ArticlePDF Available

Gastrointestinal survival of bacteria in commercial probiotic products

Authors:
  • Bio-K Plus International

Abstract and Figures

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.
Content may be subject to copyright.
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.






 
    
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).


  
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.
REFERENCES
Anonymous (1995). Simulated gastric uid and simulated
intestinal uid, TS. In: e United States Pharmacopeia 23,
e National Formulary 18. (Rockville: Maryland: e United
States Pharmacopeial Convention, Inc.), p. 2053.
Araya, M., Morelli, L., Reid, G., Sanders, M.E. and Stanton,
C. (2002). Guidelines for the evaluation of probiotic in foods.
(Ontario: Canada: FAO/WHO), pp.1-11.
Berny, J.F. and Hennebert, G.L. (1991). Viability and stability
of yeast cells and lamentous fungus spore during freeze-
drying: Eects of protectants and cooling rates. Mycologia
83:805-815.
Bolla, P.A., Serradell Mde, L., de Urraza, P.J. and De Antoni,
G.L. (2011). Eect of freeze-drying on viability and in vitro
probiotic properties of a mixture of lactic acid bacteria and yeasts
isolated from ker. e Journal of Dairy Research 78:15-22.
Bosnea, L.A., Kourkoutas, Y., Albantaki, N., Tzia, C.,
Koutinas, A.A. and Kanellaki, M. (2009). Functionality of
freeze-dried L. casei cells immobilized on wheat grains. LWT -
Food Science and Technology 42:1696-1702.
Chapman, C.M., Gibson, G.R. and Rowland, I. (2011).
Health benets of probiotics: are mixtures more eective than
single strains? European Journal of Nutrition 50:1-17.
De Angelis, M., Siragusa, S., Caputo, L., Ragni, A.,
Burzigotti, R. and Gobbetti, M. (2007). Survival and
persistence of Lactobacillus plantarum 4.1 and Lactobacillus
reuteri 3S7 in the gastrointestinal tract of pigs. Veterinary
Microbiology 123:133-144.
de los Reyes-Gavilan, C.G., Suarez, A., Fernandez-Garcia, M.,
Margolles, A., Gueimonde, M. and Ruas-Madiedo, P. (2011).
Adhesion of bile-adapted Bidobacterium strains to the HT29-
MTX cell line is modied after sequential gastrointestinal
challenge simulated in vitro using human gastric and duodenal
juices. Research in Microbiology 162:514-519.
Favaro-Trindade, C.S. and Grosso, C.R. (2002).
Microencapsulation of L. acidophilus (La-05) and B. lactis
(Bb-12) and evaluation of their survival at the pH values of
the stomach and in bile. Journal of Microencapsulation 19:485-
494.
7
Gibson, G.R. and Roberfroid, M.B. (1995). Dietary
modulation of the human colonic microbiota: introducing
the concept of prebiotics. e Journal of Nutrition 125:1401-
1412.
Grzeskowiak, L., Isolauri, E., Salminen, S. and Gueimonde,
M. (2011). Manufacturing process inuences properties of
probiotic bacteria. British Journal of Nutrition 105:887-894.
Gueimonde, M., Delgado, S., Mayo, B., Ruas-Madiedo, P.,
Margolles, A. and de los Reyes-Gavilán, C.G. (2004). Viability
and diversity of probiotic Lactobacillus and Bidobacterium
populations included in commercial fermented milks. Food
Research International 37:839-850.
Jacobsen, C.N., Rosenfeldt Nielsen, V., Hayford, A.E.,
Moller, P.L., Michaelsen, K.F., Paerregaard, A., Sandstrom, B.,
Tvede, M. and Jakobsen, M. (1999). Screening of probiotic
activities of forty-seven strains of Lactobacillus spp. by in
vitro techniques and evaluation of the colonization ability
of ve selected strains in humans. Applied and Environmental
Microbiology 65:4949-4956.
Kankaanpaa, P., Yang, B., Kallio, H., Isolauri, E. and
Salminen, S. (2004) Eects of polyunsaturated fatty acids in
growth medium on lipid composition and on physicochemical
surface properties of lactobacilli. Applied and Environmental
Microbiology 70:129-136.
Kankaanpaa, P.E., Salminen, S.J., Isolauri, E. and Lee, Y.K.
(2001). e inuence of polyunsaturated fatty acids on
probiotic growth and adhesion. FEMS Microbiology Letters
194:149-153.
Lo Curto, A., Pitino, I., Mandalari, G., Dainty, J.R., Faulks,
R.M. and John Wickham, M.S. (2011). Survival of probiotic
lactobacilli in the upper gastrointestinal tract using an in
vitro gastric model of digestion. Food Microbiology 28:1359-
1366.
Lodato, P., Se govia de Huergo, M. and Buera, M.P. (1999).
Viability and thermal stability of a strain of Saccharomyces
cerevisiae freeze-dried in dierent sugar and polymer matrices.
Applied Microbiology and Biotechnology 52:215-220.
Long, M. and Chen, Y. (2009) Drug Release Test Methods
for Enteric Coated Products. In: Qiu, Y., Chen, Y. and
Zhang, G.G.Z. (Eds), Developing solid oral dosage forms:
pharmaceutical theory and practice. (New York: New York:
Academic Press), p.331.
Mainville, I., Arcand, Y. and Farnworth, E.R. (2005). A
dynamic model that simulates the human upper gastrointestinal
tract for the study of probiotics. International Journal of Food
Microbiology 99:287-296.
Masco, L., Crockaert, C., Van Hoorde, K., Swings, J. and
Huys, G. (2007). In vitro assessment of the gastrointestinal
transit tolerance of taxonomic reference strains from human
origin and probiotic product isolates of Bidobacterium.
Journal of Dairy Science 90:3572-3578.
Millette, M., Luquet, F.M. and Lacroix, M. (2007). In
vitro growth control of selected pathogens by Lactobacillus
acidophilus- and Lactobacillus casei-fermented milk. Letters in
Applied Microbiology 44:314-319.
Millette, M., Luquet, F.M., Ruiz, M.T. and Lacroix, M. (2008).
Characterization of probiotic properties of Lactobacillus
strains. Dairy Science & Technology 88:695-705.
Oozeer, R., Leplingard, A., Mater, D.D., Mogenet, A.,
Michelin, R., Seksek, I., Marteau, P., Dore, J., Bresson, J.L.
and Corthier, G. (2006). Survival of Lactobacillus casei in the
human digestive tract after consumption of fermented milk.
Applied and Environmental Microbiology 72:5615-5617.
Priya, A.J., Vijayalakshmi, S.P. and Raichur, A.M. (2011).
Enhanced survival of probiotic Lactobacillus acidophilus by
encapsulation with nanostructured polyelectrolyte layers
through layer-by-layer approach. Journal of Agricultural and
Food Chemistry 59:11838-11845.
Ronka, E., Malinen, E., Saarela, M., Rinta-Koski, M.,
Aarnikunnas, J. and Palva, A. (2003). Probiotic and milk
technological properties of Lactobacillus brevis. International
Journal of Food Microbiology 83:63-74.
Siro, I., Kapolna, E., Kapolna, B. and Lugasi, A. (2008).
Functional food. Product development, marketing and
consumer acceptance-a review. Appetite 51:456-467.
Sumeri, I., Arike, L., Adamberg, K. and Paalme, T. (2008).
Single bioreactor gastrointestinal tract simulator for study
of survival of probiotic bacteria. Applied Microbiology and
Biotechnology 80:317-324.
Talwalkar, A. and Kailasapathy, K. (2004). A review of oxygen
toxicity in probiotic yogurts: inuence on the survival of
probiotic bacteria and protective techniques. Comprehensive
Reviews in Food Science and Food Safety 3:117-124.
Tompkins, T.A., Mainville, I. and Arcand, Y. (2011). e
impact of meals on a probiotic during transit through a model
of the human upper gastrointestinal tract. Benecial Microbes
2:295-303.
Tormo Carnicer, R., Infante Pina, D., Rosello Mayans, E.
and Bartolome Comas, R. (2006). Intake of fermented milk
containing Lactobacillus casei DN-114 001 and its eect on
gut ora. Anales de Pediatria 65:448-453.
8
Verdenelli, M.C., Ghel, F., Silvi, S., Orpianesi, C., Cecchini,
C. and Cresci, A. (2009). Probiotic properties of Lactobacillus
rhamnosus and Lactobacillus paracasei isolated from human
faeces. European Journal of Nutrition 48:355-363.
... This suggests that Kapi1 can be induced by mitomycin C through the traditional SOS pathway in response to DNAdamaging agents, although there appears to be a basal level of spontaneous induction even in the absence of mitomycin C. The classical mechanism of SOS-mediated prophage induction as set by the widely studied phage l is dependent on an activated form of recA, which mediates autocleavage of the l repressor, cI (47). recA is activated by the presence (50), or LB supplemented with 0.5 ng/ml mitomycin C for 24 h. After 24 h, the number of cells was enumerated by spotting on LB plates, and the number of phages were enumerated by spotting on soft-agar overlays prepared with KP121. ...
... Since MP1 was recently isolated from the feces of a healthy mouse (11) and is more host adapted than our standard laboratory strains of E. coli such as MG1655 or MC4100, we wondered if Kapi1 might be important to the biology of commensal E. coli in the GI tract. To investigate the biology of Kapi1 under more physiologically relevant conditions, we repeated the same experiments following the numbers of phage and host cells in media composed of 50% LB and 50% simulated intestinal fluid (SIF) as well as 50% LB and 50% simulated gastric fluid (SGF) (50). We found lower ratios of Kapi1 PFU to host CFU in SIF than LB (Fig. 5A), although the strains grow to nearly identical cell densities. ...
... To monitor the CFU and PFU in cultures of MP13 Kapi1 lysogens over time, three colonies of MP13 and three colonies of KP7 (nonlysogenic control) were picked and grown overnight. The next day, cultures were adjusted to an OD 600 of 1.0 to ensure equal cell numbers and then subcultured 1:100 into LB, LB with 0.5 ng/mL mitomycin C, LB mixed 50:50 with simulated intestinal fluid (SIF; 6.8 g KH 2 PO 4 , 1.25 g pancreatin, 3 g bile salts in 1 L distilled water [dH2O], pH adjusted to 7 [50]), or LB mixed 50:50 with simulated gastric fluid (SGF; 2 g/L NaCl, 3.2 g/L porcine mucosa pepsin, pH adjusted to 3.5 [50]). After 24 h incubation, an aliquot was taken from each culture. ...
Article
Full-text available
Although research exploring the microbiome has exploded in recent years, our understanding of the viral component of the microbiome is lagging far behind our understanding of the bacterial component. The vast majority of intestinal bacteria carry prophages integrated into their chromosomes, but most of these bacteriophages remain uncharacterized and unexplored.
... According to Millette et al. (2013), for a probiotic culture to provide benefits to the individual it is necessary that it survives and reaches the gastrointestinal tract with counts of at least 10 6 and 10 7 CFU g -1 . Therefore, at the end of the SGIC exposure ( Figure 2D -enteric phase II), the Control formulation would not be able to provide probiotic health benefits on any day evaluated. ...
... Viability of commercial probiotics under the GI conditions was reported to be low (Millette et al., 2013). The effect of LbL coating on the viability of single-cell probiotics and its protective properties against harsh conditions are still under study and not hugely investigated. ...
Article
Full-text available
Probiotics and prebiotics are widely used as functional food ingredients. Viability of probiotics in the food matrix and further in the digestive system is still a challenge for the food industry. Different approaches were used to enhance the viability of probiotics including microencapsulation and layer-by-layer cell coating. The of aim of this study was to evaluate the viability of coated Lacticaseibacillus rhamnosus using a layer-by-layer (LbL) technique with black seed protein (BSP) extracted from Nigella sativa defatted seeds cakes (NsDSC), as a coating material, with alginate, inulin, or glucomannan, separately, and the final number of coating layers was 3. The viable cell counts of the plain and coated L. rhamnosus were determined under sequential simulated gastric fluid (SGF) for 120 min and simulated intestinal fluid (SIF) for 180 min. Additionally, the viability after exposure to 37, 45, and 55°C for 30 min was also determined. Generally, the survivability of coated L. rhamnosus showed significant (p ≤ 0.05) improvement (<4, 3, and 1.5 logs reduction for glucomannan, alginate and inulin, respectively) compared with plain cells (∼6.7 log reduction) under sequential exposure to SGF and SIF. Moreover, the cells coated with BSP and inulin showed the best protection for L. rhamnosus under high temperatures. Edible films prepared with pectin with LbL-coated cells showed significantly higher values in their tensile strength (TS) of 50% and elongation at the break (EB) of 32.5% than pectin without LbL-coated cells. The LbL technique showed a significant protection of probiotic cells and potential use in food application.
... Low stomach pH and high bile acid concentrations are the major factors in reducing probiotic viability (Sahadeva et al., 2011;Millette et al., 2013). Thus delayed-release delivery systems such as DRcaps® capsules or VC-in-DR, targeting colonic delivery, may improve probiotic performance in modulating gut microbial function and potentially its diversity and composition, as observed in this study in vitro, leading to various health benefits. ...
Article
Full-text available
Oral administration of active pharmaceutical ingredients, nutraceuticals, enzymes or probiotics requires an appropriate delivery system for optimal bioactivity and absorption. The harsh conditions during the gastrointestinal transit can degrade the administered products, hampering their efficacy. Enteric or delayed-release pharmaceutical formulations may help overcome these issues. In a Simulator of Human Intestinal Microbial Ecosystem model (SHIME) and using caffeine as a marker for release kinetics and L. acidophilus survivability as an indicator for protection, we compared the performance of ten capsule configurations, single or in DUOCAP® combinations. The function of L. acidophilus and its impact on the gut microbiota was further tested in three selected capsule types, combinations of DRcaps® capsule in DRcaps® capsule (DR-in-DR) and DRcaps® capsule in Vcaps® capsule (DR-in-VC) and single Vcaps® Plus capsule under colonic conditions. We found that under stomach and small intestine conditions, DR-in-DR and DR-in-VC led to the best performance both under fed and fasted conditions based on the slow caffeine release and the highest L. acidophilus survivability. The Vcaps® Plus capsule however, led the quickest caffeine and probiotic release. When DR-in-DR, DR-in-VC and single Vcaps® Plus capsules were tested through the whole gastrointestinal tract including under colonic conditions, caffeine release was found to be slower in capsules containing DRcaps® capsules compared to the single Vcaps® capsules. In addition, colonic survival of L. acidophilus was significantly increased under fasted conditions in DR-in-DR or DR-in-VC formulation compared to Vcaps® Plus capsule. To assess the impact of these formulations on the microbial function, acetate, butyrate and propionate as well as ammonia were measured. L. acidophilus released from DR-in-DR or DR-in-VC induced significant increase in butyrate and decrease in ammonia, suggesting a proliferation of butyrate-producing bacteria and reduction in ammonia-producing bacteria. These data suggest that L. acidophilus included in DR-in-DR or DR-in-VC reaching the colon is viable and functional, contributing to changes in colonic microbiota composition and diversity.
... The probiotic association Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2 has demonstrated a great gastrointestinal survival (Millette et al., 2008(Millette et al., , 2013 and multiple health benefits. Indeed, it has been proved it can prevent and reduce the incident of antibiotic associated diarrhea (Beausoleil et al.,2007), and Clostridioides difficile associated diarrhea (McFarland et al., 2018), reduce symptoms of irritable bowel syndrome (Preston et al., 2018) and it also has anticancer properties (Baldwin et al., 2010;Desrouillères et al., 2015). ...
Article
Full-text available
The objective of this study was to develop probiotic beverages, enriched with plant proteins, with high nutritional value. A rice-based beverage fermented with a specific probiotic formulation comprised Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R and Lactobacillus rhamnosus CLR2 has been enriched with a combination of pea and rice proteins (PR) or pea and hemp proteins (PH) at 13 and 11% total protein, respectively. These protein associations have been selected because their amino acid ratio was >1, as recommended by the FAO. The beverage enriched with protein significantly increased its viscosity by more than 10 times thanks to the enrichment, while the fermentation reduced it by 50% for PR and 20% for PH. In vitro protein digestibility results showed that the protein enrichment and the fermentation treatment significantly increased digestibility values of the beverages with value of 72.7% for fermented PR beverage and 61.4% for unenriched fermented control beverage (p ≤ 0.05). Peptide profiles of PR and PH enriched beverages indicated that the fermentation led to a reduced level of high molecular weight (HMW) peptides of about 60% and an increase of low molecular weight (LMW) peptides by over 50%. Therefore, both the fermentation and the enrichment in protein increased the nutritional value of the rice-based beverages. Practical Application Good quality of probiotics formulation and high-protein products are in increasing demand and plant proteins as an alternative of animal protein are popular. This study has permit to develop rice-based commercial probiotic beverages enriched in a combination of pea and rice or pea and hemp proteins in order to obtain a complete protein in terms of amino acids composition. The lactic acid fermentation and the enrichment with a plant protein combination led to a better protein digestibility of beverage.
... Proteolytic activity of lactic acid bacteria (LAB) allows to generate more peptides and free amino acids, leading to a better digestibility (Melini et al., 2019;Raveschot et al., 2018). The combination of Lactobacillus acidophilus CL1285, Lactobacillus casei (Lacticaseibacillus) LBC80R, and Lactobacillus rhamnosus (Lacticaseibacillus) CLR2 has already demonstrated multiple health benefits such as its ability to reduce symptoms of irritable bowel syndrome (Preston et al., 2018), to prevent and reduce the antibioticassociated diarrhea (Beausoleil et al., 2007;Mcfarland et al., 2018), and Clostridioides difficile associated diarrhea (McFarland et al., 2018) and it has a good gastrointestinal survival (Millette et al., 2008;Millette et al., 2013). However, the effect of this combination of probiotics on the nutritional quality of products has never been studied. ...
Article
Full-text available
The aim of this study was to evaluate the effect of the fermentation of a probiotic beverage enriched with pea and rice proteins (PRF) on its protein quality. The protein quality was determined as the protein efficiency ratio (PER), net protein ratio (NPR), and the apparent (AD) and the true digestibility (TD) evaluated in vivo. The probiotic beverage was incorporated to a rat diet at a final concentration of 10% protein, for the evaluation of the PER, the NPR, the AD, and the TD. The protein digestibility amino acid score was also calculated. Results showed that the fermentation of beverage enriched with PRF had no effect on the TD but significantly increased the PER and the NPR (P ≤ 0.05) from 1.88 to 2.32 and from 1.66 to 2.30, respectively. Thus, the fermentation increased the protein quality of the PRF probiotic beverage. In addition, to determine if the beverage constitute in a good carrier matrix for the probiotics, the level of alive probiotics in the feces was evaluated and showed a concentration of 7.4 log CFU/g. Practical Application Plant proteins are often of lower quality compared to animal proteins. Lactic acid fermentation of pea and rice protein has allowed to reach the same protein quality as casein. A plant-based fermented beverage with high protein quality and enriched with probiotics was developed.
... By industry standards, an effective dose is generally expressed as the number of colonyforming units (CFUs). Based on this definition, some experts do not consider fecal microbiota transplant a probiotic because it is not standardized with identified microorganism strains 10 . It is important to note that the terms "live" or "active" do not imply probiotic activity. ...
Article
Full-text available
The review is to analyse the therapeutic benefits of probiotics in various chronic disorders.All the data were identified using PUBMED (2000-2020) and bibliographic reviews of recent and old articles from an English literature search. After independent analysis by co-authors of the identified articles, data were analysed and extracted for the stated purpose.The most-studied species include Lactobacillus,Bifidobacterium,and Saccharomyces.Probiotics have an essential role in maintaining immunologic equilibrium in the gastrointestinal tract through direct interaction with immune cells. There is high-quality evidencethat probiotics are effective for acute infectious diarrhoea, antibiotic-associated diarrhea, Clostridium difficileassociated diarrhoea, hepatic encephalopathy, ulcerative colitis, irritable bowel syndrome, and necrotizing enterocolitis. Probiotics are safe for infants, children, adults, and older patients.Therapeuticapplications of probiotics have been widely studied to treat and improve the gut's health; however, choosing a probiotic for a specific condition is a challenging task that requires parsing of the data for strain-specific efficacy and evaluation of product quality. It appears likely that more national organizations will be conducting evidence-based research, and the pharmacist and clinicians should pay attention to this ever-changing field
Chapter
Fermented beverages are increasing in demand to fulfill the health needs of the human population. Increasing medical costs are forcing us to find cheaper and effective resources for protecting human health. The use of probiotics has been proven in the treatment of several inflammatory conditions including arthritis, pouchitis, Crohn’s disease, and colitis. The ingredients of dairy-based fermented beverages contain protein, minerals, and vitamins that provide a favorable environment for the growth of probiotics. Modern fermented beverage production includes a defined starter culture with desirable characteristics to ensure consistency and commercial viability of the final product. The selection of defined starters depends on specific phenotypes that benefit the product by guaranteeing shelf life and ensuring the safety, texture, and flavor of the final product. Recent research revealed that the whey-based fermented beverage cosupplemented with Lactobacillus casei possesses high bactericidal activity. In the production of dairy-based fermented beverages, the industrialist is concerned about the new approaches towards the active viability of the probiotic culture involved in beverage production. Hence, the novel starter culture plays a key role in modern dairy-beverage production. In the modern production process, the industrialist is much concerned about the increasing level of bacteriocin and bioactive peptide production and minimizes the presence of biogenic amines in the final products by selecting a novel starter culture. In large scale fermented dairy-beverage production, viability, stability of starter cultures, and certain technological challenges are also faced by the industrialists. Some novel approaches to improve the stability and survival of probiotic strains include protective compounds such as glucose to energize cells on exposure to acid during protection, and cryoprotectants such as inulin to improve survivability during freeze-drying. Genetically manipulated strains have shown improved performance due to overexpression of heat shock proteins GroESL under a variety of conditions including heat, spray drying, and exposure to gastric acid. The major difficulty in probiotic dairy-beverage production is the preservation of the physical stability of the product. Process optimization of probiotic or functional dairy-based beverages needs extra care, including the selection of concentration and type of stabilizer and optimization of pretreatment conditions such as high-effect homogenization and heating regimes. This chapter concentrates on the overview of fermented dairy beverages, recent scientific, technological, and commercial development in the production of dairy beverages which includes the strain selection, processing, starter cultures and selecting appropriate foods as a vehicle. The chapter also discusses their challenges, improvements made to overcome these challenges, diversified beverages and its production, screening of novel organisms for the production of new beverages with eliminated challenges and improvement of sensory properties.
Article
The incidence of antibiotic-associated diarrhea, according to various authors, ranges from 5 to 39% and depends on the patient’s age and other contributing factors. Antibiotic-associated diarrhea can be caused by any antibiotic, regardless of dosage form or route of administration. In the pediatric population, the prevalence of antibiotic-associated diarrhea ranges from 6 to 70%. An urgent problem is the development of this disease against the background of a course of H. pylori eradication therapy, which significantly complicates tolerance and adherence to therapy. This article presents current data on the pathogenesis and risk factors of antibiotic-associated diarrhea in children. The clinical picture ranges from idiopathic enteritis to antibiotic-associated diarrhea caused by Cl. difficile - pseudomembranous colitis. The main principle of antibiotic-associated diarrhea treatment is cancellation of the antibacterial medicine that caused the diarrhea, or reducing its dose (if the course of the disease allows it). In complex treatment sorbents are used, correction of water-electrolyte balance is carried out. The use of probiotics seems quite logical for the treatment and prevention of antibiotic-associated diarrhea in terms of the pathogenesis of this condition. To correct dysbiosis, drugs are used to maintain and restore the quantitative and qualitative composition of the intestinal microbiota. Taking into account modern recommendations the main groups of drugs (probiotics, prebiotics, synbiotics) used for correction of intestinal microbiocenosis are presented. The mechanism of action of probiotics and mechanisms of their effect on intestinal microflora are considered. The basic requirements for bacterial strains that are part of the probiotic drugs are presented. The results of various randomized clinical trials and meta-analyses confirming the necessity of including probiotic complexes in antibiotic-associated diarrhea treatment regimens are presented from an evidence-based medicine perspective. The clinical effects of strains of Lactobacillus spp ., Bifidobacterium spp ., Streptococcus spp . and Lactococcus spp . on the digestive tract microbiota are considered. The role of a synbiotic containing 9 probiotic strains of 4.5 × 10 ⁹ CFU in one capsule and the prebiotic component fructooligosaccharides in the prevention of antibiotic-associated diarrhea in children is discussed separately. The results of microbiological studies confirmed the presence of microorganisms of genera Bifidobacterium , Lactobacillus , Streptococcus in the product, and the content of bacteria in one dose of the product was not less than 2 x 10 ¹⁰ CFU.
Chapter
The term “probiotic” is derived from Greek words and means “for life.” The definitions of this term were developed over time. The current definition of probiotic by FAO/WHO is “live microorganisms, when consumed in adequate amounts, confer a health effect on the host.” The selection of candidate probiotics must be based on safety, functionality, and technological aspects. The theoretical basis for the selection of probiotics can be listed as human origin, stability in acid and bile salts, adherence to human intestinal cells, production of antimicrobial substances, safety in food and clinical use, and clinically validated and documented health effects. The starting point for the selection of probiotic candidates should be careful characterization of the strain using both phenotypic and genetic techniques. Then, the use of in vitro tests to assess the potential functionality and safety of the strains may facilitate the selection of the most appropriate probiotic.
Article
Two parameters of a one step freeze-drying process, cooling rate and protecting media, are studied in an effort to improve the viability and the stability of the preserved fungal strains. Cooling rates of 1.6 C, 3 C and 40 C/min are applied on cells of Saccharomyces cerevisiae and Brettanomyces bruxellensis and on spores of Trichoderma viride and Arthrobotrys arthrobotryoides preserved in 93 suspending media containing polymers, sugars, albumin, milk, honey, polyols, amino acids alone or in combination. The viability rate of Saccharomyces cerevisiae cells is increased from 30% to 96-98% by using an appropriate protecting medium containing 10% skim milk with 2 compounds among honey, sodium glutamate, trehalose or raffinose in the freeze-drying process carried out at a 3 C/min cooling rate. In the same conditions Arthrobotrys arthrobotryoides spores, the most sensitive strain among the four tested, provides 60-65% viability, while this strain does not survive a classical freeze-drying in 10% skim milk. Moreover, with the improved method the stability of properties of Penicillium expansum is demonstrated.
Article
Objective To study the gut flora in infants who received fermented milk containing Lactobacillus casei and Streptococcus termophilus and its effect on secretory immunoglobulin levels. Material and methods An experimental, randomized, prospective, parallel group study was carried out. Thirty-five infants were included (18 in the treatment group and 17 in the control group) with a mean age of 2 years (SD: 0.6 years; range: 1-3 years). The experimental group received both fermented milk (0.5 l/day) containing L. casei and S. termophilus for 6 weeks and standard cow’s milk for the following 6 weeks. The control group received standard cow’s milk (0.5 l/day) for 12 weeks. Secretory IgA levels in saliva were evaluated in the experimental group at the start of the study (baseline levels) and 6 weeks later. In both groups, stools were collected to study gut flora at 0, 6 and 12 week. Results Secretory IgA levels significantly increased (p = 0.0063) from a mean baseline value of 2.5 mg/dl to a mean of 3.4 mg/dl at 6 weeks. Gram-negative aerobic flora were decreased in the experimental group after 6 weeks compared with the control group (p = 0.0203). The number of infants with Lactobacillus spp in their gut flora was greater in the experimental group than in the control group at week 6 and this difference was statistically significant (p = 0.028) at week 12. Conclusion The present study provides evidence of L. casei survival in the gastrointestinal tract and of its effect of increasing secretory IgA.
Article
Oxygen toxicity is considered a significant factor influencing the viability of probiotic bacteria such as Lactobacillus and Bifidobacterium spp. in yogurts. This review assesses the involvement of oxygen during the manufacture of yogurt and its diffusion into the final product. The oxygen sensitivity of Lactobacillus acidophilus and Bifidobacterium spp. is discussed. The impact of dissolved oxygen of the product on the survival of these probiotic bacteria is highlighted. Additionally, microbiological, chemical, and packaging techniques recommended to protect probiotic bacteria from oxygen toxicity in dairy products are reviewed.
Article
Lactobacillus casei cells were immobilized on wheat grains and the effect of nine cryoprotectants during freeze-drying was investigated. Survival and fermentative activity of the freeze-dried immobilized biocatalysts was studied by monitoring pH, lactic acid and lactose content in successive fermentations batches of both synthetic lactose medium and milk. Freeze-dried L. casei cells immobilized on wheat grains without using cryoprotectants resulted in high cell survival and metabolic activity. The same biocatalysts were stored at room temperature for 9 months and at 4 °C and −18 °C for 12 months. Reactivation of the stored biocatalysts was carried out in synthetic lactose medium. Storage at room and low temperatures (4 °C and −18 °C) resulted in about 5.11, 4.9 and 4.3 final pH respectively during fermentations, indicating the suitability of the immobilized biocatalysts for the production of mild and low pH dairy products. The immobilization of a probiotic microorganism, such as L. casei, on boiled wheat which contains prebiotic compounds might provide a potential synbiotic preparation.
Article
A selection of commercial fermented milks was evaluated for the presence and viability of probiotic lactobacilli and bifidobacteria. Counts of Lactobacillus spp. always remained higher than 105 CFU ml−1, whereas the population of Bifidobacterium spp. decreased below this level in two products. All the probiotics announced on the label were present in commercial products, yet in two fermented milks one additional group of microorganisms was also found. The yogurt microorganism Streptococcus thermophilus was present in all cases, whereas Lactobacillus delbrueckii ssp. bulgaricus was only detected in two products. Bifidobacterium was the most frequently isolated group, all commercial strains being identified as Bifidobacterium animalis/Bifidobacterium lactis by analysis of partial sequences of the 16 rRNA gene. The same technique allowed the identification of members of Lactobacillus casei (species Lactobacillus casei/Lactobacillus paracasei/Lactobacillus zeae) and Lactobacillus acidophilus (species L. acidophilus sensu stricto and Lactobacillus johnsonii). The analysis of macrorrestriction profiles by pulsed-field gel electrophoresis evidenced the reduced genetic variability existing among commercial Lactobacillus and Bifidobacterium strains. The combined use of macrorrestriction analysis and carbohydrate fermentation profiles enhanced the discriminatory power of the first technique for strains differentiation. None commercial Bifidobacterium strain presented the harmful β-glucuronidase activity whereas all of them displayed β-galactosidase, α-glucosidase and α-galactosidase activity.
Article
Commercial literature on various probiotic products suggests that they can be taken before meals, during meals or after meals or even without meals. This has led to serious confusion for the industry and the consumer. The objective of our study was to examine the impact of the time of administration with respect to mealtime and the impact of the buffering capacity of the food on the survival of probiotic microbes during gastrointestinal transit. We used an in vitro Digestive System (IViDiS) model of the upper gastrointestinal tract to examine the survival of a commercial multi-strain probiotic, ProtecFlor®. This product, in a capsule form, contains four different microbes: two lactobacilli (Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011), Bifidobacterium longum R0175 and Saccharomyces cerevisiae boulardii. Enumeration during and after transit of the stomach and duodenal models showed that survival of all the bacteria in the product was best when given with a meal or 30 minutes before a meal (cooked oatmeal with milk). Probiotics given 30 minutes after the meal did not survive in high numbers. Survival in milk with 1% milk fat and oatmeal-milk gruel were significantly better than apple juice or spring water. S. boulardii was not affected by time of meal or the buffering capacity of the meal. The protein content of the meal was probably not as important for the survival of the bacteria as the fat content. We conclude that ideally, non-enteric coated bacterial probiotic products should be taken with or just prior to a meal containing some fats.
Article
The encapsulation of probiotic Lactobacillus acidophilus through layer-by-layer self-assembly of polyelectrolytes (PE) chitosan (CHI) and carboxymethyl cellulose (CMC) has been investigated to enhance its survival in adverse conditions encountered in the GI tract. The survival of encapsulated cells in simulated gastric (SGF) and intestinal fluids (SIF) is significant when compared to nonencapsulated cells. On sequential exposure to SGF and SIF for 120 min, almost complete death of free cells is observed. However, for cells coated with three nanolayers of PEs (CHI/CMC/CHI), about 33 log % of the cells (6 log cfu/500 mg) survived under the same conditions. The enhanced survival rate of encapsulated L. acidophilus can be attributed to the impermeability of polyelectrolyte nanolayers to large enzyme molecules like pepsin and pancreatin that cause proteolysis and to the stability of the polyelectrolyte nanolayers in gastric and intestinal pH. The PE coating also serves to reduce viability losses during freezing and freeze-drying. About 73 and 92 log % of uncoated and coated cells survived after freeze-drying, and the losses occurring between freezing and freeze-drying were found to be lower for the coated cells.
Article
The aim of this study was to investigate survival of three commercial probiotic strains (Lactobacillus casei subsp. shirota, L. casei subsp. immunitas, Lactobacillus acidophilus subsp. johnsonii) in the human upper gastrointestinal (GI) tract using a dynamic gastric model (DGM) of digestion followed by incubation under duodenal conditions. Water and milk were used as food matrices and survival was evaluated in both logarithmic and stationary phase. The % of recovery in logarithmic phase ranged from 1.0% to 43.8% in water for all tested strains, and from 80.5% to 197% in milk. Higher survival was observed in stationary phase for all strains. L. acidophilus subsp. johnsonii showed the highest survival rate in both water (93.9%) and milk (202.4%). Lactic acid production was higher in stationary phase, L. casei subsp. shirota producing the highest concentration (98.2 mM) after in vitro gastric plus duodenal digestion.
Article
Lactobacillus sp. are important inhabitants of the intestines of animals. They are also largely used as probiotics for both humans and animals. To exert beneficial effects, lactobacilli have to survive through the gastrointestinal transit. Based on bile-salt resistance, pH tolerance, antimicrobial activity and heat resistance, Lactobacillus plantarum 4.1 and Lactobacillus reuteri 3S7 were previously selected and used as probiotic additives in pelleted feeding trials. Both strains were fed to pigs (sows and piglets) at a cell number of ca. 10(10) viable cells per day. A polyphasic approach, comprising growth on selective media, Biolog System analysis, 16S rRNA gene sequencing and RAPD-PCR typing, was used to identify and differentiate L. plantarum 4.1 and L. reuteri 3S7 from other faecal Lactobacillus sp., L. plantarum 4.1 and L. reuteri 3S7 had the capacity to survive during the gastrointestinal transit and were found in the feaces at a cell number of 6-8 log cfu/g. Their persistence was shown after 6 days from the administration. Compared to untreated pigs, the administration of L. plantarum 4.1 and L. reuteri 3S7 significantly (P<0.05) decreased the population of Enterobacteriaceae. Besides, the beta-glucuronidase activity of all pigs decreased of ca. 23.0% during administration. The findings of this study showed that L. plantarum 4.1 and L. reuteri 3S7 have the potential to be used as probiotic additives in pelleted feed for pigs.