Partial purification and characterization of novel cell ...shodhganga.inflibnet.ac.in/bitstream/10603/17837/8/13_chapter 3.pdf · Bacillus spp., B. licheniformis Me1 was selected

Chapter – 3

Partial purification and characterization of novel cell wall

active antibacterial peptide produced by

Bacillus licheniformis Me1

Chapter – 3

Antibacterial peptide of B. licheniformis Me1

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3.1. Abstract

Aim of this study was to characterize the antibacterial peptide (ABP) produced

by native Bacillus cultures isolated from food sources. Among the potent food-grade

Bacillus spp., B. licheniformis Me1 was selected for further studies as it possess

potential broad range of activity against food-borne pathogens and other promising

technological properties, such as stability in wide range of pH and temperature,

resistance to chemicals and organic solvents, and as well as proteinaceous in nature.

The purification, production, characterization and mode of action (MOA) studies of

the ABP of B. licheniformis Me1 were performed. Ammonium sulphate precipitation

and n-butanol extraction was used initially for partial purification of the ABP. The

partially purified ABP was further purified by reverse phase HPLC using C-18

column. The final specific activity of the purified ABP was evaluated to be about

68817 AU/mg and the yield was 0.4%. The apparent molecular weight (MW) of the

ABP as determined using tricine SDS-PAGE was found to be between 3 to 3.4 kDa.

The LCMS analysis of the HPLC purified ABP revealed an m/z value of 279.28 for

single active peptide. The studies, such as chromogenic assay using cell reporter

strains, FTIR and release of UV absorbing materials evidenced that the target of

inhibition of the ABP was on cell wall. Scanning electron microscopy (SEM) analysis

of the pathogens treated with ABP showed complete disintegration of the cells within

30 min of incubation and the minimum inhibitory concentration (MIC) of ABP was

found to be 1600 AU/ml against L. monocytogenes Scott A and 800 AU/ml against

other test organisms (M. luteus, Staph. aureus and Salm. typhimurium). Minimum

bactericidal concentration (MBC) of the ABP for L. monocytogenes was found to be

3200 AU/ml and for M. luteus and Salm. typhimurium was 800 AU/ml, whereas for

Staph. aureus, 1600 AU/ml was required for bactericidal effect. An economical media

was designed for the ABP production which consists of 2% corn steep liquor, 0.25%

NaCl and 0.5% yeast extract and optimum conditions for the production of ABP in

this modified media was; pH 8, temperature 37°C, incubation time 48 h and agitation

at 150 cycles/min. The production yield of ABP by B. licheniformis Me1 in this

economical media was 16 times higher than those obtained in LB broth. The

knowledge of technological properties and the MOA of the ABP of B. licheniformis

Me1 can be useful to determine its effective application as an antimicrobial agent in

food or clinical industry.

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3.2. Introduction

With an increasing incidence of antibiotic resistance in bacteria, screening of

novel antibacterial peptides (ABPs) from new sources, especially from microbes is an

interesting field of study. Furthermore, the increasing demand of bacteriocins for

biopreservation of processed food has also forced the scientific community to search

for novel, potential and safe bacteriocins. Microbes, especially bacteria produce ABPs

in order to compete for the limited space and nutrients present in natural ecological

niches, which is characterized as one among the several strategies utilized by

microbes in order to survive (Riley and Wertz 2002a,b). Thus, the ABP can find

importance in both pharmaceutical and food industries to combat pathogens and

spoilage microorganisms. The ABP, the so-called bacteriocins or bacteriocin like

substance (BLIS) are ribosomally synthesized and are a heterologous group of

proteinaceous compounds that are produced by a number of different bacteria

(Abriouel 2010; Riley and Wertz 2002a,b). The ABPs are divided into several groups

according their molecular mass, secondary and tertiary structures and presence or

absence of disulfide bridges. Based on their chemical structures, stability, and mode

of action, bacteriocins have been classified as: (i) lantibiotics, (ii) small heat-stable

peptides, (iii) large heat labile proteins, and (iv) complex proteins that require

carbohydrate or lipid moieties for activity (Klaenhammer 1993, Nes et al. 2007).

The bacteriocins produced by LAB have been extensively studied, probably

because of the GRAS status of most of the LAB and to their involvement in food

fermentation and processing (Heng et al. 2007; Jack et al. 1995; Kim 1993; O’Sullivan et

al. 2002; Pedersen et al. 2002). The susceptibility for proteolytic degradation in the

gastrointestinal tract of human and animals, suggests that ABP-producing bacteria or

purified ABPs could be used as natural preservatives in food with no harm to the

consumer (Cleveland et al. 2001; Eckner 1992). Like LAB, some representatives of

Bacillus spp. are also considered as GRAS in food industry and agriculture (Durkee 2012;

Sharp et al. 1989) and are known to produce wide array of ABPs that are active against

both spoilage and pathogenic bacteria (Abriouel et al. 2010).

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Production of ABP has been described for several Bacillus spp., such as B.

amyloliquefaciens, B. cereus, B. coagulans, B. licheniformis, etc., (Abriouel et al. 2010;

Stein 2005). B. licheniformis has been applied widely in the fermentation industry for the

production of amylases, proteases, antibiotics, and special chemicals with low risk of

adverse effects to human health or the environment ((US Environmental Protection

Agency (EPA) 1997). Furthermore, bacitracin, the first antimicrobial peptide derived

from cultures of B. licheniformis (Johnson et al. 1945), has been applied widely in the

medical and veterinary area with excellent safety (Logan 1988). Until now, several

peptide antibiotics from different strains of B. licheniformis have been isolated and

studied (Galvez et al. 1993; Korenblum et al. 2005; Mendo et al. 2004).

Many recent findings have shown that most of the ABPs produced by Bacillus

spp. are active against pathogens and can play a possible role in control of food-borne

pathogens (Sharma et al. 2009a,b), as well as in alkaline fermentation (Sarkar et al. 2002;

Terlabie et al. 2006). BLIS such as cerein 7A, 7B, MXRI and 8A from B. cereus have

attracted special attention particularly due to their possible application as natural food

biopreservatives (Bizani and Brandelli 2002; Oscariz et al. 2006; Oscariz and Pisabarro

2000; Sebei et al. 2007). However, reports describing the ABP-producing Bacillus spp.

from food sources and their application in food systems were less recognized, although,

Bacillus spp. from fermented foods have been considered to have additional advantages

(Beaumont 2002). Several factors influence the bacteriocin activity on the target bacterial

cell. They include the structure and amount of the substance, the composition of the

cytoplasmic membrane, the structure and the expression level of a protein with an

immunity function, and the chemical composition of the environment. Thus, the effective

use of ABPs in food preservation requires the understanding of their MOA and inhibitory

action under different biochemical conditions naturally occurring in food (De Vuyst and

Vandamme 1994; O’Sullivan et al. 2002).

Thus, considering the importance of Bacillus spp. and their ABPs in

pharmaceutical and food industry, an attempt was made to partially characterize the

antimicrobial compounds produced by seven identified Bacillus isolates. Among the

seven isolates, B. licheniformis Me1, an isolate from milk, was selected for further

studies. This study included production, purification, characterization and functional

properties of the ABP.

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3.3. Materials and methods

3.3.1. Chemicals, reagents and bacteriological media

Proteolytic enzymes (trypsin, pepsin, proteinase K and papain) were obtained

from SRL, Mumbai, India. Tricine, sodium dodecyl sulphate, tween, acrylamide and

bisacrylamide, were obtained from SRL, India. TEMED, ammonium persulphate,

were obtained from ICN chemicals. For protein precipitation ammonium sulphate was

obtained from Rankem, India.

3.3.2. Bacterial strains, maintenance and analysis parameters

The native Bacillus isolates used in this study included B. subtilis Ec1, B.

thuringiensis Ik15, B. licheniformis Me1, B. cereus Ik11, B. flexus Hk1, B.

megaterium Pk12 and B. amyloliquefaciens Bk1 (isolation and characterization are

described in Chapter 2). The indicator strains used in this study are listed in the Table

3.2. All the selected Bacillus strains and the food-borne pathogenic strains were

maintained and propagated as described in section 2.3.2. All the quantitative analysis

depicted below was done in triplicate and the results were presented as mean value.

3.3.3. Growth of Bacillus spp. and preparation of cell-free supernatant

Erlenmeyer flasks containing 100 ml of LB broth were inoculated with 1% of

overnight (16 ± 2 h) grown Bacillus cultures and incubated for 24 h in an orbital

shaker at 100 cycles/min and 37°C. The culture broth was centrifuged twice at 10,000

g for 20 min in a refrigerated (4ºC) centrifuge (REMI, India) and the cell-free

supernatant (CFS) was collected, and stored at -20ºC, until further use.

3.3.4. Antagonistic activity of the CFS of selected Bacillus spp.

The CFS of the Bacillus strains was assayed for the presence of an

antibacterial substance using agar well diffusion assay (Motta and Brandelli 2002)

with slight modification. Aliquots of BHI soft agar medium (0.8 % agar)

preinoculated with 104 CFU/ml of indicator organism was overlaid on 1.5% NA

plates and wells of uniform diameter (5 mm) were bored in the agar. Aliquots (50 μl)

of the CFS were dispensed in wells and then the plates were incubated for 24 h at

37°C. The minimum inhibitory activity/ residual activity were determined by serial

two-fold dilution method, previously described by Pattnaik et al. (2001). Activity was

defined as the reciprocal of the dilution after the last serial dilution giving a zone of

inhibition and expressed as Activity Unit (AU) per millilitre.

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3.3.5. Characterization of antibacterial compound present in the CFS of the isolates

3.3.5.1. Effect of proteolytic enzymes, heat and pH on antimicrobial activity of the CFS

3.3.5.1.1. Proteolytic enzymes: To study the effect of proteolytic enzymes

(trypsin, pepsin, proteinase K and papain), aliquots of CFS were treated with enzymes

(5 mg/ml) for 3 h at 37ºC and subsequently placed in 100ºC water bath for 5 min to

inactivate the enzymes. An untreated CFS and the enzymes in the buffer alone served

as the control.

3.3.5.1.2. Thermal stability and pH stability: To analyze thermal stability,

aliquots of CFS were exposed to temperatures ranging from 40 to 121ºC for 15 min.

The effect of pH on the CFS was tested by adjusting the pH of CFS to different pH

values (pH 2.0, 4.0, 6.0, 7.0, 8.0 and 10.0), incubating for 2 h at 37ºC and then again

neutralizing to pH 7 with sterile 1 N of NaOH or HCl. The pH stability of CFS was

determined as a measurement of retained activity after incubation at a given pH. An

untreated CFS served as the control.

After each treatment, the samples were tested for antibacterial activity against

the indicator M. luteus ATCC 9341 by agar well diffusion assay as described

previously (section 3.3.4). After incubation for 24 h at 37 ºC, the plates were

examined for clear inhibition zone surrounding each well. The residual activity was

determined as described in section 3.3.4.

3.3.5.2. Activity assay of antibacterial compound present in CFS of selected

isolates using Tricine SDS-PAGE

Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine

SDS-PAGE) was performed by using a 16% polyacrylamide gel and Tris-Tricine

buffer system (Schagger and von Jagow 1987). Reagents and the amount required for

the preparation of the gel are listed in Table 3.1. Anode and cathode buffer

composition are given in Appendix. Electrophoresis was carried out at 50 V initially,

until the sample entered the separating gel and after that, a constant power of 100 V

was applied until the electrophoresis was completed. At the end of the migration, the

gel was removed and assayed for detecting the protein bands with antibacterial

activity as described previously (Cherif et al. 2001). Briefly, the gel was removed

from the apparatus and was fixed for 1 h in 20% isopropanol, 10% acetic acid. The

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gel was then washed with deionised water for 2 h with intermediate steps of changing

the water after every 20 min. After washing, the gel was placed in a sterile Petri dish

and exposed to UV for 20 min for sterilization. Gel was then overlaid with 20 ml of

BHI soft agar seeded with 104 CFU/ml of M. luteus ATCC 9341. The plate was then

kept for incubation at 37ºC for 24 h and examined for zone of inhibition.

Table 3.1. Reagents for preparing the SDS-PAGE gel.

Components Unit Stacking gel (4%) Separating gel (16%)

Acrylamide-bisacrylamide

stock (AB-3/ AB-6)

ml 1 10

Gel Buffer (3×) ml 3 10

Glycerol g - 3

Add water to final volume ml 12 30

APS (10%) μl 90 100

TEMED μl 9 10

Note: AB-3 stock solution: 48 g of acrylamide and 1.5 g of bisacrylamide in 100 ml of water.

AB-6 stock solution: 46.5 g of acrylamide and 3 g of bisacrylamide in 100 ml of water.

3.3.6. The production kinetics of ABP by B. licheniformis Me1

Production of ABP by B. licheniformis Me1 was monitored during the growth

cycle of the organism. The culture B. licheniformis Me1 was aerobically grown in LB broth

as described earlier (section 3.3.3) and the growth of the culture was monitored every 1 h

interval by measuring optical densities (OD) at 600 nm using spectrophotometer

(Schimazdu, Japan) upto 16 h. Simultaneously, an aliquot of 1 ml of the culture was

centrifuged (10,000 g for 20 min) to obtain CFS. The antagonistic activity of the

resulting CFS against M. luteus ATCC 9341 was assayed by the agar-well diffusion

method as discussed earlier (section 3.3.4). The change in the pH of the culture media

was also noted during the incubation period.

3.3.7. Purification of ABP of B. licheniformis Me1

3.3.7.1. Ammonium sulphate precipitation of ABP

The ammonium sulphate precipitation of the ABP of B. licheniformis Me1 was

performed as described by Muriana and Klaenhammer (1991). CFS was collected

from 1000 ml of 24 h grown culture as described in section 3.3.3. A fractionated

precipitation of proteins in the CFS was carried out by the slow addition of

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ammonium sulphate in the solution until 65% saturation was achieved under constant

stirring at 4ºC. The stirring was continued for an additional 30 min and then incubated

for 24 h at 4ºC. The ammonium sulphate precipitate (ASP) collected by centrifugation

at 4ºC and 10000 g for 20 min was resuspended in 100 mM phosphate buffered saline

(PBS) (pH 7) and dialyzed in the same buffer using dialysis membrane (1 kDa MW

cutoff) (Sigma Aldrich, USA).

3.3.7.2. Butanol Extraction

The ASP after dialysis was further extracted with n-butanol. Five ml of n-

butanol was added to the dialyzed ASP. After addition, the mixutre was kept under

stirring for 1 h and then the upper butanol layer was removed after centrifugation. The

step was repeated thrice with addition of fresh butanol. The butanol layer thus

collected was vaporized using Rota evaporator (Buchi, India) and the residue left after

complete removal of butanol (ppABP) was dissolved in 10 ml sterile distilled water

and stored at -20ºC, until further use.

3.3.7.3. RP-HPLC

The purification of n-butanol extracted ABP was further performed by reverse

phase-high performance liquid chromatography (RP-HPLC), μ-Bondapack C18

column (3.9×300 mm column) (Waters, Ireland) with an automated gradient

controller (LC10AT, Shimadzu, Japan). The absorbance at 280 and 220 were

monitored using diode array detector (SPD-M10AVP, Shimadzu Japan). A 20 μl of

ppABP was loaded on to C-18 column and the elution was performed at a flow rate of

1 ml/min. The mobile phases used were solvent A (water and 0.01% TFA) and

Solvent B (100% acetonitrile and 0.1% TFA). The conditions for gradient elution of

solvent B against solvent A were as follows; 0.01 m: 0.3%, 0.01-10 m: 30%, 10-15 m

:40%, 15-30 m:55%, 30-40 m: 75%, 40-45 m: 100%, 45-50 m: 0.3%. All the fractions

were collected and checked for antibacterial activity by spot assay. The active

fractions were lyophilized and stored at -20 °C for further analysis.

Protein concentration of the product obtained at each step of purification was

determined according to BCA kit (Sigma Aldrich, USA) using bovine serum albumin

as standard.

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3.3.8. Characterization of the purified ABP

The antibacterial activity of the ABP obtained at each step of purification

against indicator organism M. luteus ATCC 9341 was determined by agar spot assay,

as described elsewhere (Todorov 2008). Samples of 5 μl of purified ABPs were

spotted on the lawn of the indicator organism, M. luteus ATCC 9341. After spotting,

the plates were kept at 4°C for 1 h, allowing diffusion of the samples in the agar.

Growth inhibition zone of the indicator organism were measured after incubation at 37°C

for 24 h. With this method, antibacterial activity of the partially purified ABP [n-butanol

extracted ABP (ppABP)] was also checked against all the other food-borne pathogens as

listed in Table 3.5. The antimicrobial activity titre (AU/ml) of the ABP was determined

after each step of purification as mentioned in the previous section 3.3.4.

Thermo and pH stability of the ppABP was determined according to the

method as described in section 3.3.5. The effect of different chemicals (Chemical and

working concentrations is shown in Table 3.4) on the activity of the ppABP was

determined by treting the samples of ppABP with chemicals at 1:1 ratio for 2 h at

37°C. After each treatment, the samples were tested for antibacterial activity against

indicator M. luteus ATCC 9341, as described in section 3.3.4.

3.3.9. Tricine SDS-PAGE analysis of the purified ABP of B. licheniformis Me1

Tricine SDS-PAGE analysis of the purified ABP obtained at each step of

purification was done by using the method as described earlier (section 3.3.5.2).

Following electrophoresis, the gel was cut vertically. The first part, containing the

sample and protein standards, was stained with silver nitrate staining solution by

following the standard protocol as described Yan et al. (2000). Briefly, the gel was

kept in fixative solution (50 ml methanol, 12 ml glacial acetic acid, 38 ml distilled

water, and 75 µl formaldehyde) for minimum 2 h or overnight. Following fixation, the

gel was washed with 50% ethanol and 30% ethanol each for 20 min and then with

deionised water. After washing, the gel was kept in sensitizer (Sodium thiosulphate

0.02% w/v) for exactly 60 sec. The gel was washed again three times with deionized

water for 20 sec and the impregnator solution (silver nitrate 0.2% with 50µl

formaldehyde and 100 ml water) was added to the gel, and kept in dark for 30 min.

Following this the gel was washed in deionized water thrice for every 20 sec. After

washing, the developer solution (6% sodium carbonate with 50µl of 37%

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formaldehyde) was added and stirred until bands appeared. Soon after the

visualization of the bands, the stopping solution (50% methanol and 12% acetic acid)

was added and the gel was stored in the same solution. The other part of the gel was

assayed for direct detection of inhibitory activity according to the method illustrated

previously (section 3.3.5.2).

3.3.10. Liquid Chromatography Mass Spectroscopy (LCMS)

The ABP detection was determined by LC/MS using HPLC (Waters, alliance-

2695) with C18 column (Zendo et al. 2008). Liquid Chromatographic conditions were

as described above (section 3.3.7.3). PDA photo diode detector (2996, Waters, UK)

was used for the measurement of absorbance. MS and MS/MS experiments were

performed on a Q-TOF Ultima Global mass spectrometer (Waters, UK). The

conditions of ESI-MS were as follows: ionization mode, positive; desolvation

temperature, 300°C; source temperature, 120°C; capillary voltage, 3.5 kV; cone

voltage, 50 V; cone gas, 50 L/h; desolvation gas, 500 L/h. The total ion

chromatograms were taken in a mass range from m/z 50 to 3000.

3.3.11. MIC and MBC determination

The MIC and MBC of the ppABP produced by B. licheniformis Me1 were

determined as described previously (Riffel et al. 2002). Sterile 96-well microtitre

plates (Tarsons, India) were filled with 100 μl of serially diluted ppABP

(concentrations ranging from 12800 AU/ ml to 50 AU/ml) and then 100 μl of the

freshly grown indicator organisms (~106 CFU/ml suspension), such as L.

monocytogenes Scott A, M. luteus ATCC 9341, Staph. aureus FRI 722 and Salm.

typhimurium MTCC 1251 were added into each well. A positive control was

maintained using sterile NaCl solution. Negative controls were developed with sterile

BHI medium plus the indicator bacteria. Microtitre plates were incubated at 37°C for

24 h, and then the MIC was determined as the last dilution at which no increase in

turbidity (OD600) was observed. For MBC determination, 20μl of sample from each

well was reinoculated in to another microtitre plate having 100 µl of sterile BHI broth.

Controls were also reinoculated. After incubation at 37ºC for 24 h, the turbidity

(OD600) was measured and the last dilutions were there was no growth was considered

as MBC. Three independent experiments were performed and the data are presented

as means ± SEM.

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3.3.12. Dose-response curve

A dose-response curve was determined as described by Sirtori et al. (2008),

using different concentration of ppABP of B. licheniformis Me1 (between 50 AU/ml

and 12800 AU/ml) and an initial inoculum of ~106 CFU/ml of the indicator

organisms. Viable counts were determined after incubation at 37°C for 120 min.

3.3.13. Effect of ABP on the growth of indicator organisms

Active cultures of pathogens (L. monocytogenes Scott A, M. luteus ATCC

9341 and Staph. aureus FRI 722) were obtained in BHI broth at 37°C after 18 h. A

150 μl sample of these overnight grown cultures containing 106 CFU/ml was

inoculated in tubes containing 14.85 ml of BHI broth and incubated at 37°C. After 4 h

of cultivation, ppABP (final concentration 1600 AU/ml for L. monocytogenes Scott A,

800 AU/ml for Staph. aureus FRI 722 and 400 AU/ml for M. luteus ATCC 9341) was

added to cultures which were at the exponential phase of their growth. The turbidity

(OD at 600 nm) and the number of viable cells (CFU/ml) were determined at 2 h

intervals. Pathogens without addition of ABP were also run in parallel and served as

the control. Three independent experiments were performed and the mean value

(±SEM) was calculated.

3.3.14. Effect of ABP on B. cereus spores

Inhibitory effect on spores of B. cereus ATCC 14579 was carried out as

reported by Hyronimus et al. (1998). In a microtitre plate, serial dilution of ppABP

(concentrations ranging from 12800 AU/ ml to 100 AU/ml) of 100 µl in each well

was prepared. The serially diluted ABP solution was inoculated with 100 µl of spore

suspension prepared in BHI broth and incubated for 2 h at 37°C. The viable count

(CFU/ml) and OD600 was determined after 2 h of incubation. Alternatively, 100 µl of

initial spore concentration was spread inoculated on LB agar plates amended with

ppABP at a final concentration of 1600 AU/ml. Spores were also inoculated on LB

agar plates without any ppABP to determine the starting spore concentration. Three

independent experiments were performed and the results indicated as the mean value

(±SEM).

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3.3.15. SEM analysis to determine the effect of ppABP

An overnight culture of each of the indicator organisms L. monocytogenes

Scott A and M. luteus ATCC 9341 was obtained by growing the respective bacteria in

BHI broth at 37°C for 18 h. After incubation, ppABP was added to the culture at a

final concentration of 1600 AU/ml and kept again for incubation at 37°C under

shaking. An untreated sample of the indicator culture served as the control. Samples

were withdrawn at different time intervals (5, 15, 30, 60 and 120 min) during the

incubation period. The treated and non-treated cells collected during the incubation

were processed and analysed, as described in section 2.3.4.1.2.

3.3.16. Release of UV absorbing solutes

The effect of ppABP in the release of UV absorbing material from the ppABP-

treated L. monocytogenes Scott A was studied by the method described by Motta et al.

(2008) for determining the MOA. Cell pellet collected from 24 h old culture of L.

monocytogenes Scott A was suspended in 10 mM phosphate buffer (pH 7) to a

concentration of 0.5 OD at 600 nm and then treated with 1600 AU/ml of the ppABP

for 4 h at 37°C. After incubation, the samples were filtered through 0.22-μm filter

membrane (Millipore, USA). The filtrate was then checked for absorbance at 260 nm

and 280 nm using a UV-visible spectrophotometer (UV 1600; Shimadzu, Japan).

Samples without ABP served as the negative control. For analysis, a blank was made

with appropriate dilution of the ABP. All determinations were done using three

independent experiments and the data are presented as means ± SEM.

3.3.17. Fourier transform infrared (FTIR) spectroscopy

FTIR spectroscopy analysis was carried out to determine the MOA as

described by Motta et al. (2008). The ppABP of B. licheniformis Me1 was added

(1600 AU/ml) to cell suspensions of about 106 CFU/ml of L. monocytogenes Scott A

or M. luteus ATCC 9341 and incubated at 37ºC for 1 h. After incubation, both treated

and control cells were washed three times with sterile 100 mM PBS (pH 7). After

washing, the cells were lyophilized to remove moisture and powdered. The cells were

mixed with finely grounded potassium bromide and the FTIR spectrum was recorded

using a FTIR spectrometer model 5700 (M/S Thermo Electron Corporation, USA).

The curves represent the average of three individual measurements of the same

experiment. The IR spectra were taken in the range of 4000–400 cm–1

. Thirty two

scans were taken with 4 cm–1

resolution. CO2 and H2O correction were incorporated.

Reproducibility of the normalized spectra was ± 2%.

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3.3.18. β-gal induction assay

The induction of β-galactosidase enzyme based on the stress induced by ABP

in different reporter strains were analysed in order to determine the MOA and the kind

of antibacterial substance. Freshly grown reporter strains of B. subtilis (BSF 2470,

TMB 488, TMB 299 and TMB 279) were inoculated into 50 ml of LB broth and

allowed it to grow in LB broth at 37°C, until the growth reached an OD600 of 0.5.

The cultures (2 ml each) were then dispersed in different test tubes and ppABP was

added in to the reporter strains cultures at a concentration ranging from 0.4 mg to 8

mg, as an inducer for β-galactosidase. Cell pellet collected after incubation for 30 min

at 37°C was resuspended in 1 ml of working buffer (20 mM β-mercaptoethanol, 60

mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCL and 1 mM MgSO4, pH 7.0) and

assayed for β- galactosidase activity as described previously, with normalization to

cell density (Miller et al. 1972). Three independent trials were done to calculate the

mean value. Bacitracin and nisin (1 mg/ml) were used as the controls.

3.3.19. Production of ABP by B. licheniformis Me1 in different media and

incubation conditions

Initially, the production of ABP was carried out using different commercially

available media, such as LB, BHI, MRS and LY. The production of ABP in various

cheap food products and by-products of food industry, individually, was also

determined. The cheap food products used as the sole source of nutrients for

production of ABP included grape bagasse (50g/L), soy protein (20g/L), Fish meal

(20g/L), corn steep liquor (CSL, 2%), cheese whey (10%) and soy fibrous residue

(10%). Volumes of 100 ml of each media were prepared in 250 ml Erlenmeyer flasks;

the pH was adjusted to 7 ± 0.5 and sterilized. After autoclaving, each flask was

inoculated with 1% of 18 ± 2 h old culture of B. licheniformis Me1 (106

CFU/ml) and

incubated for 24 h at 37°C. Following incubation, the CFS was collected and

antibacterial activity of the CFS was determined as described above (section 3.3.3).

Furthermore, the production of ABP was also performed by using a complex medium

(consisting of 2 different components namely yeast extract (YE) (0.5%) along with

either CSL (2%) or soy protein (2%) or cheese whey (10%). Finding that the YE +

CSL media gave better production of ABP, the effect of different salts (ammonium

nitrate and NaCl) on the production of ABP in this media (pH 7) incubated at 37°C

was also determined. After determining the better salt for ABP production, the ABP

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production was also checked in media consisting of YE (0.5%), CSL (2%), and various

concentration of NaCl (0.25-1%) and in media consisting of YE (0.5%), CSL (2%) and

glucose (1%). The influence of different incubation temperatures (30, 37 and 44°C),

and pH values (6, 7 and 8) and shaking speed of the orbital shaker (cycles/min) at 37°C

on the ABP production in the complex media consisting of YE (0.5%), CSL (2%) and

NaCl (0.2%) were also determined. The AU/ml of ABP produced during this

production study was determined as described above (section 3.3.3). Three independent

trials were performed and the results are indicated as mean value.

3.4. Results

3.4.1. Antibacterial activity of the CFS of selected Bacillus cultures

After collection of the CFS of the selected culture, the antibacterial activity of

the same was analyzed by well diffusion assay. The CFS of all the isolates showed

inhibitory spectrum against the tested food-borne pathogens as shown in Table 3.2. The

isolate B. licheniformis Me1 and B. subtilis Ec1 showed zone of inhibition against wide

range of pathogens as compared to other cultures and exhibited the highest titre of

antibacterial activity against M. luteus ATCC 9341 (3400 AU/ml) (Table 3.3).

3.4.2. Effect of proteolytic enzymes, heat and pH on antimicrobial activity of CFS

The antagonistic activity of the antibacterial substance present in the CFS of

the cultures was completely abolished by proteinase K, whereas, it was insensitive to

papain (Table 3.3). The antibacterial substance of the cultures Ec1 and Hk1 was

resistant to trypsin, while all other culture’s antibacterial substance were sensitive.

The antibacterial substance in the CFS of all the cultures showed stability at pH

values ranging from pH 4.0–10.0 (Table 3.3). However, the antibacterial substance of

the cultures Bk1, Ec1and Pk12 exhibited reduced activity at high acidic pH. There

was a total loss of antibacterial activity of the cultures Me1, Hk1, Ik11 and Ik15 at pH

below 4. The antibacterial substance of all the cultures was found to be thermally

stable for 15 min at 80°C, although, there was a reduction in residual activity. The

CFS of culture Ec1 exhibited highest stability at all the tested temperatures and even

at an autoclaving temperature of 121ºC for 15 min. The CFS of culture Me1 showed

reduced activity following temperature exposure from 100ºC for 15 min and activity

was completely abolished at 121ºC.

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Table 3.2. Antibacterial activity of the CFS of the identified Bacillus isolates

Pathogens Inhibitory activity of CFS of Bacillus isolates

Bk1 Ec1 Hk1 Ik11 Ik15 Me1 Pk12

L. innocua FB 21 + + + + + + +

L. monocytogenes Scott A - + - + + + +

Staph. aureus FRI 722 + - - - - + -

B. cereus F 4433 + + + - + + -

M. luteus ATCC 9341 + + + + + + +

Salm. typhimurium FB 231 + + - + - + +

Salm. paratyphi FB 254 + + - - - + +

(+) Positive; (-) Negative

Table 3.3. Characterization of antibacterial substance in CFS of the Bacillus cultures

Residual activity of CFS (expressed as AU/ml)

Bacillus isolates

Parameters Bk1 Ec1 Hk1 Ik11 Ik15 Me1 Pk12

Control 800 3200 400 1600 800 3200 1600

Temperature (ºC)

40 800 3200 400 1600 800 3200 1600

60 800 3200 400 1600 800 3200 1600

80 800 3200 400 800 800 3200 800

100 - 3200 - - - 1600 -

121 - 3200 - - - - -

pH

2 400 800 - - - - 800

4 800 1600 - - - 1600 800

6 800 3200 400 1600 800 3200 1600

7 800 3200 400 1600 800 3200 1600

8 800 3200 400 1600 800 3200 1600

10 800 3200 400 1600 800 1600 1600

Enzymes

Trypsin - 3200 400 - - - -

Proteinase K - - - - - - -

Papain 800 3200 400 1600 800 3200 1600

Pepsin - 3200 - 1600 800 3200 1600

*Each value is the mean of three independent experiments.

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3.4.3. Tricine SDS PAGE analysis of antibacterial substance of the seven isolates

The concentrated CFS after lyophilization was used for this study. When the

crude proteins in the CFS were fractionated by Tricine SDS-PAGE and successively

overlaid with LB soft agar containing indicator organism M. luteus ATCC 9341, a

single band exhibiting antibacterial activity was detected for all the seven cultures

(Fig. 3.1). The known bacteriocin with a molecular weight (MW) of 3.4 kDa from B.

subtilis ATCC 6633 was used as control. The protein bands that resolved from the

seven cultures using electrophoresis, showed antibacterial activity and are distributed

over a molecular weight ranges from 3 to 6 kDa.

Figure 3.1. Tricine SDS-PAGE analysis of ABP from the Bacillus cultures. The numbers on the

left indicate the positions of MW markers. Crude proteins in CFS of the strains Hk1

(lane 1), B. subtilis ATCC 6633 (lane 2), Me1 (lane 3), Ec1 (lane 4), Ik11 (lane 5),

Pk12 (lane 6), Bk1 (lane 7) and Ik15 (lane 8) were fractionated by Tricine SDS-

PAGE. B. subtilis ATCC 6633 producing subtilin (3.4 kDa) was used as a control.

Rectangle box indicate the position of the ABPs showing inhibitory activity.

3.4.4. Growth dynamics of B. licheniformis Me1, production of ABP and changes in pH

Growth kinetics was measured over a period of 24 h under shaking conditions

at 37ºC and initial inoculum concentration was 1%. Samples were removed at every 1h

and checked for antibacterial activity. The maximal ABP production (3400 AU/ml) was

recorded after 11 h of aerobic growth in LB broth (Fig. 3.2). Low levels of ABP activity

against M. luteus ATCC 9341 (approximately 400 AU/ml) were recorded initially after

2 h of growth i.e., at the starting of the exponential growth phase and maximum activity

was observed at beginning of the stationary growth phase and thereafter it was static.

Growth curve made at OD600 nm, showed lag, log and stationary phase of the growth.

The pH of the medium changed during growth, from 7.0 to 8.5 (Fig. 3.3).

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Figure 3.2. Kinetics of ABP production by B. licheniformis Me1; () AU/ml of ABP

produced at each hour of incubation, () Optical density of the growing culture

of B. licheniformis Me1. Each value is the mean of three independent

experiments

Figure 3.3. Change in pH during the growth of B. licheniformis Me1 in LB medium at 37°C.

3.4.5. Purification of ABP

The antimicrobial substance produced by B. licheniformis Me1was purified

from the culture supernatant. Partial purification of the ABP of B. licheniformis Me1

from the CFS was done by ammonium sulfate precipitation at 65% saturation,

followed by dialysis to remove salts and impurities. Subsequently the dialyzed

ammonium sulfate precipitate was further extracted with n-butanol. After extraction

with n-butanol, the recovery of ABP was 16%. The butanol extracted ABP was then

subjected to a HPLC μ-Bondapack C18 column in order to obtain a highly purified

ABP. The each absorbance peaks (220 nm) obtained after chromatography were

assayed for antimicrobial activity. The active ABP fraction (Fig. 3.4) was eluted with

42-43% of acetonitrile, showed a specific activity of 68817 AU/mg. This peak was

collected and lyophilized. The purification steps and the recovery values of the

peptide are given in Table 3.4. After HPLC purification, the yield of ABP was 0.4%.

0

500

1000

1500

2000

2500

3000

3500

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

AU

/ML

OD

600

Time (h)

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

8

8.2

8.4

0-4 h 6-10 h 12-16 h

Time (h)

pH

Chapter – 3


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Nithya V.

Table 3.4. Stepwise purification of ABP produced by B. licheniformis Me1

Steps

of purification

Protein

(mg/ml)

AU/ml

Volume

(ml)

Total

protein

(mg/ml)

Total

activity

(AU/ml)

Specific

activity

(AU/mg)

Purification

fold

Yield

(%)

CFS

444.2

3200

1000

444200

3200000

7.20

1

100

ASP

128.4

51200

10

1284

512000

398.75

55.35

16

Butanol extraction

83.0

51200

10

830

512000

616.8

85.63

16

RP HPLC

0.19

12800

1

0.19

12800

68817

9553

0.4

Note: Total protein = Protein (µg) * total volume (ml); Total activity = Activity (AU) * total volume

(ml); Specific activity = Total activity (AU)/total protein (mg); Purification fold = Specific activity of

same sample/Initial specific activity; Yield = [total activity of the sample/Initial total activity] *100.

Figure 3.4. HPLC profile of the n-butanol extracted ABP of B. licheniformis Me1 (Inset:

The arrow indicates the bioactive fraction obtained after chromatography). The

fraction which eluted at 42-43% of ACN showed antibacterial activity.

3.4.6. LCMS analysis

The rechromatography of purified ABP using LCMS gave a single active peak

with maximum absorbance similar to RP-HPLC analysis at 42-43 % of acetonitrile

concentration. This particular peak gave an m/z value of 279.28, detected by mass

spectroscopy (Fig. 3.5).

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Figure 3.5. LCMS analysis of the purified ABP from B. licheniformis Me1: a) total mass

chromatogram b) mass spectrum corresponding to the active peak.

3.4.7. Tricine SDS PAGE of ABP of B. licheniformis Me1

The MW and to certain extent the homogeneity of the ABP at each step of

purification was determined by Tricine SDS-PAGE using 16% gel. Silver staining

was used to detect the protein band and the direct detection of antibacterial activity of

the resolved band was done by bioactivity assay. The SDS PAGE analysis revealed an

active protein band of MW between 3.5 and 6 kDa. The SDS-PAGE gel also revealed

the purity of ABP after each step of purification (Fig. 3.6). As shown in the silver

nitrate stained gel, more impurities were present along with the active peptide after

ammonium sulphate precipitation of the ABP in CFS (Fig 3.6, Lane 2). However, the

butanol extraction removed all other proteins, except the ABP. After HPLC, a single

protein band corresponding to the position of the active band was observed.

RS17-May-201214:56:21GAA082ME 1 HP

Time0.00 10.00 20.00 30.00 40.00 50.00

%

-10

90

0.00 10.00 20.00 30.00 40.00 50.00

AU

0.0

5.0e-2

1.0e-1

NITHYA17051204 2: Diode Array 220

Range: 1.475e-1

50.05

16.67

10.939.72

17.88

NITHYA17051204 1: TOF MS ES+ TIC269

49.9147.93

47.8117.53

17.38

47.70

17.89

50.21

50.28

50.31

ME 1 HP

m/z250 260 270 280 290 300 310 320 330 340 350

%

0

100

NITHYA17051204 1648 (17.529) 1: TOF MS ES+ 81279.28

279.29

a)

b)

Retention time

Re

lati

ve

Inte

nsi

ty (

%)

Re

lati

ve

Inte

nsi

ty

Mass to charge ratio (m/z)

Chapter – 3


138

Nithya V.

Figure 3.6. Tricine-SDS PAGE analysis of the ABP of B. licheniformis Me1 obtained at each

step of purification. Lane 1: Marker, Lane 2: ammonium sulphate precipitated ABP

(dialyzed ASP), Lane 3: butanol extracted ABP (ppABP), Lane 4: HPLC purified

ABP, Lane 5: activity assay against M. luteus ATCC 9341 (All the samples in each

lane showed antibacterial activity in the same position as shown in the lane 5,

arrow indicates the zone of inhibition produced by the ABP.

3.4.8. Antibacterial activity of partially purified ABP of B. licheniformis Me1

The ppABP presented a large spectrum of activity, inhibiting the growth of

many food spoilage bacteria and food-borne pathogens as determined by spot assay

(Table 3.5). As indicated therein, the ppABP exhibited highest inhibitory activity than

CFS against both Gram-positive (B. cereus F 4433, Staph. aureus FRI 722, M. luteus

ATCC 9341, L. monocytogenes Scott A) and Gram-negative bacteria (Salm. paratyphi

FB 254, Salm. typhimurium MTCC 1251 and E. coli CFR 02, Shigella flexineri).

Table 3.5. Inhibitory activity of the ppABP against selected pathogens

Pathogens zone diameter (mm)

L. monocytogenes Scott A 7 ± 2

Staph. aureus FRI 722 9 ± 1.5

B. cereus F 4433 9 ± 1.5

M. luteus ATCC 9341 11 ± 3

Salm. typhimurium FB 231 8 ± 3

Salm. paratyphi FB 254 8 ± 3

Shigella flexineri (clinical isolate) 8 ± 2

E. coli CFR 02 5 ± 1

*Each value is the mean ± SEM of three independent experiments.

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3.4.9. Effect of enzymes, pH, temperature and organic solvents on antibacterial

activity of ppABP of B. licheniformis Me1

The ppABP was completely stable up to 100°C for 15 min, however when

treated at 121°C, residual activity reduced to 75% (Table 3.6). It also showed stability

over a wide range of pH. In the presence of proteolytic enzymes, the ppABP lost its

antibacterial activity as seen with CFS, indicating protein nature of the compound. It

was stable in the presence of organic solvents tested except TCA. The loss of activity

in the presence of TCA (which will precipitate protein) in the supernatant aqueous

phase further specified protein nature (Table 3.6).

Table 3.6. Residual activity of ppABP of B. licheniformis Me1 upon different treatments

Parameter Residual Activity (%)

Control 100

Temperature

60 100

80 100

100 100

121 75

pH

2 100

4 100

6 100

8 100

10 100

Enzymes

Prteinase K (5mg/ml) 0

Trypsin (5mg/ml) 0

Organic Solvents

TCA (10%) 0

DMSO 100

EDTA 100

Urea 100

Tween-20 100

SDS 100

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Nithya V.

3.4.10. MIC and MBC

The indicator organisms were incubated along with different concentration of

ppABP for 24 h in order to determine the MIC and MBC values. The MIC of the ppABP

required to inhibit the growth of pathogen L. monocytogenes Scott A was determined as

1600 AU/ml (Fig. 3.7), whereas, a dose of 3200 AU/ml was needed to achieve a

bactericidal effect (MBC) (Fig. 3.7). The MIC of ppABP for other test organisms M.

luteus ATCC 9341, Staph. aureus FRI 722 and Salm. typhimurium MTCC 1251 was

found to be 800 AU/ml (Fig. 3.7). MBC values of ppABP for M. luteus ATCC 9341 and

Salm. typhimurium MTCC 1251 was found to be 800 AU/ml, whereas for Staph. aureus

FRI 722, 1600 AU/ml was required for bactericidal effect (Fig. 3.8).

Figure 3.7. Minimal inhibitory concentration (MIC) of the ppABP of B. licheniformis on

tested pathogens; L. monocytogenes Scott A (), Staph. aureus FRI 722 (),

M. luteus ATCC 9341 (●), and Salm. typhimurium MTCC 1251(▲). Each point

is the mean ± SEM of three independent experiments.

0

0.2

0.4

0.6

0.8

1

50 100 200 400 800 1600 3400 6400 12800

OD

60

0

AU/ml (ABP concentration)

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141

Nithya V.

Figure 3.8. Minimal bactericidal concentration (MBC) of the ppABP of B. licheniformis

Me1 on tested pathogens; L. monocytogenes Scott A (), Staph. aureus FRI

722 (), M. luteus ATCC 9341 (●), and Salm. typhimurium MTCC 1251

(▲).Each point is the mean ± SEM of three independent experiments.

3.4.11. Dose response curve

The effect of different concentration (50 to 12800 AU/ml) of the ppABP of B.

licheniformis Me1 on the survival of L. monocytogenes Scott A, M. luteus ATCC

9341, Staph. aureus FRI 722 and Salm. typhimurium MTCC 1251 is shown in Figure

3.9. The number of viable cells reduced with increase in concentration of the ppABP.

Complete lysis of viable cells of pathogen was occurred at different concentrations of

ppABP for different pathogens. The complete growth inhibition was observed at 6400

AU/ml for L. monocytogenes Scott A, and at 3200 AU/ml for Staph. aureus FRI 722

and Salm. typhimurium MTCC 1251 after 2 h incubation with the ppABP. The

concentration of ppABP required to completely inhibit the growth of M. luteus ATCC

9341 was found to be 800 AU/ml. The EC50 which is considered as the concentration

that caused half-reduction of viable counts was around 1600, 800, 1600 and 100

AU/ml for L. monocytogenes Scott A, Staph. aureus FRI 722, Salm. typhimurium

MTCC 1251 and M. luteus ATCC 9341, respectively.

0

0.2

0.4

0.6

0.8

1

0 100 200 400 800 1600 3200 6400 12800

OD

60

0


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142

Nithya V.

Figure 3.9. The viable count of pathogens incubated at different concentrations of the

ppABP of B. licheniformis after 2 h of incubation; L. monocytogenes Scott A

(), Staph. aureus FRI 722 (), M. luteus ATCC 9341 (●), and Salm.

typhimurium MTCC 1251 (▲). Each point is the mean ± SEM of three

independent experiments.

3.4.12. Effect of ABP on the growth kinetics of L. monocytogenes

The effect of the ppABP on the growth kinetics of L. monocytogenes Scott A

is shown in Figure 3.10. The ppABP at a final concentration of 1600 AU/ml added to

a 4 h-old grown culture of L. monocytogenes Scott A resulted in the rapid reduction of

the viable counts compared to the control within 30 min of the incubation period.

Bacteristatic effect was observed against the growth of pathogen, as the growth

remained constant after 30 min of addition of ppABP.

3.4.13. Effect of ABP on the growth of Staph. aureus FRI 722

Similar to L. monocytogenes Scott A, addition of 800 AU/ml ppABP (as a

final concentration) to a 4 h-old culture (early exponential phase) repressed the cell

growth of Staph. aureus FRI 722 within 30 min (Fig. 3.11). The inhibition of growth

of Staph. aureus FRI 722 by the addition of ppABP resulted in a decrease in OD600

during the incubation, suggesting that cell lyses is triggered in Staph. aureus FRI 722

after cell death and the ABP has bacteriolytic effect.

0

2

4

6

8

10

12

0 50 100 200 400 800 1600 3200 6400 12800

Log

(CFU

/ml)


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143

Nithya V.

Figure 3.10. Effect of ppABP on the growth kinetics of L. monocytogenes Scott A. 1600

AU/ml of ppABP was added to the growing culture at exponential phase. CFU/

ml of control sample without ppABP () and with ppABP (). Dotted and

Cross lined bar graph bar graph represents the OD of the control (C. OD) and

the tested pathogens with added ABP (T. OD), respectively. Each point is the

mean ± SEM of three independent experiments.

Figure 3.11. Effect of ppABP on the growth kinetics of Staph. aureus FRI 722. 800 AU/ml

of ppABP was added to the growing culture at exponential phase. CFU/ ml of

control sample without ppABP () and with ppABP (). Dotted bar graph is

the OD values of the control (C. OD), Cross lined bar graph is OD values of

test sample with added ppABP (T. OD). Each point is the mean ± SEM of three


0

0.2

0.4

0.6

0.8

1

0

2

4

6

8

10

12

0 2 4 4.5 6 8 10 12

OD

60

0

log

(CFU

/ml)

Time (h)

C.OD T.OD

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0

2

4

6

8

10

12

14

0 2 4 4.5 6 8 10 12

OD

60

0

log

(CFU

/ml)

Time (h)

C. OD T. OD

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144

Nithya V.

3.4.14. Effect of ABP on the growth of M. luteus ATCC 9341

The effect of ABP on the growth kinetics of M. luteus ATCC 9341 is shown in

Figure 3.12. The ppABP added at a concentration of 400 AU/ml to exponential phase

grown culture of M. luteus ATCC 9341 and resulted in a decrease of viable counts within

30 min of incubation. The bacteriolytic effect was observed as the complete lysis of

viable cells of M. luteus ATCC 9341 was observed after incubation with ppABP.

Figure 3.12. Effect of ppABP on the growth kinetics of M. luteus ATCC 9341. 400 AU/ml

of ppABP was added to the growing culture at exponential phase. CFU/ ml of

control sample without ppABP () and with ppABP (). Dotted bar graph is

the OD values of the control (C. OD), Cross lined bar graph is OD values of

test sample with added ppABP (T. OD). Each point is the mean ± SEM of three


3.4.15. Effect of ABP on Bacillus cereus F 4433 spores

The addition of bacteriocin at different concentration to a spore suspension of

B. cereu F 4433 for 2 h caused a large decrease in the number of spores (Fig. 3.13).

The viable count reduced to 4 log, at 400 AU/ml, and the count was decreased with

further increase in concentration of ppABP. The complete reduction in OD value was

observed at 1600 AU/ml, this indicates the effectiveness of ppABP for the inhibition

of B. cereus F 4433 spores. When the spores were placed along with a 1600 AU/ml of

ppABP in BHI, no colonies were grown even after 72 h of incubation at 37°C.

0

0.1

0.2

0.3

0.4

0.5

0.6

0

2

4

6

8

10

0 2 3 4 4.5 5 5.5 6 6.5

OD

60

0

log

(CFU

/ml)

Time (h)

C. OD T. OD

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145

Nithya V.

Figure 3.13. Effect of increasing concentration of ppABP on the survival of B. cereus F

4433 spores. The viable cell count () and OD600 (bar graph) in the presence of

serially diluted ppABP after 2 h of incubation. Each point is the mean ± SEM

of three independent experiments.

3.4.16. SEM analysis

This study was carried out to determine the effect of ABP on the cell

morphology of indicator organisms. The indicator microorganisms (L. monocytogenes

and M. luteus) harvested from an early-stationary-phase culture (106

CFU/ml) were

incubated with 1600 AU /ml of ppABP. The cell pellets were collected at 5, 10, 15, 30

and 60 min of incubation. These pellets were processed for SEM and observed for

distortion in cell morphology. In both case of pathogens, the cell lysis was observed

from 5th

min of incubation onwards. As shown in Figure 3.14, the cell integrity was

lost and complete lysis was observed within 15 min.

0

0.1

0.2

0.3

0.4

0.5

0.6

0

2

4

6

8

10

0 100 200 400 800 1600 3200 6400 12800

OD

60

0

log

(CFU

/ml)


Chapter – 3


146

Nithya V.

Figure 3.14. SEM images of the cells of pathogen treated with ppABP for 1 h, at 20 K X

amplification; (a) L. monocytogenes Scott A and (b) M. luteus ATCC 9341.

3.4.17. Fourier trasform infrared (FTIR) spectroscopy

The FTIR spectroscopy was used as an additional tool to evaluate the putative

MOA of the ABP from B. licheniformis Me1. Treated cells of L. monocytogenes Scott

A showed an important increase in the frequency of absorbance was observed at 2960

cm–1

to 2850 cm–1

indicating C-H anti-symmetric and symmetric structural vibration

of the lipid acyl chains. The FTIR spectra of both L. monocytogenes Scott A and M.

luteus ATCC 9341 treated with ppABP showed an increase in intensity between

1399-1234 cm–1

(frequency absorbed by C-H bending and CH3 stretch in fatty acids

and C-N stretching), 1660-1535 (NH2 bending, C=O, C=N stretching (amide I and

II), 1450 (C–H deformation in aliphatics), 1398-1390 (C–H bending, –CH3 stretching

(fatty acids) were observed indicating the change in the cell wall components in cells

treated with ppABP compared with difference in absorbance of untreated cells (Fig.

(b)

(a)

Chapter – 3


147

Nithya V.

3.15). An absorbance increase observed between 1440-1380 and 1240- 1068

corresponding to the assignments of fatty acid and phospholipids. The increase in the

intensity of absorbance was more for the cell pellet of M. luteus ATCC 9341 rather

than L. monocytogenes Scott A treated with ppABP.

Figure 3.15. FTIR spectra of indicator organism treated with ppABP; a) L. monocytogenes

Scott A, b) M. luteus ATCC 9341; control (Red colored line), without ppABP

and the test, with ppABP (Blue colored line).

Wave numbers (cm-1)

(b)

Abs (

%)

0.02 0.04

0.06

0.08

0.10 0.12

0.14

0.16

0.18

0.20 0.22

0.24 0.26

0.28 0.30

0.32

500 1000 1500 2000 2500 3000 3500 4000

Wave numbers (cm-1)

(a)

Abs (

%)

0.65

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

500 1000 1500 2000 2500 3000 3500 4000

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148

Nithya V.

3.4.18. Release of intracellular materials

The effect of ABP on the integrity of the cell membranes of L. monocytogenes

Scott A was evaluated by quantifying the released UV-absorbing molecules from

ABP-treated cells using spectrophotometer. Treatment of L. monocytogenes cells with

1600 AU/ml of ppABP caused leakage of UV-absorbing materials measured at both

260 and 280 nm (Table 3.7).

Table 3.7. Release of intracellular UV-absorbing materials of L. monocytogenes Scott A

Parameters

Untreated cells

ppABP- treated cells

Nucleic acids (A260 nm)

0.016 ± 0.005

0.288 ± 0.033

Proteins (A280 nm)

0.033 ± 0.011

0.410 ± 0.013

*Each value is the mean ± SEM of three independent experiments.

3.4.19. Detection of specific mode of action of ppABP using reporter strains

Most of the antibacterial substances produced by Gram-positive bacteria have

a target of inhibition on cell. Reporter strains, such as B. subtilis (BSF 2470 and TMB

488) have a promoter which gets induced by cell wall acting, lipid II binding

antibacterial substance. Among these, the reporter strain, BSF 2470 is generally

induced by some of other chemical substances, such as SDS, ethanol etc., nevertheless

it can also be used for detection of cell wall stress causing antibacterial substances.

Whereas, the induction of β-galactosidase activity of the reporter strain TMB 488

specifically differentiate cell-wall acting antibacterial substance. The ppABP of Me1

was found to induce both reporter strains, BSF 2470 and TMB 488. The maximum

induction of BSF 2470 β-gal was observed at a concentration of 2 mg/ml of ppABP.

In case of TMB 488, maximum induction was given by ppABP at a concentration of 8

mg/ml (27 Miller units) (Fig. 3.16). This indicates that the ABP produced by Me1 is a

cell wall acting compound. Further, the induction of lacZ by ppABP in two other

reporter strains TMB 299 and TMB 279 were determined, in which TMB 299

specifically gets induced by cationic membrane acting lantibiotics whereas, TMB 279

is a specific reporter strain for determination antibacterial peptide, bacitracin. The

ppABP could only induce TMB 299, but not TMB 279 (Fig. 3.16), indicating the

ABP produced by B. licheniformis Me1 is not a bacitracin, but it is a cell wall acting

cationic lantibiotic similar to nisin or subtilin.

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149

Nithya V.

Figure. 3.16. β-galactosidase activity of various reporter strains when induced with different

concentrations of ppABP of B. licheniformis Me1 (expressed in Miller units).

Each point is the mean ± SEM of three independent experiments.

3.4.20. Production of ABP

The production yield of ABP was calculated and compared in terms of the

ABP titre (AU/ml) obtained at each condition of production and the results obtained

are shown in Table 3.8 and Table 3.9. In order to search an optimum media and

growth conditions for getting a higher yield of ABP from B. licheniformis Me1,

initially production study was carried out in different commercial media and then in

different food industry by-products at a particular growth conditions (pH 7,

temperature 37°C and agitation speed 150 cycles/min). Maximum ABP production

was observed in LB and BHI broth (3200 AU/ml) as compared to MRS broth (400

AU/ml). The different food industry products and by products used as a source of

nutrient for growth and production of ABP are listed in the Table 3.8. It was observed

that the CSL (2%), which is a by-product from corn wet milling, induced the

production of ABP, and resulted in high AU/ml (6400) within 24 h of incubation. The

addition of YE (0.5%) in CSL increased the activity 12800 AU/ml, suggesting that

CSL+YE media can be used for maximum production of ABP. After selecting a better

media for ABP production, the effect of salts, glucose, pH, temperature and agitation

speed on the production of ABP was also determined.

-5

0

5

10

15

20

25

30

35

BSF 2470 TMB 488 TMB 299 TMB 279

Reporter strains

Mill

er u

nit

s

0 mg

0.4 mg

2 mg

4 mg

8 mg

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Nithya V.

The addition of NaCl increased the stability of ABP during the incubation

time, while a reduction in the AU/ml was observed when ammonium nitrate was

supplemented in the modified media (YE + CSL). The addition of glucose to the

formulated media (CSL + YE + NaCl) inversely affected the production of ABP. The

ABP production was reduced to as low as 100 AU/ml. However, the turbidity was

more in this media, indicating that glucose might have enhanced the growth of the

culture alone. The difference in agitation had a significant effect on the production of

ABP by B. licheniformis Me1 grown in formulated media at 37°C. The optimum

temperature for ABP production was found to be 37°C in the formulated media,

where AU/ml of CFS was noted to be 12800 (Table 3.9). When tested at different pH

(6, 7 and 8) at 37°C, the maximum production was obtained at pH 8, with an activity

titre value of 51200 AU/ml. Hence, the optimum conditions for the production of

ABP using formulated media (CSL + YE + NaCl) as a substrate was found to be

temperature 37°C, pH 8, agitation speed of 150 cycles/min and incubation time 24 h.

Table 3.8. The production of ABP by B. licheniformis Me1 in different media at pH

7, 37°C, and agitation at a rate of 150 cycles/min

*Values are the mean of three independent experiments.

Media used AU/ml

MRS 400

LY 1000

BHI 3200

LB 3200

NB 1800

Grape Bargasse -

Fish meal -

Cheese whey 100

Cheese whey + yeast extract (YE) -

Soy fibrous residue -

Soy protein 800

Soy protein + yeast extract 2800

Corn steep liquor (CSL) 3200

CSL (2%) + YE (0.5%) 5600

CSL + YE+ NH4NO3 (1%) 6400

CSL + YE + NaCl (1%) 6400

CSL + YE + NaCl (0.5%) 7200

CSL + YE + NaCl (0.25%) 12800

CSL + YE + glucose (1%) 400

CSL + YE+ glucose (1%) + NaCl (0.25%) 100

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Nithya V.

Table 3.9. The production of ABP by B. licheniformis Me1 grown in formulated

media at different growth conditions.

Growth parameters AU/ml

Agitation speed (cycles/min) (Temp 37°C, pH 7, 24 h)

static 6400

50 9600

150 12800

Temperature (pH 7, agitation- 150 cycles/min, 24 h)

27 800

30 5600

37 12800

44

3200

pH (Temp 37°C, agitation- 150 cycles/min, 24 h)

6 9600

7 12800

8

51200

Incubation time (Temp 37°C, pH 8, agitation- 150 cycles/min)

24 51200

48 32000

72 9600

*Values are the mean of three independent experiments.

3.5. Discussion

In the last few years, a variety of bacteriocins such as those of Gram-positive

bacteria have attracted attention for their potential use in many fields, including

biopreservation (Galvez et al. 2007). However, much of the study has been conducted for

the bacteriocins from LAB, although members of the genus Bacillus are known to

produce a wide range of antimicrobial substances, including peptide and lipopeptide

antibiotics (Abriouel et al. 2010; Stein 2005). The identification and characterization of

antibacterial substances, and exploring their potential use in the biological control of

pathogenic and spoilage microorganisms, addresses a very important aspect of food

protection. Very few reports are available on bacteriocins from Bacillus species.

Furthermore, for potential application in food, it is very important to know the effect and

MOA of the novel antimicrobial compounds on different pathogens. It is also known that

isolates from food source with ability to produce ABP can find better applications in food

systems as biopreservatives. Thus, in this section, the ABP produced by one of the potent

isolate B. licheniformis Me1 was characterized and the MOA, production yield and effect

of ABP on the growth of different pathogens was carried out.

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To check whether the antibacterial substance produced by seven potent

Bacillus isolates is extracellular or intracellular, further agar well diffusion assay was

performed by evaluating the antimicrobial activity of the CFS of the seven selected

isolates. The CFS of all the tested cultures showed similar inhibitory activity against a

wide range of pathogens as shown for streak assay (section 2.4.2.3). The inhibition of

pathogens as evidenced by the well-diffusion test showed that the inhibitory

metabolites produced by these test cultures were extracellular and diffusible, as the

test was conducted via diffusion through a layer of agar. Similar observations were

reported elsewhere for bacteriocin producing Bacillus strains (Cherif et al. 2001;

Kayalvizhi and Gunasekaran 2008). Mah et al. (2001) also reported a B. brevis strain

isolated from kimchi producing a bacteriocin which is active on a broad spectrum of

pathogens and food spoilage bacterial and yeast strains.

The inhibitory effect of a bacterial cultures can be due to the production of

H2O2, organic acids, diacetyl, ammonia, bacteriocins, etc. (Jacobsen et al. 1999;

Olaoye et al. 2008). Thus, preliminary characterization of antibacterial substance was

carried out after exposure of the CFS of the cultures to different temperature, pH and

enzymes. The antibacterial activity of the antibacterial substance was lost upon

incubation of the CFS with proteolytic enzymes, such as protienase K, implying the

presence of an essential protein or peptide moiety. However, some cultures’ CFS were

resistant to papain and trypsin, and this is agreeable with the fact that some Bacillus

produce antimicrobials which are cyclic peptides containing unusual amino acids,

rendering them resistant to proteolytic treatment (von Dohren 1995). The activity of

the ABP of isolates was pH dependent and was highly maintained in the wide range

of pH (4.0-10.0). This is of great advantage from application point of view because

pH of many foods varies from very acidic to alkaline. With respect to this, it is

interesting to note that nisin, the only bacteriocin so far employed commercially as a

food additive acts in acidic conditions and it is practically not soluble at pH 8.0

(Schillinger et.al. 1996). The antbacterial peptides of the Bacillus cultures were found

to be thermally stable for 15 min at 80°C, except for the culture Ec1. A similar result

was reported for thuricin 7 produced by B. thuringiensis BMG1.7 (Cherif et al. 2001).

The interesting feature of heat stability of ABP of Ec1 and Me1 supports the fact that

they can be potentially used as a food additive in processes like pasteurization, drying,

refrigeration and freezing.

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Nithya V.

Bioactivity assay of the crude CFS of the 7 Bacillus spp. on SDS-PAGE

suggested that inhibitory activity was due to a low molecular weight peptide (ranging

from 3.0 to 6.0 kDa). The relative mobility of the seven isolates’ ABPs and the control

bacteriocin subtilin (lane2; MW 3.34 kDa) were different and distributed over a MW

ranging from 3 to 6 kDa. To the best of knowledge, there are no reports on the ABP

from B. flexus. The isolate B. flexus Hk1 (lane 1) produces an ABP of MW >4 kDa,

exhibiting stability over a wide range of temperature and pH. The MW of ABP

produced by B. licheniformis Me1 (lane 3) was less than subtilin and exhibited wide

range of inhibitory spectrum, even against Gram-negative bacteria. The MW of ABP

(lane 4) produced by B. subtilis Ec1 is less than that of subtilin and thus it may be ericin

A of MW 2.98 (Stein et al. 2002a). Also, this is the first report on ABP producing- B.

subtilis isolate from fermented food chhurpi. The culture B. cereus Ik11 (lane 5)

produces an ABP of MW greater than subtilin and is 5 kDa. Oscariz et al. (2006) has

also reported bacteriocin from B. cereus, cerein 7B having a MW of 4.89 kDa. Khalil et

al. (2009a) reported bacteriocin from B. megaterium which differs from B. megaterium

Pk12 ABP (lane 6) not only in MW but also in their other characteristics, specially the

stability in alkaline pH. B. thuringiensis is a well known producer of a number of

bacteriocins of MW < 3.4 kDa for eg, thuricin (Cherif et al. 2001). Similarly, the ABP

produced by B. thuringiensis Ik15 is also of MW <3.4 kDa. B. amyloliquefaciens Bk1

(lane7) produces ABP of MW higher than subtilin. Lisboa et al. (2006) reported

bacteriocin of 5 kDa from B. amyloliquefaciens isolated from Atlantic forest.

The inhibition zone as observed by bioassay of tricine SDS-PAGE gel gives a

rough estimation of the MW of the potential peptide (s), future experiments in relation

to MW determination and uniqueness of the ABP produced by one of the selected

isolate B. licheniformis Me1 was carried out. The heat resistance, sensitivity to

proteases, and low MW of the ABP of the cultures shows that these ABPs belong to

group I bacteriocins (Chen and Hoover 2003; Klaenhammer 1993).

The results of the antimicrobial spectrum and technological properties of the

antibacterial substances of the cultures, such as stability in different temperature and

pH conditions shows that cultures, B.subtilis EC1 and B. licheniformis Me1 were the

most promising candidates for further studies. However, there a number of reports on

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Nithya V.

the antimicrobial compounds from B. subtilis (Abriouel et al. 2010). Moreover, the

SDS-PAGE analysis showed that the apparent MW of the ABP produced by B.

subtilis Ec1 is similar to subtilin. Hence, the culture, B. licheniformis Me1 was

selected for further studies including production, purification, characterization of its

ABP. No much reports are available about the application of this culture as

biopreservative. Moreover, B. licheniformis has Generally Recognized as Safe

(GRAS) status of in food industry and agriculture (Sharp et al. 1989).

The ABP present in the CFS of B. licheniformis Me1 was purified using a

scheme of purification steps. The ABP was purified by precipitation with 60%

ammonium sulphate, followed by n-butanol extraction. These methods served to be

optimum for recovery of ABP. Previously n-butanol has been successfully used for

extraction of various bacteriocins (Bizani et al. 2005b; Messi et al. 2001; Tamehiro et

al. 2002). Reverse phase HPLC using C18 column allowed final purification of the

ABP obtained from n-butanol extraction with increased specific activity. As obvious, a

number of bands were obtained in case of ammonium sulphate precipitated protein run

on Tricine SDS-PAGE gel. However, a single band was obtained after n-butanol and

HPLC purification steps. Moreover, when the gel was overlaid with indicator organism

a single zone of inhibition was observed, on the same position in all the cases which

further suggested that inhibitory activity was due to a low MW peptide. Also, bioassay

after the electrophoresis of crude CFS also showed that the peptide is of molecular

weight less than subtilin. Hence, it can be concluded that the ABP produced by B.

licheniformis Me1 was of MW between 3 to 3.4 kDa. Low MW bacteriocins have also

been reported for other bacteriocins, including cerein (9 kDa) produced by B. cereus

(Naclerio et al. 1993), 1.4 kDa bacteriocin like compound (lichenin) produced

anaerobically by B. licheniformis isolated from water buffalo (Pattnaik et al. 2001), a 3

kDa bacteriocin from B. licheniformis ZJU12 (Lili et al. 2006) and a heat-stable

bacteriocin (1.5 kDa) from B. licheniformis MKU3 (Kayalvizhi and Gunasekaran

2008). The two peptide lantibiotic produced by B. licheniformis DSM 13, lichenicidin α

and β is of molecular weight 3.25 and 3.05 (Begley et al. 2009; Dischinger et al. 2009),

however this reported lantibiotic is cell associated (no activity in CFS) and does not get

inactivated by trypsin and chymotrypsin and also acts synergistically to produce activity

against microorganisms.

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Nithya V.

The sensitivity of the ppABP to proteinase K and trypsin is a proof of its

proteinaceous nature, which allows for its consideration as a bacteriocin or ABP for

biopreservation. Other bacteriocins produced by Bacillus, such as cerein and coagulin

I4, are often less resistant to proteolytic treatments (Hyronimus et al. 1998; Naclerio

et al. 1993). The ppABP was stable over a wide range of pH and temperature range,

which is a common feature to many bacteriocins of Bacillus (Bizani and Brandelli

2002; Hyronimus et al. 1998; Kamoun et al. 2005). Chang et al. (2001) reported a

heat labile bacteriocin (loss of activity at 80°C in 15 min) with a molecular mass of

6.5 kDa from B. licheniformis cy2. Lili et al. (2006) reported a bacteriocin from B.

licheniformis ZJU12 showing inhibitory activity against wide range of Gram-positive

bacteria and no inhibitory activity against Gram-negative bacteria. A high molecular

weight protein was reported by Cladera-Olivera et al. (2004) with 150 and 20 kDa

from B. licheniformis P40. However, our findings were of significant importance, as

the ppABP of B. licheniformis Me1 exhibited inhibitory activity against both Gram-

positive and Gram-negative food-borne pathogens.

B. licheniformis Me1 does not produce any organic acid as the pH of the

growth medium changed from 7.0 to 8.5. The maximum activity was observed at end

of the logarithmic growth phase, which suggests that the ABP may be produced as a

primary metabolite. Similar observations were documented by Yang and Ray (1994)

for the production of nisin, pediocin PA-1 from Ped. acidilactici, leuconocin Lcm1 by

Leuconostoc carnosum, and sakacin A by L. sake Lb706. They reported that

bacteriocin yield was higher in correspondence to high cell density, which is an

indicative of a primary metabolite.

Results indicate that the MIC and MBC values required to inhibit the growth of

L. monocytogenes was higher as compared to other tested food-borne pathogens. Motta

et al. (2008) reported no inhibition of growth of Salm. enteritidis by BLS P34 alone,

while a synergistic effect of EDTA and BLS P34 was required to kill Salm. enteritidis.

However, the ABP of B. lichenfiormis Me1 was effective alone at 800 AU/ml to kill all

the cells of tested Salmonella spp., indicating that the ABP had a broad range of

activity. Similar results were also observed for dose response curve study.

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Nithya V.

Different experiments were carried out in order to elucidate the MOA of the

ABP B. licheniformis Me1 on indicator microorganisms. Results suggested that the

ABP of B. liceniformis Me1 had a bactericidal affect on L. monocytogenes Scott A,

based on the decrease in the number of viable cells within 30 min after the addition of

the ABP. The bacteriolytic effect of the ABP was also indicated by the decline in

OD600 suggesting that the cells of L. monocytogenes Scott A were lysed. Furthermore,

both the biomass (measured as OD600) and viable cell accumulation was immediately

affected by the presence of ABP in stationary phase cultures. The ABP also displayed

similar trends of inhibition on the growth kinetics of other indicator strains (M. luteus

ATCC 9341 and Staph. aureus FR1 722). However, complete inhibition of M. luteus

ATCC 9341 was observed at 400 AU/ml. The rapid cell death caused by the ABP

suggests that it is targeted to the cell wall or to the cytoplasmic membrane, similar to

that observed for cerein 7 (Oscariz and Pisabarro 2000), BLS P43 (Motta et al. 2008)

and peptide P45 (Sirtori et al. 2008). These observations were further verified by

SEM image analysis. SEM images showed that the ABP disrupts the cell wall and the

cytoplasmic membrane, releasing the cellular content, while the untreated cells

showed no injury, which clearly indicated the occurrence of cell lysis after treatment

with ABP. Similarly, Bizani et al. (2005a) and Motta et al. (2008) used transmission

electron microscopy to show damaged cell envelop.

The effect of the antimicrobial compounds on the indicator bacteria depends

on certain factors, such as the amount, type and purity of the bacteriocin, the indicator

strain, and its cellular concentration (Dalmau et al. 2002). With respect to this,

bacteriostatic may become a bacteriolytic effect by simply increasing the bacteriocin

dose (Motta et al. 2002). Furthermore, the cationic bacteriocins are reported to induce

the release of autolytic enzymes from the anionic polymers of the cell wall, leading to

bacteriocin-induced lysis of the bacterial cell (Bierbaum and Sahl 1987; Jack et al.

1995). Moreover, disruption or disturbance of the cytoplasmic membrane affects the

cell wall integrity and autolysis regulation, since the cytoplasmic membrane is

involved in the cell wall synthesis and turnover process (Kemper et al. 1993). The

ABP also displayed a significant effect on B. cereus spores, a similar property to that

observed for coagulin I4, a bacteriocin-like substance produced by B. coagulans

(Hyronimus et al. 1998), cerein 8A produced by B. cereus 8A (Bizani et al. 2005a,b),

and the bacteriocin AS-48 from Enterococcus faecalis (Abriouel et al. 2002).

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The exact MOA of antimicrobial peptides on Gram-positive bacteria is not yet

completely elucidated (Motta et al. 2008). However, it has been proposed that the

peptides interact with and disrupt the cytoplasmic membrane, leading to the

dissolution of the proton motive force and leakage of the essential molecules,

resulting in the cell death (Montville and Bruno 1994; Brotz and Sahl 2000).

Although, FTIR has been used to identify microorganisms (Maquelin et al. 2002;

Mariey et al. 2001), few attempts have also been made to use this technique to

investigate antimicrobial mechanisms, study of microbial metabolism and antibiotic

susceptibility (Bizani et al. 2005a; Halami et al. 2011; Motta et al. 2008; Rodriguez

2000; Naumann 2000; Preisner et al. 2007). FTIR spectroscopy of L. monocytogenes

Scott A treated with ABP revealed major changes in assignments for phospholipids

and fatty acids, suggesting that the cell membrane is the target of the ABP. By

affecting the bacterial membrane, the ABP might have caused the dissipation of the

proton-motive force, as well as leakage of intracellular contents. Also, release of UV-

absorbing materials was detected after treatment of L. monocytogenes Scott A with

the ABP of B. licheniformis Me1.

PbceA (TMB 299) and PpsdA (TMB 299) based reporter strains are sensitive and

more specific biosensor for lipid II-binding peptide antibiotics than any of the

established cell wall antibiotic biosensors currently available, such as the PypuA and

PliaI (BSF 2470, TMB 488) derived reporter strains (Mascher et al. 2004; Urban et al.

2007). PpsdA is induced by lipid II binding lantibiotics, such as nisin, subtilin,

actagardine and gallidermin, whereas, PbecA could not be induced by nisin, sublancin,

but induced by lipid II lantibiotic bacitracin (Staron et al. 2011). Bacitracin is a type

of cyclic lantibiotic produced by B. licheniformis (Azevedo et al. 1993). Previously, in

section 2.4.2.2., initial differentiation of the MOA of native Bacillus isolates using

reporter strain BSF 2470 was carried out by chromogenic plate assay (section 2.3.5).

The reporter strains based β-gal induction study revealed that the ABP produced by

Me1 is a cell wall acting cationic lantibiotic and the culture, B. licheniformis Me1 is

not a bacitracin producer. Pascale et al. (2007) explored the use of Bacillus subtilis

strain expressing the Enterococcus faecium vanRS genes and a vanH–lacZ fusion, as

a reporter strain, which produced β-galactosidase activity in the presence of cell wall

inhibitors and lysozyme and claimed that reporter strain based antibiotic discovery is

Chapter – 3


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Nithya V.

a powerful tool with the ease of operation and specificity. Similarly, Burkard et al.

(2007) used the reporter strain, B. subtilis ΔspaS amyE::PspaS–lacZ for the

qualitative and quantitative determination of subtilin.

The determination of optimum conditions such as temperature, pH and media

for the production of novel antimicrobial substance from a culture is important to

ensure economical production and subsequent commercial use of the bacteriocin. It

has been shown that the production of antimicrobial compounds by microbial cells is

strongly influenced by the composition of the medium (Audisto et al. 2001;

Hammami et al. 2009; Kayalvizhi and Gunasekaran 2008; Kim et al. 2006; Li et al.

2002), pH of the medium, incubation temperature and agitation (Bizani and Brandelli

2004; Cabo et al. 2001; De Vuyst et al. 1996; Krier et al. 1998; Moita et al. 2005;

Olivera et al. 2004). Reports on factors affecting the production of bacteriocins of

Bacillus spp. are scarce as compared to those of LAB (De Vuyst 1995; Penna and

Moraes 2002; Yang and Ray 1994). In the study of production of ABP by B.

licheniformis Me1, a formulated medium was developed for getting maximum yield

of ABP. Dominguez et al. (2007) optimized the bacteriocin (cerein 8A) production by

B. cereus 8A using food industry waste or by-products as the sole source of nutrients

and reported maximum bacteriocin production at initial pH between 6.5 and 9.0,

temperature between 22 and 34°C and soybean protein concentration higher than 20

g/L. Similar observation was noted for the ABP production by the culture Me1 when

tested for growth at different temperature and pH. The highest production of ABP by

B. licheniformis Me1 in CSL + YE media could be explained by the fact that this

media is an excellent source of nitrogen. Higher ABP production and the stability

ABP concentration after 48 h was observed in media rich in protein supplemented

with salt (NaCl) as compared without salt, which was also in agreement with those

demonstrated by Ganzle et al. (1999). They reported that ecological factors, such as

protein content, acidic pH and NaCl increased the activity of bacteriocins (nisin,

sakacin A and sakacin P). Also, other reports suggest that the salt concentration at low

level stimulate the bacteriocin production (Kanmania et al. 2011) and also helps in the

release of bacteriocin from the producing strains by influencing the adsorption of

bacteriocins to the cell envelop ( Kayalvizhi and Gunasekaran et al. 2008).

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Nithya V.

3.5. Conclusion

Partial characterizations of ABPs of the seven different native Bacillus spp.

lead us to know some of their technological properties, such as heat and pH stability,

as well as low MW (ranging from 3 to 6 kDa) and proteinaceous nature indicating that

these isolates and the ABP produced by them has potential for application as a

biopreservatives to control pathogens in processed foods. The ABP produced by the

culture B. licheniformis was purified and characterized. The purification protocol

employed confirmed the ABP was purified to homogeneity and the SDS–PAGE

analysis of the purified ABP of B. licheniformis Me1 revealed a MW ranging between

3 to 3.5 kDa. The heat resistance, sensitivity to proteases, and unique low MW of the

ABP showed that it belongs to Class I bacteriocin and is a novel one. Chromogenic

assay using cell reporter strains, FTIR, Scanning electron microscopy and release of

UV absorbing materials used to determine the MOA showed that the ABP targets the

cell wall of indicator strains and has a bacteriolytic effect. These observations indicate

the potential use of the ABP of culture B. licheniformis Me1 as food biopreservatives.

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