APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p. 3355-3359 Vol. 58, No.

10
0099-2240/92/103355-05$02.00/0
Copyright ) 1992, American Society for Microbiology

Novel Method To Extract Large Amounts of Bacteriocins

from Lactic Acid Bacteriat
RONGGUANG YANG, MONTY C. JOHNSON, AND BIBEK RAY*
Food Microbiology Laboratory, Animal Science Department,
University of Wyoming, Laramie, Wyoming 82071
Received 4 May 1992/Accepted 21 July 1992

Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of
producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of
adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules
were adsorbed at pHs near 6.0, and the lowest (c5%) adsorption took place at pH 1.5 to 2.0. On the basis of

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this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria.
By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcml that were
potent and concentrated. This method produced a higher yield than isolation procedures, which rely on
precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large
quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.

In the search for a food biopreservative, investigations on
certain antibacterial proteins (bacteriocins) from lactic acid
bacteria have been very popular (11, 18, 25). To study their
chemical and antibacterial properties and to determine their
effectiveness in food systems, it is necessary to obtain
relatively large quantities of these peptides in a pure and
concentrated form. Currently, most methods rely on ammo-
nium sulfate precipitation of the bacteriocins from cell-free
culture liquor. This method has been used to obtain bacte-
riocins from Pediococcus spp. (4-6, 13), Lactobacillus spp.
(17, 21, 23), Lactococcus spp. (12, 15, 16, 24), and Leu-
conostoc spp. (10, 14). However, there is general agreement
that it does not yield a good product because many other
proteins from the medium can also be precipitated and the
yield is not very high (5, 9, 12). For further purification of
precipitated bacteriocins, especially in the determination of
the amino acid composition and sequence, these researchers
have used various column chromatography techniques.
Commercial nisin preparations are available in highly
purified food-grade form. However, the methods used com-
mercially are not known. Mattick and Hirsch (20) used a
combination of acid treatment of the culture followed by
removal of the cells and then solvent extraction and precip-
itation to obtain nisin with high potency. Later, other
workers used several different methods based on propanol-
NaCl or butanol-acetic acid extraction from culture super-
natant (2, 9) and breaking of cells and extraction with acid (1,
31). Although the nisin preparations had high potency, the
methods were laborious and total yields were low.
Bhunia et al. (7), while studying the mode of bactericidal
action of pediocin AcH from Pediococcus acidilactici H on
sensitive cells of Lactobacillus plantarum NCDO 955, ob-
served that adsorption of pediocin AcH to the cells is pH
dependent. Maximum adsorption occurred at pH 6.0 to 6.5,
but when the pH of the cell suspension was reduced to 2.0 or
below, the pediocin AcH was not adsorbed. It is known that,
in general, cells of a bacteriocin-producing bacterial strain
*
Corresponding author.
t Wyoming Agricultural Experiment Station journal article num-
ber 1687.
3355
adsorb the bacteriocin molecules that they produce (7, 13,
18, 25, 30). We hypothesized that if, after fermentation, the
culture broth of a producer strain was adjusted to the pH for
maximum adsorption of the bacteriocin onto the cell sur-
faces, the cells with adsorbed bacteriocin molecules could
easily be removed from the culture liquor by centrifugation.
The peptide could then be selectively released from the cells
at pH 1.5 to 2.0, and the preparation could provide large
quantities of purified bacteriocin. This hypothesis was tested
with bacteriocin-producing strains from four genera of lactic
acid bacteria and the results are presented here.
MATERIALS AND METHODS
Bacterial cultures and media. The bacterial strains used in
this study are described in Table 1. Pediococcus acidilactici
LB 42-923 (27), used to produce pediocin AcH, was grown at
370C for 18 h in TGE broth (8). Lactococcus lactis subsp.
lactis ATCC 11454, used to produce nisin, was grown at
30C for 24 h in TGE buffered broth, which consisted of TGE
broth plus 0.5% sodium citrate, 0.1% sodium acetate, and
0.05% dipotassium phosphate (pH 6.5). Leuconostoc carno-
sum Lml, used to produce leuconocin Lcml, and Lactoba-
cillus sake Lb 706, used to produce sakacin A (29), were
grown at 30C for 24 h in MRS broth (Difco). All indicator
bacteria were grown in TGE broth and transferred daily.
Antimicrobial activity assay of bacteriocin. Each bacterio-
cin preparation was serially diluted (1:10, 1:20, 1:30, etc.),
and 5 ,ul from each dilution was spotted onto a lawn of
appropriate indicator bacteria. Activity units (AU) per mil-
liliter were calculated as previously reported (7, 8). The
indicator bacteria were Lactobacillus plantarum NCDO 955
for pediocin AcH and nisin, Leuconostoc mesenteroides Ly
for sakacin A, and Enterococcus faecalis MB1 for leucono-
cin Lcml.
Preparation of cells for bacteriocin adsorption. A desired
bacterial strain (Fig. 1) was grown overnight in the appro-
priate medium, and the cells were collected by centrifuga-
tion, washed in 1/5 volume of sterile 5 mM sodium phosphate
(pH 6), resuspended in 1/5 vol of sterile 1.0 M NaCl solution
(pH 2.0, adjusted with 5% phosphoric acid), and mixed at
4C for 1 h. The cells were harvested by centrifugation,
3356 YANG ET AL.
TABLE 1. Bacterial strains, bacteriocin sensitivity, and growth conditions
Bacteriocin
Strain produced
P. acidilactici LB 42-923 Pediocin AcH
L. lactis subsp. lactis ATCC 11454 Nisin
L. sake Lb 706 Sakacin A
L. carnosum Lml Leuconocin Lcml
L. plantarum NCDO 955 Noned
L. mesenteroides Ly None
E. faecalis MB1 None
a Not sensitive to any of the bacteriocins tested.
b ATCC, American Type Culture Collection, Rockville, Md.
c Isolated from processed meat products in our laboratory.
d Nonbacteriocin producer strains were used as indicators.
e Originally from the Microbiology Department, University of Wyoming.
washed once with sterile deionized water (dH2O), and resus-
pended in sterile dH2O to 20 times the original culture
volume.
Influence of pH on bacteriocin adsorption by producing cells
and indicator cells. A cell suspension of a desired bacterial
strain (0.1 ml, about 107 cells) and a bacteriocin preparation
(0.1 ml, about 105 AU) were added to 1.8 ml of 5 mM sodium
phosphate adjusted to a pH range from 1.5 to 10.0 (with
phosphoric acid and NaOH). This mixture was incubated at
4C for 30 min and centrifuged at 17,000 x g for 5 min, and
the AU of the bacteriocin in the supernatant was assayed (7,
8). Control I consisted of 0.1 ml of dH2O instead of bacte-
riocin, and control II had 0.1 ml of dH2O instead of cell
suspension. The percentage of bacteriocin adsorbed was
calculated as follows: percentage of bacteriocin adsorbed =
100 x [1 - (AU/ml in cell-free supernatant - AU/ml in
control I)]/(AU/ml in control II).
Extraction of adsorbed bacteriocin from producer cells. For
extraction of pediocin AcH, nisin, leuconocin Lcml, and
sakacin A, each producer strain was grown to early station-
ary phase (about 5 x 108 cells per ml) in 1 liter of the
appropriate medium. The culture broths were adjusted to pH
6.5 for pediocin AcH, nisin, and sakacin A and pH 5.5 for
leuconocin Lcml. Each culture broth was heated to 70C for
25 min to kill the cells (heating can be done before pH
adjustment also), and the cells were harvested by centrifu-
gation at 15,000 x g for 15 min. Supernatant activity was
assayed to calculate the amount of bacteriocin not adsorbed
onto the cells. After the cells had been washed with 5 mM
sodium phosphate (pH 6.5), they were resuspended in 50 ml
of 100 mM NaCl at pH 2.0 (adjusted with 5% phosphoric
acid) for pediocin AcH, leuconocin Lcml, and sakacin A
and pH 2.5 for nisin and mixed with a magnetic stirrer for 1
h at 4C. Cell suspensions were then centrifuged at 29,000 x
g for 20 min, and the cells were resuspended in 5 mM sodium
phosphate (pH 6.5) and assayed to calculate the amount of
bacteriocin lost with the cells. The supematants were dia-
lyzed in 1,000-molecular-weight-cutoff dialysis bags against
dH20 at 4C for 24 h and then freeze-dried. The total dry
matter, protein concentration (19), and bacteriocin activity
of each product were determined. This process was per-
formed twice for each bacteriocin.
SDS-PAGE and identification of the activity band. Freeze-
dried extraction preparations were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE). Methods previously reported (4, 5) were used with
APPL. ENVIRON. MICROBIOL.
Bacteriocin Growth Growth
sensitivity medium conditions Source (reference)
Nisin TGE 37C, 20 h Our laboratory (26)
Nonea TGE 30C, 24 h ATCCb
Nisin MRS 25C, 24 h F. K. Lucke (28)
Pediocin AcH, nisin MRS 25C, 24 h Our laboratoryc
Pediocin AcH, nisin TGE 30C, 18 h M. A. Daeschel (5-8)
Pediocin AcH, nisin, TGE 30C, 18 h Our laboratory (7)
sakacin A
Pediocin AcH, nisin, TGE 30C, 18 h Our laboratorye
sakacin A, leuco-
nocin Lcml
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a Tris-tricine system (28). This method used a 10-cm layer of
16.5% acrylamide resolving gel and a 2-cm layer of 10%
acrylamide spacer gel. The electrophoresis was run at 15C
and 20 mA for the first 2 h and then at 30 mA for another 12
h. Sample preparation, application of samples, and gel
division after electrophoresis have been described before
(4). One-half of each gel was stained with Coomassie brilliant
blue, and the other half was used for identification of the
bacteriocin band by using growth inhibition of E. faecalis
MB1 (4). For growth inhibition, the gel half was washed in
deionized water for 2 h at 25C, placed on a TGE agar
prepoured plate, and overlaid with soft agar seeded with E.
faecalis MB1. The plate was incubated at 30C for 24 h,
examined for the location of the zone of growth inhibition,
and photographed. The stained gel half was destained and
photographed.
RESULTS
Sensitivity of bacterial strains to bacteriocins. The four
bacteriocin-producing strains and the three indicator strains
were tested for sensitivity to four bacteriocins (Table 1).
Their sensitivities varied greatly: Lactococcus lactis subsp.
lactis ATCC 11454 was not sensitive to any bacteriocin,
whereas E. faecalis MB1 was sensitive to all four. These
data allowed us to select a specific strain for measuring the
AU of each bacteriocin preparation and for measuring, in
separate experiments, the adsorption of each bacteriocin to
a sensitive strain and the producer strain (Fig. 1).
Effect of pH on adsorption of bacteriocin onto producer and
indicator cells. The influence of pH on the adsorption of
pediocin AcH, nisin, sakacin A, and leuconocin Lcml onto
their respective producer and indicator strains is presented
in Fig. 1. Adsorption of all four bacteriocins onto cells was
strongly influenced by the pH of the suspending environ-
ment. Pediocin AcH was adsorbed 100% at pH 6.0 to 6.5,
while at pH below 1.5 it was not adsorbed to either P.
acidilactici LB 42-923 or Lactobacillus plantarum NCDO
955. Maximum adsorption of nisin to both producer and
indicator bacteria occurred at pH 6.5, and complete loss of
adsorption was found at pH 3.0 and below. Maximum
sakacin A adsorption occurred at pH 5.5 and above on both
the producer and indicator strains. At pH 2.0 no sakacin A
was adsorbed to the producer cells but about 75% remained
adsorbed onto E. faecalis MB1. Leuconocin Lcml had a
sharp adsorption curve on producer cells, with a maximum
VOL. 58, 1992 BACTERIOCIN EXTRACTION FROM LACTIC ACID BACTERIA 3357

Extraction and purification of bacteriocin. On the basis of

the influence of pH on the adsorption and release of each
bacteriocin, an extraction method for bacteriocin was devel-
oped. The recovery and loss of each bacteriocin at different
A stages of purification and in the final dried product are
d
8 presented in Table 2. The loss of bacteriocin in the supema-
0
r tant following harvesting of the cells ranged from 0 to 7.7%,
b
e
and loss with the cells after extraction ranged from 0.6 to
d 2.3%. Recovery in dried preparations ranged from 44.3% for
sakacin A to 106.7% for pediocin AcH. The potency (AU per
gram) of bacteriocins ranged from 1.8 x 10 to 5.0 x 10 on
10 a dry-matter basis and from 1.1 x 108 to 4.9 x 108 on a
protein content basis (19). Although the loss of sakacin A in
supernatant and cells was very low, the total yield in the
dried preparation was only 44%. It was not clear why there
was such a discrepancy. The yields of dry matter and protein
A for both Lactobacillus sake Lb 706 and Leuconostoc camo-

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d sum Lml were higher than for P. acidilactici LB 42-923 and
a
0
r
Lactococcus lactis subsp lactis ATCC 11454.
b SDS-PAGE analysis of the preparations. A Coomassie
e
d blue-stained SDS-PAGE gel revealed a single sharp band
from both the pediocin AcH and nisin preparations, espe-
cially when they were tested within 2 weeks of preparation
(Fig. 2). After storage for 2 weeks or more, pediocin AcH
and nisin preparations produced more than one band. How-
ever, the sakacin A and leuconocin Lcml preparations
produced several bands, some of which were diffused. A
comparison of the bands in the stained gel with the gel half
A showing zones of inhibition indicated that the lowest band
d for each preparation contained the active bacteriocin (Fig. 2
0 and 3). Nisin, sakacin A, and leuconocin Lcml bands were
r very close to 2.5 kDa, while pediocin AcH was just above
b
e the 2.5-kDa standards. Also, leuconocin Lcml gave two
d
activity bands, one just below the other, and the pediocin
AcH preparation showed a streak of slight activity above the
main zone (Fig. 3).

% OVu X Adsorption of bacteriocin molecules by the Pediococcus
A (7, 13), Lactobacillus (18), Lactococcus (16), and Leuconos-
d 60
a
0 and resistant gram-positive bacteria, has been reported.
r
b
40
d 20
1 2 3 4 5 6 7 8 9 10
pH
FIG. 1. Adsorption of bacteriocins onto producing and indicator
bacteria. (A) Pediocin AcH adsorption on P. acidilactici LB42-923
(*) and Lactobacillus plantarum NCDO 955 (E). (B) Nisin adsorp-
tion on Lactococcus lactis subsp. lactis ATCC 11454 (*) and
Lactobacillusplantarum NCDO 955 (E). (C) Sakacin A adsorption
on Lactobacillus sake Lb706 (*) and E. faecalis MB1 (0). (D)
Leuconocin Lcml adsorption on Leuconostoc carnosum Lml (*)
and Leuconostoc mesenteroides Ly (0).
at pH 5.5 and a minimum at pH 3.0 and below (acid range).
Adsorption of leuconocin Lcml on Leuconostoc mesenteroi-
des Ly was relatively low, about 50% between pH 4.5 and
7.0, and the lowest was at 4.0 or below in the acid range. In
the alkaline range the adsorption varied from 0% at pH 8.0
for leuconocin Lcml to almost 100% at pH 10 for sakacin A.
DISCUSSION
toc (10, 14) producer strains, as well as by other sensitive
Also, the pH of optimum adsorption for nisin (16) and
pediocin AcH (7) by gram-positive bacteria has been re-
ported. Bhunia et al. (7) recently reported the conditions for
adsorption of pediocin AcH onto sensitive Lactobacillus
plantarum NCDO 955. This report was the basis for the
present study on the influence of pHs between 1 and 10 on
the level of adsorption of four bacteriocins from four genera
of lactic acid bacteria onto the respective producer strains
and lactic acid bacterium strains sensitive to a particular
bacteriocin. For this purpose Lactobacillus plantarum
NCDO 955 was used for pediocin AcH and nisin, Leuconos-
toc mesenteroides Ly was used for sakacin A, and E.
faecalis MB1 was used for leuconocin Lcml (Table 1; Fig.
1).
Methods for bacteriocin isolation that involved removal of
cells from culture broth, following adjusting the pH to 6.0
(13), and/or precipitation of cell-free supernatant with
(NH4)2SO4 will recover only a portion of the total bacterio-
cin present in the culture broth, because the portion ad-
sorbed on the cells is lost. Since adsorption of bacteriocins is
not affected by heating the cells, we used heat-killed cells to
avoid any loss of activity by proteolytic enzymes of the cells
(7, 8). Although the maximum and minimum adsorption with
3358 YANG ET AL. APPL. ENvIRON. MICROBIOL.

TABLE 2. Recovery of bacteriocins at different stages of extraction and purification

AUa (%) of:
Bacteriocin recovered
Pediocin AcH Nisin Sakacin A Leuconocin Lcml
Culture broth (1 liter)a 3.0 x 107 (100%) 3.0 x 107 (100%) 7.0 x 107 (100%) 2.6 x 107 (100%)
Lost in broth supernatanta 8.0 x 105 (2.7%) 2.0 x 105 (0.7%) 0.0 (0.0%) 2.0 x 106 (7.7%)
Lost with cells after extraction' 6.0 x 105 (2.0%) 2.0 x 105 (0.7%) 4.0 x 105 (0.6%) 6.0 x 105 (2.3%)
In total dried preparationb 3.2 x 107 (106.7%) 2.8 x 107 (93.3%) 3.1 x 107 (44.3%) 2.5 x 107 (96.2%)
AU/g of dry mattet 5.0 x 107 5.0 x 107 3.1 x 107 1.8 x 107
AU/g of proteinc 4.1 x 108 4.9 x 108 1.5 x 108 1.1 X 108
a
Total AU in relation to 1-liter culture broths and supernatants and cells from the broths. Percentages are based on AU in culture broths as 100%.
b AU in freeze-dried products was determined by resuspending the dry material in 50 mM SDS for better dispersion and prevention of clumping of bacteriocin
molecules.
c After freeze-drying the values for total dry matter and total protein content, respectively, were as follows: pediocin AcH, 640 and 78 mg; nisin, 560 and 57.5
mg; sakacin A, 995 and 206 mg; leuconocin Lcml, 1,400 and 230 mg. The results are the average of two extractions.

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respect to pH varied slightly among the bacteriocins, a single
protocol could usually be used, namely pH 6.0 for high
adsorption onto the cells and pH 2.0 for maximum release
from the cells. Extraction at alkaline pH was not used
because it is known that nisin (16), pediocin AcH (7), and
some other bacteriocins of lactic acid bacteria are inacti-
vated at alkaline pHs. Phosphoric acid was used to adjust the
mixtures to the desired pH; other acids could also probably
be used. We used 100 mM NaCl during extraction to prevent
the bacteriocin molecules from clumping and thus being
removed with the cells during centrifugation.
The total recoveries of pediocin AcH, nisin, and leucono-
cin Lcml in the dried preparations were relatively high. For
pediocin AcH the recovery was over 100%. This may be a
result of the dilution method used for the assay of AU.
Because only fixed dilutions are used, there may be differ-
ences in the end point, which may introduce some error.
However, for all three bacteriocins the recovery was over
90%. For sakacin A the recovery was only 44.0%, even
though losses in supernatant and cells were very low (Table
2). The reason for this discrepancy is now being investi-
gated. The dried preparations have very high bacteriocin
activity based on either dry-matter or protein concentration.
Dry-matter and protein concentrations of both sakacin A and
leuconocin Lcml were relatively high compared with those
of pediocin AcH and nisin. Thus, it is possible that Lacto-
bacillus sake Lb 706 and Leuconostoc camnosum Lml have
some other proteins that were extracted along with the
bacteriocins. Some Lactobacillus strains have been shown
to possess easily extracted surface proteins (3, 26).
The method described here produced dry preparations of
bacteriocins that were not only highly potent but, for pedi-
ocin AcH and nisin, also pure (Fig. 2). In a separate study
the pediocin AcH band from the gel was transferred to a
membrane by transblot and the membrane was used for
partial sequencing of amino acids (22). The formation of
more than one band by stored preparations of pediocin AcH
is now being studied. Both sakacin A and leuconocin Lcml
gave several bands with higher Mr than the bacteriocin.
These bands are probably surface or cell wall proteins; this
is currently being investigated. In gel overlays there was a
single inhibition zone for each bacteriocin, except for leu-
conocin Lcml, which gave two zones, suggesting that both
monomer and dimer forms of leuconocin Lcml were present
(Fig. 3).
The actual Mr of the bacteriocins as determined from the
amino acid sequences and the Mr as estimated on the basis of
their migration in relation to standards in the gel could be
quite different. Nisin, with a known Mr of 3,500 (16), forms
a band in the same line as the Mr standard of 2,500. Pediocin
AcH, with a known Mr of 4,600 (22), forms a band slightly
above 2,500.

FIG. 2. SDS-PAGE gel half, stained with Coomassie blue. (A)
Lanes: 1, Mr standards (a, Mr 16,900; b, Mr 14,400; c, Mr 8,200; d,
Mr 6,200; e, Mr, 2,500); 2, pediocin AcH (old preparation); 3, nisin;
4, sakacin A; 5, leuconocin Lcml. (B) Lanes: 1, pediocin AcH (fresh
preparation); 2, Mr standards. Arrows show respective bacteriocin
bands.
FIG. 3. SDS-PAGE gel half, showing the zone of growth inhibi-
tion of E. faecalis MB1 by the bacteriocin bands. Lanes: A, pediocin
AcH (old preparation, showing a slight streak of activity above the
main zone); B, nisin; C, sakacin A; D, leuconocin Lcml. These
zones of inhibition correspond to the respective bacteriocin bands in
Fig. 1A. For further explanation, see the text.
VOL. 58, 1992 BACTERIOCIN EXTRACTION FROM LACTIC ACID BACTERIA
The method described here for the extraction of bacterio-
cins from lactic acid bacterial strains produces products with
high potency and in concentrated form. The total loss is
quite low. This method could be an economical procedure to
produce large quantities of bacteriocins from lactic acid
bacteria for use as food biopreservatives. The extraction
procedure described here can also be used for the extraction
of proteins from biological systems in which adsorption to
and release from specific receptors are pH dependent.
ACKNOWLEDGMENTS
This investigation was partially funded by the National Live Stock
and Meat Board, Binational Agricultural Research Development
(with Israel), and Wyoming Experimental Station funds.
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