Food Research International 66 (2014) 424431

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Food Research International

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

Effect of microencapsulation of Lactobacillus acidophilus LA-5 on

physicochemical, sensory and microbiological characteristics of stirred
probiotic yoghurt
Maria Ceclia E. Ribeiro a,, Karina S. Chaves a, Clarice Gebara a, Flvia N.S. Infante b,
Carlos R.F. Grosso b, Mirna L. Gigante a
a
b

Department of Food Technology, Faculty of Food Engineering, University of Campinas, UNICAMP, 13082-862 Campinas, SP, Brazil
Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, UNICAMP, 13082-862 Campinas, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 30 July 2014
Accepted 22 October 2014
Available online 30 October 2014
Keywords:
Probiotic
Microencapsulation
Yoghurt
Viability
Simulated gastrointestinal conditions
Sensory acceptance

a b s t r a c t
The aim of this study was to evaluate the characteristics of probiotic stirred yoghurt and the survival of free and
microencapsulated cells of Lactobacillus acidophilus LA-5 during refrigerated storage. L. acidophilus LA-5 was microencapsulated by ionic gelation and complex coacervation techniques using pectin and whey protein as wall
and coating materials, respectively. The survival of probiotics subjected to conditions simulating the passage
through the gastrointestinal tract and the sensory acceptance of yoghurts were evaluated after 35 days of refrigerated storage. The yoghurt containing encapsulated L. acidophilus LA-5 showed lower post-acidication and improved probiotics survival (62%) when compared to the yoghurt containing free cells of L. acidophilus LA-5 (10%)
after 35 days of refrigerated storage. The encapsulated L. acidophilus LA-5 showed higher survival than the free
microorganism during simulated gastrointestinal conditions. After 35 days of storage, no signicant difference
was observed for the attributes appearance, aroma, avor, and overall impression for both samples. However,
with respect to the attribute texture, the yoghurt containing encapsulated L. acidophilus LA-5 was less accepted
than yoghurt containing free cells of L. acidophilus LA-5. Microencapsulation of L. acidophilus LA-5 by ionic
gelation and complex coacervation provided lower post-acidication in probiotic yoghurts, and protection to
the microorganism, during 35 days of refrigerated storage and during the passage through the simulated gastrointestinal conditions.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Probiotics are dened as live microorganisms which when administered in adequate amounts confer a health benet on the host (FAO/
WHO, 2001). The concept of probiotics has been hypothesized by Elie
Metchnikoff in the early 1900s, when he associated the longevity of
Bulgarian peasants with their high consumption of fermented dairy
products. Subsequently, it was observed that the microorganisms
present in yoghurt confer protection to the gastrointestinal tract against
the damaging effects of harmful bacteria (Tripathi & Giri, 2014). In this
context, studies have been performed with different microorganisms,
showing the benets provided by the probiotics for human use, as the
reduction of lactose intolerance, reduced cholesterol levels, stimulation
of the immune system, relief from constipation, increased minerals
absorption, as well as anti-mutagenic, anti-carcinogenic, and antihypertensive effects (Charteris, Kelly, Morelli, & Collins, 1998; Lomer,
Parkes, & Sanderson, 2008; Palomar, Galdeano, & Perdign, 2014;
Corresponding author. Tel.: +55 19 35213993.
E-mail address: cissaribeiro@gmail.com (M.C.E. Ribeiro).

http://dx.doi.org/10.1016/j.foodres.2014.10.019
0963-9969/ 2014 Elsevier Ltd. All rights reserved.

Tuohy, Probert, Smejkal, & Gibson, 2003; Vasiljevic & Shah, 2008; Zhu,
Luo, Jobin, & Young, 2011). The effect of probiotics on human health
has generated great interest for the food industry, since these microorganisms represent an important category within the functional food
segment (Stanton et al., 2001).
The market of functional foods has increased considerably, from $33
billion in 2000 to $176.7 billion in 2013, representing an increase of 5%
of the global food market (Tripathi & Giri, 2014). This growth is associated with the consumer perception, not only as a source of nutrients,
but also as promoters of health and wellness (Sanders & Marco, 2010;
Sir, Kpolna, Kpolna, & Lugasi, 2008). Yoghurts and fermented milks
are the most popular food carriers for the delivery of probiotics due to
their great acceptance by consumers and excellent nutritional value
(Antunes et al., 2007; Shah, 2000).
The genera Lactobacillus sp. and Bidobacterium sp. have been the
most studied and used in probiotic foods in recent years. To confer
their benecial effect, the probiotics must survive the processing operations and storage, as well as the gastric environment, hydrolytic
enzymes and bile salts from the gastrointestinal tract, while not
adversely affecting the physicochemical and sensory characteristics of

M.C.E. Ribeiro et al. / Food Research International 66 (2014) 424431

the product (Del Piano et al., 2006; Ding & Shah, 2007; Heller, 2001,
Liu et al., 2007). However, some strains of these genera are sensitive
to acid and bile salts (Hansen, Allan-Wojtas, Jin, & Paulson, 2002;
Mohammadi, Mortazavian, Khosrokhava, & Cruz, 2011). Food characteristics may inuence the strains survival, once they can protect the
probiotics by reducing their physical exposure during passage through
gastrointestinal tract (Ranadheera, Evans, Adams, & Baines, 2012).
However, this protection may be not sufcient for maintenance of
probiotic viability until their site of action. Thus, new strategies have
been developed for the maintenance and protection of the viability of
probiotic microorganisms, and microencapsulation has proven to be a
promising method for the protection of probiotics.
Microencapsulation is a micropackaging technology that uses thin
polymer coatings to enclose droplets of liquid, solid or gaseous material,
being used primarily to protect the encapsulated material from adverse
conditions (Annan, Borza, & Hansen, 2008; Shoji et al., 2013).
The production of microparticles by ionic gelation using natural
polysaccharides and calcium ions does not require the use of high temperatures or organic solvents (Krasaekoopt, Bhandari, & Deeth, 2004;
Patil, Kamalapur, Marapur, & Kadam, 2010). Among the encapsulating
agents, pectin that is a natural polymer extracted from pectic material
of some fruits is considered less sensitive to chemical agents and more
resistant to acids and intestinal environments when compared to alginate, a polysaccharide widely used as encapsulating agent (Brando &
Andrade, 1999; Parkar et al., 2010; Voo, Ravindra, Tey, & Chan, 2011).
Pectin has also been investigated by its prebiotic properties (Nazzaro,
Fratianni, Nicolaus, Poli, & Orlando, 2012). The resulting gel matrix is
porous, making microparticles sensitive to the acidic conditions of the
medium (Burey, Bhandari, Howes, & Godley, 2008; Mortazavian,
Razavi, Ehsani, & Sohrabvandi, 2007). To improve the characteristics of
these microparticles, researchers have used a combination of techniques to ensure a greater protective effect on microorganisms
(Chvarri et al., 2010; Gbassi, Vandamme, Ennahar, & Marchioni,
2009; Gebara et al., 2013; Krasaekoopt et al., 2004; Lambert,
Winbreck, & Kleerebezem, 2008; Souza et al., 2012). Whey proteins
are one of the coating agents that have been studied once they are
considered a versatile nutritional source. Different microorganisms,
encapsulation techniques, and wall materials have been used successfully in maintaining the viability of probiotics in yoghurt (Adhikari,
Mustapha, & Grun, 2003; Brinques & Ayub, 2011; Kailasapathy, 2006;
Kailasapathy & Sureeta, 2004; Krasaekoopt, Bhandari, & Deeth, 2006;
Pavunc et al., 2011; Pinto et al., 2012; Shoji et al., 2013; Ziar, Grard, &
Riazi, 2012). In general, studies have shown that the incorporation of
encapsulated Lactobacillus acidophilus by different techniques into the
yoghurt ensures a better survival of the probiotic microorganism
when compared to the free microorganism. Krasaekoopt et al. (2006)
found an increase of 1 log cycle on the viability of L. acidophilus 547
encapsulated in chitosan-coated alginate microparticles by extrusion
technology when compared to the free yoghurt cells after 4 weeks of
refrigerated storage. Shoji et al. (2013) observed a reduction in the
viability of L. acidophilus Lac-04 encapsulated with pectin and casein
by a complex coacervation technique (~ 1.47 log cycles) in yoghurt
made from buffalo milk, when compared to the free microorganism
(4.66 log cycles) after 5 weeks of refrigerated storage. Despite the
promising results, it is important to evaluate probiotic survival during
the passage through the gastrointestinal tract, once it is desirable that
they arrive at their site of action in adequate concentrations to confer
its probiotic effect.
The combination of two microencapsulation techniques was investigated: ionic gelation and complex coacervation, which does not expose
the microorganisms to harmful conditions. Besides that, the use of pectin as wall material and whey protein concentrate as coating material
may be effective on the protection of probiotic bacteria.
Thus, the general objective of this study was to produce and evaluate
the characteristics during refrigerated storage of probiotic stirred yoghurt containing L. acidophilus LA-5 in free and encapsulated forms,

425

using ionic gelation and complex coacervation as encapsulation techniques, and pectin and whey protein concentrate as wall and coating
materials, respectively. The survival of the free and encapsulated microorganism during the simulation of the passage through the gastrointestinal tract was also assessed.
2. Material and methods
2.1. Materials
The following ingredients were used: whole UHT milk (Shefa Ltda,
Amparo, SP, Brazil); and instant skim milk powder (Molico, Nestl
Brazil Ltda, Araatuba, SP, Brazil); culture of Streptococcus thermophilus
and Lactobacillus delbrueckii subsp. bulgaricus YF-L812 (Christian
Hansen, Valinhos, SP, Brazil); probiotic culture of L. acidophilus LA-5
(Christian Hansen, Valinhos, SP, Brazil); low methoxyl amidated pectin
GENU (CPKelco, Limeira, SP, Brazil); whey protein concentrate (80%
Lacprodan, Arla Foods Ingredients, Portea, CO, Argentina); and unsalted butter (Laticnios Aviao, So Sebastio do Paraso, MG, Brazil). MRS
agar (Difco-BD, USA), mucin (M1778), pepsin (P7012), pancreatin
(P1625) and bile (B3883) (Sigma-Aldrich Co., St. Louis, MO, USA)
were also used. All reagents were analytical grade.
2.2. Preparation of traditional lactic acid culture and cell concentrate of
L. acidophilus LA-5
Lyophilized culture of S. thermophilus and L. delbrueckii subsp.
bulgaricus (50 U DVS) was inoculated into sterile reconstituted skim
milk at 11% total solids, and incubated at 45 C for about 3 h until
coagulation. The culture was kept under refrigeration until used.
For each experiment, the cell concentrate of L. acidophilus LA-5 was
prepared for use in the microcapsules and yoghurt according to
Gebara et al. (2013).
2.3. Production and characterization of microcapsules containing
L. acidophilus LA-5
The probiotic microorganism was microencapsulated by combining
ionic gelation and complex coacervation techniques as described by
Gerez, Font de Valdez, Gigante, and Grosso (2012), Souza et al. (2012)
and Gebara et al. (2013). Pectin and whey protein concentrate were
used as wall and coating materials, respectively. First, an emulsion
was obtained using an aqueous solution of pectin (2% w/w) at pH 4.0
and melted unsalted butter (2% w/w) through homogenization
(Ultraturrax homogenizer, IKA Works Inc., Staufen, Germany) at
14,000 rpm for 5 min. After addition of the cell concentrate of
L. acidophilus LA-5 (2% v/v), the emulsion was homogenized
(6000 rpm for 1 min), and atomized in a calcium chloride solution
(2% w/v, pH 4.0) under stirring (410 rpm). The microcapsules remained
in the calcium chloride solution for 30 min (hardening time) and then
they were washed in a sieve (pore size 0.125 mm) using sterile distilled
water, pH 4.0. After ionic gelation, the microcapsules were coated by
complex coacervation by immersion (30 min) in a 4% (w/v) solution
of whey protein concentrate previously heat treated (80 C for
15 min) and adjusted to pH 4.0. Microencapsulation was carried out
at room temperature (25 C) under aseptic conditions in laminar ow
chamber.
The microcapsules were obtained the day before yoghurt manufacture, and maintained under refrigeration (b10 C) until use. Moisture
content was determined by oven drying at 105 C, and the protein
content was determined by the Kjeldahl method, using the conversion
factor 6.38 (AOAC, 2006).
The morphology of the microcapsules was observed under a scanning electron microscope (SEM Jeol JMS T300, Tokyo, Japan) at an
accelerating voltage of 20 kV. The samples were xed in stubs doublesided copper tape and coated with a thin gold layer (180 s and a current

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M.C.E. Ribeiro et al. / Food Research International 66 (2014) 424431

of 40 mA) using a Baltzer evaporator (Baltec SCD50, Liechtenstein,

Austria).

conditions (Dave & Shah, 1996). The percent survival was calculated
based on the population of viable cells expressed as CFU/g, as shown
in Eq. (1) (Adhikari et al., 2003 and Mortazavian et al., 2008):

2.4. Viability of microencapsulated L. acidophilus LA-5

The viable counts of free and microencapsulated microorganisms
were determined. The microcapsules were placed in sodium citrate solution (2% w/v) pH 7.0 under vigorous stirring for 5 min to break the
capsules and release the microorganisms (Grosso & Fvaro-Trindade,
2004; Krasaekoopt et al., 2004). Then, serial dilutions were made in
0.1% (w/v) peptone water followed by pour plating in MRS agar using
Petri plates. The plates were incubated at 37 C for 72 h in an anaerobic
jar using Anaerogen (Oxoid), with subsequent enumeration of the
probiotic microorganisms (De Man, Rogosa, & Sharpe, 1960).
2.5. Manufacture of stirred yoghurt containing free and microencapsulated
L. acidophilus LA-5
Free or microencapsulated L. acidophilus LA-5 was added to the milk
with the yoghurt starter culture. For yoghurt manufacture, whole UHT
milk (6 L) standardized to 13% total solids (using milk powder, 95.67%
total solids) was heat treated (85 C/5 min) and cooled to 42 1 C.
The volume was divided into two portions (3 L each), to which
2.5% (v/v) yoghurt starter culture (S. thermophilus and L. delbrueckii
subsp. bulgaricus) and 1% (v/v) L. acidophilus LA-5 free or 10% (w/v) microcapsules containing L. acidophilus LA-5 were added. The fermentation was carried out at 42 1 C and pH and titratable acidity were
determined every 20 min. The fermentation time was considered the
time required for the product to reach pH 4.8 0.05. After cooling
(10 1 C), the yoghurt samples were stored under refrigeration for
24 h and then were stirred slowly using a stainless steel perforated
stirrer. The stirred yoghurts were packaged in 200 mL plastic cups,
which were sealed with aluminum caps and stored in a cold cabinet
(5 1 C). Three complete replications of the experiment were
performed.
2.6. Physicochemical and microbiological characterization of yoghurts
After one day of storage (5 1 C), the yoghurt samples were
characterized for pH, titratable acidity, total solids, total protein by
the Kjeldahl method, fat by Mojonnier method and ash (AOAC, 2006).
The lactose content was calculated by difference [total solids
(protein + fat + ash)] as used by Sodini, Morin, Olabi, and JimnezFlores (2006). The pH and the viable cell count were determined after
1, 7, 14, 21, 28 and 35 days of refrigerated storage.
For microbiology analysis, the yoghurts were diluted in 2% sodium
citrate solution (w/v) pH 7.0 to break the capsules and release the microorganisms, and stirred at medium speed for 15 min (Stomacher
400, Seward Laboratory Systems, Port Saint Lucie, FL, USA). The same
procedure was performed for the yoghurt containing free microorganisms. Serial dilutions in 0.1% peptone water (w/v) were made and
pour plated in Petri dishes.
The viability of microorganisms in yoghurt was assessed by selective
methodologies. The enumeration of L. acidophilus LA-5 was determined
in MRS agar with 0.15% bile. The plates were incubated at 37 C for 72 h
under anaerobic conditions, with subsequent enumeration of the probiotic microorganisms (Vinderola & Reinheimer, 1999). The enumeration
of S. thermophilus was performed using ST (S. thermophilus) agar, and
the plates were incubated at 30 C for 72 h under aerobic conditions
(Zacarchenco & Massaguer-Roig, 2004). To prepare 1 L of ST agar, the
following ingredients were used: 10 g tryptone, 10 g sucrose, 5 g yeast
extract, and 2 g of K2HPO4 dissolved in 1 L distilled water. The pH was
adjusted to 6.8 0.1 and 6 mL of bromocresol purple solution and
12 g agar were added to the medium. For enumeration of L. delbrueckii
subsp. bulgaricus, RCA (Difco-BD, USA) was used with pH adjusted to
5.3, and the plates were incubated at 45 C for 72 h under anaerobic


% survival

f inal population C FU=g

.
initial population C FU=g


 100

2.7. Viability of L. acidophilus LA-5 in yoghurt during the passage through

the gastrointestinal tract
For the simulation of gastrointestinal conditions, simulated gastric
juice and intestinal juice were prepared according to Mozzi, Gerbino,
Font De Valdez, and Torino (2009) and Picot and Lacroix (2004), with
modications as described by Gebara et al. (2013). Simulated gastric
juice (SGJ) at pH 3.0 and simulated intestinal juice (SIJ) at pH 7.0 were
used in the experiments. The 1% bile solution (w/v) was sterilized
(121 C/15 min) and the pH was adjusted to 7.0 with 1 N NaHCO3.
The viability of L. acidophilus LA-5 in yoghurt during simulation of
gastrointestinal conditions was assessed after 35 days of refrigerated
storage (5 C) in accordance with a methodology adapted from Wang
et al. (2009). One gram of yoghurt was added to 10 mL SGJ (pH 3.0),
followed by incubation at 37 C under agitation in a metabolic bath
with stirring arrangement. Viability was assessed at time intervals of
0, 60, and 120 min. After 120 min of exposure to SGJ, pancreatin
solution was added and the pH of the samples was adjusted to 7.0,
thereby obtaining the simulated intestinal juice (SIJ). The samples
remained incubated for a further 300 min, and a new aliquot was removed for assessment of the microorganism viability. To evaluate the
resistance to bile, 1 g yoghurt was mixed with 10 mL of 1% bile solution
(pH 7.0), and incubated at 37 C under agitation in a metabolic bath
with stirring arrangement. After 0, 60, and 300 min of exposure to bile
solution, the samples were subjected to analysis of L. acidophilus LA-5
viability.
The aliquots were transferred to 2% sodium citrate solution (w/v)
pH 7.0 under vigorous stirring to break the capsules and release the
microorganisms. The same treatment was applied to the yoghurt
containing free microorganisms. The viable cell count was carried out
in MRS-bile agar and the plates were incubated at 37 C for 72 h
under anaerobic conditions, as described in Section 2.6.
2.8. Sensory evaluation
The sensory evaluation of yoghurts was performed by consumer acceptance test (Villanueva & Da Silva, 2009) based on the appearance,
texture, avor, aroma, and overall impression of the product, using a
9-point hedonic scale (1 disliked extremely; 9 liked extremely). A
team of 100 regular consumers of yoghurt was randomly recruited
to participate in the test. Samples were served in plastic recipients
coded with 3 digit random numbers, at 710 C, and the analysis
was carried out in individual booths under white light. The sensory
evaluation of the yoghurt samples was approved by the Ethics in Research Committee (University of Campinas, Campinas, SP, Brazil;
register no. 799/2009) and was performed after 1 and 35 days of
refrigerated storage.
2.9. Experimental design and statistical analysis
A randomized block design with a 2 6 factorial arrangement and
three replications was used. The factor delivery of probiotics had 2 levels
(free or microencapsulated), and the factor refrigerated storage time
had 6 levels (1, 7, 14, 21, 28 and 35 days after manufacture). The results
were assessed by analysis of variance (ANOVA) and Tukey's test at 5%
signicance level for comparison between means. The data were analyzed using the software STATISTICA 7.0 (StatSoft Inc, Tulsa, OK, USA).

M.C.E. Ribeiro et al. / Food Research International 66 (2014) 424431

427

3. Results and discussion

3.2. Characterization of probiotic yoghurts

3.1. Characteristics of microcapsules and viability of L. acidophilus LA-5

during microcapsules manufacture

During fermentation, both yoghurts exhibited the same behavior of

decreasing pH and increasing acidity over time and reached pH 4.8
0.05 in 180 min. Similar incubation time was observed for both yoghurts
with free and microencapsulated L. acidophilus LA-5 once the addition of
probiotic bacteria (free or microencapsulated) did not affect the coagulation time, suggesting the acidication time of L. acidophilus was not
sufcient to alter the fermentation time of yoghurt. Despite
L. acidophilus utilizes lactose that is the main sugar found in milk, it is
not capable of initiating rapid growth in milk (Srinivas, Mital, & Garg,
1990). Thus, the contribution of these bacteria for yoghurt fermentation
time was not signicantly.
The physicochemical and microbiological characterization of the probiotic yoghurts are presented in Table 1. All yoghurts met the parameters
required by the Brazilian legislation for fermented milks, that claim
the total viable lactic bacteria count must be at least 107 CFU/g in the
product (Brasil, 2007). The number of viable cells of S. thermophilus for
the yoghurt with free and microencapsulated L. acidophilus LA-5 was
1.41 109 CFU/g and 1.67 109 CFU/g, respectively. The counts of
L. bulgaricus were 4.46 107 CFU/g and 1.33 107 CFU/g for yoghurt
containing free and microencapsulated L. acidophilus LA-5, respectively.
Regarding the probiotic characteristics, the yoghurt containing free
L. acidophilus LA-5 and microencapsulated L. acidophilus LA-5 presented
3.02 1010 CFU/200 g, and 3.60 109 CFU/200 g, respectively, whose
values are within the limit established by legislation, which is a minimum of 108 to 109 CFU in the daily recommendation of the product
ready-to-eat, which is 200 g or mL for yoghurts (Brasil, 2003).
It was observed that the treatments signicantly affected the pH,
acidity, and the contents of protein, total solids, ash, and lactose of the
yoghurts. With regard to the acidication of the product, the yoghurt
containing free L. acidophilus LA-5 showed lower pH and higher acidity
than that produced with the microencapsulated microorganism.
Although immediately after fermentation both yoghurts with free and
microencapsulated L. acidophilus LA-5 showed pH of 4.81 0.02, a
greater decrease in pH values was observed after the rst day of refrigerated storage for the yoghurt containing free L. acidophilus LA-5 when
compared to the microencapsulated microorganism, with pH values of
4.34 0.02, and 4.50 0.02, respectively. The same behavior was
observed for acidity. The lower acidity of the product containing the microencapsulated microorganism may be an advantage for the probiotics
survival during refrigerated storage. The lower acidication behavior
was reported by Kailasapathy (2006), who compared set yoghurt

The microcapsules containing L. acidophilus LA-5, showed, on average, 96.9 0.7% moisture and 20.0 4.1% protein on a dry basis. During
the production of microcapsules, no loss in viability of L. acidophilus
LA-5 was observed. The cell concentrate of L. acidophilus LA-5 used
in the experiments presented 1.25 1010 CFU/mL, the emulsion (pectin
and butter) presented 2.5 108 CFU/mL, and the enumeration of
L. acidophilus LA-5 in microcapsules was 2.29 0.24 108 CFU/g.
These results indicate that the combination of both ionic gelation
and complex coacervation techniques, and the use of pectin as wall
material and whey protein concentrate as coating material were effective for the microencapsulation of L. acidophilus LA-5. A reduction
of the number of viable cells was observed only due to the dilution
factor.
Fig. 1 shows images obtained by scanning electron microscopy
(SEM) of the microcapsules containing L. acidophilus LA-5 obtained by
ionic gelation and complex coacervation, which evidences the presence
of probiotic microorganisms.

Table 1
Physicochemical and microbiological characterization of probiotic yoghurts on day 1
(mean SD, n = 3).
Characterization

Fig. 1. Scanning electron microscopy (SEM) of microcapsules obtained by ionic gelation and complex coacervation with L. acidophilus LA-5. (A) Microcapsules at 900
magnication, bar = 10 m; (B) Cells of L. acidophilus LA-5 (marked with arrows)
can be found randomly distributed within the microcapsules at 4000 magnication,
bar = 1 m.

Yoghurt with free

L. acidophilus LA-5

Yoghurt with
microencapsulated
L. acidophilus LA-5

Physicochemical
pH
Acidity (% lactic acid)
Protein (%)
Fat (%)
Total solids (%)
Ash (%)
Lactose (%)*

4.34 0.02b
0.80 0.02a
3.38 0.10b
3.21 0.02a
12.71 0.06a
0.89 0.01a
5.23 0.03a

4.50 0.02a
0.75 0.01b
4.04 0.05a
3.22 0.03a
12.10 0.01b
0.85 0.02b
3.99 0.02b

Microbiological
S. thermophilus (CFU/g)
L. bulgaricus (CFU/g)
L. acidophilus LA-5 (CFU/g)
L. acidophilus LA-5 (CFU/200 g)

1.41
4.46
1.51
3.02

109b
107a
108a
1010a

1.67
1.33
1.80
3.60

109a
107b
107b
109a

Calculated by difference.
a,b

Means followed by different lower case letters in the same line differ signicantly
(p b 0.05).

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M.C.E. Ribeiro et al. / Food Research International 66 (2014) 424431

samples containing free and encapsulated L. acidophilus DD910 and

Bidobacterium lactis.
Regarding the physicochemical composition, the yoghurt produced
with free microorganisms had higher total solids, lactose and ash contents, and lower protein content than the yoghurt containing encapsulated L. acidophilus LA-5. The fat content was not signicantly affected
by the treatments. The higher protein content found in the probiotic yoghurt containing the microcapsules (4.04%) as compared to the yoghurt
with free microorganisms (3.38%) may be due to the composition of the
microcapsules, which were coated with whey protein by complex
coacervation and showed 20% protein on a dry basis. Although this is
not the focus of the present study, higher protein contents can positively
affect the nutritional characteristics of the product. Several studies
(Adhikari et al., 2003; Kailasapathy, 2006; Kailasapathy & Sureeta,
2004; Krasaekoopt et al., 2006; Picot & Lacroix, 2004) on the production
of yoghurt using free and encapsulated probiotics did not compare the
physicochemical composition of the products.
With respect to the microbiological characterization, it was observed
that the treatments signicantly affected the initial population of each
microorganism (S. thermophilus, L. bulgaricus and L. acidophilus LA-5).
Although signicant, the difference in relation to the initial population
of S. thermophilus and L. bulgaricus is of little importance from a technological point of view, since the yoghurts presented total counts of lactic
acid bacteria above the minimum of 107 CFU/g required by legislation.
3.3. Effect of refrigerated storage on the characteristics of probiotic yoghurts
3.3.1. Post-acidication and viability of microorganisms
The pH of the yoghurts was signicantly affected by the treatments
(p b 0.0001), storage time (p b 0.0001), and interaction between
these factors (p b 0.0001). The yoghurt with free L. acidophilus LA-5
had the lowest pH (4.17) when compared to the yoghurt containing
the encapsulated L. acidophilus LA-5 (4.34). Fig. 2 shows that the yoghurt with free L. acidophilus LA-5 exhibited higher post-acidication
than the yoghurt with microencapsulated L. acidophilus LA-5, in
which the pH remained almost constant after 7 days of refrigerated
storage. Kailasapathy (2006) and Mortazavian et al. (2008) also observed
lower post-acidication in probiotic yoghurts containing encapsulated
L. acidophilus when compared to the free form. The post-acidication is
undesirable for both the sensory quality of yoghurt and microorganisms
viability (Walstra, Wouters, & Geurts, 2006). In this study, the lower
post-acidication of the yoghurt with encapsulated L. acidophilus LA-5
indicated that the encapsulation process decreased the activity of the
microorganisms, thus protecting the product against post-acidication.
With respect to the traditional yoghurt, Brazilian legislation states
that the total viable lactic bacteria count should be at least 107 CFU/g
in the nal product during the entire validity period (Brasil, 2007).
The number of viable cells of S. thermophilus and L. bulgaricus of the

yoghurts met the standards required by the Brazilian legislation for all
treatments. The average number of viable cells of S. thermophilus and
L. bulgaricus after 35 days of storage was 4.50 108 CFU/g and 9.77
106 CFU/g, respectively, for the yoghurt with free L. acidophilus LA-5,
and 6.92 108 CFU/g and 1.91 106 CFU/g for the yoghurt with microencapsulated L. acidophilus LA-5.
As shown in Table 2, the number of viable cells of L. acidophilus LA-5
after 35 days of refrigerated storage was 1.58 107 CFU/g for the yoghurt with free L. acidophilus LA-5, and 1.12 107 CFU/g for the yoghurt
with microencapsulated L. acidophilus LA-5. Thus, both yoghurts containing L. acidophilus LA-5 in free or microencapsulated form met the
standards established by legislation for probiotics at the end of shelf
life, with counts of 3.16 109 and 2.24 109 CFU in the portion of
200 g, respectively. A reduction of 0.98 and 0.20 log units was
observed in the viable L. acidophilus LA-5 cells for yoghurts with free
and microencapsulated microorganisms, respectively, after 35 days
of refrigerated storage when compared with the initial population
(Table 2). The percent survival of L. acidophilus LA-5 after 35 days of
refrigerated storage was 10.46% and 62.26% in the yoghurt containing
free and microencapsulated L. acidophilus LA-5, respectively.
Regarding probiotic viability, the present results corroborate with
the literature that reports that the microencapsulation process can be
suitable to maintain the microorganism viability in yoghurt for longer
periods. Several authors (Adhikari et al., 2003; Kailasapathy, 2006;
Krasaekoopt et al., 2006) investigated different microorganisms, microencapsulation techniques, and wall materials and found that the microencapsulation improved the viability of probiotic microorganisms in
yoghurt when compared to the free form, as observed in the present
study.
3.4. Viability of microorganisms after simulated gastrointestinal conditions
Table 3 shows that the treatments (p b 0.0001), the sequential incubation time (p b 0.0001), and the interaction between these factors
(p b 0.0001) signicantly affected the number of viable L. acidophilus
LA-5 cells in yoghurts. Although the number of viable cells decreased
over time, it was different for each treatment. After 2 h exposure to simulated gastric juice at pH 3.0, the population of L. acidophilus LA-5 reduced approximately 0.38 and 0.17 log cycles in the yoghurts with
free and microencapsulated microorganisms, respectively. When comparing the counts after exposure of yoghurts to the simulated gastric
juice (120 min at pH 3.0) and the simulated intestinal juice conditions
(pH 7.0), no signicant difference was observed for the population of
L. acidophilus LA-5 in both yoghurts. Fig. 3 shows that after 7 h exposure
to SGJ/SIJ, the percent survival of L. acidophilus LA-5 in the yoghurt with
microencapsulated microorganism was higher (68.49%) than in the
yoghurt with free L. acidophilus LA-5 (45.68%).
After ve hours exposure to 1% bile solution, a reduction of 3.5 log
and 1.37 log cycles was observed in the population of L. acidophilus
LA-5 in yoghurts with free and microencapsulated L. acidophilus LA-5,
respectively. Thus, the survival of L. acidophilus LA-5 in the free form
Table 2
Viability of L. acidophilus LA-5 (log10CFU/g) during 35 days of refrigerated storage time of
probiotic yoghurts (mean SD, n = 3).
Storage time (days)

Yoghurt with free

L. acidophilus LA-5

1
7
14
21
28
35

8.18
8.16
8.04
7.88
7.75
7.20

A,B

Fig. 2. pH measurement during refrigerated storage time of probiotic yoghurts (n = 3).

0.03A,a
0.03A,a
0.10B,a
0.02C,a
0.03D,a
0.05E,a

Yoghurt with microencapsulated

L. acidophilus LA-5
7.25
7.19
7.14
7.08
7.06
7.05

0.04A,b
0.03A,B,b
0.05B,C,b
0.05C,D,b
0.06C,D,b
0.06D,b

Means followed by different uppercase letters in the same column differ signicantly
(p b 0.05).
a,b
Means followed by different lower case letters in the same line differ signicantly
(p b 0.05).

M.C.E. Ribeiro et al. / Food Research International 66 (2014) 424431

Table 3
Viability of L. acidophilus LA-5 (log10CFU/g) during sequential exposure to simulated gastric juice (SGJ, pH 3.0) for 120 min and simulated intestinal juice (SIJ, pH 7.0) for 300 min,
and exposure to bile solution 1% pH 7.0 (mean SD, n = 3).
Sequential incubation

Time (min)

Yoghurt with free

L. acidophilus LA-5

SGJ pH 3.0

0
60
120
300

6.08
5.87
5.70
5.74

0
60
300

4.30 0.30A,b
2.20 0.50B,b
0.80 0.30C,b

SIJ pH 7.0
Bile 1% pH 7.0

0.09A,b
0.09B,b
0.10C,b
0.09C,b

Yoghurt with
microencapsulated
L. acidophilus LA-5
6.14
6.04
5.97
6.00

0.08A,a
0.06B,a
0.09B,a
0.10B,a

5.47 0.09A,a
4.60 0.30B,a
4.10 0.20B,a

A,B

For each treatment (SGJ, SIJ and Bile) means followed by different uppercase letters in
the same column differ signicantly (p b 0.05).
ab
For each treatment (SGJ, SIJ and Bile) means followed by different lower case letters in
the same line differ signicantly (p b 0.05).

was lower (0.03%) when compared to the yoghurt with microencapsulated L. acidophilus LA-5 (5.61%) (Fig. 3).
Therefore, the yoghurt containing microencapsulated L. acidophilus
LA-5 showed a greater resistance to simulated gastric juice, simulated
intestinal juice, and bile solution.
Studies have shown that microencapsulation can protect probiotic
microorganisms against drastic conditions during their passage through
the gastrointestinal tract. Several authors have evaluated the exposure
of probiotics to these conditions and obtained better results for the
microorganisms in encapsulated form when compared to the free microorganisms. L. acidophilus CSCC 2400 free and microencapsulated in
calcium alginate by ionic gelation technique was subjected to 2 h
exposure to pH 2.0, and presented a decrease of 5 and 3 log cycles, respectively (Chandramouli, Kailasapathy, Peiris, & Jones, 2004). FvaroTrindade and Grosso (2000) also observed that the microencapsulation
of L. acidophilus LA-5 in sodium alginate by ionic gelation protected the
microorganisms, with no signicant reduction in the population when
subjected to pH 2.0 for 3 h. After exposure to simulated gastric juice,
the L. acidophilus encapsulated with calcium alginate and a double

429

layer of sodium alginate exhibited higher viability, suggesting a decrease in pore size, thus preventing the interaction of cells with the gastric juice (Mokarram, Mortazavi, Habibi Naja, & Shahidi, 2009). The
comparison of in vitro gastrointestinal tolerance studies is complex
once there is a large variation in the parameters pH, bile concentration,
initial concentration of microorganisms, and composition of gastric uid
that may affect the viability of different probiotic strains.
3.5. Sensory evaluation
The probiotic yoghurts were subjected to sensory evaluation after 1
and 35 days of refrigerated storage after conrming that the products
met the standards required for microbiological safety established by
the Brazilian legislation for fermented milks (Agncia Nacional de
Vigilncia Sanitria do Brasil, 2001; Brasil, 2007).
As shown in Table 4, despite the higher post-acidication of yoghurt
with free L. acidophilus LA-5, no difference in avor was observed for
both periods. The attributes aroma, overall impression, and purchase
intention did not differ between the two products.
The yoghurt containing microencapsulated L. acidophilus LA-5 was
less accepted in relation to the appearance and texture on the rst day
of storage than yoghurt with free microorganism, and only the attribute
texture was different between the products after 35 days of storage.
During the refrigerated storage, whey proteins in the microcapsules
possibly have interacted with the protein matrix of yoghurt leading to
a more homogeneous appearance, not differing from yoghurt containing free L. acidophilus LA-5 after 35 days.
Regarding texture, the yoghurt containing encapsulated L. acidophilus
LA-5 remained less accepted (score 6.68) than the yoghurt containing
L. acidophilus LA-5 in free form (score 7.33). These data suggest that
the average particle size (253.3 23.8 m) was perceived by the
consumers, adversely affecting the texture of the product. This result corroborates with the ndings of Adhikari et al. (2003), who investigated
the overall acceptance and texture of stirred blackberry avor yoghurt
containing free and encapsulated cells of Bidobacterium longum, and yoghurt without probiotics (control). Although no signicant difference
was observed for overall acceptance, yoghurts containing microcapsules
exhibited a granular texture, and differed signicantly from the others,
scoring 5.2 on a 9-point scale (1 extremely granular and 9 extremely
smooth) when compared to 7.2 and 7.1 found for yoghurts with nonencapsulated cells and control, respectively.
The addition of fruit pulp or pieces in the yoghurt would be a viable
alternative to reduce the consumers' perception of particles in the product. Despite the difference in texture, no differences on purchase

Table 4
Sensory evaluation of probiotic yoghurts after 1 and 35 days of refrigerated storage
(mean SD).
Sensory attributes

Fig. 3. Survival of L. acidophilus LA-5 (%) in probiotic yoghurts after exposure to simulated
gastrointestinal conditions (n = 3).

Yoghurt with free

L. acidophilus LA-5

Yoghurt with
microencapsulated
L. acidophilus LA-5

1 day of refrigerated storage

Appearance
Aroma
Flavor
Texture
Overall impression
Purchase intention

7.41
6.50
5.38
6.84
6.21
3.24

1.21a
1.57a
2.23a
1.70a
1.84a
1.18a

6.98
6.68
5.46
6.25
6.07
3.19

1.48b
1.41a
2.01a
2.00b
1.77a
1.20a

35 days of refrigerated storage

Appearance
Aroma
Flavor
Texture
Overall impression
Purchase intention

7.58
6.87
5.66
7.33
6.52
3.32

1.30a
1.56a
1.94a
1.62a
1.68a
1.14a

7.46
7.04
6.10
6.68
6.54
3.48

1.22a
1.54a
1.90a
1.71b
1.79a
1.09a

a,b

Means followed by different lower case letters in the same line differ signicantly
(p b 0.05).

430

M.C.E. Ribeiro et al. / Food Research International 66 (2014) 424431

intention of the products were observed, suggesting that the microencapsulation of L. acidophilus LA-5 may be an alternative to maintain probiotic viability during storage, without affecting consumers' preference.
4. Conclusions
Microencapsulation of L. acidophilus LA-5 by ionic gelation and complex coacervation techniques using pectin and whey protein concentrate was considered a potential technique for probiotics delivery in
the human gastrointestinal tract, since it protected the microorganisms
during storage and consumption of the yoghurts, conferred lower post
acidication and provided a product with good overall impression.
Acknowledgments
The authors are grateful to the Foundation for Research Support of
the State of So Paulo (FAPESP) for the nancial support to the project
2009/54268-9, the Coordination of Improvement of Higher Education
Personnel (CAPES) and the National Council for Scientic and Technological Development (CNPq) for granting the scholarship.
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