Available online at www.jpsscientificpublications.com
Life Science Archives (LSA)
ISSN: 2454-1354
Volume – 4; Issue - 5; Year – 2018; Page: 1470 – 1489
DOI: 10.22192/lsa.2018.4.5.5
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Research Article
BACTERIOCIN PRODUCTION BY Lactobacillus nasuensis NAKR1
ISOLATED FROM FERMENTED UNNIAPPAM BATTER
K. Naresh Kumar1,2
, Krishna Revi1, S. Murugan
1 and Tha.Thayumanavan
2,3*
1Department of Biotechnology, Karunya Institute of Technology and Sciences, Coimbatore, Tamil Nadu,
India 2Department of Biotechnology, Dr. G.R. Damodaran College of Science, Coimbatore,
Tamil Nadu, India 3Department of Biotechnology, KIT – Kalaignar Karunanidhi Institute of Technology, Coimbatore, Tamil
Nadu, India
Abstract
Traditional fermented foods prepared from most common types of cereals (such as rice, wheat, corn
or sorghum) are well known in many parts of the world. In most of these products the fermentation is natural.
Unniappam is a small round snack prepared in all over the state of Kerala, India. We have reported here the
isolation of bacteriocin producing lactic acid bacterium - Lactobacillus nasuensis NAKR1 from the
fermented Unniappam batter. The bacteriocin produced was active against Listeria monocytogenes
MTCC657 and Acinetobacter baumannii MTCC 1425. We further reported the influence of growth
conditions on the bacteriocin production and characterized the partially purified bacteriocin.
Article History
Received : 02.05.2018
Revised : 12.06.2018
Accepted: 15.07.2018
Key words: Unniappam batter, Lactobacillus
nasuensis, Bacteriocin and Lactic acid bacteria.
1. Introduction
A wide variety of traditional fermented
foods made from ingredients like milk, cereals,
pulses, and vegetables have been developed for
the benefit of human health from ancient times
The primary microorganism responsible in
bringing about the desirable attributes in the final
products are those belonging to Lactic Acid
Bacteria (LAB). This indigenous microflora has
advantages in suppressing undesirable
microorganisms in food preservation and safety
(Vascovo et al., 1995).
* Corresponding author: Dr. Tha. Thayumanavan E.mail: [email protected]
Lactic Acid Bacteria (LAB) are found to
be associated widely in various traditional foods
throughout the world. These are a group of
bacteria that can preserve the dairy-based products
by synthesizing a number of organic compounds
that are antagonistic to other microorganisms
(Lindrren and Dobrogosz, 1990). Amongst the few
alleged benefits are strong antagonistic activities
against many microbes including food spoilage
organisms and pathogens by producing various
compounds such as organic acids, diacetyl,
hydrogen peroxide, and bacteriocins or bacterial
peptides during lactic acid fermentation
(Vandenbergh, 1993; Vossen et al., 1994;
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1471
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Zhennai, 2000). Moreover, the fermentation is
reported to enhance the nutritional quality of food
by improving the vitamin content and soluble
proteins (Sohliya et al., 2009).
The antimicrobial compounds or
substances produced by certain bacteria that are
active against the other bacteria are traditionally
defined as bacteriocins. Though, they are being
proteinaceous in nature, but differing in chemical
composition, mode of action and their specific
target organisms (Jack et al., 1995). The use of
these substances in extending the shelf life of
foods, vegetables, and milk or its products has
reported successfully (Schuenzel and Harrison,
2002). LAB are generally characterized as Gram-
positive bacteria and being deeply studied for their
production of bacteriocin-like substances (Heng et
al., 2007).
Bacteriocins are bioactive antimicrobial
peptides synthesized in the ribosome of numerous
bacteria and released extracellularly. Bacteriocins
have the ability to kill or inhibit the growth of
prokaryotes and could potentially be useful against
pathogens and antibiotic-resistant strains of
bacteria. The antimicrobial mechanisms and
relatively narrow killing spectrums of bacteriocins
distinguish them from traditional broad-spectrum
antibiotics, making them possible candidates to
replace antibiotics in the future Bacteriocins have
preservative properties and can be used as a bio
preservative which can replace chemical
preservatives which have side effect on health of
the consumer and well as the food to be preserved.
The research focused on bacteriocins from
LAB has expanded in recent decades to study their
antimicrobial activity against food and human
pathogens. Numerous alternative strategies are
employed in killing or controlling the pathogenic
organisms. The use of antimicrobial peptides
called bacteriocins has attracted highly as it is
considered as safe in the human point of view. In
recent years, researchers have discovered many
new bacteriocins from different sources.
Acinetobacter spp. was ubiquitous
inhabitants of soil, water, and sewage
environments. However, the association of LAB in
food fermentation could prevent the growth of
pathogenic and spoilage microorganisms. LAB
has been employed for centuries in the
fermentation of food, partly due to the fact that
they can prevent the growth of spoilage and
pathogenic microorganisms (Cheigh and Pyun
2005).
India is rich in fermented foods since
ancient time (Das and Deka, 2012), but the nature
of the product and their base materials vary from
region to region. Most of the traditional foods
contain beneficial bacteria named as probiotics
(Guamer and Schaafsma, 1998). Preparation and
consumption of fermented food believed to be
strongly linked with culture, tradition (Sekar and
Mariappan, 2007) and these indigenous
preparations still remain continuous as a
household art to date (Larry and Beuchat, 2008).
Unniappam, a traditional sweet snack
usually prepared during the festive seasons in the
state of Kerala of India. It is a small round snack
made from the ingredients like rice flour, wheat
flour, jaggery, ripe banana, coconut bits (grated
coconut), roasted sesame seeds, cardamom
powder, and ghee. These ingredients are mixed
and the batter is fried in oil to get unniappam. A
study was undertaken to explore the bacteriocin
producing lactic acid bacteria from the fermented
batter of ‘unniappam’ and to optimize the process
parameters for the bacteriocin production.
2. Materials and Methods
Chemicals
The media like Nutrient broth, Muller
Hinton Agar (MHA), de Man, Rogosa and Sharpe (MRS) broth, Agar agar type -1, Peptone, Tween
20, Tween 80, CTAB, SDS, EDTA, ammonium
sulphate and Cellulose membrane were procured
from Hi-Media, Mumbai. Whereas, all other
chemicals and reagents used in this study were of
the highest purity available.
Microorganisms
The strains chosen in this study were with
following characteristics: Listeria monocytogenes
MTCC 657, was known as food pathogen,
Acinetobacter baumannii MTCC 1425 and
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1472
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Methicillin-resistant Staphylococcus aureus
MTCC 1430 (MRSA) were the hospital
pathogens, Lactococcus lactis subsp. lactis MTCC
440 and Enterococcus faecalis MTCC 3159 were
bacteriocin producers. All these strains were
procured from Microbial Type Culture Collection
and Genebank (MTCC), IMTECH, Chandigarh
for the study undertaken. These strains were
propagated in appropriate media as directed by
IMTECH.
Collection and processing of ‘Unniappam’
batter
The batter for ‘Unniappam’ was prepared
by grinding the ingredients like rice flour, wheat
flour, jaggery, ripe banana, coconut bits (grated
coconut), roasted sesame seeds, cardamom
powder, and ghee. The batter was prepared by
mixing these ingredients as like a paste. The batter
was kept few hours (5 - 6 hours) allowing the
batter to ferment naturally, before the unniappam
dish preparation. This fermented unniappam batter
was collected in a sterile container and transported
to the laboratory by maintaining low temperature
and processed for further investigations within 24
hrs.
One gram of ‘unniappam’ batter was
diluted into 100 ml of 0.1 % (w/v) peptone water
and incubated at 37 °C under shaking conditions at
100 rpm for 1 - 2 hrs. The contents were serially
diluted (tenfold serial dilution) later with 0.1 %
(w/v) peptone water. An aliquot of 100 microlitres
from 10-7
dilution was spread onto sterile MRS
agar plates. The plates were incubated at 37 °C for 48 hrs under anaerobic conditions for the
development of colonies on the MRS agar. The
developed colonies were propagated further
separately on fresh MRS agar plates by quadrant
streaking. This process was repeated until
obtaining the pure culture. Every individual
colony developed was labeled as UB1, UB2, UB3
and so on for further references. The labeled
individual bacterial isolates were stored at 4 °C on
MRS agar slants for further investigations.
Preparation of cell - free culture filtrates
(CCFs)
The labeled isolates were inoculated into
100 ml of sterile MRS broth in 250 ml Erlenmeyer
flasks and incubated at 37 °C using shaking
incubator at 125 rpm for 24 hrs. The fermented
broth was centrifuged at 13,000 rpm for 15 min at
4 °C and the supernatant collected was adjusted to
pH 6.5 by using either 1 N NaOH or 1 N HCl ,
and filtered through 0.45 μm filter. This filtrate
was known as Cell - free Culture Filtrate (CCF)
which would be used for further studies.
Preliminary screening of LAB for antagonistic
activities
The CCFs of individual LAB isolates were
screened for antagonistic activity against the
indicator organisms Acinetobacter baumannii
MTCC 1425, Methicillin resistant Staphylococcus
aureus MTCC 1430 (MRSA), Listeria
monocytogenes MTCC 657, Lactococcus lactis
subsp. lactis MTCC 440 and Enterococcus
faecalis MTCC 3159 using MHA plates by well
diffusion method as described by Vignolo et al.
(1993). The MHA plates were prepared and
seeded with individual indicator organisms. The
wells with 6 mm diameter were made on the agar
plates, loaded the 50 μl of CCF and incubated at
37 °C for 24 hrs. The diameter (in mm) of the
zone of inhibition was measured and the
bacteriocin activity was determined. The LAB
isolate that showed highest antagonistic activity
was selected for further studies undertaken.
Examination of CCF for antibacterial activity
and bacteriocin assay
The selected LAB isolate was inoculated
into 100 ml of sterile MRS broth and the
bacteriocin production was carried out. Then the
CCF was processed from the broth as described
earlier. The bacteriocin activity assay was
performed according to Usmiati and Marwati
(2009) by the agar well method using the indicator
organisms. Fifty µl of CCF from the selected LAB
isolate was loaded into 6 mm diameter wells in
Mueller Hinton agar plates previously seeded with
indicator organisms like L. monocytogenes MTCC
657 and A. baumannii MTCC 1425. The plates
were examined after 24 hrs of incubation at 37 °C,
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1473
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
for the zone of growth inhibition of indicator
organisms. The bacteriocin activity was expressed
as arbitrary units per milliliter (AU/ml). One AU
of bacteriocin was defined as a unit area of
inhibition zone per unit volume of bacteriocin
added, in this case, mm2/ml. The bacteriocin
activity (AU/ml) was calculated using the
following formula:
Lz = clear zone area (mm
2)
Ls = well area (mm2)
V = volume of sample (ml)
All the assays were carried out in
triplicates for individual indicator strains L.
monocytogenes MTCC 657 and A. baumannii
MTCC 1425. The antibacterial effects of CCF of
the selected LAB isolate were evaluated by using
A. baumannii and L. monocytogenes in subsequent
assays.
Morphological characteristics of the
bacteriocin producing LAB culture
The selected bacteriocin producing LAB
isolate was evaluated for its morphological,
physiological and biochemical characteristics
according to the criteria of Bergey’s manual of
Determinative Bacteriology (1994), and the results
were recorded.
Identification of bacteriocin producing LAB
isolate by 16S rRNA sequencing and
phylogenetic relationship
Genomic DNA of the selected LAB isolate
was isolated by the method described by Galvez et
al. (2007). The 16S rRNA was amplified using
both forward and reverse primers (16S1: 5’-
GCTCACCCTTAACCC-3’ and 16S2: 5’ACCTTCCAAGGGCCTAC-3’) from genomic
DNA. The assay was performed by using Taq
DNA polymerase and buffers in the thermocycler
for 30 cycles comprising 95 °C denaturation for 30
s, 55 °C annealing for 30 s and 72 °C for the
extension for 45 s. The PCR amplified rRNA
product was purified using the quick PCR
purification kit. The analysis of alignment,
homology and the construction of the phylogenetic
tree was performed. The nucleotide sequences
determined in this study have been submitted to
GenBank for assigning the Accession No.
Optimization of process parameters for
bacteriocin production
The selected LAB isolate was used as a
starter culture for the optimization of bacteriocin
production by the conventional method.
Effect of temperature on bacteriocin
production
The selected LAB isolate was inoculated
into a 250 mL Erlenmeyer flasks containing 100
ml of sterile MRS broth (initial pH 6.5±0.2). The
flasks were maintained at different temperatures
viz., 25, 30, 35, 40 and 45 °C for 24 hrs in a shaker
cum incubator (125 rpm). All other parameters
like medium components, incubation time and pH
were kept constant. The CCF was prepared after
the incubation time and subjected to antibacterial
activity.
Effect initial pH on bacteriocin production
The variation on initial pH of MRS
medium for bacteriocin production was analyzed
by preparing MRS medium with varying or
adjusting the initial pH to 4.5, 5.0, 5.5, 6.0, 6.5,
7.0 and 7.5 using either 1 N NaOH or 1 N HCl.
The selected LAB isolate was inoculated into 250
ml Erlenmeyer flasks containing 100 ml of sterile
MRS broth and incubated at 37 °C for 24 hrs for
the production of bacteriocin under shaking
conditions (125 rpm) in a shaker cum incubator.
All other parameters like medium components,
incubation time and temperature were kept
constant. After the incubation time, the CCF was
prepared and subjected to antibacterial activity.
Effect of incubation time on bacteriocin
production
Briefly, 100 mL of MRS broth was
prepared, inoculated with the selected LAB and
incubated at different time intervals like 0, 12, 24,
36, 48, 60 and 72 hrs. After the respective time
intervals, an aliquot of CCF was prepared by
collecting broth by centrifugation to measure the
optimum incubation time of bacteriocin
production.
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1474
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Effect of carbon and nitrogen sources on
bacteriocin production
The effects of carbon and nitrogen sources
on the production of bacteriocin were evaluated
(Hoda et al., 2013). An experimental setup was
prepared for the production of bacteriocin by
varying the concentrations of carbon and nitrogen
sources (protease peptone, beef extract, yeast
extract, and dextrose) of the MRS medium as
described in Table - 1, whereas the existing MRS
broth served as control.
Three sets of the run were carried out by
inoculating the selected bacteriocin producing
isolate at 37 °C in an incubator cum shaker (125
rpm) for 24 hrs. The CCFs obtained were tested
for antimicrobial activity against the indicator
organisms L. monocytogenes MTCC 657 and A.
baumannii MTCC 1425.
Table – 1: Experimental setup for screening the
influence of carbon and nitrogen sources for
the production of the bacteriocin
S.
No
Modified ingredient
in MRS broth
Concentration
(g/L)
1 Protease peptone (g/l)
5.0
10.0
15.0
2 Beef Extract (g/l)
5.0
10.0
15.0
3 Yeast Extract (g/l)
2.5
5.0
7.5
4 Dextrose (g/l)
10.0
20.0
30.0
5 MRS broth --
Partial purification of bacteriocin from CCF by
ammonium sulphate precipitation and dialysis
The CCF obtained from the selected LAB
isolate was subjected to 60, 70 and 80 %
saturation with solid ammonium sulphate with
continuous stirring until dissolving the salt. Then
the contents were kept undisturbed at 4 °C
overnight with an occasional stirring (Yang et al.,
1992). Later the contents were centrifuged at
16,500 rpm at 4 °C for 30 min. The pellet and
supernatant were separated. The pellet was
reconstituted with sterile water and both
supernatant and reconstituted pellet sample were
dialyzed separately against 10 mM sodium
phosphate buffer (pH 6.5) using a tubular cellulose
membrane dialysis bag at 10 °C. The buffer was
changed 3 - 4 times with an interval of 6 - 7 hrs.
Bacteriocin assay was performed in all the
fractions after dialysis.
Protein Determination
The amount of protein present in the crude
bacteriocin samples like CCFs and the fractions
obtained after partial purification process was
determined using Bovine Serum Albumin fraction
- V (BSA) as a standard by Lowry’s method
(Lowry et al., 1951).
Characterization of the partially purified
bacteriocin
The partially purified bacteriocin sample
from the selected LAB isolate was evaluated for
its antimicrobial activity with respect to the
influencing factors like temperature, pH and
susceptibility to denaturation by enzymes,
surfactants, metals and different concentrations of
NaCl (Shiba et al., 2013).
Effect of temperature on bacteriocin activity
Ten milliliters of partially purified
bacteriocin sample obtained from the selected
LAB isolate was exposed to various temperatures
like 20, 35, 50, 65, 80, 95 and 110 °C for 2 hrs and
the fractions from each sample were examined for
bacteriocin activity against the indicator
organisms by agar well diffusion method.
Effect of pH on bacteriocin activity
Ten milliliters of partially purified
bacteriocin sample obtained from the selected
LAB isolate was adjusted to pH 3.5, 4.5, 5.5, 6.5,
7.5, 8.5 and 9.5 by adding either 1N sodium
hydroxide or 1N hydrochloric acid and incubated
for 2 hrs at room temperature. Residual
bacteriocin activity of every sample was determined against the indicator organisms by the
agar-well diffusion method (Rattanachaikunsopon
and Phumkhachorn, 2006).
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1475
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Effect of surfactants on bactericidal activity
The effect of surfactants on the activity of
bacteriocin was examined by mixing non-ionic
(Tween 20: 0.5 % v/v); Tween 80: 0.5 % (v/v) and
ionic (SDS 0.1 % w/v), CTAB 0.1 % (w/v)
surfactants to the partially purified bacteriocin.
The samples were incubated at room temperature
for 2 hrs and assayed for antimicrobial activity
against indicator organisms by well diffusion
assay as described by Hoda et al. (2013) and the
zone of inhibition was measured. The MRS broth
with the similar concentration of surfactants
served as control.
Effect of enzymes on bacteriocin activity
The sensitivity of partially purified
bacteriocin to enzymes was examined by treating
the bacteriocin sample with enzymes like trypsin,
papain and α-amylase to a final concentration of 1
mg/ml (in phosphate buffer at pH 6.0). The
contents were incubated at 37 °C for 2 hrs and the
remaining antimicrobial activity was determined
against the indicator organisms (Shiba et al.,
2013).
Effect of metal ions on bacteriocin activity
The impact of metal ions on bacteriocin
activity was analyzed by adding MgSO4 and
CuSO4 at 0.5 % (w/v) level to the partially purified
bacteriocin and the samples were incubated at
room temperature for 2 hrs. The bacteriocin
activity of each sample was determined against the
indicator organisms by Agar well diffusion assay
(Rushdy and Gonnaa, 2013).
Effect of NaCl on bacteriocin activity
The influence of sodium chloride
concentration at different levels on bacteriocin
activity was performed by adding 2, 4, 6 and 8 %
(w/v) NaCl to partially purified bacteriocin
samples. After 2 hrs of incubation at room
temperature, the samples were examined for
bacteriocin activity against the indicator
organisms by Agar well diffusion method (Hoda
et al., 2013).
Molecular mass determination
The molecular weight of partially purified
bacteriocin was determined by tricine-SDS-PAGE
gel electrophoresis (Hailer et al., 2012). Ten µg of
partially purified bacteriocin mixed with sample
loading buffer (15 mM Tris HCl pH 6.8, 2.3 %
SDS, 10 mM 2- mercaptoethanol, 20 % glycerol, 1
% bromophenol blue) was loaded into the wells
and was run on 10 % tricine SDS PAGE. After the
run, the gel was removed and cut into half. The
half containing sample and molecular weight
marker were stained with Coomassie brilliant blue
R 250 (Sambrook et al., 1989). The other half
containing the samples was processed as described
by Mirhosaini et al. (2006). Then, the gel was
placed in a petridish and overlaid with 7 ml of 0.6
% (w/v) agar containing the indicator organisms.
Then, the plate was incubated at 37 °C for 24 hrs
and the antimicrobial activity of bacteriocin was
screened by the clear zone of inhibitions.
3. Results and Discussion
A wide variety of traditional fermented
foods made from ingredients like milk, cereals,
pulses and vegetables have been developed for the
benefit of human health from ancient times. Most
East-Asian fermented foods are non-dairy
products featuring various other food raw-
materials such as cereals, soybeans, fruits, and
vegetables, as well as fish and other marine
products. The importance of lactic acid bacteria in
fermented non-dairy foods and beverages was
reviewed previously in the early 1990s (Lee,
1994). LAB played a vital role along with yeasts
during dough fermentation and resultant products
had higher contents of lactic acid and acetic acid
due to bacterial growth (Salim-Ur-Rehman et al.,
2006). During cereal fermentation, the nutritional
and mineral contents of raw cereals are always
enhanced (Umeta et al., 2005). The cereal based
fermented food has been further reviewed by Jyoti
Prakash Tamang (2010). The primary
microorganisms responsible for bringing about the
desirable attributes in the final products are those
belonging to Lactic Acid Bacteria (LAB).
Alternative raw materials for probiotics need to be
searched for the developing countries like India
due to economic reasons. Hence, an attempt was
carried out to explore the bacteriocin producing
LAB from the fermented unniappam batter.
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1476
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Preliminary screening of LAB for antagonistic
activities
Totally, 3 colony forming units were
identified on MRS agar plates under anaerobic
conditions during 48 hours of incubation from the
unniappam batter, labeled as UB1, UB2 and UB3
and stored at 4 °C for further analysis. These
isolates were grown separately on MRS broth and
cell-free culture filtrates were prepared. The CCFs
of individual isolates were screened for their
antagonistic behavior against the indicator
organisms and the results reported as Table - 2.
Table - 2: Antimicrobial activity (AU/ml)* of CCFs of 3 LAB isolates against the different indicator
organisms
Indicator strain
LAB isolates from fermented
Unniappam batter*
UB1 UB2 UB3
Acinetobacter baumannii MTCC 1425 - 3456
Enterococcus faecalis MTCC 3159 - - -
Lactococcus lactis subsp.lactis MTCC 440 - - -
Listeria monocytogenes MTCC 657 - - 2513
Methicillin-resistant Staphylococcus
aureus MTCC 1430 (MRSA) - - -
*Zone of growth inhibition of indicator organisms in diameter (mm) was used to calculate the antimicrobial activity.
The CCF of the UB3 isolate was found to
be active against the A. baumannii and L.
monocytogenes among the 3 LAB isolates. Hence,
the antagonistic behavior of UB3 was screened
only with these 2 indicator organisms further.
Morphological characteristics of the
bacteriocin producing LAB culture
The isolate UB3 was found to be Gram
positive, rod shaped, non-motile, non-spore
forming and catalase negative bacterium. It failed
to produce gas while fermenting glucose,
however, produced acids with D-glucose,
galactose, D-fructose and D-ribose. These reports
found to be similar to Yimin Cai et al. (2012).
Identification of bacteriocin producing LAB
isolate by 16S rRNA sequencing and
phylogenetic relationship
The 16S ribosomal RNA genome the LAB
isolate UB3 was sequenced and submitted to
GenBank with an accession number as
MH4887111. The BLAST analysis of this 16S
rRNA sequence showed that the UB3 isolate was
identified as Lactobacillus nasuensis NAKR1. The
phylogenetic tree was constructed using the 16S
rRNA sequence data and the taxonomic position
of Lactobacillus nasuensis NAKR1 was shown by
neighbor joining method as in the Figure - 1.
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1477
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Figure – 1: Neighbour-joining phylogenetic tree showing the taxonomic position of Lactobacillus
nasuensis NAKR1
Yimin Cai et al. (2012) were isolated two
strains of lactic acid bacteria, from silage prepared
with Sudan grass [Sorghum sudanense (Piper)
Stapf.] and proposed Lactobacillus nasuensis sp.
nov. as a novel species in the genus Lactobacillus
by analyzing the closest phylogenetic neighbors.
The position of this kind of species has
been documented in the Lactobacillus genus (L.
rhamnosus group) in the Lactobacillus phylogeny
using glycolysis enzyme sequences (Katelyn
Brandt and Rodolphe Barrangou, 2018).
Several strains of lactic acid bacteria from
food products as cheese and milk have been
isolated by Moreno et al. (1999) by detecting their
antagonistic activities through the well diffusion
assay on agar plates (Toro, 2005).
Bacterial fermentation of perishable raw
materials has been used for centuries to preserve
the nutritive value of food and beverages over an
extended shelf life (Deegan et al, 2006). Lactobacillus spp. played a significant role in
most of the fermented cereals (Sanni, 1993).
The presence of various microorganisms
like Lactobacillus fermentum, L. buchneri,
Streptococcus lactis, S. faecalis, and
Saccharomyces cerevisiae were found in the
fermented batter of the Indian traditional food
'Jalebi' a sweetened fermented product made out
of maida (refined wheat flour), dahi and water
(Sekar and Mariappan, 2007).
Our study reported a bacteriocin producing
LAB isolate Lactobacillus nasuensis NAKR1
acting on both Gram positive Listeria
monocytogenes MTCC 657 and Gram negative
Acinetobacter baumannii MTCC 1425.
Bacteriocins are not frequently active against
Gram negative bacteria (Stevens et al., 1991).
However, the bacteriocin activity against the
Gram negative bacteria has been reported by
remarkable researchers during the course of time
(Messi et al, 2001; Todorov and Dicks, 2004;
Todorov and Dicks, 2005). A potent bacteriocin
producer Lactobacillus delbrueckii subsp
bulgaricus isolated from yoghurt exhibited a broad
spectrum inhibition of both Gram positive and
Gram negative pathogens (Radha and Padmavathi,
2015). Usmiati and Marwati (2009) with a
Lactobacillus spp. (SCG 1223) showed that the
bacteriocin produced acted against both Gram
positive and Gram negative bacteria (E. coli, L.
monocytogenes and S. typhimurium).
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1478
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Optimization of process parameters for
enhanced production of bacteriocin by the
conventional method
Temperature
The antimicrobial activity CCFs obtained
after fermentation with Lactobacillus nasuensis
NAKR1 at various temperature zones against both
Listeria monocytogenes MTCC 657 and
Acinetobacter baumannii MTCC 1425 were
represented as Figure - 2.
Figure - 2: Effect of temperature on bacteriocin production by Lactobacillus nasuensis NAKR1 against
L. monocytogenes MTCC 657 and A. baumannii MTCC 1425
A maximum antimicrobial activity was
observed from the CCF obtained from the
fermentation broth incubated at 35 °C with
Lactobacillus nasuensis NAKR1. Beyond this, the
rise in temperatures didn’t support the bacteriocin
production. The same temperature was reported
for the highest antimicrobial activity of
Lactobacillus murinus AU06 isolated from marine
sediments against fish pathogens (Sivaramasamy
Elayaraja et al., 2014).
The optimum temperature for bacteriocin
production by Lactobacillus spp. (SCG 1223) was
reported as 33.5 °C by Usmiati and Marwati
(2009). The highest antimicrobial activity was
reported for a natural isolate of Lactobacillus
delbruecki ssp. bulgaricus CFR 202 at an
incubation temperature of 37 °C, when grown in
milk medium (Balasubramanyam and Varadaraj,
1998).
Our findings supported that the incubation
temperature 35 ± 2 °C showed the highest
antimicrobial activity of Lactobacillus spp.
studied. However, Yimin Cai et al. (2012)
reported that the optimum temperature for the
growth was approximately as 30 °C to their
strains SU 18T and SU 83 proposed as
Lactobacillus nasuensis sp. nov.
pH The initial pH of the incubation medium
had a good influence on the growth of the
microorganism under study. Highest antimicrobial
activity by Lactobacillus nasuensis NAKR1 was
observed in MRS medium with the initial pH as
6.0 (Figure - 3). Furthermore, the highest
antimicrobial activity of Lactobacillus murinus
AU06 was reported at pH 6.0 in the incubation
medium by Sivaramasamy Elayaraja et al. (2014).
They further reported that the lower pH like 5.0
yielded a lower bacteriocin production.
The optimum condition for bacteriocin
production was pH 5.0 reported for Lactobacillus
spp. (SCG 1223) by Usmiati and Marwati (2009).
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1479
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
However, the decrease in pH from an initial level
of 6.6 to around 3.7 in 48 hrs for the natural
isolate of Lactobacillus delbruecki ssp. bulgaricus
CFR 2028 was also recorded (Balasubramanyam
and Varadaraj, 1998).
Figure - 3 Effect of different initial pH on bacteriocin production by Lactobacillus nasuensis NAKR1
against Listeria monocytogenes MTCC65 and Acinetobacter baumannii MTCC 1425
Incubation period
The antimicrobial activity of CCFs
obtained during the incubation period with by
Lactobacillus nasuensis NAKR1 was screened
with both L. monocytogenes MTCC 657 and A.
baumannii MTCC 1425 up to 72 hours (Figure -
4). Highest antimicrobial activity was found
against the indicator organisms tested by the CCF
obtained during 48 hours of incubation.
Figure – 4: Production of bacteriocin by Lactobacillus nasuensis NAKR1 at various incubation periods
The antimicrobial activity of Lactobacillus
delbruecki ssp. bulgaricus CFR 2028 was high at
48 hrs of incubation when grown in milk medium.
The antibacterial activity appeared to be produced
between the late logarithmic and early stationary
phases (Balasubramanyam and Varadaraj, 1998).
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1480
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Whereas, the optimum conditions for bacteriocin
production by Lactobacillus spp. (SCG 1223) was
reported as 9-hour incubation period by Usmiati
and Marwati (2009). However, the antimicrobial
activity of Lactobacillus murinus AU06 was
reported higher at 30 hrs of incubation. Growth
beyond stationary phase resulted in the decrease in
bacteriocin production (Sivaramasamy Elayaraja
et al., 2014). Amidya Nugrahani et al. (2016)
stated that the production of bacteriocin by
Lactobacillus casei occurred at the 19th
hour of the
incubation period, and best production was
achieved at the end of the exponential phase or
early stationary phase.
Optimization of carbon and nitrogen source
medium constituents
The effect of major carbon and nitrogen
sources for the bacteriocin production using
Lactobacillus nasuensis NAKR1 was screened
through by altering the carbon and nitrogen
sources (protease peptone, beef extract, yeast
extract, and dextrose) in the MRS medium as
mentioned in Table - 1. The results were presented
as Figures - 5 to 8.
Figure – 5: Effect of protease peptone on bacteriocin production
Figure - 6: Effect of beef extract on bacteriocin production
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1481
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Figure - 7: Effect of yeast extract on bacteriocin production
Figure - 8 Effect of dextrose on bacteriocin production
Partial Purification of bacteriocin
The bacteriocin produced by Lactobacillus
nasuensis NAKR1 was partially purified from the
CCF by 60 - 80 % saturation with ammonium
sulphate. The reconstituted pellet after dialysis
showed improved antibactericidal activity. The
partially purified bacteriocin produced by
Lactobacillus nasuensis NAKR1 using 60 - 80 %
ammonium sulphate saturation with its CCF
showed improved antibactericidal activity against
A. baumannii (10057 AU/mg) and L.
monocytogenes (7425.97 AU/mg) respectively.
Various reports based on similar partial
purification procedures for bacteriocins produced
from L. plantarum and E. mundtii (Todorov et al.,
2004; Granger et al., 2005; Todorov et al., 2005).
The fold of purity of partially purified bacteriocin
was increased by 54.1 % and 53.3 % for Listeria
monocytogenes MTCC 657 and Acinetobacter
baumannii MTCC 1425 respectively. The
purification tables were summarized in Tables - 3,
3a, 3b, 3c.
Table - 3a): Purification table of bacteriocin from Lactobacillus nasuensis NAKR1 isolated from
fermented unniappam batter by ammonium sulphate precipitation method
Indicator strains
Bactericidal activity (AU/ml)
Cell-free
Culture
Filtrate
Partially purified (pellet
obtained at 60-80 % saturation
of ammonium sulphate)
Listeria monocytogenes MTCC 657 2,513 5718
Acinetobacter baumannii MTCC 1425 3,456 7744
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1482
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Table - 3b): Partially purified bacteriocin activity against Listeria monocytogenes MTCC 657
Sample Total
volume
(ml)
Protein
conc.
(mg/ml)
Total
proteins
(mg)
Total
activity
(AU)
Specific
activity
(AU/mg)
Fold
of
purity
Act.
Yield
Cell-free culture
filtrate (CCF) 97.4 18.3 1782.42 244766.2 137.3224 1 100
Reconstituted pellet
after dialysis 10.6 0.77 8.162 60610.8 7425.974 54.1 0.46
Table - 3c): Partially purified bacteriocin activity against Acinetobacter baumannii MTCC 1425
Sample Total
volume
(ml)
Protein
conc.
(mg/ml)
Total
proteins
(mg)
Total
activity
(AU)
Specific
activity
(AU/mg)
Fold
of
purity
Act.
Yield
Cell-free culture
filtrate (CCF) 97.4 18.3 1782.42 336614.4 188.8525 1 100
Reconstituted pellet
after dialysis 10.6 0.77 8.162 82086.4 10057.14 53.3 0.46
Characterization studies on partially purified
bacteriocin
The partially purified bacteriocin was
treated with various pH conditions, temperature
ranges, the presence of various metal ions,
different surfactants and its antimicrobial activity
was examined against the indicator organisms
used this study. Partially purified bacteriocin from
Lactobacillus nasuensis NAKR1 showed a good
antimicrobial activity against the test indicators
even incubating at the 85 °C for 2 hours. The
results were shown in the Table - 4.
Table – 4: Effect of temperature on the
antimicrobial activity of partially purified
bacteriocin from Lactobacillus nasuensis
NAKR1
Temperature
(°C)
Zone of inhibition (mm)
L. monocytogenes
MTCC 657
A. baumannii
MTCC 1425
20 2969 3974
35 5718 7744
50 4524 6362
65 2969 4524
80 1005 2513
95 0 0
110 0 0
The partially purified bacteriocin had
decreasing antimicrobial activity against the
indicator organisms after 2 hours at 50 °C
onwards. Our study found that the bacteriocin
from Lactobacillus nasuensis NAKR1 completely
lost its bactericidal activity at 95 and 110 °C. The
antibacterial activity of Lactobacillus delbruecki
ssp. bulgaricus CFR 2028 reported being stable at
75 °C (Balasubramanyam and Varadaraj, 1998).
Effect of pH sensitivity on bactericidal activity
The partially purified bacteriocin showed a
highest antimicrobial activity when treated with
pH 6.5 for 2 hrs. The antimicrobial activity was
found to be in a decreasing manner below and
above this pH. The antimicrobial activity of
bacteriocin was found minimal at pH 3.5 and
completely lost at pH 9.5 (Figure - 9).
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1483
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Figure- 9: Effect of pH on the activity of partially purified bacteriocin
Balasubramanyam and Varadaraj (1998) reported
that the inhibitory activity in the culture filtrate of
Lactobacillus delbruecki ssp. bulgaricus CFR
2028 remained constant at pH 4.0 to 4.5 and
completely lost at pH 6.0. Bacteriocin from
Lactobacillus casei had an optimum activity at pH
5 for Pseudomonas sp and pH 4 for Micrococcus
sp. (Nagrahani et al., 2016). Neha Gautam and
Nivedita Sharma (2009) found that the bacteriocin
of L. brevis had maximum activity at neutral pH,
though it had a wide range of activity of pH (3 -
10).
Effect of surfactants on bacteriocin activity
The influence of both non-ionic and ionic
surfactants on antimicrobial activity of bacteriocin
was reported in Table – 5
Table - 5: Effect of surfactants on the antimicrobial activities of partially purified bacteriocin
The antimicrobial activity of partially
purified bacteriocin from Lactobacillus nasuensis
NAKR1 was found to be sensitive with non-ionic
detergents like Tween 20 and Tween 80 and
cationic detergents like CTAB. The antimicrobial
activity was lost while treating the bacteriocin
with these agents including EDTA. However, the
addition of anionic detergent SDS to the partially
purified bacteriocin from Lactobacillus nasuensis
Surfactant
Zone of growth inhibition (mm)
L. monocytogenes
MTCC 657
A. baumannii
MTCC 1425
Tween 20 (0.5 % v/v) - -
Tween 80 (0.5 % v/v) - -
SDS (0.1 % w/v) 2,089 2,969
EDTA Na2 (0.1 % w/v) - -
CTAB (0.1 % w/v) - -
Control 5718 7744
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1484
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
NAKR1 was found to be retaining the
antimicrobial activity to 36.53% and 38.34 %
against Listeria monocytogenes MTCC 657 and
Acinetobacter baumannii MTCC 1425
respectively (Figure - 10). Anionic detergents
often unfold proteins by complexing to the interior
hydrophobic core of their native structure which
may affect their three-dimensional conformation
(Ivanova et al., 2000). Todorov and Dicks (2005)
reported that the bacteriocins produced by LAB
isolated from black olives were sensitive to Triton
X-100 and Triton X-114, but not with Tween 20,
Tween 80, SDS and EDTA.
Figure - 10 Effect of surfactants on partially purified bacteriocin activity
Effect of enzymes on partially purified
bacteriocin
The antimicrobial activity of partially
purified bacteriocin from Lactobacillus nasuensis
NAKR1 was found to be inhibited by the
proteolytic enzymes like trypsin and papain.
However, α-amylase didn’t affect the
antimicrobial behavior of bacteriocin.
Balasubramanyam and Varadaraj (1998) reported
that the inhibitory activity in the culture filtrate of
Lactobacillus delbruecki ssp. bulgaricus CFR
2028 was inhibited by trypsin.
Effect of metal ions on bacteriocin activity
The antimicrobial activity of partially
purified bacteriocin from Lactobacillus nasuensis
NAKR1 was found to be lost by adding the metals
like CuSO4 and MgSO4 at 0.5 % (w/v)
concentrations. The antagonistic activity of
bacteriocin produced from Bacillus subtilis was
completely lost when metal ions such as Fe2+
,
Mg2+
or Mn2+
were added to growth media
(Kabore et al., 2013). Von Mollendorff et al.
(2006) found that the addition of MgSO4 increased
bacteriocin activity in case of L. fermentum
JW11BZ.
Effect of salt (NaCl) concentration on
bacteriocin activity
The antimicrobial activity of partially
purified bacteriocin from Lactobacillus nasuensis
NAKR1 was found declined with increase in salt
concentration like sodium chloride (Figure - 11).
However, Altuntas et al. (2010) observed a better
growth of LAB at a low salt concentration (1 to 2
%) and inhibition above 3 % NaCl, while few
LAB showed more resistant. Yimin Cai et al.
(2012) observed Lactobacillus nasuensis growth
at 3 % level of NaCl and growth inhibition at 6 %
level of NaCl.
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1485
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Figure - 11 Effect of salt (NaCl) concentration on partially purified bacteriocin activity
Molecular weight of bacteriocin
The partially purified bacteriocin from
Lactobacillus nasuensis NAKR1 in Tricine-SDS-
PAGE revealed a zone-producing band with a
molecular weight of 12.5 kDa (Figure - 12). A
bacteriocin produced by P. pentosaceus with a molecular weight of 17.5 kDa was observed by
Wu et al. (2004).
Figure – 12: Molecular weight of bacteriocin by tricine-SDS-PAGE
Lane -5 bacteriocin stained with Coomassie brilliant blue R 250, Lane-7: Protein low range molecular
weight markers (9-14 kDa)
4. Conclusion
Our findings revealed that the possibility
of the presence of various LAB in the food
formulations of Indian origin. Though the studies
on Lactobacillus nasuensis are very limited,
further investigations to be carried out to explore
this organism. However, the results of the present
study will be supportive in bringing the roles of
the LAB in food fermentation and as well as
preservation in the natural way further.
K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1486
©2018 Published by JPS Scientific Publications Ltd. All Rights Reserved
Acknowledgements
The authors are grateful to the Department
of Biotechnology, Karunya Institute of
Technology and Sciences, Coimbatore, South
India and the Department of Biotechnology, Dr.
G.R. Damodaran College of Science, Coimbatore,
South India for providing necessary facilities to
carry out this work.
Conflict of Interests
The authors declare that there is no conflict
of interest
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DOI Number DOI: 10.22192/lsa.2018.4.5.5
How to Cite this Article:
K. Naresh Kumar, Krishna Revi, S. Murugan and Tha.Thayumanavan. 2018. Bacteriocin
production by Lactobacillus nasuensis NAKR1 isolated from Fermented Unniappam Batter.
Life Science Archives, 4(5): 1470 – 1489.
DOI: 10.22192/lsa.2018.4.5.5