JOURNAL OF=
CULTURE
COLLECTIONS
Volume 4, 2004-2005, =
pp.
53-60
ISOLATION AND CHARACTERIZATION OF
BACTERIOCIN PRODUCING LACTIC ACID BACTERIA FROM
A SOUTH INDIAN SPECIAL DOSA (APPAM) BATTER
Vijai Pal, Marilingappa =
Jamuna
and Kadirvelu Jeevaratnam*
Food Biotechnology Division, Defence Food Resear=
ch
Laboratory,
Siddarthanagar, Mysore-570 011, India
Summary
Lactic
acid bacteria (LAB) commonly used as starter cultures in food are known to
produce antimicrobial substances such as bacteriocins, having great potenti=
al
as food biopreservatives. The present study was aimed at isolating
bacteriocinogenic LAB from a traditional fermented food, “Appam”=
;.
Twenty five colonies of LAB were isolated and screened for bacteriocin
production potential. Ten isolates showed good antimicrobial activity again=
st
Gram-positive viz., Bacillus cereus, Staphylococcus aureus,
Listeria monocytogenes as well as Gram-negative viz., Pseudomonas aeruginosa, Vibrio parahaemolyticus and
Introduction
Isolation and screening of microorganisms from naturally occur=
ring
processes have always been the most powerful means for obtaining useful
cultures for scientific and commercial purposes [21]. This certainly holds =
true
for lactic acid bacteria (LAB), which are used throughout the world for
manufacture of a wide variety of traditional fermented foods. Since they are involved in numerous food fermentation=
s,
known to man for millennia, it is assumed that most representatives of this
group do not pose any health risk to man, and are designated as GRAS (Gener=
ally
Recognized as Safe) organisms. The LAB, generally considered as “food
grade” organisms, show special promise for selection and implementati=
on
as protective cultures. There are many potential applications of protective
cultures in various food systems [10]. These organisms have been isolated f=
rom
grains, dairy and meat products, fermenting vegetables, and the mucosal sur=
faces
of animals [15]. During lactic acid fermentation these bacteria not =
only
have their effect on food and flavour but they are also known to produce and
excrete compounds with antimicrobial activity, such as bacteriocins. Bacteriocins of LAB are considered as safe natural
preservatives or biopreservatives, as it is assumed that they are degraded =
by
the proteases in gastrointestinal tract [5]. Bacteriocins are extracellular=
ly
released peptides or protein molecules, with a bactericidal or bacteriostat=
ic mode
of action against closely related species. The inhibitory spectrum of some
bacteriocins also includes food spoilage and/or food-borne pathogenic
microorganisms [17].
The discovery of nisin, the first bacter= iocin used on a commercial scale as a food preservative dates back to the first h= alf of last century but research on bacteriocins of LAB has expanded in the last two decades, searching for novel bacteriocin producing strains from dairy, = meat and plant products, as well as traditional fermented products. Many bacteriocins have been isolated and characterized [5]. Earlier, we have reported the characterization of LAB isolates, a bacillus from appam batter= and another from vegetable pickle as well as characterization of the partially purified bacteriocins from the same [11]. Present investigation reports on = the isolation and characterization of the other bacte= riocin producing LAB from appam (special dosa) batter.
Materials and Methods
Chemicals. Analytical grade chemicals and dyes were
obtained either from SRL, India or SD Fine Chemicals, India while the
proteolytic enzymes, molecular weight markers and bacteriological media were
obtained from Sigma, USA and Hi-Media, India respectively.
Microorganisms. The indicator organisms, viz., Bacillus cereus, Sta= phylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Vibrio parahaemolyt= icus and Aeromonas hydrophila were procured from Microbial Type Cultu= re Collection (MTCC) at Institute of Microbial Technology, Chandigarh, India. = LAB were isolated from appam batter.
Isolation
and screening of LAB for antimicrobial activity. LAB were isolated from the appam batter by appropria=
te
dilutions with saline, plated on MRS (de Mann Rogosa Sharpe) agar and incub=
ated
anaerobically at 37 oC for 2-3 days. Twenty five well-isola=
ted
colonies were picked up and transferred to MRS broth. They were propagated
twice and streaked on MRS agar to check the purity of the isolates and then
stored in MRS soft agar (0.5 %) overlaid with 50 % glycerol at
-20 oC. These 25 isolates from appam batter were tested for
their ability to produce bacteriocins. The isolates maintained in frozen st=
ocks
were propagated twice in MRS broth and used for further study. These were
inoculated into MRS broth and incubated at 37 oC for 48&nbs=
p;h.
Cell free supernatants adjusted to pH 5.0 with 2 N NaOH, were concentr=
ated
to one tenth of the original volume by flash evaporator, sterilized by pass=
ing
through 0.22 mm membrane filter (Millipore, India) and evaluated for
antimicrobial activity by agar well diffusion method [20] against Characterization a=
nd
differentiation of bacteriocin producing LAB.
Bacteriocin producing strains were Gram stained and examined microscopically
for cellular morphology and Gram-stain phenotype. Catalase activity was tes=
ted
by spotting colonies with 3 % hydrogen peroxide. Growth was assayed in MRS broth at 10, 1=
5, 37
and 45 oC as well as at pH of 4.4=
, 5.0,
8.6 and 9.0 incubated at 37 oC.=
Salt
tolerance was tested with 6.5, 10 and 15 % (w/v) NaCl in MRS br=
oth. Production of acid and CO2 from glucose =
was
tested in MRS broth containing Durhams tube, with citrate omitted [18] and =
in
Gibson’s semi-solid tomato-juice medium by the method of Gibson and
Abd-el-Malek [9]. Production of ammonia in MRS broth omitting glucose and m=
eat
extract, while containing 0.3 % arginine and 0.2 % sodium citrate
replacing ammonium citrate, was monitored using Nessler’s reagent. Ho=
mo-
and hetero- fermentative differentiation test was also carried out accordin=
g to
the method reported by Zuniga et al. [24]. Assays for gelatin hydrol=
ysis
and nitrate reduction were performed in accordance with Harrigan [9]. Ability to ferment various carbohydrates was evaluated
using MRS broth supplemented with filter sterilized sugar solutions to a fi=
nal
concentration of 1 % w/v and 0.004 % chlorophenol red without glu=
cose
and meat extract [18]. The configuration of lactic acid formed from glucose=
was
determined enzymatically using D-lactate and L-lactate dehydrogenase. Antibiogram of LAB isolat=
es. The isolates were inoculated i=
nto MRS broth individually and incubat=
ed for
24 h. About 25 ml of MRS agar was seeded with the cultures of LAB isolates =
(106
CFU/ml) mixed well, poured into sterile Petri plates and stored at 4 <=
sup>oC
for 1 h to solidify the media. OCTA-discs (8 antibiotics in a single r=
ing)
were placed up side down, pressed on the top of the agar plates and kept ag=
ain
at 4 oC for 1 h. The plates were incubated at 37 =
oC
over night. Resistance was defined as the absence of a growth inhibition zo=
ne
around the discs. Cell wall protein
extraction and analysis. LAB isolates were gr=
own in
MRS broth and cells harvested and resuspended in 3 ml distilled water
(A600nm=3D2) and centrifuged. Cell wall proteins were extracted from final
pellets with 0.5 ml of Results and Discussion Ten out of 25 randomly picked colonies exhibited good antimicr=
obial
activity against Gram-positive bacteria, viz., B. cereus,
St. aureus and L. monocytogenes as well as Gram-negative bacteria<=
i>,
viz. Ps. aeruginosa, V. parahaemolyticus and A. hydrophila and w=
ere
studied further. It was observed that two of the strains resembled with oth=
er
two in all aspects of characterization and hence we were left out with 8
bacteriocin producing LAB. The investigation of 7 LAB is presented in this
study. All the 7 strains were found to be Gram-positive while
morphologically DFR JJ1, 6, 9, 10 and 11 were cocci and DFR JJ4 and DFR JJ5
were bacilli. Physiological and biochemical properties as well as carbohydr=
ate
fermentation pattern of these strains are shown in Tables 1 and 2. On the b=
asis
of morphological, physiological and biochemical characters as well as sugar
utilization pattern DFR JJ1, 6, 9 and 10 were identified as Pediococcus
pentosaceous, DFR JJ1=
1 as
Tetragenococcus halophilus, =
DFR JJ4
and DFR JJ5 as Lactobacillus plantarum. The four strains of P. pentosaceous were Gram-positive =
cocci,
arranged in tetrads and pairs. These strains were catalase-negative, did not
produce CO2 from glucose, homofermentative on HHD medium, did not
reduce nitrate and produced both L-(-) and D-(+) form of lactic acid (Table=
1).
These are also the major characters of genus Pediococcus as reported
earlier [4, 6, 7]. These four P. pentosaceous
strains produced NH3 from arginine and did not hydrolyze gelatin.
Only two species of Pediococcus genus, viz., P. acidilactici and P. pentosaceous were able to
hydrolyze arginine [3, 6, 7]. Luxurious growth was observed for all 4 strai=
ns
at 37 oC while it was weak at 10 oC and none
could grow at 45 oC (Table 1). P. acidilactici can
arbitrarily be differentiated from P. pentosaceous by its ability to
grow at 50 oC and survive even 70 oC for
10 min [7, 16]. Carbohydrate fermentation pattern of these strains
showed that all the four strains were able to ferment maltose, trehalose,
xylose, cellobiose, and galactose but could not ferment sorbitol, mannitol,
glycerol and arabinose (Table 2). Ability to ferment maltose is a special
character of P. pentosaceous,
while P. acidilactici could not ferment it [3, 6, 7]. On the
basis of all these properties the four strains were identified as P.
pentosaceous, however they were different strains since they distinguis=
hed
among themselves regarding sugar fermentation pattern as well as their
resistance and sensitivity towards various antibiotics (Table 3). T. halophilus strain cells were =
found to
be Gram-positive cocci occurring in tetrads and pairs and exhibited weak
reaction for catalase. Presence of pseudocatalase in some strains of Ped=
iococcus
and Tetragenococcus lead to catalase-positive reaction [19]. Strain =
DFR
JJ11 did not grow at pH 4.4 but grew luxuriously at pH 8.6 and in 15 %
NaCl. It was found to be homofermentative, hydrolyzed arginine and did not
produce CO2 from glucose (Table 1). This strain did not ferment
arabinose, sorbitol and glycerol but easily fermented sucrose, maltose, man=
nose
and trehalose (Table 2). Garvie [7] and Villar et al. [23] also report=
ed
similar results for P. halophilus,
which later was renamed as T. halophilus [19]. This strain belongs to family Aerococcaceae which is considered a LAB from a practical food
technology point of view [2]. T. halophilus strain was highly resist=
ant
to streptomycin and moderately resistant to nalidixic acid, cotrimazine and
colistin, while it was sensitive for all=
other
antibiotics examined (Table 3). Table 1. Morphological, ph=
ysiological
and biochemical properties of bacteriocin producing LAB isolates. Tests Isolates JJ1 JJ4 JJ5 JJ6 JJ9 JJ10 JJ11 Morphology Cocci
Tetrads & Pairs Rods Rods Cocci
Tetrads & Pairs Cocci
Tetrads & Pairs Cocci
Tetrads & Pairs Cocci
Tetrads & Pairs Growth at temperature (oC)=
10 + ND ND + + + – 15 ND + + ND ND ND ND 37 ++ ++ ++ ++ ++ ++ ++ 45 – – – – – – – Growth at pH 4.4 w w w + w w – 5.0 + + + + + + – 8.6 ++ ++ ++ ++ ++ ++ ++ 9.0 ++ ND ND ++ ++ ++ ++ Growth at NaCl (%) 6.5 w vw vw w w w ++ 10 – – – – – – ++ 15 – – – – – – ++ CO2
from Glc – – – – – – – NH3
from Arg + – – + + + + HHD
Medium Ho Ho Ho Ho Ho Ho Ho Catalase – – – – – – +1 Gelatin
hydrolysis – – – – – – + Nitrate
reduction – – – – – – + Lactate DL DL DL DL DL DL ND Legend: gr=
owth
(+), no growth (–), luxurious growth (++), weak growth (w), homoferme=
ntative
(Ho), very weak (vw), not determined (ND), weak reaction due to pseudocatal=
ase
(+1), glucose (Glc), arginine (Arg). Table 2. Carbohydrate utilization pattern of the bacteriocin
producing LAB isolates. Carbohydrates Isolates JJ1 JJ4 JJ5 JJ6 JJ9 JJ10 JJ11 Arabinose – – – – – – – Cellobiose + + + + + + – Esculin + + + + +* +* – Galactose + + + + + + + Gluconic
acid + + + + + + – Glycerol – – – – – – – Inulin + + + – + + + Lactose +* + + +* +* +* +* Maltose + + + + + + + Mannitol – + + – – – + Mannose + + + + + + + Melibiose – +* +* – – + – Raffinose – + + – – – – Rhamnose – – +* – – – – Ribose + +* + + + + – Salicin + + + + + + – Sorbitol – + + – – – – Sucrose – + + + +* + + Trehalose + + + + + + + Xylose + – – + + + – Legend: growth (+), no growth (–), delay=
ed
fermentation (+*). Both strains of =
Lb.
plantarum, viz., JJ4 and JJ5 were found to be Gram-positi=
ve,
catalase-negative bacilli. Both the strains were homofermentative and did n=
ot
produce CO2 and ammonia from glucose and arginine respectively.
Their growth was weak at 15 oC and pH of 4.4, while both gr=
ew
luxuriously at 37 oC and pH of 8.6. The two strains produced both L-(-) and D-(+) form of lactic
acid (Table 1). Among sugars examined both strains fermented mannitol,
melibiose, esculine, galactose, sorbitol, sucrose, raffinose and trehalose =
but
they could not ferment arabinose, glycerol and xylose. Lb. plantaru=
m JJ5 was able to ferment rhamnos=
e,
however, the same was not utilized by other strain JJ4 (Table 2). Similar
characters for Lb. plantarum have been observed earlier [13, 22=
].
The behavior of these two strains against various antibiotics was also foun=
d to
be similar (Table 3). Table 3. Antibiogram of bacteriocin producing LAB isolates
determined by antibiotic sensitivity OCTA-discs. Antibiotics Concen-tration
(µg) Isolates JJ1 JJ4 JJ5 JJ6 JJ9 JJ10 JJ11 Cephaloridine (Cr) 30 S S S S S S S Tetracycline (T) 10 S S S S S S S Co-Trimoxazole (Co) 25 M S S R S R S Ampicillin (A) 25 S S S S S M S Amikacin (AK) 10 S M M R M R S Colistin (Cl) 10 R R R R R R M Co-Trimazine (Cm) 25 R R R R R R M Nalidixic acid (Na) 30 M R R M R M M Nitrofurantoin (Nf) 300 S S S M S M S Streptomycin (S) 10 S M M R M R R Penicillin G (P) 2 S S S S S S S Cloxacillin (Cx) 5 S S S S S S S Erythromycin (E) 10 S S S S S S S Lincomycin (L) 10 S S S S S S S Cefuroxime (Cu) 30 S S S S S S S Norfloxacin (Nx) 10 R R R R M R S Mecillinam (Mc) 33 S S S S S S S Chloramphenicol (C) 25 S S S S S S S Gentamycin (G) 30 S S S S M M S Ceftriaxone (Ci) 30 S S S S S M S Ciprofloxacin (Cf) 10 S S S S M R S Legend: se=
nsitive
(S), resistant (R), moderate (M). Cell wall protein
extraction and analysis. Cell wall protein pr=
ofiles
for 7 bacteriocin producing LAB isolates are depicted in Fig. 1. Two m=
ajor
bands of >66 kDa were present in case of DFR JJ6, 9 and 10, while only o=
ne
band was observed for DFR JJ1, 4 and 5. However, these bands were absent in=
DFR
JJ11. One band of ~66 kDa was prominent in DFR JJ1 and DFR JJ9 but not in D=
FR
JJ6. DFR JJ1 profile shows a band of ~40 kDa while a band of ~38 kDa could =
be
seen for DFR JJ6, 9 and 10. Bands of ~21.5 kDa and 15 kDa were prominent in=
DFR
JJ6, 9 and 10, while absent in DFR JJ1. The profile of DFR JJ4 and 5 showed
much resemblance, both being Lb.
plantarum, while the strains DFR JJ6, 9, 10 showed similar profile, all
being P. pentosaceous, however,=
DFR
JJ1 being P. pentosaceous diffe=
red
considerably. The profile of DFR JJ11 was found to be differed from others =
and
this was characterized as T. halophilus. Fig.
1. Cell wall proteins profile of the LAB isolates. Lanes: MW markers (1), J=
J1
(2), JJ6 (3), In conclusion, eight LAB isolates from appam batter, capable of
producing good amount of bacteriocins have been anticipated to have enormous
potential for food applications as biopreservatives. Among these strains,
characterization of one lactobacillus isolate and its bacteriocin as well as
its potential in preserving vegetarian food systems has been reported earli=
er
[11, 12]. Studies on the production, purification and characterization=
of
bacteriocins from other isolates are in progress. Acknowledgements. =
The authors are thankful to Dr. A. S. Bawa, Director, Defence =
Food
Research Laboratory, Mysore for his keen interest and providing the facilit=
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![](3D"JCC0547NB.files/image002.jpg")
JJ9 (4), JJ10 (5), MW markers (6), JJ4 (7), JJ5 (8) and JJ11 (9).