Nakphaichit M., Plupjeen S., Phumsombat P., Nakayama J., Nitisinprasert S.
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Thailand

Vancomycin-resistant enterococci (VRE) is a major opportunistic pathogen in immunocompromised populations. VRE treatment with antibiotics remains challenging due to the robustness and ability of the pathogen to mutate under harsh conditions. The close relationships between gut microbiota and health and disease have increased interest in the use of probiotics and prebiotics to positively modulate the gut microbiota to prevent or treat infections. Lactococcus lactis KA-FF 1-4 isolated from Thai fermented fish produces a bacteriocin which is tolerant to temperatures of C for 15 min, and stable at pH 3 to 8. The purified bacteriocin has a molecular weight of 4703 Da, and inhibitory activity against a wide range of lactic acid bacterial strains and pathogens, especially VRE.

The in vitro gastrointestinal assay of strain KA-FF 1-4 was performed by following the method described by Ranadheera (2012) with some modification. The stimulated gastric juice was prepared by suspending pepsin in sterile filtered 0.5% (w/v) NaCl solution to a final concentration of 3 g/l, with the pH adjusted to 2.0. The simulated small intestinal juices were prepared by suspending pancreatin in filter sterile 0.5% NaCl (w/v) solution to a final concentration of 1 g/l and adjusting pH to 8.00 with sterile 0.1 mol/l NaOH. The probiotic strain was incubated in gastric juice at C for 1 hour and then was transferred to small intestine juice for 2 hours. To evaluate the efficiency of KA-FF 1-4 in eliminating VRE, the large intestine system was performed under anaerobic batch fermentation. The temperature was maintained at C and pH was controlled between 6.65 and 6.95. The optimum concentration of KA-FF 1-4 against VRE was investigated. In addition, commercial prebiotics including inulin, fibersol (maltodextrin), fructo-oligosaccharide (FOS), and xylo-oligosaccharide (XOS) were applied to improve the inhibition activity in gut model. The inhibition activity was determined by spot on lawn and culture based technique.

The strain KA-FF 1-4 survived 65% (6.03 Log CFU/ml) in human gastrointestinal conditions. A concentration of 108 CFU/ml of KA-FF 1-4 showed the highest activity, decreasing VRE from 104 CFU/ml to zero in 9 h in human gut model. Moreover, the KA-FF 1-4 supplement with fibersol showed the greatest inhibition and improved cell growth. The highest inhibition activity again VRE was determined after 18 hours; higher than the non-prebiotic supplement for 150 times by spot-on-lawn technique. The optimum concentration of fibersol was determined at 2%, and decreased VRE from 104 CFU/ml to zero in 6 hours.

However, probiotic and prebiotics work largely through direct or indirect effects on the gut microbiota in host function. The combination of KA-FF 1-4 and fibersol requires further study for fecal anaerobic batch culture. The molecular base technique as qPCR will be performed for gut microbiota analysis, and bacteriocin production will be analysed by enzyme-linked immunosorbent assay (ELISA). This research will be useful to apply synbiotics for therapeutic products or functional food in the future.

Keywords: Gut Microbiota, Synbiotic, Vancomycin resistance enterococcus, Gut modelling, Probiotic

Nakphaichit M., et al. (2016). Potential use of lactococcus lactis KA-FF 1-4 supplement with non-digestible oligosaccharide against vancomycin-resistant enterococci. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 52.). IPC2016

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