LWT - Food Science and Technology 62 (2015) 1162e1168

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LWT - Food Science and Technology

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Lactobacillus acidophilus La-05 encapsulated by spray drying: Effect of

mucilage and protein from flaxseed (Linum usitatissimum L.)
Mariela Bustamante a, *, Mario Villarroel a, Mo

nica Rubilar a, Carolina Shene a, b
a
Center of Food Biotechnology and Bioseparations, Scientific and Technological Bioresource Nucleus, BIOREN, and Department of Chemical Engineering,
Universidad de La Frontera, Ave. Francisco Salazar 01145, Box 54-D, Temuco, Chile
b
Centre for Biotechnology and Bioengineering (CeBiB), Universidad de La Frontera, Temuco, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Flaxseed mucilage (FM) and soluble protein (FSP) were used as wall materials for encapsulating Lacto-
Received 2 August 2014 bacillus acidophilus La-05 by spray drying. The effects of the content of FM in the encapsulating solution
Received in revised form and drying temperature on the survival of bacterium after drying and its viability during storage were
9 February 2015
evaluated. Optimal conditions for maximizing the survival of L. acidophilus (78%) predicted by the surface
Accepted 14 February 2015
Available online 21 February 2015
response methodology were 0.2% w/v of FM and 110
C. These encapsulating conditions permitted
almost a 2-fold increase of viability compared to the product encapsulated without FM. A further in-
crease in survival (90%) was obtained by supplementing the encapsulating solution with heat-treated
Keywords:
Microencapsulation
FSP. Moreover, the encapsulation of L. acidophilus enhanced its viability during incubation in simu-
Spray drying lated gastric acid and bile solutions.
Lactobacillus acidophilus © 2015 Elsevier Ltd. All rights reserved.
Flaxseed mucilage
Flaxseed soluble protein

1. Introduction rates. Nevertheless, the elevated temperatures used in spray drying

The consumers' interest in foods that confer health benefits has
led, in recent decades, to the growth of the probiotic product
market. Probiotics are defined as “live microorganisms which when
administered in adequate amounts confer a health benefit to the
host” (FAO/WHO, 2001). The species most used in functional foods
belong to the lactobacilli and bifidobacteria genera (Holzapfel,
Haberer, Geisen, Bjo€ rkroth, & Schillinger, 2001). In the develop-
ment of fermented dairy products the final cell content is a function
of fermentation conditions. However, the addition of probiotics in
non fermented products such as chocolate, probiotic capsules,
tablets, and chewables requires stages of fermentation, harvesting
and stabilization. Stabilization stages, in which moisture is
removed by hot air, are critical because process conditions deter-
mine cell survival. Several studies have established that microen-
capsulation can improve survival and viability of probiotic (Ann
et al., 2007; de Lara Pedroso, Thomazini, Jorda ~o Barrozo
Heinemann, & Favaro-Trindale, 2012; McMaster, Kokott, & Slatter,
2005). Spray drying is widely used in the food industry because it is
easily scaled-up, offers low operating cost and high production
* Corresponding author. Tel.: þ56 45 2325491; fax: þ56 45 2732402.
E-mail address: mariela.bustamante@ufrontera.cl (M. Bustamante).
can be stressful to cells affecting their survival (Boza, Barbin, &
Scamparini, 2004). The fast rate of water removal may induce
irreversible changes in the functional integrity of bacterial mem-
branes and proteins (Crowe, Crowe, Carpenter, & Aurell-Wistrom,
1987).
Several studies have shown that the composition and concen-
tration of the solids in the encapsulating solution can improve
viability of probiotics (Ann et al., 2007; Corcoran, Ross, Fitzgerald, &
Stanton, 2004; Fritzen-Freire, Prude ^ncio, Pinto, Mun
~ oz, & Amboni,
2013). Enhancement of survival and viability is explained because
cell encapsulation into a solid matrix offers protection not only
during drying but also during storage. Moreover, encapsulation of
probiotics might also improve viability during gastric transit. It has
been suggested that the use of prebiotics, as encapsulating material
could improve the survival of probiotics during spray drying. Pre-
biotics that have been used as encapsulating materials include
polydextrose (Corcoran et al., 2004), inulin and oligofructose
(Fritzen-Freire et al., 2013), lactulose and raffinose (Ann et al.,
2007). Today, research is directed to find alternatives having
similar or better characteristics.
Flaxseed (Linum usitatissimum L.), also known as linseed, is a
source of bioactive phenolic compounds, polyunsaturated fatty
acids, dietary fiber and proteins (Bozan & Temelli, 2008; Oomah,

http://dx.doi.org/10.1016/j.lwt.2015.02.017
0023-6438/© 2015 Elsevier Ltd. All rights reserved.
 M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168
2001). Bioactivity of flaxseed proteins includes the control of blood
glucose level stimulating insulin secretion (Nuttall, Mooradian,
Gannon, Billington, & Krezowski, 1984). The flaxseed mucilage
(FM) contains a mixture of rhamnogalacturonan I and arabinox-
ylan; prebiotic properties of arabinoxylan has been described
(Naran, Chen, & Carpita, 2008; Pastell, Westermann, Meyer,
Tuomainen, & Tenkanen, 2009; Sørensen, Pedersen, & Meyer,
2006). Besides, the intake of FM has been suggested for the
reduction blood glucose and cholesterol in diabetics (Thakur, Mitra,
Pal, & Rousseau, 2009), the increase of fecal fat excretion in human
(Kristensen et al., 2012), and for suppressing postprandial lipemia
and hunger in young men (Kristensen et al., 2013). Nevertheless, in
spite the bioactive properties of the mucilage and protein of flax-
seed, its use in the food industry is limited.
The aim of this work is to evaluate FM and soluble protein from
flaxseed (FSP) as wall materials for the spray drying encapsulation
of Lactobacillus acidophilus La-05. Survival of the lactic acid bacte-
rium (LAB) after spray drying and viability of the encapsulated
bacterium during storage at 4
C were evaluated. The viability of
encapsulated and free LAB in simulated gastric juice and bile so-
lution was determined. Morphology of the encapsulated products
was investigated. The impact of FM on the growth of the LAB was
also evaluated.
2. Materials & methods
2.1. Strains, chemicals, and culture media
L. acidophilus La-05 (La-05®) was from Chr. Hansen, (Ho

nsholm,
Denmark). Malt dextrin (MD) of food grade with a dextrose
equivalent (DE) of 15 was purchased from PRINAL®. Flaxseeds were
purchased in the local market. Pepsin (0.7 FIPeU mg1) was ob-
tained from Merck (Germany). Bile bovine was obtained from
Sigma (USA). Lactobacilli broth (MRS broth, de Man, Rogosa, &
Sharpe, 1960) and agar were from Difco (USA). All the chemicals
were of analytical grade.
2.2. Extraction of FM and FSP
The FM was extracted according to the Esparza, Leyton, Rubilar,
and Shene (2011). Briefly, seeds were mixed (30 min) with hot
distilled water (90e95
C), pH 5.0 at a ratio 1:10 w/v; the extraction
cycle was performed twice. The extract was spread on a tray and
dried at 60
C in an air convection heat oven. Mucilage-free seeds
were dried (60
C), milled and passed through a 0.5 mm mesh. The
meal was defatted with hexane twice (ratio 1:10, w/v) in a shaker
(160 rpm, 8 h at 10
C). The defatted meal was recovered by
filtration (Whatman® N
1) and the solvent was evaporated. The
FSP was extracted from the defatted meal with 0.10 M NaCl in
0.10 M Tris buffer at pH 8.6 (ratio 1:16 w/v) (Li-Chan & Ma, 2002).
The extraction was carried out in a shaker at 160 rpm for 16 h at
4
C. The extract was passed through a double layer of cheesecloth
and centrifuged (7000  g, 4
C, 20 min). The supernatant was dried
(60
C), milled and stored at 20
C until use. For the drying ex-
periments the FSP was dispersed in distilled water and heated at
90
C for 10 min to destroy microorganisms and to allow protein
denaturation and aggregation (Kiokias, Dimakou, & Oreopoulou,
2007). The chemical composition of FM and FSP were determined
following the AOAC methods (AOAC, 1995).
2.3. Bacterial culture preparation
The LAB was sub-cultured twice with 5% v/v inoculum in 5 mL of
MRS broth (37
C, 12 h). For the spray drying assays 200 mL of MRS
broth were inoculated (5% v/v) with the grown culture and
1163
incubated under the same conditions. Cells in late-log phase were
harvested by centrifuging (5000  g, 4
C, 5 min); the pellet was
washed with sterile distilled water and re-suspended in sterile
distilled water.
2.4. Effect of FM on the growth of the LAB
The MRS broth (5 mL) without glucose was supplemented with
FM to a final concentration of 0.2 and 1% w/v. After sterilization
(121
C, 15 min) the medium was inoculated (5% v/v) with the LAB
from a second sub-culture and incubated at 37
C for 24 h. The
growth was followed at 600 nm after proper dilution. Titratable
acidity (TA) was determined (AOAC, 1995) and expressed as percent
of lactic acid. Acid production after 24 h of incubation was deter-
mined by HPLC. The assay was performed in triplicate.
2.5. Spray drying
Drying runs were performed in a laboratory spray dryer unit
(LabPlant SD-05 dryer, Huddersfield, England) equipped with a
1.5 mm diameter nozzle, a spray chamber (500 mm length, 215 mm
height) and a peristaltic pump. Bacterial suspensions (total viable
counts between 108 and 109 CFU mL1) fed to the spray dryer
contained MD (15% w/v) and different concentrations (0, 0.1 and
0.2% w/v) of FM and FSP (2, 6, and 12% w/v). Feeding rate was
6 g min1. The dry powder was collected in sterile glass bottles
attached to the bottom of the cyclone, cooled to room temperature
and stored at 4
C until their characterization. The experimental
design corresponded to the matrix of response surface methodol-
ogy (RSM) for two variables at two levels (Table 1). Each drying run
was carried out in duplicate except for the central point which was
made in triplicate. Viability of the encapsulated LAB during the
storage at 4
C was followed for 45 days.
2.6. Viability of the free and encapsulated LAB in simulated gastric
juice and bile solution
The methodology described by Rajam, Karthik, Parthasarathi,
Joseph, and Anandharamakrishnan (2012) with modifications was
used. The simulated gastric juice was prepared in MRS broth
adjusting the pH to 2.0 with sterile 1 M HCl. Pepsin solution was
filtered through a 0.2 mm sterile membrane and added to the acidic
MRS broth to reach a final concentration of 0.3% v/v. Bile tolerance
was evaluated in MRS broth containing bovine bile (2% v/v). In both
assays 0.1 g of the encapsulated LAB or 0.1 mL of LAB suspension
were added to 4.9 mL of sterile simulated gastric juice or bile so-
lution. Viable cell count was determined in samples taken imme-
diately after mixing and every 2 h during the incubation at 37
C.
Results were expressed as relative viability given by the ratio be-
tween viable cells at time “t” and that at time zero.
2.7. Analysis
Concentrations of butyric, lactic, acetic, pyruvic and formic acids
in culture samples (filtered under 0.2 mm) were determined by
HPLC, using a Shodex KC-811 (Showa Denko KK, Tokyo, Japan)
column kept at 40
C in an Alliance Waters e2695 Separation
Module (Waters Inc, Mass, USA), detection was made at 210 nm
(Waters Inc, Mass, USA). The sample (10 mL) was eluted with
phosphoric acid 0.1% (v/v) at a rate of 1 mL min1. Calibration
curves were constructed using acetic acid, formic acid, propionic
acid, sodium butyrate, and pyruvic acid sodium salt (Merck, Ger-
many) and L-(þ)-lactic acid (Scharlau Chemie S.A.).
Survival after drying and viability during storage of the LAB
were determined by the standard plate count method. Briefly, the
1164 M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168

Table 1
Effect of the flaxseed mucilage (FM) content in the encapsulating solution and drying inlet air temperature (T) on the survival of L. acidophilus La-05, logarithmic reduction of
viable cells (LRVC), and viscosity of encapsulating solution (m). Response given by the lineal model derived from the experimental results is also shown.

Independent variables ma (mPa s) Experimental survivalb (%) Predicted survival (%) Student residual LRVC Log10 (N0/N)

FM (% w/v) T (
C)

0.0 110 3.61 ± 0.01 69.84 ± 1.40 69.04 0.49 2.96 ± 0.12
125 62.37 ± 1.73 60.32 1.11 3.69 ± 0.10
140 47.12 ± 3.33 51.61 2.75 5.07 ± 0.12
0.1 110 5.48 ± 0.11 73.05 ± 3.88 74.61 0.84 2.62 ± 0.35
125 67.02 ± 2.57 65.89 0.55 3.09 ± 0.50
140 58.62 ± 0.63 57.18 0.78 4.02 ± 0.10
0.2 110 7.87 ± 0.03 78.43 ± 0.29 80.18 1.07 1.92 ± 0.08
125 71.02 ± 0.19 71.46 0.24 2.71 ± 0.00
140 63.30 ± 4.75 62.75 0.34 3.57 ± 0.42
a
Mean values of four experiments.
b
Mean values of two replicates, except in central point in which were three replicates.

sample (0.1 g) was diluted in sterile buffered peptone water 0.1% w/
v (4.9 mL); the suspension was kept at 4
C for 30 min to release the
cells. The appropriate dilution of the suspension was seeded on
MRS agar and incubated at 37
C for 48 h. Viability of LAB during
storage was expressed as colony forming units per gram (CFU g1)
of dry power. The results were expressed as Log10 CFU g1. The
percent survival after drying was calculated from (Simpson,
Stanton, Fitzgerald, & Ross, 2005):
 
N was able to grow in medium containing FM as carbon source, a high
Survivalð%Þ ¼  100
N0
where, N is Log10 CFU per g of the spray-dried powder immediately
after drying and N0 is Log10 CFU per g of dry matter in the sus-
pension fed to the dryer.
Reduction of viable cells with respect to counts after drying was
calculated from (Boza et al., 2004):
 
N0
Logarithmic reduction of viable cells ðLRVCÞ ¼ Log10
N bacteria; these can act as antimicrobials displacing pathogenic
where, N0 is Log10 CFU per g of dry matter in the suspension fed to
the dryer and N is Log10 CFU per g of the spray-dried powder.
The viscosity of feed solutions was determined at 20
C using an
AND Vibro Viscometer SV-10 (A&D Company, Limited, Japan).
2.8. Physical properties of the spray dried product
The outer structure of dried particles was observed with a
scanning electron microscope (SEM). The dry particles containing
L. acidophilus La-05 were stored at 4
C until microscopic analysis.
Then, the samples were dispersed over the sample holder which
was equipped with a double sided carbon tape. The dried particles
were gold-coated with Edward S150 Sputter Coater. Morphology
was observed with a microscope JEOL JSM-6380LV (Jeol, Tokyo,
Japan) at 10 kV and filament current at 50 mA. The particle size of
dried particles was measured by laser diffraction (Zetasizer model
Nano-ZS90, Malvern, UK) at 25
C using isopropyl as the continuous
phase.
2.9. Statistics
Statistical analysis was carried out using the Design Expert 6.0
statistical software (Stat-Ease, Minneapolis, Mn, USA). Analysis of
variance (ANOVA) was used to determine significance of the effects
(P < 0.05). Differences between means were detected using Duncan
test (SPSS version 15.0).
3. Results and discussion
3.1. Effect of FM on the growth of the LAB
Changes in growth, pH, and TA during fermentation of MRS
broth supplemented with FM (0.2 and 1% w/v) as the carbon source
are shown in Fig. 1. Main component in FM was the non-nitrogen
extract (67.37%); contents of other components were 14.89% pro-
tein, 0.75% lipids, 2.50% fiber and 11.36% ash. Even though the LAB
cell concentration was obtained at 0.2% w/v FM; the slow growth of
the LAB could be due to the increase of medium viscosity when FM
was tested at 1% w/v. It has been shown that FM has prebiotic ac-
tivity increasing the concentration of L. acidophilus and Bifido-
bacterium lactis when kefir formulations were prepared with 2 g of
FM per liter of milk (HadiNezhad, Duc, Fong Han, & Hosseinian,
2013). The pH decrease was explained by the synthesis of lactic,
formic, and propionic acids (2.09, 1.13, and 0.11 g L1, respectively).
Production of organic acids is one of the properties of probiotic
bacteria (Gibson, Probert, Van Loo, Rastall, & Roberfroid, 2004).
3.2. Effect of FM on the survival of the LAB after spray drying and
viability during storage
The effect of FM as a component of the encapsulating solution
on the survival of the LAB after spray drying was tested at different
inlet air temperature. The RSM was used to determine the sig-
nificance of the effects on the survival of the LAB after drying; the
results obtained in the 11 drying runs are shown in Table 1.
Because FM exerted an important increase on the viscosity of the
encapsulating solution (3.61 mPa s and 7.87 mPa s for FM con-
centration of 0 and 0.2% w/v, respectively) this was tested in a
small range (0.1e0.2% w/v). A high viscosity of the solution fed to
spray drying is not desirable because the size of the droplets in-
creases demanding higher air inlet temperatures. The highest LAB
survival (78.43%) was obtained with FM at 0.2% w/v and the
lowest inlet air temperature (110
C) (run 7, Table 1). Even though
drying at 140
C significantly reduced the survival of the LAB, FM
reduces this effect being this concentration dependent; compared
with the control (without FM) addition of FM at 0.2% w/v
increased 34% the survival of the LAB while FM at 0.1% w/v
increased the survival by 24%. The lineal relationship for the sur-
vival in terms of FM concentration and inlet air temperature (T)
that fits the experimental data is given by:
Sð%Þ ¼ 65:89 þ 5:57 FM  8:71 T r2 ¼ 0:94
 M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168 1165

Fig. 1. Effect of flaxseed mucilage (FM) added to MRS broth on growth of L. acidophilus La-05. (a) Cell concentration (Log10 CFU mL1), (b) pH, and (c) titratable acidity. Concentration
of FM (B) 0.2% w/v and (C) 1% w/v.

The model has predictive capability deduced from the signal to absorption (Naran et al., 2008). These properties could promote the
noise ratio (higher than 4), the small residual value and Student survival of LAB during drying.
residual (Table 1). The ANOVA (Table 2) showed that both factors
significantly contributed (P < 0.05) to the predicted survival of the
3.3. Effect of the FSP on the survival of the LAB after spray drying
LAB after drying. The response surface plot (Fig. 2) shows that the
survival of the LAB increased as the inlet air temperature decreased
Main component of FSP was protein 44.98%; contents of other
from 140
C to 110
C and the concentration of FM increased from
components were 2.33% lipid, 0.96% fiber, 14.04% ash and 36.77%
0.0% to 0.2%.
non-nitrogen extract. The effect of FSP at concentrations of 2, 6, and
Viability of the LAB encapsulated with or without FM decreased
12% w/v in the encapsulating solution on the survival of LAB after
gradually during storage at 4
C (Fig. 3). However, after one day of
spray drying was evaluated by replacing partially MD (Table 3);
drying, viability of the LAB encapsulated with 0.2% w/v of FM and
drying temperature and FM in the encapsulating solution, were
dried at 110
C (Fig. 3c) was higher than the control (without FM
110
C and 0.2% w/v, respectively. Survival of the LAB was signifi-
Fig. 3a) and with 0.1% w/v of FM (Fig. 3b). After 45 days the count of
cantly (P < 0.05) affected by FSP. Relatively low survivals were
viable LAB in the products encapsulated with 0.1 and 0.2% w/v of
obtained with 0% (78.51%) and 2% (79.00%) w/v of FSP in the
FM and dried at 110
C was 6.24 and 6.84 Log CFU g1, respectively.
These values are higher than the levels recommended
(6 Log CFU g1) for exerting health benefits to the consumer (Roy,
2005).
The enhanced survival and viability of the LAB encapsulated
with FM at 0.2% w/v suggests that this flaxseed component would
act as thermoprotector of cells undergoing the drying process. The
functions of seed mucilage are still matters of speculation. It has
been proposed that seed mucilage facilitate seed hydration or
improve resistance to desiccation during brief drought after

Table 2
ANOVA for the overall effects of flaxseed mucilage (FM) concentration in the
encapsulating solution and drying inlet air temperature (T) on the survival of the
L. acidophilus La-05 after spray drying.

Factors DFa Mean square Fexpb P>F

FM 1 186.15 40.35 0.0002

T 1 455.53 98.75 <0.0001
Fig. 2. Response surface plot showing the effects of the flaxseed mucilage (FM) con-
a
DF ¼ Degree of freedom. tent in the encapsulating material and inlet air temperature (T) on the percent survival
b
Fexp ¼ Fisher ratio. Test for comparing model variance with residual variance. of L. acidophilus La-05 after spray drying.
1166 M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168
Fig. 3. Viability during storage at 4
C of L. acidophilus La-05 encapsulated by spray drying with different concentrations of flaxseed mucilage in the encapsulating solution, (a)
control without the flaxseed mucilage (FM), (b) 0.1% w/v FM, and (c) 0.2% w/v FM. Inlet air temperature (C) 110
C, (:) 125
C, and (B) 140
C.
encapsulating solution whereas the highest survival was obtained
with 12% w/v of FSP (90.35%). In these experiments FSP was heat
denaturated before it was added to the encapsulated solution
because preliminary tests showed better results (not shown). The
major fraction of flaxseed protein is globulin (Li-Chan & Ma, 2002),
which exhibits poor mechanical properties because most of the
hydrophobic and SH groups are buried inside the molecule.
Denaturation changes the native structure of proteins allowing
protein-protein interactions, disulphide cross-linking and
hydrogen-bonding. These interactions make the denatured pro-
teins stiffer, stronger and stretchable.
3.4. Viability of the encapsulated LAB after incubation in simulated
gastric juice and bile solution
The ability to survive at the adverse environmental conditions
characteristic of the digestive tract (low pH at the stomach and bile
salts in the first portion of the intestinal tract) is a desirable prop-
erty that probiotics should meet. Cell encapsulation could enhance
survival due to the protection conferred by the wall material. Fig. 4a
Table 3
Effect of the content of flaxseed soluble protein (FSP) in
the encapsulating solution on the survival L. acidophilus
La-05 after spray drying at 110
C. Other components in
the encapsulating solution were malt dextrin (15% w/v)
and flaxseed mucilage (0.2% w/v).
FSP (% w/v) Survival (%)
0 78.51 ± 1.31c
2 79.00 ± 0.32c
6 81.86 ± 0.66b
12 90.35 ± 0.76a
Different superscript letters in the same column indicate
significant differences (P < 0.05).
shows the relative viability of encapsulated and free LAB in MRS
broth containing 2% of bile salts; viability of the LAB encapsulated
with FM (0.2% w/v) decreased after the first 2 h, remained relatively
stable the next 2 h and finally grew reaching a relative viability of
0.85. The LAB encapsulated with FM and FSP (12% w/v) grew for 4 h
and then remained constant reaching a relative viability of almost
1.20 whereas the free LAB showed a stable viability during 4 h after
which a sharp decline was recorded (0.27).
Fig. 4b shows the relative viability of the encapsulated and free
LAB in a simulated gastric juice. A sharp decline in the number of
LAB in all samples was observed. After 4 h no survival was detected
in the free LAB sample whereas the encapsulated LAB with FM
showed a decreased in relative viability which was almost constant
in the last 3 h of the test. The LAB encapsulated with FM and FSP
reached a relative viability of 0.47 after 6 h of incubation. The re-
sults obtained in other studies have shown mixed results,
depending on the methodology, composition of reagents and
strains evaluated. Pan, Chen, Wu, Tang, and Zhao (2009) deter-
mined no growth of L. acidophilus NIT after 2 h exposure to pH 2
whereas Rajam et al. (2012) determined that Lactobacillus planta-
rum encapsulated with denatured whey protein showed better
stability compared with that encapsulated with untreated whey
protein after 4 h incubation in simulated acidic and bile conditions;
this behavior was explained due to the stronger and insoluble film
forming property of denatured protein, limiting cells release. Dolly,
Anishaparvin, Joseph, and Anandharamakrishnan (2011) found that
the relative viability of L. plantarum mtcc 5422 had a gradual
decline reaching null viability after 4 h exposition to pH 2.0 and bile
salts.
3.5. Physical properties of the dry powders
Fig. 5 shows the SEM micrographs of dry particles containing the
LAB encapsulated with solutions of different composition, stored 1
 M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168 1167

Fig. 4. Effect of (a) bile salt (2% v/v), (b) acid (pH 2.0) and pepsine (0.3% v/v) on the relative viability of L. acidophilus La-05 incubated at 37
C: (C) encapsulated with flaxseed
mucilage (FM) (0.2% w/v), (:) encapsulated with FM (0.2% w/v) and flaxseed soluble protein (12% w/v), and (B) control (free LAB).

Fig. 5. The SEM micrographs of dry particles containing L. acidophilus La-05 stored at 4
C. Particles were obtained with an encapsulating solution that contained 15% w/v of malt
dextrin and 0.2% w/v of flaxseed mucilage and dried with inlet air temperature of 110
C. Dry particles after: (a) 1 day of storage, and (b) 30 days of storage. Dry particles after 1 day
of dried with flaxseed soluble protein (c) 2% w/v and (d) 12% w/v.

and 30 days at 4
C. The particles showed a spherical shape,
wrinkled surface and varied size. The external surface did not show
evidence of fissures or cracks. Fig. 5(aeb) shows the “flat ball” effect
which may be related to the heat penetration and water evapora-
tion from droplet during drying (Barbosa-Ca
novas, Ortega-Rivas,
Juliano, & Yan, 2005). Morphology of the particles did not change
during storage (Fig. 5aeb). The particles of the product encapsu-
lated with 2 or 12% FSP (Fig. 5ced) did not reveal any difference in
terms of microstructure; however, the presence of wrinkles is
lower than in the particles of the product encapsulated without FSP
(Fig. 5aeb). Sheu and Rosenberg (1998) determined that the
incorporation of whey proteins into wall material consisting of MD
with high DE (15) minimized the presence of surface wrinkles
associated with wall materials consisting of only MD. Entrapped
cells were not visible in powder. Average size of the particles was
3.4 mm, varying between 0.06 mm and 7.0 mm.
4. Conclusions
Survival of L. acidophilus La-05 encapsulated by spray drying
was highly dependent on the inlet air temperature an effect that
can be reduced when FM is added to the encapsulation solution.
The high viscosity of aqueous solution containing FM restricts its
concentration in encapsulating solutions. The positive effect of FM
and heat-treated FSP on the survival of the LAB during spray drying
and the enhanced survival against bile solution and simulated
gastric juice suggest that flaxseed could be a source of fractions for
the design of encapsulating wall.
Conflict of interest statement
The authors declare that there are not conflicts of interest.
Acknowledgments
This research was supported by funding CONICYT through
FONDECYT project 3130561.
References
Ann, E. Y., Kim, Y., Oh, S. J., Imm, J. Y., Park, D. J., Han, K. S., et al. (2007). Microen-
capsulation of Lactobacillus acidophilus ATCC 43121 with prebiotic substrates
using a hybridisation system. International Journal of Food Science and Tech-
nology, 42, 411e419.
AOAC. (1995). Official methods of AOAC international (16th ed.) Arlington, VA. USA.
1168 M. Bustamante et al. / LWT - Food Science and Technology 62 (2015) 1162e1168
Barbosa-C
anovas, G. V., Ortega-Rivas, E., Juliano, P., & Yan, H. (2005). Food Powders:
Physical properties, processing and functionality. New York: Kluwer Academic.
Boza, Y., Barbin, D., & Scamparini, A. R. P. (2004). Effect of spray-drying on the
quality of encapsulated cells of Beijerinckia sp. Process Biochemistry, 39,
1275e1284.
Bozan, B., & Temelli, F. (2008). Chemical composition and oxidative stability of flax,
safflower and poppy seed and seed oils. Bioresource Technology, 99, 6354e6359.
Corcoran, B. M., Ross, R. P., Fitzgerald, G. F., & Stanton, C. (2004). Comparative
survival of probiotic lactobacilli spray-dried in the presence of prebiotic sub-
stances. Journal of Applied Microbiology, 96, 1024e1039.
Crowe, J. H., Crowe, L. M., Carpenter, J. F., & Aurell-Wistrom, C. (1987). Stabilization
of dry phospholipid and proteins by sugars. Biochemical Journal, 242, 1e10.
Dolly, P., Anishaparvin, A., Joseph, G. S., & Anandharamakrishnan, C. (2011).
Microencapsulation of Lactobacillus plantarum (mtcc 5422) by spray-freeze-
drying method and evaluation of survival in simulated gastrointestinal condi-
tions. Journal of Microencapsulation, 6, 568e574.
Esparza, Y., Leyton, A., Rubilar, M., & Shene, C. (2011). Cine
tica de extraccio

n y
actividad prebio
tica del mucilago de linaza. In VIII Congreso Iberoamericano de
Ingeniería de Alimentos, Lima, Perú (pp. 24e26).
FAO/WHO. (2001). Evaluation of health and nutrition properties of probiotics in food
including powder milk with live lactic acid bacteria. Report of a Joint FAO/WHO
Expert Consultation, Cordoba, Argentina.
Fritzen-Freire, C. B., Prude^ncio, E. S., Pinto, S. S., Mun
~ oz, I. B., & Amboni, R. D. M. C.
(2013). Effect of microencapsulation on survival of Bifidobacterium BB-12
exposed to simulated gastrointestinal conditions and head treatments. LWT e
Food Science and Technology, 50, 39e44.
Gibson, G. R., Probert, H. M., Van Loo, J., Rastall, R. A., & Roberfroid, M. B. (2004).
Dietary modulation of the human colonic microbiota: updating the concept of
prebiotics. Nutrition Research Reviews, 17, 259e275.
HadiNezhad, M., Duc, C., Fong Han, N., & Hosseinian, F. (2013). Flaxseed soluble
dietary fibre enhances lactic acid bacterial survival and growth in kefir and
possesses high antioxidant capacity. Journal of Food Research, 2, 152e163.
Holzapfel, W. H., Haberer, P., Geisen, R., Bjo €rkroth, J., & Schillinger, U. (2001). Tax-
onomy and important features of probiotic microorganisms in food and
nutrition. The American Journal of Clinical Nutrition, 73, 365Se373S.
Kiokias, S., Dimakou, C., & Oreopoulou, V. (2007). Effect of heat treatment and
droplet size on the oxidative stability of whey protein emulsion. Food Chemistry,
105, 94e100.
Kristensen, M., Jensen, M. G., Aarestrup, J., Petersen, K. E. N., Søndergaard, L.,
Mikkelsen, M. S., et al. (2012). Flaxseed diatery fibers lower cholesterol and
increase fecal fat excretion, but magnitude of effect depend on food type.
Nutrition and Metabolism, 9, 1e8.
Kristensen, M., Savorani, F., Christensen, S., Engelsen, S. B., Bugel, S., Toubro, S., et al.
(2013). Flaxseed dietary fibers suppress postprandial lipemia and appetite
sensation in young men. Nutrition, Metabolism and Cardiovascular Diseases, 23,
136e143.
de Lara Pedroso, D., Thomazini, M., Jorda ~o Barrozo Heinemann, R., & Favaro-
Trindale, C. S. (2012). Protection of Bifidobacterium lactis and Lactobacillus aci-
dophilus by microencapsulation using spray-chilling. International Dairy Journal,
26, 127e132.
Li-Chan, E. C. Y., & Ma, C. Y. (2002). Thermal analysis of flaxseed (Linum usitatissi-
mum) proteins by differential scanning calorimetry. Food Chemistry, 77,
495e502.
de Man, J. C., Rogosa, M., & Sharpe, E. (1960). A medium for the cultivation of
Lactobacilli. Journal of Applied Bacteriology, 60, 130e135.
McMaster, L. D., Kokott, S. A., & Slatter, P. (2005). Micro-encapsulation of Bifido-
bacterium lactis for incorporation into soft food. World Journal of Microbiology
and Biotechnology, 21, 723e728.
Naran, R., Chen, G., & Carpita, N. C. (2008). Novel rhamnogalacturonan I and ara-
binoxylan polysaccharides of flax seed mucilage. Plant Physiology, 148, 132e141.
Nuttall, F. Q., Mooradian, A. D., Gannon, M. C., Billington, C., & Krezowski, P. (1984).
Effect of protein ingestion on the glucose and insulin response to a standardized
oral glucose load. Diabetes Care, 7, 465e470.
Oomah, B. D. (2001). Flaxseed as a functional food source. Journal of the Science of
Food and Agriculture, 81, 889e894.
Pan, X., Chen, F., Wu, T., Tang, H., & Zhao, Z. (2009). The acid, bile tolerance and
antimicrobial property of Lactobacillus acidophilus NIT. Food Control, 20,
598e602.
Pastell, H., Westermann, P., Meyer, A. S., Tuomainen, P., & Tenkanen, M. (2009).
In vitro fermentation of arabinoxylan-derived carbohydrates by Bifidobacteria
and mixed fecal microbiota. Journal of Agricultural and Food Chemistry, 57,
8598e8606.
Rajam, R., Karthik, P., Parthasarathi, S., Joseph, G. S., & Anandharamakrishnan, C.
(2012). Effect of whey protein e alginate wall systems on survival of micro-
encapsulated Lactobacillus plantarum in simulated gastrointestinal conditions.
Journal of Functional Foods, 4, 891e898.
Roy, D. (2005). Technological aspects related to the use of bifidobacteria in dairy
products. Lait, 85, 39e56.
Sheu, T. Y., & Rosenberg, M. (1998). Microstructure of microcapsules consisting of
whey proteins and carbohydrates. Journal of Food Science, 63, 491e494.
Simpson, P. J., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Intrinsic tolerance of
Bifidobacterium species to heat and oxygen and survival following spray drying
and storage. Journal of Applied Microbiology, 99, 493e501.
Sørensen, H. R., Pedersen, S., & Meyer, A. S. (2006). Optimization of reaction con-
ditions for enzymatic viscosity reduction and hydrolysis of wheat arabinoxylan
in an industrial ethanol fermentation residue. Biotechnology Progress, 22,
505e513.
Thakur, G., Mitra, A., Pal, K., & Rousseau, D. (2009). Effect of flaxseed gum on
reduction of blood glucose and cholesterol in type 2 diabetic patients. Inter-
national Journal of Food Sciences and Nutrition, 60, 126e136.