Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances.pdf

Comparative survival of probiotic lactobacilli spray-driedin the presence of prebiotic substances

B.M. Corcoran1,2, R.P. Ross1,3, G.F. Fitzgerald2,3 and C. Stanton1,3

1Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland, 2Department of Microbiology, University College,

Cork, Ireland, and 3Alimentary Pharmabiotic Centre, Cork, Ireland

2003/0693: received 7 August 2003, revised 18 December 2003 and accepted 26 December 2003

ABSTRACT

B.M. CORCORAN, R .P . ROSS, G.F . F ITZGERALD AND C. STANTON. 2004.

Aims: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an

effort to generate synbiotic powder products.

Methods and Results: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed

media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log

and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose

(PD) (10% w/v) mixture at an outlet temperature of 85–90�C. Stationary phase cultures survived best (31–50%)

in both feed media and were the most stable during powder storage at 4–37�C over 8 weeks, with 30–140-fold

reductions in cell viability at 37�C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus

GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10%

w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7Æ1–43%, while viability losses

of 20 000–90 000-fold occurred in these powders after 8 weeks� storage at 37�C. Survival of the Lactobacillus

culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was

only 0Æ011–0Æ45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus

E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in

either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx.

25–41% survival, yielding powders containing �109 CFU g)1, while Lact. salivarius UCC 500 performed poorly,

experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival

of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570–700-fold) during

storage at 37�C over 8 weeks compared with Lact. rhamnosus GG.

Conclusions: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most

susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder

storage.

Significance and Impact of the study: High viability (�109 CFU g)1) powders containing probiotic lactobacilli

in combination with prebiotics were developed, which may be useful as functional food ingredients for the

manufacture of probiotic foods.

Keywords: lactobacilli, prebiotic, probiotic, spray-drying, viability.

Correspondence to: C. Stanton, Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland (e-mail: [email protected]).

ª 2004 The Society for Applied Microbiology

Journal of Applied Microbiology 2004, 96, 1024–1039 doi:10.1111/j.1365-2672.2004.02219.x

INTRODUCTION

Probiotics are associated with beneficial health effects, and

may be selected for prevention and treatment of diseases

(Alvarez-Olmos and Oberhelman 2001; Shanahan 2002;

Guarner and Malagelada 2003). Such research has stimu-

lated interest in dairy products containing beneficial

bacteria for the general population, children and high risk

groups (FAO/WHO 2001). Lactic acid bacteria, specific-

ally lactobacilli and bifidobacteria are the principal repre-

sentatives of probiotics in the functional food industry

(Holzafpel and Schillinger 2002). Suitable strain selection

necessitates consideration of three essential premises,

encompassing general aspects (origin, identity, safety and

acid/bile resistance), technical aspects (growth properties

in vitro and during processing, survival during processing

and storage) and functional/beneficial features (Collins

et al. 1998; Holzafpel and Schillinger 2002; Stanton et al.

2003). The human derived Lact. rhamnosus GG is a

commercial probiotic strain with recognized health benefits

(Marteau et al. 2001) and has been exploited for use in the

development of functional foods (Erkkila et al. 2001; Ahola

et al. 2002). Spray-drying is an economical process for

preparing industrial scale quantities of viable microorgan-

isms. It offers high production rates and low operating

costs and is a method commonly used to prepare food

adjuncts, which are stable, dry and occupy small volume.

Its application to generate preparations of Lactobacillus

(Desmond et al. 2001; Desmond et al. 2002; Gardiner et al.

2002; Silva et al. 2002) and Bifidobacterium species (O’Ri-

ordan et al. 2001; Lian et al. 2002) has recently received

considerable interest. These spray-dried powders may be

applied to downstream processes, e.g. adjuncts for Cheddar

cheese manufacture (Gardiner et al. 2002), malted bever-

ages (O’Riordan et al. 2001) and the exploitation of

bacteriocin producing cultures in food products (Mauriello

et al. 1999; Morgan et al. 2001; Silva et al. 2002). How-

ever, the spray-drying of probiotic bacteria presents a

number of challenges, in particular, the requirement to

maintain culture viability, given the high processing

temperatures encountered (Daemen and van der Stege

1982; Stanton et al. 2003). Cell membrane damage is often

evident following spray-drying, and this has been attrib-

uted primarily to the effects of heat and dehydration

(Lievense et al. 1994; Teixeira et al. 1995b; To and Etzel

1997b; To and Etzel 1997a). Parameters affecting the

survival of LAB during spray-drying include process

airflow configuration (cocurrent or countercurrent), outlet

temperature of spray dryer, strain, carrier medium applied

and its solids content and pre-adaptation of culture

(Johnson and Etzel 1993; Bielecka and Majkowska 2000;

Gardiner et al. 2000; Desmond et al. 2001; O’Riordan et al.

2001; Lian et al. 2002).

Prebiotics may potentially be exploited as carrier media for

the purposes of spray-drying and may be useful for enhancing

probiotic survival during processing. The defining effect of

prebiotics concerns selective stimulation of Bifidobacterium

and Lactobacillus in the gut, thereby increasing the host’s

natural resistance to invading pathogens (Cummings and

Macfarlane 2002). Carbohydrates such as gum acacia and

soluble starch have been used as spray-drying carriers

(Desmond et al. 2002; Lian et al. 2002). Spray-drying

probiotic lactobacilli in conjunction with the soluble fibre,

gum acacia, increased Lact. paracasei NFBC 338 viability

during powder storage at both 15 and 30�C compared with

RSM control (Desmond et al. 2002).

The aims of this study were (i) to examine the effect of

growth phase on probiotic culture viability during spray-

drying and powder storage; (ii) to investigate the effects of

inclusion of various combinations of RSM and the prebiotics

PD and inulin substances on probiotic viability during

spray-drying and storage and (iii) to investigate the variation

in performance of different probiotic Lactobacillus strains

during spray-drying and storage.

MATERIALS AND METHODS

Bacterial strains and culture conditions

The probiotic strains Lact. rhamnosus VTT E-97800 (Lact.rhamnosus E800, VTT Biotechnology, Espoo, Finland),

Lact. rhamnosus VTT E-94522 (ATCC 53103, Lact.

rhamnosus GG, Valio Ltd., Finland) and Lact. salivarius

VTT E-01878 (Lact. salivarius UCC 500, University

College, Cork, Ireland) were obtained from University

College Cork, under a restricted materials transfer agree-

ment. Harvested cells of these strains were stored as stock

solutions in 50% (v/v) aqueous glycerol at )20�C. They

were subcultured at 1% (v/v) in MRS (de Man et al. 1960)

Oxoid broth (Oxoid Ltd, Hampshire, UK) for �17 h at

37�C under anaerobic conditions, obtained by placing one

activated Anaerocult A gas pack (Merck, Darmstedt,

Germany) in a jar, which was subsequently sealed.

For the enumeration of viable microorganisms in spray-

dried powders, the milk and manufactured powders were

pour plated on MRS agar (Oxoid) in independent triplicate

experiments. Powders were resuscitated in maximum

recovery diluent (MRD; 10% w/v; Oxoid) for 1 h at 37�Cand the appropriate serial dilutions were prepared prior to

pour plating on MRS agar.

Growth of probiotic cultures and acidification rate

Fresh overnight cultures (�17 h,1% v/v) of Lact. salivariusUCC 500, Lact. rhamnosus E800 and Lact. rhamnosus GG

were inoculated into 200 ml MRS broth, giving final cell

DEVELOPMENT OF PROBIOTIC SPRAY-DRIED POWDERS 1025

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 1024–1039, doi:10.1111/j.1365-2672.2004.02219.x

numbers of �107 CFU ml)1. Growth was assessed over

17 h at 37�C by pour plating on MRS agar and the rate of

acidification was analysed with a pH meter (model MP220,

Mettler-Toledo, Greifensee, Switzerland), with calibrated

electrode (Mettler Toledo InLab� 413).

Heat challenge experiments

The thermal tolerance of Lact. rhamnosus GG, Lact.

rhamnosus E800 and Lact. salivarius UCC 500 were compared

in reconstituted skim milk (RSM, 20% w/v), supplemented

with yeast extract (0Æ5% w/v). Sucrose was included for

studies involving Lact. rhamnosus GG, as this microorganism

utilizes lactose poorly (Goldin et al. 1992). Two 50-ml

volumes of RSM, contained in 100-ml bottles and agitated by

magnetic stirrer bars, were placed in a water bath at the

appropriate test temperatures of 37�C (control and to obtain

initial count), 55, 58, 59, 60 and 61�C. One bottle was used to

monitor the temperature, while, after temperature equilibra-

tion, a 1% (v/v) inoculum of an overnight culture (�17 h) of

either Lact. salivarius UCC 500, Lact. rzhamnosus E800 or

Lact. rhamnosus GG was added to the second bottle. At

appropriate intervals (between 30 s and 4 min), 1-ml aliquots

were removed from the test bottle, serially diluted in MRD

and pour plated in MRS agar. Survivors were enumerated

after 3 days of anaerobic incubation at 37�C. Tests were

conducted in duplicate and mean log survivor counts were

plotted as a function of heating time for each test tempera-

ture. At each temperature, a best fit straight line was obtained

by regression analysis, and D-values, which represent the

time (min) required to kill 90% of cells were determined by

taking the absolute value of the inverse of the slope of this

line (Stumbo 1965).

Preparation of early log phase cultures of Lact.rhamnosus GG for spray-drying

Polydextrose 2 (PD) was provided by Danisco Sweeteners

(Redhill, Surrey, UK). RSM (10% w/v) and PD (10% w/v)

were combined by stirring into sterile distilled H2O, and

heat treated at 90�C for 30 min. Similarly, RSM (20% w/v,

control feed) was prepared and heat treated under identical

conditions. Both milk-based media were inoculated at 1%

(v/v) with a fresh overnight culture of Lact. rhamnosus GG

(i.e. �8 · 106 CFU ml)1) and incubated anaerobically at

37�C to exponential phase (3Æ5–4Æ0 h) until a 0Æ2 decrease in

pH was obtained (approx. 3 · 107 CFU ml)1).

Preparation of stationary and lag phase culturesof probiotic cultures for spray-drying

Stationary phase cultures of Lact. rhamnosus GG, Lact.

rhamnosus E800 and Lact. salivarius UCC 500 were prepared

for spray-drying, using a method adapted from Silva et al.

(2002). Fresh overnight cultures (�17 h) of each probiotic

strain were inoculated at 1% (v/v) into 400-ml MRS broth

and incubated at 37�C for 17 h. Following centrifugation at

7000 g for 15 min at 4�C, the cells were resuspended in an

equal volume of either heat treated RSM (20% w/v), RSM

(10% w/v) and PD (10% w/v), or PD (20% w/v),

tempered at 37�C, in order to increase the spray-drying rate,

and immediately spray-dried. Lag phase cultures of Lact.

rhamnosus GG were prepared precisely as stipulated for

stationary phase cells, i.e. 17 h growth in MRS broth at

37�C, with the exception that cultures containing

�109 CFU ml)1 were resuspended and incubated in

spray-drying feed media for approx. 45 min at 37�C prior

to spray-drying.

Four inulin products were provided by ORAFTI Active

Food Ingredients (Tienen, Belgium), contrasting in degree

of polymerization (DP) of fructo oligomer chains and purity

(content of mono- and disaccharides). These powders were

commercially known as: RAFTILOSE� P95, (oligofructose

‡93Æ2% (DP 2–8), Glu + Fru + Suc <6Æ8%); RAFTI-

LOSE� Synergy1, (oligofructose 90–94% (DP 2–8),

Glu + Fru 4–6%, Suc 2–4%); RAFTILINE� GR, (oligo-

fructose >90% (average DP ‡10), Glu + Fru £4%, Suc

£8%); RAFTILINE� HP, (oligofructose >99Æ5% (average

DP ‡23), Glu + Fru + Suc £0Æ5%). Lact. rhamnosus GG

was used for studies to evaluate the usefulness of inulin for

protecting probiotic cell viability during spray-drying and

storage of powders. Spray-dried feed media was prepared by

stirring either RSM (20% w/v), RSM (10% w/v) and

inulin (10% w/v), or inulin (20% w/v) into sterile distilled

H2O, and heat treating at 90�C for 30 min. Stationary phase

Lact. rhamnosus GG cultures were prepared for processing

precisely as described above, and spray-dried.

Spray-drying and storage

The solids contents of the feed samples was determined before

spray-drying using a Labwave 9000% Solids Determiner

(Metrohm Ltd, Dublin, Ireland). A laboratory scale spray

dryer (model B191 Buchi mini spray dryer; Flawil, Switzer-

land) was used to process samples at a constant air inlet

temperature of 170�C. The feed solution was atomized into

the drying chamber using a two-fluid nozzle and the product

dried with a very low residence time. The outlet temperature

was maintained at 85–90�C, in order to obtain powders with

less than 4% moisture. Per cent surviving bacteria were

calculated as follows: % survivors ¼ N/NO · 100, where NO

represented the number of bacteria in the RSM before drying,

and N was the number of bacteria in the spray-dried powder.

Both N and NO were expressed per gram of dry matter.

The probiotic containing powders were placed in sealed

polythene bags, which were placed in aluminum coated

1026 B.M. CORCORAN ET AL.


paper bags, and stored at 4, 15 and 37�C. Viability of the

probiotic strains was determined on the day of powder

manufacture and during powder storage for 8 weeks, at the

temperatures described above.

Salt tolerance test

In order to study potential cellular damage arising from

spray-drying, we determined the sensitivity of cultures to

NaCl before and after processing as described previously

(Gardiner et al. 2000). Fresh cultures at different growth

phases and spray-dried powders containing cultures were

pour plated on MRS agar supplemented with NaCl (5% w/

v, Sigma). The plates were examined after 5 days of

anaerobic incubation and viable numbers were compared

with numbers obtained on MRS plates without NaCl.

Moisture content in spray-dried powders

The moisture content of spray-dried powders was deter-

mined by oven drying at 102�C. This involved determin-

ation of the difference in weight before and after drying,

expressed as a percentage of the initial powder weight,

according to the International Dairy Federation Bulletin

(IDF 1993).

RESULTS

Growth and acidification rates of probioticlactobacilli

In order to assess the acidification rates of the three probiotic

Lactobacillus strains, 1% (v/v) overnight cultures were

grown in MRS broth for 17 h, and pH and cell counts

monitored (Fig. 1). Initial culture pH (6Æ55–6Æ57), decreased

to a final pH of 3Æ70–3Æ74 for all three strains. The rate of

acidification varied among the strains with Lact. salivariusUCC 500 exhibiting the fastest rate (0Æ157 pH units

h)1 log10 CFU ml)1) in the first 6 h of growth. During

this period, Lact. rhamnosus strains acidified the medium at a

rate of 0Æ124–0Æ127 pH units h)1 log10 CFU g)1. Cell

numbers of Lact. salivarius UCC 500 were initially higher

(6Æ6 · 107 CFU ml)1) compared with Lact. rhamnosus cul-

tures (7Æ2 · 106–1Æ63 · 107 CFU ml)1), which may account

for the faster rate of acidification of Lact. salivarius UCC

500.

Thermal tolerance of probiotic lactobacilli

In order to generate spray-dried powders with high numbers

of probiotic lactobacilli, the microorganisms must withstand

the high temperatures encountered during spray-drying,

which has been shown to vary among strains of probiotic

lactobacilli (Gardiner et al. 2000). Initially, the thermal

tolerance of three strains of probiotic lactobacilli in RSM

(20% w/v), in the range of 55–61�C was compared. At 59�C,

a decrease of 1Æ92 log10 CFU ml)1 of Lact. rhamnosus GG

was obtained (Fig. 2a), while the two other strains were more

heat resistant at this temperature (Fig. 2 b, c). At 61�C, Lact.

rhamnosus E800 was most thermal tolerant (a decrease of

1Æ59 log10 CFU ml)1), while Lact. salivarius UCC 500 and

Lact. rhamnosus GG experienced decreases of 2Æ99 log10

CFU ml)1 and, 3Æ70 log10 CFU ml)1 respectively.

D-values calculated from these data confirmed Lact.

rhamnosus E800 as the most thermal tolerant of the three

Lactobacillus strains tested and following exposure to 61�C,

the D-value for Lact. rhamnosus E800 was 2Æ76 min. In

contrast, D-values for Lact. rhamnosus GG and Lact.

3·50

3·75

4·00

4·25

4·50

4·75

5·00

5·25

5·50

5·75

6·00

6·25

6·50

6·75

0 2 4 6 8 10 12 14 16 18Time (h)

pH

6·757·00

7·25

7·50

7·75

8·00

8·25

8·50

8·75

9·00

9·25

9·50

9·75

10·00

log 1

0 C

FU

ml–1

Fig. 1 Growth of, Lactobacillus salivarius

UCC 500 (j) Lact. rhamnosus E800 (u) and

Lact. rhamnosus GG (m) in MRS broth at

37�C. Closed symbols represent cell counts on

MRS agar (log10 CFU ml)1), while open

symbols represent the pH reduction of cul-

tures in MRS broth. The results are based on

data from triplicate analyses



salivarius UCC 500 were 1Æ32 and 1Æ64 min, respectively. At

59�C, there was considerable difference in D-values

obtained between Lact. rhamnosus GG and the other

probiotic lactobacilli (1Æ84 min for Lact. rhamnosus GG,

3Æ91 and 3Æ39 min for Lact. rhamnosus E 800 and Lact.

salivarius UCC 500, respectively).

Phase based survival during spray-dryingand storage

In order to establish the optimal growth phase of cultures for

spray-drying, Lact. rhamnosus GG culture was spray-dried

at different growth phases (lag, early log and stationary

1

2

3

4

5

6

7

8

0 1 2 3 4

Sur

vivo

rs lo

g 10

CF

U m

l–1

Sur

vivo

rs lo

g 10

CF

U m

l–1

Sur

vivo

rs lo

g 10

CF

U m

l–1

1

2

3

4

5

6

7

8

0 1 2 3 4

1

2

3

4

5

6

7

8

0 1 2 3 4

Time (min)

(a)

(b)

(c)

Fig. 2 Survival of Lactobacillus. rhamnosus

GG (a), Lact. rhamnosus E800 (b) and Lact.

salivarius UCC 500 (c) heated in reconstituted

skim milk (20% w/v) at 55�C (d), 58�C (u),

59�C (m), 60�C (·), 61�C (j). The results

are based on data from duplicate heat chal-

lenge experiments



phases) in RSM (20% w/v). Over 50% survival was

obtained with stationary phase cells (yielding powder with

viable counts of 2Æ9 · 109 CFU g)1), while early log phase

cultures exhibited 14% survival (2Æ1 · 107 CFU g)1)

(Fig. 3a). Lag phase cells were most susceptible to spray-

drying, exhibiting only approx. 2% survival

(2 · 108 CFU g)1). NaCl (5% w/v) was included in the

culture medium (MRS) to evaluate process associated cell

damage. Using this approach, no cell damage was observed

in spray-dried stationary phase cells, while lag phase

cultures exhibited some cell injury, and early log phase

cultures experienced most damage, with survival in the

presence of NaCl (5% w/v) reduced to 4% (Fig. 3a).

In order to evaluate the efficacy of the prebiotic PD for

enhancing survival during spray-drying and its protective

effect against cell damage, powders were subsequently

prepared from a medium consisting of RSM (10% w/v)

and PD (10% w/v) at the three growth phases, as described

above. In the presence of PD, Lact. rhamnosus GG showed

greatest survival in the early log phase (48% survival),

followed by stationary phase cultures (31% survival), and as

in RSM powders, lowest survival was obtained when lag

phase cells were spray-dried (9% survival, Fig. 3b). Early

log phase cultures exhibited higher degrees of cell damage

following spray-drying, with approx. 30% lower cell

numbers obtained when plated in the presence of NaCl

(5% w/v) (Fig. 3b). Cell damage also occurred in the lag

phase of growth (a decrease to 1Æ6% survival), while some

cell damage was evident in stationary phase cultures.

Therefore, the inclusion of PD in the media did not offer

any increased survival or protection from cell damage

associated with spray-drying of cultures at the three growth

phases studied.

Following spray-drying, all powders were placed in

polythene bags and stored at 4, 15 or 37�C, during which

probiotic viability was assessed to identify the optimal

growth phase for storage over 8 weeks, and to investigate if

PD treatment exerted a beneficial effect on probiotic

viability during storage. Stationary phase Lact. rhamnosus

GG cultures were most stable, yielding only small decreases

in viable numbers at all storage temperatures (Fig. 4).

While optimal survival occurred during storage at 4�C,

there was only a 30-fold reduction (to 1 · 108 CFU g)1), in

probiotic numbers in powders stored at 37�C for 8 weeks

(Fig. 4c). Spray-dried lag phase Lact. rhamnosus GG

cultures survived well at the lower storage temperatures

(Fig. 4a), with highest losses observed at 37�C, when 40-

fold reduction in viable numbers were recorded after

8 weeks (to 5Æ1 · 106 CFU g)1) (Fig. 4c). Storage of spray-

dried early log phase cultures yielded highest viability losses

at 37�C (Fig. 4c) (over 120 000-fold decrease in cell

numbers yielding viable counts of 1Æ7 · 102 CFU g)1),

while lower death rates (16-fold decrease) occurred at 15�C(Fig. 4b).

The effect of PD inclusion on survival of spray-dried

cultures during storage was subsequently assessed. Viable

losses of stationary phase Lact. rhamnosus GG stored at 4

and 15�C were low, while greatest reduction in viability

occurred in powders stored at 37�C (140-fold reductions,

yielding approx. 1 · 107 CFU g)1) (Fig. 4). At 37�C, PD

treated lag phase cultures suffered a 2600-fold decrease

(viable counts reduced to 2Æ4 · 105 CFU g)1), while viab-

ility of spray-dried early log phase cultures declined most

rapidly at 37�C (Fig. 4c), when 740 000-fold losses in

viability occurred (yielding viable counts of

7Æ7 · 101 CFU g)1). Early log phase cultures also experi-

enced considerable viability losses at 15�C (1500-fold

reduction of viable numbers) (Fig. 4b). At all phases of

growth studied, use of PD did not exert a beneficial effect

upon storage survival compared with RSM. Indeed, at the

highest storage temperature (37�C), viability was lower in

PD-containing powders than RSM powders (Fig. 4c).

0·01

0·1

1

10

100

Stationary Lag Early log

Stationary Lag Early log

Per

cent

sur

viva

lP

erce

nt s

urvi

val

0·01

0·1

1

10

100

(a)

(b)

Fig. 3 Per cent survival (CFU g)1) of Lactobacillus rhamnosus GG

spray-dried in the presence of (a) reconstituted skim milk (RSM) (20%

w/v) and (b) RSM (10% w/v) and polydextrose (10% w/v) at outlet

temperatures of 85–90�C, at three phases of growth. Powders were

pour plated in MRS ( ) and MRS containing NaCl (5% w/v) (().

Results are the mean of triplicate spray-drying trials and S.D.S.D. are

indicated by the vertical bars



0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8Time (weeks)

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

Sur

vivo

rs lo

g 10

CF

U g

–1S

urvi

vors

log 1

0 C

FU

g–1

Sur

vivo

rs lo

g 10

CF

U g

–1

(a)

(b)

(c)

Fig. 4 Survival of Lactobacillus rhamnosus

GG during powder storage at 4�C (a), 15�C(b) and 37�C (c). Powders were prepared

using cultures grown to the lag (¤, )), early

log (m, n) and stationary phases (j, ().

Closed symbols represent powders prepared

with reconstituted skim milk (RSM) (20% w/

v), while open symbols represent powders

prepared with RSM (10% w/v) and poly-

dextrose (10% w/v). Results are the mean of

triplicate spray-drying trials and S.D.S.D. are




Comparison of Lact. rhamnosus GG survival whenspray-dried in the presence of the prebiotics inulinand PD

In order to analyse the effect of inclusion of prebiotics on

survival of Lact. rhamnosus GG, four different products

derived from inulin were used in spray-drying trials and the

data compared with PD. The inulin products were Rafti-

lose� P95 and Raftilose� Synergy 1 (oligofructose powders

obtained by partial enzymatic hydrolysis of inulin) and

Raftiline� HP and Raftiline� GR (inulin powders obtained

by hot-water extraction from the chicory root). Stationary

phase cultures of Lact. rhamnosus GG were spray-dried in

the following feed media; RSM (20% w/v), RSM (10% w/v)

and inulin (10% w/v) or inulin (20% w/v). Lact. rhamnosus

GG exhibited highest viability in RSM powders (approx.

50% survival), and of the inulin containing powders

produced from the feed media consisting of RSM (10%

w/v) and inulin (10% w/v), greatest probiotic survival was

obtained using Raftilose� P95 (43Æ1%, Fig. 5a). Raftiline�

HP afforded least protection during spray-drying, yielding

probiotic survival of only 7Æ1% following spray-drying.

0·001

0·01

0·1

1

10

100

RSM (20% w/v) Polydextrose Raftilosesynergy 1

Raftilose P95 Raftiline GR Raftiline HP

Per

cent

sur

viva

l

0·1

1

10

100

RSM (20% w/v) Polydextrose Raftilose P95 Raftilosesynergy 1

Raftiline GR Raftiline HP

Per

cent

sur

viva

l

(a)

(b)

Fig. 5 Per cent survival (CFU g)1) of sta-

tionary phase Lactobacillus rhamnosus GG

spray-dried in a feed medium consisting of (a)

reconstituted skim milk (RSM) (10% w/v)

and polydextrose (PD) or inulin (10% w/v) or

(b) PD or inulin (20% w/v) at outlet

temperatures of 85–90�C. Powders were pour

plated in MRS ( ) and MRS containing

NaCl (5% w/v) ((). Results are the mean of

triplicate spray-drying trials and S.D.S.D. are




Furthermore, these cultures experienced the most cell

damage during spray-drying, as evidenced by the higher

sensitivity of Lact. rhamnosus GG in the Raftiline� HP

powders to NaCl (5% w/v) (Fig. 5a). Inulin (20% w/v) as

the feed media resulted in the greatest loss in survival of

Lact. rhamnosus GG, with only 0Æ011–0Æ25% survival of the

culture obtained following spray-drying (Fig. 5b). Further-

more, Lact. rhamnosus GG survival after spray-drying in PD

(20% w/v) was also low (0Æ45% survival), corresponding to

Lactobacillus counts of 5Æ5 · 107 CFU g)1 powder All of the

cultures spray-dried in the feed media consisting solely of

the prebiotics, PD or inulin experienced cell damage arising

from processing, with up to 60% of the cells affected.

Viability of Lact. rhamnosus GG in these powders was

assessed during storage at 4, 15 and 37�C. As observed

above, highest probiotic stability was obtained at lower

storage temperatures (Fig. 6). Following 8 weeks� storage at

the higher temperature, Lact. rhamnosus GG experienced

dramatic viability losses (20 000–30 000-fold) in powders

consisting of RSM (10% w/v) and partially hydrolysed

inulin (10% w/v) (Raftilose� P95 and Raftilose� Synergy

1), surviving to approx. 7 · 104 CFU g)1 at 37�C (Fig. 6c).

Survival in RSM (10% w/v) and inulin (10% w/v)

(Raftiline� HP and Raftiline� GR) powders was lower,

yielding approx. 8 · 103 CFU g)1 viable cells after 8 weeks

storage at 37�C. Even more dramatic viability losses

were obtained in powders devoid of RSM. Following only

1 week of storage at 37�C, 1100–18 000-fold reductions in

probiotic cell viability (corresponding to final counts of

4Æ67 102 to 7Æ00 · 103 CFU g)1) were observed in powders

produced with inulin preparations only (data not shown).

Lact. rhamnosus GG in powders produced with PD only

experienced a 31-fold reduction in viability to 1Æ76 ·106 CFU g)1 after 1 week storage at 37�C, while during

the same time-frame, only a 2Æ7-fold reduction in probiotic

viability was observed in powders produced from RSM

(20% w/v).

Strain selection during spray-drying

To investigate the variation in performance of different

strains during spray-drying, Lact. rhamnosus E800 and Lact.

salivarius UCC 500 were spray-dried in the stationary phase

of growth in either RSM (20% w/v), or RSM (10% w/v)

and PD (10% w/v) and survival compared with Lact.

rhamnosus GG. Compared with Lact. rhamnosus GG, Lact.

rhamnosus E800 and Lact. salivarius UCC 500 were relatively

poor spray-drying survivors. For example, while Lact.

rhamnosus GG exhibited 50% survival in powders made

with RSM only, Lact. rhamnosus E800 experienced 25Æ4%

survival (1 · 109 CFU g)1) while Lact. salivarius UCC 500

had only approx. 0Æ7% survival (6Æ2 · 107 CFU g)1)

(Fig. 7) under identical conditions of spray-drying. Fur-

thermore, Lact. salivarius UCC 500 experienced consider-

able process associated cell damage, as approx. 95%

(1Æ3 log10 CFU g)1) of surviving cultures exhibited cell

damage. While the presence of RSM did prevent cell

damage to the more robust Lact. rhamnosus GG and Lact.

rhamnosus E800 cultures, it did not prevent high degrees of

cell damage occurring in Lact. salivarius UCC 500 (Fig. 7a).

When Lact. rhamnosus E800 was spray-dried in the presence

of RSM (10% w/v) and PD (10% w/v), 41% survival was

obtained, which was higher than that achieved with RSM

alone. However, inclusion of PD resulted in greater loss of

viability of Lact. salivarius UCC 500 (0Æ12% survival)

compared with RSM only (Fig. 7b). Both Lact. rhamnosus

E800 and Lact. salivarius UCC 500 experienced comparable

levels of cell damage, when spray-dried in the presence of

PD compared with RSM alone (Fig. 7b). During subse-

quent storage of these powders at 4 and 15�C for 8 weeks,

all three probiotic cultures retained good viability (Fig. 8a

and b), but this declined more rapidly (570 to 700-fold)

during storage at 37�C (Fig 8c), with Lact. rhamnosus E800

powders yielding numbers of 1Æ5 · 106 CFU g)1 and Lact.

salivarius UCC 500 powders containing 1Æ1 · 105 CFU g)1

after 8 weeks. Similarly, little losses of viability of Lact.

salivarius UCC 500 and Lact. rhamnosus E800 were observed

in powders containing PD during storage 4 and 15�C for

8 weeks, (Figs. 8a and b). Similar to the results above,

during storage at 37�C for 8 weeks, viable numbers of both

cultures declined approx. 825 to 940-fold in PD-containing

powders (Fig. 8c).

Analysis of moisture content of powders

Powders are required to contain less than 4% H2O g)1 in

order to be categorized as stable (Masters 1985). All powders

produced in this study contained less than 4% moisture

when spray-drying was conducted at outlet temperatures of

85–90�C (Table 1).

DISCUSSION

In order to be successful candidates for functional food

applications, probiotic cultures must be capable of with-

standing the harsh conditions often encountered during food

processing. This study compared the performance of three

strains of probiotic lactobacilli during spray-drying and

powder storage, and sought to optimize culture viability by

comparing performance of cultures in different phases of

growth, and in the presence of different prebiotic substances

during spray-drying.

Initially, the thermal tolerance, an indicator of probiotic

survival during spray-drying (Gardiner et al. 2000), of three

strains of probiotic lactobacilli were compared. From the

data presented in the current study, Lact. rhamnosus E800



0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

Sur

vivo

rs lo

g 10

CF

U g

–1S

urvi

vors

log 1

0 C

FU

g–1

Sur

vivo

rs lo

g 10

CF

U g

–1

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

Time (weeks)

(a)

(b)

(c)

Fig. 6 Survival of stationary phase Lactoba-

cillus rhamnosus GG during powder storage at

(a) 4�C (b) 15�C and (c) 37�C for 8 weeks.

Powders were prepared using either reconsti-

tuted skim milk (RSM) (20% w/v) (·) or

RSM (10% w/v) and different forms of inulin

(10% w/v): RAFTILOSE P95 (¤), RAFTI-

LOSE Synergy1, (m) RAFTILOSE� GR (d)

and RAFTILOSE� HP, (j). Error bars

represent the mean of triplicate spray-drying

experiments



was the most heat resistant strain, followed by Lact.

salivarius UCC 500, with Lact. rhamnosus GG being least

heat resistant. D-values ranged from 2Æ76 min to 1Æ32 min

for Lact. rhamnosus E800 and Lact. rhamnosus GG at 61�C,

respectively. Previously, the D-value reported for Lact

paracasei NFBC 338 was 1Æ1 min and for Lact. salivarius

UCC 118 was 0Æ5 min at 61�C (Gardiner et al. 2000).

Therefore, the strains used in the current study appeared to

be relatively thermal tolerant.

On examination of the effect of spray-drying on the

different strains in the stationary phase, Lact. salivarius

UCC 500 survival contrasted with the survival of Lact.

rhamnosus strains, and such variations amongst different

probiotic species have been observed previously (Gardiner

et al. 2000; Lian et al. 2002). Lact. salivarius UCC 500 had

the fastest acidification rate in the first 6 h of growth,

however, it appeared to have no benefit because of cross-

protective effects during spray-drying. Lact. rhamnosus GG

was twice as robust following spray-drying in RSM as Lact.

rhamnosus E800 and the presence of PD in the feed

medium did not enhance survival during spray-drying.

Although Lact. rhamnosus GG exhibited the poorest

thermal tolerance of the three Lactobacillus strains studied,

it was the best survivor during spray-drying, indicating

that thermal tolerance alone is not an accurate predictor of

performance during spray-drying and that other phenom-

ena, such as dehydration affect cell viability during drying.

Dehydration inactivation was associated with cell damage

of Lact. plantarum, rather than thermal inactivation during

drying (Lievense et al. 1994). One of the most susceptible

sites to cellular injury is the cytoplasmic membrane, which

is exacerbated during spray-drying (Teixeira et al. 1995b;

Teixeira et al. 1996). Dehydration and high temperature

damage in the atomizer and subsequent droplet drying

occur (Fu and Etzel 1995). Increased sensitivity of

sublethally injured bacteria to NaCl has been associated

with cell membrane damage (Brennan et al. 1986; Teixeira

et al. 1995a; Gardiner et al. 2000). Lact. rhamnosus GG and

Lact. rhamnosus E800 exhibited lower levels of cell damage

following spray-drying with 0–10% susceptibility in the

RSM feed media and 42–44% susceptibility in RSM /PD

mixture. Spray-dried Lact. salivarius UCC 500 exhibited

the highest degree of cell damage, with approx. 90% of the

surviving cells being susceptible to the presence of NaCl.

Previous studies showed spray-dried Lact. salivarius UCC

118 suffered a similar fate, exhibiting 100% susceptibility

to NaCl when spray-dried in RSM (Gardiner et al. 2000).

The viability of Lact. rhamnosus GG during spray-drying

in different phases of growth was compared and optimal

survival was observed in cultures in the stationary phase

(50% survival), with lag phase cultures being most labile

(2% survival) and early log phase cultures exhibited 14%

survival. Survival of Lact. rhamnosus GG at the three phases

of growth studied was independent of the type of feed

carrier used, as the inclusion of PD into the spray-drying

medium did not result in major changes to survival

compared RSM alone. However, greater cell damage

occurred in cultures spray-dried in the presence of PD,

particularly in early log phase Lact. rhamnosus GG. Other

workers also reported greater loss of viability in spray-dried

log phase Lact. bulgaricus compared with stationary phase

cultures (Teixeira et al. 1995a). Good survival was found to

occur in early log phase Lact. paracasei NFBC 338 spray-

dried under identical spray-drying conditions, with 50% of

0·001

0·01

0·1

1

10

100

Lact. rhamnosus GG Lact. rhamnosus E800

Lact. salivarius UCC500

Per

cent

sur

viva

l

0·001

0·01

0·1

1

10

100

Lact. rhamnosus GG Lact. rhamnosus E800

Lact. salivarius UCC500

Per

cent

sur

viva

l

(a)

(b)

Fig. 7 Per cent survival (CFU g)1) of stationary phase Lactobacillus

rhamnosus GG, Lact. rhamnosus E800 and Lact. salivarius UCC 500

following spray-drying in (a) reconstituted skim milk (RSM) (20%

w/v) or (b) RSM (10% w/v) and polydextrose (10% w/v) at outlet

temperatures of 85–90�C. Powders were pour plated in MRS ( ) and

MRS containing NaCl (5% w/v) ((). Results are the mean of

triplicate spray-drying trials and S.D.S.D. are indicated by the vertical bars



0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8Time (weeks)

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

Sur

vivo

rs lo

g 10

CF

U g

–1S

urvi

vors

log 1

0 C

FU

g–1

Sur

vivo

rs lo

g 10

CF

U g

–1

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

(a)

(b)

(c)

Fig. 8 Survival of probiotic lactobacilli dur-

ing powder storage at (a) 4�C (b) 15�C and (c)

37�C. Powders were formulated from sta-

tionary phase Lactobacillus rhamnosus GG

(m, n), Lact. rhamnosus E800 (j, () or Lact.

salivarius UCC 500 (¤,)). Closed symbols

represent powders prepared from reconstitu-

ted skim milk (RSM) (20% w/v), while open

symbols represent powders prepared using

RSM (10% w/v) and polydextrose (10% w/

v). Error bars represent the mean of triplicate

spray-drying experiments



cells remaining viable in powders (Gardiner et al. 2000).

Good survival of actively growing cells has been reported by

Abd-El-Gawad et al. (1989) during spray-drying, and by

others during freeze-drying (Foster 1962; Steel and Ross

1963).

Storage survival was inversely related to temperature, in

parallel with observations of previous studies (Gardiner

et al. 2000; Desmond et al. 2002). All spray-dried probi-

otic cultures demonstrated good survival after 8 weeks�storage at 4 and 15�C (26–100% and 0Æ06–50% of spray-

dried cultures surviving storage at 4�C and 15�C,

respectively). Similarly, Lact. paracasei NFBC 338, when

spray-dried in RSM in the log phase and stored at 15�Cfor 7 weeks retained good viability (Gardiner et al. 2002).

Effective survival at ambient temperatures can remove the

more costly requirement of refrigeration. However, other

studies have reported substantial losses in culture viability

during storage of spray-dried powders. For example,

lactobacilli spray-dried in a whey based medium and

stored at 4�C exhibited 87Æ2–95Æ5% viability reduction

(Mauriello et al. 1999) and Teixeira et al. (1995b) reported

approx. 4 log10 CFU ml)1 reduction of spray-dried Lact.

delbrueckii ssp. bulgaricus stored at 15�C for 60 days. Early

log phase Lact. rhamnosus GG, a good spray-drying

survivor, was the poorest performer during storage,

particularly at 37�C. Similar decreases during storage of

log phase Lact. paracasei NFBC 338 at 30�C have been

reported (Desmond et al. 2002), indicating that spray-

drying cells during the early log phase is unsuitable for

high temperature storage of the powders. Stationary phase

cultures of Lact. rhamnosus GG, Lact. rhamnosus E800 and

Lact. salivarius UCC 500 suffered losses in viability during

37�C storage of approx. 1Æ45–2Æ95 log10 CFU g)1 . Teixe-

ira et al. (1995b) previously reported a 4 log10 CFU ml)1

decrease of spray-dried stationary phase Lact. delbrueckii

spp. bulgaricus following storage at 20�C for 60 days. The

application of PD into the spray-drying media provided no

increased survival during storage. Although Lact. salivarius

UCC 500 was a poor survivor of spray-drying, survival

during storage at 37�C was comparable to Lact. rhamnosus

E800. This comparative survival indicates that the high

degree of cell damage observed in Lact. salivarius UCC

500 did not adversely affect survival during storage.

Interestingly, Gardiner et al. (2000) showed that Lact.

salivarius UCC 118, a poor survivor of spray-drying,

survived better during storage at 30�C in contrast to the

more heat tolerant Lact. paracasei NFBC 338. This

indicates that good storage survival at higher temperatures

may not depend on selection of the best survivors

following spray-drying.

PD and inulin prebiotics supplemented into spray-dried

dairy products provide excellent mouth feel, texture and

taste attributes in food formulations (Booten et al. 1998).

Different carriers in the feed media resulted in varying

degrees of survival of stationary phase Lact. rhamnosus GG.

Probiotic survival is dependent on the spray-drying media

applied, as differences in thermal conductivity and diffu-

sivity can affect survival of spray-dried probiotics (Lian

Table 1 Per cent moisture (% H2O g)1) of powders harbouring probiotic lactobacilli

Strain Growth phase Reconstituted skim milk (% w/v) % H2O g)1S.D.S.D.

Lact. rhamnosus GG stationary 20 3Æ680 0Æ179

Lact. rhamnosus GG stationary 10% + 10% PD 3Æ182 0Æ183

Lact. rhamnosus GG lag 20 2Æ506 0Æ085

Lact. rhamnosus GG lag 10% + 10% PD 3Æ347 0Æ124

Lact. rhamnosus GG exponential 20 3Æ830 0Æ217

Lact. rhamnosus GG exponential 10% + 10% PD 3Æ829 0Æ353

Lact. rhamnosus VTT E800 stationary 20 3Æ771 0Æ279

Lact. rhamnosus VTT E800 stationary 10% + 10% PD 3Æ164 0Æ393

Lact. salivarius UCC 500 stationary 20 2Æ694 0Æ349

Lact. salivarius UCC 500 stationary 10% + 10% PD 3Æ347 0Æ124

Lact. rhamnosus GG stationary 10% + 10% Raftilose P 95 3Æ407 0Æ276

Lact. rhamnosus GG stationary 10% + 10% RaftiloseSynergy 1 3Æ309 0Æ310

Lact. rhamnosus GG stationary 10% + 10% Raftiline GR 3Æ383 0Æ035

Lact. rhamnosus GG stationary 10% + 10% Raftiline HP 3Æ318 0Æ219

Lact. rhamnosus GG stationary 0% + 20% PD 2Æ647 0Æ348

Lact. rhamnosus GG stationary 0% + 20% Raftilose P 95 2Æ986 0Æ171

Lact. rhamnosus GG stationary 0% + 20% RaftiloseSynergy 1 2Æ919 0Æ233

Lact. rhamnosus GG stationary 0% + 20% Raftiline GR 3Æ021 0Æ170

Lact. rhamnosus GG stationary 0% + 20% Raftiline HP 3Æ233 0Æ274

Results are the mean of triplicate spray-drying trials.



et al. 2002). Chemical characteristics of the media may also

have an effect upon survival, as skim milk is an aqueous

solution of proteins, lactose and minerals, while the

prebiotics tested in this study are carbohydrates with

differing degrees of polymerization. Differing protective

effects could therefore be anticipated. Of the inulin

products tested, the inclusion of Raftilose� P95 [an

oligofructose (FOS) compound] (a component which can

stimulate probiotic growth (Bielecka et al. 2002; Perrin

et al. 2002)) in the feed medium resulted in highest

probiotic survival. In addition, FOS can reduce the growth

rate of foodborne pathogens when used as a carbohydrate

growth source for probiotics (Fooks and Gibson 2002).

Approx. 50% of surviving Lact. rhamnosus GG cells spray-

dried in the presence of inulin exhibited cell damage,

compared with only 25% of cells spray-dried in the PD

containing medium, indicating that PD treatment offered

greater protection than inulin during spray-drying. The

inclusion of prebiotic as the sole carriers during spray-

drying did not afford protection to cells compared with

RSM, as has been previously observed (Lian et al. 2002).

Carrier selection has previously been shown to be import-

ant for optimal survival of probiotics during storage

(O’Riordan et al. 2001; Desmond et al. 2002). Inulin has

been implicated as a protective agent in plants during

drought and frost and it directly interacts with membrane

lipids and can stabilize egg phosphatidylcholine during

drying (Hincha et al. 2000; Hincha et al. 2002). However at

37�C, stationary phase Lact. rhamnosus GG survival was

poorest in inulin containing powders, while PD treated

cultures demonstrated better survival. PD may have

provided better protection at 37�C in its capacity as a

humectant (Murphy 2001). However, the presence of all

prebiotics tested offered no improved storage viability

compared with RSM. Best storage survival was obtained in

RSM powders, which was also the best spray-drying

medium in the present study. Previously, Prajapati et al.

(1987) observed that the medium providing optimal post-

spray-drying survival of Lact. acidophilus was the most

efficacious storage medium after 60 days at room tempera-

ture.

In conclusion, we found that the inclusion of the

prebiotics PD or inulin in the feed media did not result

in increased probiotic survival during spray-drying or

powder storage. Probiotic powders harbouring high num-

bers of viable microorganisms (�109 CFU g)1) were gen-

erated when stationary phase cultures were used which

contained RSM in the feed media, but not in the presence

of the prebiotic alone. Furthermore, probiotic cultures

retained good viability during storage in powders contain-

ing RSM/prebiotics at 4 and 15�C, although viability

during storage at 37�C declined rapidly. Powders consisting

of RSM and PD afforded better protection to probiotic

lactobacilli during storage than RSM/inulin combinations.

Storage survival was affected by the phase of growth of the

spray-dried culture with stationary phase best, followed by

lag and log phase. Lact. rhamnosus GG appears to lend itself

well to the spray-drying process and was relatively stable

during powder storage in comparison with other strains of

probiotic lactobacilli studied. Given the broad applicability

of skim milk powders and the health benefits associated

with both prebiotics and probiotics, it is possible that these

powders could be tailored for use in functional food

applications.

ACKNOWLEDGEMENTS

B. Corcoran is in receipt of a Teagasc Walsh Fellowship. We

are grateful to Danisco Sweeteners and ORAFTI Active

Food Ingredients for respectively supplying polydextrose

and inulin products. This work was funded by the Irish

Government under the National Development Plan 2000–

2006, the European Research and Development Fund,

Science Foundation Ireland and by EU Project QLK1-

CT-2000-30042.

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