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Original article
Viability assessment of lactic acid bacteria in commercial
dairy products stored at 4 �C using LIVE/DEAD�BacLightTM staining and conventional plate counts
Yolanda Moreno,1 M. Carmen Collado,1 M. Antonia Ferrus,1 Jose M. Cobo,2 Enrique Hernandez1 &
Manuel Hernandez1*
1 Departamento Biotecnologıa, Universidad Politecnica de Valencia, Camino de Vera 14, 46002 Valencia, Spain
2 Red INDE, Investigacion Nutricional Danone Espana, Barcelona, Spain
(Received 15 November 2004; Accepted in revised form 6 June 2005)
Summary The viability of lactic acid bacteria (LAB) from commercial fermented milks was studied
during storage at 4 �C. The enumeration of total viable bacteria was assessed using
fluorescence microscopy. Plate counting on selective media was used to enumerate LAB.
Using LIVE/DEAD� BacLightTM viability staining, it was observed that bacterial counts
decreased gradually after expiry dates, the number of viable bacteria remaining above 106
bacteria g)1 for all of the products. Viable cell counts estimated by plating onto selective
media were lower than those obtained by direct microscopic counting. The viability of
LAB contained in acid products decreased during their storage period at 4 �C. All products
contain viable LAB ranging from 108 to 109 bacteria g)1 and could be considered as
probiotic, given that the recommended minimum number of probiotic bacteria in such
food products is approximately 107 bacteria mL)1 product. The number of bifidobacteria
in commercial fermented milks declared to contain bifidobacteria varied from 104 to 107
bacteria mL)1. This study confirms the usefulness of fluorescent techniques for a rapid and
accurate evaluation of bacterial viability in probiotic products.
Keywords Fluorescent dyes, lactic acid bacteria, probiotics, viability.
Introduction
Fermented milks and yoghurts have the property
of containing living micro-organisms with organo-
leptic and nutritional qualities. These products are
considered to be probiotic because, when con-
sumed in certain quantities, they have beneficial
effects on health such as maintenance of the
intestinal gut, a better digestion of lactose,
immune and anti-carcinogenic activities and a
reduction in cholesterol levels (Sanders, 1993;
Schaafsma, 1996a,b).
Lactic acid bacteria (LAB) considered to be
probiotic include the Bifidobacterium, Lactobacil-
lus and Streptococcus species. It has been suggested
that they should reach the intestine alive and in a
sufficient number (106–107 micro-organisms mL)1)
for their benefits to be appreciated (Kurmann &
Rasic, 1991; Bouhnik, 1993). This requires their
survival in the food vehicle during its shelf-life and
their resistance to the gastrointestinal conditions.
In view of these barriers, it is regarded as essential
that dairy foods contain at least 106 viable probi-
otic organisms per mL; for this reason it is
necessary to analyse efficiently the viability of
micro-organisms probiotics because they must
remain viable in the food vehicle (dairy foods)
during their shelf-life.
A number of studies have investigated the
viability of LAB in different fermented milk
products (Nighswonger et al., 1996; Shin et al.,
2000) by conventional cultivation on a selective
growth medium. However, this approach has a*Correspondent: Fax: +34 963877429;
e-mail: mhernand@upv.es
International Journal of Food Science and Technology 2006, 41, 275–280 275
doi:10.1111/j.1365-2621.2005.01060.x
� 2005 Institute of Food Science and Technology Trust Fund
number of disadvantages, such as its time-consu-
ming nature and the underestimation of true
bacterial counts due to clumping or bound
bacteria. Fluorescence microscopy has the advant-
age of allowing a rapid and direct assessment of
cell viability and new fluorescent nucleic acid
specific dyes have been developed in order to
determine bacterial viability (Nebra & Blanch,
1999). The LIVE/DEAD� BacLightTM kit
(Molecular Probes, Eugene, OR, USA) stain
mixture distinguishes viable bacterial cells from
dead cells on the basis of membrane integrity. The
kit contains two nucleic acid stains. The green
fluorochrome (SYTO 9) is a small molecule that
can penetrate intact plasma membranes, while the
larger red fluorochrome (propidium iodide) pen-
etrates only compromised membranes. Bacterial
suspensions incubated in the presence of both
stains simultaneously will fluoresce either green
(i.e. viable) or red (i.e. dead), depending on their
viability. The excitation and emission maxima for
these dyes are about 480 and 500 nm for SYTO9
and 490 and 635 nm for propidium iodide res-
pectively.
The aim of this study was to determine LAB
viability in dairy products during refrigerated
storage at 4 �C using the LIVE/DEAD� Bac-
LightTM viability kit. LAB were also monitored by
plate counting on selective and general media.
Materials and methods
Sampling
Ten different commercial fermented milks and
yoghurts from local supermarkets were analysed.
Products A, E, F and G were fermented milks
declared to contain bifidobacteria in addition to
traditional yoghurt cultures, Streptococcus ther-
mophilus and Lactobacillus delbrueckii. C and H
were traditional yoghurts (S. thermophilus and
L. delbrueckii) and B and D were fermented milks
containing classic ferments supplemented with
L. casei or L. acidophilus respectively. All products
were stored at 4 �C during the study and we
analysed two samples for each product. Viable
LAB were stated on the labels of all the fermented
milks. The products were monitored at weekly
intervals up to 2 weeks before and 1 week after
their expiry dates.
Enumeration of LAB
For plate counting, 10 g of each sample was mixed
with 90 mL of peptone water (4%). The samples
were then serially diluted in Tween 80 (1% v/v) and
appropriate dilutions (10)5–10)8 cells mL)1) were
spread on different media. All plate counts were
determined in two independent experiments, and
each assay was performed in duplicate. Lactobacilli
were enumerated on Man Rogosa Sharpe plates
(MRS agar; Merck, Darmstadt, Germany) and
Streptococciwere enumerated onM-17 Agar plates
(Merck). Bifidobacteria were enumerated using
BFM agar plates (Nebra & Blanch, 1999) and
TPY Agar plates (Scharlau Schemie, Barcelona,
Spain). LS-Differential agar (Scharlau Schemie)
was used as a general medium to enumerate LAB
including bifidobacteria. Plates were incubated at
37 �C for 3 days in anaerobic conditions using
AnaeroGen sachets (Oxoid Ltd, Hampshire, UK).
Identification of bacteria
The API 50 CHL identification system (Bio-
Merieux, Lyon, France) was used for species
designation by comparison to their carbohydrate
profiles with known micro-organisms. Fermenta-
tion is shown by a colour change in the media for
pH change. Bifidobacteria were identified by
means of the fluorescent in situ hybridization
technique using a 16S rRNA-targeted probe as
described by Langendijk et al. (1995).
In situ viability of LAB at 4 �C
Counts of total, viable and dead bacteria were
obtained using the LIVE/DEAD� BacLightTM
Bacterial Viability stain (Molecular Probes). For
viability analysis, 1 mL of each sample (commer-
cial dairy product) was mixed in 9 mL of PBS
buffer (130 mm sodium chloride, 10 mm sodium
phosphate, pH 7.2), and was serially diluted
(1–10)4) in the same buffer PBS. An aliquot of
each dilution (1 mL) was mixed with 3 lL of a
mixture of SYTO9 and propidium iodide (1:1),
nucleic acid stains from a LIVE/DEAD� Bac-
LightTM, which were then vortexed and incubated
in the dark for 15 min at room temperature. A
5-lL aliquot was spotted on a poly-l-lysine
(Sigma, Barcelona, Spain) coated slide, covered
Survival of lactic acid bacteria at 4 �C Y. Moreno et al.276
International Journal of Food Science and Technology 2006, 41, 275–280 � 2005 Institute of Food Science and Technology Trust Fund
and sealed with vaseline to avoid evaporation. The
total bacterial population was enumerated by
counting green and red micro-organisms under
an Olympus epifluorescence microscope (BX50)
with filters U-MWB and U-MWIB (Olympus,
Hamburg, Germany). A minimum of twenty fields
were counted, all counts by viability were deter-
mined in two independent experiments, and each
assay was performed in triplicate. Ideally, healthy
(live) bacteria with intact plasma membranes
fluoresce green and the dead or injured cells with
compromised membranes fluoresce red. In accord-
ance with the manufacturer’s instructions, all
green cells were considered viable and red cells
were considered dead. Images were recorded with
a digital camera (model DP-10; Olympus).
Results and discussion
Enumeration of LAB in fermented milk
The total viable cultivable bacteria contained in
milk products were counted onLS-Differential agar
plates. Lactobacillus delbrueckii grew forming irre-
gular red colonies surrounded by a white opaque
area and S. thermophilus grew forming oval or
round surrounded by a clear area. Bifidobacteria
appeared as round red colonies. The total number
of cultivable bacteria was 107–108 CFU g)1 for all
of the dairy products analysed. The specific plate
counting on selective media is shown in Table 1.
All fermented milks studied contained L. del-
brueckii and S. thermophilus. The isolation rates
(percentages) were calculated comparing the total
plate counts obtained on a general medium
(100%) with the specific counts obtained using a
selective medium. In fermented milk products with
bifidobacteria (products A, E, F and G), the
isolation rates were between 6 and 10% for the
Bifidobacterium genus (BFM agar), 70–90% cor-
responding to Streptococcus (M-17) and 5–10%
corresponding to Lactobacillus (MRS). In yoghurt
products C, G and H containing classic lactic
ferments (L. delbrueckii and S. thermophilus), the
isolation rate was 80–90% for Streptococcus and
10–20% for Lactobacillus. The number of bifido-
bacteria in commercial fermented milks varied
from 104 to 107 bacteria g)1. The low number of
Bifidobacterium cells contained in some products
could limit their probiotic effect because the viable
bifidobacteria was lower than <106 cells mL)1.
Viability of LAB strains contained in all sam-
ples measured by traditional methods remained
above 107 cells mL)1 until their expiry dates. This
microbial concentration is the suggested recom-
mended minimum dose for a probiotic effect.
Identification of isolated strains
All isolated LAB strains were identified using the
API 50–CHL miniaturized commercial system
(BioMerieux). All lactobacilli isolated (eight
strains for eight fermented products) from dairy
products were identified as L. delbrueckii and all
streptococci isolated (one strain for each product)
were identified as S. thermophilus. These species
were found in all samples analysed. Lactobacillus
casei were also isolated in product B, and Lacto-
bacillus acidophilus was found in product
D. Bifidobacteria were isolated from all samples
(A, E, F and G products). For lactobacilli and
streptococci strains, identification levels from
Table 1 Initial bacterial counting of different dairy products using selective media 2 weeks before their expiry date
Product
Total counts LS
medium (log CFU g-1) pH
BFM
(log CFU g-1)
MRS
(log CFU g-1)
M-17
(log CFU g-1)
TPY
(log CFU g-1)
A 8.96 ± 0.01 4.51 7.74 ± 0.02 7.28 ± 0.02 8.88 ± 0.06 8.95 ± 0.01
B 8.65 ± 0.05 4.00 – 8.56 ± 0.01 8.05 ± 0.05 –
C 8.05 ± 0.02 4.02 – 7.30 ± 0.10 7.78 ± 0.01 –
D 8.02 ± 0.03 3.97 – 6.90 ± 0.09 7.54 ± 0.05 –
E 8.99 ± 0.03 4.24 6.18 ± 0.01 7.38 ± 0.03 8.86 ± 0.06 8.80 ± 0.01
F 8.40 ± 0.10 4.2 4.54 ± 0.05 7.38 ± 0.04 8.86 ± 0.10 8.80 ± 0.02
G 8.51 ± 0.09 4.37 6.56 ± 0.01 7.64 ± 0.06 8.40 ± 0.05 8.18 ± 0.01
H 8.48 ± 0.01 4.06 – 6.75 ± 0.05 8.55 ± 0.03 –
Values are mean ± SEM.
Survival of lactic acid bacteria at 4 �C Y. Moreno et al. 277
� 2005 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2006, 41, 275–280
good to excellent were obtained, but identification
levels of bifidobacteria strains were considered
doubtful or unacceptable due to atypical fermen-
tation reactions. The API system was useful for
lactobacilli and streptococci, but it was not useful
for the identification of the Bifidobacterium genus.
The reproducibility of the biochemical identifica-
tion of isolates using the API 50-CHL system was
low and imprecise because we obtained ambiguous
results and different carbohydrate profiles at
different incubation times and different inoculum
quantities. Although some authors consider this
system to be appropriate for the identification of
bifidobacteria (Nighswonger et al., 1996, Shin
et al., 2000), these biochemical tests cannot differ-
entiate this genus from other LAB.
Therefore, isolated bifidobacteria colonies were
identified by fluorescent in situ hybridization using
a 16S rRNA-specific oligonucleotide probe for the
Bifidobacterium genus (Langendijk et al., 1995).
All the strains isolated from BFM agar gave
positive signals when hybridized with the probe.
In situ viability of LAB at 4 �C
Total counts of bacteria estimated using LIVE/
DEAD� BacLightTM viability stain were between
108 and 109 bacteria mL)1 for all products at the
beginning of the study (2 weeks before expiry
date). About 80–97% of these LAB were viable by
fluorescent dyes included in the LIVE/DEAD�BacLightTM kit (Table 2). The percentages of
viability were calculated comparing the total
counts (viable and non viable) with the viable
counts obtained at different times. The results
showed that plate counts were approximately
10-fold lower than the corresponding fluorescent
(SYTO9-propidium iodide) counts (Table 2). This
suggests that not all of the viable LAB present
were culturable, as the addition of a pure culture
increased the plate counts while the fluorescent
counts remained unaffected.
Lactobacilli, streptococci and bifidobacteria
counts decreased during the period prior to the
products� expiry dates (Table 2 and Fig. 1). Sur-
vival differences also varied depending on the
product analysed (Table 2). Viability of LAB
strains contained in all samples measured by
SYTO9 and propidium iodide staining and using
Table
2Viable
cellsoflactic
acidbacteria
enumeratedusingplate
countinganddirectfluorescentcounts
usingLIV
E/D
EAD
stainingofyoghurt
samplesstoredat4�C
for
differenttimes.Resultsare
themeanofcellsenumeratedbydirectcountingunder
fluorescence
microscopybySYTO
andPIstaining
Sample
2weekbefore
expirydate
1weekbefore
expirydate
Expirydate
1weekafterexpirydate
Totalcounts
(logCFU
g-1)
pH
Viable
cells
(SYTO)
Totalcounts
(logCFU
g-1)
pH
Viable
cells
(SYTO)
Total
(logCFU
g-1)
pH
Viable
cells
(SYTO)
Total
(logCFU
g-1)
pH
Viable
cells
(SYTO)
A8.96(0.01)
4.51
9.16(0.04)
7.89(0.02)
4.40
8.80(0.02)
7.75(0.01)
4.41
8.74(0.05)
7.20(0.10)
4.40
7.81(0.09)
B8.65(0.05)
4.00
9.01(0.04)
7.90(0.01)
3.93
8.70(0.07)
7.45(0.05)
3.90
8.49(0.05)
7.30(0.04)
3.87
6.21(0.10)
C8.05(0.02)
4.02
8.77(0.09)
7.75(0.03)
3.96
8.42(0.04)
7.30(0.01)
3.89
8.36(0.07)
6.90(0.03)
3.75
7.96(0.09)
D8.02(0.03)
3.97
8.36(0.04)
7.35(0.02)
3.94
7.25(0.04)
6.75(0.02)
3.91
6.83(0.08)
6.72(0.02)
3.85
6.79(0.09)
E8.99(0.03)
4.24
9.25(0.08)
8.00(0.02)
4.23
7.89(0.07)
7.65(0.01)
4.22
7.84(0.07)
7.00(0.10)
4.18
7.02(0.04)
F8.40(0.10)
4.2
8.77(0.09)
8.20(0.05)
4.13
8.44(0.04)
7.15(0.08)
4.00
7.27(0.09)
7.10(0.12)
3.96
7.25(0.10)
G8.51(0.09)
4.37
8.72(0.05)
8.00(0.10)
4.30
8.30(0.45)
7.94(0.09)
4.19
8.24(0.06)
7.50(0.05)
4.14
7.60(0.06)
H8.48(0.01)
4.06
8.56(0.06)
8.16(0.10)
3.90
8.26(0.08)
7.92(0.02)
3.74
8.15(0.15)
7.01(0.02)
3.59
7.46(0.08)
T–
4.07
––
4.02
––
4.04
––
4.00
–
O–
4.10
––
4.06
––
4.05
––
4.06
–
Valuesare
mean±SEM.
Survival of lactic acid bacteria at 4 �C Y. Moreno et al.278
International Journal of Food Science and Technology 2006, 41, 275–280 � 2005 Institute of Food Science and Technology Trust Fund
plate counts remained above 107 cells mL)1 until
their expiry dates.
Culture methods and morphological observa-
tion following fluorochrome staining showed that
Bifidobacterium and Lactobacillus lost the survival
capacity before Streptococcus during their storage
period at 4 �C (from 2 weeks before expiry date to
1 week after expiry date) (Fig. 2).
Figure 1 Viability of bacteria contained in fermented milk
with bifidobacteria during refrigerated storage at 4 �C.(a) Two weeks before expiry date. (b) One week before
expiry date. (c) Expiry date.
9
8
7
6
52 weeksbefore
expiry date
1 weekbefore
expiry date
Expiry date 1 weekafter
expiry date
2 weeksbefore
expiry date
1 weekbefore
expiry date
Expiry date 1 weekafter
expiry date
2 weeksbefore
expiry date
1 weekbefore
expiry date
Expiry date 1 weekafter
expiry date
Log
CFU
g–1
Log
CFU
g–1
Log
CFU
g–1
ABCDEFGH
10
8
9
7
6
5
ABCDEFGH
10
8
6
4
0
2
AEFG
(a)
(b)
(c)
A Fermented milk with bifidobacteriaB Fermented milk with L. caseiC Traditional yoghurtD Fermented milk with L. acidophilusE Fermented milk with bifidobacteriaF Fermented milk with bifidobacteriaG Fermented milk with bifidobacteriaH Traditional yoghurt
Figure 2 Viability of lactic acid bacteria contained in
commercial dairy products during storage at 4 �C obtained
by plate count method. (a) Lactobacilli. (b) Streptococci.
(c) Bifidobacteria. Results shown as mean ± SEM.
Survival of lactic acid bacteria at 4 �C Y. Moreno et al. 279
� 2005 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2006, 41, 275–280
The comparison of LAB survival times from the
different dairy products analysed indicates differ-
ent viability behaviour for strains used in indus-
trial processes. This diversity of strains and
production processes may contribute to the slight
differences in the viability and activity of bifido-
bacteria as well as LAB in fermented milks.
Therefore, the initial LAB content in fermented
milks, especially bifidobacteria and lactobacilli,
must be enough to yield viable bacterial counts
between 108–109 cells mL)1.
The significant decrease in LAB viability from
the expiry date has also been observed by other
authors (Nighswonger et al., 1996, Shin et al.,
2000) by traditional methods. In accordance with
other studies (Auty et al., 2001) the number of
bacteria by plate counting was lower than direct
counts due to the presence of viable but non-
cultivable bacteria.
Likewise, the selection of acid-resistant strains is
of great interest for the elaboration of milk
products. The results obtained show that further
research is required to identify and establish the
characteristics of probiotic strains used in the
dairy products industry such as acid tolerance,
survival to gastrointestinal conditions, survival
during refrigerate storage, antimicrobial activity,
adhesion to mucus, etc.
The results of this study demonstrate that only
intrinsic acid-resistant cells can survive their pas-
sage through the human intestine. Therefore, the
selection of acid-resistant strains is necessary for
the elaboration of probiotic products. The amount
of initial inoculums to be added should vary
depending on the acid-resistant profiles of the
strain.
In conclusion, these results indicate that in situ
LIVE/DEAD� BacLightTM viability staining may
be of great use for the reliable and rapid estima-
tion of viable bacteria in dairy products, which
could take over 3 days using the plate count
method.
Acknowledgment
An FPU/MEC scholarship to M. C. Collado is
acknowledged.
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International Journal of Food Science and Technology 2006, 41, 275–280 � 2005 Institute of Food Science and Technology Trust Fund
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