Professional Documents
Culture Documents
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
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
426
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):
% survival
.
initial population C FU=g
100
427
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
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).
428
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)
1
7
14
21
28
35
8.18
8.16
8.04
7.88
7.75
7.20
A,B
0.03A,a
0.03A,a
0.10B,a
0.02C,a
0.03D,a
0.05E,a
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).
Time (min)
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
microencapsulated
L. acidophilus LA-5
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
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
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.
References
Adhikari, K., Mustapha, A., & Grun, I.U. (2003). Survival and metabolic activity of microencapsulated Bidobacterium longum in stirred yogurt. Journal of Food Science, 68,
275280.
Agncia Nacional de Vigilncia Sanitria do Brasil (2001). Regulamento tcnico
sobre padres microbiolgicos para alimentos. Resoluo RDC No. 12-02/01/
2001. Accessed Jul. 10, 2014. http://portal.anvisa.gov.br/wps/wcm/connect/
a47bab8047458b909541d53fbc4c6735/RDC_12_2001.pdf?MOD=AJPERES
Annan, N.T., Borza, A.D., & Hansen, L.T. (2008). Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bidobacterium adolescentis
15703 T during exposure to simulated gastro-intestinal conditions. Food Research
International, 41, 184193.
Antunes, A.E.C., Marasca, E.T.G., Moreno, I., Dourado, F.M., Rodrigues, L.G., & Lerayer, A.L.S.
(2007). Desenvolvimento de buttermilk probitico. Cincia e Tecnologia de Alimentos,
27, 8390.
AOAC (2006). Ofcial method of analysis (18th ed.). Gaithesburg: Association of Ofcial
Analytical Chemists.
Brando, E.M., & Andrade, C.T. (1999, Jul/Sett). Inuncia de fatores estruturais no
processo de gelicao de pectinas de alto grau de metoxilao. Polmeros: Cincia e
Tecnologia, 3844.
Brasil (2003, 12/26). ANVISA Agncia Nacional de Vigilncia Sanitria. Resoluo RDC
n 359, de 23/12/2003. Regulamento Tcnico de Pores de Alimentos Embalados para
Fins de Rotulagem Nutricional. Braslia: Dirio Ocial da Unio (Available in: http://
www.anvisa.gov.br).
Brasil (2007, 10/24). Ministrio da Agricultura Pecuria e Abastecimento. Instruo
Normativa n 46, de 23/10/2007. Regulamento Tcnico de Identidade e Qualidade de
Leites Fermentados (pp. 5). Braslia: Dirio Ocial da Unio (Seo 1, Available in:
http://www.agricultura.gov.br).
Brinques, G.B., & Ayub, M.A.Z. (2011). Effect of microencapsulation on survival of
Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration and
yogurt. Journal of Food Engineering, 103, 123128.
Burey, P., Bhandari, B.R., Howes, T., & Godley, M.J. (2008). Hydrocoloid gel particles:
Formation, characterization, and application. Critical Reviews in Food Science and
Nutrition, 48, 361377.
Chandramouli, V., Kailasapathy, K., Peiris, P., & Jones, M. (2004). An improved method of
microencapsulation to protect Lactobacillus spp. in simulated gastric conditions.
Journal of Microbiological Methods, 56, 2735.
Charteris, W.P., Kelly, P.M., Morelli, L., & Collins, J.K. (1998). Ingredient selection criteria
for probiotic microorganisms in functional dairy foods. International Journal of Dairy
Technology, 51, 123136.
Chvarri, M., Maran, I., Ares, R., Ibez, F.C., Marzo, F., & Villarn, M.C. (2010). Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves
survival in simulated gastro-intestinal conditions. International Journal of Food
Microbiology, 142, 185189.
Dave, R.I., & Shah, N.P. (1996). Evaluation of media for selective enumeration of
Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus
acidophilus, and Bidobacteria. Journal of Dairy Science, 79, 15291536.
De Man, J.C., Rogosa, M., & Sharpe, M.E. (1960). A medium for the cultivation of
lactobacilli. Journal of Applied Microbiology, 23, 130135.
Del Piano, M., Morellic, L., Strozzi, G.P., Allesina, S., Barba, M., Deidda, F., et al. (2006).
Probiotics: From research to consumer. Digestive and Liver Disease, 38, S248S255.
Ding, W.K., & Shah, N.P. (2007). Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria. Journal of Food Science, 72, 446450.
FAO/WHO. Food and Agriculture Organization of the United Nations; World Health
Organization (2001). Evaluation of health and nutritional properties of probiotics in
food including powder milk with live lactic acid bacteria. (Crdoba, 34 pp.).
Fvaro-Trindade, C.S., & Grosso, C.R.F. (2000). The effect of the immobilisation of
Lactobacillus acidophilus and Bidobacterium lactis in alginate on their tolerance to
gastrointestinal secretions. Milchwissenschaft, 55, 496499.
Gbassi, G.K., Vandamme, T., Ennahar, S., & Marchioni, E. (2009). Microencapsulation of
Lactobacillus plantarum spp. in an alginate matrix coated with whey proteins.
International Journal of Food Microbiology, 129, 103105.
Gebara, C., Chaves, K.S., Ribeiro, M.C.E., Souza, F.N., Grosso, C.R.F., & Gigante, M.L. (2013).
Viability of Lactobacillus acidophilus LA-5 in pectinwhey protein microparticles during exposure to simulated gastrointestinal conditions. Food Research International, 51,
872878.
Gerez, C.L., Font de Valdez, G., Gigante, M.L., & Grosso, C.R.F. (2012). Whey protein coating
bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low
pH. Letters in Applied Microbiology, 54, 552556.
Grosso, C.R.F., & Fvaro-Trindade, C.S. (2004). Stability of free and immobilized Lactobacillus acidophilus and Bidobacterium lactis in acidied milk and of immobilized B. lactis
in yoghurt. Brazilian Journal of Microbiology, 35, 151156.
Hansen, L.T., Allan-Wojtas, P.M., Jin, Y.L., & Paulson, A.T. (2002). Survival of Ca-alginate
microencapsulated Bidobacterium spp. in milk and simulated gastrointestinal
conditions. Food Microbiology, 19, 3545.
Heller, J.K. (2001). Probiotic bacteria in fermented foods: Product characteristics and
starter organisms. American Journal of Clinical Nutrition, 73, 374S379S (Suppl.).
Kailasapathy, K. (2006). Survival of free and encapsulated probiotic bacteria and their
effect on the sensory properties of yoghurt. LWT Food Science and Technology, 39,
12211227.
Kailasapathy, K., & Sureeta, B.S. (2004). Effect of storage on shelf life and viability of
freezedried and microencapsulated Lactobacillus acidophilus and Bidobacterium
infantis cultures. Australian Journal of Dairy Technology, 59, 204208.
Krasaekoopt, W., Bhandari, B., & Deeth, H. (2004). The inuence of coating materials on
some properties of alginate beads and survivability of microencapsulated probiotic
bacteria. International Dairy Journal, 14, 737743.
Krasaekoopt, W., Bhandari, B., & Deeth, H. (2006). Survival of probiotics encapsulated in
chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated
milk during storage. LWT Food Science and Technology, 39, 177183.
Lambert, J.M., Winbreck, F., & Kleerebezem, M. (2008). In vitro analysis of protection
of the enzyme bile salt hydrolase against enteric conditions by whey proteingum Arabic microencapsulation. Journal of Agricultural and Food Chemistry, 56,
83608364.
Liu, Z., Jiang, Z., Zhou, K., Li, P., Liu, G., & Zhang, B. (2007). Screening of bidobacteria with
acquired tolerance to human gastrointestinal tract. Anaerobe, 13, 215219.
Lomer, M.C.E., Parkes, G.C., & Sanderson, J.D. (2008). Review article: Lactose intolerance in
clinical practice myths and realities. Alimentary pharmacology & therapeutics, 27,
93103.
Mohammadi, R., Mortazavian, A.M., Khosrokhava, R., & Cruz, A.G. (2011). Probiotic ice
cream: Viability of probiotic bacteria and sensory properties. Annals of microbiology,
61, 411424.
Mokarram, R.R., Mortazavi, S.A., Habibi Naja, M.B., & Shahidi, F. (2009). The inuence of
multi stage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice. Food research international, 42, 10401045.
Mortazavian, A.M., Ehsani, M.R., Azizi, A., Razavi, S.H., Mousavi, S.M., Sohrabvandi, S., et al.
(2008). Viability of calcium-alginate-microencapsulated probiotic bacteria in Iranian
yogurt drink (Doogh) during refrigerated storage and under simulated
gastrointestinal conditions. Australian Journal of Dairy Technology, 63, 2429.
Mortazavian, A.M., Razavi, S.H., Ehsani, M.R., & Sohrabvandi, S. (2007). Principles and
methods of microencapsulation of probiotic microorganisms. Iranian Journal of
Biotechnology, 5, 118.
Mozzi, F., Gerbino, E., Font De Valdez, G., & Torino, M.I. (2009). Functionality of
exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system.
Journal of Applied Microbiology, 107, 5664.
Nazzaro, F., Fratianni, F., Nicolaus, B., Poli, A., & Orlando, P. (2012). The prebiotic source
inuences the growth, biochemical features and survival under simulated gastrointestinal conditions of the probiotic Lactobacillus acidophilus. Anaerobe, 18, 280285.
Palomar, M.M., Galdeano, C.M., & Perdign, G. (2014). Inuence of a probiotic lactobacillus strain on the intestinal ecosystem in a stress model mouse. Brain, Behavior, and
Immunity, 35, 7785.
Parkar, S.G., Redgate, E.L., Wibisono, R., Luo, X., Koh, E.T.H., & SchroDer, R. (2010). Gut
health benets of kiwifruit pectins: Comparison with commercial functional polysaccharides. Journal of Functional Foods, 2, 210218.
Patil, J.S., Kamalapur, M.V., Marapur, S.C., & Kadam, D.V. (2010). Ionotropic gelation and
polyelectrolyte complexation: The novel techniques to design hydrogel particulate
sustained, modulated drug delivery system: A review. Digest Journal of Nanomaterials
and Biostructures, 5, 241248.
Pavunc, A.L., Beganovic, J., Kos, B., Buneta, A., Beluhan, I., & Suskovic, J. (2011). Inuence of
microencapsulation and transglutaminase on viability of probiotic strain Lactobacillus
helveticus M92 and consistency of set yoghurt. International Journal of Dairy
Technology, 64, 254261.
Picot, A., & Lacroix, C. (2004). Encapsulation of bidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt.
International Dairy Journal, 14, 505515.
Pinto, S.S., Fritzen-Freire, C.B., Munoz, I.B., Barreto, P.L.M., Prudencio, E.S., & Amboni,
R.D.M.C. (2012). Effects of the addition of microencapsulated Bidobacterium BB-12
on the properties of frozen yogurt. Journal of Food Engineering, 111, 563569.
Ranadheera, C.S., Evans, C.A., Adams, M.C., & Baines, S.K. (2012). In vitro analysis of
gastrointestinal tolerance and intestinal cell adhesion of probiotics in goat's milk ice
cream and yogurt. Food research international, 49, 619625.
Sanders, M.E., & Marco, M.L. (2010). Food formats for effective delivery of probiotics.
Annual review of food science and technology, 1, 6585.
431
Villanueva, N.D.M., & Da Silva, M.A.A.P. (2009). Comparative performance of the ninepoint hedonic, hybrid and self-adjusting scales in the generation of internal preference maps. Food quality and preference, 20, 112.
Vinderola, C.G., & Reinheimer, J.A. (1999). Culture media for the enumeration of
Bidobacterium bidum and Lactobacillus acidophilus in the presence of yoghurt
bacteria. International dairy journal, 9, 497505.
Voo, W.P., Ravindra, P., Tey, B.T., & Chan, E.S. (2011). Comparison of alginate and pectin
based beads for production of poultry probiotic cells. Journal of Bioscience and
Bioengineering, 111, 294299.
Walstra, P., Wouters, J.T.M., & Geurts, T.J. (2006). Dairy science and technology (2nd ed.).
Boca Raton: CRC Press, Taylor & Francis Group (744 pp.).
Wang, J., Guo, Z., Zhang, Q., Yan, L., Chen, W., Liu, M.X., et al. (2009). Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and
bovine milk during storage. Journal of Dairy Science, 92, 24682476.
Zacarchenco, P.B., & Massaguer-Roig, S. (2004). Enumeration of Streptococcus thermophilus
in the presence of Bidobacterium longum and Lactobacillus acidophilus Effect of incubation temperature and atmospheric conditions. Milchwissenchaft, 59, 370372.
Zhu, Y., Luo, T.M., Jobin, C., & Young, H.A. (2011). Gut microbiota and probiotics in colon
tumorigenesis. Cancer letters, 309, 119127.
Ziar, H., Grard, P., & Riazi, A. (2012). Calcium alginate-resistant starch mixed gel improved the survival of Bidobacterium animalis subsp. lactis Bb12 and Lactobacillus
rhamnosus LBRE-LSAS in yogurt and simulated gastrointestinal conditions.
International Journal of Food Science and Technology, 47, 14211429.