BACTERIOCIN PRODUCTION BY Lactobacillus nasuensis … · 2019-06-23 · Hinton Agar (MHA), de Man,...

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;

http://www.jpsscientificpublications.com/

K. Naresh Kumar/Life Science Archives (LSA), Volume – 4, Issue – 5, 2018, Page – 1470 to 1489 1471


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



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,



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.



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).



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.



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.



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).



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).



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).



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



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



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).



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


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



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



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



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



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.



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.



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

5. References

1) Altuntas EG, Cosansu S, and K.

Ayhan.2010, Some growth parameters and

antimicrobial activity of a bacteriocin-

producing strain Pediococcus acidilactici

13. Int J Food Microbiol. 141(1-2):28–31.

2) Amidya Nugrahani, Hardoko, and Anik

Martinah Hariati, 2016, Characterization of

Bacteriocin Lactobacillus casei on

Histamine-Forming Bacteria, J. Life Sci.

Biomed. 6(1): 15-21.

3) Balasubramanyam, B.V., and M. C.

Varadaraj, (1998), Cultural conditions for

the production of bacteriocin by a native

isolate of Lactobacillus delbruecki ssp.

bulgaricus CFR 2028 in milk medium,

Journal of Applied Microbiology, 84, 97–

102.

4) Holt, JG. NR. Kreing. PHA. Sneath, and

JT. Stanely,1994, In : Bergey's Manual of

Determinative Bacteriology. 9/e, Williams

and Wikins, Maryland.

5) Cheigh CI, and Pyun YR. 2005, Nisin

biosynthesis and its properties. Biotechnol

Lett . 27:1641–1648.

6) Das, A. J. and Deka, S. C., 2012,

Fermented foods and beverages of the

North-East India, International Food

Research Journal, 19(2): 377-392.

7) Deegan, L. H.; Cotter, P. D.; Hill, C. and

Ross, P. 2006, Bacteriocins: Biological

tools for biopreservation and shelf-life

extension. Int. Dairy J., 16, 1058-1071.

8) Galvez, A., H. Abriouel, RL. Lopez, and

N. Ben Omar,2007, Bacteriocin-based

strategies for food biopreservation. Int J

Food Microbiol., 120(1-2):51-70.

9) Granger, M., Todorov, S.D., Matthew,

M.K.A., and Dicks, L.M.T., 2005, Growth of

Enterococcus mundtii ST15 in medium

filtrate and purification of bacteriocin ST15

by cation exchange chromatography. Journal

of Basic Microbiology, 45: 419–425.

10) Guarner F, and Schaafsma GJ. 1998.

Probiotics. Int J Food Microbiol.,

17;39(3):237-8.

11) Haider, S.R., H. J. Reid and B. L.

Sharp,2012, Chapter-8; Tricine-SDS-

PAGE, In: Protein Electrophoresis:

Methods and Protocols, Methods in

Molecular Biology, Biji T. Kurien and R.

Hal Scofi eld (eds.), , Humana Press, 869

:81-91.

12) Nicholas C. K. Heng, Grace A.

Burtenshaw, Ralph W. Jack, and John R.

Tagg, 2007, Ubericin A, a Class IIa

Bacteriocin Produced by Streptococcus

uberis, Appl. Environ. Microbiol., 73 (23)

7763-7766.

13) Hoda Mahrous, Abeer Mohamed, M. Abd

El-Mongy, A. I. El-Batal, and H. A.

Hamza, 2013, Study bacteriocin

production and optimization using

new isolates of Lactobacillus spp.

isolated from some dairy products under

different culture conditions. Food Nutr.

Sci., 4(3): 342-356.

14) Ivanova, I., Kabadjova, P., Pantev, A.,

Danova, S., and Dousset, X., 2000, Detection,

purification and partial characterization of a

novel bacteriocin substance produced by

Lactococcus lactis subsp. lactis B14 isolated

from boza-Bulgarian traditional cereal

beverage. Biocatalis — Vestnik Moskova

Uniiversiteta, Seria Kimia, 41: 47–53.

15) Jack, R W., Tagg, J R., and B Ray, 1995,

Bacteriocins of gram-positive bacteria,

Microbiol. Rev., 59(2):171.

16) Jyoti Prakash Tamang, 2010, Ch-2,

Diversity of Fermented Foods, In

Fermented Foods and Beverages of the

World, (edited by Jyoti Prakash Tamang

and Kasipathy Kailasapathy) , CRC Press,



Taylor & Francis Group, New York, : 41-

85.

17) Kabore D, Dennis SN, Hagretou SL,

Brehima D, Mamoudou H, Mogens J, and

Lin T. 2013, Inhibition of Bacillus cereus

growth by bacteriocin producing Bacillus

subtilis Isolated from fermented baobab

seeds (maari) is substrate dependent.

International Journal of Food

Microbiology, 162: 114- 119.

18) Katelyn Brandt and Rodolphe Barrangou.,

2018, Using glycolysis enzyme sequences

to inform Lactobacillus phylogeny,

Microb Genom., 4(6) e000187.

19) Larry, R,. and G. Beuchat, 2008, Ch: 13,

Indigenous Fermented Foods. In:

Biotechnology Set, Second Edition, USA.

20) Lee CH, 1994, Importance of lactic acid

bacteria in non-dairy food fermentation, In;

Lactic Acid Fermentation of Non-dairy

Food and Beverages. (Edrs) Lee, C.H.,

Adler-Nissen, J. and Barwald, G.,

HarnLim Won.

21) Lindgren, S.W. and Dobrogosz, W.J. 1990.

Antagonistic activities of lactic acid

bacteria in food and feed fermentations.

FEMS Microbiol. Rev., 87: 149-164.

22) Lowry, O.H., Rosebrough, N. J., Farr, A.

L., and Randall, R. J., 1951, Protein

measurement with the folin phenol reagent.

J. Biol. Chem., 193:265-275.

23) Messi, P., Bondi, M, Sabia, C.,

Battini, R. and Manicardi, G., 2001,

Detection and preliminary

characterization of a bacteriocin

(plantaricin 35d) produced by a

Lactobacillus plantarum strain. Int. J.

Food Microbiol., 64, 193–198.

24) Mirhosaini, M., I. Nahvi , R. Kasra

Kermanshahi and M. Tavassoli , 2006,

Isolation of Bacteriocin Producing

Lactococcus lactis Strain from Dairy

Products. International Journal of Dairy

Science, 1: 51-56.

25) Moreno, I., Lerayer, A. S. L., and Leitão,

M. F. F., 1999, Detection and

characterization of bacteriocin-producing

Lactococcus lactis strains. Rev. Microbiol.,

30: 130-136.

26) Nugrahani A, Hardoko, and Martinah

Hariati A. 2016. Characterization of

Bacteriocin Lactobacillus casei on

Histamine-Forming Bacteria. J. Life

Sci.Biomed., 6(1): 15-21.

27) Neha Gautam and Nivedita Sharma, 2009.

Purification and characterization of

purified bacteriocin of Lactobacillus brevis

isolated from traditional fermented food of

H.P. Indian Journal of Biochemistry and

Biophysics, 46: 337-341.

28) Radha.K.R, and Padmavathi Tallapragada,

2015, Purification, characterization and

application of bacteriocin for improving

the shelf-life of sprouts: An approach to

Bio preservation, International Journal of

ChemTech Research, 8(8): 265-277.

29) Rattanachaikunsopon, P., and

Phumkhachorn, P., 2006, Isolation and

preliminary characterization of bacteriocin

produced by Lactobacillus plantarum

N014 isolated from Nham, a traditional

Thai fermented pork. J Food Prot.,

69(8):1937–1943.

30) Rushdy, A.A. and E.Z. Gomaa, 2013,

Antimicrobial compounds produced by

probiotic Lactobacillus brevis isolated

from dairy products. Ann. Microbiol., 63:

81-90.

31) Salim-ur-Rehman, Alistair Paterson and

John R. Piggott, 2006, Flavour in

sourdough breads: a review, Trends in

Food Science & Technology, 17 : 557-566.

32) Sambrook, J. E.F. Fritsch, and T. Maniatis,

1989, Molecular Cloning: A Laboratory

Manual, 2nd ed.; CSH Laboratory Press,

Cold Spring Harbor, NY, USA.

33) Sanni, A. I., 1993, The need for process

optimization of African fermented foods

and beverages, International Journal of

Food Microbiology, 18 : 85-95.

34) Schuenzel, K.M. and M.A. Harrison, 2002.

Microbial antagonists of food borne

pathogens on fresh, minimally processed

vegetables. J. Food Prot., 65: 1909-1915.

35) Sekar, S., and S. Mariappan, 2007, Usage

of traditional fermented products by Indian

rural folks and IPR. Indian Journal of

Traditional Knowledge, 6(1): 111-120.



36) Shiba Prosad Banerjee, Krushna Chandra

Dora and Supratim Chowdhury, 2013,

Detection, partial purification and

characterization of bacteriocin produced by

Lactobacillus brevis FPTLB3 isolated

from freshwater fish. J. Food Sci.

Technol., 50(1):17–25.

37) Sivaramasamy Elayaraja, Neelamegam

Annamalai, Packiyam Mayavu,and

Thangavel Balasubramanian, 2014,

Production, purification and

characterization of bacteriocin from

Lactobacillus murinus AU06 and its broad

antibacterial spectrum, Asian Pac J Trop

Biomed., 4(1): S305-S311.

38) Sohliya, I., Joshi, S. R., Bhagobaty, R. K.

and Kumar, R., 2009, Tungrymbai- A

traditional fermented soybean food of the

ethnic tribes of Meghalaya. Indian J. Trad.

Knowl., 8:559-561.

39) Stevens, K. A., Sheldon, B. W.,

Klapes, N. A. and Klaenhammer, T. R.,

1991, Nisin treatment for inactivation of

Salmonella species and other Gram-

negative bacteria. Appl. Environ.

Microbiol., 57, 3613-3615.

40) Toro, C. R., 2005, Uso de bactérias láticas

probióticas na alimentação de camarões

Litopenaeus vannamei como inibidoras de

microrganismos patogênicos eestimulantes

do sistema imune. PhD Theses,

Universidade Federal do Paraná, Curitiba,

Brazil

41) Todorov, S. D. and Dicks, L. M. T., 2004,

Effect of medium components on

bacteriocin production by Lactobacillus

pentosus ST151BR, a strain isolated from

beer produced by the fermentation of

maize, barley and soy flour. World J.

Micobiol. Biotechnol., 20, 643-650.

42) Todorov, S. D., and Dicks, L. M. T., 2005,

Lactobacillus plantarum isolated from

molasses produces bacteriocins active against

Gram-negative bacteria. Enzyme and

Microbial Technology, 36: 318-326.

43) Todorov, S.D., Vaz-Velho, M., and Gibbs, P.,

2004, Comparison of two methods for

purification of plantaricin ST31, a bacteriocin

produced from Lactobacillus plantarum

ST31. Brazilian Journal of Microbiology, 35:

157–160.

44) Todorov, S.D., Wachsman, M.B., Knoetze,

H., Meincken, M., and Dicks,

L.M.T.,2005, An antibacterial and

antiviral peptide produced by Enterococcus

mundtii ST4V isolated from soy beans.

International Journal of Antimicrobial

Agents, 25, 508–513

45) Umeta, M., C.E. West, and H. Fufa, 2005,

Content of zinc, iron, calcium and their

absorption inhibitors in foods commonly

consumed in Ethiopia. Journal of Food

Composition and Analysis, 18: 803–817.

46) Usmiati. S, and T. Marwati, 2009,

Selection and optimization processes of

bacteriocin production from Lactobacillus

sp., Indonesian Journal of Agriculture,

2(2): 82-92.

47) Vandenbergh, P. A., 1993, Lactic-acid

bacteria, their metabolic products and

interference with microbial-growth,

FEMS Microbiology Reviews, 12: 221-

238.

48) Vescovo, M., C. Orsi, G. Scolari and S.

Torriani, 1995. Inhibitory effect of selected

lactic acid bacteria on microflora

associated with ready to use vegetables.

Lett. Applied Microbiol., 21: 121-125.

49) Vignolo, G. M., F. Suriani, A. P. Holgado,

and G. Oliver,1993, Antibacterial activity

of Lactobacillus strains isolated from dry

fermented sausages. Journal of Applied

Bacteriology, 75: 344-349.

50) Von Mollendorff J.W., Todorov S.D., and

Dicks L.M.T., 2006, Comparison of

Bacteriocins Produced by Lactic Acid

Bacteria Isolated from Boza, A cereal

Based Beverage from Balkan Peninsula.

Current Microbiology, 54: 209-216.

51) Van der Vossen, J. M. B. M., van

Herwijnen, M. H. M., Leer, R. J.,ten Brink,

B., Pouwels, P. H. & Huis in 't Veld, J. H.

J. 1994. Production of acidocin By a

bacteriocin of Lactobacillus acidophilus

M46 is a plasmid encoded trait: plasmid

curing, genetic marking by in vivo plasmid

integration, and gene transfer. FEMS

Microbiol Lett., 116, 333-340.



52) Wu CW, Yin LJ, and Jiang ST., 2004,

Purification and characterization of

bacteriocin from Pediococcus pentosaceus

ACCEL. J Agr Food Chem., 52:1146–

1151.

53) Yang R., Johnson M C., and Ray B., 1992,

Novel method to extract large amounts of

bacteriocins from lactic acid bacteria, Appl.

Environ. Microbiol., 58(10): 3355-3359.

54) Yimin Cai, Huili Pang, Maki Kitahara and

Moriya Ohkum, 2012, Lactobacillus

nasuensis sp. nov., a lactic acid bacterium

isolated from silage, and emended

description of the genus Lactobacillus,

International Journal of Systematic and

Evolutionary Microbiology, 62: 1140–

1144.

55) Zhennai, Y., 2000. Antimicrobial

compounds and extracellular

polysaccharides produced by lactic acid

bacteria: Structures and properties.

Academic Dissertations. Department of

Food Technology, University of Helsinki.

Access this Article in Online

Quick Response Code

Website www.jpsscientificpublications.com

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

http://www.jpsscientificpublications.com/

BACTERIOCIN PRODUCTION BY Lactobacillus nasuensis … · 2019-06-23 · Hinton Agar (MHA), de Man,...

Documents

Transcript of BACTERIOCIN PRODUCTION BY Lactobacillus nasuensis … · 2019-06-23 · Hinton Agar (MHA), de Man,...