Isolation and Partial

Purification of

Bacteriocins from

Milk & Milk Products

-SANYA SHANGARI

The use of non-pathogenic micro-organisms and their metabolites to improve microbiological safety and extended shelf life of foods is defined as bio preservation.

Bacteriocins are ribosomally synthesized polypeptides possessing bacteriocidal activity that are rapidly digested by proteases in the human digestive track.

◦ Bacteriocins are proteinaceous toxins produced by bacteria to inhibit growth of similar or closely related producer bacterial strain(s) (producer cells are immune to their own bacteriocin) a natural competitive means to overcome other micro-organism sharing same niche among them.

Bacteriocins have a narrow host range, and is likely to be most

effective against related bacteria with nutritive demands for

the same scarce resources.

INTRODUCTION

Bacteriocins versus

Antibiotics

BACTERIOCINS ANTIBIOTICS

Applied in food sector. Applied clinically.

Synthesized ribosomally Synthesize as secondary

metabolites.

Narrow spectrum activity. Varying spectrum.

Host cell are immune. Host cell are not immune.

Mechanism of target cell

resistance is usually adaptive of

affecting cell membrane

composition.

Mechanism of target cell resistance

is genetically transferable

determinant affecting different

sites.

Interactions require docking

molecules.

Interaction require specific target.

Mode of action is pore formation

usually, but in some case cell wall

biosynthesis.

Cell membrane or intracellular

targets.

No side effects are known. Side effects are known.

Classification CLASS CHARACTERISTICS EXAMPLES

Class 1 •Small membrane active peptides (<5kDa)

containing Lanthionine.

•Induce pore formation

•Fairly broad inhibitory spectrum

•Lactobacillus lactis- nisin

Lactobacillus sake- lactocin S

Bacillus subtilis- mersacidin

• Inhibit Enterococcus, Lactobacillus,

Lactococcus, Leuconostoc,

Pediococcus and Streptococcus. Also

Bacillus cerus, Clostridium

botulinum, Staphylococcus aureus.

Class 2 •Small heat stable, non-lanthionine containing

and membrane active peptides (<10kDa).

•Function by creating barrelstave like pores or

a carpet mechanism whereby peptides orient

parallel to the membrane surface and interfer

membrane structure.

•Narrow activity spectra

•Pedicoccus acidilactici- pedocin

Lactobacillus sake – sakarcin A and

sakacin P

Leuconstoc mesenteroids-

mesentericin

•Inhibit Enterococcus, Lactobacillus,

Pedococcus. (Lactococcus are

resistant)

Class 3 Heat liable protein of large molecular mass Lactobacillus heleveticus –

helveticin T

Class 4 •Complex protiens, associated with other

lipid and carbohydrate moities.

•Relatively hydrobhobic and heat stable

•Bacteriocin producing bacteria have been isolated from a variety of habitats including

soil, food, and the human body .

•Gram-negative and gram-positive species of bacteria have been found to produce

them.

•Bacteriocins that contain the modified amino acid lanthionine as a part of their

structure are called lantibiotics.

•Bacteriocin producing LAB have attracted significant attention because of their GRAS

status and potential use as safe additives for food preservation.

•Nisin, produced by Lactococcus lactis, is the most thoroughly studied bacteriocin to

date and has been applied as an additive to certain foods worldwide. Substantial work

has been done on the effectiveness of nisin on various spoilage and pathogenic

microorganisms such as Listeria monocytogenes and its application in different food

products .

Method

Collection of

samples

Subculture of

test pathogens

Isolation and

development of pure

culture of bacterial

isolates

Identification

of bacterial

isolates

Preparation of

cell free

extract

Antagonistic

activity

Partial

purification of

Bacteriocin

Materials & Method • Milk, curd and buttermilk streaked on

nutrient agar plate.

• Twelve unidentified samples revived.

Collection of

samples

• Bacillus subtilis, Escherichia coli,

Klebsiella pneumonia and Staphyloccus

aureus subcultured in nutrient broth

and incubated at 37◦C.

Subculture of test

pathogens

• Serial dilution & Inoculated on nutrient

agar and incubated at 37◦C for 24 hours.

• Pure cultures developed by subsequent

sub culturing.

Isolation and

development of

pure culture of

bacterial isolates

Identification of

bacterial isolates

• Gram staining

• Methyl red test

• Voges- Proskauer test

• Catalase test

• Oxidase test

Colonies Isolated after Serial Dillution

Pure Isolates on Nutrient Agar Plate

Gram Staining

A smear of bacteria was prepared

on clear and sterile drop of

distilled water

Smear was heat fixed and slide

was processed for Gram staining

i.e. Crystal violet, iodine, ethanol

and safranin .

The stained slide was observed

u der light icroscope for Gra ’s: reaction, cell shape, size etc.

Gram Positive Rods

Gram Negative Rods

Methyl Red Test The test needs 2 days of

incubation to ensure the

presence of stable acids

The test needs 2 days of

incubation to ensure the

presence of stable acids.

Methyl red is red at pH less

than 5 and yellow at pH

greater than 6

Methyl Red Test

Voges-Proskauer Test 5ml Glucose phosphate broth was

inoculated with respective isolates and

incubated at 37 ̊C for 48 hours.

One ml of 40% Potassium Hydroxide

containing 0.3% Creatine and 3ml of 5%

solution of Alpha-naphthol in absolute

alcohol was added to each tube.

Appearance of pink in 2-5 min indicated

positive reaction. Voges-Proskauer Test

Catalase Test

Few drops of 3% H2O2 were

mixed in culture tubes

containing cell suspension.

Appearance of air

bubble will be

consider it positive

Catalase Test

Oxidase Test Place a drop of freshly prepared 1%

oxidase reagent (Tetramethyl-p-phenyl

diamine hydrochloride) on a piece of

filter paper.

Few colonies of the isolated microbes

were rubbed over it with help of a

sterile loop.

Development of deep blue colour

within 10 seconds indicated a positive

reaction

Oxidase Test

Preparation of Cell Free Extract Isolates were inoculated in nutrient broth at 37 ̊C for 24

hours.

The broth was centrifuged at 15000 rpm for 15 min.

The supernatant was then neutralized by 1N NaOH and 1N HCl

followed by membrane filtration (using 0.45 µm filter) to obtain

cell free extract

This solution was further used as the crude bacteriocin

for assay of antagonistic activity.

Antagonistic Activity of Crude Bacteriocin against selected Pathogens

Antagonistic activity of the crude bacteriocin was assayed by using the agar well diffusion method

10µl of selected test pathogens namely

Escherichia coli, Bacillus subtilis,

Staphylococcus aureus, Klebsiella

pneumoniae were spread on solidified

nutrient agar and then wells were

created by using 10 mm borer.

200µl of crude bacteriocin was added

in the well.

The plates were examined for clear

zone after 24 hours of incubation

Antagonistic Activity of Crude Bacteriocin against Selected Pathogens

Antagonistic activity in Escherichia coli Antagonistic activity in Klebsiella pneumonia

Antagonistic activity in Bacilus subtilis Antagonistic activity in Staphylococcus aureus

Partial purification of Bacteriocin

24 hour broth culture was centrifuged at 15000

rpm for 15 min, subsequently the filtrate was

neutralized by using 1N NaOH and 1N HCl and

filtered.

Ammonium Sulphate was mixed to the filtrate for

saturation purpose and incubated for 18 hour with

continuous stirring at 4 ̊C.

The mixture was further centrifuged at 15000 rpm

for 30 min. The pellets were re-suspended in 10 ml

of 10 M Sodium Phosphate buffer (pH- 7) and

taken in dialysis membrane.

The dialysis membrane was inserted in beaker

containing 2L of same buffer and was kept for

overnight incubation Partial Purification by Dialysis

Preparation of Media Composition Used S.no. Media Constituents Amount

1 Nutrient agar

(pH-6.8)

Peptone 5g

Beef extract 3g

Agar 15g

NaCl 5g

Distilled water 1000ml

2 Nutrient broth Beef extract 1g

Yeast extract 2g

Peptone 5g

Sodium chloride 5g

Distilled water 1000ml

3 Glucose phosphate peptone

water

Peptone 5g

K2HPO4 5g

Glucose 5g

Distilled water 1000ml

Preparation of Reagents Used 1. Crystal Violet (a) crystal violet 1 g

Absolute alcohol 10 ml

Distilled water 100 ml

(b) ammonium oxalate 5 g

Distilled water 100 ml

Take 30 ml of (a) and 8 ml of (b)

2. Mordant (Iodine) Iodine 1 g

Potassium iodide 2 g

Distilled water 100 ml

Dissolve 2 g of (a) in 25 ml of distilled water and add 1 g of iodine. Than make the volume up to 100 ml.

3. Counter Stain

(Basic Fuschin)

Basic Fuschin 0.05 g

Distilled water 100 ml

4. Hydrogen Peroxide (3%) Hydrogen peroxide 3 ml

Distilled water 97 ml

5. Oxidase Reagent (1%) Tetramethyl-p-phenylene-

diaminedihydrochloride

1 g

Distilled water 100 ml

6. Phosphate Buffer

Stock solution (A) Monobasic sodium phosphate 31.2gm in 1L

Stock solution (B) Dibasic sodium phosphate 28.39gm in 1l

RESULTS

Antagonistic Activity of Crude Bacteriocins against Pathogenic Bacteria

S.no Samples Pathogenic Strains

Zone diameter

(size in mm)

Borer size =10mm

Bacillus subtilis Klebsiella pneumonia Escherichia coli

1 Standard

antibiotic

(Ciprofloxacin)

15mm 16mm 17mm

2 MRL 1 0mm 16mm 0mm

3 MRL2 21mm 18mm 0mm

4 MRL3 0mm 0mm 0mm

5 MRL4 16mm 13mm 0mm

6 MRL5 0mm 0mm 0mm

7 MRL6 13mm 0mm 0mm

8 MRL7 0mm 10mm 10mm

9 MRL8 13mm 0mm 0mm

10 MRL9 0mm 12mm 07mm

Biochemical Test for Bacterial Isolates S.no. Samples Gram’s

Reaction

Morphology Methyl Red

Test

Voges-Proskauer

Test

Catalase Test Oxidase Test

1 MRL 1 Gram +ve Rods -ve -ve -ve -ve

2 MRL2 Gram +ve Coccus -ve -ve -ve +ve

3 MRL3 Gram –ve Rods -ve +ve -ve +ve

4 MRL4 Gram +ve Rods -ve -ve -ve -ve

5 MRL5 Gram +ve Ovococcus +ve -ve -ve -ve

6 MRL6 Gram +ve Coccus -ve -ve -ve -ve

7 MRL7 Gram +ve Coccus -ve -ve -ve +ve

8 MRL8 Gram +ve Rods -ve -ve -ve -ve

9 MRL9 Gram +ve Coccus -ve -ve -ve +ve

10 MRL10 Gram +ve Coccus +ve -ve -ve +ve

Cell Weight of The Given Samples S.no. Samples Wet Weight(g) Dry Weight(g)

1 MRL 1 0.0817 0.0245

2 MRL2 0.1074 0.0384

3 MRL3 0.0643 0.0181

4 MRL4 0.1027 0.0613

5 MRL5 0.0664 0.0054

6 MRL6 0.1263 0.0013

7 MRL7 0.1002 0.0004

8 MRL8 0.1264 0.0492

9 MRL9 0.1458 0.0027

10 MRL10 0.0680 0.0015

Discussions

• Klebsiella pneumonia and Bacillus subtilis are the causative agent of

chronic debilitating pulmonary infection, pneumonia and food

poisoning respectively.

• Studies also suggest that consumption of milk and milk products

could boost population of lactic acid bacteria and helpful in

prevention of adherence of pathogenic bacteria to intestinal wall.

• Lactobacilli are considered as beneficial bacteria, exert a strong

antagonistic activity against many food contaminating micro-

organism.

• The probiotic bacteria used in commercial products today are mainly

members of the genera Lactobacillus and Bifidobacterium.

• Bacteriocins from probiotic seem to be most active against

Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae.

• Svetlana, Seatovic et al. examined the effect of the bacteriocins from

Lactobacillus plantarum G2 on other micro-organism. They also

tested, the neutralized cell free supernatant for antimicrobial activity

against gram positive and gram negative bacteria by agar well

diffusion test and showed that, the bacteriocin was active against all

the tested strains of the genra Lactobacillus: Lactobacillus acidophilus,

Lactobacillus rhamnosus, Lactobacillus leishmany, Lactobacillus

plantarum G1 and Lactobacillus casei G3 and also inhibited the

growth of the pathogenic bacteria Saphylococcus aureus and

Salmonella abony.

• The isolated LABs exhibiting excellent probiotic characteristics can be

used in controlling and improvement of intestinal microbial flora and

thus can contribute in health benefits to consumers

Discussions contd.

Conclusion The milk and milk products contain LAB which produce

bacteriocins having potential to cover a broad field of

application including, food and medical sectors. The

strong antagonistic effect of bacteriocin against the

food borne pathogens has been seen indicating its

usefulness in the preservation of different food

products enhancing their shelf life. With respect to

medical applications, antimicrobials produced by

probiotic LAB might play role during in vivo interactions

occurring in human gastrointestinal tract, hence

contributing to gut health.