1. Materials and Methodology
INHIBITORY ACTIVITY OF POSTBIOTIC PRODUCED BY LAB USING RECONSTITUTED MEDIA SUPPLEMENTED WITH INULIN
K. Y. Kareem1,5, Loh, T. C.1, 2, Foo, H. L.3, 4 Asmara, A.1, Akit, H.1 and Abdulla. N. R.1,5
1Department of Animal Science, Faculty of Agriculture, University Putra Malaysia, Serdang, Selangor, Malaysia
2Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. 3Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.4Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. 5Department of Animal Resource, University of salah al-din, Erbil, Iraq.
Corresponding
Introduction
The act of feeding antibiotics to livestock has been practiced for over fifty years (Choe et. al., 2013). However, the use of antibiotics has been criticized for having negative impacts on animal production and health as it could have residual effects on tissues long after withdrawal and microbial resistance. The bacteria considered as the main cause for food poisoning are L. monocytogenes, Campylobacter, Salmonella, and pathogenic E. coli. Because of these consequences, there is increasing public awareness and pressure to search for alternatives to antibiotics (McCartney, 2002). Prebiotics, probiotics, postbiotics, and synbiotics are common natural feed additives recently used in poultry industries to promote the immune response and the performance of birds. Postbiotics are substances produced in the final or intermediate stage of metabolic process in Lactic acid bacteria (LAB), while prebiotics are defined as indigestible carbohydrates that leave a desired effect on the host by selective growth stimulation or activation of one or more beneficial bacteria in gut (Gibson and Roberfroid,1995). The objective of this study is to determine the inhibitory activity of postbiotic produced by 6 strains of L. plantarum using reconstituted media supplemented with different levels of inulin (a prebiotic) and to select the best combination based on the modified inhibitory activity against pathogens and an indicator bacterium.
Materials and Methodology
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 1-2hrs at room temperature
Overlay the agar plates with 3ml of soft agar seeded with 1% (v/v) indicator
Incubate at 30 ̊C for 24hrs
The highest dilution factor with the clear zone was measured and the modified bacteriocin activity was calculated based on the formula as below
Agar well diffusion assay
Production of postbiotics
Reconstituted media for RG11,
RG14, RI11, UL4, TL1 and RS5
were prepared
Different level of inulin was added & mixed
0.2 % (w/v), 0.4 % (w/v), 0.6 % (w/v),
0.8 % (w/v) and 1.0 % (w/v)
Autoclave at 1180C, 15min.
Fermented by L. plantarum strains
respectively
Modified bacteriocin activity (MAU/ml) = * diameter of zone (mm)
The highest dilution factor
Incubate at 300C for 24hrs
Volume of postbiotic (mL)
Centrifuge at 10,000 xg, 15min.
to obtain the postbiotics 3
Results 3 1 4 1 4
Table 1. Result modified bacteriocin activity (MAU/ml) score rank of top 10 combinations of postbiotics
produced by using reconstituted media supplemented with inulin against pathogens 2 5 2 5
Trts.
P. Acidilactici
VRE
L. monocytogenese
S. enterica
E. coli
Score4
MAU/mL
Rank3
Rank
P31.I52
±133.33a 1
±88.88a
2240.0±0.00bc 3
433.33±3.33g 7 _ 6
162
P3.I6
7200.0±0.00bc 4
±88.88cd 5
±53.33a
157
P2.I5
±0.00ab 2
±26.66d
193.33±1.66k 12
154
P2.I1
186.66±1.66k 13
153
P3.I1
±133.33c
±88.88bc
±53.33c
380.0±0.00hi 9
152
P3.I4
±88.88f
±53.33b
386.66±3.33f 8
151
P3.I2
6800.0±0.00cde
150
P2.I6
±133.33b
1120.0±0.00de
149
P2.I3
±133.33b
±26.66de
170.0±0.00l 14
148
P4.I5
±0.00d 10
±26.66g
446.66±3.33f 3 1 4 2 5
Figure 1: Inhibitory activity of postbiotic produced by
LAB using reconstituted media supplemented
with inulin against P. acidilactici
Discussion
The inhibitory effect, exhibited the postbiotics and inulin combinations which were observed by the formation of clear and distinct zones around the wells, may be due to the presence of several antimicrobial compounds such as bacteriocins or organic acids (Labioui et. al., 2005). Bacteriocin from L. plantarum is a natural antimicrobial compound capable of inhibiting the growth of pathogens at molecular and cellular levels. fermentation of inulin and FOS leads to a considerable production of organic acids. It is also able to increase acidification of gut contents (Yang et. al., 2009).
a-o Means (mean of modified bacteriocin activity ± SEM) in the same column with common superscripts are non-significantly different. 1P1-P6=different postbiotics (RG11,RG14,RI11,UL4,TL1 and RS5), which were numbered 1,2,3,4,5,6. 2I1-I6=Inulin levels (0, 0.2, 0.4, 0.6, 0.8 and 1%). 3Rank of modified bacteriocin activity against single indicator strain, 4Score is the sum of single indicator score as a subtraction of 36 and rank number (score=36-rank). The treatment with higher score has stronger inhibitory activity against 5 above-mentioned indicator strains. It was arranged in descending order in the column. Trts. (treatments)
Conclusion
The combinations have a stronger inhibitory activity than the postbiotic alone due to the synergistic effect of postbiotic and inulin. The results of this study show that postbiotics and inulin supplementation enable to inhibit proliferation of pathogenic bacteria. Among the 36 treatments, P3.I5, P3.I6, and P2.I5 showed a higher level of modified bacteriocin activity
Acknowledgements
References
This project was supported by Long-Term Research Grant Scheme (LRGS) from Ministry of Education Malaysia.
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Project leader: Prof. Dr. Loh Teck Chwen
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