The prebiotic effect of Molkosan® on Escherichia. coli and Lactobacillus. planterum growth, pH and antibiotic sensitivity and morphology.

Abstract

Background: Probiotic bacteria like Lactobacillus. plantarum (L. plantarum) can promote a healthy gut through inhibitory effects on Escherichia. coli (E. coli). Molkosan® is a prebiotic drink featuring lactofermented whey and L(+) lactic acid which can improve the gastrointestinal tract environment for growing probiotic gut bacteria. Aims & Objectives: To determine the optimum concentration of Molkosan® for inhibiting and promoting both independent and symbiotic growth of E. coli and L. plantarum respectively. Molkosan® concentration effect on pH, antibiotic sensitivity and morphology was also investigated. Methods: Molkosan® concentrations and a tryptone soya broth (TSB) 0% control was inoculated with E. coli or L. plantarum and spread plated in triplicate on tryptone soya agar (TSA) following serial dilutions with phosphate buffered saline for 24 hour pilot growth and without dilutions for antibiotic sensitivity testing. Independent culture broths and a combined broth featuring both bacteria were spread plated on MacConkey (MAC) and de Man, Rogosa, Sharpe (MRS) agar for selective growth of E. coli and L. plantarum respectively between 0-24 hours where pH strips were used for the combined broth. Independent culture pH was tested at 24 and 48 hours. Gram staining was carried out on colonies grown at various Molkosan® concentrations as described by Delfiner et al (2016) before microscopy at X1000 (oil immersion). Results: Molkosan® concentrations ≥5% increased and decreased growth of L. plantarum and E. coli respectively overall compared with controls (p<0.001). Pilot use of 25% Molkosan® on L. plantarum and E. coli produced a mean growth of Log10(CFU/ml) 10.16 and 4.49 respectively (p<0.001). The 2nd experiment with Molkosan® between 5%- 10% produced greater reduction in independent E. coli growth (Log10(CFU/ml) 12 to 9.34) compared to the combined culture (Log10(CFU/ml) 12.64 to 10.47). The 2nd growth experiment reduced L. plantarum 24 hour growth between 5%-10% Molkosan® where greater reduction was seen in the combined broth (Log10(CFU/ml) 11.12 to 7.23) compared with independent L. plantarum (Log10CFU/ml 11.4 to 8.66) Comparing the combined group with MRS:MAC ratio saw significantly higher growth on MRS agar at 10% Molkosan® (Log10(CFU/ml) 11.12:10.47) (p<0.005). The 3rd experiment displayed differences between the combined culture on MRS and MAC agar at 24 hour growth with 12.5% Molkosan (Log10(CFU/ml) 9.19 and 5.15 respectively) (p<0.001). Over 24 hours the combined culture with 5% Molkosan® slowly increased growth followed by a log phase at 6-24 hours on MRS and MAC. At 12.5% Molkosan® the combined culture broth on MRS showed no difference in growth over time compared to 5% (p>0.05) but MAC increased between 2-4 hours then decreased between 4-24 hours (p<0.001). Between 24-48 hours for Molkosan® concentrations there was no major difference in pH for all bacteria broths. The combined bacteria broth showed no major changes over time but all concentrations ≥5% altered pH to ≤6 up to 24 hours. E. coli remained resistant to Penicillin G, Sulphatriad and Ampicillin while increasing Molkosan® concentration increased sensitivity to all other antibiotics (p<0.05). L. plantarum remained resistant to Penicillin G and Ampicillin whilst reducing sensitivity to other antibiotics at ≥12.5% Molkosan®. Morphological changes were identified through microscopy of combined cultures exposed to Molkosan® concentrations ≥5%. Conclusion: Molkosan® at ≥12.5% displays effective inhibition and stimulation of E. coli and L. plantarum growth independently and symbiotically whilst affecting pH, antibiotic sensitivity and morphology of both bacteria. Keywords: Escherichia. coli, Lactobacillus. plantarum, Molkosan®, Growth, pH, Antibiotic Sensitivity