Mortazavian, Probiotic bacteria in Doogh

427

MILCHWISSENSCHAFT

MILK SCIENCE INTERNATIONAL

ZEITSCHRIFT FR ERNHRUNGSFORSCHUNG UND LEBENSMITTELWISSENSCHAFTEN

JOURNAL OF NUTRITION RESEARCH AND FOOD SCIENCE
ORGAN DER GESELLSCHAFT FR MILCHWISSENSCHAFT / ASSOCIATION OF MILK SCIENCE
WISSENSCHAFTLICHE REDAKTION / SCIENTIFIC EDITOR: PROF. DR. W. HEESCHEN

Vol. 63

Nr / No 4

Kempten

MILCAD 63 (4) 349 472 (2008)

Survival of encapsulated probiotic bacteria in Iranian yogurt drink

(Doogh) after the product exposure to simulated gastrointestinal
conditions
By A.M. MORTAZAVIAN1, A. AZIZI2, M.R. EHSANI1, S.H. RAZAVI1, S.M. MOUSAVI1, S. SOHRABVANDI1 and
J.A. REINHEIMER3
1

Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/National Nutrition and Food
Technology Research Institute, Beheshti University of Medical Sciences and Health Services, P.O.Box 19395-4741,
Tehran, Iran. E-mail: mortazvn@ut.ac.ir
2
Ministry of Jihad-e-Agriculture, Agricultural Research and Education Organization, Agricultural Engineering Research
Institute, Food Engineering and Post-Harvest Technology Research Department, Karaj, Iran
3

Instituto de Lactologia Industrial, Facultad de Ingenieria, Quimica , Universidad Nacional del Litoral, Santiago del
Estero, 2829, 3000, Santa Fe, Argentina

Microencapsulation of probiotic cells with hydrocolloid materials, as an efficient method of viability increase of probiotics, has been recently under special concern and investigation. In present study, the protective effect of microencapsulation with calcium alginate on survival of probiotic cells (Lactobacillus acidophilus La-5 and Bifidobacterium lactis Bb-12)
incorporated in Iranian yogurt drink (Doogh) was studied after the product exposure to simulated gastrointestinal conditions. The 3 simulated gastrointestinal conditions were: reinforced/strict gastrointestinal condition (pH1.5-90 min/2%
bile-90 min), semi-alleviated gastrointestinal condition (pH1.5-90 min/1% bile-90 min) and real-alleviated gastrointestinal
condition (pH2.0-30 min/0.6% bile-60 min). The viability of free probiotic cells increased from 0.6 and 0.2% (L. acidophilus and bifidobacteria, respectively) to 18.0 and 9.5% at reinforced gastrointestinal conditions, after encapsulation. At
real-alleviated conditions, the cell survival rates were 16.1% for L. acidophilus and 21% for bifidobacteria before encapsulation, whilst these amounts improved to 26.3 and 34.0% (L. acidophilus and bifidobacteria, respectively) after encapsulation.
berleben verkapselter probiotischer Bakterien in einem iranischen Joghurtgetrnk (Doogh) nach Exposition
des Produktes gegenbersimulierten gastrointestinalen Bedingungen
Die Mikroverkapselung probiotischer Bakterien mit Hydrokolloiden gilt als wirksame Methodik zur Verbesserung des
berlebens probiotischer Keime. Diese Technologie fand in jngster Zeit besondere Beachtung und wurde eingehend
untersucht. In der vorliegenden Studie wurde der protektive Effekt der Mikroverkapselung mit Kalziumalginat auf das
berleben probiotischer Keime (Lactobacillus acidophilus La-5 and Bifidobacterium lactis Bb-12) nach Exposition des
Produktes gegenber simulierten gastrointestinalen Bedingungen geprft. Drei gastrointestinale Bedingungen wurden
simuliert, und zwar verstrkte/strikteBedingungen (pH-Wert 1,5 - 90 min / 2% Galle - 90 min), leicht abgeschwchte
gastrointestinale Bedingungen (pH-Wert 1,5 - 90 min / 1% Galle - 90 min) und der Realitt angepasste Bedingungen
(pH-Wert 2,0 - 30 min / 0,6% Galle - 60 min). Die Lebensfhigkeit der freien probiotischen Zellen erhhte sich von 0,6
und 0,2% (L. acidophilus bzw. Bifidobakterien) nach der Verkapselung auf 18,0 und 9,5% bei den ntensivierten
gastrointestinalen Bedingungen. Unter
gastrointestinalen Bedingungen betrugen die Keimberlebensraten
16,1% bei L. acidophilus und 21% bei den Bifidobakterien vor der Verkapselung, und diese Werte verbesserten sich auf
26,3 und 34,0% (L. acidophilus bzw. Bifidobakterien) nach der Verkapselung.
62 Yogurt drink (doogh, encapsulation of probiotics)
62 Joghurtgetrnk (Doogh, Verkapselung von Probiotika)

1. Introduction
Microencapsulation is a process in which the bacterial cells are entrapped within coatings of hydrocolloidal

Milchwissenschaft 63 (4) 2008

materials in order to be segregated from adverse environmental conditions (9). Microencapsulation has been
recently used as an efficient practice to improve the

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Mortazavian, Probiotic bacteria in Doogh

Table 1: Survival percentage of free and encapsulated probiotic cells in Doogh after their successive exposure
to different simulated conditions of stomach and small intestine.*
Gastrointestinal
conditions
pH1.5-90 min/
2% bile-90 min

Encapsulated
state
Free
Encapsulated

pH1.5-90 min/
1% bile-90 min

Free
Encapsulated

pH2.0-30 min/
0.6% bile-60 min

Free
Encapsulated

Probiotics
L. acidophilus
bifidobacteria
L. acidophilus
bifidobacteria
L. acidophilus
bifidobacteria
L. acidophilus
bifidobacteria
L. acidophilus
bifidobacteria
L. acidophilus
bifidobacteria

Viability before and after

exposure (log cfu/mL)
6.54-4.32
6.90-4.20
6.54-5.79
6.90-5.88
6.54-5.58
6.90-5.08
6.54- nT**
6.90- nT
6.54-5.75
6.90-622
6.54-5.96
6.90-6.43

Percentage
of survival
h
0.6hi
0.2 d
18.0f
9.5 f
11.0g
1.5
nT
nTde
16.1 c
21.0b
26.3a
34.0

* Means in the same column with different letters are significantly different (p<0.05). ** Not tested

viability of probiotic bacteria in fermented milks (1, 4, 8,

13) as well as gastrointestinal tract (3, 5, 7, 11, 14). The
most widely used encapsulating material is alginate.
We recently reported the effective impact of encapsulation of probiotic cells with calcium alginate on their
viability during a 42-day refrigerated storage period of
Iranian yogurt drink, i.e. "Doogh" (8). Following that research, present study was designed to investigate the
protective efficiency of alginate micro-capsules on entrapped probiotic cells against the gastrointestinal conditions, when the cells are ingested in Doogh medium.

2. Materials and methods

2.1 Starter cultures
The DVS pouches of commercial lyophilized cultures
including Y-type (mixed culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus),
A-type (single culture of L. acidophilus La-5) and B-type
(single culture of B. lactis Bb-12) were supplied by Chr.
Hansen (Horsholm, Denmark). These starter cultures
are widely used by dairy industry to produce fermented
milks.

2.2 Enumeration of probiotic bacteria

Lactobacillus acidophilus and bifidobacteria were
enumerated selectively using MRS-bile agar (MRS
agar: Merck, Darmstadt, Germany and Bile: Sigma,
Reyde, USA) by "Subtractive Enumeration Method" according to MORTAZAVIAN et al. (10).
Encapsulated bacteria were enumerated after the
capsules were digested in 0.1 M solution of phosphate
buffer with pH7.000.02 (Merck, Darmstadt, Germany)
and incubated at 37C for 90 min.

2.3 Microencapsulation procedure

Microencapsulation of probiotic bacteria was accomplished according to MORTAZAVIAN et al. (8). Briefly, 150
mL of sterile 2% (W/V) sodium alginate (Provisco,
Swiss) was mixed with 30 mL of concentrated cell culture. The mixture was dispended by a pipette into a
beaker containing 600 mL of pure corn oil and 1 g of
tween 80 while stirring using at 200 rpm. Calcium chloride (0.1 M) was gently added at the side of the beaker
until the emulsion was broken. After 1 h, gel-beads
which contained a mixture of L. acidophilus and bifido-

Milchwissenschaft 63 (4) 2008

bacteria cells were separated, washed and incorporated into the thermized Doogh (pH 4.500.02).

2.4 Doogh preparation

Doogh milk with 4% of dry matter was made by reconstitution of skim milk powder. The mixture also contained 0.5% of sodium salt.

2.5 Evaluation of microencapsulated cells survival

at simulated gastrointestinal conditions
The Doogh samples contained encapsulated or free
cells were added into different gastric solutions with
pH1.5 or pH2 and 0.2% of sodium salt. The sample solutions were incubated at 37C for 90 min or 30 min and
then transferred into different intestinal solutions consisted of phosphate buffer with pH7.000.02 (Merck,
Darmstadt, Germany) and 2 or 1 or 0.6% of bile salts
(Sigma, Reyde, USA), after their pH were adjusted on
7.00.2 by adding a few droplets of concentrated sodium hydroxide solution. Then, Incubation was carried
out at 37C for 90 or 60 min. Gentle shaking of the samples was done over the gastric and intestinal incubations. The condition of pH2.0-30 min/0.6% bile-60 min
was considered as "real-alleviated gastrointestinal condition", i.e. the situation in gastrointestinal tract of a
normal and healthy person after consumption of a probiotic-containing dairy drink when the stomach has not
been free for a relatively long time.

2.6 Statistical analysis

Experiments were performed in quadruples and the
significant differences among the means were analyzed
using Anova test from Minitab software (version 13,
2002).

3. Results
The survival rates of free and encapsulated probiotic
cells in Doogh after their successive exposure to different simulated conditions of stomach and small intestine
have been presented in Table 1. As is appeared, at
strict/reinforced conditions (pH1.5-90 min/2% bile, 90
min), the survival percentage of probiotic cells were 0.6
and 0.2 for L. acidophilus and bifidobacteria, respectively. These data increased at semi-alleviated conditions (pH1.5-90 min/1% bile-90 min) to 11.0 and 1.5%
and at real-alleviated conditions (pH2.0-30 min/0.6%

Mortazavian, Probiotic bacteria in Doogh

bile-60 min) to 16 and 21% for L. acidophilus and bifidobacteria, respectively. Regarding these results, bifidobacteria cells were primarily susceptible to very low pH
value of stomach rather than the bile concentration,
whereas L. acidophilus cells are substantially sensitive
to bile salts concentration. The same fact found in the
results reported by KRASAEKOOPT et al. (5). As can be
understood from Table 1, alginate micro-beads markedly increased the survival of free cells of L. acidophilus
by 17.4% and bifidobacteria by 9.3% under strict gastrointestinal conditions and by 10.3 and 13.0% under realalleviated conditions. The complementary experiments
revealed that alginate beads were degraded by 27.2
and 28.4% (details not shown; data obtained by applying light scattering method) after 30 and 90 min of exposure to gastric conditions (pH1.5). Therefore, hydrogen
ions can partially affect integrity of the beads. Relative
digestion of the beads leads to the much higher exposure of bacterial cells to the great concentration of hydrogen ions in the gastric juice and as a result, sharp
loss in viability of entrapped cells. Apart from this reason, natural micro-pores present in a gel-bead matrix
permit diffusion of hydrogen ions inwards, which would
be magnified in the case of cracked beads. This explains why for example viability of Bifidobacterium free
cells, as hydrogen ion-sensitive cells, at strict GI conditions increased by 9.3% after encapsulation compared
with real-alleviated conditions with the greater value of
13.0%, in which the higher survival was due to the alleviated ambient situations rather than more adequate
protection of alginate capsules. Higher pH value along
with shorter exposure time at latter situations causes
less diffusivity of hydrogen ions inward the capsules and
subsequently greater viability of the cells. Evidences
have proved that the size distribution of the beads as
well as the cell load within them significantly influences
survivability of encapsulated cells. Regarding investigation of CHANDRAMOULI et al. (2) and SHEU and MARSHAL
(12), the higher protective efficiency of micro-capsules
resulted in parallel with increase in capsule size and cell
load in each capsule. However, very large beads (about
1000 m or larger) cause a coarseness of texture in
mouthfeeling and weakness of the coated structure (3,
6). In our study, the size distribution of the capsules was
20.10-1096.48 m in range with a peak size of 339.60
m. In sensory evaluation tests, no sense of coarseness was identified (data not shown). The size of encapsulated starter cells was about 1.5-6.0 m (including
both single and attached cells) in range with a peak size
of 2.36 m. Hence, most of the capsules should have
been loaded with several cells and presumably noticeable aqueous portion. Such conditions inside the beads
slow down the micro-flux of hydrogen ions form ambient
inward the capsules at least for the central cells, which
result in the considerably higher survival of the cells particularly if the exposure time is shorter (e.g. 30 min
compared to 90 min). Our previous relevant study (8)
revealed that the best size range of micro-beads to obtain the highest protective efficiency of encapsulation on
probiotic cells at a 42-day refrigerated storage period
was 300-500 m. Comparing the results obtained from
present study, it is evident that this range can be also

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429

successfully applied for in vivo objectives. It seems that

the protection efficiency of encapsulation would be
more markable if the severity of gastrointestinal stress
factors alleviated. For example, according to KRASAEKOOPT et al. (5), encapsulated cells of L. acidophilus
were more viable (0.62% vs. 0.15%) compared to free
cells when exposed to gastric juice with pH1.55 for 90
min. Reducing the exposure time by 30 min caused
considerable greater difference among the viability of
encapsulated and free cells (10.47% vs. 1.15%). In present research, to some extent, the similar results were
observed, as previously explained.

4. Conclusions
This work demonstrated that the survival of encapsulated probiotic bacteria in Doogh was significantly
higher than free cells when subjected to simulated in
vivo conditions, especially at alleviated conditions
(pH2.0-30 min/0.6% bile-60 min). Because also in our
previous relevant study, microencapsulation with Caalginate has been successfully performed to increase
the viability of L. acidophilus and bifidobacteria in Doogh
throughout the refrigerated storage time, the applied
encapsulation procedure is suggested for production of
probiotic Doogh.
Acknowledgements

This study was supported by the R&D Center of

Zam-Zam Iran Co. (Tehran, Iran).

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