Gastrointestinal survival of bacteria in commercial probiotic products

Abstract

This work compared bacterial gastrointestinal (GI) resistance of commercial probiotic products (capsules, fermented milk and powder). To simulate GI transit, the probiotic products were subjected to gastric fluid for 120 min then to intestinal fluid for 180 min. Gastric and intestinal fluids were prepared according to United States Pharmacopeia protocols. Bacterial enumeration was compared before and after the GI transit to evaluate the protective effect of the vehicle or the food matrix. Bacteria of the four probiotic capsules covered with an enteric coating had a higher survival rate (>1 log10 CFU reduction) than uncoated. Eight encapsulated but non enteric coated probiotic products showed limited GI resistance (between 1 and 5 log10 CFU reduction) while five products showed no GI survival. For probiotic fermented milk, two products demonstrated excellent or good protective property (>1 log10 CFU reduction) while the other four showed no resistance. Only one of six powdered probiotic strains had excellent GI survival. This study demonstrated that GI survival varies from one probiotic product to another. It reiterates the importance of manufacturing probiotic strains using the appropriate vehicle for the bacteria to reach its site of action and produce the expected beneficial effects.

Full text

International Journal of Probiotics and Prebiotics Vol. 8, No. 4, pp. xx-xx, 2013
ISSN 1555-1431 print, Copyright © 2013 by New Century Health Publishers, LLC
www.newcenturyhealthpublishers.com
All rights of reproduction in any form reserved
GASTROINTESTINAL SURVIVAL OF BACTERIA IN COMMERCIAL PROBIOTIC PRODUCTS
1,2Mathieu Millette, 2Anne Nguyen, 1Khalie Mahamad Amine and 1Monique Lacroix
1INRS-Institut Armand-Frappier, Research Laboratories in Sciences Applied to Food, Institute of Nutraceuticals and
Functional Foods, Canadian Irradiation Centre, 531, Boulevard des Prairies, Laval, Québec, Canada, H7V 1B7; and
2Bio-K Plus International Inc., 495, Boulevard Armand-Frappier, Laval, QC, Canada, H7V 4B3
[Received Month XX, 2013; Accepted October 11, 2013]
ABSTRACT: is work compared bacterial gastrointestinal
(GI) resistance of commercial probiotic products (capsules,
fermented milk and powder). To simulate GI transit,
the probiotic products were subjected to gastric fluid for
120 min then to intestinal fluid for 180 min. Gastric
and intestinal fluids were prepared according to United
States Pharmacopeia protocols. Bacterial enumeration was
compared before and after the GI transit to evaluate the
protective effect of the vehicle or the food matrix. Bacteria
of the four probiotic capsules covered with an enteric
coating had a higher survival rate (<1 log10 CFU reduction)
than uncoated. Eight encapsulated but non enteric coated
probiotic products showed limited GI resistance (between
1 and 5 log10 CFU reduction) while five products showed
no GI survival. For probiotic fermented milk, two products
demonstrated excellent or good protective property (<1 log10
CFU reduction) while the other four showed no resistance.
Only one of six powdered probiotic strains had excellent GI
survival. is study demonstrated that GI survival varies
from one probiotic product to another. It reiterates the
importance of manufacturing probiotic strains using the
appropriate vehicle for the bacteria to reach its site of action
and produce the expected beneficial effects.
KEY WORDS: Acid Tolerance, Bile Salts, Gastrointestinal,
Probiotic
Corresponding Author: Professor Monique Lacroix, INRS-Institut
Armand-Frappier, Research Laboratories in Sciences Applied
to Food, Institute of Nutraceuticals and Functional Foods,
Canadian Irradiation Centre, 531, Boulevard des Prairies, Laval,
Québec, Canada, H7V 1B7; Tel.: +311 450-687-5010 #4489;
Fax: +311 450-686-5501; E-mail: monique.lacroix@iaf.inrs.ca
INTRODUCTION
Probiotics are dened as «live microorganisms which, when
administered in adequate amounts, confer a health benet on
the host» (Araya et al. 2002). A good probiotic strain should
preferably be of human origin, possess a generally recognized
as safe (GRAS) status, the capacity to survive through the
gastrointestinal (GI) tract and colonize the gut (Ronka et al.
2003). A wide range of probiotics ready for consumption
are currently available on the market. However, the ecacy
of commercially available probiotic products diers a lot,
since their properties and characteristics are dierent from
a probiotic strain to another. In most cases, marketing has
preceded scientic control (De Angelis et al. 2007). In fact,
the GI survival of several strains of probiotics has not been
supported by scientic evidence. In order for the bacteria to
exert their benecial eects on the host, they must be able to
survive and reach the GI tract in sucient numbers, at least
106-107 CFU/g (Bosnea et al. 2009). e ability of a probiotic
to survive through the GI system depends mainly on their
acid and bile tolerance. During GI passage, the strains are
required to tolerate the presence of pepsin and the low pH of
the stomach, the presence of enzymes in the duodenum and
the antimicrobial activity of bile salts (Masco et al. 2007).
erefore, it is indispensable to demonstrate their survival
by in vitro experiments that simulate the human GI tract
conditions before conducting expensive in vivo tests.
e most studied probiotic are the lactic acid bacteria
(LAB), especially Lactobacillus and Bidobacterium (Verdenelli
et al. 2009). ey are also the most commonly found in
probiotic products for human consumption (Gueimonde et
al. 2004; Masco et al. 2007). Lactobacilli are non-pathogenic
microorganisms in human and animal intestine. Studies have
shown that lactobacilli possessed inhibitory eect towards
enteropathogens and produce several antimicrobial compounds
(Jacobsen et al. 1999; Millette et al. 2007). Bidobacterium
strains have also various health benets, from inhibition
of enteric pathogens to amelioration of lactose digestion,
immune system modulation, and reductions of symptoms
related to allergy and hepatic encephalopathy (Talwalkar and
Kailasapathy 2004).

2
e biggest issue regarding many in vitro studies is that
these experiments do not evaluate the GI survival rate of
probiotic strains in commercial products. In 2008, Sumeri et
al. reported that the same probiotics in dierent food matrix
behaved dierently. is, together with variations in bile
excretion between individuals and with the food, could clarify
the contradictory results obtained between in vitro and in vivo
experiments.
A recent study demonstrated that Lactobacillus
casei Shirota, L. casei Immunitas and L.
acidophilus subsp. johnsonii were able to survive in
vitro gastric and gastric plus duodenal digestion
by using a dynamic gastric model (DGM) of
digestion followed by incubation under duodenal
conditions, with milk and/or water as vehicle.
L. acidophilus johnsonii was found to be the best
probiotic strain because of its highest survival in
both tested foods (milk and water) (Lo Curto et
al. 2011). A dynamic model with two reactors
simulating gastric and duodenal conditions was
designed by Mainville in 2005 (Mainville et al.
2005). A food matrix was included in the design
to better represent the pH levels found in vivo
before, during and after meal consumption.
Two strains (Bidobacterium animalis ATCC
25527 and Lactobacillus johnsonii La-1 NCC
533) exhibited good survival through the GI
tract with and without the food matrix. Another
simple and non expensive way to assess the GI
survival of bacteria is to use static simulated
gastric and intestinal uids. In fact, another
recent study demonstrated that bile-adapted
Bidobacterium strains were able to better survive
in vitro in human gastric and duodenal uids
than the wild strain (de los Reyes-Gavilan et al.
2011). Moreover, Millette et al. (2008) used this
model to demonstrate the GI survival of various
probiotics.
erefore, the aim of the present study was to
establish the GI resistance in vitro of the bacteria
contained in 29 commercially available probiotics.
To our knowledge, this is the rst study verifying
the GI survival of probiotic bacterial strains in
nished commercial product as available in the
market. is is of importance because viability is
part of the WHO/FAO probiotic denition. To
mimic the GI conditions, simulated gastric and
intestinal uids have been used.
MATERIAL AND METHODS
Commercial probiotic products
Twenty-nine commercially available probiotic
products were purchased from natural health food
stores, supermarkets or drugstores in USA and
Canada. All tests were performed using the commercial product
(fermented milk, powder, capsules and yogurts) as purchased.
e probiotic products were stored as recommended on their
label (room temperature or refrigerated) until utilization.
Strains labelled on the probiotics are presented in the Table
I (capsules) or in Figures 2 (fermented milks or probiotic-
enriched yogurts) and 3 (powders).
TABLE 1. Ability of capsules to remain intact after 2 h in simulated gastric solution, pH 1.5.
Probiotic
Capsule
Number
of capsules
resistant to
gastric acidity
after 2 h
Strains
1 6/6 L. acidophilus CL1285, L. casei LBC80R
2 6/6
B. bidum, B. breve, B. longum, L. acidophilus, L.
rhamnosus, L. casei, L. plantarum, Lc. lactis, L.
bulgaricus, L. salivarius
3 6/6
L. bidus, L. acidophilus, L. helveticus 8781, L.
plantarum, L. casei, B. longum, B. infantis, B. breve, S.
thermophilus, L. bulgaricus
4 6/6
L. rhamnosus R0011, L. casei R0215, L. plantarum
R1012, L. acidophilus R0052, B. longum BB536, B.
breve R0070, P. acidilactici R1001, Lc. lactis R1058
5 0/6
B. bidum HA-132, B. longum HA-135, B. breve
HA-129, L. acidophilus HA-122, L. casei HA-108, L.
rhamnosus HA-111, L. rhamnosus HA-114, L. plantarum
HA-119, Lc. lactis HA-136, S. thermophilus HA-110
6 0/6
L. acidophilus R0052, L. rhamnosus R0011, S.
thermophilus R0083, Lc. lactis R1058, B. breve RR0070,
B. longum R0175, P. acidilactici R1001, L. delbrueckii
R9001
7 0/6
Saccharomyces boulardii, L. plantarum, Bacillus subtilis, L.
paracasei, L. brevis, L. acidophilus, L. casei, L. rhamnosus,
L. salivarius, B. longum, B. bidum, B. breve, B. lactis
8 0/6 L. acidophilus, L. acidophilus, B. bidum, B. lactis
9 0/6 L. acidophilus, L. casei, L. rhamnosus, Enterococcus
faecium
10 0/6 L. rhamnosus, L. casei, L. acidophilus, B. longum, B.
bidum
11 0/6
L. acidophilus, L. rhamnosus, S. thermophilus, L.
plantarum, B. bidum, L. bulgaricus, B. longum, L.
Salivarius
12 0/6
L. casei, L. rhamnosus, B. breve, B. longum, L.
acidophilus, L. plantarum, L. rhamnosus, B. bidum, Lc.
Lactis, L. bulgaricus, L. helveticus, L. salivarius
13 0/6 L. rhamnosus GG
14 0/6 L. acidophilus KS-13, B. bidum G9-1, B. longum MM-2
15 0/6 L. acidophilus, L. plantarum, L. rhamnosus, L. casei, L.
paracasei, L. salivarius, B. bidum, B. longum
16 0/6 L. acidophilus, L. rhamnosus, S. thermophilus, Lc. lactis,
B. bidum, B. longum, L. bulgaricus
17 0/6 L. acidophilus LA-5, B. lactis BB12, S. thermophilus STY-
31, L. delbrueckii LBY-27

3
Preparation of simulated gastric and intestinal uids
To test the GI survival of encapsulated probiotic bacteria, a
simulated gastric solution (SGF #1) at pH 1.5 was prepared
(Anonymous 1995). is solution was prepared by dissolving
2.0 g of NaCl (Laboratoire MAT, Quebec, QC, Canada)
and 3.2 g of porcine mucosa pepsin (1100 U/mg of protein;
P-7000; Sigma-Aldrich Canada Ltd, Oakville, ON, Canada)
in 900 mL of water. e pH was then adjusted by HCl (1
N; Fisher Scientic Company, ON, Canada) to obtain a nal
pH of 1.5. e solution was completed with water for a nal
volume of 1000 mL. e second simulated gastric solution
(SGF #2) was needed for the treatment of probiotic fermented
milk or yogurts and powders because all bacteria were killed by
SGF at pH 1.5 as demonstrated in preliminary experiments.
e formulation was similar as SGF #1, but the nal pH was
adjusted at 2.0 with HCl.
Finally, a simulated intestinal solution (SIF) was prepared
by dissolving 6.8 g of KH2PO4 (Laboratoire MAT) in 250
mL of water. en, 77 mL of NaOH (0.2 N) and 500 mL
of water,1.25 g of pancreatin (activity equivalent to 8 times
the specications of USP; P-7545; Sigma-Aldrich) and 3 g of
bile salts (Oxgall; P-8381; Sigma-Aldrich) were added to the
solution. Eventually, the pH was adjusted to 6.8 ± 0.1 with
NaOH (0.2 N) or HCl (0.2 N). e SIF was completed with
water to obtain 1000 mL.
All the solutions were tested for sterility on MRS (EMD
Chemicals inc, Mississauga, ON, Canada) and Plate Count
agar (BD Biosciences, Mississauga, ON, Canada) and the plates
were incubated for 72h at 37°C under anaerobic atmosphere.
Treatment of probiotic capsules in SGF
e SGF was incubated at 37°C for 60 min before the
experiment to simulate the body temperature. A probiotic
capsule was added to 25 mL of SGF #1 and then the solution was
incubated at 37°C with stirring (200 rpm) using an incubator-
shaker (Environmental Shaker G24, New Brunswick Scientic
Co. Inc.; Edison, NJ, USA) to simulate the bowel movements.
After 120 minutes, the capsule was removed and added to the
SIF. If the capsule was dissolved, 1 mL of the gastric uid was
transferred to the SIF.
Treatment of probiotic fermented milk, powder or yogurts
in SGF
e SGF #2 was incubated at 37°C for 60 min before
the experiment to simulate the body temperature. One g of
probiotic yogurt, fermented milk or powder was added to 24
mL of SGF #2, and the solution was incubated at 37°C under
stirring (200 rpm) using an incubator-shaker (Environmental
Shaker G24) to reproduce the bowel movements. After 120
minutes, 1 mL of the SGF#2 was transferred to the SIF.
Treatment of the probiotic products in SIF
e SIF was incubated at 37°C for 60 min before the
experiment to simulate the body temperature. Following
the gastric treatment, the 1 mL of SGF or the capsule taken
previously was transferred in 24 mL of SIF. e intestinal
suspensions were incubated at 37°C under stirring (200 rpm)
for 180 minutes and 1 mL of each suspension was withdrawn
and the evaluation of bacteria survival was performed as
described below.
Assessment of bacterial survival
To determine the initial count of bacteria contained in the
capsules, each non treated capsule was opened and rehydrated
in 9 ml of MRS for 30 minutes at 37°C to allow optimal
suspension of bacteria mixed with the excipients. en, a
series of tenfold dilution was performed in sterile peptone
water (0.1% w/v) and appropriate dilutions were pour plated
into MRS agar and incubated 72 h at 37°C under anaerobic
conditions. e incubation time of 30 min did not allowed
cell division of bacteria. erefore, there was no risk of false
results.
When powder, fermented milk or yogurts were evaluated,
11 g of product was added to 99 mL of sterile peptone water
(0.1% wt/vol) in a sterile bag and homogenized using a Lab-
blender 400 stomacher (Laboratory Equipment, London, UK)
for 1 min. e suspension was diluted, plated and incubated
as described above. e colonies were then enumerated using a
Dark eld Quebec Colony Counter.
After GI treatment, 1 mL of intestinal uid was withdrawn
then diluted in sterile peptone water, plated, incubated and
enumerated as described above.
Statistical analysis
For each probiotic product, total bacterial concentration was
evaluated from three independent samples before GI transit
while six samples were subjected to GI uids and analyzed for
bacterial concentration per capsule or gram. Values are given as
means ± standard deviation. Data were analyzed with the SPSS
software (version 19; IBM-SPSS, Chicago, Ill, USA). Student’s
t-test for two paired samples was used to compare the mean
of bacterial concentration of each probiotic product before
GI treatment to the mean after the treatment. Dierences
between means were considered signicant at P ≤ 0.05.
RESULTS
Survival of probiotic capsules under GI conditions
To assess the resistance of probiotic capsules to gastric
acidity, the products were added to SGF (pH 1.5) for 2 h. To
determine the survival level of bacteria under GI conditions,
the assessment of their survival was performed at the initial
time (T = 0) and at the end of the intestinal time treatment.
e dierence between the two values was evaluated. Results
showed that only probiotic capsules #1 to 4 were able to resist
gastric acidity (< 1 log10 CFU reduction). Eight encapsulated
but non enteric coated probiotic products showed limited GI
resistance (between 1 and 5 log10 CFU reduction) while the last
ve products showed no GI survival. e other capsules were
all dissolved under gastric condition (Table I and Figure 1).

4
Survival of fermented milk or probiotic-enriched yogurt
under GI conditions
As for the fermented milk, only one out of the eight
products evaluated (#18) demonstrated an excellent survival
rate with an initial bacteria count of 8.98 log CFU/g and a
nal count of 9.00 log CFU/g (Figure 2). Another probiotic
product showed a good survival (#19) with an initial count
of 8.77 log CFU/g and a nal count of 8.11 log CFU/g.
e products #20-22 had a moderate GI survival with a
respective initial value of 7.58, 7.23 and 6.47 log CFU/g
and nal counts of 5.47, 5.37 and 5.46 log CFU/g. e
last fermented milk (#23) had a bad survival rate because
its initial and nal bacteria count was from 4.07 to 3.8 log
CFU/g.

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



 
    
FIGURE 1. Survival of encapsulated probiotic bacteria after 2 h in simulated gastric uid (pH 1.5) and 3h in simulated intestinal uid
(pH 6.8). An asterisk means signicant dierence between bacterial before and after GI treatment (P ≤ 0.05). Please see Table 1 legend for the
type of bacteria in each capsule numbered 1 to 17.
FIGURE 2. Survival of bacteria in fermented milk or probiotic-enriched yogurt after 2 h in simulated gastric uid (pH 2.0) and 3h in
simulated intestinal uid (pH 6.8). 18: L. acidophilus CL1285 and L. casei LBC80R; 19: L. casei DN-114001; 20: B. lactis DN-173010; 21:
L. acidophilus NCFM and B. lactis HN 019; 22: B. lactis and L. acidophilus; 23: B. lactis, Streptococcus thermophilus, L. bulgaricus, L. casei and L.
acidophilus. An asterisk means signicant dierence between bacterial before and after GI treatment (P ≤ 0.05).


5
Survival of probiotic powder under GI conditions
Six probiotic powders were evaluated for their GI survival
(Figure 3). Results showed that the product #24 was the only
one showing an excellent survival rate with an initial count of
11.08 log CFU/g and a nal count of 10.98 log CFU/g. e
samples #25 and #26 had a moderate survival rate showing an
initial count of 10.87 and 8.55 log CFU/g and a nal counts of
7.93 and 5.83 log CFU/g respectively. e last three probiotic
powders (#27-29) demonstrated a bad survival rate by having
a respective initial value of 9.11, 8.56 and 8.3 log CFU/g and
a nal count of under the limit of detection (3.8 log CFU/g)
for each of them.
DISCUSSION
Although many scientists agree on the importance of the
probiotics bacteria survival in vivo, many products available
on the market don’t meet the requirements. is study
demonstrated that not all probiotic products were able to
survive GI conditions in vitro, and showed that among the
probiotic capsules evaluated, only those that were enteric
coated were able to resist to the degradation caused by
stomach conditions. e results demonstrate the importance
of protecting the bacteria by adding an enteric coating to
the capsules. ese data also support those found by Priya et
al. (2011). ese authors showed that the GI survival of L.
acidophilus increased when the probiotic was encapsulated. In
fact, the uncoated bacteria were almost completely destroyed
under GI conditions. Moreover, the encapsulated bacteria
are freeze-dried to increase the bacterial concentration and
the stability of the probiotic products. is study conrm
also that enteric coating protect the bacteria during their
passage through the GI tract because its ingredients resist
dissolution under acidic conditions, but are soluble under the
alkaline conditions of the intestine (Long and Chen 2009).
However, several studies have reported that the conditions
under which samples are freeze-dried (e.g. phase of growth,
suspending uid, cell concentration, drying and freeze-drying
technique) could strongly aect the bacterial viability (Berny
and Hennebert 1991; Lodato et al. 1999; Bolla et al. 2011).
erefore, it is important to assess the survival of probiotic
strains by evaluating the nal product.
For the probiotic powders, only one product had an
excellent survival rate (#24). Compared to the other samples,
that product contained a higher level of bacteria, with 450
billion live bacteria per package. It could be hypothesized
that the large amount of bacteria in the product may have a
protective eect, which would explain the great survival of the
probiotic strains.
One probiotic milk (#18) stood out from the others
because of its excellent rate of GI survival. is product was
a fermented milk unlike other products that were probiotic-
enriched yogurt. e advantage of fermented substances is
that the exogenous bacteria reach the large intestine in an
intact and viable form, which allows them to exert their eect
immediately upon consumption. erefore, this protective
and nourishing environment could ensure optimal bacterial
activity (Gibson and Roberfroid 1995). In addition, some
studies have shown that probiotic strains survived better when
stored in milk (Lo Curto et al. 2011; Tompkins et al. 2011).
FIGURE 3. Survival of probiotic powder after 2 h in simulated gastric uid (pH 2.0) and 3h in simulated intestinal uid (pH 6.8).
24: L. casei, L. plantarum, L. acidophilus, L. delbrueckii subsp. bulgaricus, B. longum, B. breve, B. infantis and Streptococcus salivarius subsp.
thermophilus; 25: L. acidophilus; 26: L. acidophilus and L. bidus; 27: B. longum BB536; 28: L. acidophilus LAC361 and B. longum BB536;
29: L. plantarum and B. lactis. An asterisk means signicant dierence between bacterial before and after GI treatment (P ≤ 0.05).
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6
is result could be related by the buering eect of milk
which could protect the strains against harmful eect of gastric
and duodenal environment (Siro et al. 2008).
Grzeskowiak et al. (2011) have demonstrated that dierent
isolates of the same strain (L. rhamnosus GG) had dierent
properties that could inuence their in vivo eects. is study
emphasized the importance of controlling the manufacturing
process and the food matrix since previous studies have
indicated that the vehicle could aect the strain properties
(Kankaanpaa et al. 2001; Kankaanpaa et al. 2004). Moreover,
in a recent review, they reported that some studies have shown
that a probiotic mixture was not more eective than a single
strain. e hypothesis is that a greater variety of strains reduce
the eectiveness of a multi-strain probiotic. e many species
could inhibit each other by production of antagonistic agents
or by competition for the nutrients or binding sites in the GI
tract (Chapman et al. 2011). erefore, it is primordial not
only to choose strains that coexist, but also act synergistically.
is, combine with the manufacturing process and individual
variability, could explain the dierent results obtained between
the probiotic products evaluated in this study.
Millette et al. (Millette et al. 2008) demonstrated that
the probiotic mixture of L. acidophilus CL1285 and L. casei
LBC80R could resist the gastric conditions at pH ≥ 2.5, which
is consistent with the ndings in this study. For the probiotic
strain, L. rhamnosus GG, large losses (up to 6 log) were observed
with the addition of bile salts in another study (Sumeri et al.
2008). ese results conrm those of this study because the
probiotic capsule #13 contained only L. rhamnosus GG and
its initial count was 10.06 log CFU/g with a nal count lower
than 3.8 log CFU/g after the intestinal treatment, which is
a loss of more than 6 log. Clinical studies also demonstrated
that L. casei DN-114001 could survive the GI tract in infants
and adults (Oozeer et al. 2006; Tormo Carnicer et al. 2006).
is eect was conrmed in this study with the #19 having
a good survival rate. Favaro-Trindade and Grosso (2002)
showed that free L. acidophilus La-05 and B. lactis Bb-12 were
tolerant to bile acid in vitro even when the concentration was
greater than the normal concentration found in the human
intestine. Moreover, these strains underwent a slight reduction
of concentration at pH 2, but were completely destroyed at
pH 1 after one hour. In this study, the probiotic capsules #17
was not able to survive the gastric conditions at pH 1.5 and the
intestinal conditions.
In conclusion, our study showed the importance of evaluating
the survival of probiotic strains in the nished product since
their viability could be modied during the manufacturing
process. It also showed that all probiotic products were
not similar and that some could not even survive the harsh
environment of the GI tract in order to exert their benecial
eects. erefore, because we observed that the majority of the
probiotic products have failed to protect the GI survival of the
strains, it would be important for manufacturers to develop
technologies to ensure this ability. quality and the ecacy of
the products. Finally, the use of enteric coating of encapsulated
probiotic bacteria seem to be eective to preserve bacterial
viability during the GI passage.
ACKNOWLEDGEMENTS
M. Millette received an industrial R&D fellowship (IRDF)
from NSERC. Financial support by Bio-K+ International Inc.
(Laval, Quebec, Canada) and NSERC.
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