위장관내 조건에서 이중코팅 처리 된 프로바이오틱 비피도박테리움의...
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Korean Journal of Microbiology (2013) Vol. 49, No. 3, pp. 275-281DOI http://dx.doi.org/10.7845/kjm.2013.3042Copyright ⓒ 2013, The Microbiological Society of Korea
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Dual Coating Improves the Survival of Probiotic Bifidobacterium
Strains during Exposure to Simulated Gastro-Intestinal Conditions
Joo Yeon Kang1, Do Kyung Lee
2, Jae Eun Park
1, Min Ji Kim
2, Joong-Su Lee
3, Jae-Gu Seo
3,
Myung Jun Chung3, Hea Soon Shin
1, and Nam Joo Ha
2*
1College of Pharmacy, Duksung Women’s University, Seoul 132-714, Republic of Korea2College of Pharmacy, Sahmyook University, Seoul 139-742, Republic of Korea
3R&D Center, Cell Biotech Co., Ltd., Gimpo 415-871, Republic of Korea
위장관내 조건에서 이중코팅 처리 된 프로바이오틱 비피도박테리움의 생존력 향상
강주연1․이도경2․박재은1․김민지2․이 수3․서재구3․정명 3․신혜순1․하남주2*
1덕성여자 학교 약학 학, 2삼육 학교 약학 학, 3㈜쎌바이오텍 연구소
(Received June 14, 2013 / Accepted September 25, 2013)
Probiotics have been reported to benefit human health by modulating immunity, lowering cholesterol, improving lactose tolerance, and preventing some cancer. Once ingested, probiotic microorganisms have to survive harsh conditions such as low pH, protease-rich condition, and bile salts during their passage through the gastro-intestinal (GI) tract colonize and proliferate to exert their probiotic effects. The dual coating technology, by which the bacteria are doubly coated with peptides and polysaccharides in consecutive order, was developed to protect the ingested bacteria from the harsh conditions. The aim of the study was to evaluate the viable stability of a doubly coated blend of four species of Bifidobacterium by comparing its bile/acid resistance and heat viability in vitro with that of the non-coated blend. After challenges with acid, bile salts, heat, and viable cell counts (VVCs) of the dual coated and non-coated blend were determined by cultivation on agar plates or flow cytometric measurement after being stain with the BacLigtht kitTM. The results showed that the dual coated blend was much higher resistant to the acidic or bile salt condition than the non-coated blend and heat viability was also higher, indicating that the dual coating can improve the survival of probiotic bacteria during their transit through the GI tract after consumption.
Keywords: Bifidobacterium, dual coating technology, duolac, probiotics
*For correspondence. E-mail: [email protected]; Tel.: +82-2-3399-1607; Fax: +82-2-3399-1617
In today’s society, there has been increasingly interested in
their personal health and functional food. Probiotic products are
an important functional food as they represent about 65% of the
world functional food market, and the market for probiotic
products continues to expand (Agrawal, 2005; Jankovic et al.,
2010). Probiotics are defined as ‘live microorganisms which,
when administered in adequate amounts, confer a health benefit
on the host’ (FAO/WHO, 2002). Researchers have reported to
play a therapeutic role by modulating immunity, lowering
cholesterol, improving lactose tolerance and preventing some
cancer (Kailasapathy and Chin, 2000; Sanders et al., 2007).
Probiotics are orally administrated and are available in various
forms such as food products, capsules, sachets, or tablets.
Ingested probiotics have to survive adverse conditions such as
low pH, protease-rich condition, and bile salts during their
passage through the gastro-intestinal (GI) tract to be able to
influence the human gut microflora (Weichselbaum, 2009; Burgain
et al., 2011). However some probiotic bacteria are sensitive to
oxygen, and many require media ingredients or modified gas
environments to enable their growth (Dave and Shah, 1996;
Talwalkar and Kailasapathy, 2004). Especially, Bifidobacterium
strain that are the most widely used probiotic bacteria and are
included in many products and functional foods vary greatly in
their sensitivity to the harsh acidic environment of the stomach
and many foods (Clark and Martin, 1994; Lankaputhra and
276 Kang et al.
Fig. 1. Matrix structure of dual coated bacteria.Fig. 2. Field emission scanning electron microscope images of the non-coating (A, C) and dual coating (B, D) Bifidobacterium blend.
Shah, 1997; Charteris et al., 1998; Truelstrup et al., 2002; Guarner
and Malagelada, 2003; Annan et al., 2008). For this reason,
many researchers reported that there is poor survival of probiotic
bacteria in products containing free probiotic cells (De Vos et al.,
2010). Various technologies of encapsulation such as emulsification,
spray drying, spray cooling, and freeze drying have been developed
for protection live cells in the food industry (De Vos et al., 2010;
Burgain et al., 2011), and it has been reported these technologies
of encapsulation improved the viability of probiotic bacteria in
the GI tract (Krasaekoopt et al., 2003; Picot and Lacroix, 2004;
Sohail et al., 2011; Su et al., 2011; Saarela et al., 2011).
Dual coating technology is so-called fourth generation coating
technology for protection of LAB during the passage through
the GI tract and manufacturing process and was patented in
Korea (patent no. 0429495), Japan (patent no. 3720780), and
Europe (patent no. 1514553B) (Burgain et al., 2011; Cha et al.,
2011). The technology was developed to protect the ingested
bacteria from the harsh conditions. In the technology, bacteria
cells are coated with peptides and subsequently with polysaccharide
matrix (Figs. 1 and 2). A polypeptide behaves in a pH-dependent
way, and there exists a pH gradient along the stomach and intestine.
Thanks to the coating layers, the doubly coated cells remain
mostly uncoated at pH 4.0, which is the pH of stomach after
meal and begin to be released from the coats at pH 6.0 and are
fully released at pH 7.0, the pH in the intestine. The polysaccharide
matrix protects from moisture, heat, and physical pressure, and
so stability is increased. Therefore the dual coated bacteria after
ingestion are able to reach the intestine alive and in good condition
to colonize and proliferate while uncoated bacteria that is prone
to be damaged in the gastro-intestinal environment.
In this study, we evaluated in vitro acid/bile resistance and
heat viability of a probiotic blend consisting of four Bifidobacterium
species which were doubly coated or non-coated and compared
the resistances of the dual coated blend with those of the non-
coated blend.
Materials and Methods
Bacteria
Dual coated Bifidobacterium bifidum BF3 (KCTC 12199BP),
B. infantis BT1 (KCTC 12859BP), B. longum BG7 (KCTC
12200BP), and B. rhamnosus BR3 (KCTC 12201BP) were blended
in equal proportions and tested to assess the resistance to acid
or bile salts. The same tests were applied to a blend containing
the same kind of species of non-coated Bifidobacterium. Comparison
of acid, bile, and heat resistance between the two formulations
was made.
Resistance to acid and bile salt
For acid tolerance test, the blend of dual coated Bifidobacterium
or non-coated Bifidobacterium was inoculated in BL broth (around
107-8 CFU/ml) which was adjusted to pH 2.0, 3.0, 5.0, or 7.0
using 0.1 M HCl or 0.1 M NaOH. Samples were taken at various
time points (0, 0.5, 1, 3, and 8 h) and subjected to viable cell
count using BL agar plate with 0.005% bromocresol purple or
LIVE/DEAD BacLight kitTM (Invitrogen, USA).
For bile tolerance test, the blends were inoculated into acidified
BL broth (pH 4.0) (approximately 107 CFU/ml) containing oxgall
(BD, USA) at the concentration of 0, 0.1, 0.3, or 1% (w/v).
Samples were taken at various time points (0, 3, 6, 9, 12, and
24 h) and subjected to viable cell count with the same methods,
as described above.
Staining of bacterial cells
Cells collected at the time points were adjusted to be 106-7
CFU/ml in potassium phosphate buffer (PBS, pH 7.2), treated
with the reagents in the BacLight kit as recommended by the
manufacturer, and gently shaken for 15 min in dark condition.
Dual coating improves survival of probiotic Bifidobacterium strains 277
Fig. 3. Time course analysis of viable cell counts of the dual coated Bifidobacterium and non-coated Bifidobacterium blend under different acidic conditions (pH 7.0, 5.0, 3.0, and 2.0). Viable cell counts were determined by collecting cells at the time points, which were then plated on BL agar plates and incubated at 37℃anaerobically.
First labeling was confirmed by the use of a fluorescent microscopy
(Olympus, Japan) set to accept fluorescence intensity at a wavelength
(emission 1; green and emission 2; red).
Flow cytometric measurements (FCM)
Flow cytometric measurements were performed on a FACSCalibur
flow cytometer (Becton Dickinson Immunocytometry Systems,
USA) equipped with a 15-mW, 488-nm, air-cooled argon ion
laser and a cell-sorting catcher tube. Cell samples were diluted
to approximately 106 cells/ml and delivered at the low flow rate,
corresponding to 150 to 500 cells/sec. FSC, SSC, and three
fluorescence signals were measured. A band pass filter of 530
nm (515 to 545 nm) was used to collect the green fluorescence
(FL1), a band pass filter of 585 nm (564 to 606 nm) was used
to collect the yellow-orange fluorescence (FL2), and a long
pass filter of 670 nm was used to collect the red fluorescence
(FL3). FSC was collected with a diode detector. SSC and the
three fluorescence signals were collected with photomultiplier
tubes. All signals were collected by using logarithmic amplifications.
A combination of FSC and SSC was used to discriminate bacteria
from background.
Heat stability
For stability test, we studied the differences in the viability
values between non-coated and dual coated Bifidobacterium
during 2 weeks storage period at 40℃. The blend of dual coated
Bifidobacterium or non-coated Bifidobacterium were kept for 2
weeks at 40℃ for subsequent microbial counts. Later, samples
were randomly taken before and after 1, 3, 7, and 14 days during
the 2 weeks storage period. Test samples were rehydrated to the
original volume with 0.1% peptone for 10 min at room temperature,
and appropriated dilutions were poured in BL agar (Difco,
USA). Plates were incubated anaerobically at 37℃ for 48 h.
After that, the number of CFUs was counted.
Results
Acid tolerance
To assess acid tolerance of the four bifidobacterial species in
each blend, the cells were challenged with various acidic
conditions (pH 2.0, 3.0, 5.0, or 7.0) up to for 8 h and sampled at
0, 0.5, 1, 3, and 8 h after incubation. There was little difference
in viable cell counts (VCCs) determined using agar plate
between the blends of dual coated or non-coated bifidobacteria
under conditions of pH 7.0 and 5.0 over the test period (Figs.
3A and 3B). Differences in acid tolerance between them began
to appear when incubated for 30 min at pH 3.0 during which
the VCCs of the non-coated blend dropped by 1.4 log-fold
compared to the initial counts whereas the dual coated blend
showed a small decrease in the VCCs, from 7.7±0.12 log-
CFU/ml to 7.5±0.14 log-CFU/ml (Fig. 3C). Incubation at pH
2.0 severely affected the VCCs of both blends. For the non-coated
blend, the initial viable counts dropped from 7.3±0.19 log-
CFU/ml to 5.3±0.11 log-CFU/ml (2 log-fold drop) only after
30 min and to under the lower detection limit, 2.3±0.0 log-
CFU/ml, after 8 h (5 log-fold drop or greater). By contrast, the
dual coated blend was much less affected under the same pH
condition. Its VCCs were determined to be 6.9±0.19 log-
CFU/ml after 30 min incubation, showing a small decrease, and
4.9±0.06 log-CFU/ml even after 8 h incubation (Fig. 3D).
LIVE/DEAD BacLight kitTM was used to monitor visually
the fraction of live or dead bacterial cells in the test samples as
live and dead cells fluoresce green and red lights, respectively,
when stained with the kit. When the cells in the dual coated or
non-coated blend were incubated for 3 h in pH 7.0 condition,
most of the cells were live (green) and no distinct differences
between the samples were observed (Figs. 4A and 4B). However,
most of the non-coated bacteria incubated for 3 h in pH 2.0
condition were severely damaged (yellow and orange) or dead
(red). By contrast, half of the dual coated bacteria were visually
found to be live (Figs. 4C and 4D).
In order to quantify the live and dead cells in the samples,
the green and red bacterial counts were analyzed by FCM (Fig. 5).
The percentages of green and red bacteria for the non-coated
blend at 3 h in pH 3.0 condition were 28.3% (live cell) and
278 Kang et al.
Fig. 5. Flow cytometric analysis of bifidobacterial cells incubated in acidic conditions. All bacteria were previously stained with STYO Green I and propidium iodide. Gates indicate the position and concentration of intact cells on the plots. The strains of non-coated Bifidobacterium blend incubated at pH 3.0 (A) or at pH 7.0 (B) for 3 h. The dual coated Bifidobacterium blend incubated at pH 3.0 (C) or at pH 7.0 (D) for 3 h. Q1 and Q2, dead cell; Q3, injured cell or un-staining cell; Q4, live cell. The counts of live cells (E) and dead cells (F) for the dual coated blend were compared with non-coated blend.
A B
C D
Fig. 4. Fluorescent microscopic images of cells of the non-coatedBifidobacterium blend or dual coated Bifidobacterium blend stained with the Live/Dead Baclight kit after challenge with acid. The non-coated bacteria (A, C) and dual coated bacteria (B, D) were incubated at pH 7.0 (A and B, respectively) or at pH 2.0 (C and D, respectively) for 3 h. Live cells, green; dead cells, red; yellow and orange, injured.
64.3% (dead cell), respectively (Fig. 5A). Under the same
condition, live and dead cells for the dual coated blend were
determined to be 53.7% and 34.7% respectively (Fig. 5C). In
the 3 h samples at pH 7.0, the percentages of live and dead cells
in the non- coated blend were 87.1% (live cell) and 2.5% (dead
cell), respectively (Fig. 5B). On the other hand, the respective
percentages for the dual coated blend were 89.0% for live cells
and 0% for dead cells (Fig. 5D). Therefore the result further
supports that the dual coated bacteria are more resistant to acid
than the non-coated bacteria.
Bile tolerance
Bile tolerance of the bacteria in the non-coated or dual
coated blend was investigated by incubating them in BL broth
(pH 4.0) containing oxgall at the final concentration of 0, 0.1,
0.3, or 1.0% (w/v). The pH of BL broth was adjusted to 4.0
because such acidic condition is assumed to be closer to the
human proximal intestinal condition (Succi et al., 2005). At
0.1% oxgall, bacterial cells of both blends proliferated over
time, and no large differences were observed in the VCCs
between the blends (Fig. 6). However, the growth of the non-
coated bacteria began to be inhibited at 0.3% oxgall, and the
VCCs dropped by 1.7 log fold after 24 h incubation while the
growth of the dual coated bacteria was barely affected (Fig.
6C). Distinct differences in the VCCs were seen when they
were incubated at 1.0% oxgall. Under the condition, the viability
of the non-coated bacteria dropped sharply from 7.6±0.10
log-CFU/ml to 2.3±0.0 log-CFU/ml after 24 h incubation whereas
the dual coated bacteria were less affected and their VCCs after
24 h were much higher than those of non-coated bacteria
(5.0±0.28 log-CFU/ml vs 2.3±0.0 log-CFU/ml) (Fig. 6D).
Bacterial cells in the non-coated or dual coated blend
incubated at 1.0% oxgall for 3 h were subjected to staining with
the BacLight kit and examined under the fluorescent microscope.
Dual coating improves survival of probiotic Bifidobacterium strains 279
Fig. 8. Flow cytometric analysis of bifidobacterial cells incubated under different bile salt conditions. All bacteria were previously stained with STYO Green I and propidium iodide. Gates indicate the position and concentration of intact cells on the plots. The non-coated bacteria incubated at 0% oxgall (A) or at 1.0% oxgall (B) for 3 h. The dual coated bacteria incubated at 0% oxgall (C) or at 1.0% oxgall (D) for 3 h. P1, total percents (live, dead, injured cell, and debris); P2, total percents (live, dead, and injured cell); Q1 and Q2, dead cell; Q3, injured cell or un-staining cell; Q4, live cell. The counts of live cells (E) and dead cells (F) for the dual coated blend were compared with non-coated blend.
Fig. 6. Time course analysis of viable cell counts of the non-coatedBifidobacterium blend and dual coated Bifidobacterium blend incubated at different concentrations of oxgall (0, 0.1, 0.3, and 1%). Viable cell counts were determined by collecting cells at the time points, which were then plated on BL agar plates and incubated at 37℃ anaerobically.
A B
Fig. 7. Fluorescent microscopic images of the dual coated and non-coated bacteria stained with Live/Dead Baclight kit after challenge with oxgall. The non-coated bacteria (A) and dual coated bacteria (B) were incubated at 1.0% oxgall for 3 h. Live cells, green; dead cells, red; yellow and orange, injured.
The result was that a great number of the non-coated bacteria
were found to be dead (red in color) from the fluorescent
observation while half of the dual coated bacteria were found to
be live (green) (Fig. 7).
When the stained bacteria were further analyzed by FCM,
the percentages of live and dead cells of the non-coated
Bifidobacterium blend incubated at pH 4.0 and 0% oxgall for 3
h were 71.5% (live cell) and 20.3% (dead cell), respectively
(Fig. 8A). Under the same condition, live and dead cells of the
dual coated blend were 78.2% and 11.3%%, respectively (Fig.
8C). In the 3 h samples at 1.0% oxgall, the percentages of live
and dead cells of the non-coated blend were 26.2% and 66%,
respectively (Fig. 8B). By contrast, the respective percentages
of the dual coated bacteria were 67.5% for live cells and 21.0%
for dead cells (Fig. 8D).
Heat stability
To measure the heat resistance, non-coated or dual coated
bacteria were kept at 40℃ for 14 days. Figure 9 shows the
viability of non-coated or dual coated bacteria at the end of
storage at 40℃ for 14 days. The initial counts of non-coated
280 Kang et al.
Fig. 9. Survival of non-coated and dual coated Bifidobacteriumblend during 2 weeks storage period at 40℃.
and dual coated bacteria ranged from 10.57±0.08 log-CFU/ml
and 10.13±0.05 log-CFU/ml and there was little difference in
viable cell counts between them (Fig. 9). However, non-coated
bacteria showed a tendency to decrease highly throughout the
storage period. After 1 day, non-coated blend counts decreased
from 10.57±0.08 log-CFU/ml to 9.38±0.2 log-CFU/ml and
kept decreasing at the end of storage period (Fig. 9). The viable
counts of non-coated blend declined by about 44% after 2
weeks compared to the initial viable count. By contrast, dual
coated blend showed a small decrease. Its VCCs were determined
to be 10.10±0.02 log-CFU/ml after 1day and 9.31±0.11 log-CFU/ml
even after 2 weeks (Fig. 9). These results suggested that dual
coated bacteria are more resistant to heat than non-coated
bacteria.
Discussion
The viability of probiotics in functional foods is the most
important requirement, because it has a direct bearing on
effects of functional foods. For this reason, many researchers
studied methods for protection of live cells and various technologies
of encapsulation have been developed. The demands of successful
encapsulation are protection against adverse environmental
conditions such as low pH, biliary salts, and proteases during
passage through the GI tract and efficient release of the probiotics
bacteria. The encapsulation technologies depend on the capsule
material, particle size, and bacterial strain (Burgain et al., 2011).
It has been reported that various probiotics encapsulation
technologies improve the viability of bacteria during exposure
to simulated gastro-intestinal conditions such as alginate-coated
gelatin microspheres (Annan et al., 2008), alginate–human-like
collagen (Su et al., 2011) and in alginate gel microbeads
(Sohail et al., 2011). Alginate is a representative encapsulating
material and extensively used, but they are sensitive to the
acidic environment and very difficult to scale up (Mortazavian
et al., 2008). Many researchers have attempted to remedy
disadvantages in various materials and ways. Among the probiotic
encapsulation technologies, the spray coating technology is
easy to scale up and is adapted to give multilayer coatings
(Burgain et al., 2011).
We have developed the dual coating technology which is
so-called fourth generation coating technology. The dual coating
system is based on a pH-dependant release mechanism which
protects the cells against acidic environments in the stomach
and releases the bacteria from coating in the neutral pH environment
of the intestines. The dual coated blend consisting of B. bifidum
BF3 (KCTC 12199BP), B. infantis BT1 (KCTC 12859BP), B.
longum BG7 (KCTC 12200BP), and B. rhamnosus BR3 (KCTC
12201BP) was found to be highly resistant to acid or bile salt
compared to the non-coated counterpart in this study. These
results indicate that the dual coating technology can improve
the survival of probiotic bacteria during their transit through the
GI tract after consumption. In the case of probiotic encapsulation,
the objective is not only to improve the survival of probiotic
bacteria through the GI tract, but also to protect the cell against
adverse environment. It is estimated that daily consumption of
107 CFU/ml of live probiotic cells are needed to confer health
benefits to the consumer (Ouwehand and Salminen, 1998; Shah,
2000). However, probiotic is affected by storage environments
such as temperature, and humidity, and it might contribute to
reductions in viable cell counts during storage period. Weinbreck
et al. (2010) reported that viable counts of unencaposulation bacteria
declined dramatically during 2 weeks when stored at 37℃,
whereas encapsulation bacteria were much less affected under
the same condition. Likewise, the role of the encapsulation also
is important at the end of storage period. The dual coating
technology improved the viability of Bifidobacterium blend
during 2 weeks storage period at 40℃ compared to the non-
coated counterpart in this study. These findings demonstrate the
dual coating technology can improve stability and viability of
probioitc bacteria more effectively from harsh environments.
요
프로바이오틱 박테리아는 면역력 활성 조절, 콜레스테롤 수
치 억제, 유당내성 강화, 항종양 활성 등의 다양한 생리활성 기능
으로 건강 증진 효과가 있는 것으로 보고되고 있다. 프로바이오
틱 박테리아는 일단 섭취하게 되면 위장관을 통과하는 동안 산
도가 낮거나 단백질분해 효소가 많은 열악한 환경에서 생존해야
하며 프로바이오틱 효과를 발휘하기 위해 증식해야 한다. 이중
코팅 기술은 펩타이드와 다당류의 이중코팅으로 섭취된 프로바
이오틱 박테리아를 열악한 조건으로부터 보호하기 위해 개발되
었다. 본 연구에서는 이중 코팅 된 4종의 비피도박테리움 혼합물
의 생존 안정성을 평가하기 위해 코팅이 되지 않은 비피도박테
리움 혼합물과 담즙, 산 저항성 및 열 안정성을 비교⋅평가하였
다. 이중 코팅 된 균주와 코팅이 되지 않은 균주를 산과 담즙 조
Dual coating improves survival of probiotic Bifidobacterium strains 281
건 및 40℃에 노출 시킨 후 한천배지에 배양하여 생존생육 세포
수를 측정하였으며, BacLigtht 키트를 이용하여 염색 한 후 유세
포 분석기를 이용하여 생균과 사균의 세포수를 평가하였다. 이
중코팅 된 균주 혼합물의 경우 코팅이 되지 않은 균주 혼합물 보
다 산, 담즙 내성이 더 높았으며, 열 안전성 또한 코팅 되지 않은
균주 혼합물보다 높은 것으로 나타났다. 이 같은 결과들로 이중
코팅 기술은 프로바이오틱 박테리아의 안정성 및 섭취 후 위장
관 트랙을 통과하는 동안 균주의 생존률을 향상시킬 수 있음을
확인하였다.
Acknowledgements
The authors are grateful to the financial support by Sahmyook
University Research Fund. This work (Grants No.000449270111)
was supported by Business for Cooperative R&D between
Industry, Academy, and Research Institute funded by the Korea
Small and Medium Business Administration.
References
Agrawal, R. 2005. Probiotics: an emerging food supplement with health benefits. Food Biotechnol. 19, 227–246.
Annan, N.T., Borza, A.D., and Truelstrup Hansen, L. 2008. Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions. Food Res. Int. 41, 184–193.
Burgain, J., Gaiani, C., Linder, M., and Scher, J. 2011. Encapsulation of probiotic living cells: From laboratory scale to industrial applications. J. Food Engineer. 104, 467–483.
Cha, M.K., Chung, M.J., Kim, J.E., Lee, K.O., and HA, N.J. 2011. Comparison of dual coated(DuolacTM) and uncoated lactic acid bacteria from potential probiotics. Biotechnol. Biotechnol. Eq. 25, 2489–2493.
Charteris, W.P., Kelly, P.M., Morelli, L., and Collins, J.K. 1998. Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J. Appl. Microbiol. 84, 759–768.
Clark, P.A. and Martin, J.H. 1994. Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: III – Tolerance to simulated bile concentrations of human small intestines. Cultur. Dairy Products J. 29, 18–21.
Dave, R.I. and Shah, N.P. 1996. Evaluation of media for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and Bifidobacteria. J. Dairy Sci. 79, 1529–1536.
De Vos, P., Faas, M.M., Spasojevic, M., and Sikkema, J. 2010. Encapsulation for preservation of functionality and targeted delivery of bioactive food components. Int. Dairy J. 20, 292–302.
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization) 2002. Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada. April 30 and May 1, 2002.
Guarner, F. and Malagelada, J.R. 2003. Gut flora in health and disease.
Lancet 361, 512–519.Jankovic, I., Sybesma, W., Phothirath, P., Ananta, E., and Mercenier, A.
2010. Application of probiotics in food products – challenges and new approaches. Curr. Opin. Biotechnol. 21, 175–181.
Kailasapathy, K. and Chin, J. 2000. Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp.. Immunol. Cell Biol. 78, 80–88.
Krasaekoopt, W., Bhandari, B., and Deeth, H. 2003. Evaluation of encapsulation techniques of probiotics for yoghurt. Int. Dairy J. 13, 3–13.
Lankaputhra, W.E.V. and Shah, N.P. 1997. Improving viability of Lactobacillus acidophilus and bifidobacteria in yogurt using two step fermentation and neutralized mix. Food Australia 49, 363–366.
Mortazavian, A.M., Azizi, A., Ehsani, M.R., Razavi, S.H., Mousavi, S.M., Sohrabvandi, S., and Reinheimer, J.A. 2008. Survival of encapsulated probiotic bacteria in Iranian yogurt drink (Doogh) after the product exposure to simulated gastrointestinal conditions. Milchwissenschaft 63, 427–429.
Ouwehand, A.C. and Salminen, S.J. 1998. The health effects of cultured milk products with viable and non-viable bacteria. Int. Dairy J. 8, 749–758.
Picot, A. and Lacroix, C. 2004. Encapsulation of Bifidobacteria in whey protein-based microcapsules and survival in stimulated gastrointestinal conditions and in yoghurt. Int. Dairy J. 14, 505–515.
Saarela, M., Alakomi, H.L., Mättö, J., Ahonen, A.M., Puhakka, A., and Tynkkynen, S. 2011. Improving the storage stability of Bifidobacterium breve in low pH fruit juice. Int. J. Food Microbiol. 149, 106–110.
Sanders, M.E., Gibson, G., Gill, H.S., and Guarner, F. 2007. Probiotics: their potential to impact human health. CAST issue paper No. 36, October 2007.
Shah, N.P. 2000. Probiotic bacteria: Selective enumeration and survival in dairy foods. J. Dairy Sci. 83, 894–907.
Sohail, A., Turner, M.S., Coombes, A., Bostrom, T., and Bhandari, B. 2011. Survivability of probiotics encapsulated in alginate gel microbeads using a novel impinging aerosols method. Int. J. Food Microbiol. 145, 162–168.
Su, R., Zhu, X.L., Fan, D.D., Mi, Y., Yang, C.Y., and Jia, X. 2011. Encapsulation of probiotic Bifidobacterium longum BIOMA 5920 with alginate–human-like collagen and evaluation of survival in simulated gastrointestinal conditions. Int. J. Biol. Macromol. 49, 979–984.
Succi, M., Tremonte, P., Reale, A., Sorrentino, E., Grazia, L., Pacifico, S., and Coppola, R. 2005. Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol. Lett. 244, 129–137.
Talwalkar, A. and Kailasapathy, K. 2004. A review of oxygen toxicity in probiotic yogurts: influence on the survival of probiotic bacteria and protective techniques. Compr. Rev. Food Sci. Food Safety 3, 117–124.
Truelstrup Hansen, L., Allan-Wojtas, P.M., Jin, Y.L., and Paulson, A.T. 2002. Survival of Ca-alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiol. 19, 35–45.
Weichselbaum, E. 2009. Probiotics and health: a review of the evidence. Nutrition Bulletin 34, 340–373.
Weinbreck, F., Bodnár, I., and Marco, M.L. 2010 Can encapsulation lengthen the shelf-life of probiotic bacteria in dry products? Int. J. Food Microbiol. 136, 364–367.