CHARACTERISATION OF LACTIC ACID BACTERIA ISOLATED FROM HUMAN BREAST MILK AND INFANT FAECES AS POTENTIAL PROBIOTICS

Abstract

Infant mortality, particularly in developing countries; remains a concern and one of its major causes is diarrhoea. Human breast milk and faeces contain lactic acid bacteria (LAB), which could have probiotic potential. However, there is a paucity of information on the effect of LAB from faeces of healthy infants and mothers’ breast milk on disease caused by diarrhoeagenic E. coli. This study was undertaken to evaluate the potential of LAB from human breast milk and infant faeces to contribute to the management of infantile diarrhoea. Lactic acid bacteria were isolated from the breast milk of 16 mothers and faeces of 13 infants (healthy volunteers) from Ado-Ekiti, Ekiti State. The LAB strains were identified by partial sequencing of the 16S rRNA gene. The antimicrobial activities of LAB viable cells and Cell-Free Supernatant (CFS) against enterohaemorrhagic, enterotoxigenic, Shiga-toxin producing, enteroinvasive and enteropathogenic E. coli were tested by agar overlay and diffusion methods, respectively. Production of bacteriocin-like substances was evaluated by ammonium sulphate precipitation of CFS. The kill rate of LAB was determined by co culture with diarrhoeagenic E.coli pathotypes. Organic acid production by selected LAB was quantified by HPLC, while the MICs wer/e determined by VITEK®2. The ability of selected strains to withstand simulated gastric conditions (consecutive pH 3 and 0.3% bile) was determined. Autoaggregation, co-aggregation and hydrophobicity abilities of selected strains in n-hexadecane and xylene were assessed by UV spectrophotometry, while anti biofilm effects of1:1, 1:9 and 1:99 dilutions of selected LAB CFS were evaluated by crystal violet assay. The level of IL-6 and IL-10 cytokines in groups of immunosuppressed mice (n=5, 22±4 g) treated with LAB and levamisole hydrochloride (positive control) groups were evaluated in blood and spleen by ELISA as recommended by FAO guidelines. Data were analysed by one-way ANOVA at α0.05. Ninety-three LAB belonging to five genera and 15 species were identified. Lactobacillus (44) was the predominant genus, while L. plantarum (27) was the most abundant species. Inhibition zones of CFS against the tested pathogens ranged from 8.0 to 25.0±1 mm, while viable cell inhibition zones were 12.0 to 20.0±3 mm. Eight LAB strains produced bacteriocin-like substances. Complete inhibition of E.coli strains were observed between iii 16 and 24h. The highest concentration (76.8 mg/mL) of the most prominent organic acid (lactic) was produced by L. rhamnosus A012 (from faeces). L. rhamnosus A012 was susceptible to all tested antibiotics while L. plantarum A011 (from faeces) was resistant to only one of the antibiotics. L.rhamnosus A012 and L. plantarum A011 had the highest resistance to gastric conditions with one log10 reduction in CFU/mL. L. rhamnosus A012 had hydrophobicity of 25.0% in n-hexadecane, 15.0% in xylene, autoaggregation of 32.0% at 5th h and co-aggregation of 6.8%. All dilutions of L. plantarum A011 CFS showed antibiofilm activity. There was a significant decrease in IL-6 and an increase in IL-10 levels in immunosuppressed mice treated with LAB compared to control groups. L. rhamnosus A012 demonstrated antimicrobial activity against diarrhoeagenic E. coli strains and immunomodulatory properties. It is, therefore, a potential probiotic candidate.