Document [original]

NFS

37,3

184

Nutrition & Food Science

Vol. 37 No. 3, 2007

pp. 184-200

#Emerald Group Publishing Limited

0034-6659

DOI 10.1108/00346650710749080

Flow cytometry assessment of

Lactobacillus rhamnosus GG

(ATCC 53103) response to

non-electrolytes stress

E.O. Sunny-Roberts, E. Ananta and D. Knorr

Department of Food Biotechnology and Process Engineering, Berlin University

of Technology, Berlin, Germany

Abstract

Purpose –Lactobacillus rhamnosus GG, a probiotic of human origin, known to have health beneficial

effects can be exposed to osmotic stress when applied in food production as important quantities of

sugars are added to the food product. The aim of this study is to assess the mode of action of non-

electrolytes stress on its viability.

Design/methodology/approach – Investigations were carried out on stationary phase cells treated

with 0-1.5 M sugars, by means of flow cytometric method (FCM) and plate enumeration method.

Osmotically induced changes of microbial carboxyfluorescein (cF)-accumulation capacity and

propidium iodide-exclusion were monitored. The ability of the cells to extrude intracellularly

accumulated cF upon glucose energization was ascertained as an additional vitality marker, in which

the kinetics of dye extrusion were taken into consideration as well. Sugar analysis by HPLC was also

carried out.

Findings – The results of FCM analysis revealed that with sucrose, only cells treated at 1.5 M

experienced membrane perturbation but there was a preservation of membrane integrity and

enzymatic activity. There was no loss of viability as shown by plate counts. In contrast, the majority

of trehalose-treated cells had low extent of cF-accumulation. For these samples a slight loss of

viability was recorded on plating (log N/N

0.45). At 0.6 M, cells had similar extrusion ability as

the control cells upon glucose energization. However, 20 per cent of sucrose-treated cells and 80 per

cent of trehalose-treated cells extruded the dye in the first 10 min.

Originality/value – This finding pointed out the importance of trehalose to enhance the dye

extrusion activity, which is regarded as an analogue of the capability of cells to extrude toxic

compounds. Sugars exert different effects on the physiological and metabolic status of LGG but none

caused a significant viability loss. LGG can be a choice probiotic bacterium in sugar-rich food

production e.g. candies, marmalade etc., in which exposure to high osmotic pressure is be expected.

Keywords Food products, Food testing, Bacteria

Paper type

Introduction

Lactic acid bacteria (LAB) constitute a heterogeneous group of bacteria that are

traditionally used to produce fermented foods. The industrialization of food bio-

transformations increased the economical importance of LAB as they play a crucial

role in the development of the organoleptic and hygienic quality of fermented foods

(van de Gutche et al., 2002). The use of micro-organisms as probiotic products is being

given an adequate importance in the industrial world and moreover, researches on

probiotics are gaining more interest in the scientific communities based on the

The current issue and full text archive of this journal is available at

www.emeraldinsight.com/0034-6659.htm

The first author appreciates the financial magnanimity of DAAD in pursuing her research

programme. All authors appreciate the enormous assistance of Irene Hemmerich during the

flow cytometric measurements.

Flow cytometry

assessment

185

numerous advantages associated with these group of organisms. Probiotics are defined

as live organisms that are used as dietary supplements with the aim of benefiting the

health of the hosts by positively influencing the intestinal microbial balance (Fuller,

1989). The genera Bifidobacterium and Lactobacillus are the main focus of probiotic

interest.

The production, storage, and use of LAB impose environmental stresses on the

bacterial cells (Bunthof et al., 1999) and it is well known that during industrial

fermentation, LAB encounter a number of stress conditions such as low temperature,

low pH, and low water activity (Sandine, 1996; Baati et al., 2006). Though the

application of physical stress to micro-organisms, is the most widely used method to

induce cell inactivation and promote food stability. To survive, micro-organisms have

evolved both physiological and genetic mechanisms to tolerate some extreme

conditions. This is clearly of significance to the food industry in relation to survival of

pathogens or spoilage organisms (Beales, 2004). Interestingly, of recent, the application

of these stresses to improve the viability and stability of probiotics is being given a

keen interest.

In their various applications in the food and feed industry, LAB can be exposed to

osmotic stress when important quantities of salt or sugar are added to the product

(Poolman and Glaasker, 1998). Thus, they need to adapt to such a change in their

environment in order to survive so that adequate quantities of cells can be made

available to the consumer for health benefits purposes. It has been reported that they

do so by accumulating compatible solutes (uptake or synthesis) under hyper osmotic

conditions and releasing (or degrading) them under hypo osmotic conditions.

Compatible solutes may also stabilize enzymes and thereby provide protection not only

against osmotic stress but also against high temperature, freeze thawing, and drying

(Kets et al., 1996; Poolman and Glaasker, 1998; Panoff et al., 2000; Gouesbet et al., 2001;

Leslie et al., 1995; Conrad et al., 2000).

Determination of the impact of treatment on bacterial strains have been made

mainly by the use of classical plate count methods, however, this method bears a major

draw back in the sense that it only indicates how many cells replicate under the

conditions provided for growth and its long-term determination (Ritz et al., 2001; Ben

Amor et al., 2002). Moreover, bacteria may occur in chains and clumps, resulting in

underestimation of bacterial numbers. In addition, cell injury and dormancy may result

in low viable counts (Barer and Harwood, 1999; Kell et al., 1998).

However, flow cytometric method (FCM) is a rapid and sensitive technique that can

determine cell numbers and measure various physiological characteristics of each

individual cell using appropriate probes. The differentiation of the viable states of

cultures are made possible by the use of specific fluorescent probes, into four classes

viz a viz reproductively viable, metabolically active, intact, and permeabilized (Hewitt

and Nebe-von-Caron, 2001). The applied probes include nucleic acid probes such as

propidium iodide (PI), SYTO9, carboxyflourescein diacetate (cFDA), and bis-(1,3-

dibutylbarbituric acid) trimethine oxonol (DiBAC

4(3)

) (Ananta et al., 2004; Ananta and

Knorr, 2004; Alakomi et al., 2005; Auty et al., 2001).

Many researches have been conducted on the effect of salt stress (and few on sugar

stress) on LAB probably due to the requirements of a high quantity of sugar needed to

obtain an equiosmolar with salt (amongst other factors). However, it is expedient to

assess the extent to which probiotics remain viable in the presence of the commonly

used sugars in our food products.

NFS

37,3

186

The subject of this study was the assessment, by FCM, of survival and viability of

LGG when exposed to osmotic stress (sugars). Plate counts were performed to ensure

that the populations indicated as live by FCM viability assay were indeed culturable.

Materials and methods

Test strains and growth conditions

The bacterial strains used in this study, Lactobacillus rhamnosus GG (ATCC 53103)

was obtained from Valio R & D, Helsinki, FL. The culture which was sent in freeze-

dried form in glass ampoule was later stored as glass beads cultures (Roti

(R)

_Store,

Carl-Roth, Karlsruhe, D) in a 80 C freezer (U101, New Brunswick Scientific,

¨rtingen, D) for long-term maintenance.

One bead was transferred into 10 ml MRS broth (Oxoid, Basingstoke, UK) and

incubated overnight at 37 C. For growth analysis, an overnight culture was inoculated

into MRS broth (50 ml) at OD

600

of 0.1 and samples were collected at intervals for

optical density determination (Graphicord uV-240, Schimadzu, Jpn) for 24 h.

For flow cytometry measurements, stationary phase cells were washed in Ringer’s

solution and subsequently concentrated to a theoretical value of 10 (cell concentration

~3 10

) with Ringer’s solution.

Determination of sublethal and lethal levels of osmotic stress

MRS broth (10 ml) containing 0-1.5 M trehalose (Roth) and sucrose (Merck) was

prepared and the osmotic strength of the media was measured (51308 Vapor Pressure

Osmometer Wescor Inc.). Inoculation was done at an initial OD

600

of 0.1 and incubated

at 37 C for 24 h. Samples were collected at intervals and optical density determined.

Assessment of response of exponential-phase cultures against osmotic stress

Exponential-phase cultures (OD

600

of 0.5) were harvested by centrifugation

(1,600 g 10 min) and re-suspended in MRS broth containing 0-1.5 M sugars.

Incubation was carried out for a period of 1 h at 37 C after which OD and CFU were

determined. Kinetics of sugar treatment was carried out by withdrawing samples at 0,

15, 30, 45, and 60 min.

Flow cytometry

Stress conditions. Equal volume of the cell concentrate was treated with equal volume

of the sugar solutions (0-1.5 M) at 37 C for 30 min, and 60 min. After incubation, the

cells viability was assessed by plate count enumeration and flow cytometric

measurements.

Plate enumeration method. Treated samples were serially diluted in Ringer’s

solution (No. 15525, Merck, Darmstadt, D) and drop plated in duplicate on MRS agar

(Oxoid, Basingstoke, UK). The viable cell numbers were determined after 48 h of

incubation at 37 C under anaerobic conditions produced by anaerobic kits

(Anaerocult

A, Merck, Darmstadt, D).

The impact of osmotic treatment on cell viability, as assessed by plate count method

was expressed as the logarithmic value of relative survivor fraction (log N/N

). Nrefers

to the bacterial count following osmotic exposure, while N

refers to the initial count

before osmotic exposure.

Flow cytometry

assessment

187

Fluorescence labelling

Esterase activity and membrane integrity. Osmotically treated cells were incubated

with 50 mm cFDA (Molecular Probes Inc., Leiden, NL) at 37 C for 10 min. cFDA is an

esterase substrate that yields the fluorescent carboxyfluorescein (cF) upon hydrolysis.

Cells were washed to remove excess cFDA, 30 mm PI (Molecular Probes Inc., Leiden,

NL) was added and incubated in ice bath for 10 min to allow labelling of membrane-

compromised cells (Ananta et al., 2004).

cF extrusion activity. cF-stained cells were further incubated together with 20 mM

glucose for 20 min at 37 C in order to measure the performance of treated cells in

extruding intracellular accumulated cF (Bunthof et al., 1999).

Kinetics of cF extrusion. cF labelled cells were incubated at 37 C in the presence of

20 mM glucose and samples were withdrawn every 5 min for 20 min (Ananta et al.,

2004) to monitor the kinetics of cF release from glucose energized cells.

Flow cytometric measurement. Analysis was performed on a Coulter

EPICS

XL_MCL flow cytometer (Beckman Coulter Inc., Miami, FL, USA) equipped with a

15 mW 488 nm air-cooled argon laser. Cells were delivered at the low flow rate,

corresponding to 400-600 events. Forward scatter (FS), side scatter (SS) green (FL1) and

red fluorescence (FL3) of each single cell were measured, amplified, and converted into

digital signals for further analysis. cF emits green fluorescence at 530 nm following

excitation with laser light at 488 nm, whereas red fluorescence at 635 nm is emited by

PI-stained cells.

A set of band pass filters of 525 nm (505-545 nm) and 620 nm (605-635 nm) was used

to collect green fluorescence (FL1) and red fluorescence (FL3), respectively. All

registered signals were logarithmically amplified. A gate created in the density plot of

FS vs SS was preset to discriminate bacteria from artefacts. Data were analysed with

the software package Expo32 ADC (Beckman-Coulter Inc., Miami, FL, USA). All

detectors were calibrated with FlowCheck

Fluorospheres (Beckman-Coulter Inc.,

Miami, FL, USA).

Data analysis

Density plot analysis of FL1 vs FL3 was conducted as described by Ananta et al.

(2004). Density plot was used to resolve the fluorescence properties of the population

measured by flow cytometer (Figures 1-5). The population was differentiated and gated

as described in Table I.

Residual esterase activity following osmotic treatment was calculated using

equation (1):

EAð%Þ¼ðA4p=A4ctrlÞ100 ð1Þ

where EA is the residual enzymatic activity in response to a particular osmotic

treatment, A4

is the percentage of population in gate A4 following osmotic treatment

and A4

ctrl

is the percentage of population in gate A4 prior to osmotic treatment.

The performance of cells in extruding intracellularly accumulated dye is calculated

using equation (2):

cFAð%Þ¼ð1A4Glu=A4Þ100 ð2Þ

where cFA is the measure of performance in extruding cF, A4

Glu

is the percentage of

population in gate A4 following glucose addition and 20 min incubation and A4 is the

percentage of population in gate A4 prior to glucose addition.

NFS

37,3

188

Figure 1.

Flow cytometry density

plots of FL1 vs FL3 of

Lactobacillus rhamnosus

GG for evaluating the

impact of incubation in

sucrose solution at

different molarities on

their membrane integrity

and cF-accumulation

capacity

Flow cytometry

assessment

189

Figure 2.

Flow cytometry density

plots of FL1 vs FL3 of

LGG for evaluating the

impact of inclubation in

trehalose solution at

differnet molarities on

their membrane integrity

and cF-accumulation

capacity

NFS

37,3

190

Figure 3.

Flow cytometry density

plots of FL1 vs FL3 of

Lactobacillus rhamnosus

GG to assess the impact

of incubation in sucrose at

different molarities on

their cF-extrusion activity

Flow cytometry

assessment

191

Figure 4.

Flow cytometry density

plots of FL1 vs F13 of

Lactobacillus rhamnosus

GG to assess the impact

of incubation in trehalose

at different molarities on

their cF-extrusion activity

NFS

37,3

192

Figure 5.

Flow cytometric

assessment of the kinetics

of cF-efflux upon

energization by glucose

addition as shown by the

density plot fluorescence

pattern (FL1 vs FL3) of

the untreated, 0.6 M

sucrose-treated, and 0.6 M

trehalose-treated cells

Flow cytometry

assessment

193

The kinetics of relative number of population extruding the intracellularly

accumulated dye is calculated in equation (3) thus:

RcFð%Þ¼ðA4tGlu=A4t¼0Þ100 ð3Þ

where RcF is the relative number of cells still stained with cF in gate A4 following

glucose addition, A4

tGlu

is the percentage of cells still stained with cF in gate A4

following glucose addition and incubation tmin and A4

t=0

is the percentage of cells

still stained with cF in gate A4 prior to glucose addition.

Statistical analysis. The correlation between the cell viability and osmotic-induced

changes on the physiology of LGG was tested by one-way ANOVA test. Differences

were considered significant at p< 0.05 level of probability. This was performed with

Origin7 software package (Origin Lab, Northampton, MA, USA).

Results and discussion

Growth kinetics of L. rhamnosus GG

LGG was cultivated in batch culture to determine the times taken to reach the

exponential and stationary phases of growth. These were the required stages needed to

determine the stress responses of this organism. LGG reached the exponential phase at

approximately 4 h and the stationary phase at about 12 h (data not shown).

Determination of the sublethal and lethal osmotic conditions

The measurement of growth of cells signified a decrease with increase in molarity of the

medium. At the end of the incubation period, an appreciable level (though less than the

control) of growth was observed up to 1.2 M. As shown by the sugar fermentation

pattern (API 50 CH system) our study strain utilizes trehalose and not sucrose but both

had same influence on survival rates. The environmental factors that signal to the

bacteria the transition from the logarithmic phase to the stationary phase may have a

considerable effect on the survival rates during the stationary phase (Wetzel et al., 1999),

thus a starvation signal, triggered by depletion of carbon sources, appears to be much

more favourable for survival than a low pH in the presence of sufficient carbon source

(Heller, 2001; Corcoran et al., 2005). However, the cells adjusted to the environmental

stress by accumulation of sugars to balance their internal turgor pressure. Sugars

prepared at 0.6 M were thus taken as the sublethal condition while 1.5 M was taken as the

lethal because at this level, no growth was observed (data not shown).

Table I.

Gate designation of cells

stained with cF and PI

Gate

Fluorescence properties

of cells collected

in each gate

Possible explanation of the

status of involved cellular

mechanism

A1 CF



and PI

Esterase activity not detectable;

membrane compromised

A2 CF

and PI

Active esterase; membrane

minimally damaged

A3 CF



and PI



Esterase inactivated or cF extrusion

out of the cells; intact membrane

A4 CF

and PI



Active esterase; intact membrane

Source: Extracted from Ananta et al. (2004)

NFS

37,3

194

Though the organism was cultivated in MRS broth containing glycine betaine

(a compatible solute) derived from yeast extract, the uptake of sugar together with

accumulation of betaine eventually might have resulted in the hyperosmolarity of the

cytoplasm, which would then be compensated by net exit of glycine betaine. Glycine

betaine does not protect against sugar stress (Glaasker et al., 1998).

Response of exponential-phase cells to osmotic stress

Subjection of these cells to osmotic shock resulted in a slight loss of viability, even at

the highest molarity, log N/N

0.6 was recorded (Figure 6). Kinetically, cells were

able to balance their internal pressure with that of their environment after 30 min

incubation. Several investigations showed that the logarithmic phase are more

susceptible to environmental stresses than are bacteria from stationary phase (Saarela

et al., 2004; Kim et al., 2001; Gouesbet et al., 2001), however in this study, there was no

significant difference on the reaction of the two phases of growth to the stress (Figures

6 and 7). Similar observation was made on stress response of Lb. plantarum to NaCl

stress (Kim et al., 2001). HPLC analysis of sugars revealed that the cells responded by

sugar uptake. It was assumed that the period of treatment might give little or no

opportunity to sugar metabolism.

Esterase activity and membrane integrity

The percentage of cF-stained cells was used to estimate the viability of osmotic

stressed cells (Figures 7-11). Sucrose (0.1-1.2 M) treated cells possessed residual

esterase activity as the control cells. This is shown in Figure 9 by the presence of a high

Figure 6.

Effect of increasing

sucrose and trehalose

concentration in MRS

growth medium on LGG

viability

Flow cytometry

assessment

195

Figure 7.

The impact of osmotic

treatment on the viability

of LGG assessed by plate

count method and

exhibited as the

logarithmic value of the

surviving cells (N/N

)

Figure 8.

Relative changes of

esterase activity as

affected by trehalose

treatments at different

molarities

NFS

37,3

196

Figure 9.

Relative changes of

esterase activity as

affected by sucrose

treatment at different

molarities

Figure 10.

Graphical representation

of cF-extrusion activity as

affected by osmotic

treatments

Flow cytometry

assessment

197

percentage of the population in gate A2 and A4. Cells solely labelled by cF were found

in gate A4. Insignificant percentages of cells were found in gate A2 of 0.1-1.2 M treated

cells whereas about 25 per cent was encountered in A2 of 1.5 M.

The presence of such double-stained population at 1.5 M indicated that the cell

membranes were irreversibly damaged to a low extent, under which penetration of PI

into the cells was allowed but intracellular accumulated cF could still be retained.

There was no esterase inactivation despite the perturbation of the cell membranes.

The calculation of the residual esterase activity based on the total percentage of

population in gate A2 and A4 showed that despite the membrane damage, cells were

still able to accumulate cF thus signifying the preservation of membrane integrity and

enzymatic activity. Major part of the population did not accumulate PI but were mostly

encountered in gate A4 irrespective of the presence of a double-stained population at

this molarity (Figure 1). There was no significant loss in the viability recorded on

plates; the organism must have made use of a repair mechanism on the membranes

(Figure 7). This physiological status was considered as a transient phase in the

progressive change towards cell death. However, death is not irreversible and double-

stained cells may recover (Bunthof, 2002).

Treatment of cells with trehalose resulted in the extrusion of cF out of the cells as

shown by the movement of cells from gate 4 to gate 3 (Figure 2). Though high

fluorescence at FL3 pointed out that in these cells, membranes were not damaged, it

was revealed by further investigations that there was a release of cF out of the cells

(data not shown), which could have been made possible by membrane

permeabilization. The percentage of cF-stained cells (Figure 8) showed same trend as

the results obtained by plate counts (Figure 7).

Figure 11.

Graphical representation

of the relative number of

cF-stained cells at

increasing incubation

period as derived from the

density plots in Figure 5

NFS

37,3

198

The subjection of LGG to heat treatment at 60 C for 300 s also revealed the absence

of PI labelling accompanied by increasing loss of cF accumulating activity. In this case,

the occurrence of esterase inactivation was reported (Ananta, 2005).

Extrusion of intracellular accumulated dye

This probe efflux could be put to use as an additional measure of cell vitality. cF

labelling and, subsequently, the efflux could be measured to assess multiple aspects of

cell viability, upon energizing. These combined methods could give more information

about the physiological condition than cF labelling alone does (Bunthof et al., 1999).

In FL1-FL3 density plot analysis, the extrusion ability of sugar-stressed cells upon

glucose addition was determined as a result of the shift of initially stained population

from gate 4 to gate 3 by intracellular fluorescence loss (Figures 3 and 4). Sucrose-

treated cells up to 0.6 M had a similar extrusion activity as the control samples (Figure

10). Lower percentages were recorded at 1.2 M and 1.5 M but cells treated with

trehalose up to 0.9 M had a comparable ability as the control. Culture counts correlated

well with the extrusion activity only by trehalose treatment.

Determination of the rate of cF extrusion

A value of 0.6 M sucrose-treated cells exhibited no significant difference (p> 0.05) with

the control sample in respect to the reproductive, capacity esterase activity, and cF

extrusion performance. The kinetics of the movement of cF-stained cells from gate A4

to A3 showed that both the control cells and treated cells had similar extrusion rate

(Figures 9 and 5). They completed extrusion in 20 min. This contradicts the report of

Ananta et al. (2004) on extrusion rate in control cells. This could be variability within

the strain due to loss or gain of plasmid leading to inconsistency in the metabolic traits

or activity. However, 0.6 M trehalose-treated cells had a higher extrusion rate than both

the control cells and sucrose-treated cells. Extrusion was completed in 15 min.

Conclusion

Our results clearly showed the additional value of using fluorescent stains to assess the

effect of osmotic treatment on micro-organisms, for example, changes in metabolic

activities and cellular events resulting from osmotic treatment which could not be

assessed by culture techniques were made explicit.

Survival and viability of our study strain, as shown by plate counts method,

revealed a similar protective effect of the stress agents used. The observation made on

trehalose-treated cells, as shown by flow cytometry method, could either be explained

as its inability to protect esterase activity or an extrusion of cF out of the cells. We

confirmed that the movement of cells from gate 4 to gate 3 was as a result of a low

degree of membrane permeabilization resulting in the leakage of cF out of the cells into

the surrounding medium but PI could not have an access into the cells. This suggests

the high level of membrane integrity thus, PI penetration was not permitted to

intercalate with nucleic acids. Trehalose was able to give more stabilization to the

membrane than sucrose as shown clearly by cF extrusion activity. The activities of the

survivors in extruding intracellular accumulated dyes were perturbed beyond 0.6 M

sucrose, however, no perturbation was observed in trehalose-treated cells. The higher

extrusion rate exhibited by trehalse-treated cells pointed out its importance in

enhancing the dye extrusion activity, which is regarded as an analogue of the

capability of cells to extrude toxic compounds.

Flow cytometry

assessment

199

These findings underlined the differences in the protection provided to cells

subjected to osmotic stress by compatible solutes of same osmotic strength. The

determination of the viability of cells from different metabolic and physiological

parameters as shown above is useful in enhancing the stability of starter cultures,

probiotics, etc. in food processing and storage.

References

Alakomi, H.-L., Matto, J., Virkajarvi, I. and Saarela, M. (2005), ‘‘Application of a micro-plate scale

fluorochrome-staining assay for the assessment of viability of probiotic preparation’’.

Journal of Microbiological Methods, Vol. 62, pp. 25-35.

Ananta, E. (2005), ‘‘Identification of environmental factors involved in viability and stability of

probiotic bacteria Lactobacillus rhamnosus GG (ATCC 53103) during spray drying and

application of high pressure pre-treatment for the improvement of stress tolerance’’,

Department of Food Biotechnology and Process Engineering, Berlin University of

Technology, Germany, pp. 227.

Ananta, E. and Knorr, D. (2004), ‘‘Evidence on the role of protein synthesis in the induction of heat

tolerance of Lactobacillus rhamnosus GG by pressure pre-treatment’’, International Journal

of Food Microbiology, Vol. 96, pp. 307-13.

Ananta, E., Heinz, V. and Knorr, D. (2004), ‘‘Assessment of high pressure induced damage on

Lactobacillus rhamnosus GG by flow cytometry’’, Food Microbiology, Vol. 21, pp. 567-77.

Auty, M.A.E., Gardiner, G.E., McBrearty, S.J., O’Sullivan, E., Mulvihill, D.M., Collins, J.K.,

Fitzgerald, G.F., Stanton, C. and Ross, K.P. (2001), ‘‘Direct in situ viability assessment

of bacteria in probiotic diary products using viability staining in conjunction with

confocal scanning laser microscopy’’, Applied Environmental Microbiology, Vol. 67,

pp. 420-425.

Baati, L., Fabre-Gea, C., Auriol, D. and Blanc, P.J. (2006), ‘‘Study of the cryotolerance of

Lactobacillus acidophilus: effect of culture and freezing conditions on the viability and

cellular protein levels’’, International Journal of Food Microbiology, Vol. 59, pp. 241-47.

Barer, M.R. and Harwood C.R. (1999), ‘‘Bacterial viability and culturability’’, Advanced Microbial

Physiology, Vol. 41, pp. 93-137.

Beales, N. (2004), ‘‘Adaptation of microorganisms to cold temperature, weak acid preservatives,

low ph, and osmotic stress: a review’’, Comprehensive Reviews in Food Science and Food

Safety, Vol. 3, pp. 1-20.

Ben Amor, K., Breeuwer, P., Verbaarschot, P., Rombouts, F.M., Akkermans, A.D.L., De Vos, W.M.

and Abee, T. (2002), ‘‘Multiparametric flow cytometry and cell sorting for the assessment

of viable, injured and dead Bifidobacterium cells during bile salt stress’’, Applied

Environmental Microbiology, Vol. 68 No. 11, pp. 5209-16.

Bunthof, C.J. (2002), ‘‘Flow cytometry, fluorescent probes, and flashing bacteria’’, Department of

Agro-Technology and Food Science, Wageningen University, Wageningen, p. 160.

Bunthof, C.J., Vanden Braak, S., Breeuwer, P., Rombouts, F.M. and Abee, T. (1999), ‘‘Rapid

fluorescence Assessment of the viability of stressed Lactobacillus lactis’’, Applied

Environmental Microbiology, Vol. 65 No. 8, pp. 3681-89.

Conrad, P.B., Miller, D.P., Cielenski, P.R. and de Pablo, J.J. (2000), ‘‘Stabilization and preservation of

Lactobacillus acidophilus in Saccharide Matrices’’, Cryobiology, Vol. 41, pp. 17-24.

Fuller, R. (1989), ‘‘Probiotics in man and animals’’, Journal of Applied Bacteriology, Vol. 66,

pp. 365-78.

Gouesbet, G., Gwenael, J. and Boyawal, P. (2001), ‘‘Lactobacillus delbrueckii ssp bulgaricus thermo-

tolerance’’, Lait, Vol. 81, pp. 301-9.

NFS

37,3

200

Kell, D.B., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R. and Barer, M.R. (1998), ‘‘Viability and

activity in readily culturable bacteria: a review and discussion of the practical issues’’,

Antonie Leeuwenhoek, Vol. 73, pp. 169-87.

Kets, E.P.W., Teunissen, P.J.M. and De Bont, J.A.M. (1996), ‘‘Effect of compatible solutes on

survival of lactic acid bacteria subjected to drying’’, Applied Environmental Microbiology,

Vol. 62, pp. 259-61.

Kim, W.S., Perl, L., Park, J.H., Tandianus, J.E. and Dunn, N.W. (2001), ‘‘Assessment of stress

response of the probiotic Lactobacillus acidophilus’’, Current Microbiology, Vol. 43,

pp. 346-50.

Leslie, S.B., Israeli, E.I., Lighthart, B., Crowe, J.H. and Crowe, L.M. (1995), ‘‘Trehalose and sucrose

protect both membranes and proteins in intact bacteria during drying’’, Applied

Environmental Microbiology, Vol. 61 No. 10, pp. 3592-7.

Panoff, J.M., Thammavong, B. and Gueguen, M. (2000), ‘‘Cryoprotectants lead to phenotypic

adaptation to freeze-thaw stress in Lactobacillus delbrueckii ssp. bulgaricus. Ctp 101027T’’,

Cryobiology, Vol. 40, pp. 264-9.

Poolman, B. and Glaasker, F. (1998), ‘‘Regulation of compatible solute accumulation in bacteria’’,

Molecular Microbiology, Vol. 29, pp. 397-407.

Ritz, M., Pholozan, J.L., Federighi, M., Pilet, M.F., (2001), ‘‘Morphological and physiological

characterization of Listeria monocytogenes subjected to high hydrostatic pressure’’,

Applied Environmental Microbiology, Vol. 67, pp. 2240-47.

Saarela, M., Rantala, M., Hallamaa, K., Nohynek, L., Virkajarvi, I. and Matto, J. (2004),

‘‘Stationary-phase acid and heat treatment for improvement of viability of probiotic

lactobacilli and bifido bacteria’’, Journal of Applied Microbiolgy, Vol. 96, pp. 1205-14.

Sandine, W.F. (1996), ‘‘Commercial production of diary starter cultures’’, in Cogan, T.M. and

Accolas, J.-P. (Eds), Diary Starter Cultures, VCH Publisher Inc., New York, NY, pp. 191-206.

van de Gutche, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D. and Maunguin, E. (2002),

‘‘Stress responses in lactic acid bacteria’’, Antonie von Leeuwenhoek, Vol. 82, pp. 187-216.