Journal of Biotechnology 121 (2006) 5461

Optimization of culture conditions and medium composition for

the production of micrococcin GO5 by Micrococcus sp. GO5
Mi-Hee Kim 1 , Yoon-Jung Kong, Hong Baek, Hyung-Hwan Hyun
Department of Biological Science and Biotechnology, Hankuk University of Foreign Studies, Yongin 120-749, Republic of Korea

Received 8 February 2005; received in revised form 17 June 2005; accepted 27 June 2005

Abstract

To enhance the production of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5, cultivation conditions and
medium composition were optimized. The optimal initial pH and temperature for bacteriocin production were 7.09.0 and 37 C,
respectively. Micrococcus sp. GO5 displayed the highest micrococcin GO5 activity when grown in modified MRS medium that
contained lactose or sucrose, rather than glucose, as a carbon source. The maximum bacteriocin activity was obtained in modified
MRS medium containing 0.5% tryptone and 1.0% yeast extract as nitrogen sources instead of the other nitrogen sources present
in MRS medium. Bacteriocin production was greatly affected by the concentration of K2 HPO4 ; strain GO5 produced eight-fold
more bacteriocin in medium containing 2.02.5% K2 HPO4 than in medium containing 0.2% K2 HPO4 . The optimal concentration
of MgSO4 7H2 O for bacteriocin production was 0.5%. The production of micrococcin GO5 was increased 32-fold in shake flask
culture and 16-fold in a bioreactor using the optimized medium (TY medium), compared with culturing in MRS medium.
2005 Elsevier B.V. All rights reserved.

Keywords: Bacteriocin; Micrococcin; Micrococcus sp.; Kimchi; Production

1. Introduction microorganisms, and they are therefore potentially use-

Bacteriocins, which are produced by certain strains
of lactic acid bacteria and some other prokaryotes, are
proteinaceous antimicrobial agents (Tagg et al., 1976).
Some bacteriocins inhibit food spoilage and pathogenic
Corresponding author. Tel.: +82 31 330 4279;
fax: +82 31 330 4566.
E-mail address: hhhyun@san.hufs.ac.kr (H.-H. Hyun).
1 Present address: R&D Center, Daewon Pharm. Co. Ltd., Seoul
143-150, Republic of Korea.
ful as natural replacements for synthetic food preserva-
tives. Nisin, a bacteriocin produced by certain strains
of Lactococcus lactis subsp. lactis, is one of the most
intensively studied lantibiotics, due to its industrial
application and potential for other uses. It is composed
of 34 amino acids and has a broad spectrum of activ-
ity and extreme heat stability at low pH (Choi et al.,
2000; Delves-Broughton, 1990). Nisin is generally rec-
ognized as safe (GRAS); it is commercially produced
by microbial cultivation and has been widely used as a
food preservative in many countries.

0168-1656/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2005.06.022
 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461
The production of bacteriocins is, in general, closely
associated with bacterial growth because bacteriocins
are synthesized only during the growth of the produc-
ing organism; bacteriocin activities decrease more or
less sharply at the end of the growth phase as a result
of degradation by proteases (De Vuyst and Vandamme,
1994; Green et al., 1997; Hur et al., 2000). Bacteriocin
production is also affected by the medium composition
and culture conditions, such as pH, temperature and
agitation. Therefore, the optimization of environmen-
tal conditions is very important for the enhancement of
bacteriocin production. The effect of medium compo-
sition on the production of bacteriocins, such as nisin
(De Vuyst and Vandamme, 1992, 1993; Matsusaki et
al., 1996), pediocin (Biswas et al., 1991), enterocin
(Parente and Hill, 1992), lactococcin (Parente et al.,
1993) and mesenterocin (Daba et al., 1993) has been
reported. Nisin production is affected by carbon source
regulation, nitrogen sources and phosphorus (De Vuyst
and Vandamme, 1992, 1993). The pH of the medium
plays an important role in the production of bavaricin
(Kaiser and Montville, 1993), nisin (De Vuyst and Van-
damme, 1992) and lactococcin (Parente et al., 1993,
1994).
In our previous paper (Kim et al., 2005), we reported
that Micrococcus sp. GO5, isolated from traditional
Korean kimchi prepared by cultivation of a mixture
containing green onion, spices and other ingredients,
produced a bacteriocin, which was tentatively named
micrococcin GO5. Micrococcin GO5 is very similar
to nisin in its spectrum of inhibitory activity, heat sta-
bility and pH stability, but its structure is unlike that
of nisin; it has a different molecular weight (5.0 kDa),
different amino acid composition, and different N-
terminal amino acid sequence. This paper describes
how the optimization of growth parameters, such as
temperature, medium composition and pH, resulted in
the enhancement of micrococcin GO5 production by
Micrococcus sp. GO5.
2. Materials and methods
2.1. Bacterial strains
The micrococcin GO5-producing strain used in this
study was Micrococcus sp. GO5, previously isolated
from kimchi. Micrococcus avus ATCC 10240 was
55
used as the indicator strain for the bacteriocin activ-
ity assay. Stock cultures were maintained at 70 C in
MRS medium (BD, Sparks, MD) containing 20% (v/v)
glycerol. Before use in experiments, the stock cultures
were activated by cultivation twice in MRS medium at
37 C for 6 h.
2.2. Media and culture conditions
Micrococcus sp. GO5 was cultured in MRS
medium. The indicator strain was cultured in TGY
medium (Kim et al., 2005). The seed cultures of Micro-
coccus sp. GO5 were prepared by cultivating the cells
in 500 ml Erlenmeyer flasks that contained 50 ml of
MRS medium and were shaken at 250 rpm and main-
tained at 37 C until the late exponential growth phase.
For flask culture, 0.5 ml of the seed culture was inocu-
lated into 50 ml of the appropriate medium in a 500 ml
Erlenmeyer flask. The initial pH of the flask culture
was adjusted to 6.8 with 5N NaOH, and the flasks were
incubated at 37 C with shaking at 250 rpm.
To test the effect of the initial pH on bacteriocin
production, MRS media were adjusted to different pH
values (pH 4.09.0) with 5N NaOH or 5N HCl. To test
the influence of carbon sources, each carbon source
was added as 2% of the MRS medium, replacing the
2% glucose. To study the effect of different nitro-
gen sources, a modified MRS medium was used as
a basal medium, yeast extract, beef extract, peptone
and ammonium citrate, were omitted and 2% glucose
was replaced with 2% lactose. The basal medium was
supplemented with each nitrogen source (1%) or a mix-
ture of multiple nitrogen sources. To investigate the
influence of the phosphate concentration, different con-
centrations of K2 HPO4 were added to the above basal
medium, which was supplemented with 0.5% tryp-
tone and 1.0% yeast extract. The experiments to deter-
mine the influence of magnesium sulfate concentration
were carried out using a modified basal medium that
contained lactose (20 g l1 ), tryptone (5 g l1 ), yeast
extract (10 g l1 ), K2 HPO4 (22.5 g l1 ), sodium acetate
(5 g l1 ), Tween 80 (1 g l1 ) and different concentra-
tions of MgSO4 7H2 O. All experiments to evaluate
culture conditions and medium composition except for
the influence of temperature were performed by shake
flask culture. The experiments on temperature effect
and the cultivation time course studies were conducted
using a 5 l bioreactor (Kobiotech Co., Inchon, Korea),
56 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461

of which pH and temperature were controlled, and that
contained 3 l of culture medium and was agitated at
700 rpm and aerated at 1 vvm. The temperature effect
on bacteriocin production was determined in a biore-
actor containing MRS medium, which was maintained
at different temperatures (25, 30 and 37 C) and con-
trolled at pH 7.0. The cultivation time course studies
to compare MRS medium and TY medium were con-
ducted in a 5 l bioreactor that was maintained at 37 C
and controlled at pH 6.0. TY medium contained lactose
(20 g l1 ), tryptone (5 g l1 ), yeast extract (10 g l1 ),
K2 HPO4 (22.5 g l1 ), sodium acetate (5 g l1 ), Tween
80 (1 g l1 ) and MgSO4 7H2 O (5 g l1 ).
2.3. Micrococcin GO5 assay
The antimicrobial activity of micrococcin GO5 in
culture medium was measured according to the follow-
ing procedures. Samples were adjusted to pH 6.0 with
5N NaOH, centrifuged at 8000 g for 10 min at 4 C,
and then filter-sterilized through a 0.45 m pore size,
mixed cellulose ester membrane (Advantec Mfs. Inc.,
Japan). Micrococcin GO5 activity was assayed accord-
ing to the spot-on-lawn method (Tagg et al., 1976).
Cells of Micrococcus avus ATCC 10240 were grown
in TGY medium at 37 C for 12 h, and then mixed with
TGY soft agar (0.7%). Three milliliters of the mix-
ture containing 107 cells was overlaid onto 1.5% TGY
agar plates. Ten microliters of the filter-sterilized bac-
teriocin sample was spotted on a TGY agar plate, and
the plate was incubated at 37 C for about 12 h until
the inhibition zone was clearly visible. The bacteriocin
samples to be spotted were serially diluted two-fold,
and the reciprocal of the highest inhibitory dilution was
used to calculate the arbitrary activity units (AU) per
millilitres. The un-inoculated media were also tested
for inhibitory zones as a control. All experiments were
performed in duplicate, and the results are the means
of duplicate trials.
2.4. Analytical methods
For the determination of culture turbidity, cul-
ture broths were appropriately diluted with distilled
water, and the optical densities were measured at
660 nm using a spectrophotometer (Shimadzu UV-
1601, Japan). The un-inoculated media were used as
a blank.
3. Results and discussion
3.1. The initial pH, cell growth and micrococcin
GO5 production
Table 1 shows the dependence of cell growth and
bacteriocin production in Micrococcus sp. GO5 on the
initial pH of the medium. The species displayed poor
growth at initial pH values lower than 5.0, and the final
cell concentration reached a maximum when the initial
pH was 9.0. This implies that the higher the initial pH
value in a shake flask culture without pH control, the
greater the cell growth will be before the pH drops by
acid production to below pH 5.0 and growth is curtailed.

Table 1
Influence of initial pH on cell growth and micrococcin GO5 production in Micrococcus sp. GO5a
Initial pH of medium Fermentation time (h) Final pH Growth (OD660 ) Micrococcin GO5 activity
(AU ml1 )b
4.0 8 4.14 0.7 8
5.0 8 4.91 1.8 8
6.0 8 4.90 6.2 64
6.5 8 4.86 9.5 64
7.0 7 4.93 11.0 128
8.0 7 4.93 12.0 128
9.0 7 5.21 14.0 128
Data are means of duplicates. Standard errors were less than 5% of the means.
a Cells were grown at 37 C in a 500 ml Erlenmeyer flask containing 50 ml of MRS medium, which was shaken at 250 rpm. Cell growth, pH

and bacteriocin activity were measured at time intervals, and the values when bacteriocin activity was maximum are presented.
b AU ml1 represents the reciprocal of the highest inhibitory dilution in the two-fold dilution assay of bacteriocin.
 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461
Fig. 1. Effect of temperature on cell growth (open symbols) and
micrococcin GO5 production (closed symbols) by Micrococcus sp.
GO5. Cells were grown in a bioreactor that contained 3 l of MRS
medium, was controlled at pH 7.0, and was maintained at 25 C
(
, ), 30 C (, ) and 37 C (, ). Data points are means of
duplicates. Standard errors were less than 5% of the means.
The production of micrococcin GO5 was maximized
when the initial pH was between 7.0 and 9.0. In gen-
eral, pH is known to be important to cell growth as
well as to bacteriocin production because aggregation,
adsorption of bacteriocin to the cells, and/or prote-
olytic degradation depend on pH and can affect the
bacteriocin activity in culture supernatants (Cheigh et
al., 2002; De Vuyst et al., 1996; Parente et al., 1994;
Verellen et al., 1998). To determine the optimal pHs for
growth and bacteriocin production in Micrococcus sp.
GO5, further studies in a pH-controlled culture vessel
will be necessary.
3.2. Temperature, cell growth and micrococcin
GO5 production
The effect of temperature on cell growth and micro-
coccin GO5 production was tested using a bioreactor
containing 3 l of MRS medium that was controlled at
pH 7.0 and maintained at different temperatures (25,
30 and 37 C). The results in Fig. 1 show that both
the growth rate and the production rate of the bacte-
riocin increased with an increase of temperature. The
highest activity of micrococcin GO5 (128 AU ml1 )
was obtained at 6 h after cultivation at 37 C, which
was also optimal for growth. At 40 C, cell growth and
bacteriocin activity were negligible (data not shown).
The optimal temperature for growth and bacteriocin
production were the same in the case of Micrococ-
cus sp. GO5, but differences in optimal temperatures
57
Fig. 2. Effect of carbon sources on cell growth (open bars) and micro-
coccin GO5 production (closed bars) in Micrococcus sp. GO5. Cells
were grown at 37 C in a 500 ml Erlenmeyer flask containing 50 ml of
MRS medium supplemented with 2% of each carbon source instead
of glucose. Cell concentration and bacteriocin activity were mea-
sured at time intervals, and the maximum values are presented. Data
bars are means of duplicates. Standard errors were less than 5% of
the means.
were reported for organisms producing nisin and nisin-
like bacteriocins (Cheigh et al., 2002; Matsusaki et al.,
1996).
3.3. Carbon sources and micrococcin GO5
production
The effect of the carbon source on cell growth and
bacteriocin production was determined using MRS
medium supplemented with 2% of different carbon
sources in place of glucose. As shown in Fig. 2, the
highest growth was observed in media containing mal-
tose; glucose, fructose, lactose and sucrose were also
suitable carbon sources for growth. However, the high-
est bacteriocin activity was obtained in MRS medium
with lactose or sucrose (256 AU ml1 ); the activity
was twice that obtained with glucose, maltose or fruc-
tose. Glucose and sucrose were reported to be suitable
carbon sources for nisin Z production (Matsusaki et
al., 1996), lactose for nisin-like bacteriocin production
(Cheigh et al., 2002) and glucose for Streptococcin A-
FF22 production (John and Ingrid, 1991).
3.4. Nitrogen sources and micrococcin GO5
production
In order to assess the effect of the nitrogen source on
growth and micrococcin GO5 production, yeast extract,
58 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461

beef extract and peptone were eliminated from the

MRS medium, and glucose was replaced by 2% lac-
tose. This basal medium was supplemented with each
different nitrogen source (1%). Cell growth was greatly
enhanced by the addition of beef extract, peptone, tryp-
tone, soytone or yeast extract to the basal medium
(Fig. 3). The greatest cell growth was observed in cells
grown in medium with yeast extract (OD660 = 8.4); the
cell concentration was almost two-fold that observed
in basal medium with beef extract. This implies that
yeast extract contains nitrogen sources and growth fac-
tors sufficient to support the growth of Micrococcus
sp. GO5. However, the highest activity of micrococ-
cin GO5 was observed when the cells were grown in Fig. 3. Effect of nitrogen sources on cell growth (open bars) and
micrococcin GO5 production (closed bars) in Micrococcus sp. GO5.
the tryptone-containing medium, and the bacteriocin Cells were grown at 37 C in a 500 ml Erlenmeyer flask contain-
activity was at least twice those observed with the ing 50 ml of MRS medium supplemented with 1% of each nitrogen
media containing other nitrogen sources. Because yeast source instead of peptone, yeast extract and beef extract. Cell con-
extract was the optimal nitrogen source for growth and centration and bacteriocin activity were measured at time intervals,
tryptone was most suitable for micrococcin GO5 pro- and the maximum values are presented. Data bars are means of dupli-
cates. Standard errors were less than 5% of the means.
duction, the use of multiple nitrogen sources might
enhance both growth and bacteriocin production. How-
ever, as shown in Table 2, cell growth and bacteriocin peptone and 0.5% beef extract, but the activity of micro-
production were only slightly promoted by the com- coccin GO5 in this medium was equal to that obtained
bination of multiple nitrogen sources. The highest cell with the basal medium supplemented with 0.5% tryp-
concentration was obtained when the basal medium tone and 1.0% yeast extract or with 0.5% tryptone and
was supplemented with 0.5% tryptone, 0.5% proteose 1.0% beef extract. Therefore, 0.5% tryptone and 1.0%

Table 2
Effect of the combination of multiple nitrogen sources on growth and micrococcin GO5 production in Micrococcus sp. GO5a
Nitrogen sourcesb Fermentation Growth Micrococcin GO5 Specific activity
time (h) (OD660 ) activity (AU ml1 ) (AU/OD660 )
None added 8 1.7 16 9.41
0.5% TP 8.5 5.8 128 22.07
1.0% TP 8 4.8 128 26.67
0.5% TP + 0.5% BE 8.5 6.2 128 20.65
0.5% TP + 1.0% BE 8 7.0 256 36.57
0.5% TP + 0.5% PP 8 5.8 64 11.03
0.5% TP + 1.0% PP 8 7.6 64 8.42
0.5% TP + 0.5% YE 8 5.8 128 22.07
0.5% TP + 1.0% YE 7 7.6 256 33.68
0.5% TP + 1.0% YE + 0.5% BE 7 7.6 256 33.68
0.5% TP + 0.5% PP + 0.5% BE 7 8.4 256 30.48
0.5% TP + 0.5% YE + 0.5% PP 7 7.2 128 17.78
0.5% TP + 0.5% YE + 0.5% BE 7 7.4 128 17.30
0.5% TP + 0.5% YE + 0.5% BE + 0.5% PP 7 8.2 128 15.61
Data are means of duplicates. Standard errors were less than 5% of the means.
a Cells were grown at 37 C in a 500 ml Erlenmeyer flask containing 50 ml of MRS medium that was modified to contain 2% lactose instead

of glucose and was supplemented with the nitrogen sources indicated instead of peptone, yeast extract, and beef extract. Cell growth, pH and
bacteriocin activity were measured at time intervals, and the values when bacteriocin activity was maximum are presented.
b TP, tryptone; PP, peptone; YE, yeast extract; BE, beef extract.
 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461 59

Table 3
Effect of inorganic phosphate concentration on growth and micrococcin GO5 production in Micrococcus sp. GO5a
Concentration of Fermentation Growth Final pH Micrococcin GO5
K2 HPO4 (g l1 ) time (h) (OD660 ) activity (AU ml1 )
Not added 8 5.0 5.32 64
1.0 8 6.4 5.37 128
2.0 8 8.0 5.41 256
5.0 8 8.2 5.78 512
10.0 8 6.4 5.40 1024
15.0 8 5.6 6.74 1024
20.0 7 5.4 6.83 2048
25.0 7 5.4 7.04 2048
30.0 7 4.8 7.12 512
Data are means of duplicates. Standard errors were less than 5% of the means.
a Cells were grown at 37 C in a 500 ml Erlenmeyer flask containing 50 ml of a modified MRS medium that contained 2% lactose, 0.5%

tryptone, 1% yeast extract, 0.5% sodium acetate, 0.01% MgSO4 7H2 O and different concentrations of K2 HPO4 . The initial pH was adjusted
to 7.0 with 5N NaOH. Cell growth, pH and bacteriocin activity were measured at time intervals, and the values when bacteriocin activity was
maximum are presented.

yeast extract were chosen as the nitrogen sources in the
cultivation medium for micrococcin GO5 production.
3.5. Inorganic phosphate concentration
MRS medium contains 2.0 g l1 of dipotassium
phosphate; therefore, the experiments to optimize the
inorganic phosphate concentration were conducted
using the basal medium supplemented with 0.5% tryp-
tone, 1.0% yeast extract and different concentrations
of K2 HPO4 . As shown in Table 3, the increases in the
final cell concentration corresponded to the increases
in phosphate concentration up to 5.0 g l1 ; the final cell
concentration was highest in medium with 5.0 g l1
of phosphate because it decreased in phosphate con-
centration greater than 10.0 g l1 . The production of
micrococcin GO5 also increased according to increases
in the phosphate concentration up to 25.0 g l1 . The
highest bacteriocin activity was observed with medium
containing phosphate at 20.025.0 g l1 ; the activity
(2048 AU ml1 ) was eight-fold that obtained at a phos-
phate concentration of 2.0 g l1 . Phosphate concentra-
tions greater than 30.0 g l1 inhibited both growth and
bacteriocin production. Therefore, the optimal concen-
tration of K2 HPO4 appeared to be 22.5 g l1 . The fact
that the highest bacteriocin activity was obtained at
a phosphate concentration of 20.025.0 g l1 which
growth was rather inhibited indicates that the enhance-
ment of bacteriocin activity by the increase of phos-
phate concentration was not due to the increase of
growth by a buffering effect of phosphate. These results
are consistent with a previous study by De Vuyst and
Vandamme (1993); they reported that KH2 PO4 was
the best phosphorus source for nisin production by
Lactococcus lactis subsp. Lactis, that increasing the
initial KH2 PO4 concentration from 0 to 5% stimulated
nisin biosynthesis, and that initial KH2 PO4 concentra-
tions higher than 6% decreased nisin activity levels and
caused cell lysis.
3.6. Magnesium sulfate concentration
In order to improve the bacteriocin yield further,
the effect of the magnesium sulfate concentration on
growth and micrococcin GO5 production was tested
using a modified MRS medium consisting of 2% lac-
tose as a carbon source, 0.5% tryptone and 1% yeast
extract as nitrogen sources, 2.25% potassium phos-
phate, 0.5% sodium acetate and different concentra-
tions of magnesium sulfate. Table 4 shows that the
activity of micrococcin GO5 was enhanced by the addi-
tion of magnesium sulfate and reached a maximum
(4,096 AU ml1 ) at MgSO4 7H2 O concentrations of
5.010.0 g l1 , while cell growth was affected little by
the concentration of magnesium sulfate. The effects
of other metal ions, such as Mn2+ , Zn2+ , Cu2+ , Ca2+
and Co2+ , on bacteriocin production were tested, but
no significant effects were observed (data not shown).
Growth in medium with an optimized composition
improved micrococcin GO5 production 32-fold com-
pared with that in MRS medium (4096 AU ml1 versus
128 AU ml1 , respectively). The optimized medium
60 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461

Table 4
Effect of magnesium sulfate concentration on growth and micrococ-
cin GO5 production in Micrococcus sp. GO5a
MgSO4 7H2 O Fermentation Growth Micrococcin GO5
concentration time (h) (OD660 ) activity (AU ml1 )
(g l1 )
Not added 8 6.4 512
0.1 8 6.4 2048
0.5 8 6.4 2048
1.0 8 7.4 2048
5.0 7 6.6 4096
7.0 7 6.6 4096
10.0 7 7.0 4096
Data are means of duplicates. Standard errors were less than 5% of
the means. Fig. 4. Comparison of growth (open symbols) and micrococcin GO5
a Cells were grown at 37 C in a 500 ml Erlenmeyer flask con-
production (closed symbols) profiles for Micrococcus sp. GO5 in
taining 50 ml of a modified MRS medium that consisted of 2% MRS medium (
, ) and TY medium (, ). Cells were grown in
lactose, 0.5% tryptone, 1% yeast extract, 0.5% sodium acetate, 2.25% a bioreactor in 3 l of MRS or TY medium at 37 C with agitation at
K2 HPO4 and different concentrations of MgSO4 7H2 O. Cell growth, 700 rpm and aeration at 0.5 vvm; the pH was controlled at 6.0 with
pH and bacteriocin activity were measured at time intervals, and the 5N NaOH. Data points are means of duplicates. Standard errors were
values when bacteriocin activity was maximum are presented. less than 5% of the means.

consisting of the above-modified MRS medium sup-
plemented with 5.0 g l1 of magnesium sulfate was
named tryptone yeast (TY extract) medium and was
used in further experiments including studies on culti-
vation time courses in a bioreactor.
3.7. Growth and bacteriocin production proles in
MRS and TY media using a bioreactor
Growth and bacteriocin production time courses
were determined in 3 l of MRS or TY medium in
a 5 l bioreactor that was maintained at 37 C, con-
trolled at pH 6.0, agitated at 700 rpm and aerated at
1 vvm; the results are shown in Fig. 4. The growth
rate and final cell concentration were higher in MRS
medium than in TY medium. However, micrococcin
GO5 production in TY medium was 16-fold that in
MRS medium. The production of micrococcin GO5
shows primary metabolite kinetics. The extracellular
bacteriocin activity increased rapidly during late loga-
rithmic growth phase, reached a maximum during early
stationary phase (7 h), and was maintained at the max-
imum level for 1 h. The data imply that the production
of micrococcin GO5 is closely associated with growth
but is not necessarily proportional to the growth rate
or cell concentration. Based on our data, the phosphate
concentration seems to play an important role in the
regulation of bacteriocin biosynthesis. The activity of
micrococcin GO5 decreased rapidly with prolonged
cultivation after the stationary phase. These features
of the bacteriocin production profile are common to
almost all lactic acid bacteria that produce bacteriocins
(De Vuyst and Vandamme, 1994; Green et al., 1997;
Hur et al., 2000). During the practical production of
bacteriocin, it is necessary to either stop the cultivation
process or maintain the maximum level of bacteriocin
activity by other methods. It has been suggested that
the decrease in bacteriocin activity during the station-
ary phase might be due to degradation by specific or
non-specific proteases, adsorption to the producer cells,
and/or aggregation (De Vuyst and Vandamme, 1992;
Joerger and Klaenhammer, 1986; Parente et al., 1994).
The decrease of micrococcin GO5 after the stationary
phase could be eliminated by lowering the pH below
3.0 or the temperature below 4 C, suggesting that the
activity decrease might be mainly due to the proteolytic
degradation of bacteriocin.
In a previous report (Kim et al., 2005), we described
micrococcin GO5 as a new and novel bacteriocin pro-
duced by Micrococcus sp. GO5, which has a broad
spectrum of antimicrobial activity and is heat- and pH-
stable like nisin, but has a structure that differs from
that of nisin based on molecular weight (5.0 kDa),
amino acid composition, and N-terminal amino acid
sequence. To increase the potential of micrococcin
GO5 for application as a food and feed biopreserva-
 M.-H. Kim et al. / Journal of Biotechnology 121 (2006) 5461
tive, the enhancement of the bacteriocin productivity
was needed. Optimization of the cultivation medium
and conditions, as described in this report, will make
its application more feasible. Strain improvement for
the production of micrococcin GO5 is now in progress.
Acknowledgement
This work was supported by the Hankuk University
of Foreign Studies Research Fund 2003.
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