APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 2280–2282
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0099-2240/06/$08.00⫹0 doi:10.1128/AEM.72.3.2280–2282.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Synbiotic Microcapsules That Enhance Microbial Viability during
Nonrefrigerated Storage and Gastrointestinal Transit
Ross Crittenden,* Rangika Weerakkody, Luz Sanguansri, and MaryAnn Augustin
Preventative Health National Research Flagship, Food Science Australia, Werribee, Victoria 3030, Australia
Received 19 July 2005/Accepted 31 December 2005
A Bifidobacterium infantis strain was microencapsulated within a film-forming protein-carbohydrate-oil
emulsion. This novel encapsulant incorporated prebiotics and substantially protected the bacterium during
nonrefrigerated storage and gastrointestinal transit. The dried microcapsules were small (15 to 20 ␮m), had
low water activity (0.2 to 0.3), and rapidly released the bacteria in simulated intestinal fluid.
Probiotics are live bacteria that are administered in order
to provide a health benefit to the host (6). Bacterial strains
selected as probiotics are predominantly from the genera
Bifidobacterium and Lactobacillus, which are indigenous to the
human gastrointestinal tract (21). The environmental sensitiv-
ities of many potential probiotic strains currently limit their
practical use in nonrefrigerated foods and pharmaceutical-type
supplements. Hence, technologies that can protect the viability
of probiotics during manufacture, storage, and gastrointestinal
transit are highly desired.
Numerous microencapsulation strategies have been exam-
ined for the ability to protect probiotic bacteria from environ-
mental stresses (11, 13), but there remains considerable scope
for the development of microencapsulants that fulfill the many
demands of a successful probiotic encapsulant. These include
protection against oxygen, heat, and other environmental
stresses during drying, formulation, and storage; protection
against low pH and proteases during gastric transit; efficient
release of the bacteria within the gastrointestine; production of
small capsules with suitable sensory properties; and the use of
materials that are inexpensive, stable, and of food grade.
In the current investigation, a Bifidobacterium infantis strain
was microencapsulated within a novel, film-forming protein-car-
bohydrate-lipid emulsion. Nondigestible prebiotic carbohydrates,
which specifically stimulate the growth and/or activity of benefi-
cial populations of bacteria within the gut (3, 12), formed part of
the encapsulant formulation to produce a synbiotic (9) combina-
tion. The capacity of the encapsulant to protect the viability of the
bacteria was evaluated during nonrefrigerated storage and during
simulated gastrointestinal transit.
Bifidobacterium infantis Bb-02 was obtained from C. Hansen
A/S (Hoersholm, Denmark) as a freeze-dried powder contain-
ing 3.2 ⫻ 1010 CFU/g. The freeze-dried bacteria were micro-
encapsulated within a film-forming protein-carbohydrate-oil
emulsion according to the rationale and methodologies de-
scribed by Crittenden et al. (4). Briefly, oil-in-water emulsions
were prepared, containing canola vegetable oil (Crisco; Good-
man Fielder, Australia), caseinate (Alanate 180; New Zealand
* Corresponding author. Mailing address: Preventative Health Na-
tional Research Flagship, Food Science Australia, Private Bag 16,
Werribee, VIC 3030, Australia. Phone: 61-3-9731 3200. Fax: 61-3-9731
3201. E-mail: ross.crittenden@csiro.au.
2280
Vol. 72, No.
Milk Products, New Zealand), and prebiotic fructo-oligosac-
charides (FOS) (Raftilose P95; ORAFTI, Belgium) plus either
dried glucose syrup (DGS) (Maltostar 30; Manildra, Australia)
or microfluidized resistant starch (RS) (Hylon VII; National
Starches). The emulsions were heated to 98°C for 30 min to
promote Maillard reaction products, which improve film-form-
ing and oxygen-scavenging properties (17). The mixture was
then cooled to 10°C before the addition of the probiotic bac-
teria at 8% (wt/wt) prior to spray drying. Both encapsulated
and nonencapsulated B. infantis cells were spray dried using a
Drytec (Tonbridge, United Kingdom) laboratory-scale spray
dryer with an inlet temperature of 160°C and an outlet tem-
perature of 65°C. The final formulation (wt/wt) of the dried
powders was as follows: 8% probiotic, 32% oil, 20% caseinate,
20% FOS, and either 20% DGS or 20% RS.
The water activities of powdered samples were measured after
2 months of storage in sealed aluminum foil pouches. Duplicate
samples were measured using a Decagon CX-2 water activity
meter (Decagon Devices, WA). The particle size distribution of
the microcapsules was measured using a Malvern Mastersizer
2000 instrument (Malvern Instruments Ltd., United Kingdom).
The survival of encapsulated and nonencapsulated B. infantis
cells was assessed during nonrefrigerated storage over a 5-week
period. The bacterial samples were stored in open containers
exposed to an ambient atmosphere maintained at 25°C and 50%
relative humidity. The survival of the bacteria was assessed after
2 and 5 weeks in four repeat experiments.
The ability of the encapsulant to protect B. infantis during
gastrointestinal transit was assessed using a two-stage in vitro
model simulating conditions in the human stomach and small
intestine (22). Details of the model are described by Crit-
tenden et al. (4), but briefly, 1.0-g samples of encapsulated or
nonencapsulated bacteria were mixed with 10 ml of simulated
gastric fluid (SGF) at pH 1.2 and incubated for 2 h at 37°C.
Following incubation in SGF, the pH of the samples was ad-
justed to 6.8, and the bacteria were diluted 10-fold in simulated
intestinal fluid (SIF) at pH 6.8 and incubated for a further 3 h
at 37°C. Viable counts of the bacteria were compared before
and after passage through both stages of the in vitro model.
Survival was assessed in four repeat experiments.
Viable bacteria were enumerated in duplicate samples cul-
tured anaerobically on reinforced clostridial agar for 48 h at
37°C. In cases where bacteria had to first be released from the
VOL. 72, 2006 MICROCAPSULES THAT PROTECT MICROBIAL VIABILITY 2281

FIG. 1. Scanning electron micrograph (magnification, ⫻1,000) of

spray-dried encapsulated Bifidobacterium infantis.

microcapsules, the capsules were dissolved by incubation in

SIF for 1 h at 37°C. The detection limit of the analysis was 3 ⫻ FIG. 2. Survival of Bifidobacterium infantis Bb-02 during storage
for 5 weeks at 25°C and 50% relative humidity. F, nonencapsulated
102 CFU/g of encapsulated material. The statistical signifi-
bacteria; Œ, bacteria encapsulated in caseinate-FOS-oil-DGS emul-
cance of differences between treatments was assessed using sion; E, bacteria encapsulated in caseinate-FOS-oil-RS emulsion. The
one-way analysis of variance and the Tukey test. data represent the means of four experiments. Error bars represent 1
The spray-dried microcapsules produced were small (15 to standard deviation. The survival rates of the encapsulated bacteria
20 ␮m in diameter), with low water activities (0.2 to 0.3). (both formulations) were significantly higher than that of the nonen-
capsulated bacteria (P ⬍ 0.05) after both 2 and 5 weeks.
Scanning electron micrographs of unwashed microencapsu-
lated bacterial powders showed that no free, nonencapsulated
bacteria were present, indicating that the encapsulation effi-
ciency was high (Fig. 1). such as low pH, temperature, and oxygen in comparison to many
Microencapsulation significantly protected the viability of commercial probiotics (2, 16). Encapsulation with alginate, car-
bacteria when they were stored in an open container at 25°C rageenan, and modified starch has generally failed to substan-
and 50% relative humidity (Fig. 2). Similarly, microencapsula- tially improve the survival of acid-sensitive bifidobacteria in
tion significantly protected the bacteria during simulated gas- models simulating the acidic conditions in the stomach (5, 7,
trointestinal transit (Fig. 3). The addition of the encapsulant
materials to nonencapsulated bacteria increased survival in the
gastrointestinal model but afforded substantially less protec-
tion than the formulated microcapsules. Microscopic examina-
tion of the microcapsules showed that the bacteria remained
entrapped within the capsule material in SGF and were re-
leased when transferred to SIF (Fig. 4).
The small diameters of the microcapsules produced in the
current study (⬍20 ␮m) were comparable to those of micro-
capsules produced to protect probiotics using cellulose acetate
phthalate (8) or starch (15). These microcapsules are much
smaller than those produced using gelling materials such as
alginate (7, 13, 19) or gellan and xanthan gums (20) (typically
⬎100 ␮m) and provide advantages with regard to minimizing
adverse impacts on texture and mouth feel when incorporated
into foods (1).
Numerous microencapsulation strategies have been evalu-
ated for the ability to protect probiotic viability during storage
and intestinal transit (11, 13). Unfortunately, it is difficult to
directly compare the degrees of effectiveness of different ap-
proaches due to substantial variations in the durabilities of the
probiotic strains tested and differences in the methods used to FIG. 3. Effect of microencapsulation on survival of Bifidobacterium
challenge the probiotics with environmental conditions. A infantis in an in vitro model simulating gastrointestinal transit. The
encapsulant used was the caseinate-FOS-oil-DGS emulsion. The
strain of B. infantis was selected for the current study since it is data represent the means of four experiments. Error bars represent
a species that is autochthonous to the human gastrointestinal 1 standard deviation. All mean survival rates were significantly
tract and is relatively sensitive to environmental conditions different (P ⬍ 0.01).
2282 CRITTENDEN ET AL.
FIG. 4. Light micrographs (magnification, ⫻1,000) of Gram-stained
microcapsules (caseinate-FOS-oil-DGS emulsion) containing Bifidobacte-
rium infantis after 2 h in SGF at pH 1.2 (A) and following subsequent
transfer to SIF at pH 6.8 for 3 h (B).
10, 14, 15, 18, 19). However, the encapsulant tested in the
current study was highly effective in protecting B. infantis dur-
ing simulated gastrointestinal transit. Additionally, the cap-
sules remained intact in SGF and dissolved rapidly in SIF,
suggesting that the bacteria would be released in the small
intestine. The fact that encapsulated B. infantis was also pro-
tected during nonrefrigerated storage with exposure to oxygen
and humidity is particularly encouraging, as it demonstrates
the potential of this technology to extend the application of
environmentally sensitive probiotics to nonrefrigerated, long-
shelf-life food and pharmaceutical products.
APPL. ENVIRON. MICROBIOL.
We gratefully acknowledge the advice provided by Martin Playne of
Melbourne Biotechnology, Australia, and technical support from Sieh
Ng and Jenny Rusli of Food Science Australia. We thank Christine
Coombs of CSIRO Textile and Fiber Technology, Australia, for pro-
ducing the scanning electron micrograph of the encapsulated bacteria.
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