PRODUCTION OF HEAT AND ALKALI STABLE BACTERIOCINS FROM STRESS TOLERANT PROBIOTICS

Research Article CODEN: IJPRNK IMPACT FACTOR: 4.278 ISSN: 2277-8713 Bharti P, IJPRBS, 2014; Volume 3(5): 16-37 IJPRBS

Available Online at www.ijprbs.com 16

PRODUCTION OF HEAT AND ALKALI STABLE BACTERIOCINS BY STRESS

TOLERANT PROBIOTICS FROM FAECES

BHARTI P, KUMARI K

Shaheed Udham Singh College of Research and Technology, Tangori, Mohali –140306, Punjab,

India.

Accepted Date: 11/09/2014; Published Date: 27/10/2014

Abstract: A total of six probiotic strains were isolated from faecal sample of infant and calf. These strains were analyzed and partially identified by morphological (Gram staining) and biochemical (catalase, indole production, methyl red, urease, citrate utilization and H2S production) tests. All the isolates showed a broad inhibitory action against different pathogenic microorganisms including Klebsiella pneumoniae (zone of inhibition: 7-8mm), Staphylococcus epidermidis (7-8mm), Pseudomonas aeruginosa (7-8mm), Escherichia coli (8-9mm), Enterococcus faecalis (8-9mm) and Serratia marcescens (7-9mm) as exhibited by disc-diffusion method. The isolates were found to have great potential to resist a wide variety of commonly used antibiotics like amoxycillin/sulbactam, nalidixic acid, ciprofloxacin, penicillin G, norfloxacin, oxacillin, amoxycillin and ampicillin. The probiotic isolates were found to tolerate stresses like acid (pH-2.0, 3.0, 4.0, 5.0), bile salt (0.5%, 1.0%, 1.5%, 2.0% (w/v)), lysozyme (0.5 mg ml

-1),

0.4mM hydrogen peroxide (30% (w/v)) and NaCl (1.0%, 2.0%, 4.0%, 5.0% (w/v)), thus indicating their persistence under in vivo conditions. Almost negligible haemolytic activity indicated that these probiotics were safe for human health. Moreover, the bacteriocins produced by these probiotics exhibited prominent antimicrobial activity against all the test microorganisms. Some of these bacteriocins were also found to be highly stable as they retained their activity against E. coli even at high temperatures upto 121°C and under acidic as well as alkaline conditions (pH-1.5 to 9.0). Among all the probiotic strains examined, F3 and C1 and C2 possessed most of the desirable probiotic properties and can therefore claim their potential for applications in human health.

Keywords: Antagonistic, Bacteriocins, Haemolytic, Lysozyme, Probiotics

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Bharti P, Kumari K; IJPRBS, 2014; Volume 3(5): 16-37



INTRODUCTION

Probiotics are indigenous micro-flora of humans and colonize various parts of the body1-3. The

human intestinal microflora is complex with total counts of 1011-1012 bacteria per gram of stool 4, 5. Most probiotics commercially available today belong to the genera Lactobacillus and

Bifidobacterium 6. Indeed, particular Lactobacillus strains have been found to be long-term

residents of the intestinal tracts of some humans 7, 8 and are usually spread throughout the

gastrointestinal tract (GIT) of healthy humans 9. This study emphasizes the characteristics and

potential of probiotics isolated from faecal sample.

Probiotics are defined by the WHO/FAO group, as the live microorganisms which when

administered in adequate amounts confer a health benefit on the host 10. They are known to

play an important role in the maintenance of human health especially against acute diarrhoea

in childhood by stimulating the natural immunity, inhibiting the growth of harmful bacteria and

increased resistance to infections. Probiotics thus contribute to the balance of microflora,

mainly through competitive exclusion and antimicrobial activity against pathogenic bacteria 11,

12. The acid and bile tolerance are two fundamental properties that indicate the ability of

probiotic microorganisms to survive the passage through the gastrointestinal tract, resisting the

acidic conditions of pH as low as 1.5 in the stomach and the bile acids at the beginning of the

small intestine 13, 14. Lysozyme is an enzyme found in saliva at a concentration of 180 µg ml-1

and is effective in killing gram positive bacteria by promoting cell wall disruption and

subsequent cell lysis 15. For survival in the GI tract, probiotic bacteria should be resistant to the

enzymes, mainly lysozyme in the oral cavity 16.

Among the probiotic bacteria, antibiotic resistance has been reported for strains isolated from

animals as well as humans 17. This is due to the use of the antimicrobial agents for therapy and

prophylaxis of bacterial infections and in some cases to the abuse of antibiotics in general.

Tolerance of probiotic bacteria to antibiotics is interesting due to their possible use to

reconstitute the intestinal microflora of patients suffering from antibiotic-associated colitis 18.

Antioxidative strains i.e., strains with capability to tolerate hydrogen peroxide (H2O2) have

significantly increased resistance to harsh media compared to the non-antioxidative strain;

sodium chloride concentration affects the growth of probiotics and is a physiochemical

requirement for the production of bacteriocins 19. The non-haemolytic nature of probiotics

indicate that they would not be fatal for mammalian host if entered into the blood and food

chain 20.

Probiotics are live microbial feed supplements that improve the health of man by its valuable

secondary products 21. They inhibit pathogenic bacteria by producing inhibitory substances such

as antibiotics, bacteriocins or acids among others 22. Bacteriocins are small, ribosomally

synthesized, antimicrobial peptides or proteins 23, produced by probiotics which play a role



during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to

gut health 24. Bacteriocins thus have considerable attention as natural bio-preservatives and as

potential replacement of antibiotics 19. Hence, bacteriocin producing strains have in situ

application as part of or adjuncts to starter cultures for fermented/heated food products in

order to improve safety and quality 25, 26.

The purpose of the present study was to isolate probiotic strains from faecal sample of humans

and calf and their partial characterization. The antimicrobial potential against a range of

microbes as well as their tolerance to other antibiotics and environmental stresses including

acid, bile salts, lysozyme, hydrogen peroxide and sodium chloride was determined; anti-

haemolytic property was also assessed to ascertain the safety of these probiotics under in vivo

conditions. The probiotics were also assessed for the production of inhibitory compounds i.e.,

bacteriocins; their pH and temperature stability analyzed.

MATERIALS AND METHODS

Isolation of microorganisms

Procured strains were isolated from faecal sample of infant (aged below 2 years) and calf from

Tangori village, Banur (Punjab), India. These samples were collected and stored in sterile boxes.

Samples were serially diluted up to 10-6 in sterile distilled water and 100µl was spread on MRS

agar plates (de-Mann, Rogosa and Sharpe agar; Hi Media, Mumbai, India). These plates were

then incubated at 37°C for 24 hours. After incubation, 6 well isolated typical colonies were

picked and streaked on MRS agar plates, which were then incubated at 37°C for 48 hours for

further use.

Identification of microorganisms

The isolated microorganisms were identified by morphological tests such as Gram staining 27

and various biochemical tests including catalase, indole production, methyl red, urease, citrate

utilization and H2S production 28.

Detection of antagonistic activity

The antagonistic properties of all the 6 isolated strains were determined by the disc diffusion

method 29 against the test pathogens, obtained from IMTech, Chandigarh, India. Autoclaved

filter paper discs (6mm) were impregnated with 60µl of MRS broth containing 48 hours grown

isolated cultures. These impregnated discs were dried and then placed on solidified nutrient

agar plates seeded with freshly grown culture of test pathogens, like Klebsiella pneumoniae

MTCC 9024, Staphylococcus epidermidis MTCC 6810, Pseudomonas aeruginosa MTCC 2297,

Escherichia coli MTCC 443, Enterococcus faecalis MTCC 3159 and Serratia marcescens MTCC

482; Lactobacillus plantarum was used as a positive control. The plates were then incubated at



37°C for 24 hours and zones of inhibition were estimated by measuring the diameter of the

clear area around the disc.

Acid and bile salt tolerance

Isolated strains were inoculated into MRS broth of varying pH, i.e. pH 2.0, 3.0, 4.0 and 5.0

adjusted using 0.1 M HCl; broth with pH 7.0 was used as control. Inoculation was also done in

broth with varying concentrations of bile salts i.e., 0.5%, 1.0%, 1.5% and 2.0% (w/v) (Thomas

Baker Chemicals) and the control was without any bile salt. Flasks containing these broths were

incubated at 37°C for 48 hours. Hundred microlitre of inoculum from each MRS broth (of

varying pH and from varying concentrations of bile salts) were taken, and transferred to

individual flasks containing MRS agar (at 40°C-45°C) and plating was done 30. After 24 hours of

incubation at 37°C, the growth of isolates were observed on MRS agar plates in order to

designate isolates as acid and /or bile salt tolerant.

Lysozyme tolerance

Dilutions of all the isolated strains, from the MRS broth were prepared in peptone water 31 and

loop full of culture were streaked on MRS agar plates supplemented with 0.5 mg ml-1 of

lysozyme (Hi Media Laboratories, Mumbai, India). Plates were then incubated for 24 hours at

37°C and tolerance to lysozyme was ascertained by the presence of growth of desired strains.

Resistance to antibiotics

The antibiotic resistance of the isolated strains was assessed using pre-impregnated antibiotic

discs (Hi Media Laboratories, Mumbai, India). Two hundred microlitre of each of the freshly

grown isolates were spread over the surface of individual MRS agar plates. The sterile antibiotic

discs containing amoxycillin/sulbactam (30/15mcg/disc), nalidixic acid (30mcg/disc),

ciprofloxacin (5mcg/disc), penicillin G (10 units/disc), norfloxacin (10 mcg/disc), oxacillin (1

mcg/disc), amoxycillin (30 mcg/disc) and ampicillin (10 mcg/disc) were then placed on the MRS

agar surface, spreaded with probiotic lawn and incubated for 24 hours at 37°C.

H2O2 tolerance

One millilitre of all the isolated strains cultivated in MRS broth were suspended in 1 ml of

isotonic saline (0.85 gm NaCl suspended in 100 ml of distilled water) in test tubes and incubated

with 1 ml of 0.4 mM of 30% (w/v) H2O2 solution (LOBA Chemie Ltd.). The results were recorded

by measuring the optical density (OD) (Metertech, UV/VIS spectrophotometer) at 650 nm after

a time interval of 1 hour, 2 hours, 3 hours, 4 hours and 24 hours for observing their maximum

tolerance to H2O2 19.



NaCl tolerance

For the determination of NaCl (LOBA Chemie Ltd.) tolerance of isolated strains, test tubes

containing MRS broth were adjusted to different concentrations of NaCl (1.0%, 2.0%, 4.0% and

5.0% (w/v)) and autoclaved. Each sterile tube was then individually inoculated with 1% (v/v)

freshly grown culture of the 6 isolated strains (in MRS broth) and incubated at 37°C for 24

hours. After incubation, their growth was determined by observing their OD at 600 nm 19.

Anti-haemolytic activity

The isolated strains were streaked on individual blood agar plates containing 5% human blood

and incubated at 37°C for 24 hours. Haemolytic activity was checked by the presence of clear

zones on blood agar plates around the bacterial colonies 32.

Bacteriocin/s production

For bacteriocin/s production, MRS broth containing freshly grown culture of each of the 6

isolates were taken in centrifuge tubes and centrifuged using cooling microfuge (Remi Pvt.

Limited) at 8000 rpm for 15 minutes at a temperature of 4°C. The pH of the cell free

supernatant was adjusted to pH 6.5-7.0 with 1M NaOH to neutralize the acids in broth culture

of probiotics 29 and this supernatant was further used as the source of bacteriocin/s.

Bacteriocin assay

Autoclaved filter paper discs were impregnated with 60µl of the supernatant containing

bacteriocin/s from the 6 probiotic isolates, dried and placed on MRS agar plates seeded with

test pathogens (K. pneumoniae MTCC 9024, S.epidermidis MTCC 6810, P. aeruginosa MTCC

2297, E. coli MTCC 443, E. faecalis MTCC 3159 and S. marcescens MTCC 482). The antagonistic

activity of the bacteriocin/s was determined by measuring the diameter of zones of inhibition

around the discs; supernatant from L. plantarum was used as a positive control for bacteriocin

activity.

Heat sensitivity of bacteriocin/s

To test the heat sensitivity, culture supernatant containing bacteriocin/s was heated for 15

minutes at 60°C, 80°C, 100°C and 121°C and the activity was tested against E. coli, as the test

pathogen (by disc-diffusion method), using culture supernatant at room temperature (37°C) as

the control.

Acid/alkaline sensitivity of bacteriocin/s

Sensitivity of bacteriocin/s to different pH was tested by adjusting the pH of culture

supernatant (containing bacteriocin/s) of the probiotic isolates to 1.5, 3.0 and 5.0 using 0.1M

HCl and to pH 9.0 using 1 M NaOH. The antibacterial activity of the bacteriocin/s was detected

against E. coli using culture supernatant with pH 7.0 as a control.



RESULTS

Isolation of microorganisms

A total of 6 microorganisms were isolated from faecal sample of infant and calf. Four isolates

from faecal sample of infant were named as F1, F2, F3 and F4; and 2 isolates from faecal sample

of calf were named as C1 and C2.

Morphological and biochemical identification

All the probiotic isolates F1, F2, F3, F4, C1 and C2 were identified to be Gram positive and their

biochemical characteristics have been shown in Table 1, Figure 1(a) and (b).

Table 1: Morphological and biochemical identification tests of isolates

Source Isolated strain

Gram staining

Shape Catalase

test

Indole production

Methyl red

Urease test

H2S production

Infant F1 +* Cocco-bacilli

-* - - - -

F2 + Cocci - - - + +

F3 + Cocco-bacilli

- - - - -

F4 + Bacilli - - - + +

Calf C1 + Cocci - - - + +

C2 + Cocco-bacilli

- -

-

-

-

*Note: + indicates isolates are test positive; - indicates isolates are test negative.

Figure 1: Biochemical analysis of isolates (a) H2S production test and (b) Urease test



Antagonistic activity of isolates

All the 6 probiotic isolates showed the antagonistic activity against pathogenic microorganisms

(Table 2). Probiotic isolate F1 showed the zone of inhibition in the range of 7-8mm against all

the test pathogens with maximum zone of inhibition (8mm) against E. coli, E. faecalis and S.

marcescens; isolate F2 showed the zone of inhibition in the range of 7-9mm with maximum

zone of inhibition (9mm) against E. coli and S. marcescens. The isolate F3 exhibited inhibition

zone between 7-9mm size range with maximum activity (9mm) against E. coli, while isolate F4

showed the inhibition range to be 7-8mm and E. coli, S. epidermidis, K. pneumoniae and E.

faecalis were the most susceptible with zone size of 8mm. Isolate C1 showed inhibition within a

zone size range of 7-9mm, exhibiting maximum activity (9mm) against E. coli, E. faecalis, S.

marcescens and isolate C2 also showed inhibition (range 7-8mm), with the maximum activity

against E. coli, P. aeruginosa, E. faecalis and S. marcescens (8mm).

Hence, it could be seen that out of all the test pathogens, E. coli was the most sensitive against

all the probiotic isolates. Moreover, isolates F2, F3 and C1 showed quite appreciable zone of

inhibitions against most of the test microorganisms, comparable to that obtained using the

positive control L. plantarum.

Table 2: Antagonistic activity of isolates against bacterial pathogens

Isolates E. coli MTCC 443

S. epidermidis MTCC 6810

P. aeruginosa MTCC 2297

K. pneumoniae MTCC 9024

E. faecalis MTCC 3159

S. marcescens

MTCC 482

F1 8mm* 7mm 7mm 7mm 8mm 8 mm

F2 9mm 7mm 7mm 7mm 8mm 9mm



C1 9mm 7mm 7mm 7mm 9mm 9mm


L. plant

-arum

10mm 10mm 8mm 8mm 9mm 9mm

*Note: mm denotes the diameter of zone of inhibition in millimetre; L. plantarum was taken as

control

Acid and bile salt tolerance

All the 6 isolates were able to tolerate the acidic media (pH-2.0, 3.0, 4.0, 5.0) as well as bile salts

(0.5%, 1.0%, 1.5%, 2.0 % (w/v)) upto 24 hours of incubation, as indicated by the presence of



growth under these extremely acidic pH conditions and high bile salt concentrations [Table 3,

Figure 2(a) and (b)].

Table 3: Acid and bile salt tolerance of isolates

Isolates Acid tolerance (pH-2.0) Bile salt tolerance (2% w/v)

F1 +* +*

F2 + +

F3 + +

F4 + +

C1 + +

C2 + +

*Note: + indicates presence of growth; pH 2.0 was taken as minimum and 2% w/v bile salt was

taken as maximum

Figure 2: Positive growth of isolates in media with (a) pH 2.0 and (b) Bile salt conc. 2% (w/v)

Antibiotic sensitivity test

The probiotic isolates exhibited different degree of resistance against various antibiotics. The

isolates F4 and C2 showed resistance against all the eight antibiotics tested

amoxycillin/sulbactam (AMS), nalidixic acid (NA), ciprofloxacin (CF), penicillin G (P), norfloxacin

(NX), oxacillin (OX), amoxicillin (AM) and ampicillin (AMP) [Figure 3(a) and (b)]. Isolates F1 and

F2 showed resistance against 5 antibiotics, whereas F3 and C1 showed resistance against 4 of

the antibiotics tested (Table 4).



Table 4: Antibiotic resistance test of isolates

Isolates CF AMS AM P OX AMP NX NA

F1 R* S* S R R R R S

F2 S R R R R S S R

F3 R S R S S R S R

F4 R R R R R R R R

C1 S S R R R S S R

C2 R R R R R R R R

*Note: R indicates resistance to antibiotics; S indicates sensitivity to antibiotics;

Amoxycillin/sulbactam (AMS), nalidixic acid (NA), ciprofloxacin(CF), penicillin G (P), norfloxacin

(NX), oxacillin (OX), amoxicillin (AM), ampicillin (AMP).

Figure 3: Resistance against all 8 test antibiotics exhibited by isolates (a) F4 and (b) C2

Lysozyme tolerance

All the 6 probiotic strains showed tolerance to lysozyme, as indicated by the presence of

growth in a media supplemented with lysozyme at 0.5 mg ml-1 concentration (Figure 4).



Figure 4: Lysozyme tolerance of the isolates (F1, F2, F3, F4, C1 and C2)

H₂O2 tolerance

All the probiotic isolates showed tolerance to 0.4 mM H2O2 as indicated by persistence of

growth upto 24 hours of incubation. F4 followed by F1 showed appreciable H2O2 tolerance,

since they exhibited higher OD values at 650 nm even after 24 hours of incubation (Figure 5).

Figure 5: H2O2 tolerance (0.4mM) of isolates (measured as growth in OD) after different time

intervals

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 hour 2 hours 3 hours 4 hours 24 hours

OD

at

650 n

m

Time in hours

F1 F2 F3 F4 C1 C2



Tolerance to NaCl

Among all the probiotics, isolates F3 and C1 showed tolerance to NaCl upto a concentration of

5% with maximum growth at 1% (O.D; 0.665 and 0.692, respectively) and minimum at 5%

(0.292 and 0.252, respectively) NaCl concentration. Isolate F2 showed tolerance upto 4% NaCl

with maximum tolerance at 1% (0.763), isolate F4 and C2 showed the tolerance to NaCl upto

2%, with maximum growth (0.150 and 0.249 respectively) at the same concentration while

isolate F1 showed tolerance only at 1% (0.106) NaCl concentration, as indicated in Figure 6.

Figure 6: Tolerance of isolates (measured as growth in OD) to different NaCl concentrations

(1-5%)

Anti-haemolytic activity

Anti-haemolytic activity was exhibited by most of the probiotic isolates, since no zone of

clearance could be seen around most of the isolates grown on blood agar plates. Presence of

only a small clearance zone around F3 and C2, indicated extremely weak haemolytic activity

(Table 5, Figure 7).

-1

-0.5

0

0.5

1

1.5

2

2.5

3

1% NaCl 2% NaCl 4% NaCl 5% NaCl

OD

at

60

0n

m

Concentration of NaCl (in %)

C2 C1 F4 F3 F2 F1



Table 5: Anti- haemolytic activity of the isolates

Isolates Haemolytic activity (Positives/weak/negative)

F1 Negative*

F2 Negative

F3 Weak*

F4 Negative

C1 Negative

C2 Weak

*Note: Weak activity indicates very few clear zones around bacterial colonies on blood agar

plates; negative indicates absence of clear zones around bacterial colonies.

Figure 7: Anti-haemolytic activity of the isolates (F1, F2, F3, F4, C1, C2)

Bacteriocin assay

Activity of the bacteriocin/s present in the supernatant was observed against different

pathogenic microorganisms, by measuring zones of inhibition. The supernatant containing

bacteriocin/s obtained from the 6 isolates showed activity against all test pathogenic strains

including E. coli, S. marcescens, P. aeruginosa, S. epidermidis, K. pneumoniae and E. faecalis.

Among the probiotic isolates, bacteriocins produced by F3 exhibited large inhibition zone sizes.

E. coli was found to be the most sensitive pathogen to the bacteriocin/s produced by isolates



F1, F2, F3, C1 and C2 as exhibited by the zone of inhibition (12mm, 13mm, 20mm, 10mm and

10mm respectively). Bacteriocin/s produced by F4 isolate showed maximum inhibition against

K. pneumoniae (zone size-10mm); bacteriocin/s from C2 along with exhibiting maximum

inhibition against E. coli, also inhibited K. pneumoniae to an equal extent (zone size10mm);

along with inhibiting E.coli, bacteriocins from isolate C1 also showed maximum activity against

P. aeruginosa (zone size-10mm) (Table 6, Figure 8).

Table 6: Antibacterial activity of bacteriocin/s of probiotic isolates against pathogens

Isolates E. coli MTCC 443

S. epidermidis MTCC 6810

P. aeruginosa MTCC 2297

K. neumoniae MTCC 9024

E. faecalis MTCC 3159

S. marcescens

MTCC 482







L.plantraum (control)

9mm 8mm 10mm 9mm 10mm 8mm

Figure 8: Bacteriocin assay of probiotic isolates (F1, F2, F3, F4, C1, C2)



Heat sensitivity of bacteriocin/s

Bacteriocin/s produced by probiotic strain C1 exhibited maximum heat tolerance at even high

temperatures of upto 121°C when exposed for 15 minutes, as indicated by the presence of their

inhibitiory activity (zone size of 9mm) against E. coli as test pathogen (Table 7); bacteriocin/s

from C1 also exhibited tolerance at boiling temperature (at 100°C) with zone size of 9mm.

Probiotic strains F2 and F3 produced bacteriocin/s which could also withstand boiling (for 15

minutes) as exhibited by the presence of zone of inhibition (8mm and 7mm, respectively).

Bacteriocins from isolate F1 exhibited high temperature tolerance of upto 80°C with a zone size

of 8 mm; while those froms strains F4 and C2 retained activity only upto 60°C (8mm each).

Table 7: Effect of heat treatment on bacteriocin/s from probiotic isolates

Isolated strains Temperature (°C)

60°C 80°C 100°C 121°C

F1 *8mm 8mm - -

F2 12mm 10mm 8mm -

F3 11mm 8mm 7mm -

F4 8mm - - -

C1 10mm 10mm 9mm 9mm

C2 8mm - - -

Note: E. coli was taken as test organism

pH sensitivity of bacteriocin/s

Bacteriocin/s produced by the probiotic strain C1 were stable in the entire pH range (pH-1.5-

9.0), as exhibited by their activity of 7-14mm zone size against E. coli, under extremely acidic as

well as alkaline conditions (Figure 9). Bacteriocin/s produced by probiotic strains F2 and F3 also

showed marked acidic and alkaline stability over a major part of the pH range i.e., 3.0-9.0 (zone

size: 10-15mm for F2; and 9-15mm for F3). Bacteriocin/s produced by isolate C2 were stable

more in the acidic side of pH range (1.5-7.0), as exhibited by their persistent activity of 8-11mm

zone size. Probiotic strains F1 and F4 produced bacteriocin/s stable in 3.0-7.0 pH range (zone

size: 10-12mm for F1; and 10-13 mm for F4).



Figure 9: pH sensitivity of bacteriocin/s from probiotic isoltes against E. coli

DISCUSSION

The aim of the present research was to isolate the probiotics along with the inhibitory

compounds produced by them (bacteriocins) and to evaluate their persistence under extremely

stressful conditions, as encountered under in vivo conditions. This is one of only a few studies

focusing on the isolation of the probiotics from both human and calf faeces. Some of the earlier

studies have been done on either the faecal flora of child 33 or that on the veal calves 34.

Lactobacilli and Bifidobateria are the most commonly detected microorganisms in faecal

samples 8. In our study, all the isolated probiotics were identified to be Gram positive. The

biochemical tests revealed these isolates to be catalase negative, rod, cocci or coccobacilli,

which might fall into the category of Bifidobacteria or lactic acid bacteria. The antimicrobial

activitiy of six isolates as seen against different pathogenic strains like E. faecalis, E. coli, S.

epidermidis, K. pneumoniae, P. aeruginosa and S. marcescens, revealed that isolates F1, F3 and

C1 were the most effective and exhibited large inhibition zones (7-9mm). Among the test

pathogens, E. coli was found to be the most susceptible to majority of the isolated probiotics.

Earlier studies on probiotics 35 also demonstrated maximum antagonistic potential of probiotics

against E. coli as exhibited by large diameter of inhibition zones.

Acid tolerant strains have an advantage for survival in stomach (upto extreme pH 2.0), to be

able to resist the digestion process in the stomach, where hydrochloric and gastric juices are

secreted 36. In our study, all the six isolates showed tolerance to extremely acidic medium with

pH values 2.0-5.0, as exhibited by the growth of all the strains. Majority of the earlier studies

have shown tolerance of probiotic isolates to acidic medium of pH upto 3.0 37; one of the only

few reports suggested the survival of Lactobacillus strains, isolated from Moroccan traditional

dairy products under extremely acidic gastric conditions (pH 2.0 and pH 3.0) 35.The next barrier

after gastric juice in the stomach is bile, which is present in the upper part of small intestine 38

pH-1.5 pH-3.0 pH-5.0 pH-7.0 pH-9.0

0

2

4

6

8

10

12

14

16

pH

Zon

e of

inh

ibit

ion

(dia

met

er i

n m

m)

F1

F2

F3

F4

C1

C2



and for a bacteria to be effective as a probiotic, they should have the ability to survive the

passage through the upper digestive tract to the large intestine, where its beneficial action is

expected 22, 39-41. Most of the earlier studies, report tolerance of probiotics like Leuconostoc

mesenteroides and Lactobacillus species to bile salt concentration upto 1.0% (w/v) after 24

hours of incubation 35, 42. In our study, all of the 6 probiotic isolates showed tolerance to 0.5%

(w/v), 1.0% (w/v) bile salts as well as to higher concentrations of 1.5% (w/v) and 2.0 % (w/v)

after 24 hours of incubation. It was notable that, in this study, 100% of the isolates were shown

to be capable of growth at low pH and high bile salts’ concentrations, which is a mandatory

requirement for a probiotic culture 43.

Antibiotic resistance can occur in two ways in a bacterial population; mutation of an

endogenous gene or acquisition of a resistance gene from an exogenous source 44, 45. Thus,

intestinal bacteria can acquire resistance to antibiotics used for therapy, either by mutation or

by horizontal transfer of resistance genes from another intestinal species or any species that

passes through the colon 46, 47. Some probiotic strains with intrinsic antibiotic resistance could

be useful for restoring the gut microbiota after antibiotic treatment 48. In our study 2 isolates

(F4 and C2) showed resistance against all the eight antibiotics tested; three isolates F1, F2 and

C1 showed resistance against 5 antibiotics, whereas F3 showed resistance against 4 of the test

antibiotics. This is in accordance with the previous studies 49, 33, emphasizing the “selective

antibiotic resistance” of probiotics isolated from human faeces. Bacteria used as probiotic

adjuncts are commonly delivered in a food system and, therefore, begin their journey to the

lower intestinal tract via the mouth and hence, probiotic bacteria should be resistant to the

enzymes such as lysozyme present in the oral cavity 50. Like some previous studies 31, all our 6

isolated strains showed lysozyme tolerance, since they exhibited the growth in the presence of

0.5 mg ml-1 lysozyme.

Haemolytic property of probiotics is a preliminary approach to address whether they are safe to

mammalian system or not and is an important consideration as far as the food safety is

concerned 20. Similar to some previous studies 20, 32, 51, our probiotics exhibited negative

haemolysis, with only 2 strains showing negligible haemolytic activity. The anti-haemolytic

activity indicated that our probiotics could be safely administered to humans. Antioxidative

potential of probiotics is a desirable characteristic, being able to scavange any peroxides. In the

present study, all 6 isolated probiotics could tolerate 0.4 mM H2O2. Since our isolates could

tolerate hydrogen peroxide for long duration upto 24 hours, hence our study is a step forward

to earlier studies, emphasizing tolerance (0.4 mM concentration of H2O2) upto 4 hours and 6

hours 19.

Sodium chloride has been found to be a requirement by the probiotics for the production of

inhibitory compounds including bacteriocins. The effect of NaCl on growth of 2 probiotic

isolates in a medium was earlier seen, wherein, both the organisms tolerated 1% (w/v) NaCl,



but less growth was found at 5% (w/v) NaCl 19. In our study, 2 isolates (F3 and C1) showed the

maximum tolerance of NaCl upto 5% (w/v) concentration with persistent growth (0.292 and

0.252, respectively).

Bacteriocins, the peptides produced by probiotics contain bactericidal activity and enhance the

survival of these species in a complex ecological system, thus focusing on prevention of growth

of harmful bacteria in the intestinal tract and gut 29.Various previous works have emphasized

the antagonistic potential of bacteriocins produced by probiotics against many pathogenic

microorganisms 19, 36, 52, 53. In our study, the supernatant containing bacteriocins showed the

inhibitory effect against a broad spectrum of test pathogens such as E. coli, P. aeruginosa, E.

faecalis, S. epidermidis, K. pneumoniae and S. marcescens. Highly significant activity was

exhibited by the bacteriocins produced by the most effective isolate F3 against E. coli and S.

marcescens (zone of inhibition-20mm and 13mm respectively).

Heat and pH tolerance by bacteriocin/s have been seen as a desirable characteristic 26. In the

present study, the bacteriocins obtained from 6 probiotic isolates showed varied stability at

different temperatures (60°C, 80°C, 100°C and 121°C) for 15minutes, when tested against E. coli

as the test pathogen. The bacteriocin/s obtained from the isolated strain C1 showed tolerance

upto 121°C, as exhibited by the zone of inhibition (9mm), while bacteriocin/s obtained from

probiotic isolates F2 and F3 showed stability upto 100°C. Our study is in accordance with some

previous studies emphasizing production of heat stable bacteriocin/s (121°C upto 10 minutes

and 15 minutes respectively) 26, 30.In our study, bacteriocin/s isolated from isolate C1 were

stable in the entire pH range of 1.5-9.0 and bacteriocin/s from isolate C2 were stable in the pH

range of 1.5-7.0, while bacteriocin/s from F2 and F3 exhibited stability in the range of 3.0 to 9.0.

This property of pH tolerance by the metabolites produced by our probiotic isolates, ascertains

its effectiveness in the highly acidic conditions (pH- 1.5-4.0) of the GIT.

CONCLUSION

Probiotics are of great interest and are generally recognized as safe organisms for human use.

The usual selection procedure needs to be revised, since passage through the stomach is the

first 'hurdle' that potentially probiotic bacteria face on their way to the intestines 54 and acid

tolerance is regarded as a very important pre-requisite in the selection of cultures. All our

isolates exhibited remarkable tolerance to the extreme conditions, as could be encountered

under in vivo conditions in the GIT. It is also desirable to continue to expand our understanding

of the influences that environmental factors have on the survival of bacteriocin producing

strains and the activity of their bacteriocins 55. The bacteriocins of the probiotic strains isolated

in the present study showed excellent activity, along with possessing tolerance to extreme

physical stresses. These properties are crucial in order to qualitatively estimate their efficacy for



future applications in food model systems and in the clinics and also, to establish adequate

means of application of these bio-preservatives.

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PRODUCTION OF HEAT AND ALKALI STABLE BACTERIOCINS FROM STRESS TOLERANT PROBIOTICS

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