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Applied and Environmental Microbiology, December 2000, p. 5360-5367, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Hydrolysis of Sequenced 
-Casein Peptides Provides New Insight
into Peptidase Activity from Thermophilic Lactic Acid Bacteria and
Highlights Intrinsic Resistance of Phosphopeptides
Stéphanie-Marie
Deutsch,1
Daniel
Molle,1
Valérie
Gagnaire,1
Michel
Piot,1
Danièle
Atlan,2 and
Sylvie
Lortal1,*
Institut National de la Recherche
Agronomique, Laboratoire de Recherches de Technologie
Laitière, 35042 Rennes Cédex,1
and Unité de Microbiologie et de Génétique
(CNRS-UMR 5577), Université de Lyon I, 69622 Villeurbanne
Cédex,2 France
Received 5 May 2000/Accepted 26 August 2000
 |
ABSTRACT |
The peptidases of thermophilic lactic acid bacteria have a key role
in the proteolysis of Swiss cheeses during warm room ripening. To
compare their peptidase activities toward a dairy substrate, a
tryptic/chymotryptic hydrolysate of purified 
-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the -casein hydrolysate, as
shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG
LL14 and ITG LL51, L. delbrueckii subsp.
bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus
thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized
in protein. The peptidase activities were assessed with the -casein
hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm
room ripening) by (i) free amino acid release, (ii) reverse-phase
chromatography, and (iii) identification of undigested peptides by mass
spectrometry. Regardless of strain, L. helveticus was the
most efficient in hydrolyzing -casein peptides. Interestingly, cell
extracts of S. thermophilus were not able to release a
significant level of free proline from the -casein hydrolysate,
which was consistent with the identification of numerous dipeptides
containing proline. With the three lactic acid bacteria tested, the
phosphorylated peptides remained undigested or weakly hydrolyzed
indicating their high intrinsic resistance to peptidase activities.
Finally, several sets of peptides differing by a single amino acid in a
C-terminal position revealed the presence of at least one
carboxypeptidase in the cell extracts of these species.
| |
INTRODUCTION |
Thermophilic lactobacilli such as
Lactobacillus helveticus or Lactobacillus
delbrueckii along with Streptococcus thermophilus constitute essential lactic starters in Swiss-type cheese (400,000 tons
of Emmentaler produced per year worldwide). The three species have a
key role in the acidification step and in cheese proteolysis (13). Adequate proteolysis is essential for acceptable
quality of the ripened cheese. Whole caseins are first hydrolyzed by
milk protease (plasmin) and/or rennet (chymosin and pepsin) into large and intermediate-sized peptides. The proteinases and peptidases from
starters subsequently hydrolyze them in small peptides and amino acids,
which are known to be aroma precursors. In the three species
considered, peptidases are intracellular enzymes released upon lysis in
the curd, where they remain active for several weeks (14, 40,
41). Because of their predominant role in proteolysis, studies on
peptidases of thermophilic lactobacilli and S. thermophilus have expanded greatly in recent years (5, 18, 35, 36). Several aminopeptidases, dipeptidases, and peptidases
specific to proline-containing peptides (prolidase, prolinase,
X-prolyl-dipeptidyl aminopeptidase, and prolyl aminopeptidase) were
isolated, cloned, and sequenced (20); only the general
aminopeptidase(s) and the X-prolyl-dipeptidyl-aminopeptidase of
thermophilic lactobacilli were shown to be essential to cheese flavor
(27, 33). To date, no carboxypeptidase has been isolated.
The role of each species in cheese proteolysis is still unclear since
their peptidases were mainly characterized on synthetic substrates. Few
reports compared the overall potential of the different species; all
results showed that peptidase activities are generally highest in
L. helveticus strains (17, 36).
The objective of this work was to better assess how L. helveticus, L. delbrueckii, and S. thermophilus contribute to
proteolysis in Swiss cheese in order to improve the choice of starters
and to control this essential process. For that purpose, we compared the intracellular peptidase potentials of these species, using a dairy
substrate and the pH and temperature used for warm room ripening. The
substrate chosen was a tryptic/chymotryptic hydrolysate of -casein,
mimicking the initial proteolysis as proposed by Lemée
et al. (24). We characterized it by tandem
mass spectrometry (MS-MS) and then subjected it to the action of cell
extracts, allowing quantitative and qualitative analyses of the
peptidase activities.
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MATERIALS AND METHODS |
Strains and culture conditions.
Origins of the strains used
are summarized in Table 1. L. helveticus, L. delbrueckii subsp. lactis, and L. delbrueckii subsp. bulgaricus were stored at 
80°C
in MRS broth (Difco, Sparks, Md.) (7), and S. thermophilus was kept in M17 broth (Difco) supplemented with 5%
(wt/vol) lactose (38). The media were supplemented with 15%
glycerol prior to freezing. For the propagation of strains, media
without glycerol were used, and growth at 37°C was monitored by the
optical density at 650 nm. (OD650).
Preparation of cell extracts.
Cells were collected during
the exponential growth phase (OD650 = 1) by
centrifugation (7,000 × g, 20 min, 7°C) and washed twice with cold distilled sterile water (15% of the initial culture volume). The pellets were stored 24 h at 18°C before being
resuspended in cold sterile water (6% of initial culture volume) and
submitted to a precooled French press apparatus at 138 MPa for 3 min
(one run). Suspensions were centrifuged at 4,000 × g
at 4°C for 30 min to eliminate unbroken cells. The supernatants
representing cell extracts were filtered (0.45-µm-pore-size Sartorius
filter) and stored at 18°C until used. Protein content was assayed
according to Bradford method (Bio-Rad S.A., Ivry-sur Seine, France),
using bovine serum albumin (Sigma, Saint Quentin, Fallavier, France) as
a standard. Depending on the strain, the protein content was between 1 and 5 mg per ml of cell extract.
Assay of peptidase activity.
The lyophilized -casein
hydrolyzate used as the substrate was obtained as described by
Lemée et al. (24). Briefly, pure -casein
(kindly supplied by EURIAL Poitouraine, Nantes, France) (23)
was dissolved in sterile distilled water at a final concentration of 10 g/liter. Hydrolysis was performed using a mixture of trypsin and
chymotrypsin (enzyme/substrate ratio, 1/1,000 [wt/wt]). After 3 h of incubation at 37°C, the -casein was completely converted to
peptides. Both enzymes were inactivated by heating at 80°C for 20 min, and the hydrolysate was lyophilized and stored at 4°C.
Five hundred fifty micrograms of protein of thawed cell extract
adjusted to 500 µl with sterile distilled cold water was added
to 3.5 ml of
-casein hydrolysate solution (0.8 mg of
-casein
hydrolysate
per ml in 20 mM ammonium acetate buffer [pH 5.5]),
sterilized by
filtration (0.22-µm-pore-size filter), and incubated
at 24°C.
Samples were collected at different times. Enzyme activity
was stopped
by one of two methods, depending on the nature of
subsequent analysis:
(i) by adding 1% (vol/vol) trifluoroacetic
acid (TFA) or (ii) by
adding 80% (vol/vol) absolute ethanol and
incubating the mixture for
6 h at 4°C to eliminate precipitated
proteins and large peptides
by centrifugation (5,000 ×
g, 15 min,
4°C). After
inactivation, samples were stored at
18°C until use.
Controls were
made without cell extract and with heat-treated
(100°C, 20 min) cell
extracts. For each strain, two independent
assays were carried
out.
Free amino acids.
Free amino acids were quantified using the
cadmium ninhydrin reagent by the method of Baer et al.
(2), with methionine as a standard. Triplicate samples at
each time, inactivated by ethanol treatment, were thawed at 4°C.
Fifty microliters of each sample was mixed with 550 µl of distilled
water, and 1.25 ml of Cd-ninhydrin solution was added. Free amino acids
were also analyzed by cation-exchange chromatography with an automatic
amino acids analyzer (Pharmacia LKB Alpha Plus; Amersham Pharmacia
Biotech Europe GmbH, Orsay, France) as previously described by Spackman et al. (37), using lithium citrate buffer for elution.
Before analysis, samples inactivated with 1% TFA were thawed at room temperature and centrifuged (5,000 × g, 15 min,
4°C). The supernatant was recovered, and sulfosalicylic acid (30 mg/ml) was added. The solution was then incubated for 1 h at 4°C
and centrifuged (5,000 × g, 15 min, 4°C). The free
amino acids were assayed in the supernatant fraction.
Amino acids compositions of the
-casein peptides were determined
after acid hydrolysis in sealed tubes with 6 N HC1 at 110°C
for
24 h (
6). Hydrolysates were evaporated to dryness in
vacuum
over KOH pellets, washed twice with distilled water, and
analyzed
with the amino acid analyzer described above but using sodium
instead of lithium citrate for
elution.
Peptides.
Peptides were separated by reverse-phase
high-pressure liquid chromatography (RP-HPLC) on a Waters (Milford,
Mass.) 625LC system, using solvent A (0.106% TFA in MilliQ water) and
solvent B (0.100% TFA, 80% acetonitrile, 20% MilliQ water). The
samples were analyzed on a Nova-Pack C18 column (39- by
150-mm inside diameter 4-µm particle size, and 60-Å pore size;
Waters, Saint Quentin en Yvelines, France) with a linear gradient from
0 to 55% solvent B for 60 min followed by 55 to 80% solvent B for 2 min, at a flow rate of 0.8 ml/min at 40°C. The eluted peaks were detected by absorbance at 214 nm using a photodiode array detector spectrometer (Waters 991 series). Before injection, samples inactived with TFA were thawed at room temperature and centrifuged (10,000 × g, 15 min, 4°C); 50 µl of the supernatant were
directly injected into the column.
MS.
Mass spectra were recorded on a PE Sciex Api
III Plus triple-quadruple mass spectrometer (Sciex, Thornhill, Ontario,
Canada) equipped with an atmospheric pressure ionization source. The
masses of the peptides were determined by HPLC-electrospray ionization (ESI)-MS analysis as follows. Online with the separation by RP-HPLC, a
postcolumn flow splitter was used to introduce 10% of the HPLC eluate
into the mass spectrometer (25). The ion source voltage was
set at 4.8 kV, and the orifice voltage was set at 80 V. The quadruple
mass analyzer (Q3) was scanned to an m/z range of 200 to
2,000 Da, with a step size of 0.3 Da and a dwell time of 0.5 ms per
step. The molecular masses were determined from these data by using the
software supplied by Sciex (Biomultiview 1.2). The amino acid sequence
of each peptide was determined by MS-MS as follows. The peptides
collected after RP-HPLC separation were continuously infused with a
pump model 22 (Harvard, South Natick, Mass.) at a flow rate of 3 µl/min. Collision-actived dissociation experiments were performed
with argon, and the collision energies were set around 30 eV in
accordance with the molecular mass and the charge state of the parents
ions (29). The resolution was set to pass a 2-Da window
around the parent ion on the Q1 and was adjusted a unit m/z
for analysis of the product ions on the Q3.
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RESULTS |
Characterization of the -casein hydrolysate.
RP-HPLC
coupled with MS-MS showed that the tryptic/chymotryptic
-casein hydrolysate contained 34 peptides from 3 to 35 amino acids (373.2 to 3,864.4 Da) including three phosphorylated
peptides [Arg1-Arg25],
[Phe33-Lys48], and
[Phe33-Phe52] (Table
2). Figure 1 shows the complete sequence of the
-casein (209 amino acids) as well as the 34 peptides obtained in the
hydrolysate. Two iterations of the tryptic/chymotryptic hydrolysis led
to the same 34 peptides, showing the reproducible preparation of this
substrate. The bonds hydrolyzed corresponded to those predicted in the
review by Pelissier (32), with two additional sites:
Leu125-Thr126 and
Met144-His145. The amino acid composition was
determined after complete acid hydrolysis of the -casein peptides.
The total amino acid content, 5.9 mM, was 96% of the expected
theoretical value (6.1 mM).
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FIG. 1.
-Casein sequence (209 amino acids; one-letter amino
acid code). Phosphorylated serines are indicated by the letter "U"
and highlighted in grey; every 10th amino acid is framed in bold. The
34 initial peptides of the hydrolysate are represented in black
rectangles above the -casein sequence. After 72 h of
hydrolysis, nonhydrolyzed peptides were identified by MS-MS and are
presented below the sequence with different patterns, depending on the
cell extract used:  for L. helveticus ITG LH1,
 for L. delbrueckii subsp. lactis ITG
LL14, and  for S. thermophilus TA 060.
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Kinetics of -casein peptide hydrolysis by cell extracts.
The release of free amino acids (Fig. 2)
showed that all strains were able to degrade -casein peptides at the
pH (5.5) and temperature (24°C) used. No release of amino acids was
noted in the absence of cell extracts or with heat-treated extracts,
indicating no subsequent degradation of the -casein hydrolysate by
residual tryptic or chymotryptic activity.
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FIG. 2.
Time course of hydrolysis of the -casein hydrolysate
by cell extracts of the following strains: (a) L. helveticus
ITG LH1 (  ), ITG LH77 ( ), and CNRZ 32 (--- ---), (b)
L. delbrueckii subsp. lactis ITG LL14 ( )
and ITG LL51 ( ), and L. delbrueckii subsp.
bulgaricus NCDO 1489 ( ) and CNRZ 397 ( ); and (c)
S. thermophilus CNRZ 385 ( ), TA 060 ( ), and CIP
(Pasteur Institut Collection, Paris, France) 102303 (--- ---). The enzyme reactions
were performed for 72 h at 24°C in 20 mM acetate ammonium buffer
(pH 5.5) with 137.5 µg of cell extract proteins per ml for 0.7 mg of
lyophilized -casein hydrolysate per ml. Blanks (······,
······, and ······) were assayed with the same conditions
but with boiled cell extracts.
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The time course of hydrolysis exhibited a high initial rate which
decreased drastically after 8 to 10 h of incubation. After
72 h of incubation, no significant increase of free amino acids
was
observed for most of the strains. This can be explained by
either
peptidase inactivation or resistant peptides. The addition
at 72 h
of fresh
-casein hydrolysate led to new hydrolysis with
a similar
high initial rate, showing that the peptidases were
still active (Fig.
3). Thus, the decreased rate was due to peptides
resistant to
hydrolysis. This was further confirmed by the addition
at 72 h of
fresh cell extracts; in this case, no increase of amino
group was noted
(Fig.
3).
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FIG. 3.
Hydrolysis of the -casein hydrolysate by cell
extracts () of L. helveticus ITG LH77 (a) and
S. thermophilus CIP 102303 (b): effect of the addition at
72 h of fresh -casein hydrolysate () or fresh cell extracts
().
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The initial rate and final extent were species and strain dependent.
L. helveticus showed the most efficient peptidase activity
regardless of strain, with a mean initial rate of 0.12 mM/h (±0.01
[standard deviation {SD}], depending on the strain) and final
amount of 2.93 ± 0.25 mM free amino acids. These values were,
respectively, 0.10 ± 0.02 mM/h and 2.1 ± 0.13 mM for the
S. thermophilus strains.
L. delbrueckii showed
the largest intraspecies variability:
L. delbrueckii subsp.
bulgaricus NCDO (National Collection of
Dairy Organisms)
1489 exhibited a higher initial rate than all
of the
L. helveticus strains studied, but the final content of
free amino
acids was lower.
L. delbrueckii subsp.
lactis (Institut
Technique du Gruyère, Gruyère,
France) ITG LL51 and ITG LL14
and
L. delbrueckii subsp.
bulgaricus CNRZ (Centre National de
la Recherche
Zootechnique collection, Institut National de la
Recherche
Agronomique, Jouy-en-Josas, France) 397 had the lowest
initial rate
(0.064 ± 0.01 mM/h) and the lowest final content
(only 1.86 ± 0.15 mM free amino acids after 72
h).
Analysis of amino acids released from -casein peptides by cell
extracts.
At zero time, free amino acids represented less than
2.5% of the total amino acid content. After 24 h, 42 to 53% was
released by L. helveticus cell extracts depending on the
strain; corresponding values were 24 to 36% for S. thermophilus and 20 to 48% for L. delbrueckii. After
72 h of incubation, cell extracts of L. helveticus ITG LH1, S. thermophilus CNRZ 385 and TA 060, and L. delbrueckii subsp. lactis ITG LL14 were able to
liberate 69, 44, and 57% of the total amino acid content,
respectively. These results are in complete agreement with the kinetics
presented in Fig. 2 in terms of relative peptidase efficiencies
of the strains. The profiles shown in Fig.
4 were
qualitatively similar for L. helveticus, L. delbrueckii
subsp. lactis, and L. delbrueckii subsp.
bulgaricus, with the following predominant amino acids: Gln,
Pro, Leu, Val, and Lys. Again the species L. delbrueckii
exhibited the highest variation from strain to strain. Interestingly,
the cell extracts of S. thermophilus were not able to
release free proline from the -casein peptides, except for the very
low amount detected for strain CNRZ 385 (4% of the total proline
content).
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FIG. 4.
Free amino acids released after 24 h by hydrolysis
of -casein peptides by cell extracts of the following strains: (a)
L. helveticus ITG LH1
(&cjs3744;), CNRZ 32 ( ), and ITG LH77
( ); (b) L. delbrueckii subsp. bulgaricus NCDO 1489 (&cjs3744;), and CNRZ 397 ( and L. delbrueckii subsp. lactis ITG
LL14 (), and ITG
LL51 ( ); and; (c)
S. thermophilus CNRZ 385 (&cjs3744;), TA 060 (), and CIP 102303 (). Curves indicate
values obtained after 72 h of incubation for L. helveticus ITG LH1 (a; ), L. delbrueckii subsp.
lactis ITG LL14 (b;  ), and S. thermophilus CNRZ 385 ( ), and TA 060 ( ) (c).
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Chromatographic analysis of peptides released from -casein
peptides by cell extracts.
Following the addition of cell
extracts, the peptide profile at time zero was identical to that for
-casein hydrolysate alone. After 24 h, the profiles were
species dependent; therefore, only one profile per species is reported
in Fig. 5. The peptides with a retention
time higher than 38 min were completely hydrolyzed by L. helveticus; concomitantly the area of the peaks with retention times between 22 and 38 min decreased and new peaks with retention times of less than 22 min appeared. The hydrolysis of peptides by cell
extracts of S. thermophilus and L. delbrueckii
was less extensive than that observed with L. helveticus.
These results are in agreement with the kinetics of -casein
hydrolysis. Surprisingly, three peaks, at retention times of 22, 35, and 37 min, appeared to be particularly resistant to the peptidase
activity of the three species.
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FIG. 5.
RP-HPLC profiles of -casein peptides at time zero
and after incubation for 24 h at 24°C with cell extracts of
L. helveticus ITG LH1, L. delbrueckii subsp.
lactis ITG LL14, and S. thermophilus TA 060.
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Identification by MS-MS of peptides undigested by cell
extracts.
After 72 h of incubation of the -casein
hydrolysate with extracts from L. helveticus ITG LH1,
S. thermophilus TA 060, and L. delbrueckii subsp.
lactis ITG LL14, several peptides remained undigested. They
were identified by MS-MS and are indicated in Fig. 1, except for some
dipeptides in the case of S. thermophilus (PP, GP, MP,
YP, and LP), for which several locations in the -casein sequence
were possible.
Some specific features are noteworthy. The phosphorylated peptides
[Phe
33- Lys
48] and
[Phe
33-Phe
52] were not hydrolyzed by
the
three species. The phosphorylated peptide
[Arg
1-Arg
25] was
not hydrolyzed by
S. thermophilus but could be degraded by an
endopeptidase activity of
thermophilic lactobacilli, followed
by aminopeptidase processing in the
case of
L. helveticus. The
cleavage at the peptide bond
[Glu
11-Ile
12] reflected an endopeptidase
activity. Peptides shorter than 12 amino acids, particularly from
the
N- and C-terminal regions of the
-casein, were completely
hydrolyzed
by all of the cell extracts. The short peptides located
in the central
region of the
-casein sequence,
[Gly
94-Lys
97],
[Tyr
114-Pro
119], and
[Thr
126-Leu
133], were more or less hydrolyzed
depending on the species. The long peptides (longer than 12 amino
acids) rich in proline residues and containing in particular a
Pro-Pro sequence, i.e, [Pro
75-Pro
76],
[Pro
85-Pro
86],
[Pro
152-Pro
153]
and
[Pro
158-Pro
159] were only partially
hydrolyzed; the one exception
is the sequence
[Pro
136-Pro
137] included in the peptides
[Thr
126-Leu
139],
[Thr
126-Trp
143], and
[His
134-Leu
139]. The prolyl aminopeptidase
(PepIP) is the only enzyme able to hydrolyze this bond. This enzyme
is
active mainly on di- and tripeptides (
15) and PepIP from
L. helveticus is active on tetrapeptides (
28),
implying the
preliminary involvement of endopeptidases and/or
aminopeptidases
in forming the long peptides. PepIP is not synthesized
in
S. thermophilus,
explaining the numerous dipeptides
containing proline identified
by MS-MS (Pro-Pro, Gly-Pro, Met-Pro,
Tyr-Pro, and Leu-Pro). The
presence in
L. helveticus of
undigested di- and tripeptides containing
Pro-Pro,
[Ile
74-Pro
76] and
[Leu
150-Pro
152], was surprising considering
its high PepIP activity and could indicate an inhibition by some
peptides released. Finally, the presence of a carboxypeptidase
in
L. helveticus and in
S. thermophilus was strongly
supported
by the presence of the two sets of peptides differing by a
single
amino acid in a C-terminal position:
[Gly
64-Pro
69],
[Gly
64-Asn
68],
and
[Gly
64-Ser
67] in
L. helveticus and
[Asp
129-Leu
133],
[Asp
129-Asn
132],
and
[Asp
129-Glu
131] in
S. thermophilus.
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DISCUSSION |
This work presents new insight into the peptidase activity of
various lactic acid bacteria by using a dairy substrate, a hydrolysate of -casein. The cell extracts of 10 strains belonging to
thermophilic starters, L. helveticus, L. delbrueckii, and
S. thermophilus hydrolyzed efficiently most of the
-casein peptides, at a pH and temperature close to cheese ripening conditions.
Phosphorylated peptides were shown to be much more resistant than other
peptides to hydrolysis by peptidases from thermophilic lactic acid
bacteria, in particular to the aminopeptidases, providing the first
experimental evidence for a hypothesis previously proposed (13,
22). This is in agreement with the numerous phosphorylated peptides identified by MS in various cheese aqueous extracts or cheese
juices at the end of the ripening (Grana, Emmentaler, Cheddar, etc.)
(12, 26, 34). The phosphorylated N-terminal part of -casein is also very resistant to the cell surface proteinases of
lactococci (20, 21). This mechanism of resistance is not dependent on the number of phosphorylated residues: the fragment [Phe33-Lys48] with one phosphorylated
residue is resistant to all peptidases. By contrast, the fragment
[Arg1-Arg25] with four phosphorylated
residues can be partially hydrolyzed by endopeptidases. Several
endopeptidases have already been purified from L. helveticus, L. delbrueckii subsp. bulgaricus, and
S. thermophilus (3, 10, 35).
The accumulation of phosphorylated peptides raised the question of
bacterial phosphatases. To our knowledge, little is known in this
regard. Acid phosphatase activity was detected in Lactococcus lactis and L. delbrueckii subsp. bulgaricus
(22), but under our conditions, this activity may not be
maintained at an efficient rate. It cannot be ruled out that the
degradation of the phosphorylated peptides is controlled by a mechanism
involving bacterial phosphatases.
Another original result was the identification of peptides with
successive removal of C-terminal amino acids in L. helveticus and S. thermophilus which strongly suggested
the presence of at least one carboxypeptidase in lactic acid bacteria.
This carboxypeptidase is not able to remove a proline in the
C-terminal position, which suggests that no carboxypeptidase P is
synthesized by these species. The presence of a
carboxypeptidase has been reported for Lactobacillus casei (8, 9) and L. helveticus (36), but this point was controversial
because of the synthetic substrate used. Carboxypeptidase activity was
also recently suspected from the identification of peptides in
Emmentaler juice at the end of the ripening (26). Some of
the -casein peptides produced in this work could constitute valuable substrates for further purification of this carboxypeptidase.
Some species-specific features can also be highlighted from that work.
L. helveticus exhibited the highest activity regardless of
strain, in agreement with previous data obtained with synthetic substrates or cheese models (9, 36). This supports the idea of the essential role of L. helveticus in Swiss cheese
proteolysis and highlights the importance of its efficient lysis for
the release of peptidases in curd. Lysis of L. helveticus
was shown to occur during cold room ripening (40), to be
strain dependent, and to have an obvious impact on the final extent of
proteolysis (41). L. delbrueckii exhibited a
large strain dependence with a threefold difference in free amino acid
content between the less efficient and the more efficient strain. This
should be considered in selecting strains of L. delbrueckii,
particularly when it is used in Swiss cheese without L. helveticus. Interestingly, no strain of S. thermophilus was able to release significant amounts of free proline from the -casein peptides. The peptidases of S. thermophilus were
recently reviewed by Rul and Monnet (35). In lactic acid
bacteria, the major peptidase activity specific of proline-containing
peptides corresponds to PepX, releasing dipeptides, X-Pro, from the N
terminus of peptides. A prolidase (PepQ), specific for X-Pro
dipeptides, is then required to release free proline and has been
purified from several lactococci and lactobacilli (4, 11,
30). Another means of proline release would involve a PepIP that
removes proline in an N-terminal position. PepIP has been purified and
the corresponding gene has been cloned from various lactobacilli
including L. helveticus and L. delbrueckii
(1, 16, 19, 31, 42). The absence of PepIP in S. thermophilus (35) and a weak PepQ activity can explain the inability of S. thermophilus to release free
proline from -casein peptides. With regard to the accumulating di-
and tripeptides containing Pro-Pro sequences in the presence of
L. helveticus extracts, PepIP activity may possibly be
controlled by inhibitory peptides, resulting from peptidase action
toward -casein. This hypothesis is supported by the inhibitory
effect of peptides derived from the sequence 58-72 against PepN, PepX, and endopeptidase activities (39). Swiss cheeses are known
to be especially rich in free proline, which is associated with the sweet flavor. As S. thermophilus cannot release it, the
amount of free proline will directly depend on the activity from the thermophilic lactobacilli peptidases (PepIP and PepQ) and on the efficiency of their release through lysis (41). This result can be of great technological value in terms of starter selection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
National de la Recherche Agronomique, Laboratoire de Recherches de
Technologie Laitière, 65 rue de Saint-Brieuc, 35042 Rennes
Cédex, France. Phone: 33 2 99 28 53 34. Fax: 33 2 99 28 53 50. E-mail: lortal{at}labtechno.roazhon.inra.fr.
| |
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Applied and Environmental Microbiology, December 2000, p. 5360-5367, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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[Abstract]
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Abstract
Introduction
