Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestines using a single reactor model
© Ritter et al; licensee BioMed Central Ltd. 2009
Received: 17 December 2008
Accepted: 08 May 2009
Published: 08 May 2009
Probiotic bacteria are thought to play an important role in the digestive system and therefore have to survive the passage from stomach to intestines. Recently, a novel approach to simulate the passage from stomach to intestines in a single bioreactor was developed. The advantage of this automated one reactor system was the ability to test the influence of acid, bile salts and pancreatin.
Lactobacillus gasseri K7 is a strain isolated from infant faeces with properties making the strain interesting for cheese production. In this study, a single reactor system was used to evaluate the survival of L. gasseri K7 and selected bifidobacteria from our collection through the stomach-intestine passage.
Initial screening for acid resistance in acidified culture media showed a low tolerance of Bifidobacterium dentium for this condition indicating low survival in the passage. Similar results were achieved with B. longum subsp. infantis whereas B. animalis subsp. lactis had a high survival.
These initial results were confirmed in the bioreactor model of the stomach-intestine passage. B. animalis subsp. lactis had the highest survival rate (10%) attaining approximately 5 × 106 cfu ml-1 compared to the other tested bifidobacteria strains which were reduced by a factor of up to 106. Lactobacillus gasseri K7 was less resistant than B. animalis subsp. lactis but survived at cell concentrations approximately 1000 times higher than other bifidobacteria.
In this study, we were able to show that L. gasseri K7 had a high survival rate in the stomach-intestine passage. By comparing the results with a previous study in piglets we could confirm the reliability of our simulation. Of the tested bifidobacteria strains, only B. animalis subsp. lactis showed acceptable survival for a successful passage in the simulation system.
Probiotics, especially lactic acid bacteria have beneficial effects on consumers health as suggested in 1907 . It was believed that bacteria mainly controlled infections caused by enteric pathogens and regulated toxoaemia, thereby improving health and influencing mortality. Meanwhile it has been known that some of the positive effects on consumers health are the improvement in the microflora balance in the gut, the stimulation of the immune system, and aiding the organism to fight pathogenic microorganisms . A large part of interest was concentrated on the use of strains of the genera Lactobacillus and Bifidobacterium, even if there are also other bacteria with probiotic effects, e.g. some propionibacteria.
The above mentioned properties are also the basis for a microorganism to be labelled probiotic. There are different definitions worldwide but they are similar in content. One of the criteria for a probiotic strain is its resistance to acidity and gastric solutions in the human gastrointestinal tract . It is therefore important, to evaluate the resistance of a potential probiotic strain to the acidic and gastric environment in the intestine.
Because of high costs and ethical as well as safety regulations for clinical studies, screening survival is easier to simulate in vitro. A simple test is to incubate the bacterial cells in acidic or bile salt solutions for a defined period and count the number of surviving cells. In a further step, the simulation is carried out in agitated flasks, combining acidity and gastric solutions followed by an estimation of surviving cells over the entire simulation. This is a more realistic replication of the conditions in the intestine . Another system, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), consists of 5 to 6 serially connected pH controlled bioreactors [5–7]. The setup is quite complex and demands absolute anaerobic conditions. Furthermore, the absorption of metabolites and water is not simulated. This was overcome by using dialysis membranes as described by Marteau et al. .
Recently, a new system using a single bioreactor was developed to study the stomach-intestine passage . The system allowed the pH to be altered inside a single reactor and was adapted to the retention times in the different regions of the stomach-intestine passage.
Lactobacillus gasseri K7 was recently isolated from infant faeces . It produces a bacteriocin which is active against Clostridium sp. and their spores. L. gasseri belongs to the so called "acidophilus"-group and several independent studies identified these strains as inhabitants of the skin and intestine [11–13]. In previous experiments, it has already been shown in vitro that L. gasseri K7 survived in an acidic environment and with 0.3% bile salts . These findings make the strain interesting as a possible probiotic.
In this study, a single bioreactor system based on the work of Sumeri et al.  was used to evaluate the survival of Lactobacillus gasseri K7 and eight Bifidobacterium strains from our collection. We were able to compare the results for L. gasseri K7 with a study performed in piglets  which allowed the assessment of a correlation between the in-vitro study with results from in-vivo experiments.
pH values in the human intestinal tract, measured with the Heidelberg capsule.
Retention times in the small intestine cited in literature.
Huang and Adams 2004 
Van Den Driessche et al. 2000 
Stomach and small intestine
Mojaverian 1996 
Picot and Lacroix 2004 
Selected maximum time of the simulation
Fallingborg et al. 1990 
Fallingborg et al. 1989 
Alander et al. 1998 
Simulation in the SHIME Reactor
Thews et al. 1991 
Acid resistance screening
All the other tested Bifidobacterium strains (B. longum, B. breve, B. longum subsp. infantis and B. adolescentis) showed a similar but different pattern from B. animalis subsp. lactis (Figures 2, 3 and 4). They had a short survival time below pH 2.5 and survived in higher numbers above pH 3.5.
The left-hand column of Figure 5 shows the same strain without added skim milk. At a pH above 3.5, there was no influence on the survival of the bacteria. However, below pH 3.5 the survival decreased depending on the duration of incubation. Between pH 3.0 and 3.5 the strain had already decreased by about log 5. After 30 min incubation, there was almost a linear decrease in survival with decreasing pH from 3.0 to 2.5.
Simulation in the bioreactor
Most systems described in the literature consist of several reaction vessels, e.g. the SHIME . Other studies used immobilized cells with three reactors  or a dialysis system . Based on the work of Sumeri et al.  and the collected data of the conditions in the intestinal passage we were able to limit the simulation to one vessel. Together with the data from the acid resistance screening, the selection of a possible starting pH and broth composition in the simulator could be chosen. The resulting simulation parameters are shown in Figure 1 and described in the Material and Methods section. During the experimental stage of this study, Sumeri et al.  developed a similar system to evaluate Lactobacillus sp. in a stomach-intestine passage simulation.
Strains tested in the simulation.
Identification number of ALP strain collection
Bifidobacterium longum subsp. infantis
Bifidobacterium animalis subsp. Lactis
FAM-14382, -14383, -14406
Lactobacillus gasseri K7
Bifidobacterium adolescentis was inoculated as described above at an initial concentration of 107 cfu ml-1 and decreased almost linearly to below 104 cfu ml-1 after 5 hours. B. breve and B. longum strains had an initial concentration between 107 and 108 cfu ml-1 and diminished to below 102 cfu ml-1 within the first 30 minutes. B. animalis subsp. lactis 14403 survived to approximately 15% of the initial average cfu of 5 × 108 cfu ml-1. There was a rapid decrease in survival of B. longum subsp. infantis over the first 30 min. Afterwards the survival decreased only slowly from 105 to 104 cfu ml-1.
The preparation of the inoculum of L. gasseri K7 in a 100 ml culture volume was also evaluated. The results of the experiments are shown in Figure 7. With 250 ml culture the decrease in living cells was about log 2 whereas the decrease with a 100 ml culture was only log 1 over the whole incubation time. However, 2 h after addition of bile salts and pancreatic juice, the decrease in cell counts was similar for both volumes.
When harvesting a culture after a given incubation time, the growth phase of each bacterial strain can be different since all have different growth dynamics. In order to obtain cells at approximately the same growth phase, preliminary experiments were performed (data not shown). An incubation time of 15 h for the pre-culture was suitable for all tested strains except Bifidobacterium longum subsp. infantis which needed to be incubated for only 12 h.
The acid tolerance screening (Figures 2, 3 and 4) was performed to evaluate the effect of pH independently of other conditions. Bifidobacterium dentium was highly sensitive to acid and therefore would possibly not survive the passage through the stomach. The strain was therefore not included in the simulation experiments. The B. longum strains (Figure 2) did not yield much better results than B. dentium (Figure 3). However, close to pH 4 they were more resistant than B. dentium.
B. longum subsp. infantis is one of the first species to populate the human intestine shortly after birth . Based on the experiments in this study, however, the tested B. longum subsp. infantis strain would only be able to pass the infant stomach in high numbers if the transition time in the acidic stomach was very short. The survival of the selected strain in the tested environment was too low for successful passage in high numbers. When the strain was resuspended in skim milk, survival increased (Figure 5). This could be an indication that human milk helps B. longum subsp. infantis strains to pass the stomach-intestine passage with at a higher survival rate.
The protective effects of milk proteins in the digestive system have already been described in the literature . Protection with milk proteins has also been shown in this study (Figure 5). With the appropriate matrix or even a carrier, probiotic bacteria could safely pass through the stomach to the intestines to reach their site of action.
B. adolescentis strains that populate the human intestine at a later age, had slightly higher resistance than B. longum subsp. infantis which may explain the reduction of the latter during the progress of the human infant to adulthood .
The most interesting strain was B. animalis subsp. lactis, which was the least sensitive strain in our study. This pH-resistant strain has a great potential for use in foods as a probiotic supplement since a higher number of bacterial cells would survive the passage. However, to use this strain as probiotic, more studies have to be performed in order to achieve the probiotic status according to the definition of Klaenhammer .
In our study, the ingestion of a food matrix was simulated in an initial environment of acidified milk and growth medium. The added simulated gastric solution and oxygen during the stomach phase increased the stress. During the simulated passage to the small intestine the oxygen was replaced by nitrogen and the medium was neutralized to pH 6.3. The addition of the pancreatic solution and bile salts completed the passage into the small intestine. This in-vitro system did not take into account that in in vivo digestion, enzymes are activated and inactivated and other substances, e.g. bile salts are reabsorbed. Sumeri et al.  found a partial solution to bypass this problem. They diluted the content of the reactor with a specially designed dilution medium. Another possibility would be to precipitate the bile salts at the end of simulation of the small intestine to imitate the enterohepatic circuit. This could be performed with calcium ions [28–30]. Removing the bile salts would better simulate the environment of the colon and might even allow bifidobacteria to proliferate.
In our study, the remaining bile salts and pancreatic juice in the simulation led to an additional stress on bacteria which probably altered the true characteristics of the strains in vivo.
The starting cfu in the simulation varied within one log cfu even though the adjustment of OD650 of the inoculum was previously tested with the Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis strains. The bifidobacteria used in this study showed a tendency to form clusters that may result in reduced cfu (visual observations, data not shown). In another study, the formation of clusters could be related to decreasing pH during growth . These clusters are usually counted as one colony on a plate.
Figure 6 shows the results of the stomach-intestine passage simulation over 7 h of seven tested Bifidobacterium strains. The concentration of living cells of bifidobacteria decreased immediately after incubation due to the low pH (pH 3.0). However, B. animalis subsp. lactis remained stable. This confirmed the results of previous experiments discussed above (Figure 4). This resistance could be extended to bile salts and pancreatic juice although the cell counts of B. animalis subsp. lactis decreased by about 85% of the initial value (Figure 6). Compared to the other strains used in this study, however, this decrease was almost negligible.
All B. longum and B. breve strains died very rapidly at the beginning of the simulation and were below the detection limit of the plating method within a few hours (Figure 6) which was to be expected from the results of the screening experiment above (Figures 2 and 4).
On the other hand, B. longum subsp. infantis 14390 decreased rapidly at the beginning of simulation but after the addition of pancreatic juice and bile salts and a change to an anaerobic environment, the reduction rate decreased. Our study suggests that this strain is well adapted to the conditions in the intestine but needs to be ingested in high numbers to survive the conditions in the stomach (oxygen, low pH). As mentioned above, B. longum subsp. infantis strains belong to the first group of bacteria populating the intestine of infants .
In contrast to B. longum subsp. infantis, B. adolescentis decreased almost linearly during the 7 h simulation. There was no detectable interruption when the conditions in the fermenter changed. Based on the experiments for the acid tolerance screening, this result was unexpected.
However, this might be related to the testing conditions where the bile salt and gastric juice concentrations remained at the initial level and were not diluted as they would be in vivo. In a future experiment, it should be evaluated whether the dilution method developed by Sumeri et al.  would stabilize the cell counts of B. adolescentis during the 6 h simulation period in the intestine.
In our study, we also evaluated the stomach-intestine passage of Lactobacillus gasseri K7. The strain has already been evaluated for survival in vivo in piglets . Therefore, it was possible to compare our in-vitro results with data from in vivo experiments.
Bogovic et al.  fed piglets over a period of 14 days with 5*1010 cfu day-1 of L. gasseri K7. This resulted in approx. 7*104 cfu g-1 in the faeces during the feeding period. It has to be taken into account that the concentration of bacteria was diluted before it finally arrived at the stomach-intestine passage. In a rough approximation, we estimated that about 1% arrived at the passage. This allowed us to compare the results of this piglet study with the end of our simulation.
As shown in Figure 5, L. gasseri K7 had a cell concentration of approximately 5*104 cfu ml-1 after the 7 h simulation period (with a pre-culture of 250 ml) which is similar to the concentration in the faeces of the piglets. This suggests that the simulation model used in this study could be a helpful tool to estimate the effects of the passage in an in-vitro model prior using expensive in vivo models. The model could be further optimized by diluting the bile salts and pancreatic juice as described by Sumeri et al. . To simulate the activation and deactivation of enzymes a suitable method has still to be found.
When only 100 ml medium was used for the inoculum of L. gasseri K7, the culture survived the simulation better (Figure 7). Both volumes had a similar initial cell count. Both volumes were inoculated by 1 ml. Therefore, the culture with 250 ml volume was in an earlier stage of growth than the 100 ml culture. These results were an indication of the growth phase dependency of the culture for during stress.
In this study, we were able to show that the system to simulate the stomach-intestine passage developed by Sumeri et al.  was suitable for the assessment of survival of 8 Bifidobacterium strains and Lactobacillus gasseri K7 even though we did not simulate the removal of gastric juice and bile salts. For L. gasseri K7 we were able to compare the results with an in-vivo study on piglets and obtained similar results.
The single reactor system presented here allows a more straightforward identification of the ideal growth phase for any possible probiotic strain which is required to pass the stomach-intestine passage than if it had to be performed with other systems with a difficult setup.
The study also showed that all tested Bifidobacterium strains, except for B. animalis subsp. lactis, would require protective agents to survive the passage through the stomach-intestine in high numbers. This could be done using an appropriate food matrix or encapsulation of the cells.
All bifidobacteria strains were selected from the strain collection of Agroscope Liebefeld-Posieux ALP Research Station Switzerland, isolated by ALP from human sources. Lactobacillus gasseri K7 originated from the ZIM Collection of Industrial Microorganisms of University of Ljubljana, Biotechnical Faculty (ZIM 105)  and was also deposited in the ALP strain collection. The tested strains and their identification numbers of the ALP strain collection are listed in table 3. All bifidobacteria strains are the property of ALP.
Media and growth conditions
For pre-cultures, 1 ml frozen conserves of the strains were inoculated in 250 ml Wilkins-Chalgren broth (WC CM0643, Oxoid, Hampshire, UK) supplemented with 9 g l-1 additional lactose-monohydrate (Bifidobacteria) or De Man-Rogosa-Sharpe (MRS; Biolife, Milano, Italy) medium (Lactobacillus gasseri K7) . For L. gasseri K7, a trial with a 100 ml pre-culture was also performed. All strains, except Bifidobacterium longum subsp. infantis, were incubated at 37°C for 15 hours under anaerobic conditions. Bifidobacterium longum subsp. infantis was incubated for 12 h since it was very sensitive to extended incubation periods. The pre-cultures were centrifuged for 15 min at 3500 rpm and the pellets resuspended in 10 ml of phosphate-buffered physiological sodium chloride solution (PBS).
Determination of cell count
The cell count was determined by 10-fold serial dilution of the culture in physiological saline solution. The two highest dilutions were then plated on MRS agar (Biolife, Milano, Italy) using a spiral plater (IUL Instruments, Barçelona, Spain) and evaluated by an automated colony counter with the corresponding software (IUL Instruments, Barçelona, Spain).
Screening for acid resistance
For the acid resistance screening the concentrated cell suspension from the pre-culture was pipetted into 20 ml of PBS until an OD650 of 1.0 was reached. 4 ml of this cell suspension were then inoculated in 16 ml of citrate-HCl buffer (tri-Na-Citratex2 H2O 7.35 g and 250 ml distilled H2O, adapted to the corresponding pH with 1 M HCl) at pHs of 2.0, 2.5, 3.0, 3.5 and 4.0. The incubation was done at 37°C and samples were taken every 30 min over 120 min. 1 ml of samples were mixed with 9 ml 0.25 M phosphate buffer at pH 7.0 at the first step of the dilution series. For the acid resistance test in a food matrix, the same amount of pre-culture as used above (adjusted to an OD650 of 1.0) was pipetted into 20 ml of UHT skim milk. 4 ml of this cell suspension in milk were inoculated into 16 ml of citrate-HCl buffer. All chemicals were purchased from Merck (Darmstadt, Germany). The data for the screening experiments was visualized in contour plots using the Sigmaplot 11.0 software (Systat Software Inc., Chicago IL, USA).
Simulation in the bioreactor
All solutions were freshly prepared for each experiment. Simulated stomach solution was made of 50 mg pepsin porcine gastric mucosa (Sigma-Aldrich P7012, Buchs, Switzerland) in 20 ml of 0.1 M HCl. For the simulated pancreatic juice 2 g pancreatin (Sigma-Aldrich P7545) were dissolved in 50 ml of 0.02 M phosphate buffer at a pH of 7.5. Simulated bile salt solution was made of 7.5 g bovine bile (Sigma-Aldrich B3883) made up to 50 ml with distilled H2O. The broth for the simulation was either 1 l WC or MRS broth with 29.41 g tri-sodium citratex2 H2O. During testing of survival in a food matrix, 500 ml of UHT skim milk were added and the pH adjusted to 3.0 with 5 M HCl shortly before the simulation. 1 l medium was added to the bioreactor (NewMBR Mini, NewMBR, Switzerland), previously sterilized with water (121°C, 20 min), and heated to 37°C. During the stomach simulation, aeration was implemented. The fermentation was controlled and recorded using the integrated process management software Lucullus (Biospectra, Schlieren, Switzerland). The concentrated cell suspension from the pre-culture was pipetted into 40 ml of PBS to an OD650 of 1.5. Shortly before the inoculation of 40 ml cell suspension, 20 ml of the simulated stomach solution was added to the medium (1 l) in the bioreactor. The pH was adjusted using 2 M NaOH.
Sixty minutes after the inoculation of the cells, the oxygen was replaced by nitrogen to obtain an anaerobic atmosphere. This was performed by flushing the headspace and making the system air-tight. After attaining a pH of 5.0 (after approx. 1 h fermentation time), 34 ml of the bile salt solution and 50 ml pancreatic juice were inoculated. Samples were taken every 20 minutes during the first hour and then only every 60 minutes. The total simulation time was set to 7 hours with an average stomach pH of 3.0. The time in the stomach was set to one hour, followed by rapid neutralization to 6.3 and a slow increase to 7.5 over the remaining 5 hours and 40 minutes (Figure 1).
This work was financed by Agroscope Liebefeld-Posieux. We thank Vincent O'Reilly for his support on the work with L. gasseri K7. We also would like to thank Dr. M. Casey for his English proof reading of the manuscript.
- Metchnikoff E: The prolongation of life. 1908, New York, PutnamGoogle Scholar
- Cleusix V, Lacroix C, Vollenweider S, Le Blay G: Glycerol induces reuterin production and decreases Escherichia coli population in an in vitro model of colonic fermentation with immobilized human feces. FEMS Microbiol Ecol. 2008, 63: 56-64.PubMedView ArticleGoogle Scholar
- Klaenhammer TR, Kullen MJ: Selection and design of probiotics. Int J Food Microbiol. 1999, 50: 45-57. 10.1016/S0168-1605(99)00076-8.PubMedView ArticleGoogle Scholar
- Picot A, Lacroix C: Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. Int Dairy J. 2004, 14: 505-515. 10.1016/j.idairyj.2003.10.008.View ArticleGoogle Scholar
- Alander M, De Smet I, Nollet L, Verstraete W, Von Wright A, Mattila-Sandholm T: The effect of probiotic strains on the microbiota of the Simulator of the Human Intestinal Micobial Ecosystem (SHIME). Int J Food Microbiol. 1998, 46: 71-79. 10.1016/S0168-1605(98)00182-2.View ArticleGoogle Scholar
- Molly K, Woestyne V, Verstraete W: Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem. Appl Microbiol Biotechnol. 1993, 39: 254-258. 10.1007/BF00228615.PubMedView ArticleGoogle Scholar
- Tir Touil Meddah A, Yazourh A, Desmet I, Risbourg B, Verstraete W, Romond MB: The regulatory effects of whey retentate from Bifidobacteria fermented milk on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). J Appl Microbiol. 2001, 91: 1110-1117. 10.1046/j.1365-2672.2001.01482.x.View ArticleGoogle Scholar
- Marteau P, Minekus M, Havenaar R, Veld JHJ: Survival of Lactic Acid Bacteria in a Dynamic Model of the Stomach and Small Intestine: Validation and the Effects of Bile. J Dairy Sci. 1997, 80: 1031-1037.PubMedView ArticleGoogle Scholar
- Sumeri I, Arike L, Adamberg K, Paalme T: Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria. Appl Microbiol Biotechnol. 2008, 80: 317-324. 10.1007/s00253-008-1553-8.PubMedView ArticleGoogle Scholar
- Bogovic-Matijasic B, Rogelj I: Bacteriocinogenic activity of lactobacilli isolated from cheese and baby faeces. Food Technol Biotechnol. 1999, 37: 93-100.Google Scholar
- Bergonzelli GE, Blum S, Brussow H, Corthesy-Theulaz I: Probiotics as a treatment strategy for gastrointestinal diseases?. Digestion. 2005, 72: 57-68. 10.1159/000087638.PubMedView ArticleGoogle Scholar
- Olivares M, Diaz-Ropero MP, Martin R, Rodriguez JM, Xaus J: Antimicrobial potential of four Lactobacillus strains isolated from breast milk. J Appl Microbiol. 2006, 101: 72-79. 10.1111/j.1365-2672.2006.02981.x.PubMedView ArticleGoogle Scholar
- Pavlova SI, Kilic AO, Kilic SS, So JS, Nader-Macias ME, Simoes JA, et al.: Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences. J Appl Microbiol. 2002, 92: 451-459. 10.1046/j.1365-2672.2002.01547.x.PubMedView ArticleGoogle Scholar
- Bogovic Matijasic B, Stojkovic S, Rogelj I: Survival and in vivo adhesion of human isolates Lactobacillus gasseri LF221 and K7 in weaned piglets and their effects on coliforms, clostridia and lactobacilli viable counts in faeces and mucosa. J Dairy Res. 2006, 73: 417-422. 10.1017/S0022029906001890.View ArticleGoogle Scholar
- Fallingborg J: Intraluminal pH of the human gastrointestinal tract. Danish Med Bull. 1999, 46: 183-196.PubMedGoogle Scholar
- Fallingborg J, Christensen LA, Jacobsen BA, Ingeman-Nielsen M, Rasmussen HH, Abildgaard K, et al.: Effect of olsalazine and mesalazine on intraluminal pH of the duodenum and proximal jejunum in healthy humans. Scan J Gastroenterol. 1994, 29: 498-500. 10.3109/00365529409092461.View ArticleGoogle Scholar
- Fallingborg J, Pedersen P, Jacobsen BA: Small intestinal transit time and intraluminal pH in ileocecal resected patients with Crohn's disease. Digestive Dis Sci. 1998, 43: 702-705. 10.1023/A:1018893409596.View ArticleGoogle Scholar
- Andres MR, Bingham JR: Tubeless gastric analysis with a radiotelemetering pill (Heidelberg capsule). Can Med Assoc J. 1970, 102: 1087-1089.PubMed CentralPubMedGoogle Scholar
- Fallingborg J, Christensen LA, Ingeman-Nielsen M, Jacobsen BA, Abildgaard K, Rasmussen HH: pH-profile and regional transit times of the normal gut measured by a radiotelemetry device. Aliment Pharmacol Ther. 1989, 3: 605-613.PubMedView ArticleGoogle Scholar
- Fallingborg J, Christensen LA, Ingeman-Nielsen M, Jacobsen BA, Abildgaard K, Rasmussen HH, et al.: Measurement of gastrointestinal pH and regional transit times in normal children. J Ped Gastroenterol Nutr. 1990, 11: 211-214.View ArticleGoogle Scholar
- Huang Y, Adams MC: In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Int J Food Microbiol. 2004, 91: 253-260. 10.1016/j.ijfoodmicro.2003.07.001.PubMedView ArticleGoogle Scholar
- Mojaverian P: Evaluation of Gastointestinal pH and Gastric Residence Time via the Heidelberg Radiotelemetry Capsule: Pharmaceutical Application. Drug Devel Res. 1996, 38: 73-85. 10.1002/(SICI)1098-2299(199606)38:2<73::AID-DDR1>3.0.CO;2-H.View ArticleGoogle Scholar
- Thews G, Mutscheler E, Vaupel E: Anatomie, Physiologie, Pathophysiologie des Menschen (4. Auflage). 1991Google Scholar
- Driessche Van Den M, Van Malderen N, Geypens B, Ghoos Y, Veereman-Wauters G: Lactose-[13C]Ureide Breath Test: A New, Noninvasive Technique to Determine Orocecal Transit Time in Children. J Ped Gastroenterol Nutr. 2000, 31: 433-438. 10.1097/00005176-200010000-00019.View ArticleGoogle Scholar
- Cinquin C, Le Blay G, Fliss I, Lacroix C: New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota. FEMS Microbiol Ecol. 2006, 57: 324-336. 10.1111/j.1574-6941.2006.00117.x.PubMedView ArticleGoogle Scholar
- Ley RE, Peterson DA, Gordon JI: Ecological and Evolutionary Forces Shaping Microbial Diversity in the Human Intestine. Cell. 2006, 124: 837-848. 10.1016/j.cell.2006.02.017.PubMedView ArticleGoogle Scholar
- Charteris WP, Kelly PM, Morelli L, Collins JK: Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J Appl Microbiol. 1998, 84: 759-768. 10.1046/j.1365-2672.1998.00407.x.PubMedView ArticleGoogle Scholar
- Baruch E, Lichtenberg D, Barak P, Nir S: Calcium binding to bile salts. Chem Phys Lipids. 1991, 57: 17-27. 10.1016/0009-3084(91)90045-D.PubMedView ArticleGoogle Scholar
- De Boever P, Verstraete W: Bile salt deconjugation by Lactobacillus plantarum 80 and its implication for bacterial toxicity. J Appl Microbiol. 1999, 87: 345-352. 10.1046/j.1365-2672.1999.00019.x.PubMedView ArticleGoogle Scholar
- Meer Van der R, De Vries HT: Differential binding of glycine- and taurine-conjugated bile acids to insoluble calcium phosphate. Biochem J. 1985, 229: 265-268.PubMed CentralPubMedView ArticleGoogle Scholar
- von Ah U, Mozzetti V, Lacroix C, Kheadr E, Fliss I, Meile L: Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum. BMC Microbiol. 2007, 7: 79-10.1186/1471-2180-7-79.PubMed CentralPubMedView ArticleGoogle Scholar
- de Man J, Rogosa M, Sharpe ME: A medium for the cultivation of Lactobacilli. Journal of Applied Microbiology. 1960, 23: 130-135. 10.1111/j.1365-2672.1960.tb00188.x.Google Scholar
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