Survival of Lactobacillus casei in the Human Digestive Tract after Consumption of Fermented Milk

A human trial was carried out to assess the ileal and fecalsurvival of Lactobacillus casei DN-114 001 ingested in fermentedmilk. Survival rates were up to 51.2% in the ileum and 28.4%in the feces. The probiotic bacterium has the capacity to surviveduring its transit through the human gut.

INTRODUCTION

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Introduction

The probiotic strain Lactobacillus casei DN-114 001 (CNCM numberI-1518) reduces the frequency or the duration of episodes ofacute diarrhea in young children (20, 21), increases the lactobacillusconcentration in the gut microbiota of infants (10), and canmodulate ex vivo production of proinflammatory cytokines inCrohn's disease (4). Using mouse models harboring human microbiota,our group recently established that this bacterial strain caninitiate new protein synthesis during transit, suggesting thatthe bacterium is metabolically active (18, 19).

In the present study, a human trial was carried out to assessthe survival of L. casei DN-114 001 by culture analysis of ilealand fecal samples from healthy subjects consuming fermentedmilk. The test product (supplied by Danone Vitapole, Palaiseau,France) consisted of yogurt cultures (Streptococcus thermophilusand Lactobacillus delbruekii subsp. bulgaricus) supplementedwith a rifampin-resistant spontaneous variant of L. casei DN114 001, here referred to as strain DN-114 001^Rif. This stablevariant was isolated according to previously described methods(14) and led to the same growth kinetics and organoleptic propertiesas the original strain.

Ten volunteers (eight males and two females) with a median ageof 25.5 years (range, 22 to 38 years) were included in the studyafter giving their written consent and were remunerated fortheir participation in the trial. They had no history of gastrointestinaldisorders, no antibiotic treatment during the 2 months precedingthe trial, and no laxative treatment for the week prior to thestudy. During the whole investigation period, the only restrictionwith regard to diet was the exclusion of fermented dairy products.The study was approved by a local Institutional Ethics Committee(Comité Consultatif de Protection des Personnes dansla Recherche Biomédicale, Necker) and was conducted atNecker Hospital, Paris, France. A blind review of data was performedto identify major protocol deviation and to define intention-to-treatand per-protocol populations. In per-protocol population analysis,all patients who had failed to complete the whole study protocolwere excluded. Discontinuous variables were compared using theWilcoxon rank test. For multiplicity of significance testingin the main analysis, the homogeneity of the two groups wastested using the chi-square test. The confidence interval wascalculated as 95% (two tailed). The significance level usedwas 5%.

The trial consisted of two distinct steps, with an 8-day washoutperiod between steps. The first step corresponded to the ileal-survivalstudy, while the second focused on the analysis of L. caseiDN-114 001^Rif fecal survival.

Design of the ileal-survival study.

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Design of the ileal-survival...

Seven out of 10 volunteers consented to intestinal intubation,which was performed after a 7-day period during which fermenteddairy products were excluded from the diet. Each subject wasnasally intubated with a triple-lumen weighed tube (MarquatSA, Boissy-Saint Léger, France), and once the pyloruswas passed, a bag placed at the distal end of the first lumenwas inflated in order to hasten tube progression. When the baghad reached the cecum, as confirmed fluoroscopically, subjectswere asked to stay in a semirecumbent position. The second lumenwas used to sample the ileal contents 35 cm above the ileocecaljunction, while the third lumen, 25 cm proximal to the aspirationport, was used for infusion of polyethylene glycol (PEG).

On the morning of the following day, an infusion of 10 g PEG4000 in 154 mmol NaCl · liter^–1 at 37°C wasstarted at the rate of 2 ml · min^–1. After 1 hof equilibration, fasting subjects ingested a standard meal(125 g mashed potatoes, 1 hard boiled egg, 30 g jam, 100 g bread,30 g butter, 10 g sugar, 250 ml tea or coffee, and a portionof melted cheese) and 3 bottles (3 x 100 ml in a single dose)of fermented milk containing about 10⁸ CFU · ml^–1of L. casei DN-114 001^Rif and 10⁶ spores per ml of thermoresistantBacillus stearothermophilus (Merck, Darmstadt, Germany) as atransit marker (15). Since the tube failed to go below the jejunumlevel in three volunteers, only four subjects were subsequentlyanalyzed (the ileal per-protocol population). The ileal contentswere continuously collected on ice by manual aspiration, withthe aim of collecting as much fluid as possible over the 8 hfollowing ingestion of the meal. The aspirated samples werepooled into 1-h aliquots, which were processed immediately forbacterial analysis. The remaining fluids were stored at –20°Cfor subsequent measurement of the PEG concentrations by meansof turbidimetry (11), making it possible to calculate the populationof bacteria transiting over time (22). For bacterial enumeration,10-fold serial dilutions of homogenized ileal fluids were prepared,and each dilution was plated on Man-Rogosa-Sharpe agar mediumcontaining 100 µg · ml^–1 rifampin. The plateswere incubated anaerobically at 37°C for 2 days before L.casei DN-114 001^Rif CFU were counted. B. stearothermophilusspores were germinated and enumerated as previously reported(6).

Design of the fecal-survival study.

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Design of the fecal-survival...

After a 7-day period during which fermented dairy products wereexcluded from the diet, the 10 initial volunteers were enrolledin the second step of the trial (the fecal intention-to-treatpopulation). No L. casei DN-114 001^Rif bacteria could be recoveredfrom stools at the end of this washout period.

Each volunteer was asked to consume three bottles (3 x 100 ml)of the test product daily (1 bottle at each meal) for an 8-dayperiod. As for the first step of the trial, 10⁸ spores of B.stearothermophilus were added to each dose of the test productfor the last 3 days of consumption. Stools were collected twiceduring the ingestion period (the fourth and eighth days) andtwice after completion of intake of the test product (the thirdand seventh days after consumption ceased). The stool sampleswere processed within 30 min for bacterial enumeration accordingto the method described for ileal samples. Survival was calculatedfor samples obtained at the end of the consumption period, atwhich time full data were available for only six volunteers(four volunteers had missing or invalid data).

L. casei recovery from ileal fluid.

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L. casei recovery from...

A peak in the population of L. casei DN-114 001^Rif was observedin the ileal fluid during the 3 hours after volunteers consumedthe test meal and the single dose of fermented milk (Fig. 1).In contrast, the transit marker persisted longer, reaching aplateau from 2 to 6 h after ingestion. The highest concentrationof L. casei DN-114 001^Rif detected in this compartment represented10 to 100 times more bacteria than the resident microbiota (3),and it largely exceeded the concentration of probiotics thatwas previously suggested by some authors for the recovery ofa clinical effect (at least 6 log₁₀ CFU · ml^–1in the small bowel, according to Marteau et al. (15). Basedon PEG measurements (Table 1), the total recovery of L. caseiDN-114 001^Rif in the ileum over the entire 8-hour period wasestimated at 9.2 ± 0.5 log₁₀ CFU, corresponding to around3.6% of the total ingested quantity.

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FIG. 1. Transit of L. casei (solid line) and spores (dashed line) in the ileal lumens of four volunteers after ingestion of a meal with 300 ml of fermented milk containing L. casei (10.9 ± 0.5 log₁₀ CFU per meal) and spores (8.4 ± 0.5 log₁₀ CFU per meal). The results are presented as the number of bacteria transiting over time (mean plus standard error of the mean; n = 4). The percentages indicated at the different collection times correspond to L. casei apparent survival based on the ratio between the L. casei organisms and the spores (Table 2 shows the calculation method). When the percentage is not indicated, it is below 0.1%.

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TABLE 1. Bacterial recovery from ileal fluid collected during 8 h following ingestion of a standard meal and 300 ml of the test product^a

The apparent survival of L. casei DN-114 001^Rif was greaterduring the first 2 h of collection (51.2% to 10.0%) than whathad been previously reported for other lactobacilli in the ileum(e.g., 0.5% for Lactobacillus fermentum KLD [24]; 1.5% for acommercial strain of Lactobacillus acidophilus [16]) or in thesmall intestine (e.g., 7% and 11.8% for Lactobacillus plantarumNCIMB 8826 [24] and for Lactobacillus salivarius UCC118 [5],respectively).

L. casei survival in stools.

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L. casei survival in...

In the stool-survival step of the trial, fermented-milk intakewas continued long enough for L. casei to reach a steady state.Indeed, a plateau of about 7.6 log₁₀ CFU · g^–1of stool was observed from 4 to 7 days following the fermented-milkconsumption, while this level decreased rapidly after discontinuation(data not shown). The fecal concentrations of L. casei DN-114001^Rif reported here are substantially higher than the concentrationof L. casei strain Shirota, estimated at 7 log₁₀ CFU ·g^–1 of feces with a similar inoculum (25), suggestinglower survival abilities of the latter strain. The fecal populationof L. casei DN-114 001^Rif, corresponding to about 0.1% of theautochthonous microbiota, may be a key factor in its probioticactivities (3, 15), since it has been demonstrated that probiotictransit can be associated with transient variations of the totalpopulation of lactobacilli, enterococci, or bifidobacteria inthe microbiota (5, 13, 23), as well as with fluctuations suchas beta glucuronidase activity or short-chain fatty acid concentrations(9, 13, 23).

The apparent L. casei DN-114 001^Rif survival was approximately28.4% in the feces (Table 2). The difference between apparentsurvival rates observed in the ileum and in the feces mightbe related to the ileal sampling procedure. Indeed, we suspectthat secretions, such as bile or defensins (8), could have affectedthe survival of L. casei DN-114 001^Rif during the 1-h periodseparating the beginning of sampling and sample quantification.The apparently higher fecal survival could also be explainedby Lactobacillus multiplication in the colon, as previouslysuggested (1, 12). Since the consumption protocols were differentfor ileal (300 ml once) and fecal (100 ml three times a dayfor 8 days) studies, a cumulative effect could be consideredin the fecal experimentation. According to several studies,some Lactobacillus strains may attach transiently to the ilealmucosa (2, 17). Thus, a longer stay by lactobacilli consumedduring the previous days could account for the apparently highersurvival rate measured in the feces.

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TABLE 2. Bacterial recovery from fecal samples collected on day 7 of the experiment^a

Conclusions.

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Conclusions.

The present trial led us to conclude that L. casei DN-114 001^Rifsurvives well during gastrointestinal transit. In the ileum,the high population level of L. casei DN-114 001^Rif might becompatible with a probiotic effect. In the feces, it correspondedto the subdominant population (3).

The questions as to whether L. casei DN-114 001 is metabolicallyactive and which functions are specifically activated in thehuman gut still remain to be answered in order to understandthe digestive tract adaptation and mechanisms of action of thisprobiotic.

ACKNOWLEDGMENTS

This work was supported in part by VITAPOLE, Danone, France.

FOOTNOTES

* Corresponding author. Mailing address: National Institute of Agronomic Research, UEPSD, CRJ, INRA, Batiment 440, 78350 Jouy en Josas, France. Phone: 33-134 652 467. Fax: 33-1 34 65 24 62. E-mail: gerard.corthier{at}jouy.inra.fr.

R. Oozeer and A. Leplingard contributed equally to this work.

Present address: Syndifrais, 42 rue de Châteaudun, 75314Paris cedex 09, France.

REFERENCES

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