Food Research International 96 (2017) 19–26

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Food Research International

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

The cell wall compound of Saccharomyces cerevisiae as a novel wall

material for encapsulation of probiotics
Samira Mokhtari, Seid Mahdi Jafari ⁎, Morteza Khomeiri, Yahya Maghsoudlou, Mohammad Ghorbani
Faculty of Food Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

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

Article history: Yeast cell wall is known as a food grade ingredient which is recently being used increasingly as a novel coating for
Received 17 December 2016 encapsulation of different materials in the food industry. This application is limited to core materials smaller than
Received in revised form 28 February 2017 yeast in size. In this study, we have tried to encapsulate larger particles by crushing yeast cells. Hence, probiotic
Accepted 10 March 2017
bacteria of Lactobacillus acidophilus and Bifidobacterium bifidum were encapsulated firstly by calcium alginate
Available online 12 March 2017
using the emulsion method and these microbeads were coated again by Saccharomyces cerevisiae cell wall com-
Keywords:
pound and another layer of calcium alginate. The average diameter of microcapsules for single layer microbeads
Yeast cell (M), microbeads coated by two layers of alginate (MCA), and microbeads coated by a layer of yeast cell and two
Probiotics layers of alginate (MCYA) were 54.25 ± 0.18, 77.43 ± 8.24 and 103.66 ± 13.33 μm, respectively. In simulated
Encapsulation gastrointestinal conditions, there was a significant (P b 0.05) enhancement in resistance of L. acidophilus when
Alginate applying a layer of S. cerevisiae cell wall compound. For MCA and MCYA after 2 h exposure to simulated gastric
Gastrointestinal conditions juice, it was revealed a log reduction of 1.53 ± 0.1 and 1.1 ± 0.02 with pH 1.55 and in simulated intestinal
juice, 2.92 ± 0.04 and 2.42 ± 0.06 with 0.6% bile after previous 1 h incubation in gastric conditions, respectively.
It can be concluded that the cell wall compound of S. cerevisiae is a suitable protective coating for probiotics and it
can improve the survival of probiotics within food products.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction drying, fluidized bed coating, extrusion, emulsification, coacervation,

Encapsulation may be defined as a process to entrap one substance
within another substance, thereby producing particles with diameters
of a few nm to a few mm. The substance that is encapsulated may be
called the core material, the active agent, fill, internal phase, or payload
phase. The substance that is encapsulating may be called the coating,
membrane, shell, carrier material, wall material, external phase, or ma-
trix (Mokhtari, Khomeiri, Jafari, Maghsoudlou, & Ghorbani, 2017;
Mokhtari, Jafari, & Assadpour, 2017). Microencapsulation has been rec-
ognized as an alternative for increasing microorganism resistance to
low acid environment during their storage and/or transit through the
gastrointestinal tract (GIT) (Anal & Singh, 2007). Externally, microen-
capsulation prolongs the shelf life of probiotics under the constantly
changing storage conditions. Since the stability and release properties
of microcapsules are highly dependent on wall material composition,
various carriers including sugars, cyclodextrins, maltodextrins, modi-
fied starches, gums, proteins, or liposomes have been used. Different
microencapsulation technologies such as spray-drying and freeze-
⁎ Corresponding author.
E-mail address: smjafari@gau.ac.ir (S.M. Jafari).
and electrostatic methods result in mean output capsule sizes of 10–
400, 20–5000, 5–5000, 150–8000, 10–1000, 10–800 μm, respectively
(Annan, Borza, & Truelstrup Hansen, 2008; Zuidam & Shimoni, 2010).
To exert health benefits, the minimum concentration of live probiot-
ic bacteria at point of delivery should be above 107 cfu mL−1 (Lee &
Salminen, 1995). To perform the best function, the viability of probiotics
must be maintained during manufacturing, storage, and passage
through adverse gastrointestinal environments of the stomach and
small intestine (Ying et al., 2010). Less than 10% of the strains tested
can grow in the GIT (Coeuret, Gueguen, & Vernoux, 2004).
Generally, probiotic bacteria do not survive well during the temper-
ature and osmotic extremes to which they are exposed during spray
drying and fluidized bed processes (Selmer-Olsen, Sorhaug, Birkeland,
& Pehrson, 1999). The most commonly reported microencapsulation
procedure is based on the calcium alginate gel capsule formation
(Nualkaekul, Lenton, Cook, Khutoryanskiy, & Charalampopoulos,
2012). The dispersion can be performed either by extrusion or by emul-
sification. Extrusion is a simple and cheap method that uses a gentle op-
eration which causes no damage to probiotic cells and gives a high
probiotic viability (Krasaekoopt, Bhandari, & Deeth, 2004). The most
important disadvantage of this technique is its inappropriateness in
large scale productions due to slow formation of the microbeads
(Burgain, Gaiani, Linder, & Scher, 2011). The emulsion technique results

http://dx.doi.org/10.1016/j.foodres.2017.03.014
0963-9969/© 2017 Elsevier Ltd. All rights reserved.
20 S. Mokhtari et al. / Food Research International 96 (2017) 19–26
in smaller diameter beads, and is better suited to scale up applications
(Kailasapathy, 2002).
The most widely used encapsulating material is alginate, a linear
heteropolysaccharide of D-mannuronic acid and L-guluronic acid ex-
tracted from various species of algae, which is cheap, simple to use
and biocompatible (Raei, Rajabzadeh, Zibaei, Jafari, & Sani, 2015;
Sandoval-Castilla, Lobato-Calleros, Garcia-Galindo, Alvarez-Ramirez, &
Vernon-Carter, 2010). Nevertheless, the use of alginate alone has been
widely criticized for “burst release” under acidic conditions (pH b 2.0)
(Cook et al., 2011) as a cross-linked alginate matrix system at very
low pH values is reported to undergo a reduction in alginate molecular
weight causing a faster degradation and release of active ingredients
(Krasaekoopt et al., 2004). Although the alginate matrix is resilient to
acidic condition, severe acidic environments (pH values under 2) de-
grade the matrix and cause the release of its continents. Alternatively,
applying alginate in combination with other wall materials prevents
the in situ burst of the capsule. Cells entrapped in beads also leak, escape
from the gel matrix, and grow in the medium solution. As a result, there
is a pressing need for a better microencapsulation system that can allow
release of highly viable probiotics specifically in the intestine. There are
researches reporting protective effect of multilayer coating of capsules
on survivability of probiotic bacteria in GIT and functional products
(Mokarram, Mortazavi, Najafi, & Shahidi, 2009; Nualkaekul et al.,
2012). A suitable coating should be selected based on the encapsulation
process, cost, stability during storage, legal or religious constraints and
functionality in the final product (Paramera, Konteles, & Karathanos,
2011a).
The eukaryotic structure of yeast cells, along with their presence in
human nutrition (recognized as GRAS material), natural, low cost,
health benefits and non-thermally decomposable properties have
made them an attractive excellent potential and novel encapsulation
wall material (biological vehicle) for the food industry (Paramera,
Konteles, & Karathanos, 2011a, b, Bishop, Nelson, & Lamb, 1998;
Nelson, 2002; Mokhtari, Khomeiri et al., 2017). Also, it has been
shown that dead cells of S. cerevisiae have positive effect against cancer
cells (breast, tongue, colon) in experiments with mice, with no adverse
side effects (Ghoneum, Badr El-Din, Noaman, & Tolentino, 2008). The
yeast cell wall polysaccharides and particularly the water soluble β-
1,3 and β-1,6 D-glucans have shown significant protective antibacterial,
wound-healing, antioxidant, antimutagenic, and antigenotoxic activi-
ties with potent application in anti-infective and anticancer therapy
(Kogan et al., 2008).
S. cerevisiae cell wall is approximately 70–100 nm thick (Osumi,
1998) which is composed of β-glucans, a mannoprotein layer and a
small amount of chitin (Normand, Dardelle, Bouquerand, Nicolas, &
Johnston, 2005). It has been demonstrated that yeasts are stable to
temperatures as high as 250 °C (Bishop et al., 1998). More recently,
using a multi-technique physico-chemistry approach, it has been
shown that flavors encapsulated in empty yeast cells leaked out only
at temperatures higher than 243 °C (Dardelle et al., 2007). By their
phospholipid membranes, yeast cells can behave as liposomes and
have been used for the encapsulation of both hydrophobic and hydro-
philic molecules (Shi et al., 2008). In addition to S. cerevisiae, other
yeasts including Candida utilis, Kluyveromyces fragilis, Torulopsis
lipofera, and Endomyces vernalis have also been applied (Ciamponi,
Duckham, & Tirelli, 2012).
Although encapsulation in yeast cell is a promising approach for
protecting bioactive compounds, to our knowledge, there is no study
about applying yeast cell wall compound for encapsulation of ingredi-
ents larger than the size of yeasts; 4–40 μm (Walker et al., 2002). In
this study, we introduced this idea by physical disintegrating of S.
cerevisiae cell and utilizing the broken wall materials for encapsulation
of probiotic bacteria which are bigger in size. Therefore, the main goal
of this study was to evaluate the feasibility of cell wall compound of
the yeasts as a novel coating material for probiotics and improving the
in vitro survivability of probiotic bacteria in GIT.
2. Materials and methods
2.1. Materials
S. cerevisiae was purchased from Iran Molasses (Mashhad, Iran).
L. acidophilus (PTCC 1643) and B. bifidum (PTCC 1644) were purchased
from Persian Type Culture Collection (IROST, Tehran, Iran). Sodium algi-
nate (viscosity: 15–20 cP, 1% in H2O [L]; ratio of mannuronic
acid:guluronic acid: 3.3 ± 0.3 was obtained from Sigma, UK. MRS
broth and MRS agar and sodium citrate were purchased from Merck,
Germany. All other reagents used in this work were of analytical grade
and purchased from Sigma–Aldrich (St. Louis, USA).
2.2. Preparation of probiotic bacteria
Freeze-dried cells were rehydrated in 20 mL MRS broth and for acti-
vation, incubated at 37 °C for 72 h. For B. bifidum, MRS broth was supple-
mented with filter sterilized 0.05 g/100 g L-cystein hydrochloride
incubated under anaerobic conditions using the Gas Pak system
(Merck, Germany). Bacteria were then activated by growing three
times using a 1% (v/v) inoculum under the same conditions for 16 h
for L. acidophilus and 18 h for B. bifidum according to their assessed
growth curve right after ending logarithmic phase and before starting
stationary phase. The active cultures were used likewise during the pro-
cess to obtaining a cell density of about 109–1010 cfu mL− 1 for each
treatment.
2.3. Microencapsulation of probiotics
Harvesting of cells was done by centrifugation (3K30, Yourlab, Ger-
many) at 3000 × g for 5 min at 25 °C, and the cell pellet was washed
twice with sterile 0.1% peptone solution. Microencapsulation of
probiotics using the internal gelation method was done according to
our previous study (Mokhtari, Khomeiri et al., 2017). Briefly, 80 mL of
sterile (121 °C for 15 min) sodium alginate 2% (v/w) was mixed with
20 mL of initial cell suspension. This suspension of sodium alginate
and probiotic organisms was gently dispensed using a 5 mL plastic sy-
ringe into a beaker containing 500 mL sterile canola oil and 5 g/L Span
80 as the emulsifier and stirred at 200 rpm for 20 min using a magnetic
stirrer (Falc F60, Italy). Then 500 mL calcium chloride (0.1 M) was gent-
ly added until the emulsion was broken and after 30 min, the alginate
microbeads were harvested with two subsequent washings by filter
paper (Whatman No. 4, Fisher Scientific) and stored at 4 °C until ready
for use.
2.4. Double layer coating of alginate microbeads with calcium alginate
Monolayer coated beads were obtained by adding 15 g of previously
prepared alginate microspheres into 100 mL of 0.5% sodium alginate
and stirring 20 min by a magnetic stirrer. The final microspheres were
filtered, collected, and resuspended in 75 g oil containing 5 g/L Tween
80 and 65.5 mmol/L CaCl2 for 20 min to initiate the external Ca2 +
cross-linking of the peripheral alginate layer and obtaining double
layer microcapsuls (Mokarram et al., 2009).
2.5. Triple layer coating of alginate microbeads with S. cerevisiae cell wall
and calcium alginate
For this purpose, S. cerevisiae cells in shape of compressed bakery
yeast were firstly milled by a lab-scale miller device (Moulinex AR100,
France) to produce a completely powdered form (crashed yeast pow-
der). Based on initial trial and error tests, the milling time was 3 min.
White powder stuck to the cap of miller was removed and used; incom-
pletely crashed dried granules were supplied with another small
amount of new granules for the next batch of 3 min. To check the
 S. Mokhtari et al. / Food Research International 96 (2017) 19–26
physical destruction, a dilute cell suspension was prepared by compact
yeast, stained by Crystal Violet color and evaluated by image analysis
(Fig. 1).
A 0.04 (w/v) suspension of milled powder then was prepared in dis-
tilled water and centrifuged at 6000 g force for 10 min, in order to re-
move anything except cell wall patches including filler compounds of
the initial compressed bakery yeast. The pellet was then washed five
times, sterilized (121 °C for 15 min) and stored at 4 °C until ready for
use. For coating, 12 g of previously prepared alginate beads were dis-
solved in 50 mL yeast wall compound by magnetic stirrer at 100 rpm
for 10 min. Finally, obtained microcapsules were coated again by calci-
um alginate to form the third layer as shown schematically in Fig. 2.
The abbreviations used for treatments are given in Table 1.
2.6. Release and enumeration of entrapped cells
To analyze release of bacteria from microbeads, 1 g of each freshly
prepared sample was added into 99 mL of 1% (w/v) sterile sodium cit-
rate solution at pH 6.0 at room temperature and mixed for 10 min by
a magnetic stirrer at 100 rpm. Then, serial dilution was prepared and
enumerated by pour plate counts in MRS agar after incubation at 37 °C.
2.7. Efficiency of encapsulation
To measure encapsulation efficiency, total count of bacteria before
added to sodium alginate solution was determined. 0.1 mL of bacterial
Fig. 1. Microscopic images of Saccharomyces cerevisiae analyzed by light microscopy
(100× magnified). (a) Before crushing and (b) after crushing.
21
suspension was cultured and the Total Viable Cells (TVC) was calculat-
ed. This suspension of bacteria after centrifugation was encapsulated;
entrapped bacteria in microspheres (2.2 mL) were released and enu-
merated according to Section 2.6 and the Entrapped Viable Cells (EVC)
then was calculated. Encapsulation efficiency (EE) was obtained based
on the following equation:
EE ¼ ðEVC=TVCÞ  100:
2.8. Image analysis of capsules
The size and morphology of the beads was observed in an Olympus
BX45 microscope (Olympus Optical Co., Ltd., Japan) using scaled lam
(Erma, China) with a resolution of 0.01 mm (Hosseini, Jafari, et al.,
2015). Images were taken and size analysis was performed as mean di-
ameter ± standard deviation by Image J software in triplicates.
2.9. Determination of acid tolerance of encapsulated bacteria
In vitro studies were based on the method described by Gbassi,
Vandamme, Ennahar and Marchioni (2009). 1 g of freshly prepared en-
capsulated samples were completely dispersed in 10 mL sterile simulat-
ed gastric juice (SGJ) (0.08 M HCl containing 0.2% NaCl, pH 1.55)
without pepsin and incubated for 30, 60, 90 and 120 min at 37 °C.
After incubation, the beads were removed and rinsed with distilled
water. The viable cells of each bacterium were then enumerated in trip-
licates according to Section 2.6, and the counts were expressed as mean
log cfu per gram capsule ± standard deviation. For the free cells, 1.0 mL
of washed cell suspension of each bacterium was added into 10.0 mL SGJ
under the same conditions, and the enumeration of live bacteria in each
sampling time was done by serial dilution and pour plate methods. Each
treatment was performed in triplicate.
2.10. Determination of bile tolerance of encapsulated bacteria
To evaluate survivability of probiotic bacteria in intestinal juice, after
incubating 1 g of each sample in 10 mL SGJ, the samples were neutral-
ized with NaOH solution and then removed and placed in 9 mL sterile
simulated intestinal juice (SIJ) (0.05 M KH2PO4, containing 0.6% bile
salt, pH 7.25) sterilized by a syringe using 0.45 μm Millipore filter. The
tubes were then incubated at 37 °C for 30, 60, 90 and 120 min. The
strong bond between alginic acid and calcium ions become loose in al-
kaline environment which results in fractionation of the capsule struc-
ture. At each sampling time, 1 mL of SIJ containing dissolved beads
was used for serial dilution and bacteria enumerated in triplicates on
MRS agar (Mokarram et al., 2009). Data expressed as mean log cfu per
mL of SIJ ± standard deviation. Each treatment was performed
triplicate.
2.11. Statistical analysis
Results are presented as mean ± standard deviation (SD) of three
replicated determinations. Data were subjected to one-way analysis of
variance (ANOVA) and multiple comparisons were performed by
Duncan's test. Statistical significance was set at P N 0.05. All analyses
were performed using SPSS version 17.0 (SPSS, Chicago, Illinois, USA).
3. Results and discussion
3.1. Size analysis of produced microcapsules
Fig. 3 shows the microscopic images of prepared samples. The aver-
age size of single layer (M), double layer (MCA) and triple layer (MCYA)
encapsulated probiotic capsules were measured 54.25 ± 0.18, 77.43 ±
8.24 and 103.66 ± 13.33 μm, respectively, which were significantly
22 S. Mokhtari et al. / Food Research International 96 (2017) 19–26
Fig. 2. Schematic representation of alginate microbeads (Treatment 1); micorbeads coated by another alginate layer (Treatment 2), and microbeads coated by another two layers of yeast
wall and alginate (Treatment 3).
different (P b 0.05), and the type of bacteria had no significant difference
(P N 0.05) on the result. As shown in size distribution graph (Fig. 4), the
range of size distribution increases by adding extra coating layers so that
M treatment indicates the most focused range followed by MCA and
MCYA, respectively. The results of size analysis were in agreement
with previous studies (Annan et al., 2008; Antunes, Liserre, Coelho,
Menezes, & Moreno, 2013; Mokarram et al., 2009). According to
Sandoval-Castilla et al. (2010), different factors determine particle size
in encapsulation by alginate including alginate solution concentration,
agitation, addition of calcium chloride, calcium chloride solution molar-
ity and emulsifier type.
Sheu and Marshal (1993) found that survival of L. bulgaricus
entrapped in alginate beads and added to frozen desserts was signifi-
cantly higher when beads size fell in-between 30 and 102 μm, than
when beads had a diameter of 15 μm. Hansen, Allan-Wojtas, Jin, and
Paulson (2002) reported that small beads of a size b 100 μm do not sig-
nificantly protect the bacteria in simulated gastric fluid as compared to
Table 1
Treatments used in this study for the encapsulation of probiotics.
Trial Abbreviation
Free Lactobacillus acidophilus F. La.
Free Bifidobacterium bifidum F. Bb.
Single layer microbeads of Lactobacillus acidophilus M. La
Single layer microbeads of Bifidobacterium bifidum M. Bb
Double layer alginate coated microbeads of Lactobacillus acidophilus MCA. La
Double layer alginate coated microbeads of Bifidobacterium bifidum MCA. Bb
Microbeads coated by one layer yeast wall and two layers of MCYA. La
alginate containing Lactobacillus acidophilus
Microbeads coated by one layer yeast wall and two layers of MCYA. Bb
alginate containing Bifidobacterium bifidum
free cells. The optimum size of a bead remains debatable as it varies de-
pending on the applications (Rosas-Flores, Ramos-Ramírez, & Salazar-
Montoya, 2013). Generally, larger bead size provides better protection
against detrimental environment (Sandoval-Castilla et al., 2010). How-
ever, the latter microsphere sizes are too big for incorporation into food
products without affecting the texture (Champagne & Fustier, 2007)
and diameters below 100 μm are preferred for most applications
(Annan et al., 2008).
3.2. Encapsulation efficiency (EE)
Initial total cell population of L. acidophilus and B. bifidum was adjust-
ed in the range of 9–10 log total cfu. The encapsulation efficiency (EE)
was calculated 88.33% and bacterial strain did not have a significant ef-
fect on EE (P N 0.05). The same as capsule size, EE could be affected by
many factors such as wall material, encapsulation technique, concentra-
tion of agents and intensity of procedure. As the population of probiotics
may be affected during the microencapsulation process itself, it is essen-
tial to ensure that the selected microencapsulation technique is gentle
to sensitive probiotic organisms (Pimentel-Gonzales et al., 2009). Vari-
ables such as alginate concentration, biomass concentration and hard-
ening time, have been evaluated for their individual and combined
effects on optimum microencapsulation yields (Trabelsi, Bejar, Ayadi,
Chouayekh, & Kanoum, 2013). In case of using alginate coating, alginate
concentration is one of the most effective factors on encapsulation yield.
Chan (2011) encapsulated oil in calcium alginate and found that the al-
ginate concentration had a significant direct influence on EE. Our results
were in agreement with Krasaekoopt and Watcharapoka (2014), Annan
et al. (2008) and Nag et al. (2011) who encapsulated probiotics and re-
ported EE values of 79.4%, 83% and 89.5%, respectively.
 S. Mokhtari et al. / Food Research International 96 (2017) 19–26
Fig. 3. Microscopic images of single layer alginate microbeads (A), micorbeads coated by
double layer alginate (B) and triple layer microbeads coated by two layers of alginate
and middle layer of yeast wall (C), scaled line indicates 1000 μm.
3.3. Survival of free and encapsulated bacteria in simulated gastric juice
These experiments aimed at evaluating the tolerance of encapsulat-
ed probiotics towards simulated stomach pH, and bile salts present in
the upper GIT. Smith (1995) reported that food remains in the stomach
for between 2 and 4 h while liquids empty from the stomach in only
about 20 min. In this study, we considered 2 h as an average and the re-
sults showing the in vitro viability of probiotics when exposed to the SGJ
is presented in Table 2.
The initial percentage of viable F. La declined by 31% (to 7.63 ±
0.02 log cfu g−1) and 42% (to 5.57 ± 0.02 log cfu g−1) however, F. Bb
showed a decline of 46% (to 5.32 ± 0.05 log cfu g− 1) and 62% (to
3.72 ± 0.04 log cfu g−1) after incubation in the SGJ for 60 and
120 min, respectively. The microencapsulated probiotics, on the other
hand, showed significantly (P b 0.05) enhanced survivability for both
bacteria and all three encapsulation procedures so that number of viable
cells after 120 min were N107 cfu g−1. Mituoka (1992) reported that L.
acidophilus is most active in the small intestine and B. bifidum is most ac-
tive in the large intestine of humans. B. bifidum presented more sensitiv-
ity to acidic conditions by higher log reductions in both free and
23
Fig. 4. Size distribution of different microcapsules.
encapsulated forms compared with L. acidophilus which confirms the
necessity for protection of this strain by encapsulation. Optimum
growth conditions of B. bifidum is under pH 6.5 to 7.3 (Biavati,
Vescovo, Torriani, & Bottazzi, 2000), while L. acidophilus grows readily
at rather low pH values below 5.0 (Bâati, Fabre-Gea, Auriol, & Blanc,
2000). Trains of probiotic bacteria differ with respect to their survival
in acidic environment. Specifically, in the experiment of Ding and
Shah (2008). Bifidobacterium strains were the most acid-sensitive pro-
biotic strains, which was observed in this study too.
For B. bifidum all four treatments showed decline in viable cell count
over 120 min exposure in pH 1.5 at 37 °C. The decrease of F. Bb was sig-
nificantly (P b 0.05) higher than all other treatments by 6.08 ± 0.09 log
reduction. No significant difference was observed between encapsula-
tion techniques (P N 0.05) as the final log reduction of all encapsulated
B. bifidum samples were in the same range by log reduction of 2.48 ±
0.09, 2.38 ± 0.03 and 2.31 ± 0.07 for M, MCY and MCYA treatments,
24 S. Mokhtari et al. / Food Research International 96 (2017) 19–26

Table 2
Viability of probiotics subjected to simulated gastric juices at different times (log cfu g−1).

Probiotic Encapsulation treatments Time (minutes)

0 30 60 90 120

L. acidophilus F. La 9.54 ± 0.11 8.14 ± 0.03 7.63 ± 0.02 6.80 ± 0.04 5.57 ± 0.02
M. La 9.70 ± 0.02 9.59 ± 0.10 9.04 ± 0.09 8.44 ± 0.02 7.93 ± 0.03
MCA. La 9.67 ± 0.04 9.61 ± 0.09 9.56 ± 0.02 9.07 ± 0.13 8.14 ± 0.00
MCYA. La 9.73 ± 0.01 9.68 ± 0.07 9.54 ± 0.06 9.16 ± 0.09 8.63 ± 0.05
B. bifidum F. Bb 9.8 ± 0.02 6.88 ± 0.16 5.32 ± 0.05 5.02 ± 0.12 3.72 ± 0.04
M. Bb 9.55 ± 0.03 9.11 ± 0.01 8.53 ± 0.03 7.47 ± 0.05 7.07 ± 0.14
MCA. Bb 9.55 ± 0.01 8.85 ± 0.02 8.0 ± 0.04 7.91 ± 0.12 7.17 ± 0.02
MCYA. Bb 9.63 ± 0.04 9.44 ± 0.09 8.57 ± 0.01 8.54 ± 0.08 7.32 ± 0.03

Values are average ± standard error.

respectively (Fig. 5). Generally, different encapsulation formulations
showed following trend in reduction of B. bifidum during SGI condition:
MCYA ¼ MCA ¼ MNF:
In accordance with present findings, alginate bead has been previ-
ously reported to result in higher survival of these bacteria (Mandal,
Puniya, & Singh, 2006; Sohail, Turner, Coombes, Bostron, & Bhandari,
2011; Su et al., 2011). Chandramouli, Kailaspathy, Peiris, and Jones
(2004) and Iyer and Kailasapathy (2005) have shown that only the mi-
croencapsulated probiotics were able to maintain viability in gastroin-
testinal conditions, indicating the necessity of encapsulation to protect
probiotics through upper GIT. Significant enhancement of L. acidophilus
survivability by alginate beads coated with 2nd layer of alginate in SGJ
has been reported by Mokarram et al. (2009). According Mokarram et
al. (2009) the coating of beads provides better protection in the SGJ be-
cause a reduced pore size forms in the double layer membrane and as a
result, the diffusion of gastric juice into the beads may be limited. Annan
et al. (2008) have reported an effectively increase in resistance of B.
adolescentis to the SGJ too.
As shown in Fig. 5, the viability of MCYA. La was 88% after 120 min of
treatment. When compared to MCA. La and M. La, applying a 2nd layer
of alginate and 3rd layer of S. cerevisiae cell wall compound has signifi-
cantly (P b 0.05) enhanced the survivability by at least 4% and 7%, re-
spectively. The relative superiority of MCYA. La in preserving the L.
acidophilus following 120 min exposure to pH 1.55 is therefore clear.
The order was as following:
MCYANMCANMNF:
For the first time, we found in this study that presence of S. cerevisiae
cell wall compound as a coating layer around L. acidophilus alginate
beads exerted a significant (P b 0.05) protective effect when exposed
Fig. 5. Efficacy of coating layers on death rate of probiotics through simulated gastric juice
at 37 °C for 2 h. Values with the same letters are not significantly different (P N 0.05). F:
Free probiotics, M: single layer alginate microbeads, MCA: micorbeads coated by double
layer alginate, and MCYA: triple layer microbeads coated by two layers of alginate and a
middle layer of yeast wall.
to GIT in comparison to the free cells or cells entrapped in alginate
beads uncoated and coated by alginate. It could be explained by the
structural preservative impact of the yeast cell wall compound on resis-
tance of entrapped probiotic bacteria by retarding gastric fluid perme-
ation into the capsules.
For B. bifidum however, the log reduction of bacteria in the form of
single, double, and triple layer coated beads were not significantly dif-
ferent (P N 0.05). It indicates that S. cerevisiae cell wall compound as
extra coating layer could not rebate rate of population reduction of en-
capsulated B. bifidum under pH 1.55. As previously also mentioned, this
may be due to low resistance of this strain to low pH values which did
not permit extra layers to exhibit their protection.
Annan et al. (2008) reported that there is a strong need to coat the
pepsin-sensitive gelatin matrix with a biocompatible non-toxic and
pepsin-resistant polymer like alginate, which is insoluble at acidic pH
but dissolves in the alkaline pH of the intestine.
3.4. Survival of free and encapsulated bacteria is simulated intestine juice
Both free and encapsulated L. acidophilus and B. bifidum exposed to
the SGJ previously for 120 min and were subjected to SIJ sequential in-
cubation containing bile salts (pH 7.25) for 2 h at 37 °C. The results are
illustrated in Table 3.
In the case of F. La, the initial viable count of 9.19 ± 0.12 log cfu mL−1
was reduced to 2.08 ± 0.14 log cfu mL−1 after 120 min exposure to the
SIJ. In general, the log reduction of all treatments for L. acidophilus incu-
bated in SGJ was lower than the test when they were incubated in SIJ
with bile salt. This may be due to the fact that the environmental resis-
tant of lactic acid bacteria is determined by many factors such as their
medium and cytoplasmic membrane composition (Begley, Cormac, &
Hill, 2005). Therefore, alkaline pH of the intestinal juice (7.25) is not
suitable for this strain to grow readily than rather low pH values
below 5.0 (Bâati et al., 2000). Bifidobacterium has been counted as an
important portion of human gut microbiota and is the most resistant
genera to bile as its differential media is supplemented by bile. F. Bb
showed significantly (P b 0.05) higher survivability than F. La by final
log reduction of 6.65 ± 0.1 and 7.11 ± 0.12, respectively. This difference
was not significantly recognized between M. Bb and M. La and both
treatments significantly (P b 0.05) enhanced bile resistance of free bac-
teria when exposed to this condition (Fig. 6). It has been reported that
survival of microencapsulated B. bifidum (Chávarri et al., 2010) and L.
acidophilus (Chandramouli et al., 2004) in the SIJ is enhanced by encap-
sulation in calcium alginate.
According to Su et al. (2011), release of alginate beads with a mean
diameter of 400 μm when exposing to the SIJ reached a peak at
30 min then changed slowly. Alginate beads getting weakened results
in the penetration of bile salt into the capsules which affects probiotic
cells. In our experiment, capsules were apparently dissolved before
30 min of exposure to the SIJ. After quite release of bacteria, they
would be freely exposed to environmental liquid, therefore practically
the helpful examination time is before liquidation of microsphere or
capsule walls, accordingly, we evaluated the efficacy of alginate and S.
 S. Mokhtari et al. / Food Research International 96 (2017) 19–26 25

Table 3
Viability of probiotics subjected to simulated intestinal juices at different times (log cfu mL−1).

Probiotic Encapsulation treatments Time (minutes)

0 30 60 90 120

L. acidophilus F. La 9.19 ± 0.12 5.03 ± 0.16 4.62 ± 0.16 4.03 ± 0.04 2.08 ± 0.14
M. La 9.49 ± 0.21 7.67 ± 0.03 6.81 ± 0.01 5.90 ± 0.24 6.09 ± 0.13
MCA. La 9.10 ± 0.10 8.87 ± 0.09 7.55 ± 0.02 7.25 ± 0.03 6.18 ± 0.1
MCYA. La 8.51 ± 0.15 8.05 ± 0.03 7.91 ± 0.04 7.20 ± 0.05 6.09 ± 0.10
B. bifidum F. Bb 8.14 ± 0.14 6.36 ± 0.02 4.11 ± 0.06 2.87 ± 0.16 1.49 ± 0.04
M. Bb 8.69 ± 0.05 8.54 ± 0.04 8.41 ± 0.11 7.85 ± 0.20 5.96 ± 0.21
MCA. Bb 8.16 ± 0.03 7.92 ± 0.02 7.71 ± 0.14 6.70 ± 0.03 5.54 ± 0.02
MCYA. Bb 8.78 ± 0.02 8.53 ± 0.15 8.15 ± 0.11 7.53 ± 0.07 6.17 ± 0.05

Values are average ± standard error.

cerevisiae cell wall compound layer on retarding this time. For double
layer alginate coating, both MCA. La and MCA. Bb indicated a significant
(P b 0.05) enhancement in survivability of probiotics compared with
their single layer form and free bacteria treatments (Fig. 6).
Krasaekoopt et al. (2004) illustrated that uncoated alginate beads con-
taining L. acidophilus provide less protection of bacteria than coated
beads by extra layer of alginate in 0.6% bile SIJ (pH 7.43) for 2 h after in-
cubation in the SGJ (pH 1.55) for 2 h. They also reported after incubation
in 0.6% bile salt solutions (pH 8.25) at 37 °C for 2 h, there was no signif-
icant enhancement in viability of B. bifidum encapsulated in uncoated
and coated beads; however, survivability of L. acidophilus improved by
applying 2nd layer of alginate on its microsphere. Chaikham,
Chunthanom, and Jirasatid (2015) also reported that L. acidophilus and
B. animalis entrapped in coated alginate beads with alginate sodium
had higher viability than uncoated alginate, demonstrating that the
technique was effective for the protection of probiotics. Similarly,
Annan et al. (2008) reported that coated microspheres are physically
stronger as indicating by their resistance to degradation in the SIJ and
require longer homogenization times for complete disintegration.
As shown in Fig. 6, in case of B. bifidum, there was no significant dif-
ference (P b 0.05) between MCA. Bb and MCYA. Bb, however, applying S.
cerevisiae cell wall compound layer in MCYA. La significantly (P b 0.05)
prolonged the population reduction of L. acidophilus in comparison with
MCA. La by log reduction of 2.42 ± 0.06 and 2.92 ± 0.04, respectively.
Generally, different encapsulated treatments showed following trend
in reduction of probiotics during the SIJ condition:
L:acidophilus : MCYANMCANMNF
B:bifidum : MCYA ¼ MCANMNF:
Fig. 6. Efficacy of coating layers on death rate of probiotics through simulated intestinal
juice at 37 °C for 2 h after previous incubation in gastric juice for 1 h. Values with the
same letters are not significantly different (P N 0.05). F: Free probiotics, M: single layer
alginate microbeads, MCA: micorbeads coated by double layer alginate, and MCYA:
triple layer microbeads coated by two layers of alginate and a middle layer of yeast wall.
4. Conclusion
The present study revealed that it is possible to apply yeast cell wall
for protection of large scale capsulants; an extra layer of S. cerevisiae cell
wall compounds significantly offers a protective means for delivery of
viable bacterial cells in higher levels to the colon after passing through
the adverse conditions of the gastrointestinal track. S. cerevisiae cell
wall was able to strengthen acid tolerance of L. acidophilus as a relatively
acid resistant bacterium but could not enhance this feature for B. bifidum
as a highly sensitive probiotic. Therefore, it possesses a protective ability
when applying in probiotic capsules which depends on the type of bac-
teria. Further case studies are required to extend this application to
other potential fields. As prospective studies, evaluating the application
of this method for dried components as well as its stability throughout
the shelf life of the products are suggested. The assessment of its appli-
cation for encapsulating other food bioactive components can be con-
sidered too.
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