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 Enzyme and Microbial Technology 41 (2007) 397–406

Fed-batch pediocin production on whey using different feeding media

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Nelson P. Guerra, Paula Fajardo Bernárdez, Lorenzo Pastrana Castro ∗
Departamento de Bioquı́mica, Xenética e Inmunoloxı́a, Facultade de Ciencias de Ourense,
Universidade de Vigo, As Lagoas, 32004 Ourense, Spain

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Received 13 June 2006; received in revised form 16 March 2007; accepted 21 March 2007

Abstract
Pediocin production by Pediococcus acidilactici NRRL B-5627 was followed in both batch and re-alkalized fed-batch fermentations on diluted
whey (DW) supplemented with 2% (w/v) yeast extract (DWYE2 medium). In the first fed-batch culture, the fermentor was fed with a mixture of

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a 400 g/L concentrated lactose and concentrated whey (CW) supplemented with 2% (w/v) yeast extract. The maximum pediocin concentration
obtained was only 1.2-fold higher than in batch fermentation. Since supplementing the DW with increasing amounts of glucose inhibited pediocin
production, a second re-alkalized fed-batch culture was carried by using a 400 g/L concentrated glucose as feeding substrate. The pediocin
concentration obtained was 2.2-fold higher than that in the first fed-batch culture. The third fed-batch fermentation was carried out by feeding the
on
growing culture with a mixture of a 400 g/L concentrated glucose and CW medium supplemented with 4% (w/v) yeast extract. The concentration
of biomass and the volumetric pediocin productivity obtained in this last culture were higher than those obtained in both the batch and the two
fed-batch cultures (P < 0.05). Therefore, the use of feeding substrates containing glucose instead of lactose could be an appropriate alternative for
increasing fed-batch production of pediocin in DWYE2 medium.
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© 2007 Elsevier Inc. All rights reserved.

Keywords: Pediococcus acidilactici; Biomass; Pediocin; Whey; Re-alkalizations; Fed-batch fermentation

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1. Introduction Because of these characteristics, pediocin can potentially be

Bacteriocins are biologically active proteins with antibacte-
rial activity against Gram-positive bacterial species related to
the producer strain. Some have a very narrow spectrum of activ-
r's
used as a non-toxic food preservative to improve the quality,
naturalness and safety of food products [5–7].
To produce the large amounts of bacteriocins needed for stud-
ies of their effectiveness in controlling the growth of undesirable

ity but others have a relatively broad spectrum of antibacterial
activity [1]. In recent years there has been an increased interest
in using broad-spectrum bacteriocins as natural food preserva-
tives and antimicrobial agents in veterinary and pharmaceutical
o
bacteria to preserve functional foods and health products, the
use of cheaper culture media [1] and fermentations techniques
enhancing the growth and bacteriocin concentrations are needed
[7].

areas [2]. Pediocins family, that are produced by Pediococcus Whey, which is a by-product of the dairy industry, has
strains, organisms generally recognized as safe (GRAS), have a been widely used for various productions including organic
th

wide inhibitory spectrum of activity against Gram-positive bac-
teria, including both spoilage and pathogenic organisms, such
as Listeria monocytogenes, Enterococcus faecalis, Staphylococ-
cus aureus and Clostridium perfringens [3]. This bacteriocin
Au
acids, single-cell protein, enzymes, ethanol [8] and bacteriocins
[1,9–14]. With regard to the fermentation technique, it is well
known that fed-batch processes provide higher amounts of bacte-
riocin than batch cultures [7,15,16], especially when the cultures

in combination with other stress inducing processes (such as
heating, freezing, acid treatment, chelating agents, high hydro-
static pressure and electroporation) can also be effective against
Gram-negative or pediocin-resistant Gram-positive bacteria [4].
∗ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001.
E-mail address: pastrana@uvigo.es (L.P. Castro).
were also stressed by periodical re-alkalizations [7,13,17,18].
This fermentation technique has been used to reduce or pre-
vent substrate inhibition [19], carbon catabolite repression [20]
or nutrient depletion [21], phenomena which produce harmful
effects on both cell growth and bacteriocin production [1].
In the present work, the batch production of pediocin by
Pediococcus acidilactici NRRL B-5627 was studied on diluted
whey supplemented with 2% (w/v) yeast extract (DWYE2

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2007.03.009
398 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406
medium). Secondly, the production of pediocin was followed
in a re-alkalized fed-batch culture, using a mixture of a 400 g/L
concentrated lactose and concentrated whey supplemented with
2% (w/v) yeast extract (CWYE2 medium) as feeding substrates.
Subsequently, we evaluated the effect that the addition of other
carbon source (glucose) produces on pediocin production. Thus,
the effects of initial concentrations of glucose and yeast extract
on the batch pediocin production were determined in order to
optimize the composition of the fermentation (DW) medium.
From the results obtained, other two re-alkalized fed-batch cul-
tures were carried out in DWYE2 medium. In the first, the
feeding substrate used was a 400 g/L concentrated glucose. The
last re-alkalized fed-batch culture was fed with a mixture of
concentrated lactose (400 g/L) and concentrated whey supple-
mented with 4% (w/v) yeast extract (CWYE4 medium).
2. Materials and methods
2.1. Bacterial cultures
Pediocin-producing strain Ped. acidilactici NRRL B-5627 was obtained
from the Northern Regional Research Laboratory (NRRL, Peoria, Illinois, USA).
Indicator strain Carnobacterium piscicola CECT 4020 was obtained from the
Spanish Type Culture Collection (CECT). Stock cultures were maintained at
4 ◦ C on agar slants (MRS). Working cultures were grown in MRS broth at 30 ◦ C
and 200 rpm.
rs
2.2. Culture media and fermentation conditions
Whey obtained from a local dairy plant, was used in two forms: as concen-
trated whey (CW: the liquid remaining after the first cheese pressing) and as
pe
diluted whey (DW: CW mixed with wash waters). Before being used as culture
media, both wastes were deproteinized as described previously [1]. The result-
ing diluted whey medium contained (g/L): lactose, 20.06; total nitrogen, 0.45;
total phosphorous, 0.25 and soluble proteins, 2.04. The resulting concentrated
whey medium contained (g/L): lactose, 48.51; total nitrogen, 1.05; total phos-
phorous, 0.43 and soluble proteins, 5.02. Both media were supplemented with
yeast extract, adjusted at pH 7.0, sterilised at 121 ◦ C for 15 min and used as
culture media.
r's
Batch cultures of Ped. acidilactici were performed in 250 mL Erlenmeyer
flasks containing 50 mL of DW medium supplemented with 2% (w/v) yeast
extract (DWYE2 medium), on a rotary shaker (Innova 4330, New Brunswick
Scientific Co., Inc., New Jersey) at 30 ◦ C and 200 rpm, during 52 h. Samples
were withdrawn at regular intervals to perform analytical determinations.
o
Fed-batch fermentations were carried out in a 6 L bench top fermentor (New
Brunswick Scientific, New Jersey) with a 4 L working volume of medium at a
controlled temperature of 30 ◦ C and at a controlled constant agitation of 200 rpm.
th
A constant aeration flow rate of 0.5 L/h was used during the fermentations.
Fed-batch fermentations were carried out as a batch fermentation without
pH-control during the first 12 h of culture, when the lower steady pH was reached.
Then, a sample of 100 mL was taken from the fermentation medium to perform
Au
analytical determinations. After determining the total sugars concentration in
the sample withdrawn, the medium was re-alkalized up to a set pH of 7.0 with
5 M NaOH. Then, the necessary volumes of feeding substrates to restore the
initial total sugars concentration (∼23 g/L) in the fermentation medium were
calculated by applying mass balance equations for the total sugars around the
fermentor. In these equations, the volumes of NaOH added to the fermentor in
each re-alkalization cycle were also taken into account, as indicated below.
In the first re-alkalized fed-batch culture (fed-batch fermentation 1), the
fermentor was fed with a mixture of a 400 g/L concentrated lactose and CW
medium supplemented with 2% (w/v) yeast extract (CWYE2 medium). In the
second fed-batch culture (fed-batch fermentation 2), a 400 g/L concentrated
glucose was used to feed the growing culture. In the third fed-batch culture
(fed-batch fermentation 3), the feeding substrate consisted in a mixture of a
400 g/L concentrated glucose and CW medium supplemented with 4% (w/v)
yeast extract (CWYE4 medium).
These sampling, feeding and re-alkalization strategies were repeated every
12 h until the producer strain was unable to bring about the decrease of pH. The
inoculum in both batch and fed-batch cultures consisted in 2% (v/v) of a 12-h
culture in DWYE2 medium.
2.3. Analytical determinations
The concentrations of biomass (X) total phosphorous (TP), nitrogen (TN),
py
protein (Pr) and sugars (TS) were determined by methods described in a previous
work [13]. Lactose (L), glucose (G), lactic acid (LA), acetic acid (AA) and
ethanol (Et) concentrations were measured by HPLC (Gilson, Inc., USA) using
an ION-300 Organic Acids column (300 mm × 7.8 mm i.d.) with a pre-column
IONGUARDTM (Tecknokroma S. Coop. C. Ltda, Barcelona, Spain) equipped
co
with a RI detector. The eluent consisted of 3 mM H2 SO4 with a flow rate of
0.4 mL/min at (60–65) ◦ C.
2.4. Bacteriocin assay
Culture samples were adjusted to pH 3.5 and then heated for 3 min to kill
the cells. Subsequently they were centrifuged at 27,200 × g for 15 min at 4 ◦ C
al
to obtain the cell-free supernatants containing the total antibacterial activity.
Pediocin activity was determined using 3 mL of convenient dilutions of the
cell-free supernatants in sterile and distilled water and 3 mL of an overnight
culture of the sensitive strain (C. piscicola) previously adjusted to an optical
on
density of 0.2 at 700 nm. Inoculated tubes were incubated at 30 ◦ C with an
agitation speed of 200 rpm for 6 h, after which, the optical density of each tube
was recorded. Dose–response curves were constructed from these data. One
activity unit (BU) per millilitre was expressed as the inverse of the dilution that
produced a 50% growth inhibition (inhibitory dose 50), which was obtained
from the dose-response curves [13,22].
2.5. Effects of the carbon (glucose) and nitrogen (yeast extract)
sources on the batch production of pediocin
A multifactorial composite rotatable design [23] based on five levels and two
variables was used to study the combined effect of the glucose and yeast extract
concentrations on the bacteriocin and biomass productions by Ped. acidilactici.
The designs consisted of 13 experiments with four (22 ) factorial points, four axial
points to form a central composite design with α = 1.267 and five centre points
for replication. Response surfaces were depicted from the empirical equations
derived of design. The range and coding of the two variables are shown in Table 1.
2.6. Mass balance equations in the re-alkalized fed-batch
fermentations
In this work, the volume of the fermentation medium (V) in the fed-batch
fermentations was maintained constant ((dV/dt) = 0) by matching the volumes
Table 1
Experimental domain and codification of the variables used in the experimental
design analysis for the combined influence of glucose (G) and yeast extract (YE)
concentrations on pediocin production in DW medium
Codified values Natural values
G (g/L) YE (g/L)
−1.267 0.00 0.00
−1 1.05 2.10
0 5.00 10.00
+1 8.95 17.89
+1.267 10.00 20.00
Increments
1 3.95 7.89
 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406 399

added to the fermentor (feeding volume (VF) plus the volume of NaOH 5 M) ( [Sadded ]) to the fermentation medium with the feeding substrates were cal-
with the sampling volume (VS): culated as follows:
 VStn · [S]tn
VStn−1 = VFtn + VNaOHtn = (VCWtn + VCStn ) + VNaOHtn (1) [Sext ] = + [Sext ]tn−1 (10)
tn V
where VCW and VCS are, respectively, the volumes (in L) of concentrated whey
 VCW · [SCW ] + VCS · [SCS ]
(CWYE2 or CWYE4) and concentrated solutions (CS) of lactose (CL) or glucose [Sadded ]tn = + [Sadded ]tn−1 (11)
(CG) added to the fermentor at the beginning of each feeding cycle. VNaOH V
is the volume (in L) of NaOH 5 M added to the fermentor for re-alkalizing the
where [S]tn is the nutrient concentration (in g/L) in the fermentor at the end of
medium up to the initial pH value of 7.0. The sum of the volumes of feeding
each feeding cycle.
substrates that must be added for restoring the initial total sugars (TS) in the

py
The accumulated concentrations  of products (biomass, lactic acid, acetic
fermentation medium are
acid, ethanol and pediocin) extracted ( [Pext ]) at the end of each feeding cycle
VCWtn + VCStn = VStn−1 − VNaOHtn (2) were calculated by the following mass balance equations:
 VStn · [P]tn
from which it follows that [Pext ]tn = + [Pext ]tn−1 (12)

co
V
VCWtn = VStn−1 − VNaOHtn − VCStn (3)
where [P]tn is the concentration of product (in g/L) at the end of each feeding
The reduction in the amounts (in g) of TS in the medium due to the joint effect cycle. 
of the extraction of samples and the consumption of TS by the growing strain Then, the accumulated concentrations of products formed ( [P]) at the
(TSC+E ) can be calculated by applying a mass balance equation for the total end of each feeding cycle were calculated as the sum of the concentrations of
sugars: product synthesized at the end of each feeding cycle and the total amounts of
products extracted in the previous feeding cycle:
TSC+E = (Vtn−1 · [TS])t − (Vtn−1 − VStn )[TS]tn (4) 

al
n−1 VStn−1 · [Pext ]tn−1
[P]tn = + [P]tn−1 (13)
where [TS]tn−1 and [TS]tn are the total sugars concentration (in g/L) at the V
beginning and at the end of each feeding cycle. The difference (Vtn−1 − VStn )
represents the remaining volume (in L) in the fermentor after the extraction of
on
2.7. Statistical analyses
samples.
Therefore, the amounts of TS that must be added to the fermentor to restore Individual experiments were performed in triplicate. All data points are rep-
the initial TS concentration in the fermentation medium can be calculated by resented by the mean, with the standard deviations indicated by error bars. The
the following expression: differences between means of biomass, pediocin and metabolites (lactic acid,
rs

acetic acid and ethanol) productions in batch cultures and in the three re-alkalized
VCW · [TSCW ] + VCS · [TSCS ] = TSC+E (5) fed-batch fermentations were determined by analysis of variance (ANOVA) on
SPSS 8.0 for Windows (Statsoft, Inc., 2001).
where [TSCW ] and [TSCS ] are the total sugars concentration in the CW media
pe

(CWYE2 or CWYE4) and in the concentrated solutions of lactose (CL) or

glucose (CG), respectively. 3. Results
Substituting Eq. (3) into Eq. (5) gives

(VStn−1 − VNaOHtn − VCStn ) · [TSCW ] + VCStn · [TSCS ] = TSC+E (6) 3.1. Batch culture of Ped. acidilactici NRRL B-5627 in
DWYE2 medium
Thus, the VCS can be calculated as
TSC+E − VS · [TSCW ] + VNaOH · [TSCW ] Before starting fed-batch fermentations, a simple batch fer-
VCStn = (7)
[TSCS ] − [TSCW ]
r's

mentation in DWYE2 medium was performed to obtain data

Now, the VCW can be obtained by introducing the values of VCS and VNaOH for comparison. Results are shown in Fig. 1. Pediocin was pro-
into Eq. (3). duced during the exponential growth phase, so that, its synthesis
 the accumulated concentrations of substrates (TS, TN,
On the other hand, would be considered growth linked as occurred for other bacte-
TP, Pr) consumed ( [Scons ]) in each re-alkalization cycle were calculated by riocins [18,24,25]. The maximum values for biomass (1.42 g/L)
o

using the following mass balance equation:

and pediocin (189 BU/mL) concentrations reached at the end of
(Vtn−1 − VStn ) · [S]tn−1 + (VCW · [SCW ]tn−1 ) the fermentation, while still significantly higher (P < 0.05) than
th

 + (VCS · [SCS ]tn−1 ) − (Vtn−1 · [S]tn )tn in batch fermentation in DW medium (0.17 g/L and 58 BU/mL)
[Scons ]tn = + [Scons ]tn−1 [1], were lower than the levels obtained in MRS broth (1.76 g/L
V
(8) and 493 BU/mL) [26].
Au

The pH decreased rapidly after 8 h of incubation in concomi-

where [S]tn−1 and [S]tn are the nutrient (TS, TN, TP and Pr) concentrations (in
g/L) in the fermentor at the beginning and at the end of each feeding cycle,
tance with the rapid increase in the productions of biomass and
respectively. [SCW ]tn−1 and [SCS ]tn−1 are the nutrient (TS, TN, TP and Pr) con- lactic acid. This fact presumably influenced the production of
centrations (in g/L) in the feeding substrates (CWYE2 or CWYE4 and CL or the bacteriocin, which also increased rapidly in the first 8 h of
CG). incubation, decreasing afterwards in parallel with the decrease
Since the values for VS, VCW and VCS are zero in the first feeding cycle, the in the rates of biomass production and pH drop (Fig. 1). Acetic
concentrations of nutrients consumed in this period were calculated as follows:
acid concentration increased linearly throughout the fermenta-
V ([S]0 h − [S]12 h ) tion and reached a maximum level of 1.80 g/L at the end of
[Scons ]12 h = (9)
V the culture. However, lactic acid and ethanol concentrations
 increased in parallel with the increase in biomass concentra-
The accumulated concentrations of nutrient extracted ( [Sext ]) from the fer-
mentation medium as well as the accumulated concentrations of nutrient added tion, and reached values of 4.12 and 1.30 g/L, respectively, after
400 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406
rs
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Fig. 1. Time course of the batch culture of Ped. acidilactici NRRL B-5627 on
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DWYE2 medium. X: biomass concentration; Ped: pediocin concentration; LA:
lactic acid; Et: ethanol; AA: acetic acid; TS: total sugars; TP: total phosphorous;
TN: total nitrogen; Pr: proteins; t: time. Data reported are means ± standard
deviations of three replicates.
o
52 h of incubation. This prevailing mixed acid fermentation pat-
tern is a phenomenon that has been observed before in cultures
th
under carbon-excess conditions with lactose [13,27], galactose
[27] and maltose [28]. High concentrations of nutrients (total
sugars, phosphorous, nitrogen and protein) remained at the end
Au
of the culture, although they were intensively consumed in the
first 12 h of incubation (Fig. 1).
3.2. Re-alkalized fed-batch culture of Ped. acidilactici on
DWYE2 medium with feeding with CWYE2 medium and a
400 g/L concentrated lactose (fed-batch fermentation 1)
Since fed-batch and re-alkalized fed-batch cultures yielded
higher levels of bacteriocins than in batch fermentations
[15–18], the fed-batch production of pediocin on whey supple-
mented with a growth-stimulating nitrogen source (like yeast
py
co
al
on
Fig. 2. Time course of the re-alkalized fed-batch culture of Ped. acidilactici
NRRL B-5627 on DWYE2 with feeding with CWYE2 medium and a 400 g/L
concentrated lactose (fed-batch fermentation 1). X, Ped, LA, Et, AA: concentra-
tions of biomass, pediocin, lactic acid, ethanol and acetic acid accumulated (),
remaining () and extracted (♦); Pr, TS, TP, TN: concentrations of proteins,
total sugars, phosphorous and nitrogen supplemented (), remaining (), con-
sumed () and extracted (♦). Data reported are means ± standard deviations of
three replicates.
extract) should offer a good chance to increase the growth and
pediocin production by Ped. acidilactici.
The growth kinetic of Ped. acidilactici in the first fed-batch
and re-alkalized culture (fed-batch fermentation 1) on DWYE2
medium fed with a mixture of CWYE2 medium and a concen-
trated lactose (400 g/L) is shown in Fig. 2. The concentrations
of lactic acid, acetic acid and ethanol peaked at about 4.76, 2.22
and 1.41 g/L, respectively, by 48 h of incubation. From a kinetic
point of view, it can be noted that pediocin production increased
quickly in the first re-alkalization period in parallel with the
rapid increase in biomass production and the highest pH drop
generated in the culture. However, from the 12 h of fermentation,
pediocin synthesis rate began to decrease in concomitance with
the decrease in both the biomass production and pH drop rates.
This suggested that the pediocin was produced as a pH depen-
dent primary metabolite in this culture [7,26]. In addition, after
60 h of fermentation, the pediocin-producing bacterium com-
pletely lost its capacity to recover the acidic pH at the end of
 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406 401

each re-alkalization cycle. The nutrients (total sugars, nitrogen,
phosphorous and proteins) consumption rates were also higher in
the first 12 h of incubation, decreasing progressively afterwards.
After 24 h of incubation, the concentrations of biomass and
bacteriocin reached values of 1.45 g/L and 182 BU/mL, which
represent the 80% and the 82% of the total amounts of biomass
(1.82 g/L) and pediocin (223 BU/mL) produced at the end of
the culture. In fact, the biomass and pediocin concentrations
obtained at the end of this fed-batch culture were only 1.28 and
tion medium (DW) was optimized by determining the optimal
concentrations of glucose or yeast extract needed to supple-
ment the medium. Therefore, the joint effects of initial levels
of glucose and yeast extract on biomass (X) and pediocin (Ped)
production were determined in batch fermentations by using
response surfaces methodology and empirical modelling.
Table 2 summarizes the responses for biomass and pediocin
production along with the final pH values reached in the cultures.
The model equations fitted by regression analysis are given by

py
1.18 times higher than those obtained in the previous batch fer-
mentation (P < 0.05). From a practical point of view, these results X (g/L) = 0.955 − 0.096G + 0.381YE − 0.088YE2 (14)
indicated the convenience of stopping the culture after 24 h of
incubation, since the lower amounts of biomass and pediocin Ped (BU/mL) = 125.62 − 12.65G + 54.04YE − 5.57YE2

obtained from this period do not justify the extension of the
incubation period. Thus, the low ability of Ped. acidilactici to
ferment the lactose [1,9,12,29] presumably led to the collapse
of the culture (after 60 h of fermentation), thus counteracting the
stimulant effect that the supplements with yeast extract produced
on biomass and pediocin production.
co
(15)
In both cases, the model terms G, YE and YE2 were found
to be significant according to the Student t-test (α = 0.05)
and P-values, meanwhile the interaction term between G and

3.3. Joint effect of glucose and yeast extract concentrations
on growth of Ped. acidilactici on DW medium in batch
on
cultures
The results obtained in the previous culture suggested
the convenience of using other carbon source, like the glu-
rs
al
YE (G·YE) and the quadratic term for G (G2 ) were found
to be non-significant in both equations (Table 2). Since the
P-values in the ANOVA table (Table 3) are less than 0.05 for
both models, there is a statistically significant relationship
between the response variables and the independent variables
at the 95% confidence level. The high value of the adjusted
determination coefficient (adj r2 = 0.993) for both models

cose to enhance the growth and pediocin productions on (Table 3) also indicated their high significance. All of the above
whey. For designing an adequate strategy of fermentation to considerations indicate an excellent adequacy of the quadratic
enhance pediocin production, the composition of the fermenta- models to the experimental data.
pe

Table 2
Values of X (biomass concentration) and Ped (pediocin concentration) and final pH and significance of the model coefficients obtained according to the experimental
design defined in Table 1
G YE X (g/L) Ped (BU/mL) Final pH

1 1 1.19 163.22 5.03

1 −1 0.39 51.64 6.35
r's

−1 1 1.37 180.54 4.73

−1 −1 0.55 76.96 6.08
1.267 0 0.81 107.06 5.66
−1.267 0 1.09 144.60 5.18
0 1.267 1.25 186.77 4.92
o

0 −1.267 0.36 49.46 6.40

0 0 0.96 125.68 5.41
th

0 0 0.95 124.82 5.42

0 0 0.99 129.66 5.36
0 0 0.93 122.16 5.45
0 0 0.95 124.64 5.42
Au

Biomass model (Eq. (1)) Bacteriocin model (Eq. (2))

Coefficient ± S.E.a t(7) P-value Coefficient ± S.E.a t(7) P-value

Significance of factors
Constant 0.955 ± 0.015 61.68 0.000000b 125.62 ± 1.45 86.65 0.000000b
G −0.096 ± 0.013 −7.45 0.000143b −12.65 ± 1.22 −10.27 0.000018b
YE 0.381 ± 0.013 29.25 0.000000b 54.04 ± 1.22 44.28 0.000000b
G·YE −0.004 ± 0.017 −0.26 0.804857 2.00 ± 1.64 1.22 0.261134
G2 0.002 ± 0.015 0.12 0.904179 −0.78 ± 1.44 −0.54 0.606144
YE2 −0.088 ± 0.015 −5.75 0.000696b −5.57 ± 1.44 −3.87 0.006102b
a Standard error.
b Significant at P < 0.05.
402 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406

Table 3
Summarized data of analysis of variance (ANOVA) of the empirical models obtained for biomass and bacteriocin production according to the experimental design
defined in Table 1

Source SSa dfb MSc F-value P-value r2 adj r2

Biomass production model (Eq. (1))

Regression 1.15 4 0.287 192.47 0.000001d 0.993 0.990
Residual 0.01 8 0.001
Total 1.16 12

py
Bacteriocin production model (Eq. (2))
Regression 21954.68 4 5488.67 102.57 0.000001d 0.993 0.990
Residual 428.08 8 53.51
Total 22382.76 12

co
a Sum of squares.
b Degrees of freedom.
c Mean of squares.
d Significant at P < 0.05.

The response surfaces obtained from the empirical mod- bacteriocin (494 BU/mL) concentrations by 2.0- and 2.2-fold
els (14) and (15) are depicted in Fig. 3. Maximum biomass higher (P < 0.05) than the previous re-alkalized fed-batch fer-

al
(1.42 g/L) and pediocin (201 BU/mL) productions were obtained mentation (Fig. 2). The concentrations of biomass, pediocin
for the highest concentration of yeast extract (YE = 20 g/L) and and nutrients described waved profiles (similar than those of
the lowest concentration of glucose (G = 0). Although glucose
on
is considered to be a better carbon source than lactose for Ped.
acidilactici [2], supplementing the DW medium with glucose
led to an inhibition in both biomass and bacteriocin production.
These results indicated not only that the optimum fermentation
rs

medium was the DWYE2 without glucose supplements but also,

the convenience of adding the supplements with glucose in the
feeding media.
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3.4. Re-alkalized fed-batch culture with feeding with a

400 g/L concentrated glucose (fed-batch fermentation 2)

Taking into account the results obtained in the previous exper-

iment, a new fed-batch fermentation was carried out using the
DWYE2 medium as the fermentation substrate and a 400 g/L
r's

concentrated glucose as the feeding medium (fed-batch fermen-

tation 2). With this approach, glucose could be supplemented at
regular intervals, keeping low its concentration in the fermenta-
tion medium, thus, reducing its inhibition effect on biomass and
o

bacteriocin production.
The results obtained (Fig. 4 and Table 4) showed that the
th

feeding with glucose improved both the biomass (3.48 g/L) and
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Fig. 4. Time course of the re-alkalized fed-batch culture of Ped. acidilactici

Fig. 3. Response surfaces plot showing the effect of glucose (G) and yeast extract
(YE) concentrations on the production of biomass (X) and pediocin (Ped) by Ped.
acidilactici on DW medium.
on DWYE2 with feeding with a 400 g/L concentrated glucose (fed-batch fer-
mentation 2). X, Ped, LA, Et, AA: concentrations of biomass, pediocin, lactic
acid, ethanol (open symbols) and acetic acid (closed symbols) accumulated (),
remaining () and extracted (♦). G, L, Pr, TP, TN: concentrations of glucose,
lactose, proteins, total phosphorous and nitrogen supplemented (), remain-
ing (), consumed () and extracted (♦). Data reported are means ± standard
deviations of three replicates.
 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406 403

Table 4
Growth and fermentation parameters obtained in batch and re-alkalized fed-batch (1, 2 and 3) fermentations of Ped. acidilactici in DW, MRS and DWYE2 media at
the end of the fermentations
Parameters Batch cultures Re-alkalized fed-batch cultures

DW MRS DWYE2 (1) (2) (3)

Biomass (g/L) 0.17 1.76 1.42 1.82 3.48 8.63

Pediocin (BU/mL) 58 493 189 223 494 712
Lactic acid (g/L) 1.12 4.27 4.12 4.76 45.40 56.45

py
Acetic acid (g/L) 0.35 ND 1.80 2.22 6.49 8.88
Ethanol (g/L) 0.16 ND 1.30 1.41 13.36 17.06
YPed/TSc 22.85 75.18 25.15 6.34 3.32 6.45
YPed/TNc 1050.90 601.47 163.74 200.43 239.2 165.66
YX/TSc 0.07 0.27 0.19 0.05 0.02 0.08

co
YX/TNc 3.04 2.16 1.23 1.61 1.74 2.27
YPed/X 340.00 280.23 133.18 122.54 141.95 82.50
ETS 0.10 0.34 0.32 0.57 0.87 0.77
ETN 0.12 0.27 0.48 0.37 0.82 0.72
ETP 0.41 0.19 0.40 0.36 0.42 0.45

YPed/TSc , YPed/TNc , and YPed/X are the pediocin yield coefficients (amounts of pediocin (Ped) produced (BU) per g of total sugars (TSc) or nitrogen (TNc) consumed
and biomass (X) produced). YX/TSc and YX/TNc are the biomass yield coefficients (g of X per g of TSc or TNc). ETS , ETN and ETP are the efficiencies (g of nutrients
(total sugars, nitrogen or phosphorous (TP)) consumed per g of nutrients supplied). ND: not detected.

a diauxic growth pattern), which had been observed in previous

al
veniently added to restore the initial total sugars concentration
fed-batch cultivations with some Lactococcus lactis subsp. lac-
on in the fermentation medium.
tis strains [13,17,18] and Ped. acidilactici NRRL B-5627 [7,18]. The results obtained in this fed-batch culture are shown in
The active period (204 h) and the concentrations of lactic acid Fig. 5 and Table 4. As it can be seen, the joint feeding with
(45.40 g/L), acetic acid (6.49 g/L) and ethanol (13.36 g/L) were glucose and CWYE4 medium reduced the lactose consumption,
higher than those obtained in the first fed-batch fermentation. thus, contributing to an accumulation of this carbon source in
rs

Glucose consumption was rapid in this culture and residual glu- the fermentation medium (Fig. 5). Increased amounts of biomass
cose dropped to very low values between feedings. Initial lactose (8.63 g/L), pediocin (712 BU/mL), lactic acid (56.45 g/L), acetic
concentration tended to decrease upon extended incubation as a acid (8.88 g/L) and ethanol (17.06 g/L) were obtained at the end
pe

consequence of a low consumption by Ped. acidilactici and the
successive extraction of samples every 12 h.
The feeding with glucose also increased (P < 0.05) both
the efficiencies (ETS , ETN and ETP ) in nutrient (total sug-
ars, nitrogen and phosphorous) consumptions and the yield
coefficients YPed/X , YX/TNc and YPed/TNc with respect to the for-
mer re-alkalized fed-batch fermentation (Table 4). However, a
r's
of the fermentation. On the other hand, the pediocin-producing
strain was able to bring about the decrease of pH for a period of
time longer than that observed in the fed-batch fermentations 1
and 2. In addition, higher values of the yields YPed/TSc , YX/TNc
and YX/TSc and lower values of the yields YPed/TNc and YPed/X
were obtained in this culture as compared with the two previ-
ous fed-batch cultivations. Although the efficiencies (ETS , ETN

decrease in the yields YPed/TSc and YX/TSc was observed. At the and ETP ) obtained in this culture were higher than in the first
end of the culture both the nitrogen and proteins were almost fed-batch fermentation, the efficiencies ETS and ETN obtained
depleted. were lower than those observed in the second fed-batch fer-
mentation (Table 4). However, after 312 h of incubation, the
o

3.5. Re-alkalized fed-batch culture with feeding with a concentration of pediocin obtained was 1.45-fold higher than
mixture of concentrated whey supplemented with 4% yeast the concentration obtained in the second fed-batch culture. In
th

extract and a 400 g/L concentrated glucose (fed-batch addition, high amounts of proteins (∼3.3 g/L) not consumed by
fermentation 3) the pediocin-producing strain remained in the medium at the end
of the culture.
Au

In an attempt to continue increasing pediocin production, a
third fed-batch fermentation was carried out. In this case, the cul-
ture was fed with a mixture of substrates composed by the same
concentrated glucose (400 g/L) used in the previous culture and
a CW medium supplemented with a yeast extract concentration
(4%) double (CWYE4 medium) that used in the first fed-batch
fermentation. This last approach could contribute to improve
both the biomass and pediocin productions by supplementing
the fermentation medium with amounts of glucose, nitrogen,
vitamins or minerals [26] higher than those supplemented in the
first fed-batch cultivation. Thus, the feeding substrates were con-
From the comparison between the three re-alkalized fed-
batch fermentations, it can be noted that the volumetric pediocin
productivities obtained in each re-alkalized fed-batch culture
were different (P < 0.05). Thus, the pediocin productivities after
72 h of fermentation (when the first fed-batch culture stopped),
were calculated as 3.09, 3.73 and 3.75 BU mL−1 h−1 , for the fed-
batch fermentations 1, 2 and 3, respectively. In the same way,
the volumetric pediocin productivity obtained at the end of the
second fed-batch culture (240 h) was 2.04 BU mL−1 h−1 , which
was lower than that (2.64 BU mL−1 h−1 ) obtained in the third
fed-batch culture (P < 0.05) at the same period of incubation.
404 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406
rs
Fig. 5. Time course of the re-alkalized fed-batch culture of Ped. acidilactici
on DWYE2 with feeding with CWYE4 medium and a 400 g/L concentrated
pe
glucose (fed-batch fermentation 3). X, Ped, LA, Et, AA: concentrations of
biomass, pediocin, lactic acid, ethanol (open symbols) and acetic acid (closed
symbols) accumulated (), remaining () and extracted (♦). G, L, Pr, TP, TN:
concentrations of glucose, lactose, proteins, total phosphorous and nitrogen sup-
plemented (), remaining (), consumed () and extracted (♦). Data reported
are means ± standard deviations of three replicates.
r's
The pediocin productivity in this last culture decreased after pro-
longed incubation, reaching a final value of 2.28 BU mL−1 h−1
after 312 h of fermentation. From the detailed observation of the
cultures, it can be noted that the observed progressive reduction
in pediocin productivities with the increase in the incubation
o
time, was concomitant with the decrease in the pH drop rate
(Figs. 1, 2, 4 and 5).
th
4. Discussion
Au
Pediocin was produced during the active growth phase in the
batch culture of Ped. acidilactici on DWYE2 medium, in which
the highest pH drops were produced. This indicated that the vol-
umetric bacteriocin production depended on both the biomass
production and the pH drop generated in the culture. Further-
more, both the biomass and pediocin production rates began
to decrease after 8 h of incubation. Neither bacteriocin autoin-
hibition nor the accumulation of by-products with antibacterial
activity (lactic and acetic acid and ethanol) seems to be the causes
for the cessation of growth, because the producing strains pos-
sess specific immunity to their own bacteriocin [30–32] and the
amounts of metabolites produced were lower than those consid-
ered damaging for the growth of the pediocin-producing strain
[7].
The presence of high amounts of total sugars in the fermen-
tation medium at the end of the fermentation (16 g/L), indicated
that another factor (e.g. some compounds such as amino acids,
peptides, vitamins or cations supplied with the yeast extract)
became growth limiting or that the growth was severely inhibited
by the final pH of about 4.6 reached at the end of the fermen-
py
tation. According to some investigators [33–35], the optimum
pH for nutrient transport usually varies between 6.0 and 6.5,
decreasing rapidly at higher or lower pH values. For this reason,
the failure to growth at acidic pH is likely to be caused by a
co
limitation of cytoplasmic processes such as acidification of the
cytoplasm and the collapse of the motive force [33–35].
A possible alternative to solve this problem could be the use
of a batch fermentation at a controlled constant pH of 7.0, how-
ever, this could inhibit the production of pediocin due to the
need of low pH values (or high pH drops) for high pediocin
al
productions [7,36,37]. For this reason, other alternative was
evaluated to enhance the bacteriocin levels. This consisted in
extending the time that the culture is maintained in a metabol-
on
ically active period by converting the batch fermentation into
repeated fed-batch fermentation and stressing the culture with
periodical re-alkalizations [7,13,17,18].
The first fed-batch cultivation was carried out on DWYE2
medium using a mixture of CWYE2 medium and a 400 g/L con-
centrated lactose as feeding media. However, the biomass and
pediocin concentrations obtained at the end of the culture (Fig. 2)
were only slightly higher than in batch culture on the same fer-
mentation medium (Fig. 1). This indicated that the additional
supplements of lactose and yeast extract with the feeding media
did not significantly improve the biomass and pediocin produc-
tion by Ped. acidilactici in a medium (DW) containing lactose
as a main carbon source. Thus, the following alternative was to
supplement the DW medium with glucose, which is a better car-
bon source than the lactose for the Pediococcus species [1,9,12].
Due to the possible existence of interactions between the carbon
and the nitrogen sources [1,2], the joint effect of the initial con-
centrations of glucose and yeast extract on biomass and pediocin
productions was studied using a composite rotatable design.
The increase in glucose concentration inhibited both the
growth and pediocin production, in contrast with the stimulant
effect that the increase in yeast extract concentration produced
on biomass and pediocin synthesis (Fig. 3). The inhibitory
effect of increasing glucose concentrations on pediocin pro-
duction could be due to substrate inhibition [19] or due to the
carbon source regulation involved in the bacteriocin biosynthe-
sis [20]. A similar inhibitory effect by the carbon source on
biomass and pediocin production was observed when the DW
medium was supplemented with increasing concentrations of
lactose [1].
The second fed-batch fermentation was then performed using
the DWYE2 medium without glucose supplements as fermen-
tation medium, being supplemented the glucose in the feeding
medium (a 400 g/L concentrated glucose). The higher concen-
trations of pediocin obtained in this culture in comparison with
 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406
the first fed-batch culture (P < 0.05) indicated that the strategy
of feeding the cultures with glucose is an effective way for
enhancing pediocin production in DWYE2 medium.
The increase in the yield coefficients (YPed/TNc , YX/TNc and
YPed/X ) in the second fed-batch fermentation as compared with
the first fed-batch culture, indicated a more efficient utilization
of the nitrogen source for biomass and pediocin production as
well as the production of a more productive biomass. On the
other hand, the decrease in the yields YPed/TSc and YX/TSc was
probably due to a higher total sugars consumption to produce
the increased amounts of mixed acid metabolites (lactic acid,
acetic acid and ethanol) obtained in this culture in comparison
with the previous fed-batch fermentation.
Taking into account these observations, the new strategy was
focussed on feeding the growing culture with other mixture of
substrates, which consisted in the same concentrated glucose
(400 g/L) and the CW medium supplemented with a yeast extract
concentration two-fold higher than that used in the first fed-batch
culture. The main objective of this procedure was to increase
the positive effect that the feeding with glucose produced on
biomass and pediocin production. Using this approach, the active
period and the productions of biomass, pediocin, lactic acid,
on
acetic acid and ethanol were higher than those obtained in the
first and second fed-batch fermentations (P < 0.05). However,
supplementing the CW medium with a high yeast extract con-
centration (40 g/L) led to an accumulation of proteins at the end
rs
of the culture. On the other hand, the increase in the amounts of
bacteriocin obtained in the third fed-batch culture as compared
with the second, did not seem to be as sufficiently high to justify
the use of high levels of yeast extract in the CW medium. From
pe
a practical point of view, the use of the second fed-batch alter-
native (fed-batch fermentation 2) could be not only an effective
way of producing high amounts of pediocin at a low produc-
tion cost, but also an alternative to reduce the initial amounts
of nutrients in DWYE2 medium. This could be considered as
a way for pretreating the whey before being discharged to the
r's
environment.
In general, both biomass and pediocin concentrations in the
fed-batch cultivations 2 and 3 were higher than those obtained
in batch cultures in DW [1] and DWYE2 media, although the
amount of pediocin reached in the MRS medium [26] was only
o
surpassed in the third fed-batch fermentation. The amounts of
sugars consumed in the fed-batch fermentations 2 and 3 were
th
much higher than in batch fermentations, so that the yields
of biomass and pediocin from substrate consumed (YX/TSc and
YPed/TSc ) were lower. This suggests that a significant amount
Au
of the carbon source must be used for cell maintenance or for
metabolic processes that do not yield biomass and pediocin.
Although the biomasses produced in the batch cultures in DW
and MRS media were more productive than those produced in
the fed-batch fermentations 2 and 3, these last cultures were
characterized with higher efficiencies (ETS , ETN and ETP ) with
regard to the nutrients consumption.
Prolonged incubation led to a further decrease in both the
pH drops and the volumetric pediocin productivities in the three
re-alkalized fed-batch cultures on DWYE2 medium. This reduc-
tion in the pH drops with the incubation times is a common
405
phenomenon, which has been observed in other re-alkalized
fed-batch cultures for the production of pediocin and other
bacteriocins [7,13,17,18,22]. Since higher pH drops favoured
the production of pediocin [7,36,37], the observed progressive
reduction in the pH drops (Figs. 2, 4 and 5) probably led to a
decrease in the volumetric pediocin productivities. Although the
re-alkalized fed-batch fermentation 3 was found to be the most
productive process, the final pediocin productivities obtained
in this culture were significantly lower (P < 0.05) than those
py
obtained in two re-alkalized fed-batch cultures in a mussel pro-
cessing wastes (MPW, ∼5.3 g of glucose/L) medium [7]. In
these last cultures, which were respectively fed with a 240 g/L
concentrated glucose and with a concentrated MPW medium
co
(∼100 g of glucose/L), the volumetric pediocin productivities
obtained were 9.23 and 11.63 BU mL−1 h−1 after 112 and 120 h
of fermentation, respectively.
The possible reason by which the pediocin production rates
in the re-alkalized fed-batch cultures in DWYE2 medium were
lower than those observed in the MPW cultures [7] could be
al
due to the poor utilization of the lactose present in the DW
medium by the Ped. acidilactici strain [1]. Therefore, high levels
of lactose remained in the fermentation medium at the end of
each re-alkalization cycle. Consequently, low amounts of glu-
cose and/or yeast extract were added with the feeding substrates
to the growing culture in each feeding cycle, thus, limiting the
growth and pediocin production in DWYE2 medium.
5. Conclusions
The main contribution of this research is the development
of an adequate strategy for producing high concentrations of
biomass, pediocin and other antimicrobial metabolites (lactic
acid, acetic acid and ethanol) by using fed-batch fermentation
techniques and a food waste like whey as a base of the cul-
ture media. However, the results obtained showed that these
productions in DW medium could be only increased by: (i)
supplementing the DW medium with yeast extract and (ii)
employing a well-designed re-alkalized fed-batch fermentation
using feeding substrates containing glucose instead of lactose.
With this approach, the concentrations of biomass, pediocin,
lactic acid, acetic acid and ethanol obtained were significantly
higher than those obtained in the conventional batch cultures
in the same fermentation medium (P < 0.05), as it was also
observed by other researchers [7,13,15–17].
The culture of Ped. acidilactici obtained from the fed-batch
fermentation with feeding with glucose (fed-batch fermentation
2) are being assayed in our laboratory for two important potential
applications: (i) as a probiotic additive in feeds for piglets and
chickens in an attempt for minimizing antibiotic use and (ii) to
produce active antimicrobials films by adsorbing the pediocin
on food contact surfaces to develop suitable food packaging
materials.
Acknowledgements
The research presented in this paper was financially supported
by the Instituto Nacional de Investigación y Tecnologı́a Agraria
406 N.P. Guerra et al. / Enzyme and Microbial Technology 41 (2007) 397–406
y Alimentaria (INIA), Spain (project CAL01-045-C2-2) and The
Xunta de Galicia, Spain (project PGIDT00BIO1E).
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