International Food Research Journal 18(4): 1515-1522 (2011)

Survival of commercial probiotic strains to pH and bile

Sahadeva, R.P.K., Leong, S.F., Chua, K. H., Tan, C.H., Chan, H.Y.,
Tong, E.V., Wong, S.Y.W. and *Chan, H.K.

Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI

University. No. 1, Jalan Menara Gading, UCSI Heights, 56000, Kuala Lumpur,
Malaysia.

Abstract: This study was performed to enumerate the total viable cell count of probiotic in five brands (A to E)
of commercially cultured milk drinks that are available in the Malaysian market as well as to test their tolerance
to various pH and bile concentrations by simulating the human gastrointestinal pH and bile concentration. The
acid tolerance test was studied under pH 1.5 and 3.0 with 7.2 as control. The cell count for the acid tolerance
test was obtained at an interval of 0, 1.5 and 3 hours respectively and was plated onto duplicate MRS agars to be
incubated at 37C for 48 hours. All cells recovered after 3 hours of pH treatment were selected for bile tolerance
test in MRS broth containing bile concentrations of 0% (control), 0.3% and 2.0% and cell counts were recorded
after 24 hours of incubation. The probiotic strains in products A, B, C & D met the suggested initial count of
106 CFU/ml with brand C recording the highest at 9.19 0.14 log CFU/ml. Strains in product A, B & C showed
good tolerance to pH 3.0 and 7.2 recording a count of >106 CFU/ml after 3 hours with a range of 6.60 9.04
log CFU/ml. The higher bile concentrations resulted in lower growth of strains in all the brands. Upon pH 1.5
treatment, only brand C recorded growth in all bile concentrations. After pH 3.0 treatment, all brands except
brand E met the requirement of survival at 0.3% bile concentration. Results showed probiotics in product A, B
& C met the initial count requirement, and exhibited good acid and bile tolerance therefore being a potentially
good source of probiotic.

Keywords: Acid and bile tolerance, cultured milk drink, Lactobacillus, probiotic, total viable cell count
Introduction 2006). The amount required to gain any therapeutic
benefits is a minimum of 106 viable probiotic cells
Recent increase of awareness towards human per millilitre during storage until the expiry date
nutrition over the past few decades, especially in (Samona and Robinson, 1994; Lourens-Hattingh and
the developed countries has seen a shift from the Viljoen, 2002; FAO/WHO, 2006).
concept of adequate to optimal nutrition. This is In the past two decades, there has been renewed
evident in the rapid growth of interest in probiotics to interest in the study of the nutritional and therapeutic
promote better health and well-being which shows a aspects of the mentioned products (Parvez et al.,
substantial promise to expand the food industry into 2006). It is widely accepted that probiotics may exert
new fields. Strains from genera of Lactobacillus and positive influence on the host through modulation
Bifidobacterium species, both of which are indigenous of the endogenous ecosystem and stimulation of
to the human intestine, are predominantly selected immune system as well as maintaining a healthy
for use although some other species have also been intestinal microflora (Silva et al., 1987; Goldin et al.,
used (Holzapfel and Schillinger, 2002). Probiotics, 1992; Lee and Salminen, 1995; Lu and Walker, 2001;
also termed as functional foods, are commonly found Marteau et al., 2001; MacFarlane and Cummings,
in dairy products such as yogurt and cultured milk 2002). However, research suggests that health benefits
drinks or even in the form of health supplements. are strain specific and vary by amount ingested and
The notion of probiotics evolved from a theory duration administered.
first proposed by Nobel laureate, Elie Metchnikoff, Several studies have revealed that some probiotic
who associated longevity with the consumption products in the market have deficiencies in the
of fermented milk products. He postulated that the viability of probiotic strain(s), especially in products
bacillus present could positively modify the bacterial containing bifidobacteria (Fasoli et al., 2003; Masco
community structure of the colon, thus contributing et al., 2005). This may be due to storage conditions,
to human health status (Vasiljevic and Shah, 2008). manufacturing or food technologies setbacks such as
Probiotics were later termed as live micro-organisms inappropriate packaging materials that could affect
which, when administered in adequate amounts, probiotic stability through variations in oxygen
confer a health benefit to the host (FAO/WHO, permeability (Miller et al., 2002).

*Corresponding author.
Email: [email protected] All Rights Reserved
Tel: 603 9101 8880/3370; Fax : 603 9102 3606
1516 Sahadeva, R.P.K., Leong, S.F., Chua, K. H., Tan, C.H., Chan, H.Y., Tong, E.V., Wong, S.Y.W. and Chan, H.K.

The viability of these cells after consumption ice box to preserve the optimal storage environment
remains obscure as the bacteria are also subjected before transporting it to the laboratory. They were
to unfavourable physiological conditions of the then stored at 4oC and were utilized two weeks before
Gastrointestinal (GI) tract such as acidic environment its expiry date.
and bile secretions (Holzapfel et al., 1998). These Table 1. List of species and presence of prebiotics contained in
include variation in the level of acidic conditions and each cultured milk brand
bile secretion at different incubation time simulating Brand Species Prebiotics* Amount of prebiotics
Inulin and
the physiological aspects of human digestive A Lactobacillus acidophilus polydextrose Not Stated
B Lactobacillus acidophilus None -
system. Viability of these bacteria upon ingestion Lactobacillus casei Shirota
C None -
and sufficient survival through the transit to GI tract strains
Lactobacillus acidophilus,
is crucial to confer any health benefits to the host D Streptococcus
thermophilus, None -
(Salminen et al., 1998; Hou et al., 2003; Krasaekoopt Bifidobacterium;
Lactobacillus acidophilus,
et al., 2003; Kailasapathy, 2006). Consequently, E Lactobacillus casei, None -
Bifidobacterium.
the survival of commercial probiotic strains when : Prebiotics are stated in the ingredients of the cultured milk.
**

subjected to various pH and bile conditions similar to

that of human GI conditions should be assessed and De man, Rogosa, Sharpe (MRS) (DIFCOTM,
substantiated accordingly. Therefore, it is reasonable USA) agar was selected as the growth medium for the
to enumerate the viable cell counts in commercial probiotics in the samples. On the other hand, MRS
cultured milk drinks upon reaching consumers in broth (OXOID, UK) was used for determination of
this study. Hence five brands of commercial cultured bile tolerance. The MRS broth used has the exact
milk drinks were selected off the shelves in a local formulations as MRS agar, without the agar as a
hypermarket in Malaysia. solidifying agent.
Most studies evaluating the resistance of potential The samples (cultured milk drinks) were shaken
probiotic strains to gastric and bile secretion have been to ensure the homogeneity of the contents. Serial
conducted in vitro (Del Piano et al., 2006) although dilutions were performed to enumerate the total
some discrepancy between in vitro and in vivo number of viable cells upon consumption. Plating
observations does exist due to the complex nature of with duplicates was done in the laminar flow and
the human system (Morelli 2000). However, in two the plates were incubated aerobically for brand A, B,
seperate studies conducted by Marteau et al. (1997) and C and anaerobically for brand D and E at 37oC
and Lin et al. (2006); no significant differences were for 48 hours. Anaerobic condition was achieved
observed between in vitro and in vivo data, indicating by enclosing the plated cultures with an activated
the predictive value of the model for the survival of the Anaerocult A gas pack (Merck, Germany) in a sealed
bacteria. Thus, in this study, an in vitro methodology anaerobic jar (Merck, Germany).
was employed to assess the transit tolerance of the
probiotic samples. The effects of bile on probiotic Acid tolerance test
strains are more difficult to assess by in vitro due to The method used to evaluate the viability of the
variation of bile concentration in the system at any cells under acidic stress in this study was adapted from
given moment (Marteau et al., 1997). Hence, various Conway et al. (1987), Brashears et al. (2003) and
concentration of bile are considered and used in this Tsai et al. (2007). Various simulated GI conditions
study to mimic the physiological conditions closely. were achieved by subjecting the samples to different
pH levels at a designated incubation time. Jacobsen
Materials and Methods et al. (1999) and Huang and Adams (2004) had
described the use of sterile PBS adjusted to different
Five commercial cultured milk drinks labeled pH to study the acid tolerance of the microorganisms.
as A, B, C, D, and E were obtained from a local Hence, the pH of PBS was adjusted to pH 1.5, 3.0
hypermarket at Taman Connaught for this study and 7.2 (control) respectively using 1M HCl in this
(Table 1). Each batch of the sample consists of five study. In addition to that, the test was also subjected
bottled cultured milk drinks of the same flavour. to three incubation periods of 0, 1.5 and 3.0 hour.
Therefore, the sample selected for each repeated test Initially at 0 hour, 1.0 ml of the sample was
was of the same flavour to ensure consistency. These inoculated into the universal bottle containing 9 ml
cultured milk drinks with respect to each brand are of PBS, pH 1.5 and was mixed thoroughly. It was
expected to be the same in terms of its composition, then serial diluted with sterilized PBS pH 7.2 in
quality and quantity of probiotics. Thus, the sample microcentrifuge tubes. The appropriate dilution
was randomly selected and stored immediately in an factor was determined and plating of serial dilutions

International Food Research Journal 18(4): 1515-1522

 Survival of commercial probiotic strains to pH and bile 1517

of the culture was performed on MRS agar plates. the lowest was recorded by Brand E with a count of
Each assay was performed in duplicates. These plates 2.40 x 105 CFU/ mL (Table 2). Judging by this initial
were then incubated aerobically/ anaerobically at count, Brand A, B, C and D are considered good
37oC for 48 hours. The same procedure was repeated probiotic sources except for Brand E. Amongst the
for pH 3.0 and pH 7.2 under the same experimental reasons for the low count in Brand E could be affected
conditions for the 0 hour. The aforementioned by the temperature during the fermentation process
process was performed for 3 incubation periods of as well as during the inoculation period and most
0, 1.5 and 3.0 hour. Acid tolerance was estimated by importantly during transportation (Shah and Dave,
comparing the growth of viable cell counts in all the 1998). Oxygen that is dissolved in the product during
MRS agar plates after 48 hours. manufacturing could stress the probiotics as too much
oxygen will delay their growth (Klaver et al., 1993;
Kailasapathy and Supraidi, 1996; Lankaputra et al.,
Bile tolerance test 1996; Shah and Dave, 1998; Godward et al., 2000;
The effects of bile on the growth of probiotic Vinderola and Reinheimer, 2000; Vinderola et al.,
strains were examined using methods modified 2003) especially since Brand E is strictly anaerobic.
from those of Gilliland and Walker (1990) and Tsai The inulin and polydextrose contained in Brand A
et al. (2007). A series of bile concentrations were may have caused it to increase the initial count of
employed in this study considering the fluctuation of the probiotics as they acted as prebiotics which aid
bile concentration at different times. Broth with 0% the growth of probiotics especially of Lactobacilli
bile concentration serves as a control of the study. genera (Huebner et al., 2006).
Bile tolerance test was commenced at the end
Table 2. Total Plate Count (TPC) for five commercially cultured
of the third hour of acid pretreatment where 5 ml milk products on MRS agars under aerobic/anaerobic conditions
of sample was each pipetted out from the universal at 37C for 48 hours
Product TPC (log10CFU/ mL)a
bottles incubated earlier (pH 1.5, pH 3.0 and pH 7.2)
A 7.92 0.02
into three pH-labeled centrifuge tubes. Centrifugation
B 7.04 0.09
was carried out at 4000 rpm for 10 minutes at 25oC. Aerobic
C 9.19 0.14
The supernatants were discarded and the pellets D 6.84 0.34
were washed with PBS of pH 7.2. Centrifugation Anaerobic E 5.38 0.28
was repeated and the supernatants were once again a
: Results were expressed as mean standard deviation (SD) with each data point an
average of two repeated measurements from a total of three independently replicated
discarded. The three remaining concentrates were experiments; n=3.

then re-suspended with MRS broth. Acid tolerance

The next procedure involves the inoculation of Another important criterion to be a good source
1 ml of pH 1.5 suspensions into 9 ml of three MRS of probiotics is the tolerance of high acid levels,
broths with different bile concentrations (0%, 0.3% which is present in our stomachs. The lowest pH
and 2.0%). The step was repeated for pH 3.0 and pH recorded has been pH 1.5 (Huang and Adams, 2004;
7.2 mixtures as well. The MRS broth containing cells Lin et al., 2006). Good probiotic sources should
were incubated aerobically/ anaerobically at 37oC withstand at least pH 3.0 (Fernandez et al., 2003).
for 24 hours. Subsequently, 0.1 ml was pipetted out High concentrations of acid reach pH 1.5 during
from each of the MRS broth and serial dilutions were fasting. Generally there is a reduction in probiotic
performed for plating (duplicates). All the plates were count, as they were exposed to pH 1.5 and pH 3.0
incubated aerobically/ anaerobically at 37oC for 48 and the count is fairly constant at pH 7.2 (control)
hours. Bile tolerance was determined by comparing (Table 3).
the viable cell counts on MRS agars with and without All brands recorded a count except for Brand
bile salt. All samples were analysed in duplicates, all B which had no growth during the initial phase
experiments were repeated thrice. Data obtained from of inoculation (zeroth hour). After 1.5 hours of
the study was expressed in terms of log10 CFU/ml and incubation, all the brands showed no growth except
analysed as mean standard deviation (SD). for Brand D albeit a low count of 2.52 1.97 log10
CFU/ mL. After 3 hours of incubation, all brands
Results and Discussions showed no growth of probiotics suggesting most
probiotics were killed by this harsh pH (Table 3).
Initial counts of probiotics According to Zavaglia et al. (2002), acid such as
All brands have met the minimum requirement set the hydrochloric acid (HCl) that is found in the human
by FAO/WHO (1 x 106 CFU/ml) except for brand E. stomach is a strong oxidizer. Thus, it can oxidize many
The highest number of live probiotics was recorded important biomolecular compounds in the cells and
by Brand C with a count of 1.55 x 109 CFU/ mL and

International Food Research Journal 18(4): 1515-1522

1518 Sahadeva, R.P.K., Leong, S.F., Chua, K. H., Tan, C.H., Chan, H.Y., Tong, E.V., Wong, S.Y.W. and Chan, H.K.

Table 3. Total plate counts for five commercially cultured milk are always strains dependent (Huang and Adams,
products on MRS agars at different pH values of 1.5, 3.0 and
7.2(control) over 1.5 hour intervals 2004; Lin et al., 2006).
Total plate counts (log10 CFU/ mL) # Based on the results obtained (Table 3), the
pH value Brand
1.5 A
0 hour
6.09 0.13 3
1.5 hour
-
3.0 hour
-
survival of probiotic differed considerably when
B
C
-
8.53 0.64 4
-
-
-
-
compared at 0 and 3.0 hour for Brand D, with the
D
E
5.93 0.15 a,2
2.32 2.03 1
2.52 1.97 b
-
-
-
former achieving higher viability than the latter. With
3.0 A
B
7.17 0.05 a,3

6.82 0.04 a,2

7.08 0.03 b,3
6.73 0.05 ab,2
6.94 0.03 c,4
6.60 0.06 b,3
the number of counts of log10 5.92 CFU/ ml, the sample
C
D
9.06 0.06 a,4

6.83 0.44 a,2

9.06 0.03 a,4
6.17 0.23 ab,2
9.04 0.06 a,5
5.92 0.25 b,2
has proven to be unsuccessful to meet the minimum
7.2
E
A
5.25 0.02 a,1
7.57 0.05 a,4
4.10 0.70 b,1
7.51 0.04 ab,4
3.59 1.12 b,1
7.46 0.04 b,3
requirement of 106 viable probiotic cells per ml at pH
B
C
7.02 0.08 a,2

9.12 0.08 a,5

6.89 0.05 ab,3
9.05 0.07 a,5
6.68 0.08 b,2
9.04 0.07 a,4
3 after exposure for 3 hours. Having multiple strains
D
E
7.24 0.14 a,3

5.16 0.11 a,1

6.73 0.09 b,2
5.48 0.29 b,1
6.58 0.22 b,2
5.43 0.20 ab,1
in this brand, has indeed affected the overall viability
#
: Each value in the table represents the mean value Standard Deviation (SD). Each
data point is the average of two repeated measurements from 3 independently replicated
count. It is well established that L. acidophilus, is
experiments, n = 3
abc
: Mean value with different superscripts in the same row differs significantly (P< 0.05).
more resistant compared to Bifidobacterium spp.
-
: The hyphen symbol represents plate counts of < 1 log10CFU/ mL on the MRS agar
plates. in terms of high acidity (Boylston et al., 2004).
: The period of 0 hour is the time when the samples from milk cultures were plated
Comparatively, the intrinsic resistance of acid of
*

immediately for assay upon being exposed to PBS with different pH values.
: Growth increases numerically. Mean value with different superscripts for each pH
S. thermophilus has been reported to be fairly poor
123

value in the same column differs significantly (P<0.05).

(Conway et al., 1987; Vinderola and Reinheimer,

disrupt them while it will undergo reduction. Amongst 2003). However, the addition of S. thermophilus
the important biological compounds that acid can may also increase the survival of certain strains of
destroy includes fatty acids, proteins, cholesterol Bifidobacterium through the reduction of oxygen
and the DNA (Pan et al., 2008). As demonstrated by pressure (Ishibashi and Shimamura, 1993; Nogueira
Sultana et al. (2000) and Chan and Zhang (2005), et al., 1998). Nevertheless, the results obtained in this
aciduric members such as L. acidophilus, generally in vitro study may not truly reflect their performance
could not survive in low pH environment as these in situ as many other physiological conditions that
cells were proven to be vulnerable at pH 2.0 and might affect the survival of the strains (Morelli,
below. Low pH environments are thought to inhibit 2000).
the metabolism activity and growth of L. acidophilus,
thus reducing the probiotics viability. Another study Bile tolerance
conducted by Mandal et al. (2006) also confirmed The survival rate of bacteria supplemented
that the viability count of the bacteria declined with bile was similar to the trend portrayed in acid
tremendously when exposed to simulated gastric juice tolerance test, with higher inhibition of growth seen
of pH 1.5 after an incubation period of 3 hours. The as the bile concentrations increased for all the brands.
threshold point to determine acid resistance was set 0% bile acted as control for all experiments and it
at pH value of 3.0 and incubation period of 3 hours in recorded the highest growth. Upon exposure to bile
this in vitro study as it simulates the residence time acids, cellular homeostasis disruptions causes the
in the stomach (Prasad et al., 1998; Haddadin et dissociation of lipid bilayer and integral protein of
al., 2004). This is in accordance with findings from their cell membranes, resulting in leakage of bacterial
Liong and Shah (2005) which stated that resistance at content and ultimately cell death.
pH 3 is set as standards for acid tolerance of probiotic Data obtained from the acid tolerance study
culture. Therefore, result in Table 3 indicates the indicates that bacteria from all samples that cannot
strong inhibition on the viable bacteria numbers at survive at high pH 1.5, also failed to grow in this
pH 1.5 was well supported. subsequent bile test except Brand C (Figure 1). As
As for pH 3 (Table 3) in Brand A and B, there for pH 3 and 7.2 (Figures 2 and 3), there is a gradual
were strains of L. acidophilus that survived perhaps decline in viable count as the bile concentrations
because the pH was not too high so as to cause increased.
complete destruction of all the cells. At any given
time, there were cells that were still dying at pH 3
and there were those that survived, but after 3 hours
the rate of cells dying outnumbers the rate of survival
cells.
For Brand C (Table 3), the number of cell
counts remained significantly unchanged during all
the interval transit at pH 3 and pH 7.2. Good acid Figure 1 Cell growth in different probiotic brands at different
tolerance properties exhibited by the bacteria are bile concentration upon pH 1.5 treatment during 24 hours of
closely related to their strains specification as they incubation at 37C

International Food Research Journal 18(4): 1515-1522

 Survival of commercial probiotic strains to pH and bile 1519

agar medium. As bile is a detergent, the addition of

Tween 80 is postulated to enhance the stability of
the cell membrane and thus contributing to the bile
tolerance observed in some strains (Kimoto et al.,
2002).
Apart from that, the revival of these bacteria
after 24 hours of incubation with bile may also be
attributed to the pH of the bile itself. The pH of the
Figure 2. Cell growth in different probiotic brands at different
bile concentration upon pH 3 treatment during 24 hours of bile was expected to be in the range of 7.0 and higher.
incubation at 37C After acid pre-treatment, the revival of the bacteria
might be associated with the higher pH encountered
in bile tolerance test. This is postulated to be a positive
factor that contributes to the increased revival of
these probiotics after prolonged incubation with
bile solution. However, the actual pH of the oxgall
used in this study was not determined as it was not a
part of the research design. Thus, it is suggested that
future study may need to investigate the relationship
Figure 3. Cell growth in different probiotic brands at different of the biles pH and the revival of probiotics when
bile concentration upon pH 7.2 treatment during 24 hours of assessing the sensitivity of probiotic strains.
incubation at 37C
The probiotic strains proved to exhibit an
The findings in this study are in accordance to excellent quality of bile tolerance. Another important
Chou and Weimer (1999) which also combined two factor is the bile salt hydrolase (BSH) activity which
selection criteria, acid and bile in their assessment. accounts for the bile salt resistance. It is observed in
A common observation among these studies, some strains where BSH hydrolyse conjugated bile,
despite major design differences, is that acid and thus reducing its bactericidal effect (Du Toit et al.,
bile have separate and combined effects on the 1998). Later a study by Suskovic et al. (2001) proved
growth of bacteria. As bile stress takes place after that BSH was active when tested on lactobacilli and
pH stress in the stomach, Leyer and Johnson (1993) showed a relatively strong resistance to bile such as
and Lin et al. (2006) postulated that sub-lethally in Brand A, B and C. This may explain the sensitivity
injured microorganisms may have a different and of some strains which lacked this BSH activity.
unpredictable resistance to new stress factors. However, sensitivity to bile may also have positive
Overall in the results, bile did not inhibit the consequences as lysis of cells during passage through
growth of the bacteria completely as even when GI tract may result in the release of -galactosidase
subjected to 2% of bile, there is still a high number (Marteau et al., 1997). Yazid et al. (1999) reported
of bacterial count (8.51 0.03 log units)( Brand that bile acid had a greater inhibitory effect on the
A, Figure 2). The high growth of the bacteria at lactobacilli than bifidobacteria strains.
2% bile could be that stress adaptation mechanism
may serve as a logical rationalization to explain the Conclusion
increased growth with longer incubation hours after
pre-exposure to acid stress. The enhanced survival All brands except brand E has met the minimum
capabilities appeared to be due to the acclimatization initial count requirement as set by WHO/FAO
of the bacteria to the low pH environment, therefore 2006. The acid tolerance for all brands is the same
minimising the relative toxicity to glycoconjugates where increasing level of acidity has a negative
in the intestine (Begley et al., 2005; Martoni et al., impact on the viability of the probiotics. This trend
2007). The protective effect of food matrix also may is noticeable for all intervals tested (0, 1.5 and 3.0
prevent the bacteria from bile exposure and hence, hour) as findings observed a decline in viable counts
giving rise to the increased bile resistance of the when the sample was subjected to increasing acidity
strains (Begley et al. 2005). levels. Only brand A, B and C can survive pH 3.0
Besides that, some of the cells that were not and have met the criteria to be considered as good
entirely killed off and upon being exposed to the sources of probiotics. The bile tolerance for all the
MRS broth, they proliferate. Polysorbate 80, also brands show the same trend where increasing levels
commercially known as Tween 80, is a non ionic of bile shows more inhibitory effect. Brand A, B, C
surfactant and emulsifier commonly found in MRS and D shows good bile tolerance with growth more

International Food Research Journal 18(4): 1515-1522

1520 Sahadeva, R.P.K., Leong, S.F., Chua, K. H., Tan, C.H., Chan, H.Y., Tong, E.V., Wong, S.Y.W. and Chan, H.K.

than 6 log units at 0.3% of bile. Brand C shows the Fernandez, M.F., Boris, S. and Barbes, C. 2003. Probiotic
best tolerance towards bile, surviving with more than properties of human lactobacilli strains to be used
6 log units even after pH 1.5 and pH 3.0 treatments in the gastrointestinal tract. Journal of Applied
with the highest count amongst all the samples. Microbiology 94: 449-455.
Gilliland, S.E. and Walker, D.K. 1990. Factors to consider
when selecting a culture of Lactobacillus acidophilus
Acknowledgements as a dietary adjunct to produce a hypocholesterolemic
effect in humans. Journal of Dairy Science 73: 905-
This work was financially supported by UCSI 911.
University. Godward, G., Sultana, K., Kailasapathy, K., Peiris,
P., Arumugaswamy, R. and Reynolds, N. 2000.
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