A BBREVIATIONS
C. Class III
4. Finally, a salient achievement of the study was the application of the primer in AP-PCR based fingerprinting of select anti-listerial bacteriocin producing LAB strains and
5.3. Results and Discussion
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A 2.0 µl aliquot of treated cells was spotted on carbon coated TEM grid (Pacific Asia, USA) and air-dried in laminar hood. Samples of untreated cells were given similar treatment without pediocin CRA51. The treated and untreated cells of Listeria monocytogenes Scott A were examined through a transmission electron microscope (Jeol JEM 2100, Japan) operating at 200 kV.
Results and Discussion Chapter 5
Table 5.1
Sugar fermentation and biochemical tests for anti-listerial LAB isolates
Characteristics Anti-listerial LAB isolatesa,b
DF14 CRA21 CRA51
Acid production from:
Ribose + - -
Lactose - - +
Xylose - - -
Maltose + + +
Fructose + + +
Dextrose + + +
Galactose + - +
Mannose + + +
Raffinose - - -
Rhamnose - - -
Trehalose + + +
Melibiose + - -
Cellobiose + + +
Melezitose - - +
Sucrose + + +
L-Arabinose - - -
D-Arabinose + - -
Sorbose - - -
Inulin + + +
Salicin + + +
Glycerol - - -
Dulcitol - - -
Inositol - - -
Sorbitol - - -
Mannitol + - -
Adonitol - - -
Xylitol - - -
Sodium gluconate - - -
Glucosamine + - +
α-Methyl-D-glucoside - - -
α-Methyl-D-mannoside - - +
ONPG decarboxylase - - -
Esculin hydrolysis + - -
Citrate utilization - - -
Malonate utilization - - -
a Biochemical properties of LAB isolates were determined with HiCarbohydrateTM Kit KB009, HiMedia, Mumbai, India. b DF: dried-fish; CRA: salt-fermented cucumber
The 16S rRNA sequence based phylogenetic analysis for isolates DF14, CRA21 and CRA51 shown in Figure 5.3 reiterate the species level identification of these LAB isolates.
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Figure 5.3 16S rRNA gene sequence based phylogenetic tree constructed for anti-listerial LAB isolates Lactobacillus sp. CRA21 (A), Pediococcus sp. CRA51 (B), and Enterococcus sp. DF14 (C).
Phylogenetic tree was constructed by using neighbour-joining algorithm (www.phlogeny.fr).
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The cell morphology of isolates CRA21, CRA51 and DF14 were ascertained by SEM analysis. The images of SEM analysis are depicted in Figure 5.4. From the figure it is evident that Lactobacillus plantarum CRA21 was rod-shaped (Figure 5.4A). It can also be observed that the typical cocci-shape in case of Pediococcus pentosaceus CRA51 and Enterococcus faecium DF14 was conspicuous (Figure 5.4B and C).
Figure 5.4 Scanning electron micrograph of anti-listerial LAB isolates. (A) Lactobacillus plantarum CRA21; (B) Pediococcus pentosaceus CRA51; (C) Enterococcus faecium DF14. Magnification: 2.5 KX; Voltage: 15 kV.
5.3.2. Bacteriocin purification
Purification of bacteriocin produced by select anti-listerial LAB isolates (DF9, DF14, CRA21, CRA28, CRA51 and CRA61) by cell-adsorption method resulted in appreciable yield and purification fold. Bacteriocin, being a cationic peptide could be readily adsorbed on producer cells at a pH of 6.0 wherein the bacterial cell surface bears a net negative charge.
Subsequently bacteriocin molecule could be selectively desorbed at an acidic pH. An analogous method has been adopted earlier by other research groups (Yang et al., 1992;
Halami et al., 2005). A schematic representation of the principle steps involved in the purification of bacteriocin molecule is depicted in Figure 5.5.
A B C
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Figure 5.5 Schematic representation of cell adsorption based purification of bacteriocin produced by anti-listerial LAB isolate.
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A representative result for the purification of pediocin CRA51 produced by Pediococcus pentosaceus CRA51 is shown in Table 5.2. Bacteriocin activity in the purified sample for the anti-listerial LAB isolates ranged from 102400-204800 AU/ml.
Table 5.2
Summary of pediocin CRA51 purification from Pediococcus pentosaceus CRA51
Tricine SDS-PAGE analysis of purified pediocin produced by Pediococcus pentosaceus CRA51 revealed a single band corresponding to an approximate molecular size of 4.6 kDa (Figure 5.6A), with corresponding biological activity against L. monocytogenes Scott A (Figure 5.6B).
Figure 5.6 (A) Tricine-SDS-PAGE for purified pediocin produced by Pediococcus pentosaceus CRA51; (B) Gel overlay assay of purified pediocin showing anti-listerial activity. Lane M is a low molecular weight marker (Sigma-Aldrich, USA). Arrow ‘1’ indicates purified pediocin and arrow ‘2’
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168 5.3.3. MIC and MKC of purified bacteriocin
The average MIC of purified bacteriocin from 6 anti-listerial LAB isolates varied from 83.3- 266.6 AU/ml whereas MKC ranged from 133.3-533.3 AU/ml (Table 5.3). The varying levels of MIC and MKC observed amongst the LAB strains reflect the differences in the susceptibility of the target strain of Listeria and corroborates with earlier results of colony overlay and agar well diffusion assay (Chapter 4a). On the basis of ANOVA analysis, bacteriocin produced by Enterococcus faecium DF14, Lactobacillus plantarum CRA21, Pediococcus pentosaceus CRA51 and Lactobacillus plantarum CRA52 exhibited significant anti-listerial activity (P<0.05). Results of ANOVA test are elaborated in Appendix (section 2.3). The bacteriocin gene in these isolates were earlier identified as enterocin (DF14), pediocin (CRA51) and plantaricin A (CRA21, CRA52), which are categorized as Class IIa bacteriocins. Compelling anti-listerial activity of Class IIa bacteriocin is a well documented phenomenon (Drider et al., 2006).
Table 5.3
MIC and MKC for purified anti-listerial bacteriocins
LAB strain Bacteriocin Listeria monocytogenes Scott Aa
MIC (AU/ml) MKC (AU/ml)
Lactobacillus plantarum DF9 Plantaricin A 266.6 533.3 Enterococcus faecium DF14 Enterocin A 83.3* 166.6*
Lactobacillus plantarum CRA21 Plantaricin A 83.3* 166.6*
Pediococcus acidilactici CRA28 Pediocin 266.6 533.3
Pediococcus pentosaceus CRA51 Pediocin 83.3* 166.6*
Lactobacillus plantarum CRA61 Plantaricin A 266.6 533.3
a MIC and MKC for L. monocytogenes is represented as an averaged value for triplicate samples.
*Asterisk and boldface denotes significantly higher activity against target strain (P<0.05) compared to other isolates.
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5.3.4. Functional assessment of anti-listerial bacteriocin 5.3.4.1. Flow cytometric analysis
Flow cytometric analysis were carried out to study the effect of bacteriocin treatment on target cells of Listeria monocytogenes Scott A. Flow cytometry has been used earlier by other research groups to study effect of bacteriocin treatment on target cells (Budde et al., 2001) and viability of lactic acid bacteria (Bunthof et al., 2001; Volker et al., 2008). Figure 5.7 shows the distribution of fluorescence intensities for cells of Listeria monocytogenes Scott A after exposure to purified pediocin CRA51 (6400 AU/ml) for 6 h (Figure 5.7, Panel B) compared to control sample without bacteriocin treatment (Figure 5.7, Panel A).
Figure 5.7 Flow cytometric analysis of the effect of pediocin CRA51 on cFDA-SE labelled Listeria monocytogenes Scott A cells. (A) Untreated cells and (B) cells treated with 6400 AU/ml of pediocin CRA51. Data represented as fluorescence histogram of cFDA-SE labelled Listeria monocytogenes Scott A cells. M1: Marker 1; M2: Marker 2
It is quite evident from figure 5.7 that in case of control samples (Panel A) the fluorescence histogram and statistical analysis revealed the presence of an overwhelming population of viable Listeria cells exhibiting strong cFDA fluorescence (M2: 92.6%). Following treatment