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 Research 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 potential 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 against Gram-positive viz., Bacillus cereus, Staphylococcus aureus, Listeria
monocytogenes as well as Gram-negative viz., Pseudomonas aeruginosa, Vibrio
parahaemolyticus and Aeromonas hydrophila microorganisms. During characterization
of these strains two of them were found to resemble two other strains. One of the bacilli
has been reported earlier. Therefore, in this study characterization of 7 isolates through
morphological, physiological, biochemical and carbohydrate fermentation tests as well
as cell wall protein profiles have been reported. Five of them were cocci, identified as
Pediococcus pentosaceous (4 strains) and the other as Tetragenococcus halophilus,
while the remaining two being rods and were found to be Lactobacillus plantarum.

Introduction
Isolation and screening of microorga- assumed that most representatives of this
nisms from naturally occurring processes group do not pose any health risk to man, and
have always been the most powerful means are designated as GRAS (Generally Recog-
for obtaining useful cultures for scientific and nized as Safe) organisms. The LAB, generally
commercial purposes [21]. This certainly holds considered as “food grade” organisms, show
true for lactic acid bacteria (LAB), which are special promise for selection and imple-
used throughout the world for manufacture of mentation as protective cultures. There are
a wide variety of traditional fermented foods. many potential applications of protective cul-
Since they are involved in numerous food tures in various food systems [10]. These or-
fermentations, known to man for millennia, it is ganisms have been isolated from grains, dairy

53
and meat products, fermenting vegetables, The discovery of nisin, the first bacteriocin
and the mucosal surfaces of animals [15]. used on a commercial scale as a food pre-
During lactic acid fermentation these bacteria servative dates back to the first half of last
not only have their effect on food and flavour century but research on bacteriocins of LAB
but they are also known to produce and ex- has expanded in the last two decades, sear-
crete compounds with antimicrobial activity, ching for novel bacteriocin producing strains
such as bacteriocins. Bacteriocins of LAB are from dairy, meat and plant products, as well
considered as safe natural preservatives or as traditional fermented products. Many bacte-
biopreservatives, as it is assumed that they are riocins have been isolated and characterized
degraded by the proteases in gastrointestinal [5]. Earlier, we have reported the characteriza-
tract [5]. Bacteriocins are extracellularly relea- tion of LAB isolates, a bacillus from appam
sed peptides or protein molecules, with a batter and another from vegetable pickle as well
bactericidal or bacteriostatic mode of action as characterization of the partially purified
against closely related species. The inhibitory bacteriocins from the same [11]. Present inve-
spectrum of some bacteriocins also includes stigation reports on the isolation and charac-
food spoilage and/or food-borne pathogenic terization of the other bacteriocin producing
microorganisms [17]. LAB from appam (special dosa) batter.

Materials and Methods

Chemicals. Analytical grade chemicals batter were tested for their ability to produce
and dyes were obtained either from SRL, bacteriocins. The isolates maintained in frozen
India or SD Fine Chemicals, India while the stocks were propagated twice in MRS broth
proteolytic enzymes, molecular weight mar- and used for further study. These were
kers and bacteriological media were obtained inoculated into MRS broth and incubated at
from Sigma, USA and Hi-Media, India res- 37 oC for 48 h. Cell free supernatants adjusted
pectively. to pH 5.0 with 2 N NaOH, were concentrated
Microorganisms. The indicator organisms, to one tenth of the original volume by flash
viz., Bacillus cereus, Staphylococcus aureus, evaporator, sterilized by passing through
Listeria monocytogenes, Pseudomonas aeru- 0.22 µm membrane filter (Millipore, India) and
ginosa, Vibrio parahaemolyticus and Aeromonas evaluated for antimicrobial activity by agar
hydrophila were procured from Microbial Type well diffusion method [20] against B. cereus,
Culture Collection (MTCC) at Institute of St. aureus, L. monocytogenes, Ps. aeruginosa,
Microbial Technology, Chandigarh, India. LAB V. parahaemolyticus and A. hydrophila as indi-
were isolated from appam batter. cator organisms.
Isolation and screening of LAB for Characterization and differentiation
antimicrobial activity. LAB were isolated of bacteriocin producing LAB. Bacteriocin
from the appam batter by appropriate dilu- producing strains were Gram stained and
tions with saline, plated on MRS (de Mann examined microscopically for cellular morpho-
Rogosa Sharpe) agar and incubated anaero- logy and Gram-stain phenotype. Catalase
bically at 37 oC for 2-3 days. Twenty five well- activity was tested by spotting colonies with
isolated colonies were picked up and trans- 3 % hydrogen peroxide.
ferred to MRS broth. They were propagated Growth was assayed in MRS broth at 10,
twice and streaked on MRS agar to check the 15, 37 and 45 oC as well as at pH of 4.4, 5.0, 8.6
purity of the isolates and then stored in MRS and 9.0 incubated at 37 oC. Salt tolerance was
soft agar (0.5 %) overlaid with 50 % glycerol tested with 6.5, 10 and 15 % (w/v) NaCl in MRS
at -20 oC. These 25 isolates from appam broth. Production of acid and CO2 from glu-

54
cose was tested in MRS broth containing dually and incubated for 24 h. About 25 ml of
Durhams tube, with citrate omitted [18] and in MRS agar was seeded with the cultures of
Gibson’s semi-solid tomato-juice medium by 6
LAB isolates (10 CFU/ml) mixed well, poured
the method of Gibson and Abd-el-Malek [9]. into sterile Petri plates and stored at 4 oC for
Production of ammonia in MRS broth omitting 1 h to solidify the media. OCTA-discs (8
glucose and meat extract, while containing antibiotics in a single ring) were placed up
0.3 % arginine and 0.2 % sodium citrate side down, pressed on the top of the agar
replacing ammonium citrate, was monitored plates and kept again at 4 oC for 1 h. The
using Nessler’s reagent. Homo- and hetero- plates were incubated at 37 oC over night.
fermentative differentiation test was also carried Resistance was defined as the absence of a
out according to the method reported by growth inhibition zone around the discs.
Zuniga et al. [24]. Assays for gelatin hydrolysis Cell wall protein extraction and ana-
and nitrate reduction were performed in lysis. LAB isolates were grown in MRS broth
accordance with Harrigan [9]. Ability to ferment and cells harvested and resuspended in 3 ml
various carbohydrates was evaluated using distilled water (A600nm=2) and centri-fuged.
MRS broth supplemented with filter sterilized Cell wall proteins were extracted from final
sugar solutions to a final concentration of 1 % pellets with 0.5 ml of 0.01 M Tris-HCl, 0.01 M
w/v and 0.004 % chlorophenol red without EDTA, 0.01 M NaCl, 2 % SDS, pH 8.0, at
glucose and meat extract [18]. The confi- 100 oC for 5 min [8]. After treatment sus-
guration of lactic acid formed from glucose was pensions were centrifuged at 11 600 g for
determined enzymatically using D-lactate and 10 min and supernatant fluids were examined
L-lactate dehydrogenase. using Tris-Glycine SDS-PAGE [14] with sta-
Antibiogram of LAB isolates. The iso- cking gel of 5 % and separating gel of 20 %
lates were inoculated into MRS broth indivi- acrylamide – bisacrylamide.

Results and Discussion

Ten out of 25 randomly picked colonies mical characters as well as sugar utilization
exhibited good antimicrobial activity against pattern DFR JJ1, 6, 9 and 10 were identified
Gram-positive bacteria, viz., B. cereus, St. au- as Pediococcus pentosaceous, DFR JJ11 as
reus and L. monocytogenes as well as Gram- Tetragenococcus halophilus, DFR JJ4 and
negative bacteria, viz. Ps. aeruginosa, V. para- DFR JJ5 as Lactobacillus plantarum.
haemolyticus and A. hydrophila and were The four strains of P. pentosaceous were
studied further. It was observed that two of the Gram-positive cocci, arranged in tetrads and
strains resembled with other two in all aspects pairs. These strains were catalase-negative,
of characterization and hence we were left out did not produce CO2 from glucose, homo-
with 8 bacteriocin producing LAB. The investi- fermentative on HHD medium, did not reduce
gation of 7 LAB is presented in this study. nitrate and produced both L-(-) and D-(+)
All the 7 strains were found to be Gram- form of lactic acid (Table 1). These are also
positive while morphologically DFR JJ1, 6, 9, the major characters of genus Pediococcus
10 and 11 were cocci and DFR JJ4 and DFR as reported earlier [4, 6, 7]. These four P. pen-
JJ5 were bacilli. Physiological and bioche- tosaceous strains produced NH3 from argi-
mical properties as well as carbohydrate nine and did not hydrolyze gelatin. Only two
fermentation pattern of these strains are species of Pediococcus genus, viz., P. aci-
shown in Tables 1 and 2. On the basis of dilactici and P. pentosaceous were able to
morphological, physiological and bioche- hydrolyze arginine [3, 6, 7]. Luxurious growth

55
was observed for all 4 strains at 37 oC while it the four strains were identified as P. pento-
was weak at 10 oC and none could grow at saceous, however they were different strains
45 oC (Table 1). P. acidilactici can arbitrarily be since they distinguished among themselves
differentiated from P. pentosaceous by its abi- regarding sugar fermentation pattern as well
lity to grow at 50 oC and survive even 70 oC for as their resistance and sensitivity towards
10 min [7, 16]. Carbohydrate fermentation pat- various antibiotics (Table 3).
tern of these strains showed that all the four T. halophilus strain cells were found to be
strains were able to ferment maltose, treha- Gram-positive cocci occurring in tetrads and
lose, xylose, cellobiose, and galactose but pairs and exhibited weak reaction for cata-
could not ferment sorbitol, mannitol, glycerol lase. Presence of pseudo-catalase in some
and arabinose (Table 2). Ability to ferment strains of Pediococcus and Tetragenococcus
maltose is a special character of P. pento- lead to catalase-positive reaction [19]. Strain
saceous, while P. acidilactici could not ferment DFR JJ11 did not grow at pH 4.4 but grew
it [3, 6, 7]. On the basis of all these properties luxuriously at pH 8.6 and in 15 % NaCl. It was

Table 1. Morphological, physiological and biochemical properties of bacteriocin producing LAB isolates.

Isolates
Tests
JJ1 JJ4 JJ5 JJ6 JJ9 JJ10 JJ11
Cocci Rods Rods Cocci Cocci Cocci Cocci
Morphology Tetrads Tetrads Tetrads Tetrads Tetrads
& Pairs & Pairs & Pairs & Pairs & Pairs
10 + ND ND + + + –
Growth at
15 ND + + ND ND ND ND
temperature
(oC) 37 ++ ++ ++ ++ ++ ++ ++
45 – – – – – – –
4.4 w w w + w w –
5.0 + + + + + + –
Growth at pH
8.6 ++ ++ ++ ++ ++ ++ ++
9.0 ++ ND ND ++ ++ ++ ++
6.5 w vw vw w w w ++
Growth at
10 – – – – – – ++
NaCl (%)
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: growth (+), no growth (–), luxurious growth (++), weak growth (w), homofermentative
(Ho), very weak (vw), not determined (ND), weak reaction due to pseudocatalase (+1), glucose
(Glc), arginine (Arg).

56
 Table 2. Carbohydrate utilization pattern of the bacteriocin producing LAB isolates.

Isolates
Carbohydrates
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 (–), delayed fermentation (+*).

found to be homofermentative, hydrolyzed argi- negative bacilli. Both the strains were homo-
nine and did not produce CO2 from glucose fermentative and did not produce CO2 and am-
(Table 1). This strain did not ferment arabinose, monia from glucose and arginine respectively.
sorbitol and glycerol but easily fermented suc- Their growth was weak at 15 oC and pH of 4.4,
rose, maltose, mannose and trehalose (Table 2). while both grew luxuriously at 37 oC and pH of
Garvie [7] and Villar et al. [23] also reported 8.6. The two strains produced both L-(-) and D-
similar results for P. halophilus, which later was (+) form of lactic acid (Table 1). Among sugars
renamed as T. halophilus [19]. This strain be- examined both strains fermented mannitol, meli-
longs to family Aerococcaceae which is consi- biose, esculine, galactose, sorbitol, sucrose,
dered a LAB from a practical food technology raffinose and trehalose but they could not
point of view [2]. T. halophilus strain was highly ferment arabinose, glycerol and xylose. Lb. plan-
resistant to streptomycin and moderately re- tarum JJ5 was able to ferment rhamnose, how-
sistant to nalidixic acid, co-trimazine and co- ever, the same was not utilized by other strain
listin, while it was sensitive for all other anti- JJ4 (Table 2). Similar characters for Lb. plan-
biotics examined (Table 3). tarum have been observed earlier [13, 22]. The
Both strains of Lb. plantarum, viz., JJ4 and behavior of these two strains against various
JJ5 were found to be Gram-positive, catalase- antibiotics was also found to be similar (Table 3).

57
 Table 3. Antibiogram of bacteriocin producing LAB isolates determined by antibiotic sensitivity
OCTA-discs.

Concen-
Isolates
Antibiotics tration
(µg) 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: sensitive (S), resistant (R), moderate (M).

Cell wall protein extraction and ana- all being P. pentosaceous, however, DFR JJ1
lysis. Cell wall protein profiles for 7 bacte- being P. pentosaceous differed considerably.
riocin producing LAB isolates are depicted in The profile of DFR JJ11 was found to be
Fig. 1. Two major bands of >66 kDa were differed from others and this was charac-
present in case of DFR JJ6, 9 and 10, while terized as T. halophilus.
only one band was observed for DFR JJ1, 4 In conclusion, eight LAB isolates from
and 5. However, these bands were absent in appam batter, capable of producing good
DFR JJ11. One band of ~66 kDa was pro- amount of bacteriocins have been anticipa-
minent in DFR JJ1 and DFR JJ9 but not in ted to have enormous potential for food ap-
DFR JJ6. DFR JJ1 profile shows a band of plications as biopreservatives. Among these
~40 kDa while a band of ~38 kDa could be strains, characterization of one lactobacillus
seen for DFR JJ6, 9 and 10. Bands of ~21.5 isolate and its bacteriocin as well as its
kDa and 15 kDa were prominent in DFR JJ6, 9 potential in preserving vegetarian food sys-
and 10, while absent in DFR JJ1. The profile tems has been reported earlier [11, 12]. Stu-
of DFR JJ4 and 5 showed much resemb- dies on the production, purification and cha-
lance, both being Lb. plantarum, while the racterization of bacteriocins from other iso-
strains DFR JJ6, 9, 10 showed similar profile, lates are in progress.

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 Fig. 1. Cell wall proteins profile of the LAB isolates. Lanes: MW markers (1), JJ1 (2), JJ6 (3), JJ9 (4),
JJ10 (5). MW markers (6), JJ4 (7), JJ5 (8) and JJ11 (9).

Acknowledgements. The authors are thank- Research Laboratory, Mysore for his keen
ful to Dr. A. S. Bawa, Director, Defence Food interest and providing the facilities.

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ИЗОЛИРАНЕ И ХАРАКТЕРИЗИРАНЕ НА БАКТЕРИОЦИН

ПРОДУЦИРАЩИ МЛЕЧНОКИСЕЛИ БАКТЕРИИ ОТ ЮЖНОИНДИЙСКИЯ
СПЕЦИАЛЕН ПРОДУКТ “APPAM BATTER”

Виджай Пал, Марилигапа Джамуна, Кадирвелу Джееваратнам*

Резюме
Известно е, че млечнокиселите бактерии (LAB), използвани обикновено
като стартерни култури в храните, продуцират антимикробни вещества -
бактериоцини, които имат голям потенциал като хранителни биоконсерван-
ти. Представеното проучване е насочено към изолиране на бактериоциногенни
LAB от традиционния ферментирал продукт “Appam”. Изолирани са 25 колонии
на LAB, които са изпитани за продуциране на бактериоцини. Десет изолата
показват добра антимикробна активност срещу Грам-положителни (Bacillus
cereus, Staphylococcus aureus, Listeria monocytogenes) и Грам-отрицателни
бактерии (Pseudomonas aeruginosa, Vibrio parahaemolyticus, Aeromonas hydro-
phila). В процеса на изследването е установено, че два от щамовете са сходни
на други два. За един от бацилите е докладвано по-рано. Затова, в настоя-
щата работа се представя характеризирането на 7 изолата чрез морфо-
логични, физиологични, биохимични и ферментационни тестове, както и чрез
белтъчните профили на клетъчните стени. Пет от тях са коки, иденти-
фицирани като Pediococcus pentosaceous (4 щама) и един като Tetragenococcus
halophilus. За останалите два пръчковидни щама е установено, че са Lacto-
bacillus plantarum.

60