Karwan Yaseen Kareem (GS34295) Supervisory committee Inhibitory activity of postbiotic produced by Lactobacillus plantarum RS5 using reconstitute media supplemented with different level of Inulin Karwan Yaseen Kareem (GS34295) Supervisory committee Prof. Dr. Loh Teck Chwen Dr. Anjas Asmara Dr. Henny Akit

Overview Introduction Objectives Materials and Methodology Preparation of inoculum Preparation of sample Agar Well Diffusion Assay Results Conclusion

Introduction The practice of feeding livestock with antibiotics has been in use for over fifty years. Antibiotics affect microflora by altering the metabolism of microorganisms, and suppressing microbial growth in the gut (Gadd, 1997) Usage of antibiotics has negative effects on animal’s health and production such as residual in tissues, development of resistance in microorganisms, and harmful effects on human health by development of microbial resistance to specific products (Markovicv, 2005)

Introduction Probiotic, postbiotic, prebiotic and medicinal plants as natural feed additives are recently used in poultry diet to enhance the performance and the immune response of birds Postbiotics are substances which are produced in the final or intermediate stage of metabolic process in Lactic acid bacteria

Introduction Recently, postbiotic have been shown to have many beneficial probiotic effects on animal growth performances and particularly in the gut health when used as additive in animal diet (Thanh et al., 2009; Loh et al., 2010; Thu et al., 2010) Postbiotics also display a wide inhibitory activity against various species of pathogens such as E. coli, S. typhimurium and L. monocytogenes (Savadogo et al., 2006; Gaggìa et al., 2010; Thanh et al., 2010)

Introduction Prebiotics, which includes Inulin, is defined as indigestible carbohydrates that leave a desired effect on the host, by selective growth stimulation or activation of one or more bacteria in a large part of the GI tract (Gibson and Roberfroid, 1995) Fermentation of inulin and FOS leads to a considerable production of organic acids. It is also able to increase acidification of gut contents

Objective To determine the inhibitory activity of the postbiotic production by Lactobacillus plantarum RS5 using reconstitute media supplemented with different level of Inulin

Materials and Methodology

Preparation of inoculum Pipette 100µl into 10ml MRS broth incubate 48 h at 30˚C Thaw the frozen stock culture Subculture Pipette 100µl into 10ml MRS broth incubate 24 h at 30˚C Streak plate and incubate for 48 h at 30˚C Pick colony into 10 ml broth, incubate for 48 h at 30˚C Subculture : Pipette 100µl into 10ml MRS broth incubate 24 h at 30˚C Culture is active

Preparation of inoculum (cont.) If the inhibitory activity of culture achieved 800 AU/ml The culture is used as inoculum after the OD(600nm) adjusted to 1.0 using sterile NaCl Inoculate 1% (v/v) inoculum into the respective reconstituted media supplemented with different level of inulin Subculture & incubate for 24h at 30˚C Check the activity using Agar Well Diffusion Assay Active culture

Production of postbiotic Different levels of Inulin was added & mixed 0.2 % (w/v) 0.4 % (w/v) 0.6 % (w/v) 0.8 % (w/v) 1.0 % (w/v) Reconstituted media for RS5 was prepared Autoclave at 1180C, 15min. Fermented by L. plantarum RS5 Incubate at 300C for 24h Centrifuge at 10,000 xg, 15min. to obtain the postbiotic * The experiment is performed at triplicate

Preparation of sample 1ml culture is transferred Into Eppendorf tube Centrifuge at 10, 000 xg , 15min. Add in 1ml sterile NaCl (0.85%) w/v & suspend Cell pellet Centrifuge at 10,000 xg, 15min. postbiotic (Supernatant) Agar Well Diffusion Assay Remove the NaCl & add in 1ml sterile NaCl Read the OD at 600nm

Agar Well Diffusion Assay Postbiotic Two fold serial dilution of postbiotic, using sterile NaCl 0.85% (w/v) (20-2 -5) Inoculate 20 μL of postbiotic into the prepunched agar plate Let the postbiotic diffuse for 1-2h at room temperature Overlay the agar plates with 3ml of soft agar seeded with 1% (v/v) indicator Incubate at 30 ̊C for 24h The highest dilution factor with the clear zone was measured and the modified bacteriocin activity was calculated based on the formula as below Indicator : Pediococcus acidilactici 4-46 Modified bacteriocin activity (MAU/ml) = * diameter of zone (mm) Volume of postbiotic (mL) The highest dilution factor * Same assay is conducted to test against Escherichia coli, Salmonella enterica, Listeria monocytogenes, at postbiotic volume of 100 μL and Vancomycin Resistant Enterococci (VRE) at 60 μL

Results

Table 1. The modified bacteriocin activity (MAU/ml) of the postbiotic produced after treated with inulin against P. acidilactici, S. enterica, VRE, L.monocytogenes and E. coli Postbiotic Inulin levels (%) MAU/ml P. acidilactici S. enterica VRE L.monocytogenes E. coli RS5 4177.75±88.88a 760.00±0.0d 3466.67±53.33a 1120.00±0.0a 146.66±3.33a 0.2 773.33± 6.66cd 3520.00±0.0a 0.4 786.66±6.66c 1093.33±26.66a 146.66±6.66a 0.6 4266.64±0.0a 3573.0±53.33a 0.8 4444.42±88.88a 813.33±3.33b 153.33±3.33a 1.0 4355.53±88.88a 906.66±6.66a 3626.67±53.33a a-d Means (mean of modified bacteriocin activity ± SEM) in the same column with common superscripts are non- significantly different.

Table 2. Optical density of L. plantarum RS5 and pH of the postbiotic produced by using reconstitute media supplemented with Inulin Postbiotic Inulin level (%) OD pH RS5 2.21 ± 0.005a 3.83 ± 0.005a 0.2 2.22 ± 0.012a 3.83 ± 0.008a 0.4 2.23 ± 0.005a 3.82 ± 0.012a 0.6 2.23 ± 0.006a 3.81 ± 0.005a 0.8 2.23 ± 0.008a 1.0 3.81 ± 0.010a a Means (mean of OD and pH ± SEM) in the same column with common superscripts are non-significantly different.

Conclusion The results of this study show that postbiotics and inulin supplementation enable to inhibit proliferation of pathogenic bacteria The highest MAU/ml was detected when the postbiotic was tested against P. acidilactici, followed by Vancomycin-Resistant Enterococci (VRE), L. monocytogenes, S. enterica and E. coli.

Thank you for your attention

Inhibitory activity against VRE by using 60 and 100 μL Volume of postbiotic (mL) The highest dilution factor (MAU/ml) = * diameter of zone (mm) 100 1000 25 (MAU/ml) = * 14 = 4480 60 1000 25 (MAU/ml) = * 12 = 6400

Inhibitory activity of postbiotics produced by LAB using reconstitute media supplemented with Inulin against pathogens P. acidilactici E. coli Salmonella enterica Vancomycin-Resistant Enterococci (VRE) L. monocytogenese

Justification of study Previous research indicates that there are many cases of food contamination and antibiotic resistance of microbes. Furthermore, there is a possibility of horizontal spread of the resistant genes from bacteria in food animals to commensal strains in the intestinal microflora of humans (Jin et al., 1998). Therefore, it seems essential to find alternative ways to antibiotics as growth promoters. Some studies proposed the use of postbiotic and prebiotics as feed additives. Many studies have explored the effect of postbiotics and prebiotics on growth performance in broiler chicken, while there is a paucity of research on the synergistic effects of the combination of postbiotics and prebiotics on the broiler chickens.