LWT - Food Science and Technology 117 (2020) 108686

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Protective effects of the use of taro and rice starch as wall material on the T
viability of encapsulated Lactobacillus paracasei subsp. Paracasei
O. Alfaro-Galarzaa, E.O. López-Villegasb, N. Rivero-Pereza, D. Tapia- Maruric,
A.R. Jiménez-Aparicioc, H.M. Palma-Rodrígueza,∗, A. Vargas-Torresa
a
Universidad Autónoma del Estado de Hidalgo, Instituto de Ciencias Agropecuarias (ICAP), Av. Universidad km 1, Rancho Universitario, C.P. 43600, Tulancingo de
Bravo, Hidalgo, Mexico
b
Instituto Politécnico Nacional, Central de Microscopia, ENCB, Mexico City, Mexico
c
Instituto Politécnico Nacional, CEPROBI, Km 6 Carr, Yautepec-Jojutla, Calle Ceprobi No. 8, Apartado Postal 24, Yautepec, 62731, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, was evaluated the protective effect of two microencapsulation materials of different sized starch
Starch granules, taro (5.16 μm) and rice (6.99 μm), on the survival of Lactobacillus paracasei subsp. paracasei (LPSP) at
Encapsulation controlled spray-drying inlet air temperatures of 70, 115, and 135 °C. Physicochemical properties, encapsulation
Lactobacillus efficiency, and gastrointestinal viability of microcapsules were analyzed. Rice microcapsules showed a slight
Viability
increase in water activity and moisture values when compared with taro microcapsules. Viability of the mi-
In-vitro digestion
crocapsules was affected drastically with the increase in inlet temperature during spray-drying. Small-sized taro
starch granules showed a better formation of spherical aggregates for one logarithmic cycle in the initial viability
of LPSP. Electron microscopy showed less LPSP on the outside of the taro starch microcapsules. The findings in
this study indicated that use of microcapsules constructed from taro starch can offer better protection to pro-
biotic strains.

1. Introduction hydrocolloids (Avila-Reyes et al., 2014; Cortés et al., 2014) as wall

The intake of probiotics microorganisms has risen owing to the in-
creased awareness regarding their associations with multiple health
benefits, especially in the digestive system, when administered in ap-
propriate doses. FAO/WHO (2002) reported that the consumption of
probiotic microorganisms should range from 106 to 107 CFU/g or mL
(CFU, colony-forming unit). However, when bacteria are used as pro-
biotics, their viability must remain stable throughout the duration of
the shelf-life of the food product and their resistance to the acidic en-
vironment of the stomach and to bile salts in the small intestine must be
maintained. Other factors that affect bacterial viability are pH, lactic
and acetic acid, hydrogen peroxide, and dissolved oxygen content of the
product (Picot & Lacroix, 2004). Therefore, various technologies have
been developed to protect microorganisms against adverse environ-
mental factors and to improve the delivery of bioactive molecules and
living cells within foods, for example, spray-dried microencapsulation
(Avila-Reyes, Garcia-Suarez, Jiménez, San Martín-Gonzalez, & Bello-
Perez, 2014; Peredo, Beristain, Pascual, Azuara, & Jimenez, 2016).
Studies have reported the use of carrageenan, alginate, cellulose acetate
phthalate, gelatin, chitosan, starch, or combination of some these
material in microencapsulation by spray-drying to preserve probiotic
microorganisms.
Some studies have reported that starches with small granule size, or
modified starches, can also be used as wall material because they have
shown to be effective encapsulation agents (Avila-Reyes et al., 2014;
Palma-Rodríguez, Alvarez-Ramírez, & Vargas-Torres, 2018). Other
studies have reported that small granule starch has a greater protective
effect on encapsulated chemical compounds (Hoyos-Leyva, Chavez-
Salazar, Castellanos-Galeano, Bello-Perez, & Alvarez-Ramirez, 2018;
Zhao & Whistler, 1994). This improvement in the protective effect
could be explained by the greater tendency of small granule starch to
form spherical aggregates (Gonzalez-Soto, de la Vega, Garcia-Suarez,
Agama-Acevedo, & Bello-Perez, 2011). However, there is little in-
formation on the use of small granule starch in the encapsulation of
probiotics. Pankasemsuk, Apichartsrangkoon, Worametrachanon, and
Techarang (2016) reported protective effect of microcapsules made
with Hi-maize starch-alginate mixtures and showed that the increase in
alginate concentration improved the longevity of Lactobacillus casei 01.
Avila-Reyes et al. (2014) evaluated the use of native rice starch and
inulin as wall material in the microencapsulation of Lactobacillus

∗
Corresponding author.
E-mail addresses: heidi_palma9528@uaeh.edu.mx, apolovt@hotmail.com, palma.heidi@gmail.com (H.M. Palma-Rodríguez).

https://doi.org/10.1016/j.lwt.2019.108686
Received 13 December 2018; Received in revised form 20 September 2019; Accepted 24 September 2019
Available online 25 September 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
O. Alfaro-Galarza, et al.
rhamnosus. They found that microcapsules made with native rice starch
maintain the stability and viability of probiotics under storage and
gastrointestinal conditions Other authors studied the viability of using
porous starch in the encapsulation of Lactobacillus plantarum (Li, Thuy
Ho, Turner, & Dhital, 2016).
However, another factor that affects the viability of probiotic mi-
croorganisms is the air temperature during the spray-drying en-
capsulation process (Avila-Reyes et al., 2014). Usually, low air tem-
peratures during drying and encapsulation processes provoke a
decrease in probiotic cellular lesions, which increases the CFU/g in the
microcapsules. Therefore, the adjustment and control of appropriate
processing conditions, the inlet and outlet air temperatures, types of
starch, modification of starches, and starch granule sizes are important
parameters to viably encapsulate probiotic microorganisms
(Kailasapathy, 2002). The aim of the present study was to evaluate the
protective effect of two starches with granules of different size on the
viability, during 30 days of storage, of the probiotic microorganism
Lactobacillus paracasei subsp. paracasei (LPSP) encapsulated by spray-
drying.
2. Materials and methods
2.1. Materials
Taro tubers were purchased from the Valle National Region,
Tuxtepec, Oaxaca, México. Rice grain of the variety A-98 was acquired
from the Buena vista rice farm in Cuautla Morelos. A Total Starch Assay
Kit (K-TSTA-50A) was purchased from Megazyme International
(Ireland, Wicklow, Ireland). The content of total starch and apparent
amylose in the taro and rice starch was 17.34–9.44% and
82.60–86.68%, respectively. The particle sizes of the taro starch (TS)
and rice starch (RS) granules were 5.16 μm and 6.99 μm, respectively.
The probiotic bacteria LPSP (LBC81 LYO 10 D) was obtained from
the “food ingredient solutions for the food industry – DuPont – Danisco”
(DANISCO®, Mexico) and grown by fermentation using MRS broth (de
Man, Rogosa and Sharpe) at a controlled temperature of 30 °C. The
microorganisms were stored in vials with added glycerin (30%) in an
ultra-freezer (−80 °C) for later use. Prior to use, the LPSP were re-
activated using MRS broth (DIBICO, Mexico).
2.2. Isolation of taro starch
A tuber in a fresh physiological state was washed, peeled, cut into
small pieces (3 × 3 cm), placed in a 40 L container with 3 quarts of
water, then liquefied in an industrial blender (model CB15, 4 L capa-
city) at low speed for 40 s. The obtained mixture was first passed
through a sieve to remove the excess fiber and then sieved through
mesh No. 40 (0.425 mm) and No. 100 (0.15 mm). The mixture of water
and starch was allowed to settle for 2 days, and then repeatedly washed
until there was a clear liquid on the surface and on the bottom the solids
(starch). The liquid was decanted and discarded, while the starch was
recovered by centrifuging it using a Rotina 420 Hettich centrifuge at
878×g for 8 min and dried in a tray dryer (APEX brand model SSE
17 M, USA) at a temperature of 45 °C for 24 h. The dried starch was
ground in a mortar, then sieved again with a No. 100 mesh and finally
stored in a plastic container with a hermetic seal at 25 °C for later use.
2.3. Isolation of rice starch
A head rice sample was soaked in 0.1% w/v NaOH (1:4 ratio)
overnight at room temperature (25 °C). The soaked sample was then
wet-milled in an Osterizer blender for 4 min at speed 6, filtered suc-
cessively through US standard 40 (425 μm) and 100 (250 μm) sieves,
and centrifuged at 1500×g for 15 min. The supernatant and top yellow
layer were discarded, the prime starch was then washed with 0.1% w/v
NaOH, centrifuged under the same conditions as before, and again the
2
LWT - Food Science and Technology 117 (2020) 108686
supernatant and top yellow layer were discarded. The pH of the residue
was then adjusted to 6.5 with 0.2 M HCl and with 40 mL distilled water.
A final centrifugation at 830×g for 2 min was performed to recover the
dewatered rice starch, which was then dried in a convection oven at
40 °C for 24 h, ground into powder with a mortar and pestle, passed
through a standard 100-mesh sieve, and stored in a sealed container
until use.
2.4. Growth curve of LPSP
Following the methodology proposed by Jurado-Gámez, Calpa-
Yamá, and Chaspuengal-Tulcán (2014) with some modifications, the
growth curve of LPSP was calculated to corroborate its growing stage
and to determine the timing when the microorganism should be mixed
with the wall material. The microorganism was initially cultured on
solid medium (MRS agar), and a loop was taken from this medium
(~1 × 103 CFU/mL) and placed in 100 mL of liquid medium (MRS
broth) that was subsequently incubated at 30 °C for 25 h. The microbial
concentration was determined over 25 h, at times 0, 5, 10, 12, 14, 15
16, 18, 20, 25 h. The viability of the probiotics was then determined
using the plate count method.
2.5. Inoculum standardization
The LPSP was first incubated with 5 mL of MRS broth at 30 °C for
24 h (Ding & Shah, 2007). The technique described by Picot and Lacroix
(2004), with some modifications, was then conducted whereby 50 μL of
inoculum was taken to first reactivation (for 15 h). Studies showed that
this duration is necessary to reach the exponential growth phase max-
imum. The LPSP was recollected by centrifugation at 4732×g for
15 min at 4 °C and re-suspended in 1 mL of sterile water prior to being
added to the dispersion mixture for the encapsulation process.
2.6. Spray drying process
Prior to microencapsulation, the wall material was prepared by
making a dispersion mixture of TS or RS (10% w/w), and the micro-
organism was immediately added at the slurry suspension stage. The
blends of wall material and LPSP were spray dried in a laboratory scale
dryer (Mod. B-290, Büchi, Switzerland). The air-flow was equivalent to
450–565 L/h. The blends were fed into the drier at a rate of 10 g/min at
different air-inlet temperatures of 135, 115, and 70 °C; the air-outlet
temperatures were 77, 66, and 43 °C, respectively. Prior to feeding the
blends into the dryer, a conditioning step with sterile distilled water
was performed to maintain stability in the input and output tempera-
tures. Dried powders were collected and immediately placed in sterile
containers and stored at 4 °C.
2.7. Encapsulation efficiency (EE)
Entrapped microorganisms were released from the microcapsules
according to the methods of Avila-Reyes et al. (2014), whereby, 100 mg
of the powder was dissolved in 10 mL of a 1% Peptone water solution.
The solution was kept in constant agitation for 15 min at 500 rpm and
finally the viability count on MRS agar was performed after 48 h of
incubation at 30 °C.
The efficiency of encapsulation (EE) represents the survival rate of
the Microorganims during the microencapsulation process, and is cal-
culated following Martin, Lara-Villoslada, Ruiz, and Morales (2013):
N
EE % = ⎛ ⎞ × 100
⎝ No ⎠
where N is the number of viable cells (log CFU) released from the mi-
croparticles and N0 is the number of viable cells (log CFU) in the feed
solution before the spray-drying process.
O. Alfaro-Galarza, et al.
2.8. Particle size distribution
The particle size distribution of the microcapsules and the starch
granules were determined by laser diffraction using a Mastersizer 2000
(Malvern Instruments Ltd., Malvern, Worcestershire, UK). Powder
samples were dispersed using a Scirocco dry disperser (Malvern,
Worcestershire, UK), at a feed pressure of 2 bars and 40%, respectively.
The obscuration range was 0.5–5.0%. The Fraunhofer approximation
was used to calculate particle size.
2.9. Water activity and moisture content
The water activity of the samples was determined using an AquaLab
Mod. 3 TE (Decagon Devices, Inc., Pullman, WA, USA) water activity
meter. The moisture content of the microcapsules was measured via
weight loss using a forced convection hot-air oven (Drying Oven, mod.
DHG-9075A) at 130 °C for 1 h (method 44-16 AACC), immediately after
spray-drying.
2.10. Bulk density
Bulk density considers all the pores in and out of the individual
particles and can be calculated by placing a mass of particles within a
container of known volume. An empty 10 mL test tube was weighed and
filling with the bulk density powder, and then reweighed. The bulk
density was calculated as follows (Marousis & Saravacos, 1990):
m
ρb =
V
where ρb the bulk density (g/mL), m is the mass of the powder (g), and v
is the volume occupied in the tube (mL).
2.11. Stability of microencapsulated probiotic at different storage
temperatures
The powders containing different probiotics and prebiotics were
stored for 30 days at 25 and 4 °C in sealed bags. Viability of the pro-
biotic strains was assessed every 7 days. For evaluation of the survival
of the Microorganims during storage, 1 g of microencapsulated powder
was dissolved in 99 mL of peptone, and this blend was spread plated in
MRS agar. The number of viable bacteria was expressed as CFU/g.
2.12. Survival of encapsulated bacteria in gastrointestinal conditions
The simulated gastric juice (SGJ) and simulated intestinal juice (SIJ)
were used according to the method described by Valero-Cases and
Frutos (2015) with light modifications. The SGJ was prepared with an
MRS broth and 3 g/L of pepsin. The pH was adjusted using 0.1 N HCl,
pH 2 was obtained. The SIJ was prepared with 1 g/L of pancreatin
(Pancreatin from porcine pancreas P7545 Sigma) and 4.5 g/L of bile
salts (B3426 Sigma). The pH of the MRS broth was adjusted to pH 7
with NaOH 0.1 N. Both solutions were sterile filtered through a mem-
brane (0.45 mm, Millipore, Spain). The microcapsules (1 g) were
homogenized for 2 min with 9 mL of SJG at 37 °C and then incubated
for 60 min at 37 °C. The enzymatic reaction was stopped by neu-
tralization with NaOH 1 N to pH 7. The SIJ (9 mL) and MRS broth were
then added to the suspension up to a volume of 20 mL and incubated for
60 min at 37 °C. The viable count in both SGJ and SIJ was determined
using the plate count method in MRS agar and expressed as CFU/g.
2.13. SEM of microcapsules
Scanning electron microscopy (SEM) was used to determine the
morphology of the encapsulates. Microcapsules made with TS and RS
were sprinkled and fixed (using double glue) to a conductive tape of
copper that was coated with a 20 nm thick layer of coal and deposited
3
LWT - Food Science and Technology 117 (2020) 108686
in a vacuum with an evaporator in a LEO 1525 (LEO Electron
Microscopy, Oberkochen, Germany) electron microscope. Later, the
samples were covered with a 50 nm thick gold layer using a JEOL
metals ionizer. Once covered the samples were placed in a tray and
observed using an accelerating voltage of 15 kV.
2.14. Confocal laser scanning microscopy
The study samples were mounted on glass slides and viewed under a
confocal laser scanning microscopy (Carl Zeiss, model LSM 800,
Germany) at 400x, with laser excitation wavelengths of 488 and
561 nm. The samples were covered with a few drops of acridine orange
(green fluorescence) solution at a concentration of 0.005% and later
propidium iodide (red fluorescence), at the same concentration, and
placed in darkness for 25 min. The fluorescence intensity was measured
using the ZEN 2.3 Blue edition software of the LSM 800. The intensity
peak of the fluorescence emission signals was captured in the range
between 410 and 590 nm for acridine orange and 595–700 nm for
propidium iodide.
2.15. Statistical analysis
The data were analyzed using one-way and two-way analyses of
variance (ANOVA); statistical significance was evaluated using Tukey's
test (P ˂ 0.05). The program used was Sigma stat version 12.5. Each
sample was analyzed in triplicate in this study.
3. Results and discussion
3.1. Growth kinetics of L. paracasei subsp. Paracasei (LPSP)
Prior to the encapsulation process, the optimal growth kinetics of
the microorganism was determined. Fig. 1 shows that the maximum cell
growth occurred at 15 h and reached ~3.5 × 1009 CFU/mL. However,
during the recollection, a reduction in the logarithmic cycle
(4.54 × 1008 CFU/mL) of the microorganism was observed in the sec-
tion material 2.5 and in the encapsulation process. This behavior may
be due to manipulations that are required during the encapsulation
process.
3.2. Effect of spray dryer temperature on the viability of encapsulated
microorganisms
Table 1 shows the effect of the different spray dryer temperatures
used to encapsulate LPSP in TS and RS as the wall materials. As ex-
pected, an increase in the air-inlet temperature in the spray dryer de-
creased the moisture content in the microcapsules. However, the taro
starch microcapsules (TSM) showed slightly lower values than those
observed in the rice starch microcapsules (RSM).
The use of small-sized starch granules reportedly produces spherical
aggregates that bind water, as was observed in the TSM in the present
study (SEM, Fig. 4), which contributes to a higher moisture content
(Zhao & Whistler, 1994). On the other hand, the decrease in moisture
with the increase in temperature during the spray-drying process is due
to the higher air-inlet temperature, thus a higher rate of heat transfer to
the microcapsule, which causes a greater driving force for moisture
evaporation. Similar behavior was observed by Quek, Chok, and
Swedlund (2007) in microcapsules made from maltodextrin. The re-
duction in the bacterial size may be due to the thermal sensitivity of the
bacteria during heating or spray drying (See Table 1). This behavior
affects the encapsulation efficiency, due to thermo-sensitivity of the
microorganism. Ananta, Volterk, and Knorr (2005) reported similar
findings in the microencapsulation of Lactobacillus rhamnosus GG, i.e.,
inverse proportions of survival rates and moisture vs. the air-outlet
temperature. However, in the present study, the TSM showed a better
protective effect with the increase in temperature compared to the
O. Alfaro-Galarza, et al. LWT - Food Science and Technology 117 (2020) 108686

Fig. 1. Growth kinetics of Lactobacillus paracase subsp. paracasei. Data represent means ± standard errors (n = 3). Mean in the bars that show different lowercase
letters are significantly different (α = 0.05).

RSM. The formation of homogenous and higher numbers of micro- Table 2

capsules with TS were observed via SEM. This may indicate a better Physicochemical characterization of taro and rice encapsulation.
protective effect to the target microorganism. Avila-Reyes et al. (2014) Taro microcapsules Rice microcapsules
mentioned that the concentration of the suspension affects the viability.
b
Higher concentration lessens the reduction in the viability and the Water activity 0.290 ± 0.115 0.308 ± 0.230a
Moisture (%) 6.27 ± 0.55a 6.60 ± 0.63a
amount of solids in the suspension that is introduced in the spray dryer,
Particle size D [V 0.5] (μm) 5.731 7.056
limits heat transfer, and, therefore, improves the viability of micro- Bulk density (g/cm3) 0.362 0.365
organisms. The increase in the viability of TSM with the air-inlet tem-
perature showed values of Log N: 8.91, 9.43, and 9.35 at temperatures Data represent means ± standard errors (n = 3).
of 135, 155, and 70 °C, respectively. This directly influenced the sta- Mean within rows followed by different superscripts are significantly different
bility of the encapsulation efficiency. A reduction of two logarithmic (α = 0.05).
cycles after the thermal process in the preparation of microcapsules
could be considered adequate to blend with food products (Rokka & obtained with an air-inlet temperature of 70 °C, only these micro-
Rantamäki, 2010). The results obtained in this study indicated that the capsules were characterized. The TSM showed low values of water
TS could be used as wall material, because it provided adequate pro- activity and moisture content than those observed in the RSM (See
tection for the microorganism during the spray drying process at least Table 2).
in the interval of air-inlet proposed temperatures and when compared The capacities of microcapsules to trap water may be attributed to
with microcapsules made of RS. The statistical analysis (two-way several factors, such as chemical structure, size particle, and the dif-
ANOVA) showed that the factor with the most influence, i.e., more ferent chemical groups present in the polysaccharides that can interact
influence than the inlet temperature of the dryer, on the survival of with water molecules to form hydrogen bridges, e.g., COO− and SO3
microorganism was the size of the starch granules used in the wall (Peredo et al., 2016; Hoyos-Leyva et al., 2018). Palma-Rodriguez et al.
material. (2013) reported low moisture content in microcapsules made with corn
and potato modified starch, i.e., a starch with a smaller particle size.
3.3. Physicochemical characterization of taro and rice microcapsules Corcoran, Ross, Fitzgerald, and Stanton (2004) reported values of mi-
crocapsule water activity between 0.15 and 0.3. They mention that the
Because the best encapsulation efficiency of the microorganism was low values of these parameters contribute to provide greater

Table 1
Efficiency of encapsulation in the microcapsules obtained through spray drying.
Starch Spray dryer inlet Moisture in the microcapsules Microorganisms in the dispersion Microorganisms after drying Encapsulation efficiency (%) (Log
temperature (°C) obtained (%) before drying (Log No) (Log N) No/Log N)*100

Rice1 135 5.43 ± 0.16a, b

7.23 ± 0.08a 69.82 ± 0.74a
115 5.62 ± 0.16b 10.36 8.64 ± 0.15b 85.88 ± 1.47b
70 6.60 ± 0.21d 9.12 ± 0.02d 90.74 ± 0.24d
Taro2 135 5.18 ± 0.16a 8.91 ± 0.05c 86.06 ± 0.53c
115 5.34 ± 0.13a, b
10.36 9.43 ± 0.03e 90.23 ± 0.30e
70 6.27 ± 0.16c 9.35 ± 0.04e 90.33 ± 0.34f

Data represent means ± standard errors (n = 3). Superscript letters represent the effect spray dryer inlet temperature. Superscript numbers (source of starch)
represent the effect of wall material used. Mean within column followed by different superscripts are significantly different (α = 0.05). Means in the same column
that do not have the same superscript number are significantly different (α = 0.05).

4
O. Alfaro-Galarza, et al.
Fig. 2. Viability of Lactobacillus paracase subsp. paracasei encapsulated in taro and rice starch. A: Room temperature (25 °C); B: Cool temperature (5 °C).
■ = Microcapsules of taro starch; ▲ = Microcapsules of rice starch.
microbiological stability and prevent caking or recrystallization pro-
cesses. This increase in the stability of Microorganism decrease the
incidence of with numerous diseases, such as colon cancer, food allergy,
acute gastrointestinal injury, Crohn's disease and rheumatoid arthritis
(Kalliomaki, Salminen, Poussa, Arvilommi, & Isolauri, 2003). This
viability characteristic was also observed in the TSM in the present
study (See Table 1).
The bulk density in the TSM and RSM (See Table 2) showed similar
values. Thus, the size of the granules in the microcapsule preparation as
well as the solids concentration did not affect the porosity structure of
the microcapsules. Fuchs et al. (2006) reported that the density of
microcapsules has a close relationship with the particle size, because
low density values indicate small particle sizes. These characteristics
are also related to an easy solubilization.
3.4. Stability of microencapsulated probiotic
The viability of the LPSP microencapsulated with TSM and RSM at
two different storage temperatures are shown in Fig. 2. It is important
5
LWT - Food Science and Technology 117 (2020) 108686
to stipulate that this study was conducted only with the samples that
exhibited the highest encapsulation efficiency. In Fig. 2, the storage
temperature is shown to affect the survival of LPSP, i.e., a greater loss of
viability with the increase in temperature. This phenomenon occurs
because the microorganism stored a higher temperature increase their
metabolic activity, which provokes a higher substrate consumption to
maintain the viability (Avila-Reyes et al., 2014).
However, in both storage temperatures, the TSM was shown to
provide better protection for the microorganism than the RSM. This
may be explained by the smaller particle size of TS (5.16 μm) than RS
(6.99 μm), which allows a better formation of aggregates (as observed
in SEM- Fig. 4B–E) and therefore, provides better protection. Similar
findings were reported by Avila-Reyes et al. (2014) in the encapsulation
of a microorganism with materials of different particle sizes. These
results suggest that the microencapsulation of probiotics with pre-
biotics, such as TS, may offer an effective alternative for the main-
tenance of the microorganism viability during storage at both re-
frigeration and 25 °C for up to 30 days.
O. Alfaro-Galarza, et al. LWT - Food Science and Technology 117 (2020) 108686

Fig. 3. Viability of taro and rice starch encapsulated

Lactobacillus paracasei subsp. paracasei in simulated
gastrointestinal conditions. ■ = Microcapsules of
taro starch; ▲ = Microcapsules of rice starch.

Fig. 4. Scanning electron micrographs of (A, B, C) microcapsules of taro starch at 300X, 1800X, and 8000X, respectively; and microcapsules of (D, E, F) rice starch at
300X, 1500X, and 8000X, respectively.

6
O. Alfaro-Galarza, et al.
Fig. 5. Laser scanning confocal micrograph of (A, B, C, D) taro starch microcapsules; and (E, F, G) rice starch microcapsules. Micrograph taken at different depths or
cuts of the microcapsule. The A–G microcapsules were obtained at a spray dryer inlet temperature of 70 °C. H) Micrograph of the taro starch microcapsules obtained
at a spray dryer inlet temperature of 135 °C.
3.5. Survival of encapsulated probiotic in gastrointestinal conditions
simulated
The TSM and RSM obtained at a dryer air-inlet temperature of 70 °C,
were selected to evaluate the gastrointestinal conditions because of
their encapsulation efficiency. Fig. 3 showed that TSM had a better
protective effect throughout the 30 days of storage. However, RSM at
the initial time of storage showed a logarithmic cycle decrease in CFU/g
of microorganism, and after 15 days of storage, most of the micro-
organism had died. The higher protective effect of TSM may have been
attributed to the small size of the TS granules, which allows a better
formation of microcapsules by decreasing the porous structure in the
spherical aggregates, as observed by SEM in the present study. Hoyos-
Leyva et al. (2018) reported that the porous size in the microcapsules
influences in the release of ascorbic acid, with larger pore sizes having a
greater release rate. Therefore, in the case of injected encapsulated
probiotics, the acid, bile salts, and enzymes of the gastrointestinal en-
vironment may be able to enter the microcapsules via the pores. Con-
sequently, larger pores in the microcapsules allow fluids flow across the
microcapsule, which affects the survival of the encapsulated Micro-
organisms.
3.6. Microscopy electron and confocal
The morphological characteristics of TSM and RSM are shown in
Fig. 4. The panoramic photographs of microcapsules indicate that the
TSM (Fig. 4A) had a better aggregation and spherical form, that of the
RSM (Fig. 4D).
The larger pores in the microcapsules observed in Fig. 4E could
explain the lower survival of the LPSP at the different storage tem-
perature and simulated gastrointestinal conditions. Similar morpholo-
gical characteristics were reported by Avila-Reyes et al. (2014) for the
microencapsulation of L. rhamnosus using rice starch as wall material.
TSM (Fig. 4C) showed a higher encapsulation capacity that RSM
(Fig. 4F), since less Microorganims were observed on the surface. This
may explain the higher encapsulation efficiency of TS, as shown in
Table 1.
The confocal micrographs are shown in Fig. 5. The superficial mi-
crograph of TSM (5A) the green color was not as clear as that observed
in the RSM (5E), which indicates the better protective effect of the TSM.
When the micrographs were taken of the internal sections of the
7
LWT - Food Science and Technology 117 (2020) 108686
microcapsules (at different depth), the intensity of the green color in-
creased both in the TSM (5B–5D) and RSM (5F and 5G). This finding
confirmed the greater survival of LPSP within the microcapsules. Be-
cause the acridine orange (chromophore) dyes the nucleic acid in the
live cells with a green color. Fig. 5H demonstrates an example of the
survival of Microorganims from the TSM obtained at an inlet spray-
dryer temperature of 135 °C. Yao et al. (2017) reported the same be-
havior, that is, an increase in the viability of probiotics at low inlet
temperatures, in a confocal microscopy study. In the present study,
electron and confocal microscopy provided important information to
support the protective effect of rice and, especially, TSM.
4. Conclusions
Microcapsules made from starch (taro) with small-sized granules
(i.e., as wall material) offered better protection to the encapsulated
LPSP, because the viability was maintained under gastrointestinal and
under extreme temperature conditions during storage for 30 days. The
results showed that the higher air-inlet temperature in the spray dryer
affected the viability of microorganism encapsulated in taro and rice
starch. The findings of this study may be useful for future studies and in
the manufacturing process where there is an increased focus on im-
proving the functionality and viability of probiotics in microcapsules
made from different wall materials, to improve the survival of micro-
organisms through the gastrointestinal tract.
Declaration of competing interest
The authors declare that no conflict of interest exists related with
this publication.
Acknowledgments
A. G. O. acknowledges study grants from CONACYT-México.
J. A. A. R. appreciate the financial support from Instituto Politécnico
Nacional – México (National Polytechnic Institute) for the financial
support provided through the SIP Projects: 20180817.
References
Ananta, E., Volkert, M., & Knorr, D. (2005). Cellular injuries and storage stability of
spray-dried Lactobacillus rhamnosus GG. International Dairy Journal, 15, 399–409.
O. Alfaro-Galarza, et al.
https://doi.org/10.1016/j.idairyj.2004.08.004.
Avila-Reyes, S. V., Garcia-Suarez, F. J., Jiménez, M. T., San Martín-Gonzalez, M. F., &
Bello-Perez, L. A. (2014). Protection of L. rhamnosus by spray-drying using two pre-
biotics colloids to enhance the viability. Carbohydrate Polymers, 102, 423–430.
https://doi.org/10.1016/j.carbpol.2013.11.033.
Corcoran, B. M., Ross, R. P., Fitzgerald, G. F., & Stanton, C. (2004). Comparative sur-vival
of probiotic lactobacilli spray-dried in the presence of prebiotic substances. Journal of
Applied Microbiology, 96, 1024–1039. https://doi.org/10.1111/j.1365-2672.2004.
02219.x.
Cortés, R. N. F., Martínez, M. G., Guzmán, I. V., Llano, S. L. A., Grosso, C. R. F., & Bustos,
F. M. (2014). Evaluation of modified amaranth starch as shell material for en-
capsulation of probiotics. Cereal Chemistry, 91, 300–308. https://doi.org/10.1094/
CCHEM-06-13-0112-R.
Ding, W. K., & Shah, N. P. (2007). Acid, bile, and heat tolerance of free and micro-
encapsulated probiotic bacteria. Journal of Food Science, 72, M446–M450. https://
doi.org/10.1111/j.1750-3841.2007.00565.x.
FAO/WHO (2002). Health and nutritional properties of probiotics in food including
powder milk with live lactic acid bacteria. Report of a Joint FAO/WHO Expert
Consultation. Córdoba, Argentina. http://www.fao.org/3/a-a0512e.pdf.
Fuchs, M., Turchiuli, C., Bohin, M., Cuvelier, M. E., Ordonnaud, C., Peyrat-Maillard, M.
N., et al. (2006). Encapsulation of oil in powder using spray drying and fluidised bed
agglomeration. Journal of Food Engineering, 75, 27–35. https://doi.org/10.1016/j.
jfoodeng.2005.03.047.
Gonzalez-Soto, R. A., de la Vega, B., García-Suarez, F. J., Agama-Acevedo, E., & Bello-
Perez, L. A. (2011). Preparation of spherical aggregates of taro starch granules.
Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 44,
2064–2069. https://doi.org/10.1016/j.lwt.2011.06.018.
Hoyos-Leyva, J. D., Chavez-Salazar, A., Castellanos-Galeano, F., Bello-Perez, L. A., &
Alvarez-Ramirez, J. (2018). Physical and chemical stability of L-ascorbic acid mi-
croencapsulated into taro starch spherical aggregates by spray drying. Food
Hydrocolloids, 83, 143–152. https://doi.org/10.1016/j.foodhyd.2018.05.002.
Jurado-Gámez, H., Calpa-Yamá, F., & Chaspuengal-Tulcán, A. (2014). Determinación de
parámetros cinéticos de Lactobacillus casei en dos medios probióticos. Veterinaria e
Zootecnia, 8, 15–35. https://doi.org/10.17151/vetzo.2014.8.2.2.
Kailasapathy, K. (2002). Microencapsulation of probiotic bacteria: Technology and po-
tential applications. Current Issues in Intestinal Microbiology, 3, 39–48.
Kalliomaki, M., Salminen, S., Poussa, T., Arvilommi, H., & Isolauri, E. (2003). Probiotics
and prevention of atopic disease: 4-yearcfollow-up of a randomised placebo-con-
trolled trial. Lancet, 361, 1869–1871. https://doi.org/10.1016/S0140-6736(03)
13490-3.
Li, H., Thuy Ho, V. T., Turner, M. S., & Dhital, S. (2016). Encapsulation of Lactobacillus
plantarum in porous maize starch. Lebensmittel-Wissenschaft und -Technologie- Food
Science and Technology, 74, 542–549. https://doi.org/10.1016/j.lwt.2016.08.019.
LWT - Food Science and Technology 117 (2020) 108686
Marousis, S. N., & Saravacos, G. D. (1990). Density and porosity in drying starch mate-
rials. Journal of Food Science, 55, 1367–1372. https://doi.org/10.1111/j.1365-2621.
1990.tb03939.x.
Martin, M. J., Lara-Villoslada, F., Ruiz, M. A., & Morales, M. E. (2013). Effect of un-
modified starch on viability of alginate-encapsulated Lactobacillus fermentum
CECT5716. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology,
53, 480–486. https://doi.org/10.1016/j.lwt.2013.03.019.
Palma-Rodriguez, H. M., Agama-Acevedo, E., Gonzalez-Soto, R. A., Vernon-Carter, E. J.,
Alvarez-Ramirez, J., & Bello-Perez, L. A. (2013). Ascorbic acid microencapsulation by
spray-drying in native and acid-modified starches from different botanical sources.
Starch - Stärke, 65(7‐8), 584–592. https://doi.org/10.1002/star.201200200.
Palma-Rodríguez, H. M., Alvarez-Ramírez, J., & Vargas-Torres, A. (2018). Using modified
starch/maltodextrin microparticles for enhancing the shelf life of ascorbic acid by the
spray-drying method. Starch - Stärke, 70(7–8), 1700323. https://doi.org/10.1002/
star.201700323.
Pankasemsuk, T., Apichartsrangkoon, A., Worametrachanon, S., & Techarang, J. (2016).
Encapsulation of Lactobacillus casei 01 by alginate along with hi-maize starch for
exposure to a simulated gut model. Food Bioscience, 16, 32–36. https://doi.org/10.
1016/j.fbio.2016.07.001.
Peredo, A. G., Beristain, C. I., Pascual, L. A., Azuara, E., & Jimenez, M. (2016). The effect
of prebiotics on the viability of encapsulated probiotic bacteria. LWT- Food Science
and Technology, 73, 191–196. https://doi.org/10.1016/j.lwt.2016.06.021.
Picot, A., & Lacroix, C. (2004). Encapsulation of bifidobacteria in whey protein-based
microcapsules and survival in simulated gastrointestinal conditions and in yogurt.
International Dairy Journal, 14, 505–515. https://doi.org/10.1016/j.idairyj.2003.10.
008.
Quek, S. Y., Chok, N. K., & Swedlund, P. (2007). The physicochemical properties of spray-
dried watermelon powders. Chemical Engineering and Processing: Process Intensification,
46(5), 386–392. https://doi.org/10.1016/j.cep.2006.06.020.
Rokka, S., & Rantamäki, P. (2010). Protecting probiotic bacteria by microencapsulation:
Challenges for industrial applications. European Food Research and Technology,
231(1), 1–12. https://doi.org/10.1007/s00217-010-1246-2.
Valero-Cases, E., & Frutos, M. J. (2015). Effect of different types of encapsulation on the
survival of Lactobacillus plantarum during storage with inulin and in vitro digestion.
Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 64(2),
824–828. https://doi.org/10.1016/j.lwt.2015.06.049.
Yao, M., Wu, J., Li, B., Xiao, H., McClements, D. J., & Li, L. (2017). Microencapsulation of
Lactobacillus salivarious Li01 for enhanced storage viability and targeted delivery to
gut microbiota. Food Hydrocolloids, 72, 228–236. https://doi.org/10.1016/j.foodhyd.
2017.05.033.
Zhao, J., & Whistler, R. L. (1994). Isolation and characterization of starch amaranth flour.
Cereal Chemistry, 71, 392–393.