Biotechnol. Appl. Biochem.
(2002) 36, 119–125 (Printed in Great Britain)
Production of bacteriocins from Lactococcus lactis subsp. lactis
CECT 539 and Pediococcus acidilactici NRRL B-5627 using
mussel-processing wastes
Nelson Pe! rez Guerra and Lorenzo Pastrana Castro1
Departamento de BioquıT mica, XeneT tica e InmunoloxıT a, Facultade de Ciencias de Ourense, Universidade de Vigo, As Lagoas,
32004 Ourense, Spain
The growth and bacteriocin production by Lactococcus
lactis subsp. lactis CECT 539 and Pediococcus acidilactici
NRRL B-5627 were investigated on mussel-processing
wastes. Both bacteriocin productions were satisfac-
torily modelled using a modified form of the Luedeking
and Piret expression, which includes a term for the
influence of the pH reduction rate. Experimental data
from cultures buffered at different initial concentra-
tions (0, 0.03, 0.10 and 0.25 M) of both bacteria were
used to fit and verify the model. The influence of total
sugars, nitrogen, phosphorus and buffer concentration
on nisin and pediocin production was also studied
using response-surface methodology and empirical
modelling. Enhanced nisin production (33 BU/ml) was
achieved in media buffered with 0.10 M potassium
hydrogen phthalate/NaOH. However, the highest levels
of pediocin (368 BU/ml) were obtained in the non-
buffered media.
Introduction
Bacteriocins are biologically active proteins produced by
lactic acid bacteria (LAB), which display a bactericidal mode
of action against many Gram-positive bacteria, including
related LAB, food-spoiling organisms and pathogens like
Listeria monocytogenes, Clostridium spp. (including Clostridium
botulinum), Staphylococcus aureus and Bacillus cereus. In
addition, bacteriocins are not toxic and most of them are
stable over several months during frozen and refrigeration
storage and after drying. These antibacterial compounds are
active over a wide pH range (mainly in acidic pH), and their
activity can be lost by one or more proteolytic enzymes,
including gastric proteinases. With these properties, some
bacteriocins, mainly nisin and pediocin, are used as food
preservatives [1].
Since the spectrum of activity of both bacteriocins can
be considerably enhanced by combination with chelating
agents, there is considerable interest in using bacteriocins in
current and potential applications in the veterinary and
119
pharmaceutical areas [2]. Therefore one potential medical
use could be as a therapy for the cure of human ulcer disease,
which links with colonization by Helicobacter pylori, a Gram-
negative bacterium, which was found to be sensitive to nisin
in vitro in the presence of chelator [3]. Both nisin and
pediocin also show good potential as a therapeutic agent in
the treatment of bovine mastitis, topical skin infections and
multiple-drug-resistant systemic infections [2,4]. On the
other hand, a nisin-based mouth rinse appeared to prevent
both the build-up of plaque and gingival inflammation in
beagle dogs [5].
The determination of optimum parameters (tempera-
ture, pH, media composition) for both enhanced production
and purification of bacteriocins is amongst the prerequisites
for their use in the food, veterinary and pharmaceutical
industries.
Studies on factors affecting bacteriocin production in
LAB are commonly performed in rich, undefined media.
However, such media are too expensive and have high
peptone contents, which make subsequent purification of
bacteriocins difficult [6]. In recent years, residual effluents
from food industry have been used as inexpensive substrates
for bacteriocin production [1,7].
Mussel-processing wastes (average COD, 25 g O2\l ;
glycogen as main component, 5–10 g\l), an important
eutrophication factor along the coast of the galician Rias
Baixas (north-west of Spain), have been used for various
bioproductions of potential economic interest [8].
In the present study, we investigated the suitability of
this waste to support both the growth and bacteriocin
production by Lactococcus lactis subsp. lactis CECT 539 and
Pediococcus acidilactici NRRL B-5627. Then, we evaluated the
effect of different nutrients (total sugars, nitrogen and
phosphorus) and final pH (using the buffer concentration in
the medium as indirect variable) on the production of these
bacteriocins, using empirical modelling and response sur-
faces methodology.
Key words : lactic acid bacteria, nisin, pediocin, pharmaceutical, veterinary.
Abbreviations used : CDW, cell dry weight ; LAB, lactic acid bacteria.
1
To whom correspondence should be addressed (e-mail
pastrana!uvigo.es).
# 2002 Portland Press Ltd
120 N. P. Guerra and L. P. Castro
Materials and methods
Bacterial strains
L. lactis subsp. lactis CECT 539, the nisin-producing strain and
Carnobacterium piscicola CECT 4020, the target organism,
were obtained from the Spanish Type Culture Collection
(CECT, Valencia, Spain). Ped. acidilactici NRRL B-5627, the
pediocin-producing strain, was obtained from the National
Center for Agricultural Utilization Research (NCAUR,
Peoria, IL, U.S.A.). Strains were grown in MRS broth (Merck,
Germany) and maintained as frozen stock held at k40 mC in
nutrient broth plus 15 % (v\v) glycerol. Working cultures
were maintained as slants on MRS agar (Merck, Germany) at
4 mC, and subcultured twice in liquid cultures in the same
medium at 30 mC before use.
Media and culture conditions
The mussel-processing waste used as culture medium was
prepared as follows : after adjusting the pH value to 4.5 with
5 M HCl, the medium was decanted for 3 h. The precipitate
was eliminated through centrifugation (12 000 g for 15 min).
Then, the glycogen contained in the supernatant was
hydrolysed at 40 mC for 1 h, with a San Super 240L
commercial preparation of α-amylase obtained from Novo
Nordisk, Denmark. The enzyme\medium ratio was 1 : 1000
(v\v). The hydrolysed medium contains (g\l) : glucose, 5.33 ;
total nitrogen, 0.65 ; total phosphorus, 0.14 ; proteins, 1.82.
In the fermentation experiments, the media pH was adjusted
to 6.3 and sterilized at 121 mC for 15 min.
Batch cultures of Ped. acidilactici and L. lactis 1.04 were
performed in 250 ml Erlenmeyer flasks containing 50 ml of
the fermentation medium on a rotary shaker (200 rev.\min)
at 30 mC for 18 h. The inoculum contained 2 % (v\v) of
exponentially growing culture. Samples were withdrawn at
intervals during incubation periods to perform the analytical
determinations.
To study the influence of the pH decline rate in
bacteriocin production by L. lactis and Ped. acidilactici, the
hydrolysed media were buffered at initial concentrations of
0.03, 0.10 and 0.25 M. Potassium hydrogen phthalate\NaOH
was used as the buffering agent in order to avoid alterations
in the composition of the medium.
Bacteriocin activity assays
Aliquots from cultures of Ped. acidilactici and L. lactis were
adjusted to pH 3.5 with 5 M HCl to avoid the adsorption
of molecules of bacteriocin on to the producer cell sur-
faces. Thereafter they were heated for 3 min to kill the
cells and centrifuged at 27 200 g for 15 min at 4 mC. The
supernatants containing overall anti-bacterial activity (bac-
teriocin extract) were adjusted to pH 6.0 and frozen until
further use.
# 2002 Portland Press Ltd
Anti-bacterial activity of bacteriocin extracts was de-
termined by a photometric bioassay method [9] using
Carnobacterium piscicola as the target organism. Bacteriocin
extracts were diluted in distilled sterile water (this step
eliminated the need to correct the pH of bacteriocin
extracts). The diluted bacteriocin extract (2.5 ml) was added
into sterile culture tubes. Each tube was inoculated with
2.5 ml of a culture of Carnobacterium piscicola [diluted to an
absorbance of 0.2 at 700 nm with sterile-buffered MRS broth
(pH 6.3)]. Controls consisted of three culture tubes in which
the diluted bacteriocin extract was substituted by distilled
sterile water. The tubes were incubated for 6 h at 30 mC.
Growth inhibition was measured spectrophotometrically at
700 nm. Dose–response curves were obtained from these
data. Bacteriocin activity was defined as the amount of
bacteriocin needed to obtain 50 % growth inhibition (lethal
dose l 50) of the indicator strain and was expressed in
bacteriocin units (BU\ml).
Analytical methods
Cell growth was followed by measurement of the absor-
bance at 700 nm in a U-2000 spectrophotometer (Hitachi,
Japan). Cell dry weight (CDW) was calculated from a
calibration curve [1]. Cells were harvested by centrifugation
(12 000 g for 15 min at 4 mC) of culture samples and washed
twice with saline (0.8 % NaCl). Methods for the deter-
mination of residual sugars, total phosphorus and nitrogen,
and proteins were described or referred to in a previous
study [10].
Experimental designs
A first-order factorial design [11,12] based on two levels and
four variables was used to study the effect of four factors
(total sugars, nitrogen, phosphorus and buffer concentra-
tion) on the production of bacteriocins by L. lactis and Ped.
acidilactici on hydrolysed medium. The design consisted of 20
experiments with 16 (24) factorial points and four replicates
of the central treatment. A least-squares method (quasi-
Newton) with Microsoft Excel, version 8.0 (Microsoft
Corp.), was used to obtain the models (for biomass and
bacteriocin production) and the response surfaces, which
were plotted using the DeltaGraph software, version 4.0
(SPSS).
The media were inoculated with 2 % (v\v) of a 12 h-old
culture of the appropriate producer strain and incubated at
30 mC for 18 h. Bacteriocin activity (BU\ml), growth and
final pH values were measured as described above.
Modelling
The cell growth and bacteriocin production curves were
smoothed by a generalized logistic equation [13]
C(t) l K\[1+exp (a+bt+ct2)] (1)

where C(t) represents the biomass (in g\l) or bacteriocin
production (in BU\ml), and K, a, b and c are general
parameters of the model. The bacteriocin production rate
dBU\dt (in BU\ml:h) was modelled using the Luedeking and
Piret expression [14]
dBU\dt l α dX\dt+βX
or dividing by the biomass (in mg\ml) :
qBU l (αµ+β )
where qBU and µ are the specific bacteriocin (in BU\mg:h)
and biomass production rate (1\h) respectively, α is a
growth-associated constant for product production (in
BU\mg) and β is the non-growth-associated constant for
product production (BU\mg:h). The parameters in the
models were fitted to experimental data by a non-linear
least-squares fitting.
Results and discussion
Kinetic behaviour of nisin and pediocin production in
hydrolysed medium
The time course of batch fermentations of L. lactis and Ped.
acidilactici on the hydrolysed medium is depicted in Figure
1. In these cultures, it may be noted that maximum growth
Figure 1
Batch fermentation profiles of L. lactis subsp. lactis CECT 539 (A) and Ped.
acidilactici NRRL B-5627 (B) on hydrolysed medium. , CDW ; 5, residual
reducing sugars (RS) ; W, residual protein (Pr) ; #, nisin (Nis) and pediocin
(Ped) titres ; =, pH. Means for three analytical replications have been presented.
Production of bacteriocins 121
(2)
(3)
Figure 2
(A) Growth CDW, pH and nisin production (Nis) profiles by L. lactis on
hydrolysed medium ($), and on hydrolysed buffered at concentrations of
0.03 M (#), 0.10 M ( ) and 0.25 M (W) with potassium hydrogen
phthalate/NaOH. (B) Final pH, CDW* and nisin titres (Nis*) produced at the
end of the fermentation. Means for three analytical replications have been
presented.
of L. lactis was obtained after 15 h of incubation (Figure 1A).
Nevertheless, the nisin synthesis ceased when the pH of the
medium decreased to less than 4 (which occurred approx. at
9 h of incubation). On the contrary, the rate of production of
pediocin was directly proportional to the rate of pH decline
(Figure 1B). Thus during the first 3 h of incubation, a rapid
decrease in the pH value was observed and pediocin
production rate was higher in this period. However, after 3 h
of incubation, the pH decline rate was reduced and pediocin
synthesis was slower in spite of the fact that the biomass
production rate remained almost constant until 15 h of
fermentation.
Yang and Ray [15] reported that highest levels of
pediocin AcH (by Ped. acidilactici LB42-923) and nisin (by L.
lactis 11454) were produced respectively at final pH values of
3.7 and 5.8. This difference in behaviour was found to be due
to the need of high (for nisin) and low (for pediocin) final pH
value for post-translational processing of prenisin and
prepediocin to produce the active form of these two
bacteriocins [15]. In the same way, recent studies showed
that the production of bacteriocins could also be influenced
by the profile described by the pH values in the culture broth
[16].
Due to these reasons, although the exhaustion of some
essential micronutrients (vitamins or minerals) or some
# 2002 Portland Press Ltd
122 N. P. Guerra and L. P. Castro

Table 1 Parameter values of the proposed fermentation model (4) for

batch cultures of L. lactis and Ped. acidilactici on the hydrolysed media buffered
at different concentrations

Buffer L. lactis Ped. acidilactici

concentration
(M) α β δ R2 α β δ R2

0 14.7 0 5.79 0.9861 272.3 0 8.99 0.9967

0.03 68.0 0 0 0.9965 11.2 0 222.85 0.9905
0.10 56.5 0.75 0 0.9954 9.63 0 230.62 0.9957
0.25 29.6 0.50 2.79 0.9999 34.61 0 48.18 0.9999

mesenteroides Lm1 [15]. Cabo et al. [17] reported that on

Figure 3
(A) Growth (CDW), pH and pediocin production (Ped) profiles by Ped.
acidilactici on the hydrolysed medium ($), and on the hydrolysed medium
buffered at concentrations of 0.03 M (#), 0.10 M ( ) and 0.25 M (W) with
potassium hydrogen phthalate/NaOH. (B) Final pH, CDW* and pediocin titres
(Ped*) were produced at the end of the fermentation. Means for three
analytical replications have been presented.
essential amino acids in the medium could also be the cause
for the decline in nisin and pediocin production rates, it
seems more plausible to link the phenomenon to the
acidification rate in the cultures.
In an attempt to clarify the role of pH in nisin and
pediocin production, three series of cultures were per-
formed for each strain on hydrolysed media buffered at
pH 6.3 with 0.03, 0.10 and 0.25 M potassium hydrogen
phthalate\NaOH. The results depicted in Figures 2 and 3
show that, in both bacteria, the increase in buffer concen-
trations determined, as expected, different acidification
rates. Although no significant differences in cell growth were
found amongst the cultures buffered at different concentra-
tions, nisin and pediocin productions were affected in
different ways. For Ped. acidilactici, the non-buffered medium
yielded the highest pediocin titre (368 BU\ml), which
decreased significantly when the buffer molarity increased.
On the contrary, in L. lactis (Figure 2B), the highest nisin
production (33 BU\ml) was obtained in the culture medium
buffered with 0.1 M potassium hydrogen phthalate\NaOH
(final pH 4.8). This specific effect of pH on both nisin and
pediocin synthesis could be related to the final pH value
reached in the cultures.
Similar observations on the existence of an optimum
final pH value in bacteriocin production have been described
previously for leuconocim Lm1 production by Leuconostoc
increasing the pH decline gradient, there was an increase in
nisin production by a strain of L. lactis subsp. lactis isolated
from salmon sausages. In addition, it has been reported
[17,18] that the rate of acidification also had an effect on the
production of pediocin and nisin.
From these observations, it can be pointed out that the
increase in the acidification rate of the hydrolysed medium
enhances both the nisin and pediocin production, before a
final pH value unsuitable for cell growth of L. lactis and Ped.
acidilactici, and for nisin synthesis was reached.
Thus in an effort to explain this last hypothesis, we
modelled both the nisin and pediocin production using the
equation proposed by Cabo et al. [17]. This equation [eqn
(4)] is a modification of the Luedeking and Piret expression
[14], including a term for the influence of pH on bacteriocin
production :
qBU l (αµ+β )(1+δ rpH) (4)
where δ is a constant ratio to be experimentally determined,
rpH is the decrease in pH value per unit time, i.e. rpH l
∆pH\∆t.
Good adjustments (Table 1) were obtained when eqn
(4) was applied to the experimental data of Figures 2 and 3.
This indicates that this model is suitable to describe the
kinetics of the production of nisin and pediocin on buffered
hydrolysed media. Therefore it can be noted that nisin
production can be kinetically defined as primary or mixed
metabolite dependent on pH (with δ  0), except in the
culture buffered at 0.10 M.
In this last case, nisin production showed a pure primary
character (α  0, β l 0) and the variable rpH did not exert
influence on nisin production (δ l 0), probably as a conse-
quence of an optimum acidification rate that leads to a final
pH value suitable for the production of the active molecule
of nisin.
On the other hand, pediocin was produced as a primary
metabolite dependent on the pH reduction rate (α  0, β l
0 and δ  0) in all series. In this case, the parameter δ was
always positive. This suggests that an additional enhancement
of production could be obtained if a bigger gradient of pH is
generated in the medium. A lower production of pediocin

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 Production of bacteriocins 123

Table 2 Coded and actual factor levels in the complete factorial design
depicting the effect of medium composition on the production of nisin and
pediocin by L. lactis and Ped. acidilactici respectively

Codification : Vc l (VnkVo)/∆Vn ; decodification : Vn l Vo+(∆Vn Vc). Vc, coded

value ; Vn, natural value ; Vo, natural value in the centre of the experimental
domain ; ∆Vn, increment in Vn corresponding to 1 unit of Vc.

Actual values

Codified Buffer Totals sugars Total nitrogen Total phosphorus

values B (M) C (g/l) N (g/l) P (g/l)

k1 0.0 5.0 0.65 0.14

0 0.05 12.5 1.89 0.39
1 0.1 20.0 3.14 0.64

AcH in the buffered media and the need of a pH value 3.6 in

the range 3.7 for high pediocin production have been
described previously [15]. Initially, these researchers sug-
gested that pediocin AcH was produced as a secondary
metabolite, since it was still produced during the stationary
phase in cultures with sufficient decrease in pH values and in
the presence of high cell mass. Nevertheless, recent studies
showed that prepediocin (inactive form of the molecule) was
produced at pH values above 5.0, but not efficiently
processed to active pediocin AcH, which occurred at a pH
value below 5.0 [15].

Factors affecting bacteriocin production on

hydrolysed medium
Although the hydrolysed medium was capable of supporting
the growth and bacteriocin production by L. lactis and Ped.
acidilactici, the amounts of biomass and bacteriocin produced
by both strains in MRS broth [18] were not reached. Thus
we tried to improve bacteriocin production by studying the
effect of some media ingredients of inexpensive nature and
pH on the production of nisin and pediocin on the hydrolysed
medium. For this, we use a complete first-order factorial
design to study the effect of four factors [carbon (C ),
nitrogen (N), phosphorus (P) and buffer (B) concentrations]
Figure 4 Response surfaces obtained for nisin (Nis) and biomass (CDW)
on bacteriocin and biomass production by L. lactis and Ped. production by L. lactis on the hydrolysed medium, according to the
acidilactici in hydrolysed medium. The actual and coded experimental procedure defined in Table 2
factor levels are shown in Table 2.
B, buffer concentration ; C, total sugar ; N, total nitrogen ; P, total phosphorus ; N,
Glucose and the simplest amino acid glycine were used P, C and B in coded values.
as carbon and nitrogen sources respectively. KH2PO4, the
most appropriate phosphate source for nisin production
[19], was used to supplement the hydrolysed medium with Bacteriocin and biomass productions were used as
phosphorus. This nutrient was sterilized separately and objective variables. The final pH value reached in each
aseptically added to the culture media. element of the matrix and in the four replies of the centre of
Since optimal pH for bacteriocin production may also design was also measured.
be affected by the composition of culture medium [20],
possible interactions between pH and other variables (C, N Joint effects of the buffer, total concentrations of
and P) can exist. Then, the media were buffered with nitrogen, phosphorus and sugars on nisin production
potassium hydrogen phthalate\NaOH at different molarities The results of the above experimental design on both
in order to generate different acidification rates during the growth CDW and nisin production by L. lactis are shown in
fermentation. Figure 4. The empirical models obtained and their coeffi-

# 2002 Portland Press Ltd

124 N. P. Guerra and L. P. Castro

cients were found to be significant according to the Fisher F

test (α 0.05) and Student’s t test (α 0.05), respectively :

Nis (BU\ml) l 14.64+4.76Bk2.94Ck2.22BC

k2.10BNk1.48CN+2.15CP (5)
CDW (g\l) l 0.30+0.05Bk0.03Ck0.04N
k0.02Pk0.04BNk0.02CN
+0.02NPk0.03BCNk0.02BCP (6)

In Figure 4, it can be noted that the increase in buffer and

sugar concentrations had the largest effect on nisin pro-
duction. As there is a significant negative interaction between
these two variables, model (5) predicts that increased
bacteriocin production can be obtained with lower total
sugar concentrations at high initial buffer concentration. In
fact, the increase in B value produced always a relevant
stimulatory effect on nisin synthesis, mainly expressed for
C lk1 (left-hand side parts of Figures 4C and 4D). In
parallel, the increase in sugar concentration inhibited the
bacteriocin production mainly for B l 1 (left-hand side parts
of Figures 4C and 4D), in spite of the fact that this was the
only case in which the increase in glucose produced a slight
improvement in cell growth (right-hand side part of
Figure 4D). De Vuyst and Vandamme [21] reported that on
increasing the sucrose concentration from 10 to 40 g\l,
there was an increase in nisin and biomass production.
Nevertheless, the nisin yield per unit biomass (YB/X) de-
creased from 19.1 to 10.9 mg\g. This was explained by
carbon source regulation of the synthesis or activity of
prenisin-modifying enzymes.
On the other hand, it can be observed that variables P
and N produced slight positive and negative effects on both
bacteriocin and biomass production [model (6) and Figures
4A and 4B]. Therefore neither the stimulant effect of
phosphate in nisin production observed by De Vuyst and
Vandamme [19] nor the inhibitory action of glycine on cell
growth [22] was confirmed for L. lactis. Similar observations
on the void effect of KH2PO4 in nisin Z production by L. lactis Figure 5 Response surfaces obtained for pediocin (Ped) and biomass CDW
production by Ped. acidilactici on the hydrolysed medium, according to the
IO-1 have been described previously [23]. experimental procedure defined in Table 2

B, buffer concentration ; C, total sugar ; N, total nitrogen ; P, total phosphorus ; N,

Joint effects of the buffer, total concentrations of
nitrogen, phosphorus and sugar on pediocin
production
The models developed for both pediocin and biomass CDW
production were satisfactory to explain the influence of the
four input variables (C, N, P and B) on both responses
according to the Fisher F test (α 0.05) and they provided
the following empirical equations :
Ped (BU\ml) l 167.2k76.4Bk11.2Ck17.6Nk17.7P
+6.3BC+18.1BN+5.8BP+9.8CN
k14.3BCNk11.5CNP+5.6BCNP (7)
CDW (g\l) l 0.77k0.04N+0.02Pk0.04BC
+0.02CNk0.01BCP (8)
P, C and B in coded values.
The most representative response surfaces generated from
these models are represented in Figure 5. In eqn (7), it can be
noted that all the independent variables possess negative
sign, but buffer concentration displayed the strongest
negative correlation with pediocin production (left-hand
side parts of Figures 5C and 5D). In fact, an increase in B led
to a dramatic inhibition in pediocin production without
affecting biomass synthesis (right-hand side parts of Figures
5C and 5D). Therefore, at higher values of B, there was no
change in pediocin production with increasing nitrogen,
phosphate and glucose concentrations (left-hand side part of

# 2002 Portland Press Ltd


Figure 5). However, at lower buffer concentrations, a small
decrease in pediocin production was observed as nitrogen
and phosphorus concentrations were increased (left-hand
side part of Figure 5B). The increase in glucose levels only
produced slight positive and negative effects in pediocin
production (left-hand side parts of Figures 5C and 5D), as
expected from its presence in eqn (7) and forming positive
and negative interactions with the rest of the input variables.
On the other hand, the supplements and buffer had a
slight influence on the growth of Ped. acidilactici during the
fermentation [eqn (8) and all response surfaces depicted on
the right-hand side part of Figure 5]. For this reason, it may
be concluded that the action of the variables studied on
pediocin production is not determined by their influence on
the growth of Ped. acidilactici.
Lower pediocin levels recorded at the higher buffer
concentration can be attributed to higher final pH levels
reached in the media. In effect, the highest production of
pediocin was obtained at final pH values less than 4 (for
B lk1). On the other hand, the decrease in bacteriocin
production observed at higher glycine and phosphate
concentrations is most likely caused by nitrogen source
regulation and phosphate control respectively [21,24].
Since lactic acid bacteria are considered to be fastidious
micro-organisms in nutrient requirements and the nutrient
supplementation of hydrolysed media with glucose, glycine
and KH2PO4 did not enhance nisin and pediocin production,
studies based on other nitrogen sources (like cotton-seed
meal, soya-bean meal and fish meal) are needed to enhance
bacteriocin production on this medium.
Acknowledgments
We thank Miguel Anxo Murado (Instituto de Investigacio! ns
Marin4 as de Vigo, CSIC) for his help in the elaboration of this
work. N. P. G. was a recipient of ICI and Xunta de Galicia
fellowships.
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Received 12 April 2002\15 May 2002; accepted 11 June 2002
# 2002 Portland Press Ltd