World Journal of Microbiology and Biotechnology (2021) 37:61

https://doi.org/10.1007/s11274-021-03028-z

ORIGINAL PAPER

Lactic acid production using cheese whey based medium in a stirred

tank reactor by a ccpA mutant of Lacticaseibacillus casei
Mariela Verónica Catone1 · María Mercedes Palomino2 · Danilo Mario Legisa1 · Joaquina Fina Martin2 ·
Vicente Monedero García3 · Sandra Mónica Ruzal2 · Mariana Claudia Allievi2

Received: 9 November 2020 / Accepted: 2 March 2021 / Published online: 15 March 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
In lactobacilli, CcpA is known to modulate the expression of genes involved in sugar metabolism, stress response and aerobic
adaptation. This study aimed to evaluate a ccpA mutant of Lacticaseibacillus casei BL23 to increase lactic acid production
using cheese whey. The ccpA derivative (BL71) showed better growth than the L. casei wild-type in the whey medium. In
a stirred tank reactor, at 48 h, lactate production by BL71 was eightfold higher than that by BL23. In batch fermentations,
the final values reached were 44.23 g ­L−1 for BL71 and 27.58 g ­L−1 for BL23. Due to a decrease in the delay of lactate pro-
duction in the mutant, lactate productivity increased from 0.17 g (L.h)−1 with BL23 to 0.80 g (L.h)−1 with BL71. We found
that CcpA would play additional roles in nitrogen metabolism by the regulation of the proteolytic system. BL71 displayed
higher activity of the PepX, PepQ and PrtP enzymes than BL23. Analysis of prtP expression confirmed this deregulation in
BL71. Promoter analysis of the prtP gene revealed CcpA binding sites with high identity to the cre consensus sequence and
the interaction of CcpA with this promoter was confirmed in vitro. We postulate that deregulation of the proteolytic system
in BL71 allows a better exploitation of nitrogen resources in cheese whey, resulting in enhanced fermentation capacity.
Therefore, the ccpA gene could be a good target for future technological developments aimed at effective and inexpensive
lactate production from dairy industrial wastes.

Mariela Verónica Catone and María Mercedes Palomino have

contributed equally to this work.

* Mariana Claudia Allievi

mallievi@qb.fcen.uba.ar
1
Centro de Investigación y Desarrollo en Biotecnología
Industrial, Instituto Nacional de Tecnología Industrial
(INTI), Av. General Paz 5445, B1650AAC​San Martín,
Buenos Aires, Argentina
2
Departamento de Química Biológica, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires - Instituto
de Química Biológica de la Facultad de Ciencias Exactas y
Naturales (IQUIBICEN) - CONICET, Ciudad Universitaria,
C1428EGA CABA, Argentina
3
Instituto de Agroquímica y Tecnología de Alimentos-Consejo
Superior de Investigaciones Científicas (IATA-CSIC),
Av. Agustín Escardino 7, 46980 Paterna, Valencia, España

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Graphic abstract
Keywords Lactic acid · Lacticaseibacillus casei · CcpA · Proteolytic system · Cheese whey
Introduction
Lactic acid (2-hydroxypropionic acid) is an organic com-
pound with applications in the pharmaceutical, chemical and
textile industries, widely used as an acidulant/flavoring, pH
buffering agent or inhibitor of bacterial spoilage in a wide
variety of processed foods (Abdel-Rahman et al. 2013).
This acid has also received great attention as a precursor
of the biodegradable polylactic acid polymer (Narayanan
et al. 2004; John et al. 2007; Sahoo and Jayaraman 2019).
Great amounts of lactate are produced through carbohy-
drates fermentation by lactic acid bacteria (LAB), such as
those belonging to the former genus Lactobacillus, which
has been recently split into more than twenty new genera
(Zheng et al. 2020), which are GRAS (generally recognized
as safe) microorganisms. The metabolism of sugars by
lactobacilli (which refers to all microorganisms that were
classified as Lactobacillaceae until the recent taxonomi-
cal modifications (Zheng et al. 2020)) is characterized by
the production of lactate through the action of the lactate
dehydrogenase enzyme, as the main product of its strictly
fermentative metabolism (Allievi et al. 2019). Contrarily to
chemical synthesis, lactate production through fermenta-
tion allows obtaining optically pure D- and L-lactic acid,
instead of a racemic mixture. Additional advantages of using
13
World Journal of Microbiology and Biotechnology (2021) 37:61
microorganisms in lactate production reside in the fact that
it can be produced from industrial wastes and other inexpen-
sive substrates and by-products (Bernardo et al. 2016). The
most expensive component of microbial growth substrates
is usually the nitrogen source. In addition, some lactobacilli
are extremely fastidious and require more than 10 essential
amino acids for their growth. Among by-products from the
cheese industry, whey contains significant amounts of lac-
tose as a carbon source (approximately 5% w/v), and also
carries proteins, vitamins, and minerals. In addition, it con-
tains other essential nutrients necessary for the growth of
LAB, like branched-chain amino acids. Thus, the reutiliza-
tion of this agro-industrial by-product provides cost reduc-
tions and environmental sustainability (Rabaioli Rama et al.
2019; Zotta et al. 2020).
The efficient use of nutrients by microorganisms is
associated with carbon catabolite repression (CCR),
which allows microbes to select their preferred source
from multiple offerings of carbon catabolites. Production
from industrial waste often starts from mixed sugars and
can therefore be hindered by the process of CCR, which
becomes an important limiting factor (Fujita 2009). The
catabolite control protein A (CcpA) is a mediator of CCR
in low-G + C Gram-positive bacteria (Mahr et al. 2000;
Viana et al. 2005; Monedero et al. 2007; Zomer et al.
World Journal of Microbiology and Biotechnology (2021) 37:61
2007; Deutscher 2008). In several Firmicutes, the ccpA
gene is clustered with the pepQ gene, encoding an X-Pro
aminopeptidase. CcpA-dependent activation of pepQ has
been postulated for Lactobacillus delbrueckii subsp. lac-
tis (Schick et al. 1999) and demonstrated in Lactococcus
lactis (Zomer et al. 2007), a fact that indicates a link
between the regulation of carbon and nitrogen metabo-
lism. This link has also been described for Bacillus sub-
tilis (Sonenshein 2007; Fujita 2009; Wünsche et al. 2012)
and Staphylococcus aureus (Li et al. 2010), where CcpA
controls genes responsible for nitrogen assimilation and
amino acid metabolism.
LAB are likely to respond to changes in nitrogen avail-
ability by regulating the activity of the proteolytic sys-
tem. It is known that the transcriptional regulator CodY
negatively regulates the expression of several components
of the proteolytic system in B. subtilis and Lc. lactis and
responds to the intracellular pool of branched-chain
amino acids (Guédon et al. 2001; Petranovic et al. 2004;
Sonenshein 2005); however, CodY is absent in lacto-
bacilli species, (Kleerebezem et al. 2003; Wakai and
Yamamoto 2013; Alcántara et al. 2016). Recently, the
promoters located in the intergenic region of the prsA-
prtP genes, encoding the main cell-surface proteinase
(prtP) and its maturase (prsA) in Lacticaseibacillus casei
BL23, have been shown to be regulated by the repres-
sor PrcR in peptide-rich growth media (Alcántara et al.
2016; Coll-Marqués et al. 2020). PrcR would directly
regulate the metabolism of nitrogen, peptides and amino
acids, since its inactivation has been found to result in
a reduced growth rate in de Man, Rogosa and Sharpe
(MRS) medium but in a rapid acidification of milk as a
consequence of the high expression of PrtP, as the fast
milk acidifying phenotype is related to the expression
of cell-envelope proteinases (Dandoy et al. 2011). How-
ever, PrcR does not play a role exactly analogous to that
of CodY in streptococci and lactococci, suggesting that,
in L. casei BL23, other elements could be involved in
the regulation of prtP and peptidases. Recent evidence
has demonstrated the roles of the pleiotropic regulator
CcpA in non-CCR processes such as stress resistance and
aerobic metabolism in Lactiplantibacillus plantarum and
Lactobacillus delbrueckii subsp. bulgaricus, but some
molecular mechanisms remain unclear (Mazzeo et al.
2012; Zotta et al. 2012, 2017; Zhang et al. 2020). Also, it
was reported that aerobic cultivation affects the expres-
sion of the peptidase system in L. casei (Siciliano et al.
2019). In this study, we aimed to evaluate the likely role
of L. casei CcpA in the performance of this species dur-
ing fermentation of a low-cost substrate (cheese whey)
to produce lactic acid and to analyze putative molecular
targets of the nitrogen metabolism that might be modu-
lated by CcpA.
Page 3 of 13 61
Material and methods
Microorganisms and culture media
The microorganisms used in the present work were Lacticasei-
bacillus casei BL23 (Acedo-Félix and Pérez Martínez 2003)
and L. casei BL71 (BL23 ccpA::erm), gently provided by Dr.
Pérez-Martínez (Monedero et al. 1997). In these strains, the
ability to metabolize milk lactose is linked to the lactose phos-
phoenolpyruvate:phosphotransferase system and a 6-phospho-
β-galactosidase (Mazé et al. 2010). Stock cultures (1 mL) were
stored at −70 °C in MRS medium (tryptone 10 g ­L−1, yeast
­ −1, meat extract 8 g L
extract 4 g L ­ −1, sodium acetate 5 g L­ −1,
­MgSO4·7H2O 0.2 g L ­ −1, ­MnSO4·4H2O 0.05 g L ­ −1, Tween
−1 −1
80 1 mL ­L and glucose 20 g ­L , pH 6.5, Biokar, Beau-
vais, France) with 20% (v/v) glycerol. Strains were grown at
37 °C under static conditions in MRS or Chemically Defined
Medium (CDM) (Piuri et al. 2003) ± meat peptone 0.1–0.5%.
Erythromycin 5 µg ­mL−1 was added to BL71 cultures for all
determinations and when the strain was used as inoculum for
stirred tank fermentation.
Cheese whey is composed of 4.6–5.2% lactose, 0.6–0.8%
soluble proteins and free amino acids, 0.3–0.4% lipids, 8–10%
of dried extract of mineral salts. Additionally, it contains
0.02–0.05% of citric and lactic acids, non-protein nitrogenous
components, such as urea and uric acid, vitamins of group B,
and traces of galactose and glucose are found by the hydrolysis
of lactose (Panesar et al. 2007; Carvalho et al. 2013).
Cheese whey medium was composed of 5% w/v of cheese
whey powder (kindly provided by Lácteos Vidal; www.​lacte​
osvid​al.​com.​ar), 0.6 mM M­ gSO4, 0.3 mM M ­ nSO4, 1 mL L ­ −1
Tween 80, and 50 mM ­NaPO4 pH 7. Cheese whey medium
was either supplemented or not with 0.5% yeast extract (Difco)
(YE) or 1.5% corn steep liquor (CSL).
Bottle batch fermentation (uncontrolled conditions)
Fermentation assays were performed in MRS or cheese whey
medium. 18 mL cultures of both strains were grown separately
in bottles under microaerophilic conditions to an initial O
­ D600
of 0.2 and were incubated for 65 h. Samples were collected
every 2 h during the first 24 h and every 4 h between 24 and
65 h of culture. A Hanna pH meter model HI98103 was used
for pH determinations. Viable bacteria counts (CFU m ­ L−1)
were determined from serial decimal dilutions in 0.85% NaCl.
Stirred tank batch fermentation (controlled
conditions)
The cultures used as inoculum were grown in MRS medium
and incubated at 37 °C and 50 rpm for 17 h. The volume of
inoculum used was calculated to initiate the fermentation at
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61 Page 4 of 13
an ­OD600 of 0.5. The batch fermentation was carried out in
MRS and cheese whey supplemented with 0.5% YE (Difco)
in a 5-L Biostat B plus bioreactor (Sartorius) in 5 L and 1
L working volumes respectively for both strains. The tem-
perature was maintained at 37 ± 1 ºC and the pH at 6 by the
addition of 1:2 (v/v) N
­ H4OH solution, which also served as
a nitrogen source. The %pO2 set point was < 10% maintained
by ­N2 addition (1vvm). Fermentations in MRS were incu-
bated for 24 h, while cheese whey fermentations with BL23
and BL71 continued for 72 and 55 h, respectively. Base con-
sumption defined the end of these last ones. Samples were
collected every 3 h. The pH was measured using a pH elec-
trode (Mettler Toledo, Germany) and the oxygen concentra-
tion was determined with a polarographic probe (Mettler-
Toledo). Foam formation was prevented through the addition
of 5% (v/v) antifoam A (Sigma, St. Louis, MO, USA). The
cultures were sampled throughout all the bioprocess phases
to determine the number of CFU m ­ L−1 by plating on MRS
agar plates and for analytical determination.
Analytical determinations
The biomass formation was monitored by ­OD600, CFU
­mL−1 and dry weight (DW) during growth in MRS. Due to
its intrinsic turbidity, bacterial growth in the cheese whey
medium was only evaluated by determining the number of
CFU ­mL−1. The dry mass was determined by taking variable
volumes according to the ­OD600, and centrifugation at 4000
×g for 10 min at 4 °C. The pellet was washed with 30 mL of
distilled water, centrifuged again under the same conditions
and resuspended in 2 mL of distilled water. This volume
was placed on the aluminum tray of a moisture analyzer
(Sartorius Moisture Analyzer series MA100) to determine
DW. The remaining glucose concentration in MRS medium
was determined using a colorimetric reaction (Glicemia
Enzimática AA líquida Wiener Lab, Rosario, Argentina).
Samples were taken every 15 min after reaching the expo-
nential growth phase and sugar was measured according to
the manufacturer´s instructions.
The enzymatic technique developed by Gutmann and
Wahlefeld (1974) was followed for lactate determination.
This protocol allows specific determination of the L- and
D-lactate isomers. L/D-lactate was oxidized to pyruvate in
the presence of ­NAD+ by the action of L(+)-lactate dehy-
drogenase or D(+)-lactate dehydrogenase. The reaction
mixtures contained 0.5 M glycine, 0.4 M hydrazine pH 9,
40 mM ­NAD+ and 5 mg protein m ­ L−1 of lactate dehydroge-
nase. The samples were incubated for 30 min at 37 °C and
the presence of lactate was measured by the formation of
NADH in a spectrophotometer at 340 nm. Lactic acid was
measured in cheese whey fermentations both in uncontrolled
conditions in bottle and in controlled conditions. In bottle
fermentations, lactate determination was performed at 22
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World Journal of Microbiology and Biotechnology (2021) 37:61
and 65 h of culture. In stirred tank fermentations, lactate
measurements were performed every 3–4 h.
In order to estimate the kinetic parameters, samples were
collected at each time point and ­OD600, DW or CFU ­mL−1
were determined (according to each culture condition). The
cultures growth parameters were determined graphically
during the exponential phase, by calculating µ values from
­ L−1) vs time.
the slope of the linear regression of Ln (CFU m
PepQ, PepX and PrtP enzymatic activities were assayed
as described in Piuri et al. (2003) from cultures grown to
stationary phase in CDM. This medium has been previ-
ously used in species of the genera Lacticaseibacillus and
Lactococcus for the study of components of the proteolytic
system, evaluating the addition of different nitrogenous
supplements, peptides and amino acids (Morishita et al.
1974; Guédon et al. 2001; Piuri et al. 2003; Alcántara et al.
2016). The activities of PepQ, PepX and PrtP are, respec-
tively expressed as nmol of Leu and Pro released per min-
ute per milligram of protein, nmol of nitroanilide released
per minute per milligram of protein, and fluorescence units
per minute per 10 mg of protein. The data were statistically
analyzed using the Prism 5.0 software (GraphPad Inc.). The
experiments were performed in triplicate and are presented
as the mean ± standard deviation (error bars). The Student’s
test was applied to establish whether the differences could
be considered significant at p < 0.05.
Protein purification of ­His6‑CcpA
Escherichia coli FT1 carrying pET15b-ccpAL.casei was grown
in Luria–Bertani (LB) broth to an O ­ D600 of 0.6 at 37 °C
and recombinant protein expression was induced for 4 h
with 0.5 mM IPTG. The cells were lysed by sonication in a
buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl,
5% glycerol, and 0.1 mM dithiothreitol, and the clarified
supernatants were loaded onto Ni–NTA columns (Qiagen,
Hilden, Germany). The columns were washed with 30 mM
and 50 mM imidazole, and the proteins eluted with 120 mM
imidazole. ­His6-CcpA-containing fractions were pooled, dia-
lyzed at 4 °C against 50 mM Tris–HCl pH 7, 40% glycerol,
0.1 mM phenylmethylsulfonyl fluoride and stored in aliquots
at –70 °C.
Electrophoretic mobility shift assay
An electrophoretic mobility shift assay (EMSA) was per-
formed with biotinylated oligonucleotide probes. To amplify
the promoter region of the prtP gene, a PCR was performed
using GoTaq DNA polymerase (Promega, Madison, WI,
USA) by following the manufacturer’s instructions. The
probe was made using primers Promp1: 5′-TTG​GAT​CCT​
TTA​TTC​TAG​CGT​TGG​C-3′ and prt2bio: Biotin-TEG-5′-
GAT​GAT​ACC​TTA​GTT​TGC​TGC​GAG​A-3′. The unlabeled
World Journal of Microbiology and Biotechnology (2021) 37:61
probe was made under the same conditions but using an
unbiotinylated oligonucleotide (5′-GAT​GAT​ACC​TTA​GTT​
TGC​TGC​GAG​A-3′) as reverse primer. The amplification
program was: 95 °C, 5 min; 25 cycles of: 1 min at 94 °C,
1 min at 54 °C, 1 min at 72 °C; and 10 min elongation at
72 °C. Chromosomal DNA from L. casei BL23 was used
as a template. The reaction product was purified from a 1%
agarose gel in 1X TAE buffer using GFX® PCR DNA and
the Gel Band Purification Kit (Pharmacia Biosciences, NJ,
USA), and used as probe in the EMSA. Purified H ­ is6-CcpA
protein (0.35; 1.33, 2.66 and 5.32 μM final concentration)
was incubated with 1.5 ng of probe in a buffer containing
10 mM Tris–HCl, pH 7.4, 50 mM KCl, 5 mM ­MgCl2, 1 mM
dithiothreitol, 10% glycerol, and 3 μg ­mL−1 sheared salmon
sperm DNA in 20 μL at 37 °C for 30 min. When required,
3 ng of unlabeled probe was used to compete with the
labeled probe. Samples were resolved by electrophoresis in
7.5% polyacrylamide gels. The gels were electro-transferred
to nylon membranes and probes were detected by chemilu-
minescence with alkaline phosphatase-conjugated strepta-
vidin and CDP-Star (GE-Biosciences, Chicago, IL, USA).
Chemiluminescent images were taken using a Fuji LAS1000
equipment and Image Gauge 3.122 software (Fuji Film).
Supershift assay
Antibody supershift assays were performed using 375-bp
and 420-bp probes synthesized by PCR with the oligonu-
cleotide pairs Promp1/prt2bio and PrtM: 5′- GCT​ACT​TTC​
AGT​CAC​CTT​GC-3′/PromP2: 5`-TTC​TGC​AGA​GAA​CCA​
AAT​CAA​ACC​C-3′, respectively, according to Ghosh et al.
(2006). After pre-incubation of the purified protein with or
without anti-CcpA antiserum (Küster et al. 1996) for 30 min,
an EMSA was carried out as described previously.
Regulator fishing assay
A “regulator fishing” approach was used as described in Rey
et al. (2003), using streptavidin-coated M-PVA Magnetic
Beads (Chemagen Technical, GmbH, Rodgau, Germany).
Biotinylated probes which include catabolite responsive ele-
ment (cre) sequences were used (positions −211 and +10 for
the 375-bp probe and −211 for the 420-bp probe; see above
and Supplementary Figure 1. DNA fragments were immo-
bilized with streptavidin-coated magnetic particles, as rec-
ommended by the manufacturer, using DNA-binding buffer
A (50 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl). After
this, magnetic particles were resuspended in protein-binding
buffer B (20 mM Tris pH 8.0, 1 mM EDTA, 10% glycerol,
1 mM dithiothreitol, 100 mM NaCl, 0.05% Triton ×100) and
incubated with crude cell extracts (0.5 mg of protein) of L.
casei BL23 for 20 min at room temperature. Subsequently,
the magnetic particles were washed three times with buffer
Page 5 of 13 61
B. Bound proteins were eluted with elution buffer C (20 mM
Tris pH 8.0, 1 mM EDTA, 10% glycerol, 1 mM dithiothrei-
tol, 1 M NaCl, 0.05% Triton X-100). Eluted proteins were
then analyzed by SDS-PAGE and Western blot with anti-
CcpA antibody.
Western blot
Samples were electrotransferred to PVDF membranes
according to the manufacturer’s instructions (Mach-
erey–Nagel, Düren, Germany). Polyclonal antibody against
CcpA (Küster et al. 1996) was used at a 1:2000 dilution.
Detection was performed with HRP-conjugated anti-rabbit
IgG (Santa Cruz Biotechnology, Dallas, TX, USA) and
chemiluminescence using ECL (Sigma, St. Louis, MO,
USA).
Transcriptional analysis of prtP
As mentioned in the evaluation of enzymatic activities (see
Analytical determinations), to evaluate the differences in
the components of the proteolytic system, it is necessary
to use cells grown in CDM medium with or without meat
peptone. RNA was extracted using the hot phenol method
(Spatafora et al. 1995). Cells of logarithmic growth phase
were harvested and washed at 4 °C in 50 mM EDTA pH
8, centrifuged for 5 min (20,000 xg) and resuspended in
10 mM Tris–HCl pH 8, 2 mM EDTA, 20 mM ammonium
acetate pH 4 in an ice bath to a final ­OD600 of 15. Then,
0.5 mL of acid phenol pH 4.7 (Ambion, Austin, TX, USA)
warmed to 65 °C was added and mixed by vortexing. After
heating at 65 °C for 5 min, an extraction with chloroform
was performed. The RNA was precipitated from the aqueous
phase using a 1/10 volume of 5 M NaCl and 0.6 volume of
isopropanol. The samples were treated with RQ1-RNAse
free DNAse (Promega, Madison, WI, USA) and another
extraction with chloroform was performed. After centrifu-
gation, RNA was resuspended in DEPC water (Ambion,
Austin, TX, USA). The samples were kept at − 20 °C for at
least 1 h. DNA absence was confirmed by PCR. The nucleic
acid concentration was measured using a Nanodrop2000
spectrophotometer (Thermo Scientific, Waltham, MA,
USA). RT-qPCR was performed as described in Palomino
et al. (2016), using the following primers: forward primer
prtp7 5′-TCG​GCG​AAA​TCC​AAG​CAA​AGG-3′ and reverse
primer prtp8 5′-GCT​GCG​GTT​GTG​TCA​GTG​G-3′ for the
prtP gene; forward primer 16srt1 5′-GCG​AAG​GCG​GCT​
GTC​TGG​-3′ and reverse primer 16srt2 5′-GGC​ACT​GAA​
GGG​CGG​AAA​CC-3′ for the 16S rRNA gene (used as ref-
erence). cDNA was mixed in the presence of Sybr Green
as fluorophore in multiwell plates covered with film in the
IQTM 5 Optical System Software (Bio-Rad) equipment. RT-
qPCR reactions were performed under the following cycling
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61 Page 6 of 13 World Journal of Microbiology and Biotechnology (2021) 37:61

conditions: for the prtP gene: 5 min at 95 °C, 30 cycles of

20 s at 90 °C, 20 s at 58 °C, and 20 s at 72 °C; and for the
16S rRNA gene: 5 min at 95 °C, 30 cycles of 20 s at 90 °C,
20 s at 54 °C, and 20 s at 72 °C. The relative change in the
expression of individual genes was analyzed following the
standard curve method (Larionov et al. 2005). The Student’s
test was applied to establish whether the differences could
be considered significant at p < 0.05.

Results

Evaluation of the ccpA mutant in MRS or cheese

whey batch fermentation

Our first objective was to find the best nitrogen supplements

in the use of cheese whey as a substrate to produce lac-
tic acid. We used wild-type L. casei (BL23) and its ccpA
derivative mutant (BL71) in cheese whey batch fermentation
under uncontrolled conditions. We evaluated cheese whey
with or without different nitrogen sources (YE or CSL) for
lactic acid production in bottle batch fermentations with
both strains. A 40-h lag phase was observed for acidifica-
tion in the absence of nitrogen (YE or CSL) supplementation
(Fig. 1a). YE or CSL supplementation resulted in a three-
fold decrease in the lag phase duration (15 h). Results of the
number of CFU ­mL−1 were consistent with the acidifica-
tion profiles (Fig. 1b), and a higher rate of acidification was
observed for BL71 in all conditions.
Lactate concentration (Table 1) was determined for both
isomers in the cheese whey experiments. The D/L-lactate
proportions were similar for the wild type and the ccpA Fig. 1  a pH determination during Lacticaseibacillus casei growth in
mutant in all the growth media assayed and higher levels of cheese whey with different nitrogen sources (None; CSL, corn steep
lactate were observed for the mutant strain in all conditions. liquor 1.2%; YE, yeast extract 0.5%) as described in Materials and
When cheese whey medium was supplemented with YE, an Methods. b Biomass increase under the same conditions, expressed
as CFU m ­ L−1
almost two-fold increase in lactate production early in the
growth was observed for BL71 compared to BL23 (Table 1).
After choosing the best nitrogen source, the following fermentations were carried out in cheese whey plus 0.5%
step was to scale-up the fermentation in a bioreactor under YE in a stirred tank under controlled pH and oxygen condi-
controlled conditions. We compared the fermentative char- tions ­(pO2 < 10%). Lactate production was determined and
acteristics of BL23 and BL71 in MRS batch fermentations the obtained kinetic parameters are shown in the Supple-
in stirred tank reactors under controlled pH and low oxygen mentary Table 1 Figures 2a and 2b show the growth pat-
conditions ­(pO2 < 10%). The growth rates (µ), measured terns obtained. In Fig. 2a, BL23 shows a pattern compatible
through the increase in dry weight (DW) and CFU ­mL−1, with a diauxic growth, which is not observed in the mutant
for the BL71 strain (ccpA mutant) (DW 0.42 ­h−1 and CFU strain lacking the CcpA regulator. Under these conditions,
­mL−1 0.47 ­h−1) were consistently higher than those for the the ccpA mutant strain showed a shorter interval of time for
BL23 strain (DW 0.24 ­h−1 and CFU ­mL−1 0.28 ­h−1). Glu- lactate production compared to BL23. Thus, BL23 shows a
cose consumption, which was measured to determine the delay in production of more than 20 h compared to BL71.
yield of the process, showed a more efficient use of substrate The 48-h cumulative base consumption, which was 16.1
for BL71 [Y(x/s) BL71 = 0.27 g ­L−1] than for BL23 [Y(x/s) and 0.3 g ­L−1 of ­NH4OH for BL71 and BL23, respectively,
BL23 = 0.12 g ­L−1]. was used as an indicator of the ability to produce acidic
After characterizing the kinetic parameters of both compounds. According to this, the 48-h cumulative lac-
strains in MRS (without nitrogen restriction) in a bioreactor, tic acid production was 2.42 ± 0.52 g L ­ −1 for BL23 and

13
World Journal of Microbiology and Biotechnology (2021) 37:61 Page 7 of 13 61

Table 1  Lactic acid production Supplement None YE CSL

in batch cultures of cheese whey
alone or supplemented with Strain BL23 BL71 BL23 BL71 BL23 BL71
0.5% yeast extract (YE) or 1.2%
−1 a
corn steep liquor (CSL) under L- lactate (g ­L ) nd nd 2.52 ± 0.19 5.01 ± 0.35 1.40 ± 0.18 1.92 ± 0.20
uncontrolled conditions ­ −1)b
L- lactate (g L 2.89 ± 0.24 3.65 ± 0.23 8.13 ± 0.52 9.39 ± 0.51 2.43 ± 0.21 3.42 ± 0.24
D- lactate (g L­ −1)b 0.14 ± 0.03 0.15 ± 0.03 0.50 ± 0.08 0.32 ± 0.03 nd nd
% L-D 95.1–4.9 94.2–5.8 95.8–4.2 96.6–3.4 nd nd
pH 4.5 ± 0.2 4.0 ± 0.1 3.8 ± 0.2 3.5 ± 0.1 5.0 ± 0.1 4.0 ± 0.1

The pH determinations were made at 65 h for the cultures of cheese whey alone or supplemented with
0.5% yeast extract (YE) and at 42 h for the cultures supplemented with 1.2% CSL
nd not determined; BL23 (wild type); BL71 (ccpA mutant)
a
Early production (22 h)
b
Late production (65 h)

19.33 ± 1.11 g ­L−1 for BL71, i.e. an eight-fold increase in

production compared to BL23 strain (Table 2). This param-
eter correlates with the higher value of CFU ­mL−1 (8.9 × ­108
for BL23 and 4.36 × ­109 for BL71). The cumulative produc-
tion of lactate at the end of the fermentation, defined by
base consumption (72 h for BL23 and 55 h for BL71), was
higher for BL71 (44.23 g L ­ −1) than for BL23 (27.58 g L
­ −1).
Thus, under the culture conditions used, the ccpA mutation
increased the productivity of lactate from 0.17 to 0.80 g
(L.h)−1 at 55 h of culture (the peak of lactate production for
BL71). This result shows that BL71 achieves a maximum
value of production of lactate 17 h before BL23. Not only
the final amount of lactate produced was higher but also the
specific productivity was 0.38 g (L.h)−1 for BL23 and 0.8 g
(L.h)−1 for BL71. These results show that the ccpA mutation
favors utilization of the different carbon and nitrogen sources
of the culture medium.

Involvement of CcpA in the regulation

of the proteolytic system

To assess the likely role of CcpA in the regulation not only

Fig. 2  a Growth curve (CFU ­mL−1) of the L. casei wild-type strain
BL23 and the ccpA mutant strain BL71 and lactic acid production on
cheese whey-based medium supplemented with 0.5% YE under pH
controlled conditions. b Comparison of ­NH4OH consumption (in g
­L−1) in L. casei BL23 and BL71 on cheese whey-based medium with
0.5% YE
of CCR but also of the provision of nitrogen sources and
the proteolytic system in L. casei, we analyzed growth in a
CDM (Morishita et al. 1974; Piuri et al. 2003) with addition
of meat peptone (80% peptides and 20% free amino-acids) as
an extra nitrogen source. CDM can be used to assess differ-
ences triggered by increased provision of nitrogen sources
that could not be studied in complex media like MRS or
cheese whey (Morishita et al. 1974; Guédon et al. 2001;
Piuri et al. 2003; Alcántara et al. 2016). The supplementa-
tion of CDM (a medium with nutrient restrictions) with meat
peptone induced a most notorious effect on growth com-
pared to YE supplementation (data not shown). As shown
in Fig. 3, growth in CDM was similar for both strains, while
addition of meat peptone resulted in a higher stimulation
of growth in BL71. This strain was presumably able to use
the nitrogen provision immediately, whereas BL23 showed

13
61 Page 8 of 13 World Journal of Microbiology and Biotechnology (2021) 37:61

27.58 ± 2.41
37.35 ± 3.20
72

9.40 ± 1.07
44.23 ± 4.19
55
­ −1) from cheese whey-based medium supplemented with 0.5% yeast extract (YE) in bioreactor under controlled conditions

7.86 ± 0.19
30.8 ± 0.65
52

3.09 ± 0.18
24.7 ± 0.37
50

2.42 ± 0.52
19.33 ± 1.11

Fig. 3  Lacticaseibacillus casei growth in chemically defined medium

(CDM) with increasing concentrations of meat peptone. Circles cor-
48

respond to L. casei BL23, whereas squares correspond to BL71. Meat

peptone was added at 0.1 and 0.5%
1.26 ± 0.05
2.66 ± 0.04

a lag phase of 4 h (Fig. 3). The activities of the cell-wall

31

proteinase PrtP and the peptidases PepX and PepQ were

evaluated in bacterial cells grown in CDM. The three activi-
0.59 ± 0.07
1.82 ± 0.04

ties showed a significant increase in the BL71 strain (Fig. 4),

which would account for a de-repression effect.
27

Molecular analysis of the CcpA mode of action

in Lacticaseibacillus casei
1.33 ± 0.06
1.28 ± 0.04

In view of the results on the proteolytic and peptidase activi-

24

ties, we next searched for consensus cre sequences, involved

in the specific CcpA recognition, within the genomic regions
0.46 ± 0.05

of the genes encoding PrtP, PepX and PepQ (NCBI Refer-

ence Sequence: NC_010999.1; Mazé et al. 2010), using the
< 0.2

consensus sequence described by Francke et al. (2008). The

7

in silico analysis revealed two putative cre sites at positions

−211 and +10 relative to the translation initiation site of
< 0.2
< 0.2

prtP. Additional sites were observed at position +23 in the

Table 2  Lactic acid production (g L

pepX gene, with high score alignment with a cre site identi-
fied in the L. casei BL23 phosphofructokinase gene (pfk),
< 0.2
< 0.2
­ −1
Lactic acid production g L

known to be recognized by CcpA (Viana et al. 2005). In L.

0

casei, the pepQ gene is transcribed divergently to ccpA and

three putative cre sites were observed at positions -123 in the
BL23 (wild type)

orientation ccpA-pepQ and at -47 and -2 in the orientation

BL71 (ccpA)

pepQ-ccpA (Supplementary Figure 1). Depending on the

relative position of the cre sites within CcpA-responsive pro-
Hours

moters, CcpA may act either as a repressor or as an activator

13
World Journal of Microbiology and Biotechnology (2021) 37:61
Fig. 4  Proteolytic specific activities in L. casei BL23 and BL71.
Activities for PepQ, PepX and PrtP are respectively expressed as
nmol of Leu and Pro released per minute per milligram of protein,
nmol of nitroanilide released per minute per milligram of protein,
and fluorescence units per minute per 10 mg of protein. *p < 0.01,
**p < 0.001
(Marciniak et al. 2012). Activation of transcription has been
reported when putative cre sites were located upstream of
the hexameric −35 sequence, whereas repression has been
reported when the cre site was found in or downstream of
putative −35 and −10 sequences (Fujita 2009). The location
of cre sites in pepX and prtP (+23 and +10, respectively)
was compatible with a repressive effect of CcpA, whereas
the location of three cre sites in the pepQ-ccpA intergenic
region would account for both effects, being the −2 position
responsible for a repressive effect.
To further investigate the role of CcpA in the regulation
of the L. casei proteolytic system, we evaluated the expres-
sion level of the prtP gene by RT-qPCR analysis, which
showed a 2.2-fold de-repression of prtP in BL71 compared
to BL23 when bacteria were grown in CDM supplemented
with meat peptone (Fig. 5a). To investigate the nature of the
relationship between CcpA and PrtP, we assessed the ability
of CcpA to bind to the prtP promoter region by using two
different approaches: the “regulator fishing” assay (Rey et al.
2003) and an EMSA. DNA probes that included cre sites
at positions −211 and +10 (375 bp probe) and only −211
(420 bp probe) were designed (Fig. 5b) and incubated with
crude cell extracts of L. casei BL23. As shown in Fig. 5c,
CcpA binding to both probes was detected with an anti-
CcpA polyclonal antibody (Küster et al. 1996); however,
only high affinity interaction was observed when the +10
cre site was present in the probe, since high ionic strength
was needed to release the protein from the 375-bp probe
(Fig. 5c). No band was observed when extracts of BL71 were
used (data not shown). EMSA experiments were also carried
Page 9 of 13 61
out with the 375-bp probe and purified H ­ is6-CcpA from L.
casei. This promoter region has the particularity of generat-
ing a secondary structure that was evidenced in the absence
of any added protein, probably due to the presence of pal-
indromic runs of A and T sequences (Fig. 5d). Increasing
amounts of H ­ is6-CcpA showed a concentration-dependent
shift (Fig. 5d). This CcpA-triggered shift was corroborated
by a supershift analysis using anti-CcpA antibodies and the
375-bp and 420-bp probes. Only when the +10 cre site was
present in the probe, a supershift was observed, showing
CcpA binding to the prtP promoter region (Supplementary
Figure 2). Altogether, these results indicate that CcpA binds
to the prtP promoter at the cre site in position +10, compat-
ible with a repressive effect.
Discussion
Strategies to improve lactic acid production by metabolic
engineering approaches include the use of ccpA mutants
that, for instance, show reduction of CCR of the galactose
(gal) utilization operon in Lc. lactis (Singh et al. 2006). Our
results indicate that inactivation of ccpA in L. casei can be a
strategy for enhanced lactate production from cheese whey,
an important food industry by-product. The substrate is one
of the highest production costs for lactate synthesis, and the
use of refined sugar (glucose for example) makes the pro-
cess even more expensive. Since food-waste materials are a
good alternative as substrates because of their high content
of carbohydrates, many efforts have been made to use such
substrates to produce lactate by Lacticaseibacillus. Wang
et al. (2015), for example, chose soybean straw as the sub-
strate to produce lactic acid by L. casei because of its high
protein content, and found a productivity of 0.61 g (L.h)−1.
In the present study, we found that lactic acid productivity in
cheese whey-based medium with the L. casei ccpA mutant
BL71 strain was 0.80 g (L.h)−1. This productivity achieved
for BL71 in batch fermentations was in the order of that
reported by others using cheese whey and Lacticaseibacillus
rhamnosus (Alvarez et al. 2010; Rabaioli Rama et al. 2019).
In batch fermentations using cheese whey-based medium,
BL71 produced higher amounts of lactic acid and the pro-
duction was significantly faster, indicative of a more efficient
use of the substrate compared to the wild-type strain.
When comparing controlled to uncontrolled conditions,
although at initial points the lactate production in the bot-
tle culture was higher, at the end of the fermentation, the
lactate production in the uncontrolled system was 9.39 g
­L −1 and in controlled bioreactor 44.23 g L ­ −1, that was
almost 5 times higher. The differences observed between
both conditions could be explained by a difference in oxy-
gen tension which is more marked in the early phases of
fermentation. As previously reported in L. casei growth
13
61 Page 10 of 13
Fig. 5  a Expression of the prtP gene determined by RT-qPCR. Rela-
tive transcription is plotted. Data represent the level of expression for
the prtP gene normalized to that of rRNA 16S for each condition. C:
CDM; CDMP: CDM with meat peptone. Bars show standard errors
from three independent experiments, *p < 0.01. b prtP promoter
sequence. The − 35/− 10 regions of the putative promoters are in
bold italics and indicated by boxes and potential cre sequences are
underlined and aligned with consensus sequences described in Sup-
plementary Figure 1. Translation-initiation codons and putative ribo-
some-binding sites are indicated by boxes. Promoters were predicted
using the Softberry software BPROM (www.​softb​erry.​com). Primers
to amplify the 375-bp probe are displayed in open boxes, whereas
primers to amplify the 420-bp probe are displayed in gray boxes. c
in cheese whey or milk media, different oxygen tension
conditions can influence lactate production by mainly
affecting LDH activity (Ricciardi et al. 2019; Siciliano
et al. 2019). On the other hand, in bottle culture the final
product is limited by pH, while in the bioreactor system
13
World Journal of Microbiology and Biotechnology (2021) 37:61
Western blot analysis of “regulator fishing” experiments. Eluted pro-
teins were detected with an anti-CcpA antibody. “Low” and “high”
indicate the ionic strength of the elution buffer used in the “regulator
fishing” assay, corresponding to buffers B and C described in materi-
als and methods, respectively. ­His6-CcpA is the recombinant purified
L. casei CcpA protein (Viana et al. 2005). d ­His6-CcpA EMSA with
the 375-bp prtP promoter probe. Increasing amounts of ­His6-CcpA
purified protein (0.35 µM to 5.32 µM) were incubated with a 375-bp
DNA probe that includes the prtP promoter and cre sites at positions
− 211 and +10. The arrows indicate the positions of free probe, sec-
ondary structure and mobility shift. *shows lanes where shift was
visualized
this limitation is lower since the pH is controlled by add-
ing base.
All these results suggested a possible improvement on the
use of carbon and energy sources in rich medium due to the
mutation of the global regulator CcpA.
World Journal of Microbiology and Biotechnology (2021) 37:61
Since most LAB rely on peptides rather than on amino
acids for efficient nitrogen assimilation, the absence of
efficient proteolytic and peptidase systems may limit their
development in cheese whey medium. Thus, the fast milk
acidification phenotype has been linked to the presence in
LAB of surface proteases able to efficiently degrade milk
casein (Dandoy et al. 2011). In agreement with the reports of
other authors (Ricciardi et al. 2019), the growth of BL23 in
cheese whey (Fig. 2a) showed a diauxic curve. This behavior
was not observed in BL71. Diauxie was probably the con-
sequence of the presence of other residual sugars in cheese
whey, in addition to lactose. As expected, the BL71 mutant,
unregulated for the CCR process, did not show this pattern.
Although ccpA mutants from lactobacilli generally show a
diminished growth rate in laboratory media (Monedero et al.
1997), this mutation allowed faster development of L. casei
BL23 in cheese whey, reducing the lag time and increasing
lactic acid productivity. Similarly, Alcántara et al. (2016)
found that a mutant from L. casei BL23 deficient in the tran-
scriptional regulator PrcR, which displays a slower growth
in MRS than the wild type, showed faster milk acidification
as a result of a deregulated expression of the cell-envelope
proteinase PrtP. In agreement with these results, in the pre-
sent study we showed that the BL71 strain expressed higher
levels of the PepQ and PepX peptidases and the proteinase
PrtP and a direct control of prtP expression was evidenced
through CcpA binding to its promoter. Deregulation of the
proteolytic system upon a ccpA mutation is thus the likely
cause of the better performance displayed by the BL71
mutant strain during cheese whey fermentation.
Our results contrast with data obtained in Lc. lactis,
where the pepQ gene in a ccpA mutant has been described
to be downregulated by a factor of two (Zomer et al. 2007).
In analogy, while CcpA activates the transcription of the pfk
and pyk glycolytic genes in Lc. lactis (Luesink et al. 1999),
it represses them in L. casei (Viana et al. 2005). An impor-
tant difference between these two LAB is the absence of the
pleiotropic nitrogen regulator CodY in lactobacilli, which,
in Lc. lactis, controls the expression of components of the
proteolytic system (Guédon et al. 2001). Therefore, the
regulatory mechanisms controlling glycolysis and nitrogen
metabolism in lactobacilli and lactococci are different. In Lc.
lactis, coordinated action of CodY and CcpA would link car-
bon and nitrogen metabolism (Petranovic et al. 2004; Zomer
et al. 2007), whereas in Lacticaseibacillus, CcpA could act
together with PrcR in this task. However, regulation of pro-
teolysis may differ among different lactobacilli and include
new participants. In Lactobacillus delbrueckii subsp. lactis,
for example, a distinct regulator (YebC) has been shown to
control the expression of the surface proteinase PrtL and
the oligopeptide transporters OppA1 and OptS by binding
to their gene promoters (Brown et al. 2017). Our results
point to CcpA as a valuable target for strain modification
Page 11 of 13 61
in lactobacilli to improve fermentative processes in which
industrial waste products with high nitrogen content, such
as cheese whey, are used. This gene target must be evaluated
in different lactobacilli species and substrates for enhanced
production of lactic acid.
Supplementary Information The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s11274-​021-​03028-z.
Funding The present report was supported by grants from the Uni-
versidad de Buenos Aires (UBA) (UBACyT 20020170200329BA and
20020170100019BA) and Consejo Nacional de Investigaciones Cientí-
ficas y Técnicas (CONICET), Argentina. JFM is a graduate fellow of
CONICET; VMG is a member of CSIC (Spain); MVC and DML are
members of INTI (Argentina) and MMP, SMR and MCA are members
of CONICET (Argentina).
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
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