ISSN 00036838, Applied Biochemistry and Microbiology, 2015, Vol. 51, No. 9, pp. 881–886. © Pleiades Publishing, Inc.

, 2015.
Original Russian Text © V.I. Surovtsev, V.M. Borzenkov, V.P. Levchuk, 2014, published in Biotekhnologiya, 2014, No. 5, pp. 44–49.

TECHNOLOGY OF BIOPREPARATIONS

Purification of Bacteriocins by Chromatographic Methods

V. I. Surovtsev, V. M. Borzenkov, and V. P. Levchuk
State Research Center for Applied Microbiology and Biotechnology, Moskovskaya Oblast, Obolensk, 142269 Russia
email: vmborzenkov@mail.ru
Received September 25, 2014

Abstract—The bacteriocins secreted by the cells of two B. subtilis strains have been isolated by hydrophobic
or ionexchange chromatography on CMC followed by purification on membranes. The yields of electro
phoretically pure products were ~75 % of the total activity in the supernatant of culture fluid obtained after
cell sedimentation. The bacteriocin secreted by B. subtilis BSX strain cells has high hydrophobicity, and it
is mainly found on the surface of cell walls in the culture fluid. These fragments served as sorbents in hydro
phobic chromatography. A B. subtilis strain B112 bacteriocin predominantly occurs in the free state in the
culture fluid supernatant. It was purified after the removal of cell debris on CMC and was then eluted with
a glycineHCl buffer at pH 2.5. The concentration of the purified bacteriocins was determined at 205 nm;
therefore, the method did not require aromatic amino acids to be available in the macromolecule structure.
It is believed that these chromatographic techniques can be used for purification with a high yield of bac
teriocins of other producers.

Keywords: bacteriocins, hydrophobicity, ionchange and hydrophobic chromatography, membranes, purifi

cation, surface cell fragments
DOI: 10.1134/S0003683815090069

FORMUALTION OF THE PROBLEM
By the end of the twentieth century, the uncon
trolled use of antibiotics had led to the wide distribu
tion of pathogenic microorganisms resistant to differ
ent groups of these drugs. Furthermore, antibiotics
may be allergenic and are very toxic to some people.
For example, aminoglycoside antibiotics are toxic to
the kidney tissue and the inner ear. Up to a quarter of
patients receiving gentamicin experience acute renal
failure, and a third have irreversibly reduced hearing.
However, aminoglycoside antibiotics have no alterna
tive in the treatment of the infections caused by the
most resistant strains of Gramnegative microorgan
isms [1]. Therefore, the search for and the use of anti
microbials other than antibiotics are urgent tasks.
Bacteriocins can be substances capable of replacing
antibiotics in some cases. This includes a large group
of chromosome or plasmidencoded amphiphilic and
antimicrobial peptides (~2–6 kDa) secreted by Gram
positive or Gramnegative bacteria. Their low toxicity
and absence of allergenicity and reactogenicity were
shown in a few examples [2]. Nisin, a Streptococcus
lactisderived bacteriocin, has been used in the food
industry for more than 30 years to protect against
Grampositive bacteria. The nisin preparation con
tains 2.5% of this bacteriocin [3]. However, high
Abbreviations: CMC—carboxymethyl cellulose, OD—optical
density, LgG—immunoglobulin G, SDSPAG—sodium dodecyl
sulfate polyacrylamide gel.
purity bacteriocins are required for medical applica
tions (for example, when injected into the blood
stream). They are now prepared mainly for structural
studies, because the yield of the purified product
accounts for no more than a few percent of the total
content in the cell. The usual procedure involves the
precipitation of proteins and peptides from culture
fluid by ammonium sulfate, hydrophobic chromatog
raphy on OctylSepharose, ion exchange chromatog
raphy, and high performance liquid chromatography
[4]. The low yield of purified product is probably one
of the reasons for the lack of examples of medical use
of bacteriocins. In any case, we have not found exam
ples in the available literature.
Our goal was to prepare electrophoretically pure
bacteriocins secreted by the cells of two strains of
B. subtilis, with a yield of at least 75% of the total activ
ity in the culture fluid supernatant of.
MATERIALS AND METHODS
Strains and Media. The bacteriocins producers
B. subtilis BSX and B. subtilis B112 were provided by
the collection of the State Research Center for
Applied Microbiology and Biotechnology.
The composition of the nutrient medium for culti
vation of the B. subtilis BSX strain included (g/L):
K 2HPO4—10.5; KH2PO4—4.5; (NH4)2SO4—1.0;
sodium citrate, trisubstituted (⋅2H2O)—0.5; MgSO4 ⋅

881
882 SUROVTSEV et al.
7H2O—0.2; and glucose—2.0; pH 7.2. The culture
medium for B. subtilis B112 strain contained (g/L):
pancreatic fish flour hydrolysate—8.0; fermentative
peptone—8.0; NaCl—4.0; and yeast extract—5.0;
pH 7.2. All of the reagents were manufactured by
Khimmed (Russia) and were analytically pure. The
glucose solution, together with a magnesium salt pre
pared in distilled water, was sterilized separately at
0.5 atm for 30 min. The combined solutions of the rest
components were sterilized at 1 atm 30 min.
Cultivation of the B. subtilis BSX and B. subtilis
B112 Strains was performed in a 7.5 L bioreactor
(Bioflo 110, New Brunswick Scientific, United States)
containing 5 L of the culture medium at 30°C for 12
and 24 hours, respectively. Aeration was performed at
1.5 L/min. The stirring speed was regulated automati
cally by the CASKAD program to maintain a constant
concentration of dissolved oxygen (10%).
The bacteriocin activity was determined with a List
eria monocytogenes 776 test culture provided by the col
lection of the State Research Center for Applied Micro
biology and Biotechnology. The supernatant obtained
after cell precipitation was analyzed at twofold dilu
tions. Ten nanoliters of each twofold dilution were
spread on a Petri dish with fresh lawns of test culture.
The plates were incubated at 37°C for 18–24 hours. The
maximum dilution of a bacteriocin causing visualized
cell growth inhibition of the test culture was taken as the
unit of specific activity expressed in arbitrary units
(AU/mL).
Dialysis of supernatants containing bacteriocin
secreted by B. subtilis cells cultures was performed at
pH 4.0 in dialysis bags intended for high molecular
weight proteins for 1 day at room temperature, and the
dialysis solution was changed two times (the ratio of
the supernatant to dialysis solution was 1 : 500).
Ultrafiltration of solutions containing bacteriocin
was performed with the use of 50 mL Amicon stirred
cells (United States) at a nitrogen pressure of 4 atm.
Millipore regenerated cellulose (United States) with a
molecular weight cutoff more than 10 kDa was used
to remove ballast substances.
Electrophoresis. SDSPAGelectrophoresis in
16% polyacrylamide gels (standard protocol of the
manufacturer BioRad, United States) was per
formed, and a kit of 25, 15, and 10 kDa molecular
weight markers were used to determine the molecular
weight and purity degree of the bacteriocin [5].
The concentration of electrophoretically pure
bacteriocin was spectrophotometrically measured at
λ 205 nm. One milliliter of the preparation solution
obtained after ultrafiltration was loaded on a column
of Sephadex G25 (superfine, 1.5 × 40 cm) equili
brated with 5 mM KH2PO4 (with the addition of
KOH), pH 7.0, and 0.1 M K2SO4 (the reagents were
manufactured by Khimmed, Russia, and were chem
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
ically pure). Fractions containing bacteriocin were
pooled. Cuvettes were preliminarily purified with
concentrated nitric acid. The absorbance of the bac
teriocin solution at a concentration of 1mg/mL was
quantified using the equation
1 mg/mL A 280
A 205 = 27 + 120 
,
A 205
where A280 is the absorbance of aqueous solution at
280 nm, and A205 is the absorbance after relevant
dilution at 205 nm. The concentration of the bacteri
ocin purified by ultrafiltration was then determined
1 mg/mL
from the ratio of A205 in the filtrate to A 205 .
Bacteriocin Purification
Method A. Three liters of B. subtilis BSX culture
fluid was centrifuged at 10000 g for 10 min. The pellet
was removed. The supernatant was adjusted to a pH
level of 2.8 by the addition of 4 M HCl,and was kept at
4°C overnight and then centrifuged at 12800 g for
10 min. The pellet was suspended in 30 mL of water,
and 30 mL of isopropanol was added and mixed. The
suspension was kept at 4°C for 1 h with periodic shak
ing and then centrifuged at 12800 g for 10 min. The
isopropanol was evaporated on a rotary evaporator at
60°C. Thereafter, 30 mL of water and activated carbon
(3 mg/mL) were added. After 1 h, the carbon was
removed by centrifugation at 10000 g for 10 min. The
pH of the resulting solution was adjusted to 4.0 with
8M NaOH and then filtered through a cellulose mem
brane with a macromolecule mass cutoff greater than
10 kDa.
Method B. 1 L of B. subtilis B112 culture fluid was
centrifuged at 10000 g for 10 min. The pellet was
removed. The supernatant was adjusted to a pH level of
2.8 by the addition of 4 M HCl and kept at 4°C over
night. The suspension was centrifuged at 12800 g for
10 min. The precipitate was removed, and the pH of
the supernatant was adjusted to 5.5 with 8M NaOH,
after which the solution was loaded on a CMC column
(2.5 × 45 cm) equilibrated with 0.05 M acetate buffer,
pH 5.5. The column was intensely washed with 2 L of
the buffer and 3 L of water. The presence of colored
admixtures was analyzed after each use of 0.5 L of
washing solution. For this, 50 mL of the eluate was
evaporated on a rotary evaporator 10–15 times, and
the absorbance was photoelectrocolorimetrically
measured in the visible region against the buffer or
water. After removal of the admixtures, the bacteriocin
was eluted with 0.4 M glycineHClbuffer at a pH of
2.5. The active fractions were pooled, the pH was
adjusted to 4.0 with 8 M NaOH, and the solution was
filtered through a cellulose membrane with a cutoff of
macromolecules with a mass of 10 kDa or higher.
Vol. 51 No. 9 2015
 PURIFICATION OF BACTERIOCINS BY CHROMATOGRAPHIC METHODS
RESULTS AND DISCUSSION
Work on the isolation and purification of bacterio
cins has been carried out for many years, but the yield
of electrophoretically pure target products accounts
for no more than a few percent [6]. We suppose that
this is caused by the use of methods and conditions
intended for high molecular weight proteins. Bacteri
ocins are not proteins. In contrast to proteins, low
molecular weight bacteriocin peptides lack a spatial
structure. They are characterized by amphiphilic
properties and are resistant to denaturing agents, i.e.,
pH and temperature fluctuations. The purification
procedures developed for proteins [7] may be used for
polypeptides, provided that their specific properties
are taken into account; in many cases they can be
treated as organic compounds.
Typically, the first purification step is bacteriocin
precipitation from culture fluid with ammonium sul
fate [8]. This technique is commonly used for proteins
15–17 kDa or higher. Low molecular weight proteins
like insulin, the molecular weight of which is approxi
mately the same as that of bacteriocins, poorly precip
itate, even at a saturation degree of 75–80% with
ammonium sulfate. After precipitation and dissolu
tion of the precipitate, they easily pass through the
pores of dialysis bags. Therefore, this method is not
popular for low molecular weight protein purification.
However, the experiment showed that B. subtilis
BSXderived bacteriocin retained almost 100% activ
ity after centrifugation at a saturation degree of 50–
60% with ammonium sulphate (for example, this sat
uration was used in the precipitation of 150 kDa IgG
[9]) and in the subsequent dialysis. Bacteriocin poly
merization, which was observed for some peptides
under acidic conditions, did not occur. It was sup
posed that amphiphilic bacteriocin adhered to the
hydrophobic cell wall fragments formed during culti
vation in the used saline medium.
If so, it is possible to develop a method of bacteri
ocin purification by hydrophobic chromatography, in
which these fragments play the role of a “sorbent.”
The fragments are tiny and are kept by the superna
tant after centrifugation. The surface charge of the
microbial cells is usually negative at neutral pH. For
example, Micrococcus lysodeicticus superficial pK is
3.5 [10]. The adjustment of the pH to <3.0 leads to a
lowered charge, aggregation, and precipitation of the
fragments. The addition of a substance that breaks
the hydrophobic interactions (for example, an alco
hol miscible with water in any ratio (isopropanol in
our research)) leads to the isolation of the bacteriocin
into the solution. After the centrifugation of aggre
gated fragments, only bacteriocin and ballast struc
tures remain in the solution, which are also linked
with the fragments by hydrophobic interactions. In
this case, the soluble bacteriocin yield was ~90% of
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 51
883
the total activity in the culture fluid supernatant, and
its specific activity increased by 32 times. Further
purification with activated carbon and membrane
cutting substances with molecular weights over
10 kDa resulted in decreased activity by about 10%,
and thus the resulting yield of electrophoretically
pure bacteriocin was 75% of the total activity in the
culture fluid. The specific activity did not grow, but
the solution was cleared of colored admixtures and
ballast proteins (Fig. 1a, 1b).
The bacteriocin produced by B. subtilis B112 cells
did not bind to cell wall fragments and was mostly
found in the supernatant in the free state. Such bacte
riocins, after ammonium sulfate precipitation (75–
80% saturation) and dialysis for one day, may have a
total activity of 40% or less of the activity in the super
natant obtained after removal of the cells [11]. Dialysis
of the precipitate, which contains fragments and bac
teriocin secreted by B. subtilis B112 cells, showed that
the activity measured after precipitation with ammo
nium sulfate was 60%. The cell wall fragments, which
were present in the supernatant and did not bind to
bacteriocin of the culture fluid, were preliminary
removed by adjustment of the pH below 3.0 and then
centrifuged to avoid contamination of the product.
Ion exchange chromatography was the main step of
B. subtilis B112derived bacteriocin purification: a
column with the bacteriocin of the previous experi
ment containing ion exchange buffer was washed to
remove colored uncharged ballast compounds,
including the proteins and polysaccharides present in
almost any nutrient medium. The specific activity
increased during elution. Ion exchange chromatogra
phy of bacteriocin on CMC from B. subtilis B112 in
0.05 M acetate buffer at a pH of 5.5 with one molar salt
gradient elution (bacteriocin isoelectric point was
~7.2) resulted in only a yield of 15–20% of the peptide
from the column. A yield of 80% was achieved by elu
tion with 0.4 M glycineHClbuffer at a pH of 2.5 (the
pK of the COOHgroup in carboxymethyl sorbents is
~4.0 [12]). After raising the pH to 4.0, purification was
performed with the use of a membrane with a macro
molecule weight cutoff of more than 10 kDa. The tar
get product was in the electrophoretically pure state
(Fig. 2).
The product yield was 77% of the total activity of
the original supernatant. Thus, a significant increase
in the yield was achieved not when the bacteriocin was
eluted as proteins, as is traditionally done, but when
we used a pH region in which it was stable and the pro
teins were denatured. The results of bacteriocin purifi
cation are given in the table.
Small bacteriocin molecules often have no aro
matic residues in their composition; spectrophoto
metric measurement of the product at 280 nm is there
fore impossible. Thus, the concentration of purified
No. 9 2015
884 SUROVTSEV et al.

a b c d
kDa

25

15

10

Fig. 1. Electrophoregram of bacteriocin samples from B. subtilis BSX at different stages of purification: (a) sample taken from the
culture fluid; (b) carbon posttreated sample (to remove colored nonprotein admixtures); (c) postmembrane filtration sample
(bacteriocin); (d) proteins, molecular weight markers.

a b c
kDa

25

15

10

Fig. 2. Electrophoregram of purified bacteriocin from B. subtilis B112: (a) proteins, molecular weight markers; (b) 0.3 µg of bac
teriocin in the sample; (c) 1.2 µg of bacteriocin in the sample.

bacteriocins was measured at 205 nm by a method
intended for protein research [13]. This method is
rarely used for proteins, which usually contain
tyrosine and tryptophan. However, this method has a
number of advantages, including an almost a 100fold
increase in sensitivity over absorbance measurement at
280 nm. One of them is very important: no aromatic
amino acids or exact information about the molecular
weight of polypeptides are required to measure the
polypeptide concentration at 205 nm. On the other
hand, the greater susceptibility of the method to a vari
ety of environmental factors and the solvent composi
tion should be noted.
Thus, the combination of simple procedures made
it possible to prepare bacteriocins in an electrophoret
ically pure state from B. subtilis BSX and B112

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 51 No. 9 2015

 PURIFICATION OF BACTERIOCINS BY CHROMATOGRAPHIC METHODS 885

Purification of bacteriocins and their concentration as compared with their content in the original supernatant of the cul
ture fluid

Ultrafiltrate
Bacteriocin
Specific Percentage A205
Volume, Total activity, concentration
Sample activity, of the total activity A 1205mg/mL *
mL AU × 10 5
in filtrate in the superna
AU/mL × 0.01 in the supernatant
tant, mg/mL
×0.01

Supernatant of the 3000 64 192.0 100 33

B. subtilis BSX
culture fluid

Sample before 81 2048 165.9 86

the addition
of activated carbon

Centrifugate after 78 2048 159.7 83

purification with
activated carbon

Ultrafiltrate 70 2048 143.4 75 0.280 0.296

Supernatant 1000 32 32.0 100 42

of the B. subtilis
B112 culture fluid

Supernatant after 1000 32 32.0 100

the removal of cell
wall fragments

Eluate 25 1024 25.6 80

after ionexchange
chromatography

Ultrafiltrate 24 1024 24.6 77 0.275 0.370

* Derived according to Scopes’s formula in [13].

strains. The yield of the purified product comprised at
least 75% of the activity in the original supernatant of
the culture fluid.
The chromatographic methods used are believed to
be useful for the preparation of highly purified bacte
riocins from other producers.
REFERENCES
1. Jankauskas, S.S., Yankauskas, S.S., Plotnikov, E.Yu.,
Morosanova, M.A., Pevzner, I.B., Zorova, L.D.,
Skulachev, V.P., and Zorov, D.B., Mitochondriatar
geted antioxidant SkQR1 ameliorates gentamycin
induced renal failure and hearing loss, Biochemistry
(Moscow), 2012, vol. 77, no. 6, pp. 666–670.
2. Cleveland, J., Cleveland, J., Montville, T.J., Nes, I.F.,
and Chikindas, M.L., Int. J. Food Microbiol., 2001,
vol. 71, no. 1, pp. 1–20.
3. Bilková, A. and Bala’zová, A., Bacteriocins produced
by lactic acid bacteria, Ceska Slov. Farm., 2011, vol. 60,
no. 1, pp. 65–72.
4. Nicolas, G.G., Lapointe, G., and Lavoie, M.C., Pro
duction, purification, sequencing and activity spectra
of mutacins D123.1 and F59.1, BMC Microbiol.,
2011, vol. 11, no. 1, pp. 69–78.
5. Laemmli, U.K., Cleavage of structural proteins during
the assembly of the head of bacteriophage T4, Nature,
1970, vol. 227, pp. 680–685.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 51 No. 9 2015

886 SUROVTSEV et al.

6. Riley, M.A. and Wertz, J.E., Bacteriocins, evolution,
ecology and application, Annu. Rev. Microbiol., 2002,
vol. 7, pp. 129–133.
7. Scopes, R., Protein Purification: Principles and Practice,
New York: Springer, 1982.
8. CarolissenMackay, V., CarolissenMackay, V.,
Arendse, G., and Hastings, J., Purification of bacteri
ocins of lactic acid bacteria: problems and pointers,
Int. J. Food Microbiol., 1997, vol. 34, no. 1, pp. 1–16.
9. Brock, J., Poluchenie preparatov immunoglobulinov.
Immunologicheskie metody (Obtaining Immunoglobu
lin Preparations. Immunological Methods), Frimel’, G.,
Ed., Moscow: Meditsina, 1980.
10. Klesov, A.A., Rabinovich, M.L., and Berezin, I.V.,
Kinetics of enzymatic reactions in heterogeneous sys
tems, Bioorg. Khim., 1976, vol. 2, no. 6, pp. 680–688.
11. Stern, N.J., Svetoch, E.A., Eruslanov, B.V.,
Perelygin, V.V., Levchuk, V.P., and Seal, B.S., Isola
tion of Lactobacillus salivarius strain and purification
of its bacteriocin which is inhibitory to Campylobacter
jejuni in the chicken gastrointestinal system, Antimi
crob. Agents Chemother., 2006, vol. 50, pp. 3111–3116.
12. Lur’e, A.A., Sorbenty i khromatograficheskie nositeli
(Sorbents and Chromatographic Media), Moscow:
Khimiya, 1972.
13. Scopes, R.K., Measurement of protein by spectropho
tometry at 205 nm, Anal. Biochem., 1974, vol. 59,
pp. 277–282.
Translated by M. Novikova

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 51 No. 9 2015