show that groups fed probiotic bacteria had average superior weights than the control group in consonance with earlier and recent studies.The average weight of the TST group was higher than that of the C(Control)and TLB treatment groups with values of 22.96,22.37 and 22.25 g respectively.We also report that there were significant improvements(P<0.05)between the TEC and TSTEC as well as the TSA and TSTSA groups.The TG levels(mmol/L)in the control,TST,TSTLB,TSTEC and TSTSA groups were 2.46,2.41,2.23,1.60 and 2.14 respectively,indicating marked improvements.The HDL levels(mmol/L)in the control,TST,TSTLB,TSTEC and TSTSA groups were 1.92,1.94,1.89,2.15 and 2.04 respectively.A similar improvement trend was also recorded for the L.bulgaricus-fed groups compared to the control.We also observed generally better haematological parameters in probiotic groups than the control.Mice in probiotic treatment groups had better immunity levels(lymphocytes,monocytes,neutrophils,eosinophils)than those in the control and pathogen groups.Histopathological studies showed that no anomalies were associated with S.thermophilus KLDS 3.1003 and L.bulgaricus KLDS 1.0207 administration.In conclusion,we have attempted to establish the molecular basis of the antimicrobial and other potential probiotic properties of the strain,S.thermophilus KLDS 3.1003 through a series of bioinformatics analyses.We have also shown in vitro that this strains is not only a potential probiotic candidate but that it can have high antimicrobial activities against notable foodborne and vaginal pathogens.Finally,we have,in an in vivo study,shown that the administration of S.thermophilus KLDS 3.1003 can increase the weekly weights of experimental animals,improve immunity against E.coli ATCC 25922 and S.aureus ATCC 25923 infections and prevent organ damage.Further studies are thus recommended to validate these findings.