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An evaluation of probiotic effects in the human gut: Physiological, Microbial, And Safety Aspects

Project Code: G01022

31/12/2003

University of Reading, School of Food Biosciences, Food Microbial Sciences Unit.
Gibson, G ; Rouzaud, G;
Kings College London
Brostoff, J; Rayment, N

Rationale

A variety of probiotic supplements are now available for human use in the UK. These range from fermented milks to lyophilised forms, containing both single and multiple strains.

However, there is an almost total absence of comparative data on probiotic effects within the human gut and their inherent safety implications. This project aimed at providing independent information on the effects of existing probiotics in the alimentary tract. The project was designed to address the following issues:

To compare commercially available probiotic strains characterised and identified in an
independent non-FSA funded study.
To test the survival of probiotics in the gastrointestinal milieu
To determine whether probiotics affect the composition of the gut microflora

Summary of the approach and objectives

The approach used several validated in vitro models simulating the physico-chemical events arising in the stomach, upper intestine and lower intestine. A wide number of strains were isolated from commercial probiotic products and phylogenetically identified. Only probiotic strains originating from products which label matched their microbiological content were chosen for the study. All selected probiotics were treated with digesta resembling the gastric environment. Probiotic strains that were able to survive the stomach conditions were subsequently considered for testing their survival in the upper intestine. This exercise determined which probiotics had the capacity to survive transit to the lower intestine– the target organ for probiotic residence and effects. Survival in the colon was tested in an in vitro model of the human large intestine that reflects microbial events in the ascending, transverse and descending regions of the colon. The effect of probiotic on gut microbial balance was also investigated using a molecular methodology.

How closely were the objectives met?

All tasks agreed in the proposal were accomplished. Valuable information on the microbiological effect that probiotics exert were obtained from the study. The project has clarified the most robust/reliable products for gut survival particularly for Lactobacillus spp. Further studies would need to clarify the effect of Bifidobacterium spp. and Enterococcus faecium in the lower intestine.

Outline of the main findings

Microbial strains originating from eight different commercial products were isolated and sequenced. Of the products tested, not all the products displayed a profile of probiotics similar to what was stipulated on their labelling. A collection of 35 bacterial strains was gathered from the initial screening. Principal strains isolated from commercial products were from Lactobacillus spp. and Bifidobacterium spp.

The survival of thirty two probiotic strains was assessed in simulated gastric contents. After twenty minutes incubation in the presence of simulated gastric contents at pH varying between 1 and 3, Lactobacilllus spp. Bifidobacterium spp., Enterococcus sp. and Lactococcus lactis sp. showed a good survival in the gastric environment. A ratio was calculated to estimate the proportion of bacterial cells surviving the gastric milieu. Eighteen strains had a ratio of live cells greater than 0.5 at pH 2 or 3 or at both pHs indicating that 50% or more probiotic cells were still viable after 20 minutes in the stomach environment.

On the basis of their resistance to simulated gastric content, the strains selected in the stomach challenge were tested for their tolerance to bile acids. The growth of probiotic strains in MRS broth in the absence or in the presence of bile acid salts was monitored at two concentrations of bile acids 18g.L^-1and 36g.L^-1. A coefficient of growth inhibition (C_inh) was calculated for each bile concentration to characterise the effect of bile acids on probiotic growth. Probiotic strains were classified in three groups according to their coefficient of inhibition. Six strains showed a Cinh close to zero indicating little or no effect of the presence of bile salts on the growth of probiotic. Seven strains had their growth slowed down by bile acids (0.2<C_inh <0.4). Five strains showed a poor tolerance to bile acid content as shown by a C_inhgreater than 0.4 and the absence of growth. The strains of the latter group were unlikely to reach the large intestine intact. A sub-set of strains was challenged with simulated upper intestine contents containing pancreatic enzymes. In this test, Lactobacillus spp. showed a higher sensitivity to upper intestinal content than predicted from the bile acid tolerance test. On the other hand, Bifidobacterium sp. and Enterococcus faecium showed a good survival in this environment. Lactococcus lactis did not, however, survive well in both assays.

Six Lactobacilllus strains were subsequently selected for test of survival in the lower intestine. The strains studied were L. casei immunitass, L. casei shirota, L. plantarum, L. pentosus, L. reuterii, L. acidophilus subsp johnsonii and L. delbrueckii subsp bulgaricus. Each strain was studied independently. L. casei shirota, L. plantarum, L. pentosus and L. reuterii were able to survive in measurable level for five days after inoculation of the strain. L. casei immunitass, L. delbrueckii subsp bulgaricus and L.acidophilus susp. johnsonii displayed poor survival.

Results of the interaction with the residential microflora showed that addition of probiotic did not affect significantly the total number of bacteria growing in the continuous culture. Little difference were also seen on the levels of main bacterial species numerated by Fluorescent In Situ Hybridisation. Variability in the composition of the microflora was seen from one gut model run to another but Bacteroides population remained at high levels throughout the 10 days of sampling in all gut models. Bifidobacteria were generally less prevalent at the end of the wash-out period. Although probiotic from Lactobacillus spp. were added to the three stage fermentation system, Lactobacillus group remained at sub-dominant level in the three vessels of the fermentor.

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