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International Dairy Journal 18 (2008) 1057–1065

Contents lists available at ScienceDirect

International Dairy Journal

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

Production of a potentially probiotic culture of Lactobacillus casei subsp. casei

CECT 4043 in whey
Paula Fajardo Bernárdez, Isabel Rodrı́guez Amado, Lorenzo Pastrana Castro, Nelson Pérez Guerra*
Departamento de Quı́mica Analı́tica 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: Batch and fed-batch fermentations for the production of a highly concentrated culture of Lactobacillus
Received 29 October 2007 casei CECT 4043 were carried out in whey-based media. In batch fermentation, the maximum concen-
Received in revised form 14 May 2008 trations of biomass (0.33 g L1) and antibacterial activity (3.42 AU mL1) were obtained in diluted whey
Accepted 18 May 2008
medium (2% w/v lactose, initial pH 7.0) after 12 h. Based on these results, biomass and antimicrobial
metabolites production in two re-alkalized fed-batch fermentations were carried out in diluted whey
medium. The first fed-batch culture was fed with concentrated whey (5% w/v lactose) and a 400 g L1
concentrated lactose solution. The second culture, which was fed with a concentrated mussel processing
waste (10% w/v glucose) and a 310 g L1 concentrated glucose solution, provided the highest concen-
trations of biomass (1.7 g L1), viable cells (w1.7  1010 cfu mL1), lactic acid (27.8 g L1), acetic acid
(3.9 g L1) and antibacterial activity (74.2 AU mL1).
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction and labelling of some probiotic products (Fasoli et al., 2003;

In recent years there has been a considerable interest in using
some probiotic microorganisms and organic acids as an alternative
to the use of antibiotics in feeds. Probiotics are viable microor-
ganisms that, once ingested in sufficient amount by animals, pro-
duce favourable physiological effects by assisting in the
establishment of an intestinal population that is beneficial to the
host entity and antagonistic to harmful bacteria (Matijašić, Stoj-
kovic, Salobir, Malovrh, & Rogelj, 2004). Microorganisms used in
animal feed in the European Union (EU) are mainly lactic acid
bacteria (LAB) belonging to the genus Lactobacillus, Pediococcus,
Streptococcus and Enterococcus, Gram-positive bacteria belonging
to the genus Bacillus and strains of yeast belonging to the species
Saccharomyces cerevisiae and Kluyveromyces (Anadón, Martı́nez-
Larrañaga, & Aranzazu, 2006). However, production of inhibitory
substances (organic acids and bacteriocins) by many LAB has been
demonstrated to provide them with a competitive advantage over
other microorganisms in the colonization of the gut (Matijašić et al.,
2004).
During the last decade, a variety of commercial animal probiotic
preparations have been developed and marketed for veterinary
administration in the European countries (Fasoli et al., 2003). The
use of these probiotic preparations in animal feed remains ques-
tionable due to the observed deficiencies in microbiological quality
* Corresponding author. Tel.: þ34 988 387062; fax: þ34 988 387001.
E-mail address: nelsonpg@uvigo.es (N.P. Guerra).
Gardiner et al., 2004; Hamilton-Miller & Shah, 2002). These
deficiencies include the incorrect identification of some probiotic
strains, the presence of undeclared microorganisms (Fasoli et al.,
2003; Hamilton-Miller & Shah, 2002; Weese, 2002) or the low
viability of the species listed on the label (Hamilton-Miller & Shah,
2002). An appropriate alternative to solve this problem could be to
select an adequate probiotic strain and grow it on a large scale prior
to its use as an additive in animal feed.
In a previous study (Guerra & Pastrana, 2002), a total of 50 lactic
acid bacteria strains were screened to identify producers of growth
inhibitory substances in batch cultures in de Man Rogosa and
Sharpe (MRS) broth. Among them, Lactobacillus casei subsp. casei
CECT 4043, a strain isolated from a Spanish traditional variety
(Majorero) of semi-hard goats’ cheese (Requena, Pelaez, & Des-
mazeaud, 1991), was found to be a major producer of lactic acid
(21.7 g L1). Another study showed that this bacterium fulfils many
of the probiotic criteria: (i) non-pathogenic, (ii) bile and acid
tolerant, and (iii) able to survive during storage with skim milk at
20  C for 3 months and in a piglet feed at room temperature for 8
days (Guerra, Fajardo, Méndez, Cachaldora, & Pastrana, 2007).
In small scale studies, LAB is commonly cultured in commercial
laboratory media (e.g., MRS and tryptone glucose extract (TGE)
broth), but for large scale production the use of these media can be
cost-prohibitive (Laitila et al., 2004). In these cases, high-cost
complex culture media must be replaced with low-cost media
based on by-products of the food industry (Doleyres & Lacroix,
2005; Guerra & Pastrana, 2003; Guerra, Fajardo, Torrado, López, &
Pastrana, 2005; Guerra, Torrado, López, & Pastrana, 2005). In this

0958-6946/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2008.05.004
 Author's personal copy
1058 P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065
way, a whey-based medium was found to be a suitable culture
medium for the fed-batch production of biomass and antibacterial
extracellular products (AECP) by Lactococcus lactis subsp. lactis
CECT 539 (Guerra & Pastrana, 2003). In fact, the maximum con-
centrations of biomass, lactic acid, acetic acid, butane-2,3-diol and
nisin obtained in this culture were higher than those obtained in
the high-cost MRS medium (Guerra & Pastrana, 2002).
In the present study, we described the fed-batch production of
a highly concentrated probiotic culture of Lb. casei subsp. casei CECT
4043 in a whey-based medium, prior to its evaluation as a feed
additive for weaned piglets (Guerra, Fajardo, Méndez, et al., 2007).
Firstly, the batch growth of Lb. casei was followed in concentrated
(5% w/v lactose) and diluted (2% w/v lactose) whey media. Sub-
sequently, the most appropriate culture medium was used to study
the effect of the initial pH on the production of biomass and AECP
by Lb. casei. From these results, we selected the most appropriate
fermentation conditions for the fed-batch fermentations.
2. Materials and methods
2.1. Bacterial strains
Lb. casei subsp. casei CECT 4043 (the potential probiotic strain)
and Carnobacterium piscicola CECT 4020 (the target organism in the
antibacterial activity assay) were obtained from the Spanish Type
Culture Collection (CECT, Valencia, Spain). Stock cultures of both
strains were maintained at 40  C in nutrient broth (Cultimed
Panreac Quı́mica S.A., Barcelona, Spain) with 15% (v/v) added
glycerol (Cultimed Panreac Quı́mica S.A.). Working cultures main-
tained at 4  C on de Man, Rogosa and Sharpe (MRS) agar (Cultimed
Panreac Quı́mica S.A.) were prepared monthly from frozen stock
cultures.
2.2. Culture medium, fermentation conditions and inoculation
Fermentations were carried out in whey-based media, which
were obtained from a local dairy plant as concentrated whey (CW:
the liquid remaining after the first cheese pressing) and diluted
whey (DW: i.e., CW mixed with water). The preparation of these
wastes before being used as culture media was carried out as pre-
viously described (Guerra, Rua, & Pastrana, 2001). The DW medium
contained the following mean composition (in g L1): total sugars,
20.54; total nitrogen, 0.45; total phosphorus, 0.25 and soluble
proteins, 2.04. The CW medium contained (in g L1) total sugars,
48.11; total nitrogen, 1.05; total phosphorus, 0.43 and soluble pro-
teins, 5.02 (Guerra, Fajardo, Méndez, et al., 2007; Guerra et al.,
2001; Guerra, Torrado, López, Fajardo, & Pastrana, 2007).
Batch cultures were performed in 250 mL Erlenmeyer flasks
containing 50 mL of the corresponding fermentation medium (DW
or CW), on a rotary shaker (Innova 4330, New Brunswick Scientific
Co., Inc., Edison, NJ, USA) at 30  C and 200 rpm. Samples were
withdrawn at intervals during the incubation periods to carry out
analytical determinations.
Re-alkalized fed-batch cultures were carried out at a controlled
temperature of 30  C in a 6 L bench top fermentor (New Brunswick
Scientific) with a 4 L working volume of DW medium adjusted to an
initial pH of 7.0. A controlled constant agitation speed of 200 rpm
and a controlled constant aeration flow rate of 0.5 L h1 were used
during the fermentation. In the first re-alkalized fed-batch culture,
two fresh substrates were used to feed the fermentor: CW medium
and a concentrated lactose solution (400 g L1). In the second fed-
batch culture, the feeding substrate consisted of a mixture of
a concentrated glucose solution (310 g L1) and a concentrated
mussel processing waste (CMPW), which was prepared as pre-
viously described (Guerra, Torrado, et al., 2007). The resulting
CMPW medium contained (in g L1) glucose, 101.33; total nitrogen,
0.54; total phosphorus, 0.06 and soluble proteins, 3.47 (Guerra,
Fajardo, Méndez, et al., 2007; Guerra, Torrado, et al., 2007). The
feeding media were added to the fermentor using a peristaltic
pump (LKB, Pharmacia, Uppsala, Sweden). Fed-batch fermentations
were carried out as a batch fermentation without pH control during
the first 12 h of culture, when the culture reached the stationary
phase in batch fermentation. At this time, a sample of 100 mL was
taken from the fermentation medium to carry out analytical
determinations. The total sugar concentration in the withdrawn
sample was determined and subsequently, the amounts of sugars
consumed by the lactic acid-producing strain were calculated.
Then, the medium was re-alkalized up to pH 7.0 with 4 M NaOH.
From the difference between the sampling volume (100 mL) and
the volume of alkali added, the necessary volumes of feeding
substrates to adjust the fermentation medium to about its initial
value (20.54 g L1) were calculated and added into the fermentor.
These sampling, feeding and re-alkalization strategies were re-
peated every 12 h until the producer strain was unable to bring
about the decrease of pH. Additionally, a batch fermentation in DW
medium was carried out for 36 h in the fermentor (working vol-
ume: 4 L, agitation: 200 rpm, initial pH: 7.0, temperature: 30  C) to
obtain data for comparisons. The batch and fed-batch cultures were
inoculated with 2% (v/v) of a 12-h culture of Lb. casei in the corre-
sponding culture medium.
2.3. Analytical methods
Growth was monitored by absorbance at 700 nm and converted
into dry cell weight from a standard curve (Guerra et al., 2001).
Cells were harvested by centrifugation (12,000g for 15 min at
4  C) of culture samples and washed twice with saline (0.8% w/v
NaCl). Total viable cell numbers were enumerated by a pour plate
method (in triplicate) using MRS agar after serial 10-fold dilution in
sterile phosphate-buffered saline (PBS: 10 mM sodium phosphate
mono-basic, 10 mM sodium phosphate dibasic, 130 mM sodium
chloride, pH 7.2). The plates were incubated at 30  C for 48 h and
the results were expressed as colony forming units (cfu) per mL.
The culture supernatants were used for analytical
determinations. Total sugars, proteins, nitrogen and phosphorus
were determined by methods described in a previous work (Guerra
& Pastrana, 2003). The concentrations of lactose, glucose, lactic acid
and acetic acid were measured by high-performance liquid chro-
matography (HPLC) using an ION-300 Organic Acids column
(length 300 mm, internal diameter 7.8 mm) with a pre-column
IONGUARDÔ (polymeric guard column), both obtained from
Tecknokroma S. Coop. C. Ltda, Barcelona, Spain. The mobile phase
consisted of 6 mM H2SO4 at a flow rate of 0.4 mL min1 at 60–65  C
and the refractive index of the peaks was measured (Guerra &
Pastrana, 2003).
2.4. Preparation of cell-free supernatants of Lb. casei and
quantification of the antagonistic activity
Samples of Lb. casei cultures were acidified to pH 3.5 with 5 mM
HCl, heated at 80  C for 3 min and centrifuged at 27,200g for
15 min at 4  C. The cell-free supernatants (CFS) containing total
antibacterial activity were stored frozen until needed.
Using C. piscicola CECT 4020 as the indicator, the antibacterial
activities of the CFS were assayed by using a photometric assay
(Cabo, Murado, González, & Pastoriza, 1999). The CFS were serially
diluted in distilled sterile water (this step eliminated the need to
correct the pH of samples). A 2.5 mL portion of the diluted CFS was
added in sterile culture tubes. Each tube was inoculated with
2.5 mL of an overnight culture of the indicator strain, which was
diluted to an absorbance of 0.2 with sterile buffered MRS broth (pH
6.3) just before being used. Controls consisted of three culture
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P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065 1059

tubes in which the diluted CFS were replaced by distilled sterile

water. The tubes were then incubated at 30  C for 6 h. Growth
inhibition was measured spectrophotometrically at 700 nm. Dose–
response curves were obtained from these data. One antibacterial
activity (AU) was defined as the reciprocal of the dilution that
produced a 50% growth inhibition compared with control tubes.
The titres of antibacterial activity were expressed in AU mL1 cell-
free supernatant (Murado, González, & Vázquez, 2002).

2.5. Statistical analyses

Individual experiments were performed in triplicate as stated in

the figure legends. Data points represent the means  standard
deviation (S.D.). Comparisons were made between the results
(concentration of biomass, organic acids and antibacterial activity,
yield coefficients and efficiencies) of all cultures. Statistical analysis
was performed with SPSS 8.0 for Windows (Statsoft 2001, Tulsa, OK,
USA). Differences were considered as statistically significant at
P  0.05.

3. Results and discussion

3.1. Batch cultures of Lb. casei CECT 4043 in DW and CW media

To find out the suitable culture medium for cell growth and
antibacterial extracellular products (AECP) synthesis in Lb. casei
CECT 4043, two batch cultures were grown in DW and CW media
previously adjusted to an initial pH value of 7.0. The concentrations
of nutrients (total sugars, nitrogen, phosphorus and proteins)
decreased in parallel with an increase in biomass production in
both fermentations (Fig. 1). In addition, Lb. casei developed
a homolactic fermentation because the lactic acid was the unique
organic acid detected in the media. The profiles described by the
productions of lactic acid and antibacterial activity (AA) in DW and
CW media paralleled the profile described by the biomass syn-
thesis, indicating that both productions were growth-linked.
On the other hand, the concentrations of biomass (0.33 g L1),
lactic acid (2.22 g L1) and AA (3.42 AU mL1) in DW medium
were slightly higher (P < 0.05) than those observed in CW me-
dium (0.20 g L1, 1.47 g L1 and 2.06 AU mL1, respectively) after
36 h of fermentation. In the same way, the highest values of the
yield coefficients for the biomass (YX/TSc, in g of biomass produced
per g of total sugar consumed) and the antibacterial activity (YAA/
1
TSc, YAA/TNc, YAA/X, calculated as antibacterial activity (AU mL )
produced per g of total sugar (TS) or nitrogen (TN) consumed or
biomass (X) produced), and the efficiencies in lactose (EL), nitro-
gen (ETN) and phosphorus (ETP) consumption (expressed as g of
lactose (L), total nitrogen (TN) or phosphorus (TP) substrate
consumed per g of substrate supplied) were obtained in DW
(Table 1). In this medium, the productions of biomass, lactic acid
and antibacterial activity slowed down from the 12-h cultivation,
meanwhile, these productions increased linearly until the end of
the culture in CW medium (Fig. 1). These results suggest that
a prerequisite to obtain high concentrations of biomass and an-
tibacterial products is to keep low sugar concentrations in culture
medium at all times.
Similarly, maximum concentrations of biomass and AECP were
reported for Lc. lactis subsp. lactis CECT 539 and Pediococcus acid-
ilactici NRRL B-5627 in DW medium in comparison with the pro-
duction obtained in CW medium (Guerra et al., 2001). Other study
showed that increasing lactose concentrations inhibited the lactic
acid production by Lb. casei NRRL B-441 in whey-based media
(Altiok, Tokatli, & Harsa, 2006). Based on these results, the DW
medium was selected as the fermentation substrate for the re-
alkalized fed-batch cultures.
Fig. 1. Time course of batch fermentations of Lactobacillus casei subsp. casei CECT 4043
on diluted whey (DW) medium (B) and concentrated whey (CW) medium (,). X,
biomass concentration; AA, antibacterial activity; LA, lactic acid; Pr, proteins; TP, total
phosphorus; TS, total sugars; TN, total nitrogen. Results shown are means  S.D. for
three replicates.
3.2. Effect of the initial pH on Lb. casei CECT 4043 cell growth and
antibacterial extracellular products synthesis
In order to study the effect of initial pH on cell growth, AECP
production and nutrient consumption in DW medium, Lb. casei
CECT 4043 was cultivated at three different initial pH values (6.0,
6.5 and 7.5) in shaker flasks. In these cultures, both the lactic acid
and antibacterial activity increased simultaneously with cell
growth (Fig. 2), as it was observed in the previous culture in DW
medium adjusted at an initial pH value of 7.0 (Fig. 1).
Although the maximum biomass and lactic acid concentrations
were obtained at initial pH values of 7.0 and 6.5, the highest AA
concentrations were obtained at initial pH values of 7.0 and 7.5
(P < 0.05), in which the highest pH gradients (difference between
the initial and final pH values) were observed. Interestingly, the
lactic acid and the antibacterial activity described different profiles
during the fermentation process (Fig. 2). In fact, AA concentration
increased in parallel with a decrease in the culture pH, as was
observed before for the productions of nisin and pediocin in DW
medium (Guerra et al., 2001). These observations suggest that the
antibacterial activity contained in the Lb. casei CECT 4043 culture
supernatants could be the result of the synergistic action of two or
more inhibitory compounds produced by this strain (Guerra &
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1060 P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065

Table 1
Growth and fermentation parameters of non-realkalized (batch) and re-alkalized
(fed-batch) cultures of Lactobacillus casei subsp. casei CECT 4043 for biomass and
antibacterial products synthesis

Parametersa DW,b batch CW,c batch Fed-batch (1) Fed-batch (2)

X* (g L1) 0.33 0.20 0.55 1.70
LA* (g L1) 2.22 1.47 10.2 27.8
Ac* (g L1) – – 0.6 3.9
AA* (AU mL1) 3.42 2.06 10.0 74.2
YAA/TSc 0.49 0.34 0.12 0.32
YAA/TNc 25.28 6.57 30.20 192.17
YAA/X 10.93 10.30 18.56 44.21
YX/TSc 0.059 0.053 0.006 0.007
EL 0.24 0.07 0.74 0.48
EG – – – 0.89
ETN 0.36 0.16 0.54 0.92
ETP 0.29 0.16 0.50 0.62
a
X*, LA*, Ac* and AA* are the maximum biomass, lactic acid, acetic acid and an-
tibacterial activity concentrations produced in the cultures. YAA/TSc, YAA/TNc and YAA/X
are the antibacterial activity yield coefficients expressed as antibacterial activity
produced (AU mL1) per g of total sugars (TS) or nitrogen (TN) consumed and bio-
mass (X) produced. YX/TSc is the biomass yield coefficient expressed as g of biomass
produced per g of total sugars consumed. EL, EG, ETN and ETP are the efficiencies
expressed as g of substrate (lactose (L), glucose (G) or total nitrogen (TN) and
phosphorus (TP)) consumed per g of substrate supplied.
b
Diluted whey.
c
Concentrated whey.

Pastrana, 2003; Laitila et al., 2004; Niku-Paavola, Laitila, Mattila-

Sandholm, & Haikara, 1999). Since lactic acid was the unique
organic acid detected in the culture medium, antibacterial activity
also could be related to the production of bacteriocins by Lb. casei
CECT 4043, as was suggested before (Vázquez, González, & Murado,
2005). This interpretation is also supported by the observation that
bacteriocin production is favoured when higher pH gradients are
generated in the cultures (Cabo, Murado, González, & Pastoriza,
2001; Guerra & Pastrana, 2003; Guerra et al., 2001), as occurred in
the case of antibacterial activity synthesis in the present study
(Fig. 2).
With regard to nutrient consumption, it can be observed that Lb.
casei CECT 4043 consumed the lowest amounts of total sugars,
nitrogen, phosphorus and proteins at the initial pH of 6.0. However,
the amounts of nutrients consumed by Lb. casei at pH 7.5 and 6.5
were not significantly different. At the initial pH of 7.5, the meta-
bolic activity (in terms of synthesis of biomass, lactic acid and
antibacterial activity as well as the nutrient consumption) was very
low during the first 6 h of incubation, when the pH value dropped
from 7.5 to 7.0. Nevertheless, when the pH dropped from 7.0 to 5.6
(from 6 to 12 h of incubation), faster cell growth was observed in
concomitance with the rapid increase in lactic acid and AA pro-
duction and nutrient consumption (Fig. 2). Subsequently, the rates
of growth and nutrient consumption decreased when the culture
reached a pH value below 5.0, as was observed in the cultures
adjusted at the initial pH values of 7.0, 6.5 and 6.0.
In order to clarify the effect of pH time course on growth and
nutrient consumption in each culture, the mean rates of biomass
production and nutrient consumption were plotted against the pH
of the culture at each sampling time (Fig. 3). As can be observed, the
high biomass production and nutrient consumption rates were
obtained when the pH of the cultures reached values between 7.0
and 6.5 in all cultures, decreasing for higher or lower values.
According to Poolman and Konings (1988), the amino acid or
peptide transport, which is one of the growth-rate-determining
steps, depends on the culture pH. For Lc. lactis and Lactococcus
cremoris strains, the optimum pH value for amino acid transport
varied between 6.0 and 6.5, decreasing rapidly at higher and lower
pH values (Poolman & Konings, 1988). Thus, the low nutrient con-
sumption and consequently, the low production of biomass by Lb.
casei CECT 4043 during the first 6 h of incubation in the culture at
Fig. 2. Time course of batch fermentations of Lactobacillus casei CECT 4043 on diluted
whey (DW) media adjusted to initial pH values of 7.5 (6), 7.0 (C), 6.5 (B) and 6.0 (,).
Other abbreviations are the same as in Fig. 1. Results shown are means  S.D. for three
replicates.
the initial pH 7.5, could be related to a limitation in nutrient
transport. However, the observed reduction in the growth of Lb.
casei when the cultures reached a pH value below 5.0 could also be
related to the exhaustion of some essential micronutrients (vita-
mins or minerals) or amino acids in the medium (van Niel & Hahn-
Hägerdal, 1999).
To investigate the effect of low pH values on growth and
nutrient consumption by Lb. casei, three new batch cultures at
initial pH values of 5.5, 5.0 and 4.5 were grown in DW media
(Fig. 4A). The results obtained showed that at decreasing initial pH
values, the production of biomass and nutrient consumption
decreased rapidly (Fig. 4A). Again, the above mentioned reduction
in the mean rates of growth and nutrient consumption at pH values
below 5.0 was observed in each culture (Fig. 4B).
Lb. casei strains are considered to be extremely aciduric bacteria,
which alter their membrane composition to contain increased
levels of long-chained, mono-unsaturated membrane fatty acids in
response to acidification (Fozo, Kajfasz, & Quivey, 2004; Svensater,
Larsson, Greif, Cvitkovitch, & Hamilton, 1997). This may be
a mechanism commonly used by Lb. casei to increase their survival
in acidic environments (Fozo et al., 2004). Therefore, acid stress
does not seem to be an acceptable cause to explain the growth
inhibition of Lb. casei CECT 4043 in DW media adjusted at initial pH
values of 5.5, 5.0 and 4.5. Since the three cultures had the same
 Author's personal copy
P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065
Fig. 3. Changes in the absolute mean rates of growth (rX) and nutrient consumption
(total sugars (rTS), protein (rPr), nitrogen (rTN) and phosphorus (rTP)) with respect to
the culture pH in the batch fermentations of Lactobacillus casei CECT 4043 on diluted
whey (DW) media adjusted to initial pH values of 7.5 (6), 7.0 (C), 6.5 (B) and 6.0 (,).
initial composition, the reduction in growth and nutrient con-
sumption by Lb. casei at low initial pH values seems to be caused by
a limitation in nutrient transport (Hutkins & Nannen, 1993; Pool-
man & Konings, 1988) rather than by an exhaustion of some
1061
essential vitamins, minerals or amino acids in the medium. Based
on the above observations, pH 7.0 was selected as the best initial pH
value for Lb. casei growth and AECP production in DW medium.
3.3. Re-alkalized fed-batch fermentations
On the basis of batch fermentation results, a re-alkalized fed-
batch fermentation was carried out in the DW medium adjusted to
an initial pH of 7.0. The fermentor was fed with a mixture of CW
medium and a concentrated lactose solution (400 g L1) each 12 h,
when the culture reached the stationary phase. The culture was re-
alkalized up to a pH of 7.0 in each feeding cycle to facilitate the
nutrient consumption by Lb. casei CECT 4043. The feeding sub-
strates were fed into the fermentation medium to replenish the
levels of total sugars consumed and other nutrients in each feeding
cycle. This approach was used to avoid or reduce the substrate-
associated growth inhibition observed in CW medium (Fig. 1), by
appropriately controlling the carbon source concentration in the
fermentor (De Vuyst, De Poorter, & Vandamme, 1989; Guerra &
Pastrana, 2003; Guerra, Fajardo, et al., 2005; Guerra, Torrado, et al.,
2005). Consequently, increased amounts of biomass and
antibacterial products would be obtained.
The time course of the first re-alkalized fed-batch culture of Lb.
casei CECT 4043 in DW medium is shown in Fig. 5. A metabolic shift
from homolactic to heterolactic fermentation was observed after
60 h of incubation when acetic acid became detectable in the cul-
ture medium. This shift has been observed before for certain lac-
tococci and lactobacilli not only in cultures under limitation of
glucose (Borch, Berg, & Holst, 1991; Cocaign-Bousquet, Garrigues,
Lubiere, & Lindley, 1996; Thomas, Ellwood, & Longyear, 1979) or
nitrogen (Thomas et al., 1979), but also under carbon excess con-
ditions with lactose (Garrigues, Loubiere, Lindley, & Cocaign-
Bousquet, 1997; Garrigues, Mercade, Cocaign-Bousquet, Lindley, &
Loubiere, 2001; Torino, Taranto, Sesma, & Font de Valdez, 2001),
galactose (Garrigues et al., 1997; Thomas, Turner, & Crow, 1980) and
maltose (Hofvendahl, van Niel, & Hahn-Hägerdal, 1999). This met-
abolic pathway shift has been associated with a modification of
pyruvate metabolism with a decreased activity of lactate
dehydrogenase (Lopez de Felipe, Kleerebezem, de Vos, & Hugen-
holtz, 1998; Zhu & Yang, 2006).
Compared with batch culture results in DW medium adjusted at
pH 7.0 (Fig. 1), a significantly enhancement (P < 0.05) was observed
for the maximum concentrations of biomass (from 0.33 to
0.75 g L1), AA (from 3.50 to 10.01 AU mL1), lactic acid (from 2.26
to 10.15 g L1) and acetic acid (from 0 to 0.56 g L1) in this first re-
alkalized fed-batch fermentation (Fig. 5). However, the lactic acid-
producing strain completely lost its ability to recover acidic pH after
156 h of incubation (Fig. 5). At 48 h, the concentration of viable cells
reached a maximum of 4.9  109 cfu mL1 and decreased to
1.9  109 cfu mL1 upon prolonged fermentation, probably due to
the decrease in the concentration of certain micronutrients nec-
essary for biomass production during the fed-batch culture.
Comparison between fed-batch cultures of P. acidilactici NRRL B-
5627 in whey-based media fed with substrates containing glucose
or lactose, showed that the feeding with glucose was an adequate
strategy for increasing both the biomass and AECP production by
this strain (Guerra, Fajardo, & Pastrana, 2007). Taking into account
these results, a second re-alkalized fed-batch culture with Lb. casei
CECT 4043 was grown in DW medium (pH 7.0), using a mixture of
substrates containing glucose instead of lactose, as feeding media.
In this case, the growing culture was fed with a mixture of sub-
strates composed of CMPW medium (101.3 g of glucose L1) and
a 310 g L1 concentrated glucose (Fig. 6). With this approach, the
active period was higher than that observed in the previous re-
alkalized fed-batch culture. Thus, after 372 h of fermentation, the
producer strain was able to bring about the decrease of pH. The
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1062 P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065

Fig. 4. (A) Time course of batch fermentations of Lactobacillus casei CECT 4043 on diluted whey (DW) media adjusted to initial pH values of 5.5 (B), 5.0 (,) and 4.5 (6). (B) Changes
in the absolute mean rates of growth and nutrient consumption with respect to the culture pH. Other abbreviations are the same as in Figs. 1 and 3. Results shown are means  S.D.
for three replicates.

above-discussed shift from homolactic to heterolactic fermentation
was again observed in this second fed-batch culture. In each fer-
mentation phase, the biomass concentration profile displayed an
exponential growth phase followed by a stationary phase. Nitrogen,
protein and phosphorus content of the fermentation medium was
almost depleted at the end of the fermentation, achieving
a reduction in the nutrient content much higher than in the first re-
alkalized fed-batch culture (Figs. 5 and 6). These results suggest
that the glucose and the proteins contained in the CMPW medium
can be more efficiently utilized as carbon and nitrogen sources by
Lb. casei CECT 4043 than the lactose or the proteins present in the
CW medium. With regard to the carbon source utilization, glucose
has been reported to be the most efficient carbon source for bio-
mass and exopolysaccharide production by Lb. casei CG11, whereas
the lactose was not a good carbon source (Cerning et al., 1994).
Other study showed that Lb. helveticus ATCC 15807 produced sim-
ilar concentrations of biomass in media containing lactose or glu-
cose, but this strain produced the highest amounts of lactic acid in
the culture medium containing glucose (Torino, Hébert, Mozzi, &
Font de Valdez, 2005).
On the other hand, it has been reported that lactose utilization
by Lb. casei ATCC 393 is strongly repressed by the presence of
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P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065
Fig. 5. Time course of the batch (closed symbols) and re-alkalized fed-batch fermen-
tations (open symbols) of Lactobacillus casei CECT 4043 on diluted whey (DW) medium
with feeding with concentrated whey (CW) medium and a 400 g L1 concentrated
lactose solution. Other abbreviations are the same as in Fig. 1. Ac, acetic acid; L, lactose;
cfu, colony forming units. I and II are the homolactic and the heterolactic fermentation
phases, respectively. Results shown are means  S.D. for three replicates.
glucose, since this strain displayed a likely diauxie when grown in
a culture medium containing both carbon sources (Veyrat, Mon-
edero, & Pérez-Martı́nez, 1994). This phenomenon was explained
by the fact that glucose appears to exert negative control upon
structural organization, regulation and expression of the lac genes
in Lb. casei strains (Gosalbes, Esteban, Galán, & Pérez-Martı́nez,
2000; Veyrat et al., 1994; Yebra, Veyrat, Santos, & Pérez-Martı́nez,
2000). This appears to be the case in the cells of Lb. casei CECT
4043 in the second re-alkalized fed-batch culture in DW medium,
which was fed with substrates containing glucose. In this second
culture, the efficiency in lactose consumption (EL ¼ 48%) de-
creased in comparison with the first fed-batch culture (EL ¼ 74%),
probably due to the preferential utilization of glucose, which was
consumed at a high efficiency (EG ¼ 89%) by Lb. casei CECT 4043
(Table 1).
The results obtained in the second fed-batch experiment (Fig. 6)
made evident that feeding the fermentor with CMPW medium and
glucose increased the production of biomass (1.7 g L1), AA
(74.2 AU mL1), lactic acid (27.8 g L1) and acetic acid (3.9 g L1) as
compared (P < 0.05) with the previous fed-batch culture (Fig. 5). In
addition, the viable cell concentrations reached a maximum of
1.7  1010 cfu mL1 after 312 h and decreased slowly upon further
fermentation. However, prolonged fermentation allowed the pro-
duction of a probiotic culture containing high concentrations of
cells and AECP (Fig. 6) in comparison with the batch (Fig. 1) and the
first fed-batch (Fig. 5) cultures. The use of this highly concentrated
culture in animal feed could be beneficial for the animals, since the
1063
Fig. 6. Time course of re-alkalized fed-batch culture (open symbols) of Lactobacillus
casei CECT 4043 on diluted whey (DW) medium with feeding with concentrated
mussel processing waste (CMPW) medium and a 310 g L1 concentrated glucose so-
lution. G, glucose. Other abbreviations are the same as in Figs. 1 and 5. I and II are the
homolactic and the heterolactic fermentation phases, respectively. Results shown are
means  S.D. for three replicates.
AECP can act synergistically to inhibit or prevent the gastrointes-
tinal survival and colonization of pathogenic bacteria, thus giving
the competitive advantage to the Lb. casei cells in colonising the
gastrointestinal tract. This could help to keep farm animals disease-
free, which has the additional advantage of promoting their growth
because the animals would not have to expend energy in fighting
illness. For these reasons, the use of a prolonged fed-batch fer-
mentation (Fig. 6) rather than the batch fermentation, would pro-
vide significant benefits to animal production.
Comparison between batch and fed-batch fermentations
revealed that the yield coefficients for the biomass (YX/TSc) and the
antibacterial activity (YAA/TSc) in the re-alkalized fed-batch fer-
mentations were the lowest. This suggests that, in the fed-batch
cultures, the carbon sources (glucose and lactose) were over-
consumed to compensate for the stress generated as a consequence
of the rapid increase of pH at the beginning of each re-alkalization
period. A similar decrease in the values of both yield coefficients in
the fed-batch fermentations as compared with the batch cultures
has been observed before for nisin (Cabo et al., 2001; Guerra &
Pastrana, 2003) and pediocin production (Guerra, Fajardo, et al.,
2005; Guerra, Torrado, et al., 2005). In contrast, the highest values
of the antibacterial activity yield coefficients (YAA/X and YAA/TNc)
obtained in the two fed-batch fermentations indicate that the
culture was more productive in these cultivations and additionally,
that the nitrogen source was more efficiently utilized for the anti-
bacterial activity production (Cabo et al., 2001; Guerra & Pastrana,
2003).
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1064 P.F. Bernárdez et al. / International Dairy Journal 18 (2008) 1057–1065
The highest efficiency in nitrogen and phosphorus (ETN and ETP)
consumption obtained in the second re-alkalized culture demon-
strated that the system evolved in a more balanced way with re-
spect to the nutrients consumed and added (Cabo et al., 2001;
Guerra & Pastrana, 2003; Guerra, Fajardo, et al., 2005; Guerra,
Torrado, et al., 2005). This means that the fed-batch culture carried
out in DW medium with feeding with CMPW medium and con-
centrated glucose could be an adequate way for producing high
amounts of biomass and antibacterial products in inexpensive
whey-based media.
Once stopped, the second re-alkalized fed-batch fermentation,
the Lb. casei culture (containing both the cells and AECP) was ad-
justed to pH 7.0, then mixed with 30% (w/v) skim milk and stored
frozen at 20  C. The skim milk was a good cryoprotective agent
because the frozen cultures showed a good viability at 20  C
during 3 months (Guerra, Fajardo, Méndez, et al., 2007). A signifi-
cant increase in body weight gain and a significant decrease in the
viable coliform counts were observed in weaned piglets fed a diet
supplemented with this lactic acid bacterium as compared with
piglets (controls) fed the non-supplemented feed (Guerra, Fajardo,
Méndez, et al., 2007).
4. Conclusions
In this study, re-alkalized fed-batch processes using different
feeding substrates were developed, aiming at maximizing cell
growth and products concentration. The results obtained have
practical relevance showing that is possible to produce a highly
concentrated probiotic culture of Lb. casei CECT 4043 by using
waste media of low cost and a well-designed fed-batch fermenta-
tion technique. This approach allows the protection of the envi-
ronment by recycling whey in production of probiotic culture
concentrated (containing cells plus antibacterial substances) from
Lb. casei CECT 4043. Highly concentrated cultures of Lb. casei CECT
4043 and other LAB are being produced and evaluated in our lab-
oratory as a replacement for antibiotics in stimulating health and
growth of farm animals, such as piglets (Guerra, Fajardo, Méndez,
et al., 2007) and chickens.
Acknowledgements
The research presented in this paper was financially supported
by the Instituto Nacional de Investigación y Tecnologı́a Agraria y
Alimentaria (INIA), Spain (project CAL01-045-C2-2), The Xunta de
Galicia, Spain (project PGIDT00BIO1E) and the Ministerio de Edu-
cación y Ciencia (project MAT2006-11662-C03-03). We thank
COREN, S.C.L. for their collaboration with this work.
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