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 Journal of Food Engineering 82 (2007) 103–113
www.elsevier.com/locate/jfoodeng

Dynamic mathematical models to describe the growth and

py
nisin production by Lactococcus lactis subsp. lactis CECT 539 in
both batch and re-alkalized fed-batch cultures

co
Nelson P. Guerra, Ana Torrado Agrasar, Cristina López Macı́as, 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

Received 3 June 2005; received in revised form 15 November 2006; accepted 20 November 2006
Available online 21 March 2007

al
on
Abstract

The growth and bacteriocin production by Lactococcus lactis subsp. lactis CECT 539 was studied in batch and in re-alkalized fed-
batch fermentations in media prepared with whey and mussel-processing wastes. From these cultures, mathematical models were devel-
oped to describe the growth and nisin production. The growth model developed which was based on Monod kinetics, has clearly more
rs

mechanistic approach and provides a major biological interpretability of the parameters than the logistic growth equation. This is the
first study presenting a pseudomechanistic model to describe the growth kinetics of lactic acid bacteria in re-alkalized fed-batch fermen-
tations. Nisin production was modeled with a modified form of the Luedeking and Piret model, which includes a term for the effect of the
pe

optimal final pH value in bacteriocin synthesis. Both models were demonstrated for three fed-batch cultivations of L. lactis using differ-
ent culture media, feeding substrates and times of re-alkalization and feeding.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Lactococcus lactis; Nisin; Fed-batch cultivation; Monod model; Modelling

r's

1. Introduction of the production costs using cheaper cultivation substrates

and/or improved cultivation procedures.
Nisin is a bacteriocin produced by Lactococcus lactis In the last years, both deproteinized diluted whey
o

that exhibits a broad spectrum of antibacterial activity (DDW) and mussel processing wastes (MPW) have been
against Gram-positive spoilage and pathogenic bacteria assayed for nisin production in batch cultures (Guerra &
th

present in foods. This peptide is innocuous, sensitive to Pastrana, 2002a; Guerra et al., 2001). Casitone and yeast
digestive proteases and it does not produce changes in extract were excellent nitrogen sources for nisin production
the organoleptic properties of the foods. For these reasons, in both media (Guerra & Pastrana, 2001; Guerra & Pastr-
Au

it has been proved to be an effective food biopreservative
(Guerra, Rua, & Pastrana, 2001). However, its cost
remains high. Then, an important factor to improve the
commercial utilization of nisin in foods is the reduction
*
Corresponding author. Tel.: +34 88 387 062; fax: +34 88 387 001.
E-mail address: pastrana@uvigo.es (L.P. Castro).
ana, 2002b), but they are too expensive and uneconomical
for a large-scale process.
Fed-batch cultivation technology provided with an ade-
quate control-system gives the possibility to obtain higher
amounts of biomass and bacteriocin than batch cultures
through the controlled supply of fresh substrate (Callewa-
ert & De Vuyst, 2000; Lee, Lee, Park, & Middelberg, 1999).
However, the lack of accurate models describing cell
growth and product formation is one of the reasons for

0260-8774/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2006.11.031
104 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113
which the control-system development for bacteriocin pro-
duction is not straightforward (Shimizu, Miura, Shioya, &
Suga, 1993).
Different models, such as the logistic and the Gompertz
equations, have been proposed to describe the growth of
lactic acid bacteria (LAB) (Lejeune, Callewaert, Crabbé,
& De Vuyst, 1998; McKellar & Lu, 2003; Rodrı́guez,
Alcalá, Gimeno, & Cosano, 2000). These models allows
to obtain the lag period (k), the maximum specific growth
rate (lmax) and the maximum biomass (Xmax) reached in
the culture, which are parameters with biological mean-
ing. However, these models do not explicitly reflect mech-
anistic knowledge on the self-limiting growth processes
when reaching the stationary phase (Leroy & De Vuyst,
2001). Moreover, in the cultures in which, the microbial
growth rate depends on the concentration of a single
growth-controlling substrate, it seems more convenient
the use of an adequate model for describing this relation-
ship (Kovárová-Kovar & Egli, 1998; Leroy & De Vuyst,
2001; Wick, Weilenmann, & Egli, 2002) like the Monod
model. This model relates the specific growth rate (l) to
the concentration of a single growth-controlling (limiting)
on
substrate (S) via two parameters, the maximum specific
growth rate (lmax) and the substrate affinity constant
(KS).
However, growth of LAB is often inhibited not only
rs
because of nutrient limitation, but also because of substrate
inhibition and the production of organic acids (mainly lac-
tic acid and acetic acid), which have a toxic and antimicro-
pe
bial effects, even for the producer cells. In such cases, the
classic Monod model must be modified by including terms
for accounting the inhibitory effects of substrate inhibition
and end product formation (Callewaert & De Vuyst, 2000).
Although bacteriocin production has found to be
growth-associated (Callewaert & De Vuyst, 2000; Guerra
& Pastrana, 2002a, 2002c), in some cases, the lack of pro-
r's
portionality between bacteriocin production rate and bac-
terial growth rate makes difficult to fit the experimental
bacteriocin data with the classic Luedeking and Piret
model (Luedeking & Piret, 1959). This was found to be
o
due to the specific influence of several key variables of
the culture (like pH or some nutrient limitation) on bacte-
th
riocin synthesis (Guerra & Pastrana, 2002a, 2002c; Guerra
et al., 2001; Parente & Riciardi, 1999).
The aim of this study was to find the most suitable
Au
model to describe the cause or mechanism behind the
dynamic growth changes of L. lactis subsp. lactis CECT
539 in batch and in periodically re-alkalized fed-batch
cultures in media prepared with deproteinized whey and
mussel processing wastes. The effects of the initial glu-
tamic acid concentration on the production of biomass
and nisin were studied in batch cultures. Re-alkalized
fed-batch cultures were carried out using both media
and different feeding substrates. The growth-associated
nisin production by L. lactis CECT 539 has also been
modelled using a modified form of the Luedeking and
Piret model.
2. Materials and methods
2.1. Microorganisms, culture media, inoculum preparation
L. lactis subsp. lactis CECT 539, the nisin-producing
strain and Carnobacterium piscicola CECT 4020, the target
organism, were acquired from the Spanish type culture col-
lection (CECT) and was maintained at 4 °C on MRS (de
py
Man Rogosa and Sharpe) agar slants at 4 °C.
Whey and mussel processing wastes were used as a base
of the culture media in this work. Whey obtained from a
local dairy plant was used in two forms: as concentrated
co
whey (CW: the liquid remaining after the first cheese press-
ing) and as diluted whey (DW: CW mixed with wash
waters). Both media were prepared as follows (Guerra
et al., 2001): after adjusting the pH to 4.5 with 5 N HCl,
the media were heated at 121 °C for 15 min to denature
the proteins, and the precipitates were removed by centrifu-
gation (12,000g for 15 min). The supernatants were
al
adjusted to pH 6.3, sterilised at 121 °C for 15 min and used
as culture media.
The mussel processing wastes (MPW, glycogen as main
component: 5–10 g/L) were obtained from a local mussel
processing plant. Two media were obtained from this
waste: the MPW (with an average glycogen level of 5 g/
L) used as fermentation medium and a concentrated
MPW (CMPW) medium, which was used as a feeding sub-
strate in the corresponding re-alkalized fed-batch fermenta-
tion. The MPW media were prepared as follows (Guerra &
Pastrana, 2002a): after adjusting the pH value to 4.5 with
5 N HCl, the media were decanted during 3 h. The precip-
itates formed were eliminated through centrifugation
(12,000g for 15 min). To obtain the CMPW medium, a
deproteinized MPW medium (10 g of glycogen/L) was con-
centrated by ultrafiltration-dialysis at 100 kDa until
obtaining a medium with an average glycogen level of
100 g/L and a low content of NaCl, non-protein nitrogen
and phosphorous in comparison to that of the MPW med-
ium. The glycogen contained in both the MPW and
CMPW media was saccharified to glucose by enzymatic
hydrolysis at 40 °C for 1 h, using a San Super 240 L com-
mercial preparation of a-amylases obtained from Novo
Nordisk, Denmark. The enzyme:medium ratio was 1:1000
(v/v). To be used as culture media, the saccharified MPW
media were adjusted to pH 6.3 and sterilized at 121 °C
for 15 min.
The compositions of the diluted deproteinized whey
(DDW), concentrated deproteinized whey (CDW), MPW
and CMPW, which were used in both the batch and re-alk-
alized fed-batch fermentations are shown in Table 1.
A loop of cells from a 1-day old MRS slant was used to
inoculate 10 mL of MRS broth (culture tubes) that were
incubated at 30 °C/12 h (200 rpm). The inoculum for
experiments was prepared by transferring 1 mL of the
above preculture to 250 mL Erlenmeyer flask containing
50 mL of DDW or MPW medium and incubating at
30 °C/12 h (200 rpm).
 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113
Table 1
Initial concentrations (mean ± standard deviations) of total sugars (TS), nitrogen (TN), phosphorous (TP) and proteins (Pr) in the culture media prepared
with diluted deproteinized whey (DDW), concentrated deproteinized whey (CDW), mussel processing wastes (MPW) and concentrated mussel processing
wastes (CMPW)
Concentration (g/L) DDW
TS 20.54 ± 0.514
TN 0.45 ± 0.014
TP 0.25 ± 0.021
Pr 2.04 ± 0.083
The analytical determinations were performed in triplicate by using the corresponding standard methods described in Section 2.
2.2. Batch cultures
Previous batch cultures in buffered and non-buffered
whey (Guerra et al., 2001) and MPW (Guerra & Pastrana,
2002a) media showed that the higher amounts of nisin were
produced in the non-buffered DDW medium and in the
MPW medium buffered at pH 6.3 with 0.1 M potassium
hydrogen phthalate/NaOH. Therefore, the study of the
influence of the initial concentration of glutamic acid
(GA) on the growth and nisin production by L. lactis,
was performed in these media (initial pH of 6.3), which
were supplemented with GA to obtain initial GA concen-
trations of 1, 2, 3, 4 and 5 g/L. Media without GA were
used as controls. The media were sterilized (121 °C/
rs
15 min), inoculated with a 2% (v/v) of the corresponding
inoculum and incubated at 30 °C/24 h. All batch cultures
were performed in 250 mL Erlenmeyer with 50 mL of pro-
pe
duction media in a rotary shaker (200 rpm), using eight
flasks in each fermentation series. The culture samples,
which comprise an experimental unit (one flask), were
withdrawn each 3 h to perform the analytical determina-
tions.
2.3. Re-alkalized fed-batch fermentations
r's
Fed-batch fermentations were carried out in a 6 L bench
top fermentor (New Brunswick Scientific, New Jersey) with
4 L working volume of medium at a controlled temperature
o
of 30 °C and at a controlled constant agitation of 200 rpm
(Guerra & Pastrana, 2003a). Since the oxygen supply stim-
th
ulated the production of both nisin A (Amiali, Lacroix, &
Simard, 1998) and Z (Cabo, Murado, González, & Pastor-
iza, 2001), a constant aeration flow rate of 0.5 L/h was used
Au
during the fermentations.
In batch cultures of L. lactis, the highest nisin produc-
tions were obtained after 8 and 12 h of incubation in the
unsupplemented MPW and DDW medium, respectively,
when the cultures reached the lower steady pH value. For
this reason, the re-alkalized cultivations were performed
by re-alkalizing the cultures repeatedly up to the initial
pH with 4 N NaOH each 8 and 12 h in MPW and DDW
medium, respectively. Before re-alkalizing the cultures,
100 mL of medium were aseptically removed from the fer-
mentor and an equal volume of the feeding substrate was
added to bring the culture up to the initial total sugars
105
CDW MPW CMPW
48.11 ± 1.206 5.33 ± 0.211 101.33 ± 1.314
1.05 ± 0.051 0.65 ± 0.021 0.54 ± 0.024
0.43 ± 0.082 0.14 ± 0.014 0.06 ± 0.009
5.02 ± 0.111 2.09 ± 0.067 3.47 ± 0.046
py
co
concentration (21 g/L in DDW culture and 5.3 g/L in
MPW cultures).
The cultures were fed with substrates that contained the
same carbon sources as the fermentation media (DDW and
MPW). Therefore, the re-alkalized fed-batch culture in
DDW medium (fermentation I) was fed with a mixture of
concentrated lactose (400 g/L) and CDW medium. The
al
two cultures in MPW medium were fed with a 240 g/L con-
centrated glucose (fermentation II) and CMPW medium
(fermentation III), respectively.
on
The amounts of biomass, nutrients or products remain-
ing inside the reactor at the time of measurement (at
t = tn) were fitted by a mathematical model to describe
the fed-batch biomass and nisin production by L. lactis
CECT 539.
2.4. Analytical methods
Growth was monitored by absorbance at 700 nm and
converted to dry cell weight from a standard curve. Cells
were harvested by centrifugation (12,000g for 15 min at
4 °C) of culture samples and washed twice with saline
(0.8% NaCl). The culture supernatants were used to deter-
mine total sugars (phenol-sulphuric acid method (Dubois,
Giles, Hamilton, Rebers, & Smith, 1956) according to
Strickland & Parsons (1968) with glucose (Panreac, Barce-
lona, Spain) as standard), phosphorous (molybdate reac-
tion (Murphy & Riley, 1962) according to Strickland &
Parsons (1968)) with potassium di-hydrogen phosphate
((Panreac, Barcelona, Spain) as standard), nitrogen
(micro-Kjeldahl, substituting distillation for the spectro-
photometric method of Havilah, Wallis, Morris, & Wool-
nouugh (1977) with ammonium sulphate (Panreac,
Barcelona, Spain) as standard) and proteins (Method of
Lowry, Rosebrough, Farr, & Randall (1951) with bovine
serum albumin (Sigma, St. Louis, MO, USA) as stan-
dard). The concentrations of lactic and acetic acids,
butane-2,3-diol and ethanol were measured by HPLC
using an ION-300*7,8 mm column with a pre-column
IONGUARDTM. The mobile phase consisted of 0.006 N
H2SO4 as a flow rate of 0.4 mL/min at (60–65) °C and
the refractive index of the peaks was measured by a
refractometer with a RI detector (Guerra & Pastrana,
2003a). All analytical determinations were performed in
triplicate.
106 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113

2.4.1. Bacteriocin activity assays
Aliquots from cultures of L. lactis were adjusted to pH
3.5 with 5 N HCl to avoid the adsorption of molecules of
bacteriocin onto the producer cell surfaces. Thereafter they
were heated for 3 min to kill the cells and centrifuged at
27,200g for 15 min at 4 °C. The supernatants containing
overall antibacterial activity (bacteriocin extract) were
adjusted to pH 6.0 and frozen until further use.
where N is the total nitrogen concentration (g/L) over the
time. Nmin is the minimum total nitrogen concentration
(g/L) that supports the growth. KN is the corresponding
substrate affinity constant for the nitrogen source (g/L).
LA is the lactic acid concentration (g/L) and KLA is the
inhibition constant of lactic acid (g/L).
Biomass formation is hence given by the following
equation:

py
Antibacterial activity of bacteriocin extracts was deter- dX
mined by a photometric bioassay method (Cabo, Murado, ¼ ðl  k d Þ  X ð4Þ
dt
González, & Pastoriza, 1999) using the C. piscicola as target
organism. Bacteriocin extracts were diluted as needed in dis- where X is the biomass concentration (grams of cell dry
mass (CDM) per liter), t is the time (h) and kd is the cell

tilled sterile water (this step eliminated the need to correct the
pH of bacteriocin extracts). Diluted bacteriocin (2.5 mL)
extracts were added in sterile culture tubes. Each tube was
inoculated with 2.5 mL of a culture of C. piscicola (diluted
to an absorbance of 0.2 at 700 nm with sterile buffered
MRS broth (pH 6.3)). Controls consisted in three culture
tubes in which the diluted bacteriocin extract was substituted
co
death rate (h1).
Nisin production in batch cultures was modelled using
both the Luedeking and Piret model (Luedeking & Piret,
1959) (model 5) and a modified form of it (model 6), which
includes a term for the effect of the optimum final pH value
on bacteriocin synthesis (Guerra & Pastrana, 2002a;

al
by distilled sterile water. The tubes were incubated for 6 h at Guerra et al., 2001; Yang & Ray, 1994):
30 °C. Growth inhibition was measured spectrophotometri- dNis
cally at 700 nm. Dose/response curves were obtained from ¼ ða  l  X þ b  X Þ ð5Þ
dt
on   
these data. The values of nisin activity were quantified as dNis pHt  pHop

¼ ða  l  X þ b  X Þ  1  j    ð6Þ
described in Murado, Gonzalez, and Vazquez (2002). dt pHop 
2.5. Model development where Nis is the nisin concentration (BU/mL, 1 BU corre-
rs

sponds to 20 International Units (IU)), a is a growth-asso-

2.5.1. Batch cultures ciated constant for bacteriocin production (BU/mg), which
The classical Monod equation relates the specific growth corresponds to the yield product on biomass formed (YP/X)
rate (l in h1) to the concentration of a single growth-con- for growth-associated metabolites, and b is the non-
pe

trolling substrate (S in g/L): growth-associated constant (BU mg1 h1), j is a constant

of proportionality, pHop is the optimum final pH, from
 
S which bacteriocin production stopped, and pHt is the pH
l ¼ lmax ð1Þ value in each sampling time.
KS þ S

where lmax is the maximum specific growth rate (h1) and 2.5.2. Re-alkalized fed-batch cultures
r's

KS is the substrate affinity constant (g/L).
However, a weakness of this model is that it neglects the
concept of maintenance energy, which implies the utiliza-
tion of substrate even at a specific growth rate of 0 h1
o
Biomass production by L. lactis in the three re-alkalized
fed-batch cultures was modelled using Eq. (4). Since nitro-
gen was the growth limiting substrate and it seemed to be
consumed in two phases, the specific growth rate was mod-

(Wick et al., 2002). For this reason, the original Monod elled by using a diauxic Monod-type equation (Reardon,
model has been modified by introducing a Smin term, which Mosteller, & Rogers, 2000) multiplied by a dimensionless
th

denotes the minimal substrate concentration needed for
growth (Kovárová, Zehnder, & Egli, 1996). Thus, the over-
all form of the pseudomechanistic model is
Au
function (ci) for accounting the inhibition by product for-
mation (Callewaert & De Vuyst, 2000):
 
lmax1  ðN  N min 1 Þ lmax2  ðN  N min2 Þ
l¼ þ

  K N 1 þ ðN  N min1 Þ K N 2 þ ðN  N min2 Þ
S  S min
l ¼ lmax ð2Þ  ci ; being ci ð7Þ
K S þ ðS  S min Þ
 
K LA
Since the nitrogen source influenced the growth of ci ¼ cLA  cAA  cEt  cB ¼
LA þ K LA
L. lactis in batch cultures in DDW and MPW media, and      
K AA K Et KB
taking into account that product formation (Callewaert    ð8Þ
AA þ K AA Et þ K Et B þ KB
& De Vuyst, 2000) can inhibit biomass production, the spe-
cific growth rate can be modelled by the relation: where lmax1 and lmax2 are the specific growth rates (h1) of
   the exponential growth phases 1 and 2, respectively. KN1
ðN  N min Þ K LA and KN2 are the nitrogen affinity constants (g/L) in each
l ¼ lmax ð3Þ
K N þ ðN  N min Þ K LA þ LA phase of nitrogen consumption. Nmin1 and Nmin2 are the min-
 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113 107

imum nitrogen concentrations (g/L) that support the growth
in each phase. cLA, cAA, cEt and cB are the individual inhi-
bition functions for lactic acid, acetic acid, ethanol and bu-
tane-2,3-diol inhibition, respectively. KLA, KAA, KEt and
KB are the inhibition constants (g/L) for lactic acid, acetic
acid, ethanol and butane-2,3-diol, respectively. LA, AA, Et
and B are the total concentrations (g/L) of lactic acid, acetic
acid, ethanol and butane-2,3-diol over the time, respectively.
containing seven growth-stimulating amino acids (histi-
dine, isoleucine, arginine, threonine, leucine, valine and
methionine) did not allow any growth of L. lactis subsp.
lactis NIZO 22186. This fact clearly showed the absolute
requirement of glutamic acid for the growth of this strain.
However, the effect of increasing concentrations of this
amino acid on the growth of L. lactis was not assayed until
now.

The production of nisin in the three re-alkalized fed-
batch cultures was described with models (5) and (6), using
the values of X and l previously calculated from Eqs. (4)
and (7), respectively.
py
The influence of the initial glutamic acid concentration (0,
1, 2, 3, 4 and 5 g/L) on both cell growth and nisin production
by L. lactis subsp. lactis CECT 539 was assessed in DDW and
MPW media. The experimental and the modelled values (see

All models were fitted using the SigmaPlot program,
version 8.0 (Systat Software Inc.). The coefficients of the
models with P values lower than 0.05 were considered sta-
tistically significant. The criteria used to evaluate the good-
ness-of-fit of each model were the determination coefficient
(r2) and the mean relative percentage deviation modulus
(RPDM) (Lomauro, Bakshi, & Labuza, 1985):
co
below) for the concentrations of biomass and nisin are
shown in Figs. 1 and 2. From these results, it can be observed
that increasing the initial glutamic acid concentration had a
positive effect on the growth of L. lactis and consequently on
nisin production. In addition, the active period of growth
increased from 12 and 15 h in the unsupplemented DDW
and MPW media to 15 and 18 h in the supplemented media,

al
respectively. Letort, Nardi, Garault, Monnet, and Juillard
100 X
N
jX i  X pi j
RPDM ¼ ð9Þ (2002) reported that the use of glutamine (2.6 g/L) and
N i¼1 Xi methionine (1.0 g/L) to supplement milk resulted in a mod-
on
where Xi is the experimental value, Xpi is the calculated va- ification of the growth of Streptococcus thermophilus ST18.
lue and N is the number of experimental data. The RPDM This strain displayed two exponential growth phases, sepa-
and r2 parameters are widely used to determine the quality rated by a non-exponential one in the unsupplemented milk,
of the fit, being a value of RPDM below 10% and a value of while it displayed only one single exponential growth phase
rs

r2 P 0.95 (95%) indicative of a good fit for practical pur- in the supplemented milk. The above results clearly showed
poses (Aguerre, Suarez, & Viollaz, 1989; Guerra, Torrado, that the nitrogen source plays an important role in the
López, & Pastrana, 2005; Lomauro et al., 1985). In all growth of these lactic acid bacteria, which can use it as both
pe

cases, RPDM < 9% and r2 > 0.95 (Tables 2–4), and the cor-
relations according to these criteria are very satisfactory.
3. Results and discussion
3.1. Batch nisin production in media prepared with
deproteinized whey and mussel processing wastes
r's
nitrogen and carbon source (Cocaign-Bousquet, Garrigues,
Lubiere, & Lindley, 1996; Guerra et al., 2005; Thomas, Ell-
wood, & Longyear, 1979).
Although the supplements with glutamic acid stimulated
highly the nitrogen consumption by L. lactis and conse-
quently its growth, surprisingly all the cultures reached the
same final nitrogen concentration (threshold nitrogen con-

supplemented with glutamic acid
A previous study (De Vuyst, 1995) showed that the
omission of glutamic acid from a synthetic culture medium
o
centration) as the control cultures (0.33 g/L in DDW and
0.47 g/L in MPW). Thus, the growth of L. lactis increased
with the increase in the glutamic acid concentration in the
supplemented media due to a high availability of this free

Table 2
th

Variation of the estimated parameters for the growth and nisin production during batch growth of L. lactis in DDW medium supplemented with different
initial concentrations of glutamic acid
Parameter Initial glutamic acid concentration (g/L)
Au

0 1 2 3 4 5
lmax (h1) 0.224 ± 0.014 0.277 ± 0.007 0.307 ± 0.008 0.331 ± 0.008 0.349 ± 0.006 0.481 ± 0.009
KN (g/L) 0.015 ± 0.002 0.060 ± 0.008 0.106 ± 0.011 0.150 ± 0.015 0.184 ± 0.013 0.188 ± 0.021
Nmin (g/L) 0.327 ± 0.001 0.341 ± 0.001 0.334 ± 0.001 0.332 ± 0.001 0.333 ± 0.001 0.336 ± 0.002
KLA (g/L) 9.19 ± 2.041 7.73 ± 0.284 5.58 ± 0.178 4.17 ± 0.278 3.91 ± 0.154 5.31 ± 0.260
r2X 0.9946 0.9995 0.9992 0.9975 0.9987 0.9971
RPDM 3.713 3.341 3.978 4.724 3.034 6.172
a (BU/mg) 70.6 ± 3.18 139.6 ± 7.57 139.6 ± 18.29 139.5 ± 33.44 83.0 ± 8.24 93.0 ± 11.14
j 7.97 ± 0.86 8.44 ± 0.36 8.44 ± 0.64 8.44 ± 1.51 4.0 ± 0.81 4.3 ± 2.16
pHop 4.90 ± 0.018 4.94 ± 0.02 4.94 ± 0.04 4.94 ± 0.12 4.78 ± 0.11 4.78 ± 0.09
r2Nis 0.9919 0.9967 0.9970 0.9938 0.9911 0.9926
RPDM 4.968 5.515 6.944 3.977 6.410 5.909
Statistically significant coefficients (P < 0.05) are expressed as means ± standard errors.
108 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113

Table 3
Variation of the estimated parameters for the growth and nisin production during batch growth of L. lactis CECT 539 in MPW medium supplemented
with different initial concentrations of glutamic acid
Parameter Initial glutamic acid concentration (g/L)
0 1 2 3 4 5
1
lmax (h ) 0.307 ± 0.009 0.320 ± 0.012 0.340 ± 0.016 0.360 ± 0.017 0.377 ± 0.020 0.380 ± 0.040
KN (g/L) 0.032 ± 0.006 0.036 ± 0.011 0.052 ± 0.021 0.119 ± 0.028 0.177 ± 0.039 0.221 ± 0.096
Nmin (g/L) 0.495 ± 0.001 0.496 ± 0.002 0.493 ± 0.003 0.502 ± 0.003 0.499 ± 0.004 0.488 ± 0.009
KLA (g/L) 0.728 ± 0.049 0.721 ± 0.053 0.700 ± 0.062 0.918 ± 0.061 1.072 ± 0.072 1.195 ± 0.162

py
r2X 0.9999 0.9998 0.9999 0.9988 0.9950 0.9958
RPDM 2.708 4.575 3.716 2.602 2.846 5.162
a (BU/mg) 68.1 ± 1.303 55.6 ± 0.875 52.2 ± 1.071 51.4 ± 0.903 51.0 ± 1.193 49.4 ± 0.669
j 1.51 ± 0.053 0.661 ± 0.261 0.174 ± 0.030 0.768 ± 0.343 1.063 ± 0.474 0.419 ± 0.164

co
pHop 4.99 ± 0.185 4.80 ± 0.080 4.84 ± 0.069 4.83 ± 0.063 4.70 ± 0.049 4.93 ± 0.151
r2Nis 0.9958 0.9973 0.9945 0.9963 0.9950 0.9965
RPDM 2.794 3.196 4.170 3.174 2.584 2.625
Statistically significant coefficients (P < 0.05) are expressed as means ± standard errors.

Table 4
Values of the biokinetic parameters for growth (model 4) and nisin (model 6) production by L. lactis in the re-alkalized fed-batch fermentations

al
Parameter Whey MPW (I) MPW (II)
lmax1 (h1) 0.0476 ± 0.0050 0.0900 ± 0.0073 0.0787 ± 0.0160
lmax2 (h1) 0.0414 ± 0.0059 0.1167 ± 0.0117 0.0580 ± 0.0117
on
KN1 (g/L) 0.0126 ± 0.0012 0.1078 ± 0.0136 0.0400 ± 0.0106
KN2 (g/L) 0.0500 ± 0.0035 0.0322 ± 0.0085 0.0028 ± 0.0001
Nmin1 (g/L) 0.2478 ± 0.0018 0.4367 ± 0.0131 0.3061 ± 0.0189
Nmin2 (g/L) 0.1523 ± 0.0027 0.0660 ± 0.0059 0.1200 ± 0.0376
kd (h1) NS NS 0.0112 ± 0.0005
rs

KLA (g/L) 0.3981 ± 0.0092 1.5813 ± 0.1607 4.2627 ± 0.0002

KAA (g/L) NS NS NS
KEt (g/L) – – 6.8897 ± 0.0013
KB (g/L) NS NS –
pe

r2X 0.9859 0.9870 0.9766

RPDM 7.598 5.986 9.282
a (BU/mg) 40.2274 ± 9.0741 24.8855 ± 8.2086 18.2040 ± 4.3511
b (BU  mg1  h1) NS NS NS
j 2.4923 ± 0.8684 1.9491 ± 0.3949 2.4556 ± 0.9218
pHop 4.2701 ± 0.2290 3.8203 ± 0.2421 3.8130 ± 0.3671
r2Nis 0.9892 0.9891 0.9754
RPDM 3.594 4.894 7.000
r's

Statistically significant coefficients (P < 0.05) are expressed as means ± standard errors. NS: No significant (P > 0.05).

amino acid. When the cultures reached the threshold nitro- substrates as feeding media (Figs. 3–5). The cumulative
o

gen concentration, the growth stopped even though the car- concentrations of biomass (X), nisin (Nis), nutrient (total
bon sources were not completely consumed (Figs. 1 and 2). nitrogen and sugars) and product (lactic acid, acetic acid,
th

This limitation in growth can be explained by the fact that
some growth-limiting factor must begin to play an important
role when the cultures reached the threshold nitrogen con-
centration. This factor could be a final nitrogen concentra-
Au
ethanol or butane-2,3-diol) obtained in these cultures were
used not only for discussing the fed-batch nisin production
kinetics but also for calculating the fermentation yield coef-
ficients (Guerra & Pastrana, 2003a). In these cultures, the

tion unable to be directly used as nitrogen source by L.
lactis, the final pH values reached in the cultures, or the
exhaustion of a necessary medium component (one or sev-
eral amino acids, cations or vitamins) essential for the
growth (Guerra & Pastrana, 2002c; Letort et al., 2002).
3.2. Repeated re-alkalized fed-batch cultures of L. lactis in
MPW and DDW media
Three re-alkalized fed-batch fermentations of L. lactis in
DDW and MPW media were carried out using different
nitrogen source seemed to be consumed in two steps and
consequently, biomass concentration displayed two expo-
nential growth phases and two nonexponential growth
phases. In addition, biomass concentration stopped when
the cultures reached a low nitrogen concentration, even
though the carbon source levels were still sufficiently avail-
able. These observations suggested that the nitrogen source
was the growth limiting substrate in the three
fermentations.
Similar results have been reported with some strains of
L. lactis and S. thermophilus, which displayed two distinct
 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113
on
rs
Fig. 1. Batch fermentations of Lactococcus lactis subsp. lactis CECT 539
on non-supplemented DDW medium (d), and on DDW medium
supplemented with 1 (e), 2 (M), 3 (h), 4 (O) and 5 (s) g of glutamic
acid per litre of medium. The curves drawn through the experimental
pe
biomass and nisin data were obtained according to the models (4) and (6),
respectively. Nis, nisin concentration; X, biomass concentration; CDM,
cell dry mass; TS, total sugars; TN, total nitrogen; LA, lactic acid.
exponential growth phases in milk (Flambard, Richard, &
Juillard, 1997; Juillard et al., 1995; Letort et al., 2002;
Niven, Knight, & Mulholland, 1998) as a consequence of
r's
a biphasic nitrogen metabolism (Niven et al., 1998). The
free amino acids and utilizable oligopeptides originally
present in milk are reported to be the main nitrogen source
for growth during the first growth phase, whereas the case-
o
ins are the source of amino acids in the second growth
phase (Flambard et al., 1997; Juillard, Helinck, Flambard,
th
Foucaud, & Richard, 1998; Juillard et al., 1995; Letort
et al., 2002).
With regard to this biphasic nitrogen consumption, a
Au
similar behavior was also observed in batch cultures of Gib-
berela fujikuroi on MPW medium (Pastrana, González, &
Murado, 1993), in which a first step (48 h) of intense con-
sumption of inorganic nitrogen and free amino acids pres-
ent in the medium was followed by a second step of low
nitrogen consumption.
3.3. Determination of cell growth and bacteriocin production
model parameters
Biokinetic cell growth parameters were determined by
simulating the profiles of batch and fed-batch cultures
109
py
co
al
Fig. 2. Batch fermentations of Lactococcus lactis subsp. lactis CECT 539
on non-supplemented MPW medium (d), and on MPW medium
supplemented with 1 (e), 2 (M), 3 (h), 4 (O) and 5 (s) g of glutamic
acid per litre of medium. The curves drawn through the experimental
biomass and nisin data were obtained according to the models (4) and (6),
respectively. Notations are as in Fig. 1.
according to the growth models (4) and (7) described
above. Standard errors of the statistically significant coeffi-
cients (P < 0.05) are shown in Tables 2–4. High values of r2
(higher than 0.97) and low values of RPDM (below 10%) in
all cases have strengthen the usefulness of models (4) and
(7) for describing growth in batch and re-alkalized fed-
batch fermentations.
In batch cultures in DDW and MPW media, increased
values for lmax calculated from model (3) were obtained
with the increase in glutamic acid concentration, thus
showing that this nitrogen source has growth-stimulating
properties for L. lactis. In addition, the levels of Nmin are
in good agreement with the actual experimental values in
each case. The parameter kd was calculated as zero in all
batch cultures.
With regard to bacteriocin production in batch cultures,
the results showed that nisin production was clearly
growth-associated (a 6¼ 0, b = 0). In addition, the values
obtained for pHop oscillated between 4.78 and 4.99
(P < 0.05) in DDW and MPW media, respectively (Tables
2 and 3). Since the correlations of the model (6)with the
experimental values were satisfactory (r2 > 0.99;
RPDM < 7%), nisin can be classified as a pH-dependent
primary metabolite.
110 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113
on
rs
pe
Fig. 3. Experimental data (symbols) of biomass and nisin production by
Lactococcus lactis subsp. lactis CECT 539 in the re-alkalized fed-batch
culture in DDW medium with feeding with concentrated deproteinized
whey (48.11 g of total sugars/L) and a 400 g/L concentrated lactose. The
r's
solid and dashed lines drawn through the experimental biomass data were
obtained according to the pseudomechanistic growth model (4) and the
modified logistic growth (MLG) model (10), respectively. The dashed and
solid lines drawn through the experimental nisin data were obtained
according to the Luedeking and Piret (L–P) models (5) and (6),
o
respectively. AA, acetic acid; B, butane-2,3-diol. Other notations are as
in Fig. 1.
th
With respect to the re-alkalized fed-batch cultures, a
modified logistic equation was presented by Cabo et al.
Au
(2001) to describe the growth of a L. lactis strain in period-
ically re-alkalized fed-batch cultures in TGE (Tryptone
Glucose Extract) broth:
K and calculated cell growth data (dashed lines in Figs. 3–
X ¼ ð10Þ
1 þ eðclð1þb0 V pHÞtÞ
 
K
being : c ¼ ln 1
X0
where K and X0 are respectively, the maximum and the ini-
tial biomass concentration (g of CDM per litre), b0 is a con-
stant of proportionality, t is the time (h) and VpH is the
py
co
al
Fig. 4. Experimental data (symbols) of biomass and nisin production by
Lactococcus lactis subsp. lactis CECT 539 in the re-alkalized fed-batch
culture in MPW medium with feeding with a 240 g/L concentrated
glucose. The solid and dashed lines drawn through the experimental
biomass data were obtained according to the pseudomechanistic growth
model (4) and the modified logistic growth (MLG) model (10), respec-
tively. The dashed and solid lines drawn through the experimental nisin
data were obtained according to the Luedeking and Piret (L–P) models (5)
and (6), respectively. Notations are as in Figs. 1 and 3.
decrease in pH per unit of time in each re-alkalization
cycle.
However, the use of model (10) to describe the growth of
L. lactis CECT 539 in the re-alkalized fed-batch cultures in
DDW and MPW media had some major drawbacks
because it leads to an overestimation of the initial biomass
concentration and a loss of fit between the experimental
5). This was probably due to the fact that the modified
logistic model does not consider the relation between the
specific growth rate and the concentration of the growth-
controlling (limiting) substrate.
By using the pseudomechanistic model developed in this
study, the biokinetic parameters corresponding to each
growth phase were calculated (for P < 0.05) in the three
 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113
rs
pe
Fig. 5. Experimental data (symbols) of biomass and nisin production by
Lactococcus lactis subsp. lactis CECT 539 in the re-alkalized fed-batch
culture in MPW medium with feeding with CMPW (101.33 g of glucose/L)
medium. The solid and dashed lines drawn through the experimental
r's
biomass data were obtained according to the pseudomechanistic growth
model (4) and the modified logistic growth (MLG) model (10), respec-
tively. The dashed and solid lines drawn through the experimental nisin
data were obtained according to the Luedeking and Piret (L–P) models (5)
and (6), respectively. Et, ethanol. Other notations are as in Figs. 1 and 3.
o
th
re-alkalized fed-batch fermentations (Table 4). The values
obtained for the biokinetic parameters (means ± standard
errors) as well as the values of r2 and RPDM (r2 > 0.97;
Au
RPDM < 10 %) showed that model (7) described satisfac-
torily the trend observed for the experimental growth data
(Table 4).
Since, the calculated values for KAA and KB (>20 g/L)
were much higher than the concentrations of acetic acid
and butane-2,3-diol produced in the three cultures (Figs.
3–5), the values of the terms cAA and cB tended towars
1. In addition, both parameters were found to be not signif-
icant (P > 0.05) in the growth model (7). This indicates that
the increase in the concentrations of both products did not
produce an important influence on the growth of L. lactis.
Therefore, the specific growth rates in each re-alkalized
111
fed-batch culture were recalculated by suppressing the
terms cAA and cB from the model (7).
The values of Nmin predicted by the model (P < 0.05) are
in perfect agreement with the experimental values observed
in the two growth phases in each fed-batch culture (Figs. 3–
5). The solid lines in the right upper part of Figs. 3–5 are
the trajectories predicted by the models (4) after calculating
the values of l with the model (7). As it can be observed,
py
model (4) offered a more accurate description of the diauxic
growth of L. lactis subsp. lactis CECT 539 in relation with
the biphasic nitrogen consumption than the modified logis-
tic model (10).
co
With respect to nisin production, the introduction of a
term for the optimum pH value (pHop) into the bacteriocin
model led to an improved fitting of the soluble bacteriocin
activity data ((r2 > 0.97; RPDM 6 7%)), compared to that
obtained with the Luedeking and Piret model (dashed lines
in Figs. 3–5). Thus, the optimum final pHs predicted by
model (6) were 4.27 for the fermentation I and 3.82 for
al
the fermentations II and III (P < 0.05). These values are
in perfect agreement with the actual final pH values
(4.18, 4.00 and 4.06) obtained in the first re-alkalization
on
cycle for the three fed-batch fermentations, in which the
highest nisin production rates were obtained (Figs. 3–5).
Therefore, nisin production was favoured with the higher
pH drops (or the lowest final pH values) as it was observed
before (Cabo et al., 2001; Guerra et al., 2001). These obser-
vations support the proposal about the existence of an
optimum final pH for bacteriocin production (Guerra &
Pastrana, 2003b; Guerra et al., 2001; Yang & Ray, 1994).
The calculated parameters (a, b, j, pHop) from model (6)
revealed that nisin production depended on both the
growth rate (a  b) and the final pH value reached in each
re-alkalization cycle (j 6¼ 0) (Table 4). Thus, nisin was pro-
duced in these cultures as a pH-dependent primary
metabolite.
4. Conclusions
The main contribution of this paper is the introduction
of a novel pseudomechanistic model which reflects the bio-
logical phenomena influencing the growth of L. lactis dur-
ing re-alkalized fed-batch fermentations in DDW and
MPW media. The growth model was developed by using
some well-known metabolic LAB characteristics, namely,
(i) increase of the biomass according to a Monod equation,
(ii) growth-associated nitrogen consumption, and (iii)
inhibitory effects of end products (lactic acid, acetic acid,
ethanol or butane-2,3-diol). To our knowledge, it is the first
time that the diauxic growth of L. lactis is modelled as a
function of the biphasic nitrogen consumption. The param-
eters obtained are microbiologically more relevant than
those obtained with the modified logistic models. A modi-
fied form of the Luedeking and Piret model was devel-
oped to describe nisin production in response to growth
and to final pH reached in each re-alkalization cycle. The
above-described mathematical models could be applied to
112 N.P. Guerra et al. / Journal of Food Engineering 82 (2007) 103–113
simulate the growth and bacteriocin production by other
LAB in re-alkalized fed-batch fermentations.
These models improve our understanding of the com-
petitive features of L. lactis subsp. lactis CECT 539 under
repeated fed-batch and re-alkalized fermentations on both
MPW and whey medium. This makes possible the devel-
opment of sudomechanistic models to monitor and con-
trol the growth and nisin production in these types of
cultures.
Acknowledgements
The research presented in this paper was financially sup-
ported by the Instituto Nacional de Investigación y Tec-
nologı́a Agraria y Alimentaria (INIA), Spain (project
CAL01-045-C2-2) and The Xunta de Galicia, Spain (pro-
ject PGIDT00BIO1E).
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