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CHAPTER 7

PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN PRODUCED BY

LACTOBACILLUS PLANTARUM AS1

7.1. Introduction

Bacteriocins produced by lactic acid bacteria (LAB) have received special attention in recent

years due to their potential application in the food industry as natural biopreservatives (Diep &

Nes, 2002). Different bacteriocins have been reported in Lactobacillus plantarum species, which

are included in following classes: Plantaricin C and W, included in class I, plantaricin C19 and

423, included in class IIa, plantaricin EF, JK, S, and NC8, included in class IIb, and plantaricin

1.25B included in class IId (Rojo-Bezares et al., 2007).

Appam or hoppers are a common type of food in South Indian cuisine especially Tamilnadu and

Kerala. Another form of appam is Kallappam which looks like a pan cake. The name originated

from ―Kallu‖ which means toddy (Satish et al., 2010). L. plantarum AS1 was isolated from

Kallappam. It showed effective cheese biopreservative property against Salmonella typhi. L.

plantarum AS1 showed probiotic properties such as bile salt tolerance, gastric juice tolerance,

cholesterol reduction, non-pathogenesis during oral feeding of albino rats for a month period

(Satish et al., 2010). L. plantarum AS1 attached efficiently to HT-29 cells as revealed by

scanning electron microscopy and bacterial adhesion assay. The attachment to human intestinal

cells involved different combinations of carbohydrate and protein factors on the bacteria and

eukaryotic cell surfaces (Satish et al., 2011).

In the present work, antimicrobial substance of L. plantarum AS1 was purified to homogeneity

and it was characterized by various enzymes and temperature sensitivity to determine the nature

of antimicrobial substance.

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7.2. Materials and methods

7.2.1. Strains and chemicals

Lactobacillus plantarum AS1 (Genbank # GQ468312) was isolated from the Kallappam batter

and cultured in MRS broth (Himedia, Mumbai) at 37 °C for 16 h before the study. The indicator

organisms, viz. Vibrio parahaemolyticus 451, Vibrio vulnificus1145, Vibrio fischeri1738, Vibrio

anguillarum, Escherichia coli DH5α, Lactobacillus acidophilus 447, Lactobacillus rhamnosus

1408, Salmonella typhi 734, Listeria monocytogenes 1143 and Proteus vulgaris 426 were

procured from the Microbial Type Culture Collection (MTCC) at the Institute of Microbial

Technology, Chandigarh, India (Table 11). All strains were maintained as frozen stocks at -80

°C. Working cultures were maintained in the agar media and subcultured in liquid media before

use.

The bacteriological media, analytical grade chemicals, and proteolytic enzymes were obtained

from Hi-Media Mumbai, India while molecular weight marker was purchased from Bangalore

Genei, Bangalore, India.

7.2.2. Bacteriocin assay

A well diffusion assay procedure was used for antibacterial assay as described previously (5.2.2).

7.2.3. Purification of plantaricin AS1

Purification of Enterocin MC13 was carried out as described previously (5.2.4)

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7.2.4. Molecular mass determination

Tricine SDS-PAGE: HPLC purified fraction was used to determine the molecular weight of

plantaricin AS1 by Tricine SDS-PAGE (10%) (Schägger & Jagow, 1987). A low molecular

weight protein marker with sizes ranging from 3.0 kDa to 205 kDa (Bangalore Genei, Bangalore,

India) was used. Gel was stained by silver staining method (Morrissey, 1981).

MALDI-TOF Mass Spectrum: The molecular mass of plantarcin AS1 was further confirmed by a

mass-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The

mass of peptide was carried out by the Proteomic facility of Molecular Biophysics Unit at Indian

Institute of Sciences, Bangalore.

7.2.5. Bacteriocin activity in polyacrylamide gel

Tricine SDS-PAGE was run under non-reducing conditions (5.2.6).

7.3.6. Effect of heat and enzyme treatment on plantaricin AS1

Dialyzed samples of plantaricin AS1 were used in these experiments. V. parahaemolyticus was

used as an indicator organism. About 1 ml aliquots of bacteriocin sample were exposed to

temperatures of 50 - 100 °C for 5 to 30 min and tested for antibacterial activity.

Sensitivity to enzymes was determined as described previously (5.2.3).

7.2.7. Antibacterial activity spectrum

Dialyzed bacteriocin sample (semi-crude preparation) was employed for determination of the

antibacterial activity spectrum against indicator strains (Table 11). Bacteriocin assay was

performed as mentioned earlier (5.2.2).

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7.3. Results

7.3.1. Purification of Enterocin MC13

The active fraction of Sephadex G-25 purified sample was lyophilized and dissolved in

minimum volume of Solvent A (0.1% (w/v) trifluoro-acetic acid (TFA) in 5% (v/v) acetonitrile in

water) for HPLC analysis. On injection of SephadexG-25 purified sample to reverse–phase

HPLC, a distinct peak was eluted at 35% of acetonitrile, corresponding to retention time of 19.48

min (Fig. 8). The peak was shown to be active against V. parahaemolyticus.

7.3.2. Molecular mass determination

Tricine SDS-PAGE of the active fraction collected from reverse phase-HPLC analysis yielded a

peptide band of approximately 3.3 kDa (Fig. 9). This result was confirmed by MALDI-TOF

mass spectrum and a sharp peak corresponding to 3.353 kDa was obtained (Fig. 10). So, this

revealed the molecular mass of plantaricin AS1 to be 3.353 kDa.

7.3.3. Bacteriocin activity in polyacrylamide gel

A clear zone in native PAGE was observed, which was corresponding to plantaricin AS1 band

(Fig. 9). This result further validated molecular mass of plantaricin AS1 as 3.353 kDa.

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Fig. 8. HPLC chromatogram of L. plantarum AS1 bacteriocin

A distinct peak of purified bacteriocin was obtained at 19.48 min.

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Fig. 9. Tricine SDS-PAGE of L. plantarum AS1 bacteriocin

A - Bacteriocin activity in native PAGE against Vibrio parahaemolyticus (An activity zone

corresponding approximately to 3.3 kDa bacteriocin band)

B - Purified bacteriocin band of approximately 3.3 kDa

C – Molecular weight marker (3.0 - 205 kDa)

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Fig. 10. MALDI-TOF Mass Spectrometry of L. plantarum AS1 Bacteriocin

A peak of 3.35 kDa molecular mass was obtained when a HPLC purified bacteriocin sample was

subjected to MALDI-TOF mass spectrometry.

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7.3.4. Effect of heat and enzymes on plantaricin AS1

Plantaricin AS1 was found to be heat resistant until the temperature of 100 °C for 30 min (Table

12). Plantaricin AS1 was sensitive to chymotrypsin, trypsin and protease but insensitive to

catalase confirming that inhibition was due to proteinaceous molecule, i.e. bacteriocin and not

due to hydrogen peroxide or diacetyl. Its activity was not reduced by lipase indicating that there

was no structural modification by lipid moiety (Table 12).

7.3.5. Antibacterial spectrum of plantaricin AS1

Inhibition was found against a wide spectrum of bacterial species which includes both gram-

positive as well as gram-negative bacteria (Table 11). Dialyzed bacteriocin sample (semi-crude

preparation) of L. plantarum AS1 was found to be inhibitory against important pathogens of food

and livestock (fish and shrimp): L. monocytogenes, V. parahaemolyticus, V. harveyi, V.

vulnificus, V. anguillarum, V. fisheri, Salmonella typhi, and Proteus vulgaricus.

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Table 11. Antibacterial spectrum of Plantaricin AS1

Indicator bacteria Growth medium Antibacterial activity

Vibrio parahaemolyticus TSA +++

Vibrio vulnificus TSA ++

Vibrio fischeri TSA ++

Vibrio aungullarum TSA +

E.coli DH5 α TSA +

Lactobacillus acidophilus MRS ++

Lactobacillus rhamnosus MRS +

Salmonella typhi TSA +++

Listeria monocytogenes BHI ++

Proteus vulgaris TSA +

+, inhibition zone less than10mm in diameter; ++, inhibition zone 10mm - 20mm in diameter;

+++ inhibition zone larger than 20mm in diameter; -, no inhibition zone recorded.

TSA – Tryptone soy agar; BHI – Brain heart infusion; MRS – de Man, Rogosa and Sharpe agar

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Table 12. Effect of heat and enzymes on plantaricin AS1 activity

+, inhibition zone; –, no inhibition zone

* Bacteriocin activity was compared against that of V. parahaemolyticus

Treatment Bacteriocin activity*

Heat

50 °C for 30 min

60 °C for 30 min

70 °C for 30 min

80 °C for 30 min

90 °C for 30 min

100 °C for 30 min

121°C for 30 min

Enzymes

Trypsin

Catalase

Protease

Lipase

Chymotrypsin

+

+

+

+

+

+

­

-

+

-

+

-

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7.4. Discussion

For the first time, we isolated Lactobacillus plantarum from South Indian fermented food

Kallappam. L. plantarum strains were isolated earlier from various sources such as fermented

milk ‗Amasi‘(Todorov et al., 2007), kefir (Powell et al., 2007), Papaya (Todorov et al., 2011),

Mexican corn ‗Tortilla‘ (Hata et al., 2010), Sorghum beer (Verellen et al., 1998), Koumiss (Xie

et al., 2011), Bulgarian salami (Todorov & Velho, 2008), fermented cucumbers (Atrih et al.,

2001), fermented Italian sausages (Messi et al., 2001), sourdough (Todorov et al., 1999),

fermented cream ‗Jiaoke‘ (Gong et al., 2010), fermented carrot (Andersson et al., 1988),

Pineapple (Kato et al., 1994), Beloura and Chaurico (Todorov et al., 2010), fermented green

olives (Diaz et al., 1993), Munster Cheese (Ennahar et al., 1996), Grass silage (Maldonado et al.,

2003), Algerian fermented olives (Mourad et al., 2005).

L. plantarum AS1 is a potential probiotic strain. Its safety evaluation was performed by feeding

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CFU/ml/day of viable cells to Wistar rats. Also, it was tolerant to bile salt, artificial gastric

juice, and assimilated cholesterol provided in the growth medium (Satish et al., 2010). L.

plantarum AS1 effectively colonized HT-29 cell line and also prevented adhesion of

enteropathogen V. parahaemolyticus to HT-29 cells (Satish et al., 2011). Dialyzed sample (semi-

crude preparation) of L. plantarum AS1 had shown broad spectrum antibacterial activity. It was

inhibitory towards pathogens such as L. monocytogenes, Vibrio parahaemolyticus, V. harveyi,

V. vulnificus, V. anguillarum, V. fischeri, Salmonella typhi, and Proteus vulgaris. Similarly,

Suma et al., (1998), reported a broad spectrum bacteriocin Plantaricin LP84 that showed

inhibitory activity towards Bacillus cereus, E. coli, Staphylococcus aureus, B. licheniformis, B.

subtilis and Pseudomonas aeruginosa. Lash et al., (2005), described a bacteriocin produced by L.

plantarum (ATCC 8014) that showed antibacterial activity towards S. aureus, E. coli, L.

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innocua, P. aeruginosa. Xie et al.,(2011) reported a bacteriocin active against strains of Listeria,

Lactobacillus, Streptococcus, Pediococcus, and Escherichia. Plantaricin 35d produced a L.

plantarum strain showed a wide range of antimicrobial activity including S. aureus, L.

monocytogenes, and Aeromonas hydrophila (Messi et al., 2001). Plantaricin MG produced by L.

plantarum KLDSI 0391 isolated from ‗Jiaoke‘ showed a broad inhibitory activity against Gram-

positive and Gram-negative bacteria including L. monocytogenes, S. aureus, S. typhimurium, and

E. coli (Gong et al., 2010). Valenzuela et al., (2008), described a bacteriocin that inhibited food

poisoning and pathogenic bacteria B. cereus, E. coli, Salmonella enterica. Plantaricin 423

showed inhibitory activity towards B. cereus, Clostridium sporogenes, Listeria spp. and

Staphylococcus spp.(Reenen et al., 1998).

Planataricin AS1 was thermostable as it was active up to 100 °C for 30 min. Also, it was

sensitive to proteolytic enzymes, trypsin, chymotrypsin, protease but not to lipase, catalase

confirming the proteinaceous nature of antibacterial substance. These properties are reported to

be similar in all the bacteriocins of L. plantarum. Plantaricin AS1 was purified to homogeneity

by multiple step purification. MALDI-TOF mass spectrometry analysis confirmed the purity of

the sample and determined the molecular mass to be 3.3 kDa. Previous reports on molecular

mass of bacteriocins of L. plantarum strains did not exactly corresponded to plantaricin AS1

molecular mass. The molecular mass varied among L. plantarum bacteriocins e.g. Bacteriocin

AMA-K was reported to be 2.9 kDa (Todorov et al., 2007), BacST8KF was 3.5 kDa in size

(Powell et al., 2007), Bacteriocin ST16Pa from L. plantarum ST16Pa reported to be 6.5 kDa

(Todorov et al., 2011). Bacteriocin ATCC 8014‘s molecular mass assessed by SDS-PAGE and

reported to be 122 kDa which is highest among L. plantarum bacteriocins (Lash et al., 2005).

Similarly, plantaricin ASM1 was reported to be 5.05 kDa (Hata et al., 2010). Some of the

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bacteriocins were very close in molecular mass to plantaricin AS1 such as plantaricin ST8SH of

3 kDa (Todorov & Velho, 2008), plantaricin C19 of 3.8 kDa size (Atrih et al., 2001),

bacST202Ch of 3.5 kDa mass (Todorov et al., 2010), and plantaricin C and 423 of 3.5 kDa each

(Gonzalez et al., 1994; Reenen et al., 1998).

The combination of antibacterial and probiotic properties of L. plantarum AS1 suggests it to be a

putative human probiotic strain. Nevertheless, further work on identification of bacteriocin genes

and animal trials are mandatory to confirm L. plantarum AS1 to be a human probiont.