Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Direct spray drying and microencapsulation of probiotic

Lactobacillus reuteri from slurry fermentation with whey
pel and C. Beermann
M. Jantzen, A. Go
Department of Biotechnology, University of Applied Sciences Fulda, Fulda, Germany

Keywords
bacterial release, bacterial survival,
Lactobacillus reuteri, microencapsulation,
probiotic, spray drying, whey.
Correspondence
Christopher Beermann, Faculty of Food
Technology, Department of Biotechnology,
University of Applied Sciences Fulda, Marquardstrasse 35, Fulda, 36039, Germany.
E-mail: christopher.beermann@lt.hs-fulda.de
2013/0751: received 19 April 2013, revised
10 June 2013 and accepted 20 June 2013
doi:10.1111/jam.12293

Abstract
Aims: Formulations of dietary probiotics have to be robust against process
conditions and have to maintain a sufficient survival rate during gastric transit.
To increase efficiency of the encapsulation process and the viability of applied
bacteria, this study aimed at developing spray drying and encapsulation of
Lactobacillus reuteri with whey directly from slurry fermentation.
Methods and Results: Lactobacillus reuteri was cultivated in watery 20% (w/v)
whey solution with or without 0
5% (w/v) yeast extract supplementation in a
submerged slurry fermentation. Growth enhancement with supplement was
observed. Whey slurry containing c. 109 CFU g 1 bacteria was directly spraydried. Cell counts in achieved products decreased by 2 log cycles after drying
and 1 log cycle during 4 weeks of storage. Encapsulated bacteria were
distinctively released in intestinal milieu. Survival rate of encapsulated bacteria
was 32% higher compared with nonencapsulated ones exposed to artificial
digestive juice.
Conclusions: Probiotic L. reuteri proliferate in slurry fermentation with yeastsupplemented whey and enable a direct spray drying in whey. The resulting
microcapsules remain stable during storage and reveal adequate survival in
simulated gastric juices and a distinct release in intestinal juices.
Significance and Impact of the Study: Exploiting whey as a bacterial substrate
and encapsulation matrix within a coupled fermentation and spray-drying
process offers an efficient option for industrial production of vital probiotics.

Introduction
The Food and Agriculture Organization of the United
Nations and the World Health Organization underline in
their definition of probiotics that living micro-organisms
with health-promoting mechanisms have to be administered in adequate amounts to be effective (FAO and
WHO 2001). Therefore, dietary formulations of probiotics
have to be robust against industrial production processes
in a cost-effective way and should maintain a sufficient
survival rate after oral uptake and gastrointestinal tract
(GIT) transit.
Commonly accepted bacterial counts for dietary products are at least 106 CFU ml 1 (Kailaspathy and Chin
2000). A wide range of health benefits have been
described, such as immune response-modulating properties, improving gut barrier function and protecting effects

of the colon against pathogens (Anal and Singh 2007).

Lactobacillus reuteri strains are probiotic lactic acid bacteria (LAB) regularly applied to milk-related products but
also in meat, fruit and vegetable-based products. Furthermore, L. reuteri has been described to be a robust bacterium feasible for large-scale cultivation techniques and
has high viability during storage and production (Casas
and Dobrogosz 2000).
Probiotic encapsulation is a multi-stage process which
includes fermentation, harvesting, resuspension in matrix
material and encapsulation. To increase process efficiency
and improve the survival of bacteria, this study aimed at
developing a microencapsulation process for L. reuteri by
spray drying directly from slurry fermentation using whey
as a culture substrate and encapsulation matrix. Several
encapsulation procedures for bacteria have been established, predominantly emulsion, extrusion and spray

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Encapsulation of Lactobacillus from fermentation with whey

drying (Anal and Singh 2007; Rokka and Rantamaki

2010). Both extrusion and emulsion mainly generate wet
particles by cross-linking polymer systems which have to
be dried to enable handling and storage afterwards (Cook
et al. 2012).
Encapsulation by spray drying directly achieves optimal
product moisture content, which is between 4 and 7% for
storage stability (Ananta et al. 2005). Other main advantages of spray drying are its low cost and fast production of
large quantities of viable cells (De Castro-Cislaghi et al.
2012). Hereby, predominant factors for bacterial viability
loss in this process are heat, oxygen, as well as mechanical
and osmotic forces (Meng et al. 2008). In contrast, encapsulation with spray drying yields dried powder of small
particle sizes with a sufficient protection for the core (Anal
and Singh 2007; Cook et al. 2012).
Several spray-drying processes for dietary applications
have been established utilizing different polysaccharides
and protein compounds to protect probiotics. The pH
value profile of the gastrointestinal passage ranges from 1
9
to 2
5 in the stomach, which is the most harmful for orally
applied probiotics, up to pH values between 6
15 and 7
88
in the small intestine and ending up with a slightly acidic
pH value in the colon (Cook et al. 2012). Also, digestive
enzymes, such as gastric pepsin and pancreatic trypsin and
chymotrypsin, pancreatin and bile salts are further antibacterial factors (Gbassi et al. 2011; Doherty et al. 2012).
Whey is a cheap cheese by-product containing 50% of
the milk nutrients, predominantly lactose (4
9% of total
whey) and whey protein (0
7% of total whey) with the
main protein fractions b-lactoglobulin and a-lactalbumin.
With its dissimilar nutritional value, whey possesses
different interesting bio- and techno-functional properties. Aside from specific lipids, vitamins, minerals and
high-energetic lactose, whey has been discussed to be an
attractive source of functional proteins and peptides with
distinct amino acid profiles (Smithers 2008). To continue, whey is thermostable and possess excellent gelation,
water binding, emulsification as well as foam forming
properties and foam formation with thermal stability
(Foegeding et al. 2002; Smithers 2008). Recent studies
suggest that whey protein resists against acidic milieus
(Doherty et al. 2012). Gbassi et al. (2009) determined no
damaging effect at pH 1
8. The stability against pepsin is
more complex as a-lactalbumin breaks down in the presence of pepsin, while the main whey protein b-lactoglobulin stays intact (Gbassi et al. 2009).
Considering the physiological and technological potential, whey might be useful as a bacterial substrate and
encapsulation matrix within a coupled fermentation and
spray-drying process to offer an efficient option for
industrial production of vital probiotics. For this, the
growth of L. reuteri in whey was characterized in batch
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M. Jantzen et al.

slurry fermentation process coupled with direct encapsulation of bacteria by spray drying. At last, physical product characteristics and bacterial protective properties
against GIT conditions were investigated in vitro.
Materials and methods
Bacterial strain
Probiotic Lactobacillus reuteri (DSM 20016) was obtained
from the German Collection of Microorganisms and Cell
Cultures Leibnitz Institute DSMZ and cultured aerobically in 9 ml de ManRogosaSharpe (MRS) broth (pH
6
2) at 37C for 24 h.
Growth of Lactobacillus reuteri in whey
To characterize the growth of L. reuteri in whey,
approximately 103 CFU ml 1 bacteria were inoculated in
a 200-ml-volume fermentation of watery 20% (w/v) whey
solution (pH 6
0) with or without 0
5% (w/v) yeast
extract as supplement and cultivated in a agitating flask
culture at 37C. Before inoculation, whey solution was
sterilized in 80C water bath for 20 min. Bacterial cell
count was measured after 0, 24, 48 and 72 h by direct
plate counting as described below.
Batch slurry fermentation of Lactobacillus reuteri in a
continuous stirred-tank reactor (CSTR)
The growth of L. reuteri was observed in a laboratory-scale
reactor (Biostat A, Sartorius Ltd., Melsungen, Germany).
Approximately 104 CFU ml 1 bacteria were inoculated in
1 l batch slurry fermentation of watery 20% (w/v) whey
solution with 0
5% (w/v) yeast extract as supplement,
tested with adjustment at pH 5
0 and cultivated at 37C
with agitation using one six-wing rotating disc at 200 rpm,
respectively. Samples of 5 ml were taken to determine cell
counts after 0, 24, 48 and 72 h by direct plate counting as
described below.
Microencapsulation of Lactobacillus reuteri in whey
For the spray-drying process, a 48-h-incubated 200-ml
slurry culture of L. reuteri with at least 108 CFU ml 1 was
applied. The fermented slurry was spray-dried by a laboratory-scale spray dryer (B
uchi mini spray dryer B190; Flawil,
Switzerland) with two different outlet temperatures maintained at 55  2 and 65  2C and a flow rate of 500 Nl
h 1. Outlet temperatures were reached by adjusting different inlet temperatures (89  1 and 100  1C) and feed
levels from 2 to 4 ml min 1. Spray-dried powder samples
were collected from the cyclone and mixed gently.

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M. Jantzen et al.

Enumeration of surviving cells

To validate the survival rate of bacteria during drying
process, cell counts were observed before and after spray
drying. To detect the viable number of bacteria, direct
plate counting was used. Samples were serially diluted in
Ringers solution (pH 7
0) and plated on MRS agar (pH
6
2). After 48-h incubation at 37C under anaerobic conditions, the cell counts were expressed in CFU g 1.
Spray-dried samples were previously rehydrated in
Ringers solution at a solid content of 20% (w/v), and
the solution was used to determine cell survival. Cell
counts were expressed as mean values of duplicate
measurements.
Moisture content and particle size of spray-dried
powders
The moisture content of spray-dried powders was determined using the moisture analyser (MA 40, Sartorius
AG, Gottingen, Germany) at 80C drying temperature.
Data are expressed as mean value of double-tested 1-g
product.
Particle size was analysed using the Mastersizer 2000
(Malvern Instruments Ltd., Malvern, Worcestershire,
UK). Data are expressed as mean value of triple determinations.
Survival of encapsulated Lactobacillus reuteri during
storage
The spray-dried product was stored in plastic tubes at
4C. Survival of encapsulated L. reuteri during storage
was determined directly following the drying process and
after 1 week and 4 weeks of storage, by performing direct
plate counting as described above.
Survival and release of encapsulated and
nonencapsulated Lactobacillus reuteri in simulated
gastrointestinal conditions
To estimate the tolerance of encapsulated cells to simulated digestive conditions, an assay was adapted by using
a modified version of the method according to Picot
and Lacroix (2004). The cultures were exposed to simulated gastric juice (pH 1
9) and afterwards to simulated
small intestinal juice (pH 7
5) at 37C. Simulated gastric
juice preparation contained pepsin (0
304 g l 1) (porcine gastric mucosa, P7012, Sigma-Aldrich, Taufkirchen,
Germany) dissolved in sterile 0
1 mol l 1 HCl/1 mol l 1
NaOH to adjust pH to 1
9. Simulated pancreatic juice
preparation contained pancreatin (19
5 g l 1) (porcine
pancreas, P1750, Sigma-Aldrich) dissolved in sterile

Encapsulation of Lactobacillus from fermentation with whey

sodium phosphate buffer (0
02 mol l 1, pH 7
5)

adjusted with 0
1 mol l 1 HCl/1 mol l 1 NaOH to pH
7
5. A concentrated bile salt solution contained bile
extract powder (150 g l 1) (bile bovine, B3883, SigmaAldrich) in sterile distilled water.
To examine the survival of encapsulated L. reuteri,
5
0 g of the dried product containing 107 CFU g 1
bacteria was gently mixed with 30 ml of pepsin preparation (0
26 g l 1, 37C) in a 50-ml sterile plastic tube.
The resulting dispersion was incubated at 37C in a
250 rpm shaking thermo element (ThermoMixer and
BlockThermostate, HLC BioTech, Bovenden, Germany).
After 30 min, the reaction was stopped by raising the pH
to 7
5. A sample of 1
5 ml was taken and kept on ice
before viable cell counts were determined. A volume of
2
5 ml of concentrated sodium phosphate buffer
(0
5 mol l 1, pH 7
5) and 1
0 ml of the concentrated
bile salt solution were added (3
33 g l 1). After adjusting
the pH to 7
5 and filling the volume to 40
5 ml with
sterile distilled water, 4
5 ml of the simulated pancreatic
juice (1
95 g l 1) was added to make the final volume of
the tube to 45 ml. At different time intervals (1, 2, 3,
5 h), 1
5-ml aliquots were taken and placed on ice before
bacterial enumeration. Except gentle shaking, no dispersion step was conducted to estimate the release properties of the microcapsules at different time intervals.
For comparison, nonencapsulated bacteria cultured in
MRS broth (37C, 24 h) were washed and resuspended
in sterile 0
85% saline. Five millilitres of cell suspension
containing approximately 106 CFU ml 1 bacteria was
added to 30
0 ml of pepsin preparation (0
26 g l 1,
37C) in a 50-ml sterile plastic tube and maintained at
37C in a shaking thermo element, as described above.
The rest of the procedure was same as that described for
the encapsulated cells.
Enumeration of bacteria from samples taken during
simulated digestion with encapsulated and nonencapsulated cells was carried out by direct plate counting as
described above. The percentage surviving bacteria was
calculated as percentage survival = N/N0 9 100, where
N0 represents the number of bacteria in the inoculum
and N is the viable number of bacteria in the digestion
juice.
Statistical analysis
Data were evaluated by GraphPad 5 Prism software
(GraphPad Software, Inc., La Jolla, CA, USA). P-values
were determined by one-sided nonparametric Wilcoxon
MannWhitney U-test for tolerance to simulated gastric
juice and by two-sided nonparametric WilcoxonMann
Whitney U-test for storage, moisture content and particle
size. P-values 0
05 were defined as significant.

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M. Jantzen et al.

Results

1010

Growth of Lactobacillus reuteri in whey

The initial experiment was accomplished to define the
growth capacity of L. reuteri (DSM 20016) in whey. For
this, a watery 20% (w/v) whey solution with or without
0
5% (w/v) yeast extract as supplement was tested. Under
both culture conditions, the stationary phase of bacterial
growth kinetics of batch culture was reached after 48 h of
fermentation in agitating flask at 37C. After 72-h culturing, the end cell counts in pure whey solution increased 4
log cycles, whereas the bacterial counts increased 5 log
cycles by supplementing 0
5% yeast extract to whey.

CFU g1

108
106
104
102
100
Before
After
Process

7 days
28 days
Storage

Batch slurry fermentation of Lactobacillus reuteri in a

CSTR

Figure. 1 Cell counts of Lactobacillus reuteri before and after spraydrying process with (
) 55C and (
) 65C outlet temperatures
and during storage for 7 and 28 days at 4C storage temperature.
The data shown represent the mean of at least three measurements.

Yeast extractsupplemented whey solution was tested

using batch slurry fermentation in a CSTR. As sufficient
agitation of whey slurry was reached at 200 rpm, stationary growth phase was reached after 24 h of cultivation
with 1
7 9 109 CFU g 1 bacteria.

Table 1 Moisture content and particle size of spray-dried powder

directly from slurry fermentation in whey 20% (w/v) with 0
5% (w/v)
yeast extract supplementation

Survival of Lactobacillus reuteri in whey encapsulation

after spray drying

Process outlet temperature (C)

Moisture (%)

Particle size (lm)

55
65

6
1  0
8
6
8  0
3

5
5  0
3
4
9  0
3

Results for 55 and 65C are the mean of triplicate trials.

Lactobacillus reuteri was spray-dried at 55 and 65C temperatures using the whole slurry fermentation with whey
directly as feed. Loss of viable bacteria after spray drying
is illustrated in Fig. 1. For all experiments, whey solutions
with 1
6 ( 1
5) x 109 CFU g 1 bacteria were applied to
the encapsulation process. Dried product contained 2
5
( 1
7) x 107 CFU g 1 bacteria directly after processing
independently from used outlet temperatures.

To describe product stability during storage at 4C,

survival of encapsulated bacteria was determined after
1 week and 4 weeks. The bacterial counts of the produced capsules decreased by 1 log cycle after a storage
period for 4 weeks (Fig. 1).

Characterization of encapsulated particles

Survival and release of encapsulated and

nonencapsulated Lactobacillus reuteri in simulated
gastrointestinal conditions

To compare physical characteristics, moisture and particle

size of the spray-dried products were determined
(Table 1). The moisture content of dehydrated products
is of importance for product and bacterial stability while
storage. Optimal moisture content is between 4 and 7%
for storage (Ananta et al. 2005). Powder manufactured in
this study had a mean moisture content of about 6
5
(0
7)%. There is no significant difference in moisture
content of the powder produced at 55 or 65C outlet
temperature of the process. The obtained particles had a
mean diameter of 5
5 (0
3) lm at 55C and 4
9 (0
3)
lm at 65C again with insignificant differences with
regard to the outlet temperatures. Achieved size is big
enough to encapsulate rod-shaped L. reuteri cells with
about 2 lm length (Muthukumarasamy et al. 2006).

To ensure the physio-functional benefits of L. reuteri, a

sufficient bacterial survival rate along the stomach and
intestine passage is essential to enable probiotic colonization of the colon. Figure 2 examines the survival of free
and encapsulated cells after exposure to artificial digestive
juices, first 30 min in simulated gastric juice and afterwards in simulated intestinal juice for a maximum of 5 h
in vitro.
Inoculation in artificial gastric juice resulted in a
decline in viable cell counts of L. reuteri. Observed cell
counts after 30 min were 12% of the applied bacteria for
free cells and about 26% for encapsulated ones. After
changing to simulated intestinal juice, the number of viable cell counts for nonencapsulated bacteria remained
constant. In contrast, cell counts for encapsulated ones

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M. Jantzen et al.

Encapsulation of Lactobacillus from fermentation with whey

Gastric juice intestinal juice

Survival and release %

100
80
60
40
20
0
0

05

Figure. 2 Percentage release and survival of () encapsulated and ()

nonencapsulated Lactobacillus reuteri in simulated gastric juice for
30 min and afterwards in simulated intestinal juice for a maximum of
5 h. The data shown represent the mean of at least three measurements.

increased during exposure to intestinal juice due to a

distinct release of encapsulated bacteria reaching a survival rate of 54% after 3 h. Thus, after 5 h in digestive
juices, nonencapsulated L. reuteri revealed 86% loss of
living cells, whereas processed bacteria revealed a loss of
54% under simulated gastrointestinal conditions. Therefore, survival rate of L. reuteri increased approximately
32% with encapsulation in whey matrix compared with
nonprocessed ones.
The statistical analysis testified the difference between
the reduction in cell counts of free and encapsulated cells
to be significant. Results show an improved survival rate
of L. reuteri in encapsulated forms.
Discussion
Aside lactose, vitamins and several minerals, whey contains a complex protein composition predominated by
b-lactoglobulin and a-lactalbumin. These proteins possess
a distinct amino acid profile and are a significant nitrogen source for bacteria, whereas whey lactose is a relevant
carbohydrate source. In this study, the nutritive value of
whey and the distinct characteristics of solubility, denaturation, dissociation and aggregation dependent on pH
value and temperature of these components were used to
cultivate probiotic L. reuteri (DSM 20016) for a coupled
encapsulation process within this matrix by spray drying
instead of harvesting the bacteria out of the fermentation
medium and resuspending in encapsulation material. In
this study, L. reuteri reached cell counts of 2
2 9
108 CFU g 1 in pure whey. On the one hand, bacterial
b-galactosidase activity is necessary to metabolize lactose,
which is generally shown by L. reuteri (Hidalgo-Morales
et al. 2011). Most LABs are able to digest whey due to
the high proteolytic activity (Pescuma et al. 2012).

However, L. reuteri possesses low proteolytic activity

(Hidalgo-Morales et al. 2011), and therefore, supplementation with extra nitrogen sources might improve growth.
Parente and Zottola (1991) suggested supporting LAB
cultivation in native whey with nutritive complex additives such as yeast extract. Furthermore, an increased
whey protein concentration to raise nitrogen source for
the bacteria has been described to be helpful (Bury et al.
1998). In this study, the bacterial growth could be
improved to maximum cell counts of up to 2
7 9
109 CFU g 1 with yeast extract supplementation.
For encapsulation, bacteria in stationary phase with
reduced proliferation rate, less physiological activity and
increased resistance to stress seem to be most appropriate for spray drying. Corcoran et al. (2004) revealed
approximately 50% higher survival rates of L. reuteri
within spray-drying processes if bacteria were taken
from stationary-phase cultures. Further, for spray-drying
processes, matrices with 20% solid content are recommended (Desmond et al. 2001; Ananta et al. 2005). In
consequence, to couple cultivation and spray-drying process, bacterial growth up to stationary phase should be
possible in 20% whey slurry fermentation. In this study,
L. reuteri reached the stationary phase at least after 24 h
in a slurry fermentation in CSTR.
Generally, spray drying is an effective way to produce
probiotic encapsulation with high survival rates and
improved resistance during gastric transit (Malmo et al.
2011; Paez et al. 2012). One critical factor for bacterial
survival within the drying process is the exposure to hot
air which leads to heat and osmotic stress for the cells.
Otherwise, high temperatures are required to facilitate
sufficient water evaporation along the process as dried
product contains at best moisture of 4 and 7% for good
stability of dried implementations throughout storage
(Ananta et al. 2005). Hereby, heat sensitivity of the bacterial strain and hygroscopic properties of the encapsulation material are important factors (Ananta et al. 2005;
Golowczyc et al. 2011). Survival of heat-sensitive L. paracasei during spray drying ranged from 97% (outlet
temperature 7075C) to 0% (outlet temperature 120C)
(Gardiner et al. 2000). Other Lactobacilli strains spraydried in skim milk at 85C outlet temperature reached
higher survival rates (loss of 0
160
95) (Paez et al.
2012). Malmo et al. (2011) revealed a loss of 1
63
1 log
of L. reuteri (DSM 17938) after spray drying with alginate
matrix at 72C outlet temperature. Higher survival rates
of spray-dried L. reuteri (KUB-AC5) were measured at
70C outlet temperature with a variation of 8393% survival depending on the growth media (Hamsupo et al.
2005). Taking the distinct heat sensitivity of bacteria into
consideration, the viability can be enhanced by lowering
the outlet temperature (Lian et al. 2002; Meng et al.

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Encapsulation of Lactobacillus from fermentation with whey

2008). In this study, both outlet temperatures 55 and

65C lead to sufficient dry powder with a negligible viable
cell decrease of 2 log cycles (2
13% mean survival rate)
after spray drying due to dehydration and thermal
damages of bacterial cell structures (Fig. 1).
Although small-sized particles are less affecting textural
and sensorial food qualities, the particle size must at least
enable total bacterial encapsulation (Chen and Subirade
2006). In this study, the process produced small particles
sized 5 lm in mean (Table 1), which are large enough to
encapsulate L. reuteri cells (Anal and Singh 2007). Generally, decreasing moisture content correlates with an
increasing temperature in the drying process (Lian et al.
2002; Anandharamakrishnan et al. 2008). In this study,
however, neither particle size nor moisture content of
capsule parameters were significantly affected by chosen
outlet temperatures of the drying process. Desmond et al.
(2001) described the possibility that milk proteins are
useful to protect probiotic cultures during heating. Furthermore, lactose from whey protein might synergistically
improve the micro-organism-protecting properties of
these proteins. Young et al. (1993) discovered synergistic
effect of whey proteins and carbohydrates for microencapsulation by spray drying. They described higher
microencapsulation efficiency using the combination of
whey protein and carbohydrates. Additionally, an
increased thermotolerance of LAB resulting in high
survival rates during storage was observed if growth
media contained lactose (Carvalho et al. 2004). The protection of dehydrated biomaterials by sugars is mainly
due to hydrogen bonds with the proteins when water is
removed (Rokka and Rantamaki 2010). And lactose has
been discussed to stabilize the bacterial cell wall by inducing a crusty glass phase (Rosenberg and Sheu 1996). Also,
Rokka and Rantamaki (2010) underlined that skim milk
and lactose, often in combination with milk proteins, are
widely used as protective agents within spray-drying
processes.
As dietary products contain at least 106 CFU ml 1 bacteria, the survival of probiotics during storage is important to assess product applicability (Kailaspathy and Chin
2000). Different studies have illustrated that the bacterial
survival rate was most sufficient at 4C storage temperature (Corcoran et al. 2004; Silva et al. 2011). In this
study, the examined loss in viable cells was at maximum
1 log cycle after a storage period of 4 weeks at 4C
(Fig. 1). Depending on the outlet temperature, the viable
counts during storage decreased at 65C by about 1 log
cycle and at 55C by <1 log cycle, which might be due to
physiological heat stress response and disturbed metabolic
mechanisms. Also, Hamsupo et al. (2005) obtained an
approximately decreased survival rate of 1 log cycle at
4C storage for 2 months with skim milk spray-dried
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M. Jantzen et al.

L. reuteri (KUB-05). Even for sensitive, strictly anaerobic

bifidobacteria, whey possesses excellent cell-protective
properties at 4C storage (De Castro-Cislaghi et al. 2012).
To fulfil all health beneficial functions, probiotic bacteria have to reach the terminal ileum and colon alive. The
highly acidic stomach and bile salts secreted into the
duodenum harm the ingested bacteria (De CastroCislaghi et al. 2012). Whey proteins remain stable at
acidic pH due to aggregation, whereas after isoelectric
point (pH 4
6), solubility improves with increasing pH
(Pelegrine and Gasparetto 2005; Gbassi et al. 2009; Dissanayake et al. 2013). Recent studies already have shown
that whey proteinbased microencapsulation protects
bacteria against acid stress, allowing the cells to survive in
the stomach and improve cell survival of probiotic LAB
in the human GIT (Rokka and Rantamaki 2010; De Castro-Cislaghi et al. 2012). In this study, the survival rate of
whey-microencapsulated L. reuteri in simulated gastric
juices is 32% higher compared with nonencapsulated
ones (Fig. 2). In contrast, no significant increase in
survival of calcium alginateencapsulated L. reuteri under
simulated gastric conditions was found by Sultana et al.
(2000) and Malmo et al. (2011).
Interestingly, the lactose in whey protein again seems
to support this protective effect synergistically. L
opezRubio et al. (2012) underlined that bacteria encapsulated
with carbohydrate only were less protected compared
with whey proteinencapsulated ones. Different combinations of alginate with starch for extrusion and emulsion
revealed increased survival of microencapsulated L. reuteri
strains in simulated gastric juice (2 h, pH 1
5) compared
with free cells (Muthukumarasamy et al. 2006). In
contrast, Lactobacilli microencapsulated in alginate and
pectin were not protected against acidic milieu (Brinques
and Ayub 2011).
Aside from stomach and bile acids, digestive enzymes
of the duodenum are relevant. Doherty et al. (2012) have
demonstrated that whey protein capsules are pepsin resistant. Whereas b-lactoglobulin is not digestible by gastric
pepsin, distinct peptide chains of whey a-lactalbumin and
bovine serum albumin are degraded (Gbassi et al. 2009).
In this study, neither did free nor did encapsulated bacteria reveal significant loss of living cells after 5-h incubation in artificial intestinal juice. These results are in
accordance with a study by Muthukumarasamy et al.
(2006), which demonstrated that several strains of L. reuteri survived in bile juice for several hours. Dommels
et al. (2009) also reported the resistance of L. reuteri to
bovine bile.
However, increased cell counts of encapsulated L. reuteri during incubation in artificial intestinal juice revealed
a release of encapsulated bacteria due to a pH value shift
from 1
9 of gastric juice to 7
5 of intestinal juice and

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M. Jantzen et al.

probably due to whey protein degradation by pancreatin

(Fig. 2). Accordingly, Chen and Subirade (2006)
described a release of whey protein capsule core material
into simulated intestinal juice with pancreatin.
In conclusion, whey exploited as growth substrate and
encapsulation matrix within a coupled fermentation and
spray-drying process might offer an efficient option for
industrial production of vital probiotics. The resulting
microcapsules remain stable during storage and reveal adequate survival in simulated gastric juices and a distinct
release in intestinal juices. While lactose is supportive but
also problematic in this coupled process, like unwanted
caramelization or stickiness to the dryer wall, the influence
of the whey-protein-to-lactose ratio on the process and
product characteristics remains to be investigated.
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Anandharamakrishnan, C., Rielly, C. and Stapley, A. (2008)
Loss of solubility of a-lactalbumin and b-lactoglobulin
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Ananta, E., Volkert, M. and Knorr, D. (2005) Cellular injuries
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