1. 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

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

3. 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)

4. 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

5. 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)

6. 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

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

8. Materials and Methodology

9. 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

10. 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

11. 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

12. 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

13. Agar Well Diffusion Assay
Postbiotic
Two fold serial dilution of postbiotic, using sterile NaCl
0.85% (w/v) ( )
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

14. Results

15. 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
±88.88a
760.00±0.0d
±53.33a
±0.0a
146.66±3.33a
0.2
773.33± 6.66cd
±0.0a
0.4
786.66±6.66c
±26.66a
146.66±6.66a
0.6
±0.0a
3573.0±53.33a
0.8
±88.88a
813.33±3.33b
153.33±3.33a
1.0
±88.88a
906.66±6.66a
±53.33a
a-d Means (mean of modified bacteriocin activity ± SEM) in the same column with common superscripts are non- significantly different.

16. 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.

17. 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.

18. Thank you for your attention

20. 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) = * = 4480 60
1000 25
(MAU/ml) = * = 6400

21. 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

22. 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.