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