Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Potential application of the nisin Z preparation of

Lactococcus lactis W8 in preservation of milk
S. Mitra, B.C. Mukhopadhyay and S.R. Biswas
Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India

Keywords
Lactococcus lactis, milk preservation, milk
spoilage bacteria, nisin, shelf life.
Correspondence
Swadesh Ranjan Biswas, Department of
Botany, Visva-Bharati, Santiniketan 731 235,
West Bengal, India.
E-mail: swadesh_s2000@yahoo.co.in

2010 1314: received 29 July 2010, revised 28

April 2011 and accepted 3 May 2011
doi:10.1111/j.1472-765X.2011.03075.x

Abstract
Aims: The aim of the study is to evaluate the effectiveness of the preparation
of nisin Z from Lactococcus lactis W8-fermented milk in controlling the growth
of spoilage bacteria in pasteurized milk.
Methods and Results: Spoilage bacteria isolated from pasteurized milk at 8 and
15C were identified as Enterococcus italicus, Enterococcus mundtii, Enterococcus faecalis, Bacillus thuringiensis, Bacillus cereus, Lactobacillus paracasei, Acinetobacter sp., Pseudomonas fluorescens and Enterobacter aerogenes. These bacteria
were found to have the ability to survive pasteurization temperature. Except
Enterobacter aerogenes, the spoilage bacteria were sensitive to the nisin Z preparation of the L. lactis W8. Addition of the nisin Z preparation to either the skim
milk or fat milk inoculated with each of the spoilage bacteria reduced the initial
counts (about 5 log CFU ml)1) to an undetectable level within 820 h. The nisin
Z preparation extended the shelf life of milk to 2 months under refrigeration.
Conclusions: The nisin Z preparation from L. lactis W8-fermented milk was
found to be effective as a backup preservative to counteract postpasteurization
contamination in milk.
Significance and Impact of the Study: A rapid inhibition of spoilage bacteria
in pasteurized skim and fat milk with the nisin Z preparation of L. lactis W8 is
more significant in comparison with the commercially available nisin (nisin A).
The nisin Z preparation can be used instead of commercial nisin, which is not
effective in fat milk.

Introduction
The frequent contamination of milk with spoilage and
pathogenic bacteria is a matter of serious concern to the
dairy industry. Such contamination not only causes deterioration in sensory quality and shelf life of milk but may
also pose a serious public health risk (Fleming et al. 1985;
CDC 1988, 2008). To minimize the spoilage, pasteurization treatment has been used routinely in commercial
milk, which can only provide a limited shelf life. It has
been demonstrated that B. cereus, Paenibacillus spp. can
survive high-temperature, short-time pasteurization (Durak et al. 2006; Huck et al. 2008). Refrigeration is also
inadequate to keep milk fresh because many psychrotrophic spoilage bacteria like Bacillus, Listeria and Pseudomonas
can grow in milk at 4C (Huck et al. 2008; He et al.
2009). The shelf life of milk may further be reduced by the
98

degradation of milk components through heat-stable

extracellular proteases, lipases and lecithinases produced
by many strains of Pseudomonas fluorescens, P. fragi and
P. putida (Dogan and Boor 2003). The enzymes produced
by the spoilage bacteria in raw milk can survive pasteurization and ultrahigh-temperature treatments (Wiedmann
et al. 2000; Hayes et al. 2006). In tropical countries like
India, the problem of microbial contamination in milk is
very frequent because many psychrotrophic bacteria such
as B. cereus, B. polymyxa, B. laterosporus, B. circulans,
B. pumilus, B. subtilis, B. coagulans, Micrococcus luteus,
P. aeruginosa, Serratia marcescens and Staphylococcus aureus
have been detected in raw milk samples (Matta and Punj
2007; Prakash et al. 2007; Sharma et al. 2007).
In view of the above-mentioned milk spoilage and
food-borne hazards, safety and shelf life of milk is a pivotal concern. In this context, earlier attempts have been

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Letters in Applied Microbiology 53, 98105 2011 The Society for Applied Microbiology

S. Mitra et al.

made to improve the safety and shelf life of milk using

commercial nisin as a food preservative (Bhatti et al.
2004; Schmidt et al. 2009). Nisin has received a great deal
of attention as it is produced by food-grade Lactococcus
lactis strains (Gross and Morell 1971; Delves-Broughton
1990; Linares et al. 2010) and is widely used as a safe and
natural preservative worldwide over 50 countries (DelvesBroughton et al. 1996; Chen and Hoover 2003). The
effective use of nisin as an antimicrobial preservative in
various food materials such as meat, cheese, flavoured
milk, canned evaporated milk, vegetables, fish and canned
foods is well documented by many (Gross and Morell
1971; Hurst 1981; Vandenbergh 1993; Delves-Broughton
et al. 1996; Thomas et al. 2000), and the commercial
nisin has been accorded as generally regarded as safe in
1988 by the US Food and Drug Administration (FDA)
(FDA HHS 1988). Nisin is a heat-stable, cationic, 34-residue polycyclic antibacterial peptide which kills a wide
range of spoilage and pathogenic bacteria including Bacillus, Clostridium, Staphylococcus and Listeria (Klaenhammer 1993; Rodriguez 1996). However, nisin is not active
against Gram-negative bacteria because of the presence of
an outer membrane that prevents the action of nisin
(Hurst 1981). But a combination of nisinEDTA treatment makes Gram-negative bacteria sensitive to nisin
(Delves-Broughton 1993). In certain countries, addition
of nisin to milk is permitted to control spoilage problems
associated with temperature and transportation (Davies
and Delves-Broughton 1999; Thomas et al. 2000),
although several attempts have been made to use commercial nisin (nisin A) as preservative of milk, but failed
owing to the loss of antibacterial activity in fat-containing
milk (Jung et al. 1992; Schmidt et al. 2009).
In our previous study, we have shown that nisin Z (a
natural variant of nisin A) could be prepared at a low
cost from diluted (20 times) skim milk fermented with
L. lactis W8 (Mitra et al. 2009) and the same can be used
for milk preservation instead of commercial nisin. Keeping this in view, this study has been carried out to demonstrate the effectiveness of the nisin Z preparation of
L. lactis W8 in controlling spoilage and pathogenic bacteria in skim- and fat milk.
Materials and methods
Culture medium and growth conditions
Lactococcus lactis W8 isolated from naturally fermented
milk (Mitra et al. 2005) was grown in tryptoneglucose
yeast extract (TGE, all at 10 g l)1, pH 65) broth at 37C.
The yeast extractpotatodextrose (YPD) medium and
ingredients of TGE were from Hi Media (Mumbai,
India). Lactobacillus plantarum NCDO 955 was used as

Application of nisin of L. lactis W8 in milk preservation

the indicator bacterium in the assay of bacteriocin activity

(Biswas et al. 1991). The strain was our laboratory collection (Mitra et al. 2005) and grown overnight at 30C in
de Mann, Rogosa, Sharpe (MRS) medium (Hi Media).
The isolates of spoilage bacteria from milk were grown at
30C in TGE broth. Listeria monocytogenes MTCC 657
(ATCC 19111) was procured from the Microbial Type
Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. It was grown in tryptic soy
broth supplemented with 6 g l)1 yeast extract. For the
enumeration of Listeria cells, PALCAM agar medium (Hi
Media) was used. All the strains were grown in their
respective media and preserved at )20C in the presence
of dimethyl sulfoxide (100 g l)1).
Isolation of spoilage bacteria from milk
A total of 10 samples of pasteurized milk collected at random from local firms in Santiniketan city, West Bengal,
India, were brought to laboratory and kept at refrigeration temperature (8C) and 15C for 10 days to spoil and
isolate spoilers. Milk samples were then tenfold serially
diluted in sterile distilled water, and an aliquot of 100 ll
from each dilution was spread onto TGE-agar plates, and
the plates were incubated at 30C for 24 h. A few colonies
from each sample with distinct colony morphology were
streaked on TGE-agar plates to obtain pure culture. Each
pure culture grown overnight in TGE medium at 30C
was inoculated to heat-treated (100C for 5 min) milk
(30 g l)1 fat) at 1%. Following incubation at 8 and 15C
for 7 days, the inoculated milk was observed for the
appearance of spoilage symptoms. The experiment was
repeated at least three times.
Determination of lecithinase and proteolytic activity of
the spoilage bacteria
To determine the lecithinase activities, each isolate of
spoilage bacteria was streaked on TGE-agar medium containing 55% (w v) egg yolk. The plates were incubated at
30C for 3 days, and the lecithinase activity was scored as
an opaque zone around the growth of the organism. Similarly, the spoilage organism was streaked on TGE-agar
containing 10 g l)1 skim milk. The plates were incubated
for 3 days at 30C. The extracellular production of proteolytic enzyme was detected by the formation of a clear
zone around the growth of the organism. All assays were
performed at least three times.
Identification of milk spoilage bacteria
Spoilage bacteria isolated from milk were identified based
on 16S rRNA gene sequence analysis. Comparison of

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99

Application of nisin of L. lactis W8 in milk preservation

S. Mitra et al.

DNA sequences with the sequences in the GenBank was

performed by blast (Altschul et al. 1990).
DNA manipulation, PCR and DNA sequencing
Genomic DNA of the isolates from spoiled milk was isolated by lysozymeproteinase K procedure (Smith et al.
1981). Approximately 50 ng of the DNA was used as template for PCR amplification of 16S rRNA in a total volume
of 50 ll containing 125 U of Pfu polymerase (Fermentas,
Hanover, MD, USA), 800 nmol l)1 of each primer,
02 mmol l)1 of each deoxynucleoside triphosphate and
2 mmol l)1 MgCl2. The 16S rRNA gene was amplified in
35 cycles with a thermocycler (Applied Biosystems, Foster
City, CA); each cycle consisted of a denaturing step at
95C for 1 min, a primer annealing step at 60C for 1 min
and an extension step at 72C for 5 min. Bacteria-specific
universal primers used for the amplification of 16S rRNA
gene were the forward primer 27f (5-AGAGTTTGATCATGGCTC-3) and the reverse primer 1327r (5-CTAGCGATTCCGACTTCA-3) (Villani et al. 2001). The
primer pair amplifies a 13-kb fragment of DNA. PCR
products were checked by electrophoresis on 15% (w v)
agarose gel. A 100-bp DNA ladder was used as the molecular marker (Fermentas). PCR products were purified using
QIAquick PCR purification kit (Qiagen, Hilden, Germany)
and sequenced with an ABI DNA sequencer (Applied Biosystems) using the 27f primer. The PCR products were
sequenced commercially from Chromous Biotech Pvt. Ltd,
Bangalore, India.
Effect of pasteurization temperature on the survivability
of the milk spoilage bacteria
Milk spoilage bacteria were grown individually overnight
in TGE medium at 30C. Cells were harvested by centrifugation at 11 000 g for 5 min at 8C and resuspended to
the original volume in sterile milk. A 50-ml cows milk
sample (previously heat treated at 100C for 5 min) was
inoculated with each of the spoilage bacteria (about 50 log
CFU ml)1) and treated at 72C for 15 s followed by immediate cooling on ice. The pasteurized milk was then serially
diluted and plated onto TGE agar plates. The plates were
incubated at 30C for 24 h, and the colonies developed
were counted to determine the CFU per millilitre. The uninoculated heat-treated milk was also pasteurized and treated similarly. Each experiment was carried out in triplicate.
Determination of the ability of the spoilage bacteria to
grow at refrigeration temperature
Each isolate of the spoilage bacteria was grown overnight
in TGE medium at 30C. Cells from each culture were
100

inoculated in heat-treated cows milk (100C for 5 min)

at about 50 log CFU ml)1. During incubation at 8C,
samples were withdrawn aseptically on day 3 and day 6
and serially diluted. An aliquot of an appropriate dilution
was spread onto TGE-agar plates. The plates were incubated at 30C to enumerate the number of the bacterial
colonies. The uninoculated heat-treated control milk was
also incubated at 8C and spread onto TGE-agar plates.
Each experiment was carried out in triplicate.
Evaluation of antibacterial potential of the nisin Z
preparation of Lactococcus lactis W8 against milk
spoilage bacteria
The preparation of nisin Z from 20 times diluted skim
milk fermented with the L. lactis W8 strain was followed
as described earlier in our laboratory (Mitra et al. 2009).
The titre of nisin of the lyophilized whey was
60 000 AU g)1. One AU was defined as 5 ll of the highest dilution showing a definite zone of growth inhibition
(Biswas et al. 1991).
To determine the antibacterial activity of the nisin
preparation against the milk spoilage bacteria, each strain
of spoilage bacteria was grown overnight in TGE medium
at 30C prior to use the cells for the antibacterial activity
of the nisin preparation. About 106 cells of each of the
milk spoilage bacteria were mixed with melted 07%
(w v) soft agar in TGE medium and then overlaid onto
the surface of TGE agar plates. A stock solution of nisin
Z was prepared by dissolving 50 mg of lyophilized preparation of nisin of W8 in 1 ml of sterile double-distilled
water which resulted in 3000 AU ml)1. Five microlitres
(15 AU) from the stock solution was spotted onto each
plate and incubated overnight at 30C. The next day
plates were observed for the zone of growth inhibition.
As this concentration (15 AU) of nisin completely inhibited the growth of 14 of 15 spoilage bacteria, a further
dilution (1 : 15) of nisin stock solution was made and
used at 5 (1 AU), 10 (2 AU) and 15 ll (3 AU) to compare the potency of the nisin Z preparation to inhibit the
growth of spoilage bacteria as described previously.
To determine the effectiveness of the nisin preparation
to control the spoilage bacteria, nisin was added to two
categories of heat-treated milk (100C for 5 min), skim
milk and cows milk (30 g l)1 fat) at 1000 AU ml)1. The
milk samples were then inoculated with each isolate of the
spoilage bacteria including L. monocytogenes MTCC 657 at
about 5 log CFU ml)1. The inoculated samples with or
without nisin were kept at 8C for 144 h. A 1-ml aliquot
of the sample was withdrawn at 4-h intervals, immediately
chilled on ice, tenfold serially diluted with sterile distilled
water, and a 50-ll aliquot from each dilution was spread
on TGE-agar plates. The plates were incubated at 30C for

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Letters in Applied Microbiology 53, 98105 2011 The Society for Applied Microbiology

S. Mitra et al.

Application of nisin of L. lactis W8 in milk preservation

24 h, and the colonies developed were counted. Each

experiment was confirmed by repeating three times.
Determination of shelf life of milk with added nisin Z
Shelf life of milk was studied in 100 ml of pasteurized
skim milk and cows milk (30 g l)1 fat) at 8C. Nisin prepared from W8-fermented milk was added to milk at
1000 AU ml)1. During storage for 2 months at this temperature, an aliquot of 1 ml sample was aseptically withdrawn at an interval of 7 days, tenfold serially diluted
with sterile distilled water, and a 100-ll aliquot from each
dilution was spread on TGE and YPD agar plates. The
plates were incubated at 30C for 48 h. Total bacterial
counts were determined on duplicate plates by plate
counting. In control experiment, total bacterial counts
were also determined in milk without added nisin. The
experiments were repeated three times.
Results
Isolation and identification of spoilage bacteria
A total of 15 spoilage bacteria were isolated from pasteurized milk after storage for 10 days at 8 and 15C. Milk
inoculated with each pure culture of the spoilage bacteria
showed symptoms of spoilage, characterized by curdling
and development of off flavours. The spoilage bacterial
strains were identified and designated as E. faecalis SP1,
E. faecalis SP2, E. faecalis S1, E. faecalis S2, Enterococcus
italicus SL2, Enterococcus italicus SL4, E. mundtii RL,
Bacillus thuringiensis SP3, B. cereus SP6, Bacillus sp. SL7,

Lactobacillus paracasei SL5, Staphylococcus sp. SL6, Enterobacter aerogenes SP4, Acinetobacter sp. B1 and Ps. fluorescens B32. Acinetobacter sp. B1 and Ps. fluorescens B32
were isolated from milk at 8C and the rest at 15C. Of
the 15 spoilage bacteria identified, 14 were protease positive, where Enterobacter aerogenes SP4 was protease negative. Enterococcus spp. Staphylococcus sp. SL6 and
Lact. paracasei SL5 were lecithinase negative. All the species of Bacillus, Acinetobacter sp. B1 and Ps. fluorescens
B32 were positive for both the enzyme activities.
Effect of pasteurization on the survival of the milk spoilage bacteria and determination of their growth at 8C
The viability of the Gram-positive bacteria during pasteurization of milk ranged from 32 to 42 log CFU ml)1
of the initial count of 5 log CFU ml)1. Gram-negative
bacteria survived pasteurization poorly (Fig. 1). Among
these isolated spoilage bacteria, the Gram-negative Acinetobacter sp. B1 grew better in milk at 8C. The initial
counts of Acinetobacter B1 in milk (53 log CFU ml)1)
increased to 832 log CFU ml)1 within 6 days (Table 1).
Bacillus spp. and Lact. paracasei SL5 were not found to
grow in milk at 8C, but remained viable. Enterococci,
Enterobacter and Pseudomonas grew in milk at a slower
rate at 8C.
Antibacterial effectiveness of the nisin Z preparation of
Lactococcus lactis W8 against milk spoilage bacteria
The differential sensitivity of the 15 isolated spoilage bacteria from milk at 8 and 15C to nisin Z is presented in

log CFU ml1

5
4
3
2
1
0

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Letters in Applied Microbiology 53, 98105 2011 The Society for Applied Microbiology

Ps

Figure 1 Effect of pasteurization

temperature (72C for 15 s) on survivability of
the spoilage bacteria. Black bar represents log
CFU ml)1 before pasteurization and white bar
represents log CFU ml)1 after pasteurization.
Data with error bars are mean SD of
triplicate analyses.

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101

Application of nisin of L. lactis W8 in milk preservation

S. Mitra et al.

Table 1 Growth of the spoilage bacteria in milk at 8C

log CFU ml)1
Spoilage bacteria

0h

72 h

144 h

E. faecalis SP1
E. faecalis SP2
E. faecalis S1
E. faecalis S2
Enterococcus italicus SL2
Enterococcus italicus SL4
E. mundtii RL
Enterobacter aerogenes
SP4
Bacillus thuringiensis SP3
B. cereus SP6
Bacillus sp. SL7
Lact. paracasei SL5
Staphylococcus sp. SL6
Acinetobacter sp. B1
Ps. fluorescens B32

547
555
56
562
569
530
541
563

018
011
027
007
014
011
031
011

584
62
569
569
584
59
63
653

034
001
025
003
013
015
022
004

63
655
595
647
647
65
69
676

014
001
007
009
008
004
002
007

56
53
56
547
53
53
509

016
012
01
007
038
005
017

544
53
53
53
541
653
547

007
014
016
009
013
013
007

53
395
447
469
562
832
62

013
011
004
009
007
007
011

Results are expressed as mean standard deviation.

Table 2. Enterobacter aerogenes SP4 was not sensitive to

nisin Z preparation, whereas the four strains of E. faecalis
and Staphylococcus sp. SL6 were sensitive at 1 AU per
spot, E. mundtii RL and Lact. paracasei SL5 at 2 AU and
Enterococcus italicus, B. thuringiensis, B. cereus, Bacillus
spp., Acinetobacter sp. and Ps. fluorescens at 3 AU. Among
these, the four strains of E. faecalis and Staphylococcus sp.
SL6 were found to be more sensitive.
When heat-treated cows milk with added nisin Z at
1000 AU ml)1 was challenged with Enterococcus or
L. monocytogenes MTCC 657 at about 50 log CFU ml)1,

Table 2 Inhibitory spectrum of nisin Z preparation of Lactococcus

lactis W8 against spoilage bacteria
Spoilage bacteria

Zone of growth inhibition

E. faecalis SP1
E. faecalis SP2
E. faecalis S1
E. faecalis S2
Enterococcus italicus SL2
Enterococcus italicus SL4
E. mundtii RL
Enterobacter aerogenes SP4
Bacillus thuringiensis SP3
B. cereus SP6
Bacillus sp. SL7
Lact. paracasei SL5
Staphylococcus sp. SL6
Acinetobacter sp. B1
Ps. fluorescens B32

+
+
+
+
+
+
+
)
+
+
+
+
+
+
+

+, strong inhibition (15 mm and over); ), no inhibition.

102

no viable cells could be detectable at 16 h under refrigeration (Table 3). The same treatment reduced Acinetobacter
sp. Bacillus spp. Lactobacillus sp. and Pseudomonas sp. to
an undetectable level at 20 h, whereas in case of Staphylococcus sp., it was 8 h (Table 3). In all the milk samples
treated with nisin, no regrowth of the spoilage bacteria
was observed till 144 h of incubation. A similar result was
obtained in skim milk challenged with the spoilage organisms in the presence of nisin (data not shown). In control
milk, the number of viable cell counts for all of the spoilage bacteria increased slowly.
Shelf life analysis of milk with added nisin Z
Nisin Z-containing milk samples withdrawn at an interval
of 7 days during storage at 8C showed no bacterial
growth on TGE-agar plates (data not shown). The growth
of yeast and moulds on YPD plates was also not observed
in all the milk samples. As no contaminants were present
at the end of 2-months observation period, the result suggest that the shelf life of milk was extended by at least
2 months with the addition of the nisin preparation of
L. lactis W8. In control milk (without nisin), bacterial
contaminants grew to about 3 log CFU ml)1 on day 7
(data not shown).
Discussion
The present study evaluates the efficacy of the nisin Z
preparation of L. lactis W8 in controlling spoilage bacteria
in pasteurized milk. To carry out this work, we have isolated spoilage bacteria from milk stored at two different
temperatures, 8 and 15C. These isolated spoilage bacteria
were found to survive pasteurization, and most of them
had the ability to grow under refrigeration (8C). These
findings suggest the requirement of an effective food preservative for controlling spoilage bacteria in pasteurized
milk. In this context, we used our nisin Z preparation for
the preservation of pasteurized milk. In the bioassay
based on spot-on-lawn method, Gram-positive enterococci, bacilli, Lact. paracasei and Staphylococcus sp. and
Gram-negative Acinetobacter sp. and Pseudomonas sp.
were found sensitive to the nisin preparation of W8. The
inhibition of some Gram-negative bacteria by the nisin
preparation of W8 is particularly interesting as the commercial nisin is not effective against Gram-negative bacteria. It may be possible that nisin Z preparation of the
strain W8 is able to bind and permeabilize the outer
membrane of Gram-negative bacteria. Earlier studies
reported that purified nisin Z shows antibacterial activity
against Gram-negative E. coli, whereas unpurified nisin Z
fails to show any activity (Kuwano et al. 2005). They have
also reported that the antibacterial activity of purified

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Letters in Applied Microbiology 53, 98105 2011 The Society for Applied Microbiology

Application of nisin of L. lactis W8 in milk preservation

S. Mitra et al.

Table 3 Loss of viability of spoilage bacteria in milk added with the nisin Z preparation of Lactococcus lactis W8
log CFU ml)1
Organisms

0h

E. faecalis SP1
Control
534 009
Treated
538 022
Enterococcus italicus SL2
Control
562 01
Treated
564 015
E. mundtii RL
Control
555 009
Treated
553 004
Lact. paracasei SL5
Control
538 007
Treated
534 005
B. cereus SP6
Control
52 002
Treated
53 011
B. thuringienesis SP3
Control
58 00
Treated
579 011
Bacillus sp. SL7
Control
555 008
Treated
556 006
Staphylococcus sp. SL6
Control
511 002
Treated
511 024
Acinetobacter sp. B1
Control
544 014
Treated
542 009
Ps. fluorescens B32
Control
541 011
Treated
53 013
Listeria monocytogenes MTCC 657
Control
514 01
Treated
511 009

4h

8h

12 h

16 h

20 h

53 015
474 01

538 008
379 009

544 007
198 022

541 014
00

544 012
00

568 013
473 014

571 01
334 01

579 008
25 0083

579 01
00

579 01
00

564 011
374 01

579 008
262 011

573 008
141 012

574 01
00

583 008
00

534 01
441 011

542 009
371 009

541 011
234 013

55 008
151 01

554 019
00

536 009
356 012

551 012
296 024

563 013
207 012

571 017
179 012

579 008
00

596 009
602 015

594 02
527 012

579 014
334 011

602 009
214 086

676 012
00

562 009
507 016

566 011
49 038

589 013
344 005

566 011
217 032

576 012
00

556 01
39 019

565 02
00

556 006
00

577 013
00

579 009
00

53 011
490 019

547 015
377 009

56 01
247 006

577 013
19 049

594 009
00

544 009
35 009

55 01
253 009

553 029
196 021

571 014
107 013

576 009
00

541 008
556 006

503 013
427 012

549 01
338 008

562 014
00

549 013
00

The data are shown as mean standard deviation.

nisin Z against Gram-negative bacteria is abolished with

salts (NaCl). This appears that nisin Z preparation of W8
is free of any interfering substances which prevent the
action of nisin Z against Gram-negative bacteria. It is also
presumed that nisin Z produced by the strain W8 is
structurally different from the existing nisin as suggested
by a mass difference of 18 Da (unpublished). This structural change may be of importance for the binding and
activation of nisin Z against Gram-negative bacteria. The
structural change in nisin Z responsible for antibacterial
activity against specific Gram-negative bacteria is supported by the work of Yuan et al. (2004). As a consequence, contamination of milk by such Gram-negative
bacteria cannot be prevented with commercial nisin. The
nisin Z preparation of W8 can thus be used in milk preservation more advantageously than the commercial nisin.

The nisin Z preparation reduced the initial counts of

spoilage bacteria (about 5 log CFU ml)1) to an undetectable level in skim and fat milk within 820 h. The result
suggests that the nisin preparation of W8 is equally effective in both skim and fat milk. The nisin Z of W8 being
different in properties (Mitra et al. 2005) from the
reported nisin Z possibly is not affected by fat in milk.
The commercial nisin has a limitation to control spoilage
bacteria in fat milk. It has been reported that commercial
nisin losses its antibacterial activity in fat milk owing to
its interaction and adsorption to fats and the surface of
protein globules (Jung et al. 1992; Sobrino-Lopez and
Martn-Belloso 2008). The observation on ineffectiveness
of nisin in fat milk is supported by the work of Schmidt
et al. (2009) who demonstrated that the initial counts of
L. monocytogenes in nisin-treated skim milk (40 log

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Application of nisin of L. lactis W8 in milk preservation

S. Mitra et al.

CFU ml)1) reduced to <1 CFU ml)1 after 72 h of 5C

incubation, and the level of initial counts of Listeria in
whole milk remained unchanged. In our study, the nisin
Z preparation of L. lactis W8 was found to rapidly
reduce L. monocytogenes (5 log CFU ml)1) to an undetectable level in milk within 16 h at 8C. Further, it is
observed that there was no regrowth of the spoilage bacteria in milk samples treated with the nisin preparation
of W8 till 144 h. This may suggest that the rapid killing
of the spoilage bacteria in milk may be an important
cause to prevent the development of resistance to the
nisin preparation of W8. This is in contrast to the previous results which demonstrated the regrowth of nisinresistant organisms in milk and other food systems
(Schillinger et al. 1998; Vignolo et al. 2000; Chi-Zhang
et al. 2004; Schmidt et al. 2009). The inhibition of spoilage bacteria and prevention of their regrowth with the
nisin preparation of W8 helped to extend the shelf life of
pasteurized milk by at least 2 months during storage at
8C. From the overall findings reported in this study, it
is concluded that the nisin Z preparation of W8 has the
potential for use as a backup preservative to counteract
postpasteurization contamination in both skim milk and
fat milk.
Acknowledgements
The first author gratefully acknowledges the financial
assistance in the form of Research Associateship (no.
9 202 (0007) 2K8-EMR-I) from the Council of Scientific
and Industrial Research (CSIR), New Delhi, India.
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