Isolation and Characterization of Bacteriocin ...

Biological Forum — An International Journal, 3(1): 10-14(2011)

I SSN : 0975-1130

Isolation and Characterization of Bacteriocin Producing Lactic Acid Bacteria’s from Fermented Milk Products of Solan District of Himachal Pradesh Amit Pratush* , Anupama Gupta* * , I sha Mahajan* , Mamta Sharma* * *, J.K. Rana* * * , Savita Kumari* , Richa Bhardwaj* , Rupali M assey* , Preet Lal* and T.C. Bhalla* * * * * Department of Biotechnology, Shooli ni Institute of Life Sciences and Business Management, Solan, (HP) * * Depar tment of Microbiology, UHF, Nauni, Solan, (HP) * * * Department of Microbiology, Shoolini I nstitute of Life Sciences and Business Management, Solan,(HP) * * * * Department of Biotechnology, Himachal Pradesh University, Shimla, (HP)

(Received 11 June 2011, Accepted 29 June 2011) ABSTRACT : L actic acid bacter ia (LAB) i.e. Lactococcus AP-2 was isolated from the fer mented milk pr oducts of Solan distr ict of H imachal Pr adesh. T he str ain was selected and scr eened for their ability to pr oduce bacter iocin by agar well diffusion method using the super natant of centr ifuged test cultur e. T he isolate Lactococcus AP-2 exhibited highest zone of inhibition against the indicator pathogenic str ains i.e. P. aeruginosa, S. dysentriae and S. aureus. T he antimicrobial activity of Lactococcus AP-2 was fur ther char acterized for heat sensitivity, storage stability, pH stability and effect of sur factant. T he bacteriocin produced by Lactococcus AP-2 stable at low temperatur e i.e. up to 60°C for 72h and with the r ise in temperatur e its activity star t decr easing and above 60°C it becomes inactive. The bacteriocin is stable at acidic pH conditions (i.e. 2-6) and can be used as a potential biopr eser vative for the pr eservation of acidic food mater ials. K eywords : Lactic aci d bacteria (LAB), Lactococcus AP-2, Bacteriocin, Biopreservative.

I NT RODUCT I ON The fermented dairy products consti tute a si gni fi cant part of human diet in vari ous parts of worl d. The fermented mi l k and mi lk products are generally produced by the lactic acid bacteria (L AB). The l actic acid bacteria (LAB) are a group of Gram positive, non spore formi ng, cocci or rod shaped organi sms whi ch are consi dered as 'General l y Recogni zed as safe' (GRAS) organisms (Patil et al . 2010). LA B have been used as a flavoring and texturing agent as wel l as preservati ve i n foods for centuri es and now are used as starters i n f ood f ermentati on (Capl i ce E and Fitzgerald GF, 1999). These organisms produce antimicrobial molecules such as lactic acid, acetic acid, hydrogen peroxide and bacteri oci n whi ch are wi del y known to i nhi bi t the gr owth of f ood bor ne pat hogens and spoi l age mi croorgani sms (Pati l et al. 2010, Jeevaratnam et al . 2005). Si nce chemi cal preser vati ves are bei ng conti nuousl y questi oned with regard of safety, the use of LAB and their metabol i tes i s general l y accept ed by consumer s as something natural and health promoting. This offers a logical expl anati on f or the non-reduci ng i nterest of the f ood scienti sts i n the particular area and the expanding trend of appl icati ons of LA B i n the food i ndustry (Papagianni and Anastasiadou, 2009). LA B from different sources have been consi dered safe in the form of food preservati ves, si nce they can be degraded by gastrointesti nal protease (Fackl am and Elliott, 1995; Cl evel and et al. 2001). Bacteriocin produced f rom the LAB i s extracel lular and bacteri ci dal i n nature (Patil et al . 2010). The bacteri ocin

showed its bactericidal effect against most of gram posi ti ve bacteri a but i ts bacterici dal effect varies f rom species to species (Jeevaratnam et al . 2005). LA B, Lactococcus l actis produce nisin, a commercially used biopreservati ve, whi ch showed bactericidal effect against l arge number of gram posi tive organisms (Rodrui guez, 1996). Earl ier, LABs were isol ated from grains, dairy products, fermenting vegetabl es and meat products (Sharma and K apoor, 2004; Sharma et al . 2009; Adetunj i and Adegoke, 2007 and Patil et al. 2010). I n t he pr esent study we r epor t the 'I sol at i on and characteri zati on of bacter i oci n produci ng L A Bs f rom fermented mi l k products of di stri ct Sol an of Hi machal Pradesh. The bacteriocin produced by i solated Lactococcus AP-2 exhi bited very good bacteri oci dal potenti al agai nst the pathogeni c bacteri a.

M AT ERI AL AND M ET H ODS Chemicals. Analyti cal grade chemicals and dyes were obtained from Hi -medi a Laboratori es, Pvt. Ltd, I ndia and SRL India, respectivel y. Pr epar ation of L AB cultur e. L acti c aci d bacteri a produci ng bacteriocin were isol ated from fermented mi lk products (dahi, l assi etc.) from the different regi ons of Solan di stri ct of Himachal Pradesh, Indi a. I ndicat or bact er ial st r ains. Di f f er ent i ndi cator pathogenic bacterial strains were col lected from Department of Biotechnology, Himachal Pradesh University, Shimla. The indi cator strains used were Escherechia coli , Pseudomonas aer ugi nosa, Shi gel l a dysenteri es, Stapyhl ococcus aur eus and Bacill us cerus.

Pratush, Gupta, Mahajan, Rana, Kumari, Bhardwaj, Massey, Lal and Bhalla

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I solation and scr eening of L AB for antimicrobial activity

step i n both the precipitate and supernatant to know whi ch one actuall y contained the bacterioci n.

El even sampl es of fermented mil k products (dahi, lassi, etc.) were col lected from different areas of Sol an di strict of Hi machal Pradesh in steril ized vials. One ml of respected sample was serially dil uted by using steril e saline sol ution. The serial dil utions of the sampl e i n the seri es i .e. 10–1 ml to 10–6 ml were prepared. From each di lution (i .e. 10–1 ml , 10–2 ml and 10–6); 100 µl was spreaded on de Mann Rogosa Sharpe agar (modified M RS agar) plates and incubated f or 48h at 30°C. After suffici ent growth (after 48h) the selected coloni es were successivel y streaked on MRS agar pl ates for purity.

Characterization of partially purified bacteriocin

I dentification and characterization of LAB Col oni es wi th characteri sti c f eatures (such as pi n pointed, whiti sh etc.) were randomly selected from pl ates and tested for Gram-stain reaction, cell morphology, catalase reacti on as di scussed by K andl er and Wei ss, (1986). Production of crude bacteri oci n and detecti on of its antimicrobi al activity All the LAB strains were inoculated individually in 1000 ml Elliker's broth (pH 6.8) for 48h at 30°C in static incubator wi th seal ed pl ugs. For extracti on of bacterioci n, a cel l-free cul ture supernatant was obtai ned after centri fugati on of cul ture broth (10,000 'g' f or 20 mi n at 4°C). A s the bacteriocin was produced extracel lularly, the pellets were di scarded and supernatant was adj usted to pH 7.0 by means of 1M NaOH to exclude the antimicrobial eff ect of organic aci d, foll owed by fil trati on of the supernatant through a 0.22 µm pore-size fil ter. The supernatant was assayed for pri mary anti mi crobi al acti vi ty agai nst i ndi cator bacterial strains. The antimicrobial activi ty of the supernatant was determined by agar well diffusion assay (Klaenhammer, 1988) on M -H agar pl ates whi ch wer e spr eaded wi th test organi sms. 100 µl of supernatant was poured i nto well s (6 mm diameter) and the pl ates were incubated at 37°C for growth of the test organi sm. A f ter 24h, the di ameters (mil l imeters) of the growth inhibiti on zones were measured. Par tial purification of bacteriocin The bacteri oci n produced from the i sol ated LA B (i.e L actococcus A P- 2) was parti al l y pur i f i ed by usi ng Ammoni um sulphate preci pitation method (Ogunbanwo et al. 2003). The ammonium sulfate precipitation table (Scopes, 1982) was fol lowed to cal cul ate the requi red amount of ammoni um sul f ate to be added i n cel l f ree extract. Ammonium sulfate was added wi th continuous sti rring until the preci pi tates were f ormed and the preci pi tates were stored at 4°C for 45 min. The preci pitated proteins were recovered by centrifugati on at 10,000 g for 20 mi n and the pel l et obtai ned was r esuspended i n 0.3 M (pH 7.0) potassium phosphate buffer. The protei n esti mati on was carried out by foll owing the procedure of Bradford, (1976). Assay of the bacteriocin acti vi ty was carri ed out at each

The crude bacteriocin samples were characteri zed by studyi ng the effect of temperature, pH, storage stabi l ity, treatment with surfactants and UV l ight on thei r activity. H eat r esistance: The bacteri oci n was exposed to various temperatures: 30, 40, 50, 60, 80 and 121°C. An aliquot of each f racti on were removed af ter 30 mi n and then assayed for bacteriocin acti vi ty using agar wel l diffusi on method. pH sensitivity: The bacteri ocin was adj usted to pH 2, 3, 4, 5, 6, 7, 8, 9 and 10 with hydrochl ori c aci d (HCl) and sodi um hydroxi de (NaOH), i ncubated f or 4h at room temperature and assayed as described above. Stability of bacteriocin during storage: The bacteriocin (1ml) was stored at -20, 4, 37, 60, 80°C and room temperature (22°C). A t different time interval s, samples were taken from the stored material s to determi ne bacteriocin activity. Effect of surfactant on bacter iocin activity: This was carri ed out by incorporating non-ionic (Triton X100, Tween80, Tween 20) surfactants. The surfactants were added to partial ly puri fied bacteri oci n at a concentration of 0.1 ml of surfactant per ml of bacteri oci n solution. Thei r preparati ons were i ncubated at 30°C f or 60 mi n and assayed f or bacteri ocin acti vity against indi cator organi sms. Effect of UV light on bacter iocin activity: The bacteriocin (1 ml) was placed in steril e microcentri fuge tube and exposed to UV l ight under l ami nar air flow. Time of exposure was 10 min, after that bacteri ocin acti vi ty was anal yzed by well diffusion method.

RESULT S I n the present study Lacti c aci d bacteria (LAB) from the fermented milk products from rural areas of Solan district of Himachal Pradesh were i solated. From el even fermented mi l k samples onl y five i solates were procured. As most of the spread plates showed simi lar col oni es thereby i ndicati ng the presence of onl y one population of LAB. Onl y five di fferent L AB colonies are i denti fied as Lactococcus sp. which were different from other LABs on the basis of their phenotypi c characters, as gi ven in Tabl e 1. These f i ve i sol ates have been characterized for their cell morphol ogy, Gram's reaction and catal ase acti vity. These col oni es on MRS Agar pl ates were of small size and mostly looked li ke ti ny dots. Al l the i sol ates were f ound to be gram +ve, catal ase -ve, non-spore f ormi ng and spheri cal i n shape (except A P-4). These types of typical col onies are formed by L AB (Kandler and Wei ss, 1986). The characteristi cs selected in present study are generally used to characteri ze LA B (Sharpe et al . 1979).

Pratush, Gupta, Mahajan, Sharma, Rana, Kumari, Bhardwaj, Massey, Lal and Bhalla a

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Table 1: Cell morphology and other character istics of isolated strains. S. No. Strains Cell form

Cell

Gram Catalase arrangement

1.

A P-1

Spheri cal

Grouped

Posi t i v e

Negati ve

2.

A P-2

Spheri cal

Pai red

+

–

3.

A P-3

Spheri cal

Si ngl e + and chai ns

–

4.

A P-4

Rod shaped

Si ngl e + and chai ns

–

5.

A P-5

Smal l cocci

Pai r ed or grouped +

–

Table 2: Antimicrobial activity of bacteriocin produced by various L AB isolates against test pathogenic or ganisms (Well diffusion assay). S. No.

LAB

I ndicator organisms

1.

A P- 1

P. aer ugi nosa

Diameter of inhibition (mm) 8

S. dysenter i es

9

S. aur eus

9

P. aer ugi nosa

12

S. dysenter i es

11

2.

A P- 2

S. aur eus

12

P. aer ugi nosa

10

S. dysenter i es

11

S. aur eus

11

P. aer ugi nosa

8

S. dysenter i es

10

S. aur eus

10

P. aer ugi nosa

10

S. dysenter i es

10

S. aur eus

9

bacteria. The selected LABs i.e. Lactococcus sp. were used to produce extracel lular bacteriocin in Elli ker's broth. The bacteri ocin produced by these five LAB isol ates, showed i nhi bi tory acti vi ty agai nst the Gram-posi tive and Gramnegative target test strai ns (Fi g.1a,b and Table 2). Out of these f i ve i sol ates Fi g. 1, 2 Lactococcus A P-2 showed hi ghest zone of i nhi bi t i on. T he cel l f r ee extr act of Lactococcus A P-2 was subj ected to sequenti al ammoni um sulphate saturations from 0 to 80 %. The bacteri oci n of Lactococcus-AP-2 was recovered at 30-40 % ammoni um sulphate saturation l evel and the protei n was 0.08 mg/ml (Bradford, 1976). There was a significant increase in zone size of partially purified bacteriocin as compared to the crude bacteriocin (Table 3). T he ef f ect of temperature, pH, storage ti me and surfactant and UV on the partial purifi ed bacteri oci n of Lactococcus A P-2 was determined against P. aeruginosa, S. dysentr iae and S. aur eus whi ch were used as indicator organi sms. The parti all y purified bacteri oci n of Lactococcus AP-2 was exposed to vari ous temperatures (30, 40, 50, 60, 70, 80 and 121°C). The bacteriocin produced by Lactococcus AP-2 was heat labi le and the bacterici dal effect goes on decreasi ng wi th ri se i n temperature f rom 30°C to 80°C (Fig. 2a). 16

14

12

P. aerugi nosa S. dysentriae S. aureus

10

8

3.

A P- 3

4.

A P- 4

5.

A P- 5

6

4

2

0 30

Bac*

Bac* *

50

60

70

80

121

o

Temperature( C)

Table 3: Percent increase in inhibition zone size against test indicators of partially purified bacteriocin of LactococcusAP-2 over crude bacteriocin. Pathogen

40

I ncrease%

Zone size (mm)

Zone size (mm)

P. aer ugi nosa

11

14

27.27

S. dysenter i es

10

13

30.00

S. aur eus

12

14

16.66

Bac* Crude bacteriocin, Bac* * Partially purified bacteriocin The extracts of five isolates of Lactococcus gave zone of i nhibi ti on against the indicator food borne pathogenic

Fi g. 2.a Ef f ect of temperature

The bacteri oci n acti vi ty was measured agai nst P. aer ugi nosa, S. dysentr i ae and S. aur eus by usi ng wel l di ffusion assay method. The pH of the partial ly puri fi ed bacteri ocin of Lactococcus A P-2 was vari ed from pH 2-8 and its bacterioci dal effect was observed on the indicator strai ns i .e. P. aer ugi nosa, S. dysentr i ae and S. aureus (Fig. 2b). The bacteriocin i s stabl e at acidi c pH i .e. pH 2-6. Effect of storage time and temperature on bacterioci n of

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Pratush, Gupta, Mahajan, Rana, Kumari, Bhardwaj, Massey, Lal and Bhalla

Lactococcus AP-2 was studi ed (Table 4).

temperature i .e. 37°C the bacteri oci n acti vi ty started decreasing and at 60°C i t is not stabl e even up to 12h of i ncubation. The effect of three different nonionic surfactant i .e. Tween 20, Tween 80 and Triton X-100 was observed on partial ly puri fied bacteri ocin. The surfactant Tween 20 and 80 enhanced the bacterioci n activi ty up to 2 to 3 mm of i nhi bition zone size, whereas the Triton X-100 onl y rises the activity up to 1mm with reference to control (without surfactant) (Table 5). The exposure of the UV radiation to the bacteri oci n does not cause any effect on its bacteriocidal effect and i ts acti vi ty remai ns si mi lar to the control. Table 5: Effect of surfactant on bacter iocin activity of Lactococcus AP-2.

16

14 P. aerugi nosa S.dysentriae S.aureus

12

10

8

6

4

S.No

I ndicator strain Surfactant

1.

P. aer ugi nosa S. dysentri ae S. aur eus

Cont rol

11 12 11

2.

P. aer ugi nosa S. dysentri ae S. aur eus

Tween 20

15 14 14

3

P. aer ugi nosa S. dysentri ae S. aur eus

Tween 80

15 15 14

4

P. aer ugi nosa

Tri t on X -100

12

2

0 2

3

4

5

6

7

8

9

10

pH Change

Fig. 2(b). Ef f ect of pH on bacteriocin activity of Lactococcus AP-2 agai nst P. aerugi nosa, S. dysentr i ae and S. aur eus.

DI SCUSSI ON

Table 4: Effect of storage time and temperature on bacteriocin activity of Lactococcus-A against P. aeruginosa, S. dysentriae and S. aureus. S.No Temp Time (°C)(h)

Zone of inhibition (mm) P. aeruginosa S. dysentriae S. aureus

1. –20

2. 4

3. 20

4. 37

5. 6.

60 121

12 24 36 48 60 72 12 24 36 48 60 72 12 24 36 48 60 72 12 24 36 48 60 72 12 12

14 13 11 9 9 8 13 13 12 11 10 9 14 12 10 9 8 8 11 10 10 9 7 5 0 0

Zone of inhibition

13 13 10 9 8 7 14 12 10 10 11 9 13 12 11 9 8 7 10 9 8 8 6 4 0 0

14 13 11 10 9 9 14 14 13 10 10 8 13 13 12 10 9 7 11 10 8 7 7 6 0 0

The bacteri oci n of Lactococcus A P-2 was stabl e at -20, 4, 20°C up to 72h of incubation whereas wi th the rise in

I n the present study we coll ected the fermented mi lk samples from the rural area of the Solan district of Himachal Pradesh f or the isolati on and characteri zation of bacteri ocin produci ng l acti c aci d bacteri a. There were about el even different fermented milk samples from which only 5 different organisms havi ng bacteriocin acti vi ty were i sol ated. These organisms exhi bi ted the common characteristic properti es of lactic acid bacteria, whi ch are described by Sharpe et al. (1979). A l l the 5 i sol ates were characteri zed for thei r cel l morphol ogy, gram char acters and catal ase assay. T he colonies of these i solates were pin pointed and smal l sized which again confirms the presence of LAB's (Kandler and Wei ss, 1986). A l l the col oni es exhi bi ted gram posi ti ve character. Earlier a large number of gram positive bacteriocin produci ng LAB's were also identifi ed from milk products (Tagg et al. 1976, Vandenbergh et al. 1993, Sanni et al. 1999). The bacteri oci n produci ng property of al l isolated LAB's was determi ned by appl yi ng the crude extract of these organi sms agai nst the three maj or pathogeni c strai ns (P. aer ugi nosa, S. dysenter i es and S. aur eus) out of whi ch Lactococcus AP-2 showed maximum zone of inhibition. The parti al puri f i cati on of bacteri oci n produced by L A B's was car r i ed out by t he ammoni um sul phat e precipi tation. The protein i.e. bacterioci n was precipitated at the 30-40% cut of ammonium sulphate saturati on level. The protein precipitated above or below this saturation level exhi bited very l ow or nil bacteri ocin acti vity. The parti al ly puri fied bacteri ocin was heat labil e in nature and when it

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Pratush, Gupta, Mahajan, Sharma, Rana, Kumari, Bhardwaj, Massey, Lal and Bhalla a

was exposed to different temperature (30-80°C and 121°C) i ts activity started decreasi ng wi th ri se in temperature. At 80°C there was about 79% decrease in bacteriocin acti vi ty as compared to at 30°C (Fi g. 2a). Nettl es and Barefoot, (1993), studied the detail ed heat stabi li ty of L actobaci ll us sp. According to their research bacteriocin of L. brevis OG1 was stabl e for 60 min at 121°C. Temperature stabil ity is i mportant i f the bacteri oci ns are to be used as a f ood preservative because many procedures of food preparati on i nvolve a heating step. The pH al so af f ects the stabi l i ty and bacteri oci dal property of bacteriocin produced by the Lactococcus A P-2 (Fig. 2b). Earli er reported bacterioci ns e.g. bacteri ocin from L. plantarum F1 and L. brevi s OG1 exhibi ted maximum bacteri ocidal activity at pH 2 to 6 and was inactive at pH 8 to 12 (Ogunbanwo et al. 2003) and bulgaricans (type of bacteri oci n) i sol ated f rom the L. bul gari cus showed i ts hi ghest activi ty at pH 2.2 to 4.0, respecti vely (Reddy et al. 1984, A bdel Bar et al . 1987). T he bacteri oci n f rom Lactococcus AP-2 is stabl e at lower pH range and hence it has a good potential to preserve the food materi al s whi ch are aci di c i n nature.

CONCL USI ON A new bacterioci n produci ng lactic acid bacteri a i.e. Lactococcus AP-2 is i solated and bacteri ocin produced from i t, is characterized. The bacteri oci n from this organism is stable at l ow temperature (up to 72h) and at acidi c pH 2 to 6 thereby renderi ng i t to be used i n aci di c f oods as biopreservati ve.

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