Lantibiotic- a Bacteriocin from Lactobacillus casei using

PSGCAS Search: A Journal of Science and Technology Volume: 3 No. : 2, ISSN: 2349 – 5456 38

Lantibiotic- a Bacteriocin from Lactobacillus casei using Whey Substrate

and it’s Antimicrobial Activity against Common Food Pathogens

*Krishnaveni. N1, Cibi Chakravarthy

2.

1Department of Microbiology, PSG College of Arts & Science, Coimbatore, India

2Department of Plant Pathology, TNAU, Coimbatore, India

*Corresponding Author:[email protected]

ABSTRACT

Bacteriocins are proteinacious compounds produced by bacteria that show a bactericidal

mode of action against closely related species. Bacteriocins from gram-positive bacteria, the

Lactic acid bacteria (LAB) are of great industrial importance and are studied for new and safe

food preservatives. Bacteriocin producing lactic acid bacteria – LANTIBIOTIC can be used as

food additive that may inhibit some food spoilage and pathogenic bacteria. In the present study,

Lactobacillus caseiwas screened from dairy industry waste product paneer whey. The Lactic

acid bacteria obtained was characterized, and studied for bacteriocin production. The

bacteriocin protein produced was partially purified, and its protein content was estimated. The

bacteriocin protein was tested for its antagonistic action against common food spoilage and

pathogenic organisms. The bacteriocin protein content was estimated to be 300µg/ml of the

media. The bacteriocin protein was found to be effective against Pseudomonas flourescens,

Salmonella typhi, Salmonella paratyphi, Staphylococcus aureus, and Clostridium sp.

Keywords: Bacteriocin, Lantibiotic, Lactobacillus casei, bacteriocin action

INTRODUCTION

Bacteriocins from generally

recognized as safe bacteria (GRAS) have

aroused a great deal of interest as a novel

approach to food-borne pathogens in foods.

These bacteriocin proteins show bactericidal

mode of action against related species. The

bacteriocins are found to afford the producing

culture a competitive advantage of

environmental resources over other bacteria

existing in the same ecological niche.

Bacteriocins of LAB have become a major

area of study in the search for new and safe

food preservatives. These lanthionine-

containing antibiotic peptides are called

‘LANTBIOTICS’ [1]

.

Members of a physiologically related

group of gram-positive bacteria, known as the

Lactic Acid Bacteria (LAB) are of great

industrial importance, mainly in food

fementation processes. The interest in

Lactobacillus species, results from its

application as a starter culture for the

fermentation of vegetables, meat and fishes.

Organisms inhibited by these substances

include food borne pathogens and proves to

Lantibiotic - a Bacteriocin from Lactobacillus casei using Whey Substrate and it’s Antimicrobial Activity against Common Food

Pathogens


be important due to its antagonistic use

against spoilage organisms and its use as a

food preservative [2]

.

Milk is one of the most ‘complete

foods’ for mammals as well as

microorganisms. Raw milk from a healthy

cow has no microorganisms. However, these

organisms are rapidly acquired from dairy

utensils and equipment and from cow shed

environment that include organisms like

lactobacilli, streptococci, and coliforms.

Lactic acid bacteria are natural chance

contaminants from environment to milk and

dairy products, due to its rich carbohydrate,

protein and vitamin content. They have been

identified from various milk products from

dairy industries. Traditionally whey is

considered to be a waste product being used

as either animal feed or sprayed on fields as

fertilizer. Sometimes the whey is nearly

disposed off into nearest water bodies or land

with or without treatment. Hence there has

been intense interest in the use of whey as a

base for a wide variety of process yielding

different valuable products [3]

.

The present paper reports the

production, characterization and action on

bacterial cells of the bacteriocin synthesized

by Lactobacillus casei isolated from paneer

whey source. The study summarizes the focus

on biosynthesis of lantibiotic from whey

source, conditions for bacteriocin production,

partial purification and antimicrobial assay on

various test pathogens.

MATERIALS AND METHOD

Whey which is a by-product of dairy

industry is 93% water, 0.7% protein, 0.3% fat,

4-5% lactose, and 0.5-0.6% salts. This high

quality medium for the microbial growth

offers an abundant energy supply (lactose)

and fixed nitrogen (protein) source. Hence

lactose utilizing organisms could significantly

be isolated from this dairy waste source [3]

.

Isolation of Lactobacillus

All the dairy products carry lactic acid

bacteria as habitants. Hence a dairy product

was chosen as substrate for identification of

Lactic acid bacteria. Whey being a dairy

waste product, the present research has

focused to have an effective utilization from

this waste; whey was chosen as the media for

isolation of Lactobacillus species.

Preparation of Whey

Milk collected aseptically was

pasteurized at 630C for 30 min and allowed to

cool to about 400C -50

0C. To separate the

solid part from the milk 5-10% (v/v) 2% citric

acid was added directly to the milk. Thus the

solid part paneer precipitated and the liquid

part whey was separated. The precipitates

were collected by filtration through a muslin

Krishnaveni. N, CibiChakravarthy


cloth and thus clear whey wasseparated out.

The separated whey sample was allowed for

fermentation overnight and further preceded

for isolation procedures.

Enrichment of sample

To enrich Lactobacillus casei the

whey sample was used as the inoculum. MRS

medium selectively enriches the identification

of lactobacillus and hence MRS (Man,

Ragosa and Sharpe) media was used for the

isolation of lactobacillus. Since most

lactobacilli are microaerophilic, they grow

better either anaerobically or in the presence

of increased CO2 tension. Agar plates were

incubated in anaerobic jars using H2 + CO2

generating kits. 1ml of paneer whey was

transferred to 99 ml of MRS broth media and

incubated at 370C for 24 hrs. This was used as

an enriching media for identification of

lactobacillus isolates [4, 5]

.

Isolation of Lactobacillus

To isolate Lactobacillus casei, 0.1 ml of the

enriched culture obtained was spread on

precooled MRS medium and incubated

anaerobically at 370C for 24 hrs. After 24 hrs

of incubation, morphologically dissimilar

colonies were picked randomly and streaked

on SL medium for the selective isolation of

Lactobacillus species [6]

.

Identification of Lactobacillus casei

For the species identification of lactobacillus,

various biochemical tests were performed

following the procedures as described in

Bergey’s manual [7]

. In addition to Gram

staining the other suitable biochemical tests

involved for the identification were;

Growth on Selective media: Growth on

acetate medium (SL) in microaerophilic

conditions at 370C or 24 hrs was performed

and observed for characteristic growth and

morphology.

Gram staining: Each type of colony in MRS

and SL medium were taken for gram staining.

Biochemical characterization: The

biochemical sign points are properties

controlled by cells enzymatic activity and

they are responsible for bioenergetics,

biosynthesis and biodegradation and defined

to be the cellular metabolism and aids in

identification and confirmation of

Lactobacillus casei from the selective media.

Gelatin hydrolysis, H2S production, Indole

production test, Catalase test, Oxidase test,

Carbohydrate fermentation pattern were

performed. Gram staining, growth in selective

media under anaerobic conditions and these

biochemical tests like catalase test confirmed

the strain to the genus Lactobacillus. Further

tests like, growth pattern of the bacteria at

150C and 45

0C, and Raffinose utilization were


Pathogens


the tests done to confirm the species as

Lactobacillus casei[5, 8]

.

Preparation of Crude Bacteriocin

To prepare crude bacteriocin from the

Lactobacillus casei, the isolates were grown

in 1 litre of MRS broth medium with 3%

whey concentration and 5% initial inoculum,

by batch fermentation at pH 6.2 and 370C for

about 10-12 hrs (Log phase cultures). For the

separation of bacteriocins the culture was

centrifuged at 8000 rpm for 20 minutes and

the active cell free supernatants were

subjected to salt precipitation followed by

dialysis for partial purification at 40C

[9].

Protein estimation by Lowry’s method

The partially purified proteins after

desalting were estimated quantitatively for

protein by adapting the procedure given by

Lowry et al., (1951) [9]

.

Estimation of bactericidal activity

The partially purified proteinaceous

compound from Lactobacillus casei was

analysed for its bactericidal activity against

common pathogens involved in food spoilage.

The bacterial pathogens selected to test the

inhibitory effect of lantibioticbacteriocin

were; Bacillus subtilis, Clostridium

botulinum, Corynebacteriumdiphtheriae,

Escherichia coli, Pseudomonas flourescens,

Salmonella paratyphi, Salmonella typhi,

Serratiamarcescens, Shigellaflexneri, and

Staphylococcus aureus. (Pathogens were

obtained from clinical isolates from hospitals

and spoiled food).

To test the bactericidal effect of

bacteriocin various concentrations of crude

protein preparations were made using water as

a solvent. To find out the Minimal Inhibitory

concentration (MIC) of bacteriocin, the

bacteriocin were diluted to get a concentration

of 100, 200, 300, 400, 500, 600 µg/ml. After

the confirmation of MIC, further bactericidal

activity was estimated by agar disc diffusion

technique for the concentrations of 500, 1000,

1500 µg/ml of bacteriocin[10]

.

Preparation of Test culture

All the test cultures were grown in

nutrient broth medium, and incubated at 370C

for 10 hrs to get log-phase cultures.

Estimation of MIC

To estimate the MIC for the above

cultures, 1 ml of each log phase cultures were

transferred into a test tube containing 8 ml of

nutrient broth. 1 ml of appropriate

concentrations of bacteriocins (100, 200, 300,

400, 500, 600µg/ml) were made and added to

the test tube into nutrient broth. All the

cultures were incubated at 370C for 24 hrs.

Control tubes carried 1 ml of culture, 8 ml of

nutrient broth and 1 ml of sterile distilled

water. The MIC was estimated by measuring



the optical density of the cultures at 660 nm

using spectrophotometer [11]

.

Agar disc diffusion techniques

Preparation of discs at various

concentrations

To test the bactericidal activity of

bacteriocin, sterile discs were made by 6 mm

of whattman No.1 filter paper. Appropriate

concentrations of bacteriocin in the disc (500,

1000, 1500 µg/ml) was made by dipping the

sterile discs into the respective concentrations

of bacteriocin for 5 minutes. Then the discs

were removed and dried at room temperatures

for 15 minutes.

The lawn of Log phase cultures of

each test pathogen was made separately on

MRS agar plates. The discs with appropriate

concentrations were placed on the lawn and

incubated at room temperature for 24 hrs.

After 24 hrs, the bactericidal activity was

measured by the presence of inhibitory zone.

The bacteriocin showing inhibitory effect

against the specific test pathogen, by

formation of cleared zone of inhibition

surrounding the discs, were considered as

“Sensitive” and absence of inhibitory zone

observed against the test pathogen was

considered to be “Resistant” to the test

concentration of bacteriocin. The effect of

bacteriocin activity was thus estimated [12]

.

RESULTS AND DISCUSSION

The current emphasis stands to report

the isolation and characterization of the

lantibiotic producing strain Lactobacillus

caseifrom paneer whey source. The process

has evidenced production of bacteriocin from

the Lactobacillus casei isolate and the partial

purification of bacteriocin protein. The main

theme of the study reports on measures of

antibacterial activity of the bacteriocin against

common food pathogens. Reports have been

summarized as follows;

Identification of Lactobacillus:

The identification of Lactic acid

bacteria involved screening of the inoculum

by appearance of colonies and observation of

morphological features of various colonies.

The colonies appeared to be small, convex,

smooth, glistening and opaque in nature.

Suspected colonies were subjected to various

biochemical tests for identification.

Growth on Selective Media:

The morphologically similar colonies

were cultured on SL agar medium. Selective

SL medium was used for initial screening and

their appearance of growth on SL medium

was found to show small, smooth, convex

glistening, opaque colonies.

Gram Staining:

Gram staining was performed. The

suspected colonies showing long, straight or


Pathogens


slightly crescent gram positive short rods or

coccobacilli were selected, further grown on

MRS media and tested for biochemical

characteristics (Table 1).

Biochemical Tests:

(i) Gelatin Hydrolysis: The gelatin medium

remained in the solid state after 40C

refrigeration of the test organism, hence

no gelatin hydrolysis observed.

(ii) H2S production: No black colouration

was observed at the stab area in SIM agar

media. Hence no H2S production

observed.

(iii)Indole Production: No red or cherry red

coloured ring formed, hence the indole

compounds were not produced thus

proving the isolates indole negative.

(iii)Catalase Test: Upon addition of H2O2,

no effervescence was observed on the

fresh colonies on media. Thus catalase

enzyme was not produced by the isolate

proving it catalase negative.

(iv) Oxidase test: No purplish blue colour

formation was observed on oxidase discs

placed on fresh colonies on media. Thus

oxidase enzyme was not produced proving

it oxidase negative.

The biochemical characters, gram staining

and morphological appearance confirmed the

genus as Lactobacillus. Further screening for

Lactobacillus casei was done by the following

tests.

Carbohydrate Fermentation:

The carbohydrate fermentation pattern of

the lactobacillus isolate when observed, the

isolate showed fermentation of cellobiose,

fructose, galactose, glucose, lactose, maltose,

mannitol, mannose, ribose, sorbitol, starch

and sucrose. Fermentation was not observed

in raffinose, rhamnose and xylose (Table 2).

Table 1

Biochemical test results of Lactobacillus isolate from Paneer Whey

S. No Tests Done Results observed Inference

1 Gram Staining Gram-positive short rods

2 Gelatin Hydrolysis Remained solid at 40C No Gelatin Hydrolysis

3 H2S production No black colour formed No H2S production

4 Indole production No red ring observed No Indole production

5 Catalase Test No effervescence observed No Catalase production

6 Oxidase Test No Purplish Blue colour

change of the disc observed

No Oxidase production

7 Growth at 150Cand

450C

Turbid growth at both the

temperatures observed

Confirms the species as

Lactobacillus casei



Table 2

Carbohydrate fermentation test results of Lactobacillus isolate from Paneer Whey

S. No Carbohydrate

fermentation

Tests Done

Results

observed

Carbohydrate

fermentation

Tests Done

Results

observed

1 Cellobiose +ve Melezitose +ve

2 Esculin +ve Raffinose -ve

3 Fructose +ve Rhamnose -ve

4 Galactose +ve Ribose +ve

5 Gluconate +ve Sallicilin +ve

6 Glucose +ve Sorbitol +ve

7 Lactose +ve Starch +ve

8 Maltose +ve Trehalose +ve

9 Mannitol +ve Sucrose +ve

10 Mannose +ve Xylose -ve

11 Mellibiose -ve

Confirmation of the Species:

The Raffinose sugar was not utilized

by the isolate. The carbohydrate fermentation

pattern revealed the species character of the

isolate. Moreover the final distinguishing

aspect of the growth at 150C and 450C on

MRS media andraffinose non utilization

confirmed the species to be Lactobacillus

casei. The organism of interest had been thus

isolated from paneer whey source.

Bacteriocin Production:

The isolated strain of Lactobacillus casei was

subjected to bacteriocin production by log

phase culture upon MRS media at

microaerophillic condition for 48 hrs. The

proteins were separated and partially purified

by ammonium salt precipitation followed by

membrane dialysis for bacteriocin separation.

Estimation of Protein content by Lowry’s

method:

The protein content of the bacteriocin

was estimated by Lowry’s method, using

Bovine Serum Albumin as standard and the

concentration of bacteriocin protein was

found to be 300µg/ml (Figure 1).

Antibacterial activity of Bacteriocin

protein:

The partially purified bacteriocin

protein was tested for its bactericidal action

against various food pathogens.

MIC determination Observations:

Minimum Inhibitory Concentration of

bacteriocin against various pathogens was

tested, the optical density values for MIC at

660 nm have been recorded and the results

have been tabulated in Table 3.


PSGCAS Search: A Journal of Science and Technology

Figure 1. Concentration of Bacteriocin protein by Lowry et al., method

Table 3 Minimum Inhibitory Concentration of Bacteriocin

S.

No.

Concentration

of Bacteriocin

in µg/ml a b

1 100 0 0

2 200 0 0.01

3 300 0 0.05

4 400 0.01 0.07

5 500 0.01 0.1

6 600 0.02 12

a. MIC for Bacillus subtilis,

b. MIC forCorynebacteriumdiphtheriae,

e. MIC for Pseudomonas flourescens,

g. MIC for Salmonella typhi,

i. MIC forShigellaflexneri, and

The results of MIC revealed that all

bacteriocin samples tested for lower

concentration up to 600µg/ml showed very

low inhibition and turbidity. Very few

pathogens like Clostridium

paratyphi, showed specific

inhibition. As reported by Maria et al (1993)

the findings showed that for

pathogenic organisms these MIC

a Bacteriocin from Lactobacillus casei using Whey Substrate and it’s Antimicrobial Activity against Common Food

Pathogens

PSGCAS Search: A Journal of Science and Technology Volume: 3 No. : 2, ISSN: 2349

Concentration of Bacteriocin protein by Lowry et al., method

Minimum Inhibitory Concentration of Bacteriocin

MIC for pathogens (Optical Density values at 660 nm)

b c d e f g h

0 0 0 0 0 0 0

0.01 0 0 0 0 0 0

0.05 0 0 0.01 0 0.01 0

0.07 0 0 0.03 0.03 0.01 0

0.1 0 0 0.05 0.06 0.01 0

12 0 0 0.08 0.07 0.02 0

Bacillus subtilis, b. MIC for Clostridium botulinum,

Corynebacteriumdiphtheriae, d. MIC for Escherichia coli,

Pseudomonas flourescens, f. MIC for Salmonella paratyphi,

Salmonella typhi, h. MIC forSerratiamarcescens,

Shigellaflexneri, and j. MIC for Staphylococcus aureus

revealed that all the

bacteriocin samples tested for lower

µg/ml showed very

dity. Very few

Clostridium, Salmonella

showed specific pattern of

inhibition. As reported by Maria et al (1993)

the findings showed that for the test

pathogenic organisms these MIC

concentrations have to be increased to obtain

a better inhibitory activity.

Agar Disc Diffusion Technique

The agar disc diffu

increased concentrations of partially purified

bacteriocin protein were done. The

concentration of protein taken

/ml, 1000 µg/ml, 1500 µg/ml for all the test

pathogens (Table 4 and Fig.2).

a Bacteriocin from Lactobacillus casei using Whey Substrate and it’s Antimicrobial Activity against Common Food

ISSN: 2349 – 5456 45

Concentration of Bacteriocin protein by Lowry et al., method

Minimum Inhibitory Concentration of Bacteriocin

MIC for pathogens (Optical Density values at 660 nm)

h i j

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0.04 0

Clostridium botulinum,

Escherichia coli,

Salmonella paratyphi,

Serratiamarcescens,

Staphylococcus aureus

concentrations have to be increased to obtain

Agar Disc Diffusion Technique:

The agar disc diffusion tests with

increased concentrations of partially purified

bacteriocin protein were done. The

of protein taken was 500 µg

/ml, 1000 µg/ml, 1500 µg/ml for all the test

pathogens (Table 4 and Fig.2).



Table 4

Agar Disc Diffusion Assay of Bacteriocin against pathogens S.

No Observation of inhibitory zone Concentration of Bacteriocin in µg/ml

Agar Disc Diffusion Assay of

Bacteriocin against pathogens

(Zone of Inhibition)

500 1000 1500

1 a No inhibitory

zone Unclear zone of

inhibition Clear inhibitory zone

of �4mm 2 b Borderline zone

of inhibition Visible zone of


3 c No inhibitory

zone Borderline zone

of inhibition Clear inhibitory zone

4 d No inhibitory

zone No inhibitory

zone No inhibitory zone

5 e Borderline zone

of inhibition Unclear zone of


of �5mm 6 f Borderline zone

of inhibition Less inhibitory

zone Clear inhibitory zone

7 g No inhibitory

zone Less inhibitory

zone Visible zone of

inhibition 8 h No inhibitory

zone No inhibitory

zone Very less unclear

Borderline zone of

inhibition 9 i Unclear zone of

inhibition Unclear zone of

inhibition Unclear zone of

inhibition 10 j No inhibitory

zone Borderline zone

of inhibition Clear inhibitory zone

a. Bactericidal action of bacteriocin against Bacillus subtilis,

b. Bactericidal action of bacteriocin against Clostridium botulinum,

c. Bactericidal action of bacteriocin against Corynebacteriumdiphtheriae,

d. Bactericidal action of bacteriocin against Escherichia coli,

e. Bactericidal action of bacteriocin against Pseudomonas flourescens,

f. Bactericidal action of bacteriocin against Salmonella paratyphi,

g. Bactericidal action of bacteriocin against Salmonella typhi,

h. Bactericidal action of bacteriocin against Serratiamarcescens,

i. Bactericidal action of bacteriocin against Shigellaflexneri, and

j. Bactericidal action of bacteriocin against Staphylococcus aureus


Pathogens


A. Salmonella paratyphi B. Salmonella typhi A. Clostridium botulinum B. Serratriamarcescens

A. Escherichia coli B. Shigellaflexneri A. Coryunebacterium sp., B. Staphylococcus aureus

A.PseudomonasFlourescens B. Bacillus subtilus

Figure 2. Agar Disc Diffusion Assay of Bacteriocin against test pathogens

SUMMARY AND CONCLUSION

The bacteriocinic peptides are of great

importance due to their bactericidal activity

against common food pathogens. The cheaper

dairy product paneer whey acts as a superior

source for isolation of the bacteriocin

producing strain of interest Lactobacillus

casei. MRS growth media facilitated

identification and isolation and the selective

SL media and has enhanced specific

identification of Lactobacillus. Gram staining

and other biochemical characterization rooted

the confirmation of the isolated organism to

be Lactobacillus casei. The isolates were

enriched for growth in MRS broth to obtain a

log phase culture and the cell free



supernatants were collected. Supernatant was

subjected to salt precipitation followed by

membrane dialysis for partial purification of

bacteriocin protein. The partially purified

bacteriocin thus obtained were analysed for

bactericidal activity against common range of

food pathogens.

The MIC pattern for test pathogens

was identified for bacteriocin concentrations

of 100, 200, 300, 400, 500, 600 µg/ml against

test pathogens. This revealed that the

pathogens required higher concentrations of

bacteriocins for greater inhibition, thus the

concentration was increased for agar disc

diffusion method. Agar disc diffusion

technique for concentrations 500, 1000, 1500

µg/ml of bacteriocin showed inhibitoy zone

with bactericidal activity against test

pathogens. The “Sensitivity” or “Resistance”

of the pathogens to the bacterocinwere

measured. The bacteriocin LANTIBIOIC

from producing culture showed inhibitory

activity against closely related genera. As per

the results observed the higher concentrations

of 1500 µg/ml have proven to be quite good

at action showing clear zone of action upon

the lawn of test pathogen, whereas at 500

µg/ml concentrations very few organisms as

Salmonella paratyphi, Clostridium botulinum

showed borderline inhibitory activity with

less margin of inhibition surrounding the disc.

Among the common food pathogens

employed for the present study, Salmonella

paratyphi, Salmonella typhi, Clostridiun

botulinum, Corynebacterium sp.,

Staphylococcus aureus, Pseudomonas

flourescens and Bacillus subtilis showed clear

sensitivity to higher concentrations of

bacteriocin. The other gram negative forms

Escherichia coli, Serratiamarcescens,

andShigellaflexneri showed resistance to the

concentrations of bacteriocins employed.

More concentrations might improve its

antimicrobial activity.

The report confess that the

bacteriocins of gram positive bacteria are less

active on gram negative organisms, though

they show active inhibitory effect on gram

positive organisms. The bacteriocins being

inhibitive, might thus find vast field of

application as preservative or additive in food

substances or any other commercial

application as needed.

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factors produced by lactic acid bacteria.

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2. Tagg JR., Dajani AS., and

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3. William C. Frazier and Denis Westhoff C.,

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4. Daeschel MA., 1990. Applications of

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5. DaeschelMA.,McKenney MC., McDonald

L.C., 1990. Bactericidal activity of

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Lantibiotic- a Bacteriocin from Lactobacillus casei using