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Centro de Referencia para Lactobacilos (CERELA)-CONICET, Chacabuco 145, 4000 San Miguel de Tucumn, Argentina
Ctedra de Microbiologa Superior, Facultad de Bioqumica, Qumica y Farmacia, Universidad Nacional de Tucumn, San Miguel de Tucumn, Argentina
a r t i c l e
i n f o
Article history:
Received 18 January 2010
Received in revised form 9 March 2010
Accepted 12 April 2010
Keywords:
Lactic acid bacteria
Whey protein concentrate
-lactoglobulin
Essential amino acids
Whey-based beverage
a b s t r a c t
Whey protein concentrate (WPC) is employed as functional food ingredient because of its nutritional value
and emulsifying properties. However, the major whey protein -lactoglobulin (BLG) is the main cause of
milk allergy. The aim of this study was to formulate a fermented whey beverage using selected lactic acid
bacteria and WPC35 (WPC containing 35% of proteins) to obtain a fermented product with low lactose and
BLG contents and high essential amino acid concentration. Cell viability, lactose consumption, lactic acid
production, proteolytic activity, amino acid release and BLG degradation by the selected strains Lactobacillus
acidophilus CRL 636, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and Streptococcus thermophilus CRL
804, as single or mixed (SLaB) cultures were evaluated in WPC35 (10%, w/v) incubated at 37 C for 24 h.
Then, the fermented WPC35 was mixed with peach juice and calcium lactate (2%, w/v) and stored at 10 C
for 28 days. During fermentation, single cultures grew 1.73.1 log CFU/ml and produced 25.195.0 mmol/l of
lactic acid as consequence of lactose consumption (14.041.8 mmol/l) after 12 h fermentation. L. delbrueckii
subsp. bulgaricus CRL 656 was the most proteolytic strain (626 g/ml Leu) and released the branched-chain
essential amino acids Leu (16 g/ml), Ile (27 g/ml) and Val (43 g/ml). All strains were able to degrade BLG
in a range of 4185% after 12 h incubation. The starter culture SLaB grew 3.0 log CFU/ml, showed marked pH
reduction, produced 122.0 mmol/l of lactic acid, displayed high proteolytic activity (484 g/ml Leu) releasing
Leu (13 g/ml), Ile (18 g/ml) and Val (35 g/ml), and hydrolyzed 92% of BLG. The addition of calcium lactate
to WPC35 maintained the drink pH stable during shelf life; no contamination was detected during this
period. After 28 days, a decrease in cell viability of all strains was observed being more pronounced for L.
delbrueckii subsp. bulgaricus CRL 656 and L. acidophilus CRL 636 (2.3 and 1.9 log CFU/ml, respectively). The
results showed that WPC fermentation by rationally selected lactic acid bacteria might be used for
developing functional beverages with improved characteristics such as reduced BLG content and increased
branched-chain essential amino acids.
2010 Elsevier B.V. All rights reserved.
1. Introduction
Over the years numerous efforts have been made to transform
large volumes of whey generated as sub-product of the cheese
industry into a suitable product for food use (Djuri et al., 2004).
Whey constitutes about 8590% of the milk volume used for
transformation into ripened cheese, and it retains about 55% of the
milk nutrients. Liquid whey is composed of lactose (5%), water (93%),
proteins (0.85%), minerals (0.53%) and a minimum amount of fat
(0.36%). Whey proteins have high biological value superior to other
proteins such as those of egg, soy and caseins of milk (Smithers, 2008)
mainly due to the high content of branched-chain essential amino
acids (isoleucine, leucine and valine). These amino acids stimulate
specic intracellular pathways associated with muscle protein
synthesis (Katsanos et al., 2006) and may play a role in the hormonal
Corresponding author. Centro de Referencia para Lactobacilos (CERELA)-CONICET,
Chacabuco 145, 4000 San Miguel de Tucumn, Argentina.
E-mail address: gfont@cerela.org.ar (G. Font de Valdez).
0168-1605/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.04.011
74
borate buffer, pH 9.5) and 200 l of the OPA methalonic solution. The
mixture was shaken, incubated for 1 min and the reaction was
stopped by adding 400 l of 0.1 mol/l sodium phosphate buffer (pH
4.0) and ltered through 0.2 m nylon membrane (Alltech Associates
Inc., Deereld, IL, USA). The amino acids used as standards (Sigma
Chemical Co) were treated in the same way as the above samples.
The amino acid content of the samples was analyzed by reverse
phase-high performance liquid chromatography (RP-HPLC) with an
ISCO model 2360 (ISCO, Inc., Lincoln, NE, USA) tted with an Ultrasphere
ODS C18 column (4.6 25 mm, particle size 5 m, Beckman Instruments
Inc., Fullerton, CA, USA). The equipment was coupled with an ISCO
model 2350 pump and an ISCO FL-2 uorescence detector (ISCO Inc.).
The operating conditions were the following: ow rate, 1.7 ml/min;
solvent A, tetrahydrofurane:methanol:sodium acetate (1:19:80, v/v/v)
0.05 mol/l pH 5.9 (Sigma Chemical Co.) in ultra pure water; solvent B,
methanol:sodium acetate 0.05 mol/l pH 5.9 (80:20 v/v) (Sigma
Chemical Co). Elution was performed by applying a linear gradient of
100% solvent A over 1 min, then 050% solvent B over the following
20 min, and 50100% solvent B over the last 20 min. Absorbance was
recorded at 305395 and 430460 nm excitation and emission
wavelengths, respectively. The injection volume of derivatized amino
acids was 10 l. The HPLC was coupled with the software Chem
Research 150 Data System 3.0.2 (1994, ISCO Inc.). All the amino acids,
except proline, cystein and methionine, were determined under the
assayed conditions. Amino acid concentration was expressed in g/ml.
2.7. Hydrolysis of -lactoglobulin in WPC35
Degradation of -lactoglobulin was monitored by RP-HPLC using
a Knauer Smartline System (Manager 5000, pump 1000) with a UV
detector (2000) tted with a C18 column (Pursuit 4.6 250 mm, 300
A, 5 m, Varian, Lexington, USA). The method used included buffer A:
water/acetonitrile/triuoroacetic acid (90/10/0.1, v/v/v), and buffer
B acetonitrile/triuoroacetic acid (100/0.1, v/v) with a ow rate of
1 ml/min. The gradient used was 100% buffer A up to 10 min and 10 to
60% buffer B in a linear fashion between 10 and 60 min. Eluted peaks
in the chromatograms were detected at 214 nm. Samples for HPLC
were prepared as follows: WPC35 samples (fermented and nonfermented) were mixed 1:1 with reduction buffer containing urea
and 20 mmol/l dithiothreitol (DTT) and incubated for 60 min at
30 C. Prior to injection in the column, the reduced sample was
diluted 5-fold in buffer A containing 6.0 mol/l urea. BLG hydrolysis
was expressed as percentage and was calculated by measuring its
relative peak area with respect to the control (non-fermented
sample).
2.8. Strains compatibility
Strains compatibility was evaluated by the plate diffusion assay
(Parente and Zottola, 1991). Briey, overnight cultures grown in MRS
were washed twice with saline solution and suspended at the initial
volume. Plates were prepared by pouring 15 ml of MRS soft agar (MRS
plus 0.7%, w/v, agar) containing 60 l of the cell suspension on the
agar. After overlay solidication, 5 mm diameter wells made with
sterilized plastic straws were inoculated with 60 l of culture
supernatants from the other strains. After incubation at 37 C for
16 h, appearance of inhibition zones were observed.
2.9. Statistical analysis
All assays were carried out in triplicate, and results were expressed
as mean values with standard deviations. Statistical analyses were
performed using MINITAB 14 software (State College, PA, USA).
Comparisons were accomplished by ANOVA general linear model
followed by Tukey's post-hoc test, and p b 0.05 was considered
signicant.
75
3. Results
3.1. Growth and metabolite production by LAB in WPC35
The assayed strains L. delbrueckii subsp. bulgaricus CRL 656, L.
acidophilus CRL 636 and S. thermophilus CRL 804 grew between 1.7
and 3.1 log CFU/ml after 12 h of incubation in WPC35 at 37 C, the
highest cell count values being observed for L. delbrueckii subsp.
bulgaricus CRL 656 (Fig. 1ac). After this period, cell viability started to
decline specially for the L. delbrueckii subsp. bulgaricus strain. S.
thermophilus CRL 804 and L. delbrueckii subsp. bulgaricus CRL 656
showed a constant drop of pH (reaching about 4.94.5, respectively)
during the rst 8 h of incubation showing a major consumption of
lactose during this period (32.540.0 mmol/l). On the contrary, L.
acidophilus CRL 636 showed a slight decrease in pH, and lactose
reduction was only detected at 12 h. As expected, the assayed strains
released only lactic acid (25.195.0 mmol/l, at 12 h) from lactose
fermentation, L. delbrueckii subsp.bulgaricus CRL 656 being the strain
which produced the highest amount. An accumulation of galactose
(4.346.2 mmol/l, 12 h) during LAB growth in WPC35 was found
while glucose was entirely consumed by all the assayed strains.
3.2. Proteolytic activity and amino acid release by LAB in WPC35
The proteolytic activity of the studied LAB during WPC35
fermentation was strain dependent (Fig. 2). L. delbrueckii subsp.
bulgaricus CRL 656 was the most proteolytic strain (626 g Leu/ml)
while no differences in the amino group concentration were
observed for S. thermophilus CRL 804 during the incubation period.
Non-fermented WPC35 showed low OPA value (82.3 g/ml) and no
amino acids were detected by RP-HPLC (data not shown). L.
delbrueckii subsp. bulgaricus CRL 656 released 14 amino acids
(Table 1) and displayed the highest amino acid concentration
(428 g/ml, Fig. 2) at 12 h incubation. The most abundant amino
acids released by this strain were Ser-His (114 g/ml), Gly (62 g/ml)
and Glu (53 g/ml) and also the branched-chain amino acids Leu
(16 g/ml), Ile (27 g/ml) and Val (43 g/ml). L. acidophilus CRL 636
released mainly the amino acids Lys (21 g/ml), Thr-Arg (18 g/ml)
and Glu (16 g/ml) while S. thermophilus CRL 804 released only Thr-Arg
(6 g/ml) after 12 h incubation (Table 1).
3.3. -lactoglobulin degradation in WPC35
All LAB strains were able to degrade BLG (Fig. 3ch) in a range of
4185% after 12 h incubation releasing mainly hydrophilic peptides
(224 min elution); however, some small peaks corresponding to
more hydrophobic peptides (3038 min) were also observed for L.
delbrueckii subsp. bulgaricus CRL 656, the most proteolytic strain (85%
at 12 h incubation) (Fig. 3e). Although certain peaks (denoted as 14
in Fig. 3ch) were found in all hydrolysates, distinct peptides were
observed for each sample (marked with arrows).
3.4. WPC35 fermentation by a starter culture
The three studied strains were compatible as observed by the
diffusion plate assay; thus, a starter culture named SLaB was formulated
combining the strains S. thermophilus CRL 804, L. delbrueckii subsp.
bulgaricus CRL 656 and L. acidophilus CRL 636 in a CFU/ml ratio of
1:1.5:6.4.
S. thermophilus CRL 804 showed the highest cell growth
(1.9 log CFU/ml) during the rst 4 h of incubation in WPC35 as
compared with the other strains (0.591.02 log CFU/ml); however,
similar cell count values were attained by all the strains after 6 h
(Fig. 4a) indicating that L. acidophilus CRL 636 displayed a lower
growth rate since it was inoculated in a higher ratio than the other
strains.
76
lower than that found for the L. delbrueckii subsp. bulgaricus CRL 656
culture (721.0 g/ml, Fig. 2) after the same incubation period. The free
amino acid concentration of the SLaB culture was also lower to that of
the CRL 656 strain due to lower concentrations of the Glu (24%), Ser-His
(26%), Gln (45%), Gly (39%) and Ile (33%) amino acids. In contrast, a
higher amount (46%) of the amino acids Thr-Arg was observed with
respect to the L. delbrueckii subsp. bulgaricus culture (Table 1).
The starter SLaB was able to hydrolyze BLG (92%) after 12 h
incubation releasing mainly hydrophilic peptides and three peptides
eluting between 15 and 20 min (Fig. 4b).
Fig. 1. Viable cell count, pH, sugar concentration and lactic acid release by a) L. acidophilus CRL 636, b) L. delbrueckii subsp. bulgaricus CRL 656 and c) S. thermophilus CRL 804 incubated
in WPC35 at 37 C for 24 h.
77
4. Discussion
L. delbrueckii subsp.
bulgaricus CRL 656
S. thermophilus
CRL 804
Starter culture
SLaB
Asp
Glu
Asn
Ser-His
Gln
Gly
Thr-Arg
Ala
Val
Ile
Leu
Lys
ND
16.20 0.80
ND
ND
ND
1.38 0.20
18.00 1.00
3.09 0.08
1.90 0.03
8.40 0.17
8.22 0.04
21.2 1.5
7.73 0.26a
53.20 1.00
1.38 0.01
114.00 1.00
40.00 0.70
61.60 4.00
24.10 1.60
12.10 0.00
43.30 1.00
26.70 0.50
16.50 0.30
27.20 0.80
ND
ND
ND
ND
ND
ND
6.24 1.09
ND
ND
ND
ND
ND
7.06 0.14
40.40 1.50
1.35 0.04
84.70 3.60
21.70 1.10
37.80 10.20
51.60 1.00
12.70 0.30
35.10 0.60
18.50 0.70
13.50 0.50
21.00 0.20
78
Fig. 3. RP-HPLC proles of whey protein degradation from WPC35 fermented at 37 C for 24 h. a) BLG (control), b) non-inoculated WPC35 incubated at 37 C for 24 h, c) L. acidophilus
CRL 636, 12 h and d) 24 h incubation; e) L. delbrueckii subsp. bulgaricus CRL 656, 12 h and f) 24 h incubation; g) S. thermophilus CRL 804, 12 h and h) 24 incubation.
79
Fig. 4. Behavior of the starter culture SLaB in WPC35, a) Differential enumeration of each strain in the starter, pH, sugar concentration and lactic acid release, and b) RP-HPLC proles
of whey protein degradation.
Fig. 5. Stability (cell viability, pH, sugar concentration and lactic acid release) of different beverage formulations during storage. a) W (fermented WPC35 plus water), b) P (fermented
WPC35 plus peach juice), c) PL (fermented WPC35 plus peach juice and calcium lactate) and d) C (control, non-fermented WPC35).
80
The SLaB culture liberated lower amount of free amino acids such
as Ser-His (26%), Gln (45%) and Gly (39%) than the strain L. delbrueckii
subsp. bulgaricus CRL 656 solely; this fact could be due to the
consumption of the released amino acids by the S. thermophilus strain.
Also, Letort et al. (2002) showed that when a S. thermophilus strain
was grown in milk the concentration of the free amino acids Glu, Gln,
Thr, Ser, Gly, Ala, Leu and Ile was diminished during the rst hours of
incubation.
Interestingly, BLG hydrolysis by SLaB was achieved at 12 h
incubation at 37 C with WPC35 instead of the 24 h needed using
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