40

MATERIALS AND METHODS

41

Chapter-1 Purification of antimicrobial peptide, AMPs LR14

Source of strain used in the study

An environmental isolate of lactic acid bacteria, Lactobacillus plantarum strain

LR/14 isolated from the plant rhizosphere (LR: L, lactic acid bacteria; R, rhizosphere)

was procured from the lab stock. Strain LR/14 was maintained on MRS (de Man-

Rogosa-Sharpe) medium. The culture was raised at 37°C under static conditions.

Micrococcus luteus MTCC 106, which was used as an indicator strain was grown in

NB (nutrient broth) at 37°C and 200 rpm. The medium composition is given in

Annexure-I.

Raising of culture and preparation of culture supernatant

Lb. plantarum LR/14 culture was grown at 37ºC for 24h. The culture supernatant was

obtained by centrifugation at 6000 X g at 4ºC for 10 min. In order to obtain the crude

AMPs LR14, the culture supernatant was filtered through a 0.2 µm membrane filter

(mdi Advanced Microdevices, Ambala, India) to remove any cell contamination.

Antimicrobial activity assay

The main body of this work was to identify and characterize the antimicrobial peptide

(AMPs LR14) produced and externalized by Lb. plantarum strain LR/14. The crude

culture supernatant was tested for its antimicrobial action against M. luteus.

Antimicrobial action was assayed in terms of both qualitative as well as quantitative

methods.

(A) Qualitative estimation:

(i) Agar Well Diffusion Assay (AWDA) - (Tagg et al., 1976): For AWDA, aliquots

of culture supernatants (100µl) were filled in wells (6mm diameter) cut out in cooled

TGYE agar that was overlaid with soft agar (0.8%) inoculated with an indicator strain

(106 cfu/ml). The plate was incubated under optimal conditions for growth of the

target organism. Diameter of the growth inhibition zones (halo) was measured (in

terms of mm) after 24 h of incubation.

42

(B) Quantitative estimation:

(i) Activity unit per milliliter (AU/ml)-(Daba et. al., 1991):

Activity of antimicrobial peptides was quantified by a microtitre plate assay by a

critical-dilution method. Each well of the microtitre plate containing 200µl nutrient

broth (NB) was inoculated with the indicator organism (106

cfu/ml) derived from a

fresh overnight culture. An antimicrobial peptide sample was serially diluted two-fold

(1/2) in 50µl volume of nutrient broth and mixed with the bacterial sample in a

Falcon 96-well microtitre plate (Becton Dickinson Labware, NJ, USA). The

microtitre plate was incubated for 6h at 37ºC prior to assessment of growth inhibition

turbidometrically at 630nm using a Microplate Reader (Bio-Rad, USA). Activity

units are arbitrary units calculated against the standard indicator strain M. luteus. One

activity unit (AU) is derived from the reciprocal of the AMPs LR14 required to

inhibit the growth of the indicator strain by 50% in comparison to the untreated

control, and represented as per ml.

(ii) Percentage inhibition

The antimicrobial activity was also checked in terms of percentage of growth

inhibition of indicator organism. The nutrient broth was inoculated with indicator

strain (initial OD630 ~0.02) along with different concentrations of AMPs, as described

later, and incubated at 37°C for overnight. Growth in terms of cfu/ml was obtained by

plating an appropriately diluted cell suspension on nutrient agar (NA). This was

compared with the untreated control. The latter was considered as 100% growth and

the percentage of growth inhibition, if any, was calculated.

Protein estimation

Following methods were used to estimate the protein concentration in the culture

supernatant/AMPs solution:

BCA method (Bicinchoninic acid assay kit, Sigma-Aldrich, USA): This method

was used to estimate the protein concentration as per the manufacturer‘s instructions.

The principle of the BCA assay relies on the formation of a Cu2+

-protein complex

43

under alkaline conditions, followed by reduction of the Cu2+

to Cu+. The amount of

reduction is proportional to the protein present. Briefly, 2 ml of BCA working

reagent (provided with the kit) was added to 0.1 ml of each BSA protein standard,

blank and unknown sample. The solutions were vortexed gently for thorough mixing

and incubated at 37ºC for 30 minutes. The tubes were allowed to cool at room

temperature. The reaction solutions were transferred into a cuvette. The absorbance of

the solution was measured at 562 nm. A standard curve based on the BSA protein

standard concentration was drawn. The protein concentration was estimated by

comparing the absorbance of the unknown samples to the standard curve.

Protein estimation by UV absorption: The protein present in the crude AMPs

sample was measured using UV-visible spectrophotometer (Bio-Rad, USA). The

standard curve for the BSA was prepared in the same way and accordingly the

concentration of unknown protein sample was determined.

Protein activity: The antimicrobial activity of protein was measured by activity units

per milliliter (AU/ml) as mentioned earlier.

Precipitation and extraction of the protein by three phase partitioning

The optimum concentration for precipitation of AMPs was standardized by adding

different amounts of ammonium sulfate, so as to achieve 40%, 60% and 90%

saturation. The culture supernatant with added ammonium sulfate was constantly

mixed on a stirrer at 4ºC to aid in the precipitation of protein. This solution was

mixed well with tertiary butanol (Fisher scientific) in a sterile bottle in a ratio of 1:1,

finally poured into a separating funnel, and was allowed to stand till three layers were

distinctly visible. The lowermost layer was the aqueous layer, uppermost organic

layer and in between the protein rich interfacial layer was observed. The interfacial

layer was separated from the other two layers, washed a few times and the protein

was dissolved in autoclaved distilled water. It was concentrated in a Speed Vac

(Thermofisher Scientific, USA). The antimicrobial activity in terms of activity units

44

per ml (AU/ml) was checked in all the three fractions. This was taken as the partially-

purified fraction and stored at 4ºC for future use.

Gel-filtration Chromatography

To get rid of the salt present in the partially-purified fraction, Sephadex G-25

Desalting columns, (pre-packed disposable PD-10 columns, GE-Healthcare Bio-

Sciences, USA) were used.

Column Equilibration : The column was equilibrated with autoclaved distilled water

with three times its bed volume at a flow rate of 1ml/min.

Sample application : The partially-purified protein solution was passed through a

0.2µm syringe filter (mdi Advanced Microdevices, Ambala, India) to get rid of any

contaminants. The sample was loaded on the column at the rate of 250µg/ml/min to

ensure effective binding with the column matrix.

Elution : The elution was done using 20 ml of autoclaved distilled water and the

desalted protein was collected in sterile eppendorff tubes at the rate of 1ml/min on a

fraction collector. Each sample was checked for antimicrobial activity against M.

luteus and protein concentration. While the amount of protein present was determined

by BCA method (Bicinchoninic acid assay kit, Sigma), AWDA as described above

was carried out to look for the presence of any antimicrobial activity. The active

proteinaceous fractions were lyophilized (FD-5, Allied Frost, India) and resuspended

in 10 ml of autoclaved distilled water.

Reverse phase-High performance liquid chromatography (RP-HPLC)

The purity of peptide/peptides obtained after gel filtration chromatography was

analyzed on HPLC using CLASS-VP software (Shimadzu, Japan). A C18 reverse

phase HPLC column (250 X 4.6 mm, Synergy, 4µ hydro RP, Phenomenex, Japan)

was used. The solvents comprised, solvent A which was composed of HPLC grade

water + 0.1% TFA (Trifluoroacetic acid) and solvent B that contained acetonitrile +

0.1% TFA. These solvents were filtered using a filtration unit attached to a vaccum

45

pump and degassed in a bath sonicator. The temperature of the column oven was set

at 40ºC. The column (C18 5µ 250 X 4.6 mm, Phenomenex) was washed with 50%

methanol and then equilibrated with a gradient of solvent A and solvent B. After the

emergence of the baseline, 1 mg/ml of sample was loaded. The peptides were eluted

with the solvent gradient. For standardization, the chromatogram was analyzed at

different flow rates - 0.1ml/min, 0.2ml/min, 0.35ml/min, 0.5ml/min, 1ml/min and

1.5ml/min. However, maximum resolution was obtained at 0.35ml/min, so it was

finally standardized using the following program. The analysis of the sample was

done using a UV detector at a wavelength of 226 nm with CLASS-VP software

(Shimadzu, Japan). After the analysis, column was washed again with methanol and

stored at room temperature. The HPLC program used:

Time (minutes) A solvent concentration B solvent concentration

0-2 80% 20%

2-6 60% 40%

6-12 40% 60%

12-15 100% 0%

Induction effect

Two peaks were resolved by HPLC, which were collected separately. Since the

HPLC profile showed up a minor peak with low activity, studies were carried out to

assess if this peptide as an inducer peptide. For this, three sets were employed and a

time-course study (4h, 8h, 12h, 16h, 20h, 24h) was carried out. While set A

represented the uninduced LR/14 control grown in MRS, in set B, Lb. plantarum

LR/14 culture was grown in the presence of 200 AU/ml of minor peak protein, and

the set C, where culture was raised in the presence of crude culture supernatant of

strain LR/14 containing 200 AU/ml. These cultures were allowed to incubate for 18h,

and the net activity units (AU/ml) was calculated for all the sets aliquoted at different

time points.

46

Ultra performance liquid chromatography (UPLC)

To further resolve the active peptides another program was devised on UPLC

(Waters, UPLC), and the same solvents (A and B) as used in HPLC were used. The

equilibration and conditioning of the column (100 mM BEH C18, 1.7 µM, 2.1 X 100,

Waters) with solvents A and B was done before injecting the sample (10 µg/ml). The

sample consisted of TPP-precipitated and gel-filtered proteins. The eluted peptides

were collected separately and checked for their activity against the standard indicator

strain M. luteus. Also, the protein content was estimated in each using BCA kit. The

UPLC program used was follows:

Matrix absorption laser Desorption ionization (MALDI)

UPLC resolved four active peptides which were processed together for matrix-

assisted laser desorption/ionization time-of-flight-mass spectrometry, MALDI-TOF-

MS.

The work was carried out using MALDI-TOF/TOF Analyzer, AB SCIEX TOF/TOF

4800 Plus Analyzer, Applied Biosystems, USA. The peptides were diluted with 50%

acetonitrile and 0.1% TFA, and α-cyano-4-hydroxycinnamic acid (CHCA), matrix

was added to the diluted peptides in 1:1 ratio. This mixture (peptide and matrix) was

spotted on a MALDI plate, and the data was acquired in an MS reflector mode. The

selected peaks were subjected to MS/MS, and the data was blast using a MASCOT

search engine (Kaur et al., 2013).

Time (minutes) A solvent

concentration

B solvent

concentration

0-3 95% 5%

3-6 80% 20%

6-8 60% 40%

8-10 50% 50%

10-11 95% 5%

47

N-terminal sequencing

One of the UPLC-purified peptides was digested with CNBr and then subjected to

Edman degradation and sequence was obtained on ABI‘s ―Procise-492‖ model. The

sequencing was done at the Protein Sequencing Facility at INTAS

Biopharmaceuticals Ltd., Ahmedabad, India.

Identification of AMPs LR14 with AMPs produced by clones A5 and A6

The plantaricin operon of strain LR/14 has been characterized in the lab, and five

structural genes, pln E, F, J, K, and N were identified. In the same study, clones, A5

and A6, were generated which were shown to have suffered large deletions (Nitika

Ghosh, unpublished results). In order to see if AMPs identified during present

investigation were in any way affected, the same were purified from the culture

filtrate of both the clones by TPP method as described above. These were further

resolved on UPLC and UPLC chromatograms were obtained following the same

program as described above.

Similarly to compare these AMPs with plantaricins from strain LR/14, commercially

synthesized plantaricins viz. Pln E, Pln F and Pln A were subjected to UPLC after

mixing them in a ratio of 1:1 v/v.

Characterization of purified AMPs LR14

The purified AMPs LR14 were characterized for different parameters:

Sensitivity to heat, pH, storage conditions and hydrolytic enzymes

Aliquots of purified AMPs LR14 samples were exposed to heat treatment at 60°, 80°

and 100°C for 30 min in a water bath, and one aliquot was autoclaved (121°C at 15

psi, for 15 min). The treated samples were tested for residual antimicrobial activity, in

terms of AU/ml.

In parallel, aliquots of AMPs LR14 samples were adjusted to pH values ranging from

2.0 to 8.0 in 1:1 ratio of different buffers (HCl-KCl 50 mM, pH 2.0, and 4.0;

48

phosphate buffer 50 mM, pH 6.0, 7.0, and 8.0), incubated at 37°C for 4 h, and tested

for residual antimicrobial activity (AU/ml).

To determine the durability, the AMPs LR14 sample was stored at different

temperatures (-20°, 4°, R.T {room temperature: fluctuating between 10° to 30°C

night/day temperature}, and 37°C). Its subsequent activity was checked at an interval

of one month up to one year, by AU/ml, and compared with the fresh preparation.

In a separate experiment, the sensitivity of the AMPs LR14 to proteolytic enzymes

(proteinase K, pepsin, papain, α-chymotrypsin and protease), as also to lipase and α-

amylase (Sigma-Aldrich, USA) was tested at a final concentration of 1 mg/ml, at

37°C for 2 h. After incubation, the enzymes were heat-inactivated (5 min at 100°C),

and the samples were tested for the antimicrobial activity in terms of AU/ml.

Untreated samples were taken as controls in each case.

Chapter-2 Mode of action and Inhibition spectrum of AMPs LR14

Sources of strains used in the study

Micrococcus luteus and Escherichia coli K12 were available in our Lab Stock.

Listeria monocytogenes and Yersinia enterocolitica were obtained from Prof. J.S.

Virdi, Department of Microbiology, UDSC, New Delhi. The fungal stocks were

procured from Indian Agricultural Research Institute (IARI), New Delhi. These were,

Aspergillus niger ITCC 321, Rhizopus nigricans ITCC 4416, Mucor racemosus ITCC

4392, Penicillium chrysogenum ITCC 419. Plasmodium falciparum 3D7 and

Mycobacterium tuberculosis H37Rv were kindly provided by Prof. P.C. Ghosh and

Prof. Anil Tyagi, respectively, Department of Biochemistry, UDSC, New Delhi. The

work against some clinically important pathogens (Pseudomonas, Acinetobacter,

Klebsiella, Enterobacter, Salmonella typhi, Staphylococcus, and VRE) was carried

out in Dr. Rajni Gaind‘s lab, Department of Microbiology, Safdarjung Hospital, New

Delhi. The clinical pathogens were revived on Mac Conkey agar medium and

maintained on Mueller Hinton Infusion agar (MHI) medium, both obtained from

HiMedia, India.

49

The media compositions are detailed in Annexure I.

Maintenance of culture

E. coli K-12 and M. luteus were maintained in Luria broth (LB) and Nutrient broth

(NB), respectively. The cultures were raised at 37°C, 200 rpm, sub-cultured to an

initial A600 of 0.02 (~106 cfu/ml) and grown for 18h in all experiments, unless

otherwise specified. All the cultures were purified by single colony isolation on their

respective agar medium. Colonies from such plates were inoculated to raise the

cultures, depending upon experimental requirements, as described later. All cultures

were maintained on specific media and sub-cultured every 2-3 weeks. All chemicals

used were of analytical grade, obtained from Sigma-Aldrich (USA), and media were

purchased from HiMedia (Mumbai, India). The composition of the media and

solutions are detailed in Annexure I.

Preparation of Glycerol Stocks

For long-term storage of the cultures, glycerol stocks were prepared (Sambrook et al.,

1989). To 0.5 ml of overnight culture, 0.5 ml of sterile 40% glycerol was mixed and

poured in a cryo-vial, and kept at -80ºC. For reviving the bacterial strains, the frozen

surface of stock was scraped with a sterile inoculation needle and immediately

streaked on the medium plate. The plates were incubated at 37ºC.

Growth

Growth was determined by two methods:

Absorbance

An inoculum size (A600 ~0.02) of an exponentially grown culture was transferred to a

fresh medium under sterile conditions. The cultures were raised under specific

conditions for a designated period of time. The change in growth was monitored

spectrophotometrically by measuring absorbance/optical density (A/OD) at 600 nm

for both net growth as well as growth at different time intervals.

50

Determination of colony forming units (cfu/ml)

For this, 0.1 ml cell suspension was serially diluted in 0.9 ml sterile normal saline

(0.85% NaCl) and vortexed. Then 0.1 ml from the appropriate dilutions was plated on

the required agar medium and incubated at specific temperature for overnight. Viable

counts were determined as colony-forming units per ml (cfu/ml) of the culture.

Percentage inhibition

The antimicrobial activity was also checked in terms of percentage of growth

inhibition of indicator organism as described earlier.

Cell viability assay

The two strains were grown in their respective media. An inoculum of ~0.02 OD600

(~106 cfu/mL) both of M .luteus and E. coli were treated with different concentrations

of crude AMPs LR14 for 18h. The concentrations used for M .luteus were 40, 80,

120, 140, and 160 AU/ml and for E. coli 200, 400, 600, and 1000 AU/ml. Cells

without any treatment were taken as control. Cells from different treatments and the

control were serially diluted in 0.85% saline, and viability was monitored in terms of

cfu/ml after overnight incubation at 37ºC. The percent inhibition was then calculated

for each treatment set with respect to control. From this, the effective concentration

was standardized, and the subsequent studies were carried out at 120 (IC50) and 140

(IC99) AU/ml in case of M. luteus, and 400 (IC50) and 600 (IC99) AU/ml in case of

E.coli. Since, the activity units (AU/ml) is an arbitrary unit and varies from organism

to organism, the same were converted into concentration of protein. Thus, 120, 140,

400, and 600 AU/ml used in the study amounted to 75, 100, 250, and 450 µg/ml of

protein, respectively.

Light microscopy : The cells of treated (IC50 and IC99 concentrations) and control

sets of the two strains were each harvested by centrifugation (6000 X g for 10min)

and washed twice with 0.1M phosphate buffer pH 7.4. The cells were stained with

safranine, mounted on a clean glass slide, and visualized (magnification 100 X) under

a light microscope, (Olympus, BX51, Japan).

51

Dual staining method: To further distinguish the resulting growth inhibition, dual

staining (two vital dyes viz. calcein AM and propidium iodide are known to provide

green and red fluorescence to live and dead cells, respectively) of AMPs LR14-

treated and control cells was carried out, as per the manufacturer‘s instructions (Live-

Dead Cell Dual Staining kit – Sigma, USA). In brief, 10 µl solution A and 5 µl

solution B was added to 5 ml PBS to prepare assay solution. The cell suspension was

centrifuged and the cell pellet was washed with PBS several times. Prepared cell

suspension in PBS was prepared at a density of 105 to 10

6 cells/ml. 200 µl of cell

suspension and 100 µl assay solution was mixed and incubated at 37°C for 15 min.

The fluorescence of viable and dead cells was monitored simultaneously at an

excitation wavelength of 490 nm through a fluorescence microscope, Olympus,

BX51, Japan (magnification 100X).

Estimation of Membrane potential

The protocol of Breeuwer and Abee (2004) was followed. Briefly, ~107 cells of M.

luteus and E. coli were centrifuged at 2800 X g for 10 min, washed and resuspended

in potassium phosphate buffer (pH 7). The cells were stored on ice until use.

Following stock solutions were prepared: florescent probe DiSC3 (3mM in DMSO)

stored in freezer, and valinomycin (3mM in ethanol). The cuvette was cleaned with

70% ethanol and air-dried. 3 ml cell suspension was added to the cuvette and mixed

using a magnetic stirrer. Then, 5 µl DiSC3 (final concentration of 5 µM) was added

and the spectrofluorimeter measurements were done at excitation wavelength 643 nm,

slit width 10 nm, and emission wavelength 666 nm, with slit width 10 nm. Once the

fluorescence signal was stable, ~15 min, 10 mM glucose was added to energize the

cells. After another 5 min of incubation, crude and purified AMPs LR14 were added

separately, and waited again until the fluorescence signal was stable. The

concentratons used were: for M. luteus cells, crude AMPs LR14 – 75 µg/ml and

purified AMPs LR14 - 65 µg/ml, and for E. coli, crude AMPs LR14 - 250 µg/ml and

purified AMPs LR14 – 100 µg/ml). In control, 10 µl valinomycin was added and the

fluorescence signal was recorded.

52

Mode of action

AMPs produced by lactic acid bacteria are known to be membrane-active compounds.

In order to see whether killing effect of AMPs LR14 is also due to this property, the

cells of M. luteus and E. coli were treated with 75, 250 (IC50) and 100, 450 (IC99)

concentrations, respectively for 18h. The culture supernatant was prepared to assess

various parameters. An untreated control was maintained in all the cases.

Preparation of culture supernatant

The treated and untreated cultures (grown at 37°C for 18h) of two strains were

centrifuged at 6000 X g at 4°C for 10 min. The spent culture filtrate was filtered

through a 0.2 µm membrane and stored at 4°C for further use.

K+ release

The culture supernatant from both the samples was analyzed to detect the

extracellular K+ using Atomic Absorption Spectrophotometer-Inductively Coupled

Plasma Optical Emission Spectrometry (ICP-OES) at 766.5 nm with an Optima 4200

instrument (Perkin Elmer, USA) and compared with untreated control. Standard curve

was made using 1 to 5 ppm of potassium chloride. The values obtained were

expressed as µmol/l.

Measurement of inorganic phosphate (ATPase assay)

The phosphate content in the culture broth was determined according to Heinonen

and Lahti (1981). Briefly, to 1ml of culture supernatant, 2ml of freshly prepared

AAM solution (acetone: 5N H2SO4: 10mM ammonium molybdate in a ratio of 2:1:1

v/v) was added and thoroughly mixed. Then 0.1ml of 1M citric acid was added,

mixed well, and the absorbance was recorded in a UV-Vis Spectophotometer

(Shimadzu, Japan) at 355nm. A standard curve of phosphate was prepared, from a

stock solution of KH2PO4 (2,000µg/ml) in distilled water. A dilution series ranging

between 50 µg/ml and 2,000 µg/ml was prepared from the stock solution. The values

obtained were expressed as µmol/l.

53

ATP release

To determine the leakage of adenosine 5‘-triphosphate (ATP) in the culture

supernatant of the cells treated with AMPs LR14, ATP detection kit (Sigma Aldrich,

USA) was used. The assay was based on the amount of light emitted when firefly

luciferase consumes ATP to catalyze the oxidation of D-luciferin. The readings were

measured using a luminometer, (Turner Biosystems, Sunnyvate, CA). These were

represented as nanomoles of ATP released, based on the standard curve of ATP (ATP

standard was made by diluting with the dilution buffer provided in the kit) in the

range of 10-3

to 10-5

mmoles/l and assayed similarly.

Release of UV absorbing material

Leakage of UV light-absorbing material was used as an indicator for the loss of cell

membrane integrity. M. luteus and E. coli cells corresponding to OD600 of ~0.02 were

treated with AMPs LR14 at their IC50 and IC99 concentrations, respectively in 0.85%

saline and incubated at 37ºC. Samples were removed after overnight incubation,

centrifuged, and culture supernatant filtered through 0.2 μm membrane (mdi

Advanced Microdevices, India). The absorbance of the filtrates was measured at 260

nm in a 96-well microtiter-plate, keeping normal saline as blank using a Microplate

Reader (Ultramark, Microplate imaging system, Biorad, USA).

Transmission electron microscopy

Untreated and treated cells of M. luteus and E. coli harvested by centrifugation (6000

X g for 10min) and washed twice with 0.1 M phosphate buffer pH (7.4), were pre-

fixed in 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M

phosphate buffer pH 7.4 for 4h. Such cells were then post-fixed in 1% (w/v) osmium

tetraoxide for 1h. Following each fixation step, excess fixative was washed with 0.1

M phosphate buffer pH 7.4. The samples dehydrated using graded ethanol series (50-

100%) and infiltrated with a resin, were placed in an embedding mould, and

polymerized in an oven at 60ºC for 24 hours. Thin sections were cut by an

ultramicrotome and mounted on grids, covered with collodion film, and stained with

2% uranyl acetate in Reynold‘s lead citrate solution. Preparations were observed

54

using a transmission electron microscope (TEM) (FEI, Philips, Morgagni 268 D,

Holland) at 70 kV.

Assessment of resistance in M.luteus and E. coli against AMPs LR14

Approximately 107 cells of M. luteus and E. coli were treated with 100 µg/ml and 450

µg/ml (IC99 concentrations) of AMPs LR14, respectively for 18h. Different dilutions

of the cultures were plated on NB and LB agar plates, respectively and allowed to

incubate for 24h. A master plate each for treated M. luteus and E.coli cells was made

by patching the viable cells. These cells were again subjected to AMPs LR14

treatment using same concentrations for overnight and plated to check the viable cell

count. The treatment was repeated for seven successive cycles and in each viability

count was monitored.

Studies on fungi

This part of the work was directed to study the effect of AMPs LR14 against food-

spoilage fungi so as to implicate its role as a food biopreservative.

Sources of strains and maintenance of culture

Isolates of fungi viz., Aspergillus niger ITCC-321, Rhizopus nigricans ITCC-4416,

Mucor racemosus ITCC-4392, and Penicllium chrysogenum ITCC-419 were

procured from ITCC, IARI, New Delhi. All the strains were maintained on PDA

(Peptone Dextrose agar) medium. The cultures were raised at 30°C under static

conditions and sub-cultured every week. All chemicals used were of analytical grade,

and medium was purchased from HiMedia (Mumbai, India). The composition of the

media and solutions are detailed in Annexure I.

Counting of spores

Spores from a fresh plate of culture were picked up with a sterile inoculation loop

suspended in 0.85% saline and vortexed to get homogenous suspension. Spore

counting was done on a Neubaur‘s chamber or Haemocytometer.

55

Screening of antifungal activity

These experiments were carried out with all the four fungal strains selected. The

antifungal effect was visualized by dual culture assay. Lb. plantarum LR/14 was

streaked on MRS plates in two vertical streaks and allowed to incubate at 30ºC for 2

days. Approximately 105 spores/ml were mixed with soft PDA. This suspension was

poured over the LR/14 grown MRS plates and left for incubation at 30ºC for another

2 days. Any zone of growth inhibition observed was qualified as the antifungal

activity.

In a separate experiment, the antifungal effect was further confirmed by growing

these fungi on AMPs LR14-supplemented PDA plates and incubated for 48h at 30ºC.

Fungal discs (1cm X 1cm) from a fully grown colony, representing different stages of

growth were cut and kept on these petriplates. Fungi grown only on PDA without

AMPs LR14 served as controls.

Standardization of effective/lethal dose for causing fungal inhibition

Different concentrations ranging from 50, 100, 250, 500, 1,000, 2,000, 3,000 and

5,000 µg/ml of crude and purified AMPs LR14 were tested against different fungi as

mentioned above. Approximately 105 spores/ml of Aspergillus niger (taken as a test

case) were added to Peptone Dextrose (PD) medium along with the above mentioned

concentrations of AMPs LR14 in Erlenmeyer flasks. Another set of flasks with fungal

spores but without AMPs LR14 were kept as untreated control. The control and

treated cultures were incubated at 30ºC for 48h. The biomass was filtered through a

pre-weighed Whatman No. 1 filter paper pre-dried at 80ºC in an oven for 4h. The

filter papers containing the biomass were dried, weighed and dry biomass was

quantified in terms of the net weight. Percentage growth inhibition was calculated by

comparing the biomass obtained in the untreated and treated samples. For further

experiments, 3 and 5 mg/ml of crude and 1 and 2 mg/ml of purified AMPs LR14

were standardized as the lethal doses.

56

Effect of AMPs LR14 on different growth phases of fungi

For this, fungal spores were treated with crude AMPs LR14 at the standardized

concentrations (3 mg/ml and 5 mg/ml). The samples were stained with cotton blue

and mounted in lactophenol. Microscopy was done at different time intervals so as to

track the different phases of life cycle including: spore germination, hyphae formation

and mycelial ramifications and sporulation. In these experiments, biomass dry weight

was measured after 48h of incubation.

Inhibition of fungal spore germination

The cell free supernatant containing AMPs LR14 was screened against the spores of

selected fungi. Crude AMPs LR14 concentrations of 3 and 5 mg/ml were added to PD

broth containing ~105 spores/ml. The control set involved the addition of sterile MRS

broth only to the fungal spore suspension. The control and the experimental flasks

were kept at 30ºC for 48h. During this period, samples from the flasks were

withdrawn at different time intervals depending upon the time taken for various

stages of growth (under control conditions) of each fungus. After staining with cotton

blue, microscopic visualization (60X) was done for each of the treated as well as the

untreated controls. Another set comprising purified AMPs LR14 concentrations of

1mg/ml and 2 mg/ml were also tested with the spore suspension (~105 spores/ml) in

PD broth, keeping an untreated control alongwith. After 48h of incubation, biomass

dry weight was measured.

Inhibition of fungal growth (hyphae formation and mycelial ramifications)

In this assay, independent sets consisting of Erlenmeyer flasks containing ~ 105

spores per ml added in 10ml PD medium were treated with 3 and 5 mg/ml of crude

AMPs LR14, and 1 and 2 mg/ml of purified AMPs LR14 at different times. While in

one set AMPs were added at the start of the experiment, further treatments were done

after 8h for A. niger, R. nigricans and M. racemosus and after 12h for P.

chrysogenum for tracking hyphal growth, and after 16h for A. niger, R. nigricans and

M. racemosus and 24h for P. chrysogenum for tracking full vegetative mycelial

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growth. In the control experiment MRS was added to PD broth containing ~105

spores in place of AMPs LR14. The flasks were incubated at 30ºC till 48h.

While the effect on spore germination was assessed microscopically, the percentage

inhibition of dry weight was employed for comparing the growth of the control with

the other treated sets. The dry weight of the mycelium was determined after 48h of

incubation in all cases, as described earlier. The percent inhibition was calculated

using the formula:

(R1 – R2) / R1 x 100

Where, R1 is the dry weight of control and R2 is the dry weight of the treated fungal

biomass.

Fungi-static/cidal effect of purified AMPs LR14

To confirm the antifungal nature of AMPs LR14, it was important to test the viability

of all the fungi tested. An aliquot of culture (100 µl) after 18h of treatment from each

set (treatment at 1 mg/ml and 2 mg/ml and an untreated control) for all the four fungi

(A. niger, R. nigricans, M. racemosus, and P. chrysogenum) was plated on PDA

medium and incubated at 30°C for 48h. The colony growth was monitored.

Effect of AMPs LR14 against various food-spoilage bacteria

Log phase cells (~ 106 cfu/ml) of M. luteus, E. coli, Listeria monocytogenes and

Yersinia enterocolitica were added to NB/LB. E. coli was taken as a representative

Gram-negative bacteria. To this medium, 25, 50 and 100 µg/ml of purified AMPs

LR14 was added. The loss in cell viability (log10 cfu/ml) was monitored by plating

the cells on their respective media.

Evaluation of anti-plasmodial activity

Plasmodium falciparum strain 3D7, obtained from National Institute of Malaria

Research, New Delhi, was grown in RPMI-1640 with 10% human serum

(Invitrogen). The strain was maintained by serial passages in human erythrocytes

cultured at 4% hematocrit in RPMI-1640 medium (Life Technologies) supplemented

58

with 10% human serum and incubated at 37°C under the atmosphere of mixed gas

(containing 5% CO2, 5%O2, and 90% N2) in a plastic chamber. Heparinized whole O+

blood was collected from Rotary Blood Bank, New Delhi, and RBCs were separated

under sterile conditions by centrifugation to remove serum and buffy coat. The levels

of parasitemia were routinely monitored on blood smear with 5% Giemsa-azure type

B stain in phosphate buffer (20mM, pH 7.2).

Anti-plasmodial effect of the crude and purified AMPs LR14 was monitored by

studying the incorporation of [3H]-hypoxanthine in the nucleic acid of the parasite. In

brief, AMPs LR14 was serially diluted (stock concentration of crude AMPs LR14 –

1.6 mg/ml and purified AMPs LR14 – 85 µg/ml) and added to P. falciparum infected

erythrocyte suspension (2% final hematocrit and 1% parasitemia) in a 96-well tissue

culture plate along with an untreated control. After 24 h of incubation at 37°C, 20 μl

of 0.2 μCi/well of [3H]-hypoxanthine (American Radiolabeled Chemicals, Inc. with

specific activity of 25 Ci/mmol) was added to each well containing unsynchronized

parasite culture. After 18h of incubation, the cells were harvested onto a glass-fibre

filter paper using a Skatron Semi-automated cell harvester. The paper discs were

placed into 5 ml scintillation cocktail. The scintillation cocktail (1 litre) composed of

0.1 gm POPOP (1,4, bis 2-5 phenyl oxazolyl benzene), 4 gm PPO (2-5 diphenyl

oxazole), 300 ml ethanol and 700 ml toluene was kept for overnight stirring before

use. [3H]-hypoxanthine incorporation in nucleic acid was measured in a liquid

scintillation β-counter (Model: Tri-Carb 2900 TR, Perkin Elmer, USA), and

inhibition of growth was calculated by comparison with control (Control consisted of

complete medium as a substitute for the test molecule.) All data points were collected

in triplicate for each experiment. The IC50 (concentration of AMPs required to inhibit

50% of growth of parasite as measured by incorporation of [3H]-hypoxanthine) was

generated as described by Mustafa et al. (2011).

Assessment of Hemolytic Activity

In order to show whether antiplasmodial activity of AMPs LR14 is not due to the

lysis of the erythrocytes, cultured infected and uninfected erythrocytes were treated

59

with AMPs LR14 and absorbance of Hb at 540 nm was measured as per the protocol

of Aditya et al. (2010). Briefly, to prepare the negative control, a volume of 100µl

cell suspension was added to 3ml normal saline solution (normotonic conditions; no

lysis expected). Likewise, 100 µl of cells were added to a second test tube and the

volume was made up to 3ml with double-distilled water (hypotonic condition; 100%

haemolysis expected). A few concentrations of AMPs LR14 (25-100 µg/ml) were

added to P. falciparum infected (2% hematocrit and 1% parasitemia) and uninfected

erythrocytes (2% hematocrit) along with an untreated control, all in PBS for 1h at

37°C. The reaction was stopped by addition of 50 µl of 2.5% glutaraldehyde. The

blood samples were then centrifuged at 3000 X g for 15 min and the absorbance of

supernatant was measured at 540 nm using a UV/VIS spectrophotometer (UV 1800

Shimadzu).

Effect against Mycobacterium smegmatis and M. tuberculosis

Mycobacteria were grown in 7H9 broth (Difco Middlebrook) and 7H11 Agar (Difco

Mycobacteria). Approximately 107 cfu/ml of M. smegmatis cells were taken in

different sets. Three concentrations: 1 µg/ml, 10 µg/ml, 40 µg/ml of AMPs LR14 was

added to different sets. Isoniazid at a concentration of 5 µg/ml was used as a positive

control for the inhibition studies. A set without AMPs or antibiotic served as a

negative control. The incubation of cells was done at 37°C and the time of treatment

was 20h. Aliquots were withdrawn at different time-points and the cells were plated

on 7H11 medium to check for the viability.

To study the inhibition of M. tuberculosis by AMPs LR14, sterile filter paper discs

were impregnated with 50, 100, 200 µg of AMPs LR14, isoniazid, and nisin (a

commercially available bacteriocin known to cause inhibition of M. tuberculosis). M.

tb cells (~ 107 cfu/ml) were suspended in 0.85% saline, and the cell suspension was

swabbed on 7H11 plates. The discs containing AMPs LR14, isoniazid, and nisin were

placed on these plates and incubated at 37°C for three weeks. Zones of inhibition, if

produced, were recorded.

60

Effect of AMPs LR14 against clinical isolates of Gram-positive and Gram-

negative bacteria

This study was carried out at Deptt. of Microbiology, Safdarjung Hospital. A few

pathogenic strains viz. E. coli 1491, Klebsiella pneumoniae 1459, Acinetobacter sp.

1476, Staphylococcus aureus 1492, Enterobacter sp. 1481, Pseudomonas sp. 383,

Vancomycin Resistant Enterococci (VRE) 408, and Salmonella typhi, isolated from

blood and sputum of patients were employed. These were revived on Mac Conkey

Agar (HiMedia, India). Crude and purified AMPs LR14 were impregnated on

sterilized filter paper discs such that the final concentration was 3 mg and 30 mg for

purified and crude AMPs, respectively. The discs were air-dried.

Further, a loopful of cells from all the eight strains was homogenously suspended in

0.85% saline. Sterilized swabs were dipped in each of the culture solutions and

swabbing was done on Mueller Hinton Infusion (MHI) Agar (HiMedia, India) plates.

The discs impregnated with purified and crude AMPs LR14 were kept on each of

these plates. The plates were incubated at 37°C incubator for 24h. The zones of

inhibition around the discs was measured.

Chapter-3 Cytotoxicity and Applications of AMPs LR14

This part of the study was developed to test the toxicity of AMPs LR14. This

comprised both the in-vitro tests as well as those carried out with different model

systems.

MTT assay

Cell viability was measured by the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-

2,5-diphenyltetrazolium bromide) (Sigma Aldrich, USA) dye reduction assay using

the protocol of Cruz-Chamorro et al. (2006). Approximately 105 Chinese hamster

ovary (CHO pro-) cells were seeded in Dulbecco Modified Eagle‘s Minimal Essential

medium (DMEM - medium composition given in Annexure-I) in each well of a 96-

well microtiter plate. Cells were grown in a humidified incubator at 37°C, under 5%

61

CO2 and 95% air atmosphere. Different concentrations (10 µg/ml to 1 mg/ml) of

purified AMPs LR14 were added to these cells. After incubation for 48h, 20 µl of

MTT at a concentration of 5 mg/ml in phosphate-buffered saline (PBS, pH 7.4) was

added to each well. After 4 h of incubation at 37ºC in a humidified atmosphere with

5% CO2, the formazan precipitate formed was solubilized by the addition of DMSO

at room temperature for 10min. The absorbance was measured by a microplate reader

(BioRad, USA), at wavelength of 550 nm. The absorbance of the untreated cells was

set at 100%, and results were expressed as percent cell viability under different

treatments.

Hemolytic activity

The test was carried out to analyze the hemolytic activity of AMPs LR14. The

experiment was done in accordance with a method used by Aditya et al. (2010). For

this, to 5ml of whole blood sampled from a healthy volunteer the anticoagulant

EDTA was added at a concentration of 1.8 mg/ml. Blood sample was centrifuged at

3000 X g for 20 min (Sigma 2-16K, Germany). Buffy coat was removed and the

packed cells were washed thrice with normal saline. To prepare the negative control,

100 µl of cell suspension was added to 3ml normal saline. Likewise, a suspension of

cells (100 µl) was taken in a second test tube and the volume was made upto 3 ml

with double-distilled water, to serve as a positive control. Different concentrations

(100, 500, 1,000, 5,000, 10,000 and 15,000 µg/ml) of the purified preparation of

AMPs LR14 were added to 100 µl of cell suspension. All the samples were incubated

at 37°C for 1h. The reaction was terminated by addition of 50 µl of 2.5%

glutaraldehyde, since it stops the hemolysis. The blood samples were then centrifuged

at 3000 X g for 15min and the absorbance of supernatant was measured at 540 nm

using a spectrophotometer (UV-1800 Shimadzu, Japan). The experiment was

performed in three independent sets.

Cytotoxicity of AMPs LR14 on onion root tip as a model system

To study the chromosomal aberrations/anomalies (if any) caused by AMPs LR14, the

studies were done on a plant root system i.e., onion. A stock solution of 10 mg/ml

62

concentration of purified AMPs LR14 was used from which different concentrations

ranging from 2.0, 1.0, 0.5, 0.25 mg/ml were taken in different beakers. Water alone

served as a control. Onion bulbs were kept on these beakers so as to effect the

germination and root tip formation. The germination was allowed for 2-3 days. The

observations suggested that the AMPs LR14 (1 mg/ml) inhibited the formation of root

tips. Therefore in the next study the onion bulb was allowed to germinate in tap water

and once the roots of 1-2 cm in length were formed they were dipped in AMPs LR14

solution (1 mg/ml) for 3h. The root tips were fixed with Carnoy‘s fixative (glacial

acetic acid : absolute alcohol in 1:3 ratio) for overnight. Staining was done with 1%

acetocarmine and a squash preparation was made and then visualized under a bright

field microscope for different stages of mitosis.

Evaluation of in-vivo toxicity of AMPs LR14

I – Drosophila as a model system

The work was further extended to study the toxic effects of AMPs LR14 and

Drosophila melanogaster was chosen as a model system. Drosophila has been a

powerful model organism to study several aspects of development and diseases. Its

relatively small life cycle with distinct developmental stages, simple tissue

organization, and availability of sophisticated genetic tools are some of the

advantages using this insect as a model system. It is also turning out to be a good

model system for study of many other biological phenomena including human

intestinal diseases.

Fruit fly (Drosophila melanogaster): Life Cycle

Distinctive stages of the life cycle of D. melanogaster along with their associated

features are discussed below (Fig. I):

Egg: The egg of D. melanogaster is about 0.5 mm long. A female may lay as many as

400 eggs in a favorable egg-laying ground. Within 24 hours of laying, the eggs hatch

into 1st

instar larvae. At room temperature, this hatching time is as short as 15h. When

kept at room temperature, a Drosophila egg requires 8-10 days to develop into an

adult; whereas in temperatures higher than this, development time is less.

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Larva: The larval stage of Drosophila consists of three instars. Within 24h of

hatching, the 1st instar larva molts to develop into 2

nd instar larva. Again after 24h

(i.e. 48 hours after egg hatching), the 2nd

instar larva molts and matures to 3rd

instar.

During these molting stages, the larva loses its spiracles, mouth, and hooks and

acquires new ones.

During larval development, tissues known as imaginal discs grow inside the larva.

Imaginal discs develop to form most structures of the adult body, such as the head,

legs, wings, thorax and genitalia. Cells of the imaginal discs are set aside during

embryogenesis and continue to grow and divide during the larval stages—unlike most

other cells of the larva, which differentiate to perform specialized functions and grow

without further cell division.

Pupa: After 4 days of voracious feeding, the 3rd

instar larva encapsulates itself inside

a hard and dark-colored puparium. It is in this pupal stage, where the metamorphosis

of D. melanogaster take place, giving rise to wings and legs. At room temperature,

the duration of metamorphosis is ~4 days.

Adult: The adult D. melanogaster emerges through the operculum of the puparium,

and the female flies are receptive within 8 – 12h of emergence. Following mating, the

female Drosophila stores the sperms, which can be utilized for fertilization for

several days.

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Figure I. Representation of life cycle of Drosophila melanogaster

Drosophila stock

Drosophila melanogaster Oregon R+

stocks were maintained in Sussex culture

medium (Graf et al. 1992) at 24 ± 1ºC in a 250 ml culture bottles. The medium

composition is given in Annexure I.

AMPs LR14 treatment

Different concentrations were prepared from a stock of 30 mg/ml of purified AMPs

LR14. Though different concentrations (2, 5, 7 mg/ml) were tried, the final

concentrations used to study toxicity were 10 mg/ml, 15 mg/ml and 20 mg/ml). These

concentrations were mixed with the food for exposing to D. melanogaster in different

sets. To extend the above study, the food mixed with AMPs LR14 was poured in

petriplates (60 mm in diameter) and utilized for further rearing the flies at various

concentrations. Adult flies (50) were raised on these concentrations and survival was

monitored. Subsequently, 50 eggs were placed on this food supplemented with

65

various concentrations of peptide, and developmental pattern was monitored over a

period of 20 days. A total of 50 eggs were used for each experimental set up, and

each experiment was replicated three times.

Staining procedure

Phalloidin-TRITC and DAPI

To study the effect on different tissues and organs, salivary glands from late third

instar larva, and ovaries from the adult flies were dissected out from control as well as

treated (10 mg/ml) individuals. The tissues were washed thoroughly with phosphate

buffer (pH 7.4) and fixed in 4% para-formaldehyde at 4ºC for 20 minutes. Following

several washes with PBS, staining was done as described by Lee et al. (2007), at 4ºC

for overnight with 1µg/ml of phalloidin-TRITC (tetramethylrhodamine B

isothiocyanate) (Sigma-Aldrich, USA), prepared in 1X PBS. The tissues were then

washed with PBS and stained with 4', 6-diamidino-2-phenylindole (DAPI, 1µg/ml

prepared in 1X PBS) for 10 min. The tissues were washed again in PBS and mounted

in 80% glycerol. Stained samples were studied under confocal microscope (Leica

TCS SP5 Version LAS AF 2.5.1). UV diode laser (excitation 405 nm) and DPS Red

lasers (excitation 561 nm) were used to excite samples and fluorescence was collected

at 420 nm and 570 nm (λmax) for DAPI and phalloidin-TRITC, respectively. DAPI

stained preparations were also visualized under a fluorescence microscope (Olympus

BX51, Japan), separately. The experiment was repeated twice.

Staining with acridine orange

In addition, acridine orange staining was performed to study DNA fragmentation and

apoptosis. Acridine orange is a cell permeable cationic dye used for identifying

apoptotic cells with an excitation maximum at 502 nm and an emission maximum at

525 nm (green). The staining was carried out as described previously (Ianella et al.

2008) with acridine orange (1µg/ml prepared in 1X PBS) for 5-10 min. The tissues

were washed with PBS to remove unbound dye. Stained samples were individually

studied under fluorescence microscope (Olympus BX51, Japan).

66

In all the above experiments, Oregon R+ flies reared on normal diet and incubated at

24°C were utilized as untreated controls.

II – A Mammalian system

Acute oral toxicity test of AMPs LR14 was carried out at Shriram Institute for

Industrial Research, Delhi. The studies have been conducted in compliance with

Good Laboratory Practices (G.L.P) in accordance to the ‗OECD Guidelines for

testing of chemicals‘ for non-clinical laboratory studies.

Experimental Design :

A batch consisting of three female Wistar rats (Rattus rattus albanicus), each

weighing 160-180 gm were used for each test with different concentration of AMPs

LR14. The selection of animals was random and was carried out at animal facility,

Shriram Institute, New Delhi. Initially an acclimatization period of 5 days was given

to the animals. The animals were administered with the single dose of test substance

(purified AMPs LR14). One control group with vehicle i.e. distilled water was also

included in the plan of work.

Identification of animals

Each cage was tagged having the description of study number, study name, dose,

animal number, date of initiation and date of completion of the experiment. The

animals were also marked with the help of marking ink.

Husbandry and diet

All animals were randomly selected and caged in a group of 3 according to sex in

polypropylene cages fitted with wire mesh tops and having sterilized paddy husk

bedding. The room temperature was maintained at 22±3ºC with 30-70% relative

humidity. The room was ventilated at the rate of approximately 15 air changes per

hour. Lighting was controlled to give 12h artificial light (8 am- 8 pm) each day.

67

Water and standard pelleted feed (Amrut feeds Ltd.) was made freely available to the

experimental animals. There were no known contaminants in the feed and the water at

levels that would have interfered with the experimental results obtained.

Method and frequency of administration

The animals were fasted overnight prior to dosing and for 4h after dosing. A batch

consisting of three female rats was administered with the single dose of AMPs LR14

solution orally at a dose level of 50 mg/kg body weight with the help of cannula

attached with syringe. Similarly, one control group was also administered with the

vehicle i.e. distilled water. Similarly second, third and fourth doses of 300, 1000,

2000 mg/kg body weight, respectively were given to different batches of three rats

each. The test substance (AMPs LR14) was administered once only following

overnight fasting.

Sacrifice and necropsy

All the animals were subjected to necropsy at the end of the observation period, and

all the findings were duly recorded.

Statistical analysis

The test was done in replicates of three animals per batch. Parameters such as body

weight changes were tabulated and analyzed by student‘s ‗t‘- test.

Studies on generation of immune response of AMPs LR14

Purified preparation of the peptide (200 μg/ml) was used to immunize rabbit. Booster

doses (100 μg/ml) were given at an interval of four weeks. AMPs LR14 as antigen

was injected subcutaneously and the rabbit was bled after four months. Blood

collected from the animal was subjected to Enzyme linked immunosorbent assay

(ELISA) in order to detect the formation of antibodies. Different dilutions (10 ng/ml,

100 ng/ml, 1 µg/ml, 10 µg/ml) of antigen (purified AMPs LR14) were added to a

microtiter plate and kept for incubation at 4°C for overnight. The plate was washed

with 0.01 M phosphate buffer pH 7.2. Casein was added to all the wells and incubated

68

at room temperature for 1h. Casein was removed from the wells and washed with

0.01M PBS. The plate was tapped gently on a blotting sheet.

Next, primary antibodies were added row-wise, where 1/10 dilution was added in row

A, 1/100 in row B, 1/500 in row C, 1/1000 in row D, 1/2000 in row E, 1/5000 in row

F and 1/10,000 in row G. In row H, 1/10 pre-bled antiserum was taken as control.

Washing was done again with PB thrice and tapped gently every time. Further,

secondary antibodies (goat antirabbit IgG - Horse radish peroxidase (HRP) conjugate)

were added and the plates were incubated for 1h at 37°C. Again, washing was done

with PB thrice. The substrate o-Phenylenediamine (OPD) at a concentration of

10mg/ml was added to each well and plate was incubated at room temperature for 30

min. Absorbance was taken at 490 nm.

Treatment of wheat seeds with AMPs LR14

The role of AMPs LR14 as a preservative was studied on wheat grains, Triticum

aestivum var. HD 2824, procured from IARI. The grains were sterilized with 0.1%

HgCl2 followed by thorough washing with autoclaved distilled water (3-4 times).

After air drying, they were dipped in AMPs LR14 solution (purified- 1 mg/ml and 2

mg/ml) and another set was kept as untreated control. The time of treatment was

standardized to be 3h. The grains were kept for germination in sterile petriplates laid

with wet Whatman sheets/ moist cotton pads. Sterile distilled water was added

regularly for germination. The plates were kept in an incubator at 25ºC with 60%

humidity.

Following parameters were undertaken to study the effect of AMPs LR14 on wheat

grains:

(1) Seed germination / vigor; (2) Seed viability; (3) Seed health; (4) Seed

carbohydrate and protein content; (5) Residual effect of the peptide

Seed Germination – Twenty wheat grains were kept on Whatman sheets/sterilized

moist cotton pads in a petriplate and checked for germination. The grains were

69

marked as normal, abnormal (sprouting but radicles not growing further), and dead

(non-germinating).

Seed viability – The viability of seeds was arrived at by two methods :

Viability Index I = Germination (%) X seedling length (cm)

Viability Index II = Germination (%) X Dry weight (gm)

Another method used for checking the seed viability was by staining them with

tetrazolium chloride solution. One percent solution of tetrazolium salt in 0.1M

phosphate buffer (pH 7.0-7.2) was prepared and stored in a dark place in a

refrigerator to prevent photo-oxidation.

Wheat grains (treated and untreated) were kept over moist filter paper for 18-24h at

25 ± 1ºC. The grains were bisected longitudinally. The excised grains/embryos were

incubated in 1% tetrazolium chloride solution for 3h in dark such that the seed was

completely immersed into it. The excess solution was drained off, seeds were washed

in distilled water and soaked in 10ml of acetone at 35-40ºC with occasional stirring

till the extraction of colored compound was complete. The seeds were visualized for

stained embryo.

Seed Health – The untreated and treated wheat grains (concentrations: crude AMPs

LR14 - 3 and 5 mg/ml; purified AMPs LR14 - 1 and 2 mg/ml) were kept on PDA

agar plates for 5 days in an incubator at 25 ± 1ºC. The seeds were then screened for

growth of the fungus on or around the grains.

Seed Quality – The total sugar estimation was done by Anthrone method as

described by Brink et al. (1960). Anthrone solution (0.2% in conc. H2SO4) was added

to 500 µl of treated (1 mg/ml and 2 mg/ml) or untreated crushed seeds in 125 ml of

50% ethanol. The solution was heated at 100°C for 7-10 min. It was cooled and OD

was measured at 620 nm.

70

The protein was measured by BCA, as described earlier.

Residual activity of the peptide (AMPs LR14)

Treated seeds of wheat were stored in dry containers at room temperature and the

residual activity was checked after two and a half years. The treated grains were kept

on PDA plates for 6 days, and fungal growth was monitored in comparison to the

untreated control.

Application of AMPs LR14 on mungbean sprouts

The effect of AMPs LR14 on mungbean sprouts was tested as a test case. Mungbean

sprouts were prepared, sterilized with 0.1% HgCl2 and washed thoroughly with sterile

distilled water. Control sets included untreated and uninfected sprouts, and in others

sprouts were infected with ~107 cells/ml of M. luteus and sprouts infected with ~10

5

spores/ml of A. niger for 10 min. For this, the sprouts were suspended in M. luteus

culture and A. niger spore suspension, separately. In different sets, treatment sets

included sprouts treated with purified AMPs LR14 (final concentration – 2 mg/ml)

and treated sprouts were further challenged with M. luteus and A. niger. These sprouts

were kept in different zipper pouches and stored at 4ºC. After 6 days, the untreated

fungus infected and AMPs-treated mungbean sprouts were kept on PDA medium.

The sprouts from different sets were also stored (for 3 months) and the status of

infection was visually monitored as described by Bennik et al. (1999).

Role of AMPs LR14 as free radical scavengers (antioxidants)

Antioxidant compounds scavenge free reactive radicals and thus inhibit the oxidative

mechanisms that may lead to food deterioration. For the assay, 0.8 ml of crude or

purified AMPs LR14 (concentrations ranging from 100 µg/ml to 50 mg/ml) was

added to 1 ml of freshly prepared α, α- Diphenyl- β- picrylhydrazyl radical [DPPH

0.2mM in methanol]. The reaction was allowed to occur for 30 min. The blank was

made by adding HPLC (de-ionized) grade water to DPPH solution. The absorbance

was measured at 517 nm in a UV-Vis Spectrophotometer (Smart-spec plus, Bio-rad,

USA). The scavenging activity of AMPs LR14 was calculated by the formula given

below :

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Scavenging ability = [1-A517 (sample)/ A517 (blank)] X 100%

A standard curve was made with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid (Trolox), a derivative of vitamin E, in the range of 50 µg/ml to 5 mg/ml and the

antioxidant potential of AMPs LR14 and Trolox was compared as described by

Prakash, (2001).

Stability of AMPs LR14 in simulated gastric passage

The protocol of Ghosh et al. (2008) was followed. To simulate the dilution and

possible hydrolysis of AMPs in the human oral cavity, the purified AMPs LR14

(50µg/ml – 850 AU/ml) was diluted 1:1 with a sterile electrolyte solution (described

in Annexure I) to which lysozyme was added at a final concentration of 100 ppm and

incubated for 5 min at 37ºC. One milliliter aliquot was removed, serially diluted and

its activity units were calculated (as described earlier). The rest of the sample was

subsequently diluted 3:5 with an artificial gastric fluid, consisting of the electrolyte

solution, as described in Annexure I (adjusted to pH 2.0) and to which 0.3% pepsin

was added. After 1h of incubation at 37ºC, another 1 ml aliquot was removed, and

activity units calculated. To simulate the digestion in small intestine, the remaining

sample was diluted 1:4 using an artificial duodenal secretion (pH 7.2). One milliliter

aliquots were again removed after 3h, and activity units calculated to determine the

activity (if any) of AMPs LR14. The dilution of AMPs LR14 and the effect of all

lytic enzymes on its own at each step was taken into consideration while checking the

activity of control sets. Also, in the control set, lysozyme was heat inactivated so as to

nullify the inhibitory effect of lysozyme against the indicator strain, M. luteus.

Control solution devoid of pepsin was adjusted to pH 7.0.

Effect of preservative agents (in combination) on the activity of AMPs LR14

The survival of target organisms was monitored in the presence of AMPs LR14 when

combined with salt or sucrose. Approximately 107 cells of M. luteus or E. coli

cultures were grown in NB and LB medium, respectively. Different concentrations of

sucrose or NaCl (2%, 5%, 10%) with or without purified AMPs LR14 (100 µg/ml)

72

were added to these cultures. The latter served as the respective controls. Incubation

was done at 37°C for 18h. Colony forming units/ml (cfu/ml) was estimated for all the

sets. The results were expressed in terms of log10 cfu/ml.

Establishment of Lb. plantarum strain LR/14 in Drosophila gut

Sussex medium was autoclaved and poured in sterile test tubes. Oregon R+ flies were

transferred to the sterile food and incubated at 24°C. The insects were allowed to

complete the generation with visual monitoring everyday. The parental flies were

transferred to another tube to separate them from the next generation flies. From the

offspring, three flies were taken out, their gut was isolated, resuspended and crushed

in phosphate buffer (PBS, pH= 7.4). Hundred microlitre of the suspension was plated

on three media, viz., MRS, LB, and BHI (Brain Heart Infusion, HiMedia), separately.

The plates were incubated at 37°C till the colonies were visible. Some of these flies

were transferred to another tube and raised on sterile Sussex medium for the second

successive generation. Three flies from this generation were subjected to the

dissection and crushing of gut and subsequently plating of the contents on the three

media.

To the autoclaved Sussex medium, Lb. plantarum strain LR/14 cells (~107 cfu/ml)

were added and flies from the last generation were reared on this food. The flies were

visually monitored on a daily basis to look for any changes in their behavior. After

the completion of the generation, three flies were again taken out, their gut isolated

and crushed and plated on MRS medium. Parallely, a control was also taken which

included flies reared on a normal (un-autoclaved) Sussex medium. The gut isolation

and plating of the suspension on MRS medium was done in a similar manner as

described above. The plates were allowed to incubate at 37°C for 2 days. A single

colony was picked from each plate and patched on a master plate. To ensure that the

bacterial colonies appearing on MRS were due to the presence of associated bacteria

only to the gut, a non-target organ, such as ovary was also dissected and crushed and

plated on the media (LB, MRS, BHI).

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Next, genomic DNA was isolated, using Gen Elute Bacterial Genomic DNA isolation

kit (Sigma-Aldrich, USA) for both sets of gut bacterial colonies as well as Lb.

plantarum strain LR/14 as a control. They were labeled as DNA from :

(A) – Bacteria isolated from gut of Drosophila reared on normal unautoclaved food

(B) - Bacteria isolated from gut of Drosophila reared on strain LR/14-containing

food

(C) – Lb. plantarum strain LR/14

A PCR was set up using six different sets of primers. These comprised: bile salt

hydrolase (bsh), 16S rRNA gene, ABC transporter gene, mannose binding protein

domain gene (mub), pln EF, and pln JK (Table I, II, III). The amplicons were

resolved on 1% agarose gel made in 1X TAE buffer containing EtBr. The gel was run

at 70 Volts for 1.5h. The bands were visualized in a UV transilluminator and

photographed (BioRad, USA). The reaction components and conditions used are

listed in Table I and II. PCR amplification was done for 30 cycles in PCR machine

(BioRad, USA).

Table I. PCR reaction components added in 25 µl volume

Reagent Amount Final

concentration

Water Variable -

10X PCR Buffer 2.5 µl 1X

25 mM MgCl2 1 µl 1.0 mM

2.5 mM dNTP mix 0.2 µl 250 µM each dNTP

10 pmol/µl Forward primer 1 µl 0.4 pmol/µl

10 pmol/µl Reverse primer 1 µl 0.4 pmol/µl

DNA template 20 ng 2 ng/µl

DMSO 1.25 µl 5%

Taq polymearse 0.1 µl 0.05 units/µl

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Table II. Reaction conditions used for PCR

Conditions Temperature Time

Initial Denaturation 94°C 5 min

For 30-35 min :

Denaturation 94°C 15 sec- 1 min

Annealing 57-63°C 30 – 90 sec

Extension 72°C 30 sec – 2 min

Final Extension 72°C 7 min

Hold 4°C ∞

Table III. Primer sets used in the study

Name of the primer Sequence Tm

BSH (F) 5‟-TGTGTACTGCCATAACTTATCAATCTT-3‟ 60 °C

BSH (R) 5‟-TGTGCTTCTGATCGTAATGGA-3‟

ABC (TRNP)(F) 5‟-AAAGCATGCTGAGGACAACGAATCCTGAA-3‟ 61 °C

ABC (TRNP)(R) 5‟-AAATCTAGACATATTGAACGGCTTCAACG-3‟

mub (F) 5‟-CGTTGTATGGCAGGATGATG-3‟ 60 °C

mub (R) 5‟-GCGGTCGTTCCTACTGGATA-3‟

16S r RNA (F) 5‟-TGCCTAATACATGCAAGT-3‟ 57 °C

16S r RNA (R) 5‟-CTTGTTACGACTTCACCC-3‟

Pln EF (F) 5‟-ATAAAGCTTCTTGGATTTGGTATCTGTTTCG-3‟ 60 °C

Pln EF (R) 5‟-ATAGGTACCGGAAAACGCCCCTGAAATA-3‟

Pln JK (F) 5‟-ATAAAGCTTTAATCCCTTGAACCACCAAG-3‟ 60 °C

Pln JK (R) 5‟ATATCTAGATAAGTTGAACGGGGTTGTTG-3‟

* (F) - Forward

* (R) - Reverse

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Statistical analysis

All the experiments were carried out in three independent sets, each consisting of 3

replicates. Values shown here represent mean ± standard error of the mean (SEM).

SEM was calculated as follows:

where ‗s‘ is the standard deviation of the population and ‗n‘ is the sample size and ‗s‘

is given by the formula:

Where xi represents the individual readings and x represents mean of individual

readings.

Test of significance

The t-test assesses whether the means of two groups are statistically different from

each other.

where xT and xC are the readings of treated and control, respectively. VarT and VarC are

the variance of treated and control, respectively, and n is the number of readings. The

t-test was done using the software QuickCalcs (t-test calculator) at the site

http://graphpad.com/quickcalcs/ttest1.cfm.