Production, Purification, Stability and Efficacy of Bacteriocin from Isolates of Natural Lactic Acid Fermentation of Vegetables

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V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006)
ISSN 1330-9862
preliminary communication
Production, Purification, Stability and Efficacy
of Bacteriocin from Isolates of Natural Lactic Acid
Fermentation of Vegetables
Vinod Kumar Joshi1*, Somesh Sharma1 and Neerja S. Rana2
1Fermentation Technology Laboratory, Department of Postharvest Technology
2Department of Vegetable Crops, Dr. Y. S. Parmar University of Horticulture and Forestry,
Nauni, 173230 Solan (H.P.), India
Received: November 11, 2005
Accepted: March 14, 2006
The antimicrobial activity of partially purified bacteriocin produced during natural
lactic acid fermentation of carrot, radish and cucumber was assessed and characterized.
Out of ten strains, the isolated strain CA 44 of Lactobacillus genus from carrot fermentation
produced bacteriocin with maximum antimicrobial activity against Escherichia coli, Staphy-
lococcus aureus
and Bacillus cereus, though it was more effective against E. coli than others.
Bacteriocin was stable at up to 100 °C but its activity declined compared to that at 68 °C
and was completely lost at 121 °C. The maximum antimicrobial activity was retained with-
in the pH range of 4–5, but it was adversely affected by the addition of papain. Bacteriocin
was also effective against B. cereus in different fruit products (pulp, juice and wine) indi-
cating its potential application as a biopreservative in fruit products.
Key words: antimicrobial, bacteriocin, lactic acid fermentation, Lactobacillus, Staphylococcus,
Bacillus cereus
, E. coli, pathogenic microorganism, stability, biopreservative
dustry in the face of increasing consumer demand for
natural products and the growing concern about food-
Preservation of vegetables by lactic acid fermenta-
borne diseases. It has also necessitated the need to ex-
tion is an ancient practice involving lactic acid bacteria
ploit the biologically derived antimicrobial substances
(LAB), which predominantly produce lactic acid besides
produced by LAB. It is not clear if any bacteriocin is
certain compounds such as bacteriocin, which has anti-
produced in the vegetables fermented by LAB in natural
microbial activity against other groups of microorgan-
or inoculated fermentation. The bacteriocin produced by
isms. The antimicrobial activity of bacteriocins produced
the strains isolated from naturally fermented vegetables
by LAB has been detected in foods such as dairy prod-
has neither been characterized nor checked for its effi-
ucts, meats, barley, sourdough, red wine, fermented veg-
cacy in various food products. Therefore, keeping in view
etables, etc. (1–5). Therefore, the strains of lactic acid
the above objectives the present investigations were car-
bacteria have also potential to act as a biopreservative or
ried out and the results obtained are discussed here.
natural food preservative (6–8). The bacteriocins pro-
duced inhibited food spoilage and pathogenic bacteria
Materials and Methods
such as Staphylococcus aureus, Escherichia coli, Bacillus ce-
reus, B. subtilis, Listeria monocytogenes
and Clostridium per-
Fermented vegetables
fringens which are recalcitrant to traditional food preser-
vation method (9). The use of bacteriocins or the micro-
Vegetables (carrot, radish and cucumber) procured
organisms that produce them is attractive to the food in-
from the markets were washed, peeled and grated/sliced.
*Corresponding author; Fax: ++91 (0)1792 252 242; E-mail: [email protected]

V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006)
The grated carrot and radish were fermented with dry
Characterization of bacteriocin
salt 2 % (by mass) at 27 °C, whereas sliced cucumbers
were fermented in 3 % (by mass per volume) brine at 32
Heat stability
°C. Predominant microflora were isolated from these
A volume of 5 mL of bacteriocin in different test
tubes was overlaid with paraffin oil to prevent evapora-
tion and then heated at 68 and 100 °C for 10 and 20 min,
respectively, and at 121 °C for 15 min under pressure.
Pathogenic bacterial cultures
The heat-treated bacteriocin samples were then assayed
for antimicrobial activity as described earlier.
Standard bacterial cultures, viz. Escherichia coli (0165),
Staphylococcus aureus (B-43-5) and Bacillus cereus procured
Effect of pH
from Central Research Institute (CRI), Kasauli, were used
A 5-mL aliquot of partially purified bacteriocin was
in bacteriocin screening procedures and all the cultures
taken in test tubes and the pH values of the contents
were maintained as per the recommended practices.
were adjusted to 2–9 individually, using either diluted
NaOH or HCl (1 M NaOH or 1 M HCl solution). After
allowing the samples to stand at room temperature for 2 h
Isolation and identification of bacteriocin
the activity was assayed as described earlier.
producing bacteria
Effect of proteolytic enzyme (papain)
The bacteriocin producers from naturally fermented
A 5-mL aliquot of bacteriocin preparation was taken
carrot, radish and cucumber were isolated by pour plate
in test tubes and treated with papain (100 TU) 1 mg/mL
method technique as per the conventional method (10)
at pH=7. The test tubes with and without the enzyme
using MRS agar. After incubation for 24–48 h at 32 °C,
(control) were incubated for 2 h at 37 °C and heated for
typical colonies were isolated and purified. The isolates
3 min at 100 °C to denature the enzyme. Both the con-
were differentiated on the basis of their morphological,
trol and the samples were assayed for antimicrobial ac-
cultural and physiological characteristics such as oxidase
tivity by using well diffusion method.
test, utilization of citrate as a sole carbon source and ca-
talase test (10,11), and accordingly were tentatively iden-
Determination of preservative effect of bacteriocin
tified up to the genus level (12).
The food products, viz. juice (apple), pulp (apricot)
and prepasteurized wine (plum) were sterilized and ino-
Screening of isolates for antimicrobial activity
culated with Bacillus cereus at 108 CFU/mL. Initial count of
inoculated samples was recorded and bacteriocin super-
Antimicrobial activity of the bacterial isolates against
natant at a concentration of 0.05 to 0.5 % was added. Af-
all the pathogenic microorganisms was determined by
ter 24 and 72 h, the plate count was recorded and com-
well diffusion method (13–16) under aerobic conditions.
pared with the control (without bacteriocin).
Agar plates were inoculated with 100 mL of each target
microorganism after growing them in a broth and dilut-
ing appropriately. Wells (3 mm) were cut into the plates
Results and Discussion
and 100 mL of cell-free culture supernatant fluid of the
Based on morphological and biochemical tests, all the
isolated strain was placed into each well. The inhibitory
isolates were identified as belonging to lactic acid bacte-
activity against E. coli was tested on EMB agar whereas
ria (LAB) group except RA33, which was identified as
Staphylococcus aureus and Bacillus cereus were tested on
yeast. The isolate CA44 (giving maximum antimicrobial
nutrient agar. Plates were kept at cool temperature for 2 h
activity) was Gram-positive, rod shaped, negative for
and then incubated at 37 °C for 24 h. The antimicrobial
catalase and peroxidase test, having circular and white
activity was determined by measuring the diameter of
colonies on the MRS media. The strain was also positive
the inhibition zone around the wells. The bacterial isolate
for galactose, arabinose, mannitol, sorbitol, sucrose, glu-
showing the widest zone of inhibition against the target
cose, trehalose, lactose, raffinose and negative for malt-
microorganism was selected for further studies.
ose, citrate and arginine test. Isolate CA44 from carrot
produced the maximum inhibition zone against all the
tested microorganisms and was maximum against E. coli.
Partial purification of bacteriocin
The best conditions for bacteriocin production by Lac-
Isolated strain having maximum antimicrobial zone
tobacillus plantarum in batch fermentation were the salt
was grown in MRS broth at 37 °C for 24 h. After incuba-
concentration ranging from 2.3 to 2.5 % and temperature
tion, the broth was centrifuged at 5000 × g for 10 min
ranging from 22–27 °C (17). Lactobacillus plantarum strain
and the cells were separated out. Supernatant was used
isolated from fermented carrots which produced bacte-
as a crude bacteriocin. Different concentrations of am-
riocin with antibacterial activity against Staphylococcus
monium sulphate were added to the supernatant. After
aureus and spheroplasts of Gram-negative bacteria (18)
stirring on a magnetic stirrer, it was kept undisturbed at
and Lactococcus lactis ssp. cremoris was also isolated from
4 °C overnight. Precipitates formed were collected by cen-
radish fermentation (1).
trifugation at 10 000 × g for 10 min and redissolved in 20
An increase in antimicrobial activity after partial pu-
mmol sodium phosphate buffer with pH=6.0. Inhibition
rification of crude bacteriocin by ammonium sulphate pre-
zone of different fractions was recorded in comparison
cipitation took place (Fig. 1). The fraction with the high-
with the crude bacteriocin.
est bacteriocin activity was precipitated with 20–30 %

V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006)
The partially purified bacteriocin showed maximum
activity against the target microorganisms at pH=5.0
(Fig. 2), but after pH=5.0 the activity of the bacteriocin
gradually but continuously decreased. At pH=9.0, the
antimicrobial activity was drastically reduced to more
than 2.5 times that of the control. Thus, the bacteriocin
was found active over a wide pH range with the highest
o 10
activity at low pH range of 4–5. Earlier, the bacteriocin
produced by a newly isolated Bacillus species strain 8A
b 5
was found active over a pH range of 5–8 but was inacti-
vated when incubated outside these limits (9). Another
bacteriocin produced by Lactococcus lactis D53 and 23 was
20–30 30–40 40–50 50–60 60–70 70–80
active over a wide pH range with the highest activity
shown at low pH range of 3–5 (13), as was the case with
m/V (ammonium sulphate)/%
the bacteriocin from Pediococcus sp. (21). Bacteriocin ac-
Fig. 1. Increase in antimicrobial activity of bacteriocin from
tivity was completely lost when treated with proteolytic
Lactobacillus sp. isolate (CA44) using ammonium sulphate frac-
enzyme (papain), which is in agreement with the earlier
report (22). The bacteriocin pediocin ACH from Pedicoc-
cus acidilacti
was sensitive to proteolytic enzymes and was
completely inactivated by several proteolytic enzymes
(by mass per volume) ammonium sulphate. The antimi-
(22,23). The stability of bacteriocin to different condi-
crobial activity (in terms of inhibition zone diameter) in-
tions reflects that such compounds can withstand the
creased from 12 to 23 mm. There was 1.91-fold increase
conditions normally encountered in food processing, so
in the partially purified bacteriocin activity than that of
would remain effective during processing.
crude bacteriocin. Earlier, the inhibitory activity of bac-
teriocin isolated from malted barley was precipitated from
cell free supernatant using 40 % ammonium sulphate sa-
E. coli
turation, and resuspended in 2 mmol sodium phosphate
B. cereus
buffer, pH=6.0 and purified using chromatography (19).
S. aureus
te 19.0
Partially purified bacteriocin was found to be stable
e 17.0
at 68 °C for up to 20 min. At 100 °C for 10 min it could
d 15.0
retain 55 % of antimicrobial activity, while at the same
temperature for 20 min, only 28 % of activity could be
retained (Table 1). However, after incubation for 15 min
at 121 °C, the complete loss of activity took place. Com-
pared to the earlier reports on bacteriocin, residual ac-
tivity was lower in our study than reported earlier (20).
5.0 Control 2
Furthermore, since tolerance of bacteriocin to heat is
known to depend on the stage of purification, pH, pres-
Fig. 2. Effect of pH on antimicrobial activity of partially puri-
ence of culture medium, other protective components,
fied bacteriocin from Lactobacillus sp. isolate (CA44)
etc. that might have influenced the antimicrobial activity
in our findings too. The heat stability of bacteriocin dis-
cussed here indicates that it could be used as biopre-
The partially purified bacteriocin from isolate CA44
servative in combination with thermal processing to pre-
was also tested for preservative effect against B. cereus
serve the food products. Furthermore, when comparatively
(Table 2), and clearly the preservative effect in juice, wine
low temperature is employed for processing compared
and pulp increased with the increase in the concentra-
to high temperature being used at present, the retention
tion of bacteriocin. Maximum reduction of Bacillus cereus
of nutrients would be higher. However, more studies on
population of 92 % was observed in wine followed by
these aspects are needed.
juice (87 %) and pulp (63 %) at a concentration of 0.5 %.
Table 1. Effect of temperature on antimicrobial activity of partially purified bacteriocin from isolated Lactobacillus sp. (CA44)
Inhibition zone diameter/mm
E. coli
B. cereus
S. aureus
23 (100)
19 (100)
20 (95)
22 (95)
19 (100)
20 (95)
15 (65.21)
13 (68.42)
11 (55)
10 (43.47)
9 (47.36)
6 (28.57)
Control (without heat treatment)

Values in parentheses represent retention of antimicrobial activity (in %)

V.K. JOSHI et al.: Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol. Biotechnol. 44 (3) 435–439 (2006)
Table 2. Preservative effect of partially purified bacteriocin from
Lactobacillus sp. isolate (CA44) in juice, wine, and pulp against
Bacillus cereus
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Total count in control
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