This article appeared in a journal published by Elsevier.

The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
 Author's personal copy

Biochemical Engineering Journal 56 (2011) 94–106

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Enhancement of enterocin A production by Enterococcus faecium MMRA and

determination of its stability to temperature and pH
Amel Rehaiem a , Nelson Pérez Guerra b,∗ , Zouhaier Ben Belgacem a , Paula Fajardo Bernárdez b ,
Lorenzo Pastrana Castro b , Mohamed Manai a
a
Laboratoire de Biochimie et Biologie Moléculaire, Faculté des Sciences de Tunis, Campus universitaire, Tunis, Tunisia
b
Departamento de Química Analítica y Alimentaria, Facultade de Ciencias de Ourense, Universidade de Vigo, As Lagoas s/n, 32004 Ourense, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The growth, nutrient (total sugars, phosphorous, nitrogen and proteins) consumption and product for-
Received 10 February 2011 mation by Enterococcus faecium MMRA in de Man, Rogosa and Sharpe (MRS) broth was followed in
Received in revised form 16 May 2011 non-realkalized batch, realkalized batch and realkalized fed-batch cultures. In the latter fermentation,
Accepted 21 May 2011
the growing culture was fed with a mixture of MRS medium (20 g of glucose/L) and a 400 g/L concentrated
Available online 27 May 2011
glucose. In the three cultures, a typically homolactic fermentation (only lactic acid was produced) was
observed. The realkalized fed-batch culture was mainly characterized with higher biomass (2.4 g/L), lactic
Keywords:
acid (32.5 g/L) and enterocin A (35.9 AU/mL) concentrations compared with the two batch processes.
Enterococcus faecium MMRA
Enterocin A
Mathematical models were developed to describe the productions of biomass, enterocin A and lactic
Realkalized batch acid in the two realkalized cultures. The growth and lactic acid production were successfully modelled
Fed-batch with the Monod and the Luedeking and Piret models, respectively. Enterocin A was modelled by using a
Fermentation modified form of the Luedeking and Piret model, which includes a term for the effect of the pH drop rate
Modelling on bacteriocin synthesis. Furthermore, the maximum enterocin A stability was obtained at temperatures
Temperature below 100 ◦ C, at acidic pH values and short incubation times.
pH © 2011 Elsevier B.V. All rights reserved.

1. Introduction rocin A – producing strains in preservation strategies of fermented

Enterococci are associated with several fermented foods, espe-
cially artisanal dairy products from Mediterranean countries. In
these products, enterococci are thought to play an important role in
flavour quality and aroma development, and hence these bacteria
may be applied as starter or bioprotective culture [1–3]. Enterococci
are known to produce enterocins, which are bactericidal peptides
or proteins antagonistic against bacteria genetically closely related
to the producer strain [4], including food spoilage and pathogenic
bacteria such as Listeria ssp. [5,6].
During recent years enterocins have received a considerable
attention, the most prevalent of them are classified as class IIa
bacteriocins, small heat-stable non-lantibiotics belonging to the
pediocin-like Listeria-active peptides [5,7]. Among them, the ente-
rocin A produced by some E. faecium strains was the first enterocin
to be thoroughly characterized [5,7]. Its strong antilisterial activ-
ity, especially against Listeria monocytogenes, a very important
pathogen associated to food poisoning, reinforced the use of ente-
∗ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001.
E-mail addresses: nelsonpg@uvigo.es, anitarey@mundo-r.com (N.P. Guerra).
foods [5,7,8].
Over fermented product preservation potential could be
reached either by using a bacteriocin-producing starter culture or
by applying the bacteriocin itself as a food additive. The latter nec-
essarily requires an optimization of the different factors that affects
its production. Since the effects of these factors have found to be
strain specific, it was necessary to investigate each strain specifi-
cally [9–11] in order to improve the production of compounds with
potential application in foods.
Previous optimization studies were focused on the determina-
tion of the effects of pH, temperature and media composition on
bacteriocin production [10–13]. Then, after optimizing these vari-
ables, the interest has been recently focused on the optimization
of the fermentation process of the bacteriocin-producing strains,
especially using cheaper culture media [14–16] and fed-batch
culture techniques [14–17]. The use of this fermentation tech-
nique not only avoids the manifestation of the substrate inhibition
phenomenon, which represents the conventional disadvantage of
batch fermentation, but also it is especially beneficial when the
changes in nutrient concentration affect the productivity of a
desired product [18].
However, before optimizing their production in cheaper culture
media from food industry, bacteriocins should be firstly produced

1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2011.05.012
 Author's personal copy
A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106
at high concentrations using commercial culture media such as
the MRS or TGE broth [19] due to the following reasons. Firstly,
because the use of these media allows the production of high con-
centrations of bacteriocins, that are necessary for studies of their
effectiveness in controlling the growth of undesirable bacteria to
preserve functional foods and health products. Secondly, because
the concentration of bacteriocin produced in the commercial
culture media would represent the maximum bacteriocin level
to be obtained or surpassed when its production is optimized
in cheaper culture media. Unfortunately, studies dealing with
the fed-batch production of enterocin A in cheap or commercial
culture medium are lacking [5,20].
Since the pH drop generated in the cultures favoured the
production of pediocin PA-1, a class IIa bacteriocin [10–12,15],
it could be expected that the synthesis of enterocin A may
also be favoured in cultures without pH control. For this rea-
son, in the present work, enterocin A production by E. faecium
MMRA was followed in non-realkalized batch, realkalized batch
and realkalized fed-batch cultures at uncontrolled pH. Further-
more, mathematical models were set up to describe the kinetics
of E. faecium MMRA cell growth and product (bacteriocin and
lactic acid) synthesis during realkalized batch and realkalized
fed-batch fermentations. Finally, to increase the potential of ente-
rocin A for its application as a food and feed biopreservative, the
effects of temperature and pH on its stability were studied and
modelled.
2. Materials and methods
2.1. Bacterial cultures and media
Enterococcus faecium MMRA, the enterocin A-producing strain
[21], was isolated from a Tunisian dairy beverage Rayeb. Liste-
ria innocua F (the target organism in the antibacterial-activity
assay) was obtained from the INRA ENV ENITIAA, Nantes, France.
The two bacteria were respectively grown in de Man, Rogosa
and Shape (MRS, Merck, Germany) and Brain Heart Infusion (BHI,
Merck, Germany) broths and maintained as frozen stock held at
−40 ◦ C in nutrient broth plus 15% (v/v) glycerol. Working cul-
tures were maintained as slants on MRS (in case of E. faecium
MMRA) or BHI (in case of L. innocua F) agar at 4 ◦ C, and propa-
gated twice in liquid cultures in the corresponding medium at 37 ◦ C
before use.
2.2. Fermentation conditions
All fermentations were carried out in a 1L Erlenmeyer flask
containing 200 mL of MRS medium (previously sterilized at
121 ◦ C/15 min) with agitation on a rotary shaker (200 rpm) at 37 ◦ C.
Non-realkalized batch culture of E. faecium MMRA, which was
used as control, was incubated for 36 h in MRS medium. The culture
samples, which comprise aliquots of 2.5 mL of fermented medium,
were withdrawn at regular intervals to perform analytical deter-
minations.
In the realkalized batch culture, stepwise pH profiles were
obtained by repeated alkalization of cultures up to pH 7.0 with 5 M
NaOH each 12 h, when the lower steady pH value was reached in
the non-realkalized batch culture (Fig. 1). The realkalization peri-
ods were maintained for as long as the E. faecium strain was able
to bring about the decrease of pH. Samples of 2.5 mL were taken at
regular intervals to perform analytical determinations.
The realkalized fed-batch culture was initiated as a batch pro-
cess with a working volume of 200 mL of MRS medium adjusted to
an initial pH of 7.0. After 12 h, the batch fermentation was converted
into repeated fed-batch and realkalized mode by rapidly withdraw-
95
ing a volume of 10 mL of the culture medium from the Erlenmeyer
flask. After determining the total sugars concentration in the sam-
ple withdrawn, the medium was realkalized up to a set pH of 7.0
with 5 M NaOH. Then, a volume of 10 mL of the feeding substrate
was added to the fermentor to bring the culture up to the initial
total sugars concentration (20 g/L). The feeding substrate consisted
in a mixture of two substrates composed by a concentrated glu-
cose (400 g/L) and MRS medium (20 g of glucose/L). The MRS broth
was used as a feeding substrate to supplement the fermentor not
only with glucose but also with other nutrients (nitrogen, phos-
phorous, proteins, vitamins, minerals, etc.) in each feeding cycle.
The volumes of sterile concentrated glucose (400 g/L) and MRS
medium were calculated by applying mass balance equations for
the total sugars across the Erlenmeyer flask, as indicated below. In
these equations, the volumes of NaOH added to the fermentor in
each realkalization cycle were also taken into account in the mass
balances [16].
The three cultures were started with a 2% (v/v) inoculum of a
12-h culture of E. faecium MMRA in MRS medium. All fermentations
were replicated twice to assess data reproducibility with triplicate
repeated measures within each replication for each experimental
point.
2.3. Analytical assays
The concentrations of biomass, total phosphorous, nitrogen,
protein, sugars, and lactic acid were determined by methods
described in previous works [10,11,14]. All the analytical determi-
nations were performed in triplicate.
2.4. Bacteriocin activity assays
Aliquots from cultures of E. faecium MMRA were adjusted to
pH 3.5 with 5 M HCl to avoid the adsorption of molecules of
bacteriocin onto the producer cell surfaces. Thereafter they were
heated at 80 ◦ C for 3 min to kill the cells and centrifuged at
27,200 × g for 15 min at 4 ◦ C. The supernatants containing over-
all antibacterial activity (bacteriocin extract) were adjusted to pH
6.0 and frozen until further use. Antibacterial activity of bacteri-
ocin extracts was determined by a photometric bioassay method
[22] using L. inoccua F as target organism. Bacteriocin extracts
were firstly diluted as needed in distilled sterile water. Diluted
bacteriocin (0.25 mL) extracts were added in sterile culture tubes.
Each tube was inoculated with 0.25 mL of a culture of L. innocua
F (diluted to an absorbance of 0.2 at 700 nm with sterile BHI
broth previously buffered at pH 6.3). Controls consisted in three
culture tubes in which the diluted bacteriocin extract was substi-
tuted by distilled sterile water. The tubes were incubated for 6 h
at 37 ◦ C. Growth inhibition was measured spectrophotometrically
at 700 nm. Dose/response curves were obtained from these data.
Enterocin A activity was quantified as described in Murado et al.
[23].
Samples of undiluted bacteriocin extracts were taken at dif-
ferent incubation times and they were purified and identified as
described in Rehaiem [21] to detect if other bacteriocin in addi-
tion to enterocin A was produced in the cultures by the growing
strain.
2.5. Mass balance equations in the realkalized fed-batch
fermentations
In this work, the feeding volumes (VF) were matched with the
sampling volumes (VS). However, the volumes of fermentation
medium (V) in both the realkalized batch and realkalized fed-batch
fermentations were not maintained constant due to the addition
 Author's personal copy

96 A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106

24 0.6

16 0.4
TS (g/L)

X (g/L)
8 0.2

0 0

600 3

TN (g/L)
TP (mg/L)

400 2

200 1

0 0

4 4
BT (AU/mL)

LA (g/L)
2 2

0 0

12
6

Prot (g/L)
9
4
pH

2
3

0 0
0 10 20 30 0 10 20 30 40

Time (h)

Fig. 1. Time course of the non-realkalized batch culture of E. faecium MMRA in MRS medium. X, LA, BT: concentrations of biomass, lactic acid and enterocin A; TS, TP, TN,
Prot: concentrations of total sugars, phosphorous, nitrogen and proteins. Data are means ± standard deviations (SD) of two independent experiments with three replicates
each.

of the necessary volumes of NaOH (VNaOH) for realkalizing the ing the initial total sugars (TS) in the fermentation medium are
medium up to the initial pH value of 7.0. [16]:
Then, the fermentation volume at the beginning of each realka-
lization cycle was calculated as: VSt+tr = VFt+tr = (VCGt+tr + VMRSt+tr ) (II)

where VCG and VMRS are respectively, the volumes (in L) of con-
Vt+tr = Vt − VSt+tr + V NaOHt+tr (for realkalized fermentation)
centrated glucose and MRS medium added to the fermentor at the
(I.1) beginning of each feeding cycle.
Then, the VMRS can be calculated as:
Vt+tr = Vt − VSt+tr + V NaOHt+tr + VFt+tr
VMRSt+tr = VSt+tr − VCGt+tr (III)
(for realkalized fed-batch fermentation) (I.2)
The reduction in the amounts (in grams) of TS in the medium due
where t is the sampling time (h) and tr is the realkalization time to the joint effect of the extraction of samples and the consumption
(12 h). of this substrate by the growing strain (TSC+E ) was calculated by
Since VSt+tr = VFt+tr in the realkalized fed-batch fermentation, Eq. applying a mass balance equation for the total sugars:
(I.2) takes the form:
TSC+E = Vt+tr × [TS]t − (Vt+tr − VSt+tr ) × [TS]t+tr (IV)
Vt+tr = Vt + V NaOHt+tr (I.3)
where [TS]t and [TS]t+tr are respectively, the concentrations of
In the realkalized fed-batch fermentation, the sum of the total sugars (in g/L) at the beginning (20 g/L) and at the end of
volumes of feeding substrates that must be added for restor- each feeding cycle. The difference (Vt+tr − VSt+tr ) represents the
 Author's personal copy

A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106 97

remaining volume (in L) in the fermentor after the extraction of 2.6. Model parameters determination and model evaluation
samples.
Therefore, the amounts of carbon source that must be added to The model parameters and their confidence intervals were
the fermentor in each feeding cycle to restore the initial TS con- obtained by using the nonlinear curve-fitting software of SigmaPlot
centration in the fermentation medium were calculated by the (version 9.0, Systat Software, Inc., 2004), which minimised the devi-
following expressions: ations between model predictions and experimental data according
to the sum of squares of errors (SSE) of the model fit:
VMRS × [TSMRS ] + VCG × [TSCG ] = TSC+E (V)

n 
m
where [TSMRS ] and [TSCG ] are the concentrations of total sugars SSE = 2i,j (XIV)
in the MRS medium (20 g/L) and in the concentrated solution of i=1 j=1
glucose (400 g/L), respectively.
Introducing Eq. (III) into Eq. (V) gives: where i,j represents the difference between the model and the
experimental value, n and m represent the number of experimental
(VSt+tr − VCGt+tr ) × [TSMRS ] + VCGt+tr × [TSCG ] = TSC+E (VI) data points and the number of variables, respectively.
The coefficients of the models with P values lower than 0.05
Thus, the VCG were calculated as:
were considered statistically significant. Parameters were removed
(TSC+E − VS × [TSMRS ]) from the models when their asymptotic interval of confidence
VCGt+tr = (VII)
([TSCG ] − [TSMRS ]) included zero.
The criteria used to evaluate the goodness-of-fit of each model
The VMRS were obtained by introducing the values of VCG into
were the determination coefficient (R2 ) and the mean relative per-
Eq. (III).
centage deviation modulus (RPDM):
The accumulated
 concentrations of nutrients (TS, TN, TP, Prot)
 
100   Xi − Xpi 
consumed ( [Scons ]) in each realkalization cycle were calculated N

by using the following mass balance: RPDM = (XV)

N Xi
i=1
 (Vt − VSt ) × [S]t + VMRSt × [SMRS ] + VCGt × [SCG ] − Vt+tr × [S]t+tr
[Scons ]t+tr = where Xi is the experimental value, Xpi is the calculated value with
Vt+tr
the model and N is the number of experimental data. The RPDM
+ [Scons ]t (VIII) parameter is widely used to determine the quality of the fit, being
where [S]t and [S]t+tr are the nutrient concentrations (in g/L) in the a value of RPDM below 10% indicative of a good fit for practical
fermentor at the beginning and at the end of each feeding cycle, purposes [24–26].
respectively. [SMRS ] and [SCG ] are the nutrient concentrations (in
g/L) in the feeding substrates (MRS and concentrated glucose). 2.7. Combined effect of temperature and pH on the stability of
Since the values for VS, VMRS and VCG are zero in the first feed- enterocin A
ing cycle, the concentrations of nutrients consumed ([Scons ]) in this
period were calculated as follows: A multifactorial composite orthogonal design [27] based on five
levels and two variables, was used to study the combined influence
[Scons ]12h = [S]0h − [S]12h (IX) of pH and temperature on the stability of enterocin A. The design
 consisted of 13 experiments with four (22 ) factorial points, four
The accumulated concentrations of nutrient extracted ( [Sext ])
axial points to form a central composite design with ˛ = 1.414 and
from the fermentation medium as well as the accumulated concen- five centre points for replication. Duplicated samples of enterocin
trations of nutrient added ( [Sadded ]) to the fermentor with the
A extracts obtained from a 108 h realkalized fed-batch culture of E.
feeding substrates were calculated as follows:
faecium MMRA were adjusted at different pH values with 5 M HCl
 VSt+tr × [S]t+tr or NaOH and incubated for 5, 10 an 20 min at the corresponding
[Sext ] = + [Sext ]t (X)
t+tr Vt+tr temperature, according to the experimental matrix defined by the
design used. After incubation, the samples were adjusted to pH 6.0
 VMRSt+tr × [SMRS ] + VCGt+tr × [SCG ] and monitored for remaining activity (%RA). Controls consisted of
[Sadded ]t+tr = samples of untreated bacteriocin extracts.
Vt+tr
All experiments were replicated twice to assess data repro-
+ [Sadded ]t (XI) ducibility with triplicate repeated measures within each replication
for each experimental point.
The accumulated concentrations of products (biomass, lactic
acid and enterocin A) extracted ( [Pext ]) at the end of each feeding 3. Results and discussion
cycle were calculated by the following mass balance equation:
3.1. Batch fermentation of E. faecium MMRA in MRS medium
 VSt+tr × [P]t+tr
[Pext ] = + [Pext ]t (XII)
Vt+tr To produce the large amounts of bacteriocins needed to test
where [P]t+tr is the concentration of product (in g/L) at the end of their efficiency as preservatives in foods and health products, it
each feeding cycle. is necessary the use of an efficient and well defined fermentation
procedure for enhancing bacteriocin production in a defined labo-
Then, the accumulated concentrations of products formed ratory culture medium [15]. For this reason, in the present work,
( [P]) at the end of each feeding cycle were calculated as the sum
of the concentrations of product synthesized at the end of each three different fermentation procedures were assayed to select the
feeding cycle and the total amounts of products extracted in the most efficient for producing a high enterocin A concentration by E.
previous feeding cycle: faecium MMRA. In these studies, the MRS broth was used as sub-
strate of fermentation because this medium highly stimulated both
 VSt × [Pext ]t
[P]t+tr = + [P]t+tr (XIII) the productions of biomass and fermentation products by different
Vt enterococci [6,28–30].
 Author's personal copy

98 A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106

80

60

rX (mg/L/h)
40

20

-20
4 5 6 7
Culture pH

20 200

15 150

rProt (mg/L/h)
rTP (mg/L/h)

10 100

5 50

0 0

45 600

rTS (mg/L/h)
rTN (mg/L/h)

30 400

15 200

0 0
4 5 6 7 4 5 6 7
Culture pH

Fig. 2. Changes in the absolute mean rates of growth (rX) and nutrient consumption (total phosphorous (rTP), nitrogen (rTN), proteins (rProt) and sugars (rTS)) with respect
to the culture pH in the non-realkalized batch culture of E. faecium MMRA in MRS medium. Data are means ± SD of two independent experiments with three replicates each.

Before starting the fed-batch fermentation, a simple batch fer-
mentation with E. faecium MMRA in MRS medium without pH
control (Fig. 1) was performed to obtain data for comparison. In this
culture, a typically homolactic fermentation was observed, since
the lactic acid was the unique antimicrobial metabolite produced
by the growing strain.
On the other hand, the highest concentrations of biomass
(0.4 g/L), enterocin A (4.0 AU/mL) and lactic acid (3.6 g/L) were
obtained after 12 h of incubation. In this period, the culture pH
dropped from 6.47 to 4.58 and the concentration of nutrients (total
sugars, nitrogen, phosphorous and proteins) exhibited their highest
decrease. However, from the 12 h of fermentation, the culture pH
remained constant and the biomass, enterocin A and lactic acid pro-
duction stopped in parallel with a decrease in nutrient consump-
tion. In addition, after 24 h of incubation, both biomass and ente-
rocin A concentrations decreased until the end of the culture (Fig. 1).
The accumulation of lactic acid does not seem to be a cause for
the cessation of growth after 12 h of fermentation, because the con-
centration of lactic acid produced in this culture (3.6 g/L) was lower
than that considered damaging for the E. faecium strains [31]. Then,
the exhaustion of one or some micronutrients (vitamins, minerals
and amino acids) in the medium or the limitation in the nutrients
(total sugars, nitrogen and phosphorous) or micronutrients trans-
port when the cultures reached a pH value below 6.0, could be the
possible causes to explain the growth cessation [32–34].
To check whether the nearness to the threshold pH value had
some influence on nutrient consumption, the rates of growth and
nutrient consumption for each sampling time were calculated and
represented versus the culture pH (Fig. 2). As it can be observed,
both the growth and nutrient consumption rates began to decrease
when the cultures reached a pH value of 6.0 (Fig. 2). Thus, the fail-
ure to growth at this acidic pH seems to be mainly caused by a
limitation of cytoplasmic processes (acidification of the cytoplasm
and the collapse of the proton motive force) that limits the nutrient
transport [32–34].
A similar effect of pH on growth and nutrient consumption by
other Enterococcus strains has been observed before [16,30,31].
From a kinetic point of view, it can be noted that enterocin A
was produced during the active growth phase when the highest
pH drop was generated in the culture (Fig. 1). Then, enterocin A
production displayed primary metabolite kinetics and was strongly
dependent on pH drop, as occurred for other bacteriocins belonging
to the pediocin-like family, such as the pediocin PA-1 [26,35,36].
3.2. Realkalized batch fermentation of E. faecium MMRA in MRS
medium
Taking into account that enterocin A had a direct dependence
on both the biomass production and pH drop, it could be expected
that those conditions that assure a high cell density of the producer
strain as well as the generation of high pH drops should favour bac-
teriocin production. Since both conditions were obtained after 12 h
of incubation in the batch culture of E. faecium MMRA (Fig. 1), a new
batch fermentation in MRS broth was carried out with periodical
realkalizations of the culture medium up to pH 7.0 each 12 h.
The results obtained in this realkalized batch fermentation are
shown in Fig. 3. Taking into account the production of organic acids,
there was only a homolactic fermentation phase in this culture
 Author's personal copy

A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106 99

25 2.0

20
1.5

15
TS (g/L)

X (g/L)
1.0
10

0.5
5

0 0

600 3
TP (mg/L)

TN (g/L)
400 2

200 1

0 0

18 12
BT (AU/mL)

LA (g/L)
12 8

6 4

0 0

6 15

Prot (g/L)
4 10
pH

2 5

0 0
0 20 40 60 0 20 40 60 80
Time (h)

Fig. 3. Time course of the realkalized batch culture of E. faecium MMRA in MRS medium without nutrient feeding. X, LA, BT: concentrations of biomass, lactic acid and enterocin
A accumulated (), remaining () and extracted (䊉); TS, TP, TN, Prot: concentrations of total sugars, phosphorous, nitrogen and proteins supplemented (), remaining (),
consumed () and extracted (䊉). Data are means ± SD of two independent experiments with three replicates each.

(Fig. 3), as it was observed before in the non-realkalized batch
fermentation (Fig. 1). Surprisingly, the final pH values at the end
of each realkalization cycle increased progressively throughout
the fermentation and after 36 h of incubation the growth stopped
even though the concentrations of nitrogen, phosphorous and
total sugars were still sufficiently available in the medium (Fig. 3).
In fact, from the 36 h of culture, the rates of products (bacteriocin
and lactic acid) formation and nutrient consumption slowed
down. Since the medium was realkalized each 12 h, the growth
cessation from the 36 h of fermentation seems to be caused by
the exhaustion of one or some essential micronutrients (vitamins,
minerals and amino acids) in the medium rather than by a lim-
itation in the nutrients (total sugars, nitrogen and phosphorous)
or micronutrients transport [32–34] or by the accumulation of
relatively high amounts of lactic acid.
From a practical point of view, it can be noted that the succes-
sive realkalizations led to an increase in the metabolically active
period of the cells from 12 h (in the non-realkalized batch culture)
to 36 h (in the realkalized batch culture). Consequently, the con-
centrations of biomass (1.0 g/L), enterocin A (14.0 AU/mL) and lactic
acid (8.0 g/L) obtained in the latter culture after 36 h (Fig. 3) were,
respectively, 2.5, 3.5 and 2.2 times higher than those maximum
concentrations obtained in the previous batch culture (Fig. 1).
On the other hand, the experimental yield coefficients for the
biomass (YX/TSc and YX/TNc ) and enterocin A (YBT/TSc , YBT/TNc and
YBT/X ) in the realkalized culture were higher than those of the
non-realkalized culture (Table 1). These results indicated a more
efficient utilization of the sources of total sugars and nitrogen for
both biomass and bacteriocin production as well as the produc-
tion of a more productive biomass. In addition, the experimental
efficiencies (ETS , ETN , ETP and EProt ) were also higher in the realka-
lized culture (Table 1), indicating that the system evolved in a more
balanced way with respect to the nutrients consumed and added
[14,22,37,38].
3.3. Realkalized fed-batch fermentation of E. faecium MMRA in
MRS medium
Taking into account the results obtained in the previous two
cultures, a realkalized fed-batch fermentation in MRS medium
 Author's personal copy

100 A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106

Fig. 4. Time course of the realkalized fed-batch culture of Enterococcus faecium MMRA in MRS medium (20 g of glucose/L) after feeding with MRS medium and a 400 g/L
concentrated glucose. X, LA, BT: concentrations of biomass, lactic acid and enterocin A accumulated (), remaining () and extracted (䊉); TS, TP, TN, Prot: concentrations of
total sugars, phosphorous, nitrogen and proteins supplemented (), remaining (), consumed () and extracted (䊉). Data are means ± SD of two independent experiments
with three replicates each.

was carried out by using a mixture of a concentrated glu-
cose (400 g/L) and MRS medium (20 g of glucose/L) as feeding
substrates to replenish the carbon source consumed in each
feeding cycle. This approach also allowed to supplement the
culture medium with other fresh nutrients (nitrogen, phos-
phorous and proteins) and micronutrients present in the MRS
medium.
The results obtained (Fig. 4) showed that, in spite of the increase
in final pH, the growing strain was able to bring about the decrease
of pH during all the culture and both the nitrogen and the phos-
phorous sources were not completely consumed.
From a comparative point of view, it can be noted that the final
productions of biomass (2.4 g/L), lactic acid (32.5 g/L) and enterocin
A (35.9 AU/mL) obtained in the fed-batch culture (Fig. 4 and Table 1)
were considerably higher than those obtained in the previous two
cultures (Table 1). In addition, the active period (108 h) in this third
culture was longer than those of the non-realkalized (12 h) and
realkalized (36 h) batch cultures, probably as a consequence of the
additional supplements of nitrogen and phosphorous sources and
other micronutrients in each feeding with the MRS broth. These
results made evident that feeding the culture with a concentrated
glucose and MRS medium was an adequate alternative for obtain-
ing further increases in biomass and product synthesis by E. faecium
MMRA in MRS broth.
Since in the realkalized fed-batch fermentation, the feeding
substrates were mainly added to bring the cultures up to the
initial total sugars concentrations (20 g/L) in each feeding cycle,
it could be considered that there was no limitation by the carbon
source during this culture. In this case, the nitrogen source could
be considered as the unique limiting nutrient for the growth of
E. faecium MMRA, as it was proposed before for other realkalized
fed-batch cultures with other LAB strains [15,16,39]. Thus, due
to the joint effect of consumption and dilution (which occurred
due to the combined effect of sampling and nutrient feeding),
the nitrogen concentration in the culture medium progressively
decreased. This probably led to a nutrient limitation due to the
 Author's personal copy

A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106 101

Table 1 0.5 0.3

Growth and fermentation parameters of the non-realkalized batch (I), realkalized
batch (II) and realkalized fed-batch (III) fermentations of E. faecium MMRA for ente- A
rocin A production in MRS.
0.4

EP (AU/mL/h)
0.2

rpH (/h)
Fermentation (I) Fermentation (II) Fermentation (III) 0.3
X* (g/L) 0.4 1.2 2.4
BT* (AU/mL) 3.6 17.2 35.9 0.2
LA* (g/L) 4.0 10.0 32.5 0.1
YX/TSc 0.06 0.09 0.03
0.1
YX/TNc 0.73 2.10 2.12
YBT/TSc 0.60 1.21 0.51
YBT/TNc 7.74 29.18 34.23 0 0
YBT/X 10.60 13.92 16.17 0 30 60 90 120
ETS 0.33 0.58 0.81
Time (h)
ETN 0.16 0.19 0.29
ETP 0.13 0.15 0.30 0.6 0.3
EProt 0.11 0.26 0.51 B
X*, BT* and LA* are the maximum biomass, enterocin and lactic acid concentrations

EP (AU/mL/h)
produced in the cultures. YX/TSc and YX/TNc are the experimental biomass yield coef- 0.4 0.2

rpH (/h)
ficients expressed as grams of biomass produced per gram of total sugars (TSc) or
nitrogen (TNc) consumed. YBT/TSc , YBT/TNc and YBT/X are the experimental enterocin
A yield coefficients expressed as amount of enterocin A produced (AU) per grams
of TS or TN consumed and per g of biomass produced. ETS , ETN , ETP and EProt are the 0.2 0.1
experimental efficiencies expressed as grams of substrate (total sugars, nitrogen,
phosphorous and proteins) consumed per gram of substrate supplied.

0 0
0 30 60 90 120

exhaustion of one or some aminoacids essential for the growth of Time (h)
E. faecium MMRA during the incubation. 0.6 0.4
On the other hand, the experimental yield coefficients YX/TSc and C
YBT/TSc in the realkalized fed-batch culture decreased in compari- 0.3
EP (AU/mL/h)

son with those of the two batch fermentations (Table 1). This was 0.4

rpH (/h)
probably due to a higher sugar consumption to support the higher
production of lactic acid as a consequence of the increase in the 0.2
active period of the cells. However, the use of the nitrogen source
0.2
for both biomass and bacteriocin production was more economical 0.1
and the biomass was more productive in the realkalized fed-batch
culture and consequently, increased values for the experimental
0 0
yields YX/TNc , YBT/TNc and YBT/X were obtained. Also the experimental
0 30 60 90 120
efficiencies (ETS , ETN , ETP and EProt ) were higher in the third fermen-
tation (Table 1), indicating a better use of the available nutrients by Time (h)
the bacteriocin-producing strain. Fig. 5. Enterocin A productivities (EP) and pH drop rates (rpH) in the non-realkalized
With regard to the experimental enterocin A productivities (EP), batch (A), realkalized batch (B) and realkalized fed-batch (C) cultures of E. faecium
it can be noted that the observed progressive reduction in the EP MMRA in MRS medium. Data are means ± SD of two independent experiments with
values with the increase in the incubation time, was concomi- three replicates each.
tant with the decrease in the pH drop rate in the three cultures
(Fig. 5). Thus, the EP values in the non-realkalized batch culture did not induce the production of other enterocins by Ent faecium
was maximal (0.44 AU/mL/h) during the first 8 h of fermentation MMRA.
and decreased sharply afterwards (Fig. 5A). In the realkalized batch
culture, the maximum EP values (0.44 AU/mL/h) were maintained 3.4. Mathematical modelling
almost constant during a period of time longer than that of the pre-
vious culture and a final EP value of 0.29 AU/mL/h was obtained Before modelling product formation, two strategies were used
at the end of the incubation (Fig. 5B). Contrarily, in the realkalized to model the growth ([X] in g/L) of E. faecium MMRA in the
fed-batch culture, the pH drop values were almost constant during non-realkalized batch, realkalized batch and realkalized fed-batch
the incubation period and the enterocin A productivities did not cultures in MRS medium. In the first strategy, the time-series data
decreased dramatically (Fig. 5C). Then, the decrease in enterocin were modelled by using a modified 4-parameter (K, c, b, Kd ) logistic
A productivities after prolonged incubation was lower than in the model (1):
two previous fermentations and a final EP value of 0.33 AU/mL/h
K(1 + e−Kd ×t )
 2K 
was obtained in this culture after 108 h of fermentation (Fig. 5C). [X] = with c = ln −1 (1)
Thus, the recourse of realkalizing and feeding the culture 1 + e(c−b×t) [X0 ]
medium showed to be an effective way for enhancing considerably where [X0 ] is the initial biomass concentration (g/L), K is the max-
both the growth of E. faecium MMRA and enterocin A production imum biomass concentration (g/L), b is a logistic rate parameter
in MRS medium. These results are consistent with those previously (h−1 ), Kd is the cell death rate (h−1 ) and t is the time (h).
obtained for other LAB [14,15,37,38,40]. However, the results obtained with this modelling procedure
The purification and characterization studies [21] showed that (Fig. 6 and Table 2) showed that the initial biomass concentration
the antibacterial activity presents in the cell-free supernatants from was underestimated (in case of non-realkalized batch culture) or
the three cultures of E. faecium MMRA was due to the presence of overestimated (in case of the realkalized batch and fed-batch cul-
enterocin A. This indicates that the different fermentation methods tures). In addition, it can be noted that only in the non-realkalized
 Author's personal copy

102 A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106

0.5 batch culture, the RPDM value was lower than 10% (Table 2). There-
fore, model (1) was not adequate to describe accurately the growth
0.4 of E. faecium MMRA in the two realkalized cultures.
For this reason, the following alternative was focused on the
0.3 analysis of the experimental growth data obtained from the two
X (g/L)

realkalized cultures by using the classical Monod equation, which

0.2 relates the specific growth rate with the initial concentrations of
the limiting substrates [26,39].
0.1 Non-realkalized batch In the realkalized batch culture, it was difficult to select the lim-
iting substrate, therefore the specific growth rate () was related to
0
the concentrations of total sugars and nitrogen as follows [26,39]:
0 10 20 30 40
 [N] [TS]

Time (h)  = max × (2)
KN + [N] KTS + [TS]
1.5
where max is the maximum specific growth rate (h−1 ), [N] and [TS]
are the concentrations (g/L) of nitrogen and total sugars, respec-
1.0 tively. KN and KTS are the nitrogen and total sugars affinity constants
(g/L), respectively.
X (g/L)

Since the product formation can inhibit biomass production

[41], the specific growth rate was multiplied by a dimensionless
0.5
inhibition function (ıi ) for accounting the inhibition by lactic acid
Realkalized batch production [26,39,41]:
 [N] [TS]

0 ıi = max × × ıi (3)
0 20 40 60 80 KN + [N] KTS + [TS]
Time (h)
 KiLA

2.5 Being ıi =
KiLA + [LA]
2.0
where [LA] is the lactic acid concentration (g/L) and KiLA is the
inhibition constant (g/L) for lactic acid.
1.5
X (g/L)

For the realkalized fed-batch fermentation, in which the nitro-

gen source was considered to be as the limiting substrate, Eq. (3)
1.0
takes the form:
0.5
 × [N]

max
Realkalized fed-batch ıi = × ıi (4)
KN + [N]
0
0 30 60 90 120 In order to take into account the effect of dilution, which
occurred due to the sample extraction and the feeding of nutri-
Time (h)
ents, mass balance was carried out across the fermentor and the
Fig. 6. Experimental data (symbols) for biomass (X) obtained in the non-realkalized biomass accumulation rate (rX) was expressed as:
batch (data from Fig. 1), the realkalized batch (data from Fig. 3) and the realka-
d[X] ([X]t+tr − [X]t × (1 − (VSt /Vt )))
lized fed-batch (data from Fig. 4) cultures of E. faecium MMRA in MRS medium. The rX = =
solid lines drawn through the experimental data of biomass were obtained accord- dt dt
ing to the modified logistic model (1). Data are means ± SD of two independent
= ( − Kd ) × ıi × [X] (5)
experiments with three replicates each.
where [X]t and [X]t+tr are the biomass concentrations (g/L) inside
the fermentor at the beginning and at the end of each feeding cycle,
respectively. Other terms are as previously defined.
Lactic acid accumulation rate (rLA) was represented to contain
growth (˛LA ) and non-growth (ˇLA ) associated constants [42] as
follows:
Table 2
Parameter values of the modified logistic model (1) used for predicting the growth ([LA]t+tr − [LA]t × (1 − (VSt /Vt )))
of E. faecium MMRA in the non-realkalized batch (I), realkalized batch (II) and real-
rLA = = ˛LA × rX + ˇLA × [X]
dt
kalized fed-batch (III) fermentations in MRS.
(6)
Parameter Fermentation (I) Fermentation (II) Fermentation (III)
where [LA]t and [LA]t+tr are the lactic acid concentrations (g/L)
K (g/L) 0.26 ± 0.023 1.05 ± 0.017 1.85 ± 0.035
inside the fermentor at the beginning and at the end of each feeding
c (dimensionless) 3.59 ± 0.333 2.46 ± 0.178 2.12 ± 0.178
b (h−1 ) 0.62 ± 0.070 0.16 ± 0.011 0.08 ± 0.007 cycle, respectively. Other terms are as previously defined.
Kd (h−1 ) 0.03 ± 0.012 NS NS Enterocin A production rate (rBT) was modelled by using a
[X0 ]* (g/L) 0.01 ± 0.014 0.08 ± 0.019 0.20 ± 0.032 modified form of the Luedeking and Piret model (model 6), which
RX2 0.9874 0.9930 0.9829 includes a term for the specific effect that the pH drop rate produces
RPDMX 8.88 19.70 35.31 on bacteriocin synthesis [10,11,35,43]:
[X0 ]* Mean initial biomass concentration calculated according to the model (1). The
([BT]t+tr − [BT]t × (1 − (VSt /Vt )))
mean experimental [X0 ] value in each culture was 0.03 g/L. Statistically significant rBT =
coefficients (P > 0.05) are expressed as means ± standard errors. NS: not significant dt
(P > 0.05).
= (˛BT × rX + ˇBT × [X]) × (1 +  × rpH) (7)
 Author's personal copy

A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106 103

A 1.5 2.5
B
2.0
1.0
1.5

X (g/L)
X (g/L)
1.0
0.5
0.5

0 0

18 30

BT (AU/mL)
BT (AU/mL)

12 20

6 10

0 0

6 24

LA (g/L)
LA (g/L)

4 16

2 8

0 0
0 20 40 60 80 0 30 60 90 120
Time (h)

Fig. 7. Experimental data (symbols) for biomass (X), enterocin A (BT) and lactic acid (LA) concentrations corresponding to the realkalized batch (A) and the realkalized
fed-batch culture (B) of E. faecium MMRA in MRS. The solid lines drawn through the experimental data of biomass, lactic acid and enterocin A were obtained according to
the models (5), (6) and (7), respectively. Data are means ± SD of two independent experiments with three replicates each.

where [BT]t and [BT]t+tr are respectively, the enterocin A concen- lactic acid was produced as a primary metabolite (˛LA  ˇLA ) in
trations (AU/mL) inside the fermentor at the beginning and at the the two cultures (Table 3). The values of the parameters (˛BT  ˇBT
end of each feeding cycle,  is a constant of proportionality to be and  = / 0) in model (7) indicate that enterocin A can be classi-
experimentally determined, rpH is the pH drop rate over time and fied as a primary metabolite dependent on the pH drop, as it was
˛BT and ˇBT are the growth and non-growth associated constants. commented before.
The results obtained with this last modelling strategy showed The negative values for ˇ in models (6) and (7) are explained by
that the value of the parameter KTS was not statistically signifi- the fact that the biomass production rate (rX) decreased until reach-
cant at (P < 0.05), suggesting that the initial TS concentration has
not a relevant influence on the growth of E. faecium MMRA in the
Table 3
realkalized batch culture. This fact supports the hypothesis that Parameter values of the models (5), (6) and (7) used for predicting the growth
the nitrogen source was the limiting nutrient for the growth of the and product (lactic acid and enterocin A) formation by E. faecium MMRA in the
enterocin A-producing strain in this culture. In the same way, the realkalized batch (II) and realkalized fed-batch (III) fermentations in MRS.
values obtained for Kd were not statistically significant (P < 0.05), Parameter Fermentation (II) Fermentation (III)
indicating that the cell death did not influence the growth of E.
max (h−1 ) 0.35 ± 0.007 0.30 ± 0.009
faecium MMRA in the two realkalized cultures. KN (g/L) 0.23 ± 0.046 0.26 ± 0.003
Therefore, the experimental growth data in these two cultures KTS (g/L) NS –
were remodelled after deleting the parameters KTS and Kd from KiLA (g/L) 2.78 ± 0.130 1.20 ± 0.091
equations (3) and (5), respectively. Thus, the values of  in both Kd (h−1 ) NS NS
cultures were obtained with the model (4) and subsequently, the RX2 0.9984 0.9954
values of [X] were calculated according to the model (5). The sta- RPDMX 1.96 4.22
˛LA (dimensionless) 7.42 ± 1.048 6.17 ± 0.686
tistically significant (P < 0.05) values of the parameters and the
ˇLA (h−1 ) −0.57 ± 0.102 0.04 ± 0.016
trajectories predicted by model (5) are shown in Table 3 and in
RLA
2
0.9916 0.9974
Fig. 7, respectively. As it can be observed, model (5) offered a more
RPDMLA 3.96 3.91
accurate description of the experimental growth data of E. faecium ˛BT (AU/g) 10.45 ± 0.219 9.49 ± 0.519
MMRA in relation with the nitrogen consumption than the modified ˇBT (AU/g/h) −0.80 ± 0.025 −0.02 ± 0.008
Logistic model (1).  (h) 0.76 ± 0.080 0.55 ± 0.007
In addition, with the use of models (6) and (7), excellent agree- RBT
2
0.9986 0.9974
ments were found between model predictions and experimental RPDMBT 2.43 3.87
results for product (lactic acid and enterocin A) formation (Fig. 7). Statistically significant coefficients (P < 0.05) are expressed as means ± standard
The calculated parameters (˛, ˇ) from model (6) revealed that the errors. NS: not significant (P < 0.05).
 Author's personal copy

104 A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106

Fig. 8. Response surfaces corresponding to the remaining activity (%RA) of enterocin A samples after pH and temperature treatment for 5, 10 and 20 min, according to the
experimental plan defined in Table 4. Data are means ± SD of two independent experiments with three replicates each.

ing a value of zero in the last sampling times, meanwhile the rates the remaining activity (%RA) at the different incubation times
of production of lactic acid and enterocin A had constant positive were:
values (Figs. 3 and 4).
%RA (5 min) = 100.30 − 13.29pH − 21.04T − 3.55pHT
In conclusion, the R2 (higher than 0.99) and RPDM values
(lower than 7.0%) obtained for the models (5), (6) and (7) have − 15.30pH2 − 15.89T 2
strengthened their usefulness for describing the growth and
product formation by E. faecium MMRA in the realkalized batch %RA (10 min) = 100.86 − 13.74pH − 20.30T − 4.55pHT
and fed-batch fermentations in MRS (Table 3).
− 15.43pH2 − 16.47T 2

3.5. Effects of pH and temperature on the stability of the %RA (20 min) = 86.42 − 11.81pH − 16.27T
enterocin A produced by E. faecium MMRA
− 13.13pHT − 13.13pH2 − 12.67T 2
The functionality of bacteriocins under industrial food pro-
cessing conditions is still questioned. In fact, a disturbing aspect These three empirical models were highly consistent, being
when using bacteriocins in food systems is the potential reduc- acceptable both the coefficients, according to the Student’s t-test
tion of their long-term effectiveness due to pH and temperature (˛ = 0.05), and the validity of the equations according to the Fisher
changes, particularly in foods subject to ageing such as fermented F-test (˛ = 0.05). The response surfaces of %RA after 5, 10 and 20 min
foods. of treatment are illustrated in Fig. 8.
Thus, in view of the possible applications of enterocin A pro-
Table 4
duced by E. faecium MMRA as a food preservative, its stability to
Experimental domain and codification of the variables in the factorial design used
temperature and pH was studied by using a complete second-order to study the combined influence of temperature and pH on the antibacterial activity
orthogonal design. This procedure allows the determination of the of enterocin A.
optimum conditions for the stability of enterocin A from the anal-
Codified values Natural values
ysis of the empirical models obtained. With this approach, the
stability of the bacteriocin in a range of pH and temperature can T (◦ C) pH
be evaluated in order to study the limitations of its activity which −1.414 59.4 2.8
may affect its performance in inhibiting the growth of undesirable −1 70.0 3.5
0 95.5 5.3
microorganisms in real food systems.
+1 121.0 7.1
The experimental domain (Table 4) was selected regarding usual +1.414 131.6 7.7
levels existing in foods (for pH) and in their processing opera-
Increments 1 25.5 1.8
tions (for both variables). The empirical equations obtained for
 Author's personal copy
A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106
Table 5
Optimum temperature and pH values for enterocin A activity calculated by deriva-
tion of the empirical models obtained for the remaining activity at different
incubation times.
5 min 10 min
T (◦ C) 86.2 86.3
pH 4.16 4.26
Maximum RA (%) 100.0 100.0
Although enterocin A showed almost a similar behaviour at
the three incubation times, its stability in the optimum conditions
decreased after 20 min of incubation (Table 5). The results obtained
also showed that the stabilities were maximal at temperatures
below 100 ◦ C and low pH values (Table 5, Fig. 8). From a practical
point of view, these results indicated that in foods with a pH around
4.0 and thermally treated at a temperature around 80 ◦ C/15 min,
the stability of enterocin A and consequently its inhibitory activity
could be highly favoured.
The heat-stability at acid pH observed for enterocin A has
been described before for most bacteriocins [44,45]. Their high
content of glycine and the formation, at a molecular level,
of globular structures and strong hydrophobic interactions in
the molecule [46] could be used as reasons to explain this
behaviour.
This result reinforces the hypothesis that stability over a
wide range of pH values was a common feature shared between
all enterocins [47], and suggests that the enterocin A could be
used as a preservative in both low and medium-acid fermented
food products (fermented and ripened dairy and meat prod-
ucts), with final pH values within the pH interval used in this
study. This can be an alternative to reduce both the intensity of
heat treatments and addition of chemical preservatives (mainly
those that have a safety risk to consumers), without compro-
mising microbial inactivation, with the additional advantage of
producing low losses in organoleptic and nutritional properties
of foods. This could be a way to satisfy the increasing con-
sumer demands for safe, fresh-tasting and minimally processed
foods.
4. Conclusion
The main contribution of the present work is the enhancement
and modelling the enterocin A production during realkalized fed-
batch fermentation in MRS broth. The concentration of bacteriocin
obtained in this culture was 10 and 2 times higher than those
obtained in the non-realkalized batch and the realkalized batch
cultures, respectively. To our knowledge, it is the first time that the
enterocin A production system by E. faecium MMRA is satisfactory
modelled by taking into account the growth inhibition due to the
depletion of the nitrogen source, product (lactic acid) formation and
culture pH drop. This modelling procedure allowed enterocin A to
be classified as a primary metabolite dependent on the pH drop. The
use of these models to predict the growth, and product (enterocin
A and lactic acid) formation by E. faecium MMRA may lead to the
development of better strategies for the optimization and control
of the enterocin A production. Furthermore, the stability of ente-
rocin A at low pH values in concomitance with its thermostability
at temperatures around 80 ◦ C are important data to be taken into
account with regard to the use of this bacteriocin as a preservative
in foods.
Further studies based on the optimization of enterocin A produc-
tion in cheaper culture media from food industry are being carried
out in our laboratory. These studies could contribute to make a
cost-effective commercial use of enterocin A.
105
Acknowledgements
The research presented in this paper was financially supported
by the Spanish Agency for International Cooperation (AECI), Spain
20 min
within the Inter-University Cooperation Programme Cooperation
84.0 Programme and Scientific Research (PCI-Mediterranean) between
4.12 Spain and Tunisia (project A/5664/06).
94.4
References
[1] C.M. Franz, W.H. Holzapfel, M.E. Stiles, Enterococci at the crossroads of food
safety? Int. J. Food Microbiol. 47 (1999) 1–24.
[2] C.M. Franz, M.E. Stiles, K.H. Schleifer, W.H. Holzapfel, Enterococci in foods—a
conundrum for food safety, Int. J. Food Microbiol. 88 (2003) 105–122.
[3] G. Giraffa, Functionality of enterococci in dairy products, Int. J. Food Microbiol.
88 (2003) 215–222.
[4] T.R. Klaenhammer, Genetics of bacteriocins produced by lactic acid bacteria,
FEMS Microbiol. Rev. 12 (1993) 39–86.
[5] T. Aymerich, H. Holo, L.S. Havarstein, M. Hugas, M. Garriga, I.F. Nes, Biochemical
and genetic characterization of enterocin A from Enterococcus faecium, a new
antilisterial bacteriocin in the pediocin family of bacteriocins, Appl. Environ.
Microbiol. 62 (1996) 1676–1682.
[6] T. Aymerich, M. Garriga, J. Ylla, J. Vallier, J.M. Monfort, M. Hugas, Application
of enterocins as biopreservatives against Listeria innocua in meat products, J.
Food Prot. 63 (2000) 721–726.
[7] C.M. Franz, M.J. van Belkum, W.H. Holzapfel, H. Abriouel, A. Gálvez, Diversity
of enterococcal bacteriocins and their grouping in a new classification scheme,
FEMS Microbiol. Rev. 31 (2007) 293–310.
[8] L.H. Deegan, P.D. Cotter, C. Hill, P. Ross, Bacteriocins: biological tools for bio-
preservation and shelf-life extension, Int. Dairy J. 16 (2006) 1058–1071.
[9] M.A. Daeschel, Applications and interactions of bacteriocins from lactic acid
bacteria in foods and beverages, in: D.G. Hoover, L.R. Steenson (Eds.), Bac-
teriocins of Lactic Acid Bacteria, Academic Press, Inc., New York, 1993,
pp. 63–91.
[10] N.P. Guerra, M.L. Rua, L. Pastrana, Nutritional factors affecting the production
of two bacteriocins from lactic acid bacteria on whey, Int. J. Food Microbiol. 70
(2001) 267–281.
[11] N.P. Guerra, L. Pastrana, Production of bacteriocins from Lactococcus lactis
subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 using mussel-
processing wastes, Biotechnol. Appl. Biochem. 36 (2002) 119–125.
[12] S.R. Biswas, P. Ray, M.C. Johnson, B. Ray, Influence of growth conditions on the
production of a bacteriocin, pediocin AcH, by Pediococcus acidilactici H, Appl.
Environ. Microbiol. 57 (1991) 1265–1267.
[13] M.V. Leal-Sánchez, R. Jiménez-Díaz, A. Maldonado-Barragán, A. Garrido-
Fernández, J.L. Ruiz-Barba, Optimization of bacteriocin production by batch
fermentation of L. plantarum LPCO10, Appl. Environ. Microbiol. 68 (2002)
4465–4471.
[14] N.P. Guerra, L. Pastrana, Enhancement of nisin production by Lactococcus lac-
tis in periodically re-alkalized cultures, Biotechnol. Appl. Biochem. 38 (2003)
157–167.
[15] N.P. Guerra, P. Fajardo, L. Pastrana, Fed-batch pediocin production on whey
using different feeding media, Enzyme Microb. Technol. 41 (2007) 397–406.
[16] N.P. Guerra, P. Fajardo, C. Fuciños, I. Rodríguez, E. Alonso, A. Torrado, L. Pastrana,
Modelling the biphasic growth and product formation by Enterococcus faecium
CECT 410 in realkalized fed-batch fermentations in whey, J. Biomed. Biotechnol.
2010 (2010), doi:10.1155/2010/290286.
[17] L. De Vuyst, E.J. Vandamme, Microbial manipulation of nisin biosynthesis and
fermentation, in: G. Jung, H.G. Sahl (Eds.), Nisin and Novel Lantibiotics, ESCOM
Science Publishers, Leiden, The Netherland, 1991, pp. 398–409.
[18] Y. Tang, M. An, K. Liu, S. Nagai, T. Shigematsu, S. Morimura, K. Kida, Ethanol
production from acid hydrolysate of wood biomass using the flocculating yeast
Saccharomyces cerevisiae strain KF-7, Proc. Biochem. 41 (2006) 909–914.
[19] P. Fajardo, C. Fuciños, J. Méndez, L. Pastrana, N.P. Guerra, Performance and
intestinal coliform counts in weaned piglets fed a probiotic culture (Lacto-
bacillus casei ssp. casei CECT 4043) or an antibiotic, J. Food Prot. 71 (2008)
1797–1805.
[20] E. van den Berghe, L. de Vuyst, T. De Winter, Enterocin A production by
Enterococcus faecium FAIR-E 406 is characterised by a temperature- and pH-
dependent switch-off mechanism when growth is limited due to nutrient
depletion, Int. J. Food Microbiol. 107 (2006) 159–170.
[21] A. Rehaiem, B. Martínez, M. Manai, A. Rodríguez, Production of enterocin A by
Enterococcus faecium MMRA isolated from ‘Rayeb’, a traditional Tunisian dairy
beverage, J. Appl. Microbiol. 108 (2010) 1685–1693.
[22] M.L. Cabo, M.A. Murado, M.P. González, L. Pastoriza, Effects of aeration and pH
gradient on nisin production. A mathematical model, Enzyme Microb. Technol.
29 (2001) 264–273.
[23] M.A. Murado, M.P. Gonzalez, J.A. Vazquez, Dose–response relationships: An
overview, a generative model and its application to the verification of descrip-
tive models, Enzyme Microb. Technol. 31 (2002) 439–455.
[24] C.J. Lomauro, A.S. Bakshi, T.P. Labuza, Evaluation of food moisture isotherm
equations. Part I. Fruit, vegetable and meat products, LWT – Food Sci. Technol.
18 (1985) 111–117.
 Author's personal copy
106 A. Rehaiem et al. / Biochemical Engineering Journal 56 (2011) 94–106
[25] R.J. Aguerre, C. Suarez, P.E. Viollaz, New BET type multilayer sorption isotherms.
Part II: Modelling water sorption in foods, LWT – Food Sci. Technol. 22 (1989)
192–195.
[26] N.P. Guerra, A.T. Agrasar, C.L. Macías, P.F. Bernárdez, L.P. Castro, Dynamic math-
ematical models to describe the growth and nisin production by Lactococcus
lactis subsp. lactis CECT 539 in both batch and re-alkalized fed-batch cultures,
J. Food Eng. 82 (2007) 103–113.
[27] N.P. Guerra, L. Pastrana, Modelling the influence of pH on the kinetics of both
nisin and pediocin production and characterization of their functional proper-
ties, Proc. Biochem. 37 (2002) 1005–1015.
[28] P. Sarantinopoulos, F. Leroy, E. Leontopoulou, M.D. Georgalaki, G. Kalant-
zopoulous, E. Tsakalidou, L. de Vuyst, Bacteriocin production by Enterococcus
faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta
cheese making, Int. J. Food Microbiol. 72 (2002) 125–136.
[29] P. Sarantinopoulos, L. Makras, F. Vaningelgem, G. Kalantzopoulos, L. de Vuyst,
E. Tsakalidou, Growth and energy generation by Enterococcus faecium FAIR-E
198 during citrate metabolism, Int. J. Food Microbiol. 84 (2003) 197–206.
[30] F. Leroy, L. de Vuyst, Bacteriocin production by Enterococcus faecium RZS C5
is cell density limited and occurs in the very early growth phase, Int. J. Food
Microbiol. 72 (2002) 155–164.
[31] C. Herranz, J.M. Martínez, J.M. Rodríguez, P.E. Hernández, L.M. Cintas, Optimiza-
tion of enterocin P production by batch fermentation of Enterococcus faecium
P13 at constant pH, Appl. Microbiol. Biotechnol. 56 (2001) 378–383.
[32] B. Poolman, W.N. Konings, Relation of growth of Streptococcus lactis and Strep-
tococcus cremoris to amino acid transport, J. Bacteriol. 170 (1988) 700–707.
[33] B. Bibal, G. Goma, Y. Vayssier, A. Pareilleux, Influence of pH, lactose and lactic
acid on the growth of Streptococcus cremoris: a kinetic study, Appl. Microbiol.
Biotechnol. 28 (1988) 340–344.
[34] L.M.D. Gonçalves, A. Ramos, J.S. Almeida, A.M.R.B. Xavier, M.J.T. Carrondo, Elu-
cidation of the mechanism of lactic acid growth inhibition and production
in batch cultures of Lactobacillus rhamnosus, Appl. Microbiol. Biotechnol. 48
(1997) 346–350.
[35] N.P. Guerra, L. Pastrana, Influence of pH drop on both nisin and pediocin pro-
duction by Lactococcus lactis and Pediococcus acidilactici, Lett. Appl. Microbiol.
37 (2003) 51–55.
[36] J.A. Vázquez, M.L. Cabo, M.P. González, M.A. Murado, The role of amino acids
in nisin and pediocin production by two lactic acid bacteria. A factorial study,
Enzyme Microb. Technol. 34 (2004) 319–325.
[37] N.P. Guerra, A. Torrado, C. López, L. Pastrana, Modelling the fed-batch pro-
duction of pediocin using mussel processing wastes, Proc. Biochem. 40 (2005)
1071–1083.
[38] N.P. Guerra, P. Fajardo, A. Torrado, C. López, L. Pastrana, Fed-batch pediocin
production by Pediococcus acidilactici NRRL B-5627 on whey, Biotechnol. Appl.
Biochem. 42 (2005) 17–23.
[39] N.P. Guerra, P.F. Bernárdez, L.P. Castro, Modelling the stress inducing biphasic
growth and pediocin production by Pediococcus acidilactici NRRL B-5627 in re-
alkalized fed-batch cultures, Biochem. Eng. J. 40 (2008) 465–472.
[40] P. Fajardo, I. Rodríguez, L. Pastrana, N.P. Guerra, Production of a potentially
probiotic culture of Lactobacillus casei subsp. casei CECT 4043 in whey, Int. Dairy
J. 18 (2008) 1057–1065.
[41] R. Callewaert, L. de Vuyst, Bacteriocin production with Lactobacillus amylovorus
DCE 471 is improved and stabilized by fed-batch fermentation, Appl. Environ.
Microbiol. 66 (2000) 606–613.
[42] R. Luedeking, E.L. Piret, A kinetic study of the lactic acid fermentation. Batch
process at controlled pH, J. Biochem. Microbiol. Technol. Eng. 1 (1959) 393–412.
[43] R. Yang, B. Ray, Factors influencing production of bacteriocins by lactic acid
bacteria, Food Microbiol. 11 (1994) 281–291.
[44] W. Kozak, J. Bardowski, W.T. Dobrzanski, Lactostrepcin, a bacteriocin produced
by Streptococcus lactis, Bull. Acad. Sci. 45 (1977) 217–221.
[45] M.P. Ryan, M.C. Rea, C. Hill, P. Ross, An application in cheddar cheese man-
ufactured for a strain of Lactococcus lactis producing a novel broad-spectrum
bacteriocin, lacticin 3147, Appl. Environ. Microbiol. 62 (1996) 612–619.
[46] L. de Vuyst, E.J. Vandamme, Nisin, a lanthibiotic produced by Lactococcus lactis
subsp. lactis: properties, biosynthesis, fermentation and applications, in: L. de
Vuyst, E.J. Vandamme (Eds.), Bacteriocins of Lactic Acid Bacteria: Microbiology,
Genetics and Applications, Blackie Academic and Professional, London, 1994,
pp. 151–221.
[47] G. Giraffa, E. Neviani, G.T. Tarelli, Antilisterial activity by enterococci in a model
predicting the temperature evolution of Taleggio, an Italian soft cheese, J. Dairy
Sci. 77 (1994) 1176–1182.