ORIGINAL ARTICLE
A R andomised Controlled Trial of a Probiotic Lactobacillus Strain in Healthy Adults: Assessment of its Delivery, Transit and In uence on Microbial F lora and Enteric Immunity J. Kevin Collins1, Colum Dunne1, Lisa Murphy1, Darrin Morrissey1, Liam O’Mahony1, Eilõ´s O’Sullivan1, Gerald Fitzgerald1, Barry Kiely1, Gerald C. O’Sullivan1, Charles Daly1, Philippe Marteau2 and Fergus Shanahan1 From the 1Departments of Microbiology, Medicine and Surgery, Cork Cancer Research Centre, National Food Biotechnology Centre, University College Cork, National University of Ireland, Cork, Ireland and 2 Service d’He´pato-Gastroente´rologie et Endoscopies Digestives, Hoà pital Europe´en Georges Pompidou, Paris, France Correspondence to: Professor Fergus Shanahan, Department of Medicine, Clinical Sciences Building, Cork University Hospital, Cork, Ireland. Tel.: »353-21-4901226 ; Fax: »353-21-4345300 ; E-mail:
[email protected] e
M icrobial Ecology in Health and Disease 2002; 14: 81 – 89 In severa l intestinal disease states, altered micro ora, impaired gut barrier and:or intestinal in ammation offer a rationale for the effective therapeut ic use of probiotic microorganisms. However , for most candidat e probiotic organisms there is a lack of evidence detailing their characterisation and effects on host ora and immunity. We have previously reported the isolation and characterisation, from surgically resected segment s of the huma n gastrointestinal tract (GIT), of potential probiotic lactic acid bacteria (LAB). We have also described subsequen t animal experimen ts that evaluated the establishment , persistence and localisation of speci c probiotic Lactobacillus strains within the murine intestinal tract, in addition to their ability to in uence the developmen t of murine in ammatory disorders. In these studies, transit and surviva l of Lactobacillus salivarius U CC118 at the ileum was demonstrat ed using enteral tube sampling of six healthy volunteer s following consumption of a single dose (150 ml) of fermented milk-born e probiotic (108 colony forming units per ml (CF U :ml)). Subsequently, we performed a randomised controlled trial of 80 volunteer s fed strain UCC118 (108 CF U:day for 21 d), using two oral delivery vehicles (fresh milk, n ¾ 20 vs. fermented milk, n ¾ 20; controls, n ¾ 20 for each). Throughout this feeding period , and for up to 100 days following cessation of feeding, the number s of total culturable lactobacilli and of the administered Lactobacillus U CC118 present in faeces were monitored. F ive subjects (5:40; fresh milk, four; fermented milk, one) were still excretin g the probiotic lactobacilli 21 days post-cessatio n of feeding, while one subject (fermented milk) was still colonised up to 100 days after feeding. Consumpt ion of fermented milk-born e UCC118 cells resulted in signi cantly increased levels of faecal-borne enterococci and lactobacilli. N umber s of bi dobacteria, coliforms and bacteroides were not signi cantly altered. In addition, changes in salivary IgA levels against U CC118 cells and increased granulocyt e phagocytic activity were observed following consumption of the fermented milk-borne probiotic. In summary, Lactobacillus UCC118 was found to effectively transit (and persist within) the human intestinal tract, to modify the faeca l ora and to engage the immune system. Key words : controlled trial, probiotic, Lactobacillus salivarius, transit, ora, enteric immunity.
INTRODUCTION The intestinal micro ora is characterised by its density, diversity and complexity of interactions and is both an asset and a potential liability in health and disease, depending on genetic and other host susceptibility factors (1). There are up to 10-fold more bacteria in the human gut than cells in the body and this accounts for about 1 –2 kg of intestinal contents composed of an estimated 50 genera, belonging to over 400 separate species (2). With a metabolic activity rivalling that of the liver (3, 4), the gastrointestinal micro ora has been described as a metabolically adaptable and rapidly renewable organ of the body, the composition and activities of which can © Taylor & F rancis 2002. ISSN 0891-060 X
affect both intestinal and systemic physiology (5, 6). The ora represents a central component of mucosal defence, has a priming effect on mucosal immune function, and has a conditioning effect on mucosal structure and function (7– 10). This is strikingly evident in germ-free animals that exhibit reductions in epithelial turnover, motility, smooth muscle function, vascularity and gut-associated lymphoid tissue (11). Alterations in bacterial ora are associated with susceptibility to pathogens such as Clostridium dif cile and there is persuasive evidence that the normal ora may participate in the pathogenesis of in ammatory bowel disease in genetically susceptible individuals (12– 14). This has prompted various strategies to fortify or otherwise Microbial Ecology in Health and Disease
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modify the enteric ora using dietary supplements. Although the theoretical rationale for modifying the enteric ora in speci c circumstances appears sound and requires scienti c pursuit, the eld has been clouded by wildly exaggerated claims from some quarters. Simplistic notions of ‘bowel cleansing’ are conceptually appealing as portrayed in the popular lay press; have been exploited commercially, often without impressive evidence for ef cacy; and have been adopted by some enthusiasts of alternative medicine. However, the use of biotherapeutic agents such as probiotics has recently attracted scienti c input from disparate traditional disciplines, including microbiology and gastroenterology (14). Probiotics are live microbial food ingredients (or pharmaceutical-type preparations) that alter the enteric micro ora and have a bene cial effect on health (12, 14, 15). Probiotic research promises much, and ambitious claims have been made for their potential contribution to health maintenance and treatment of disease. At present, however, many of the purported bene ts have not yet been matched with persuasive supporting evidence (16). To overcome the scepticism of some members of the scienti c community regarding probiotic products, their safety and their associated probiotic claims (17), the characterisation and assessment of candidate probiotic organisms must be rigorous. H owever, to-date, detailed comparisons of probiotic performance amongst different strains have not been performed in vivo in healthy humans or under clinical trial conditions and the level of scienti c characterisation of individual probiotic organisms has been variable. F urthermore, the heterogeneity of clinical disorders such as Crohn’s disease and ulcerative colitis implies that it is unlikely that any single probiotic will be equally suitable for all individuals and that strain-speci c properties may be required for subset-speci c categories of patients (12). In previous reports, we and others have described strategies adopted speci cally to comply with criteria recommended (18, 19) for the selection of potential probiotic microbes for use in humans. These approaches resulted in the isolation of Lactobacillus strains from healthy human adults (20, 21) and their subsequent assessment in murine models (22). In this study, our objective was to further characterise the probiotic Lactobacillus salivarius strain U CC118 and to focus speci cally upon: (i) effective delivery of the probiotic micro-organism to the human gut using fermented and non-fermented delivery vehicles; (ii) evaluation of the ability of the strain to survive transit through (and persist within) the human intestinal tract; (iii) accepting the complexity of the hostile intestinal and faecal environments, assessment of a method of enumerating the introduced bacterial strain using conventional microbiological techniques; (iv) determining the effect of probiotic consumption on the numbers of faecal-borne
selected indigenous bacteria; and (v) evaluation of immune recognition of the consumed probiotic bacteria. MATERIALS AND METHODS Production of probiotic test products F ermented milk (yoghurt-like) and fresh milk products containing the L. salivarius UCC118 strain were prepared to Good M anufacturing Practice (G M P) standards. F ermented milk production involved inoculating pasteurised cows’ milk with a spontaneous rifampicin-resistant derivative (22) of L. salivarius U CC118 (L. salivarius UCC118 Rif) and a commercial strain of Streptococcus thermophilus. Prior to inoculation into milk, the Lactobacillus strain was grown in liquid deM ann, Rogosa & Sharpe (M RS) medium (Oxoid, U K ) overnight at 37°C, centrifuged, and washed twice in sterile PBS. The inoculated milk was incubated at 37°C until Lactobacillus numbers reached 108 colony-forming units per g (CF U :g). F resh milk test product was produced by adding L. salivarius U CC118Rif, following incubation and washing as described above, to achieve numbers of 108 CF U :ml. The control fermented milk product was inoculated with S. thermophilus alone while the control fresh milk product was untreated beyond the typical pasteurisation process. Human intestinal transit and survival of L. salivarius UCC 118 Rif To establish kinetics and transit of the L. salivarius U CC118 Rif strain at the human ileum sampling from the human ileum was performed after single dose feeding using an enteral tube. This aspect of the study was approved by the Ethics Committee at Hopital St. Lazare, Paris, F rance, and was conducted at the same location. Six healthy human volunteers consented to intestinal intubation with a triple-lumen tube, after an overnight fast, as previously described (23). This permitted sampling of ileal contents 35 cm above the ileo-cecal junction. Equilibration was accomplished by infusing 10 g polyethylene glycol (PEG ) 4000 in 154 mM N aCl at 37°C at a rate of 2 ml:min. After 1 h of equilibration, each subject ingested a standard meal of 150 ml fermented milk containing L. salivarius U CC118Rif (108 CF U :ml). Before feeding, 108 bacterial spores of Bacillus stearothermophilus (M erck, D armstadt, F R G ), which germinate only at 65°C, were added to the fermented milk as a bacterial marker (23). Ileal content was continuously collected on ice by manual aspiration for 8 h after fermented milk consumption. Another standard meal was then ingested and two further 5 ml samples of ileal uid collected 10 and 24 h after fermented milk consumption. The ileal uid samples were pooled into 1 h aliquots and microbial counts were performed immediately at the end of each hour of collection. The samples were serially diluted 10-fold in sterile phosphate-buffered saline (PBS) and 0.1 ml of each dilution spread-plated onto freshly
Controlled trial of a probiotic Lactobacillus
prepared media. The medium used was M R S agar-containing rifampicin (50 mg:ml). Plates were incubated in anaerobic jars using CO 2-generating kits (Anaerocult A; M erck) for 2 days at 37°C. Spores of B. stearothermophilus were quanti ed on plate count agar (PCA; Bio-M erieux, Craponne, F rance) after rstly incubating aerobically at 65°C for 24 h. Double -blind placebo-controlled feeding trial A randomised double-blind, placebo-controlled feeding trial was undertaken to compare two oral delivery vehicles (fresh and fermented milk) for UCC118Rif and to assess the in uence of the probiotic Lactobacillus strain on the faecal microbial ora and health status of consumers. The trial was conducted at N ational University of Ireland, Cork. Eighty healthy human volunteers (29 male, 51 female) aged from 20 to 65 years were recruited on the basis of no recent history of antibiotic therapy, no chronic in ammatory or viral illness, no current drug therapy, no known allergies, no participation in other clinical trials. In addition, candidates for the trial were excluded if pregnant or if suffering from IgA de ciency. All subjects gave written informed consent and the study protocol was approved by the Cork University H ospitals Ethics Committee. The duration of the study was 9 weeks (F ig. 1). D uring this period, all volunteers were requested to exclude fermented dairy products from their diet. Week 1 –3 acted as a stabilisation period. Consumption of test product occurred from week 4 –6. D uring week 7– 9, trial products were again removed from the diet. Prior to commencement of the study, volunteers were randomly assigned to one of four groups (A, B, C, D; n ¾ 20 each). G roup A were fed pasteurised milk (120 ml:day) containing U CC118Rif (108 CF U :ml; i.e. 1010 CF U :day); group B were fed control pasteurised milk (120 ml:day); group C were fed fermented milk (120 g:day) containing U CC118 Rif (108 CF U :ml; i.e. 1010 CF U :day); group D were fed control fermented milk (120 g:day). On the rst day of feeding (T0), the day after the feeding period (T1) and 21 days after termination of feeding (T2), samples of faeces, blood and saliva were collected. During the feeding period two volunteers (both from consumer group D) voluntarily withdrew from the study.
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Analysis of faecal micro ora M icrobial analysis was performed on faecal samples within 6 h of collection. Each sample was weighed and resuspended in 10 ml of sterile PBS. The samples were serially diluted 10-fold in sterile PBS and either pour-plated or spread-plated in appropriate dilutions on suitable media, and incubated in anaerobic jars (BBL) using CO 2-generating kits (Anaerocult A; M erck) for 2– 5 days at 37°C. The selective media employed for the culture of lactobacilli, bi dobacteria, enterococci, bacteroides and coliforms, respectively were as follows: deM ann, Rogosa & Sharpe (M R S) agar, Trypticase:Peptone:Yeast extract (TPY) agar, Slanetz & Bartley agar, Wilkins & Chalgren agar and Violet R ed Bile agar (VRBA), respectively (all Oxoid, U K ). M R S supplemented with rifampicin (50 mg:ml) was used for the selective culture of L. salivarius U CC118Rif. M icrobial numbers were calculated as CF U :g of wet weight faeces. Salivary and serum antibodies speci c for L. salivarius UCC 118 Rif Salivary IgA speci c for the probiotic lactobacilli was quanti ed by ow cytometry as follows. Saliva samples were stored at ¼ 80°C to reduce viscosity, and concentration was standardised based on total protein concentration in each sample. A fresh overnight culture of L. salivarius U CC118 Rif was washed and resuspended in PBS at a concentration of 108 cells:ml. One hundred microlitres of bacterial suspension was mixed with 100 ml saliva (1:10– 1:100 in PBS) and incubated for 1 h at room temperature. The bacterial cells were then centrifuged and washed in PBS, resuspended in 100 ml F ITC-labelled a chain-speci c rabbit anti-human IgA monoclonal antibody (1:40 diluted) (DAK OPATTS, Denmark) and incubated for 30 min at room temperature. The cells were washed again in PBS and resuspended in 200 ml isotonic carrier uid (Isoton 2; Coulter, Luton, U K ). The uorescence of the cells was then measured by ow cytometry using a Coulter ‘Epics Elite’ F luorescence-Activated Cell Sorter (F ACS) (Coulter, Luton, UK ). M ean uorescence intensity was recorded and standardised against salivary protein concentration. Standard curves were constructed and mean uorescence
Fig. 1. Protocol design for a feeding trial assessing the deliver y and probiotic ef cacy of L. salivarius UCC118 Rif in huma n volunteers. Key: T0¾starting day of feeding, T1¾day after termination of feeding, T2¾21 days after termination of feeding; F ¾faeca l sample, B ¾blood sample, S ¾ saliva sample.
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index (M F I) was directly proportional to salivary concentration in a linear fashion. A positive antibody response was interpreted as a ] 75% increase in the M F I. F low cytometry was similarly employed to measure serum IgG speci c for U CC118Rif lactobacilli. The protocol was identical to that used in salivary detection; each serum sample was diluted 1 in 10 in PBS and F ITC-labelled goat g-chain speci c anti-human IgG monoclonal antibody (1:32) (Sigma:Aldrich Chemical Co., Dorset, U K ) was employed as the secondary antibody. Serum immunoglobuli n and cytokine measurements The overall immune status of participating volunteers was monitored by measuring the following parameters. Total serum IgG, total serum IgM and total serum IgA were assessed by immunoturbidometric assay (Tina-quant; Boehringer M annheim, East Sussex, U K). Serum IgE levels were quanti ed by ELISA. In ammatory cytokines, i.e. interleukin-(IL)-1a, IL-1b, IL-4, soluble IL-2 receptor, soluble IL-6 receptor, tumour necrosis factor-(TN F )-a, and interferon-(IF N )-g, were quanti ed in serum employing enzyme linked immunosorbent assay (ELISA) immunodetection (Quantikine; R & D Systems, Oxon, U K) Peripheral leukocyte phagocytic activity Leukocyte phagocytic activity in peripheral blood was quanti ed by ow cytometry employing uoroscein isothiocyanate (F ITC)-labelled opsonised Escherichia coli (PH AG OTEST; Becton Dickinson, Belgium). F resh heparinized blood (100 ml) was mixed with 20 ml of an E. coli suspension (1 ½ 109 bacteria per ml) so that that the ratio of bacteria to leukocytes was approximately 20:1 (v:v). The mixture was then incubated for 10 min at 37°C in a water bath. After quenching, to eliminate bacteria not ingested by leukocytes, the red blood cells in the sample were lysed, and the remaining cells xed and stained with a propidium iodide solution. F luorescence measurements were made ow cytometrically with blue– green excitation light (488 nm). Employing L YSI S II data analysis software (Becton Dickinson), a live gate was set so that only events that were positive for propidium iodide staining and, hence, human diploid cells were considered. In analysis of the data, the phagocytic activity of individual granulocyte and monocyte populations, represented by internalised bacterial uorescence, were determined as expressed as a percentage of total cells. Statistical analysis Comparisons of fresh and fermented milk delivery systems, and L. salivarius UCC118 Rif and placebo treatments, were made by assessing all parameters for signi cant changes between baseline (T0) and either T1 (3 weeks of probiotic feeding) or T2 (3 weeks after cessation of probiotic feeding). F or the faecal microbial counts, primary statistical analysis was performed on log-equality factors using
AN OVA. F or UCC118Rif concentrations, the Wilcoxan R ank Sum test was used. U CC118Rif -speci c serum antibody titers and serum cytokine levels were analysed by a Wilcoxan two-sample test. U CC118Rif -speci c salivary IgA levels were assessed by F isher’s Exact Test (twotailed). All statistical analyses were devised and performed by Independent D ata M anagement Ltd., Cork, Ireland. RESULTS Transit :survival of L. salivarius UCC118 Rif in the human gut In the six individuals who volunteered for preliminary enteric intubation feeding analysis, L. salivarius U CC118Rif was found to be delivered in signi cant numbers to the terminal ileum and to have a strong capacity for survival. Within 2 h of ingestion of fermented milk, signi cant numbers of rifampicin-resistant lactobacilli (mean: \ 105 CF U :ml) were isolated in the ileal uid of each subject (F ig. 2). At 3 h after dose consumption, the ow of probiotic lactobacilli through the ileum was at its highest, with the strain detectable at numbers of º 2.0½ 106 CF U : ml (F ig. 2). The survival capacity of the Lactobacillus U CC118 Rif strain until the terminal ileum, calculated as a percentage of original number of bacteria consumed, was approximately 11.8%. Viable UCC118Rif cells were still detectable in the subjects at º 105 CF U :ml for up to 7 h after dose consumption. N one of the strain persisted in the lumenal content of the ileum after 8 –10 h. Human feeding trial of L. salivarius UCC 118 Rif Of 80 subjects enrolled in the feeding trial, all except two completed the study; none developed adverse effects and no evidence of systemic in ammatory activity was detected. U CC118Rif cells were delivered in high numbers to the gastrointestinal tracts (G ITs) of the study subjects by both fresh milk and fermented milk products. Prior to the feeding period, no bacteria were isolated from subject faeces on M RS medium containing rifampicin. After feeding, 39 of the 40 volunteers fed with UCC118Rif-containing product (1010 CF U :day, for 21 days) exhibited signi cant faecal excretion (103 –107 CFU :g wet weight faeces) of rifampicin resistant lactobacilli (F ig. 3). These bacteria were con rmed to be L. salivarius U CC118Rif on the basis of the ability to inhibit the growth of the B. coagulans 1761 indicator strain on solid media, as described previously (22). This also con rmed general compliance with the feeding regimen. As expected, excretion of L. salivarius U CC118 Rif was not detected in any of the control-fed subjects. Based on faecal excretion levels, fresh milk was found on average to deliver 15 times more U CC118Rif Lactobacillus cells (F ig. 3; mean; 3.7 ½ 106 CF U:g faeces) than fermented milk (F ig. 3; mean; 2.4½ 105 CF U :g faeces). Of the volunteers fed test product (n¾ 40), ve individuals (12.5%) were found to have signi cant faecal
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Fig. 2. Transit and surviva l of L. salivarius U CC118Rif to the ileum in healthy subject s (n ¾ 6). F ermented milk (150 ml) containing UCC118 Rif (108 bacteria per ml) was consumed , and uid from the terminal ileum collected by enteric sampling at hourly intervals post-feeding. M ean concentrations (CF U:g) of U CC118Rif and a marker bacterial strain B. stearother mophilus owing at the terminal ileum at various time-points after ingestion of fermented milk are shown .
concentrations of the Lactobacillus UCC118 Rif strain 21 days after termination of feeding (F ig. 3). F our of these subjects were in the fresh milk delivery group, while the other one was a fermented milk consumer. H undred days after the feeding period nished, one individual in the fermented milk delivery group was still excreting detectable faecal levels of strain U CC118Rif (F ig. 3). Modulation of faecal ora in response to L. salivarius UCC 118 Rif feeding F aecal concentrations of total lactobacilli were also elevated (mean 11.5-fold) compared with pre-feeding levels (F ig. 4), in the group fed fermented milk containing the U CC118 Rif probiotic, and these reverted to baseline levels when assessed 21 days post-feeding. In contrast, in subjects fed fresh milk containing the UCC118Rif probiotic and in both control groups, the change in faecal concentrations of both enterococci and total lactobacilli was not statistically signi cant. F aecal enterococci were elevated 200-fold in subjects after 3 weeks of consumption of the Lactobacillus U CC118 Rif-fermented milk compared with baseline (F ig. 5). H owever, 3 weeks after cessation of feeding, the faecal enterococci numbers, although still elevated, returned toward baseline levels in almost all subjects. Additionally, no signi cant change occurred in the faecal populations of either bi dobacteria, bacteroides, coliforms as a result of probiotic product ingestion (data not shown) Engagement of the mucosal immune response by L. salivarius UCC118 Rif Out of the 78 volunteers who completed the feeding trial 49 (63%) had detectable pre-existing serum IgG against the L. salivarius U CC118 Rif strain at baseline. After feeding, there was no increase in the number of volunteers exhibit-
ing a UCC118 Rif-speci c systemic serologic response, and no elevation in the titer of U CC118Rif-speci c serum IgG (agglutination index) amongst those who were serologically positive at baseline. In contrast to the lack of systemic antibody response, mean levels of mucosal (salivary) IgA against L. salivarius U CC118 Rif were elevated following consumption of the probiotic (Table I). At week 3, the number of subjects with positive shifts from baseline of \ 75% in salivary IgA speci cally against U CC118Rif was signi cantly greater for those receiving the probiotic either by fermented milk or milk than for the controls (12:40 vs. 2:38; p B 0.01). D ifferences at week 6 were similarly signi cant (12:40 vs. 2:38; pB 0.01). When analysed by sub-groups, at week 3 post feeding, the number of subjects with positive shifts from baseline of \ 75% in salivary U CC118Rif -speci c IgA concentrations was signi cantly different for those who received the probiotic via fermented milk compared with control fermented product (7:20 vs. 0:18; p¾ 0.009). The difference in the proportions of positive shifts between the two groups, at week 6, i.e. 3 weeks following cessation of probiotic consumption, was also signi cant (6:20 vs. 0:18; p ¾ 0.021). Although a greater proportion of those consuming milk containing UCC118 Rif had a \ 75% shift in U CC118 Rif-speci c IgA (5:20 vs 2:18), the difference was not statistically signi cant at week 3 or 6. Systemic immune parameters and phagocytic activity in response to L. salivarius UCC 118 Rif N o systemic immune or in ammatory activity was found in association with probiotic consumption. There was no signi cant difference in change from baseline serum concentration for subjects receiving probiotic U CC118Rif via fermented milk or fresh milk versus respective controls at
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DISCUSSION
Fig. 3. F aeca l excretion of L. salivarius UCC118 Rif by human volunteers. L. salivarius UCC118 Rif number s isolated from faeces (CF U :g wet weight) before (T0), immediately after (T1) and 21 days after (T2) consumption of test product are indicated for each subject . F aeca l L. salivarius U CC118R if number s at 100 days after cessation of product consumption (T3) are indicated for those ve volunteer s who exhibited persisten t L. salivarius U CC118Rif at T2.
week 3 or 6 for any of the cytokines measured, including IL-1a, IL-1b, IL-4, soluble IL-2 receptor, soluble IL-6 receptor, TN F -a, and IF N-g (p \ 0.05 in all cases). The group that consumed the UCC118Rif strain in a fermented milk vehicle but not those who consumed it in a fresh milk vehicle exhibited a signi cant increase in granulocyte phagocytic activity (Table II). H owever, a similar shift of phagocytic activity in the monocyte subpopulation was not evident for any of the test subjects (data not shown).
The concept of health promotion through the ingestion of functional foods, including those that act as vehicles for potentially bene cial micro-organisms, has received considerable attention (15, 24, 25). H owever, some of the probiotic strains currently employed in the health food industry may not possess desirable traits such as being of human origin, resistance to technological processes (i.e. viability and activity in delivery vehicles), resistance to gastric acidity and bile toxicity, capacity to adhere to gut epithelial tissue, ability to colonise the host GIT, and production of antimicrobial substances (26, 27). This study describes the behaviour and effects, following its administration to healthy adult humans, of L. salivarius U CC118, a strain which was originally isolated from the ileal– caecal region of the intestinal tract of a healthy human adult (20, 21). Previously, speci c criteria were employed to determine the suitability of the strain for use as a probiotic. These included the ability of the strain to survive in human gastric acid at pH 2.5 and growth at physiological concentrations of human bile (0.03% v:v) (20, 21). Strain U CC118, originally isolated as crypt-adhering bacteria, was also previously con rmed to be strongly adhesive to the human adenocarcinoma cell lines Caco-2 and H T-29 (20, 21). The human gastrointestinal environment is a complex, interactive system involving the host itself, ingested dietary components, and many microbial species. M easurement of ingested probiotic micro-organisms from faecal material is problematic if the administered bacterial strains do not possess speci c characteristics that facilitate their differentiation from indigenous, closely related, strains (28). F or the purposes of this study, a previously selected rifampicinresistant derivative of L. salivarius UCC118 (22) was used in order to facilitate enumeration of the strain from faecal samples, and as a de ned method of distinguishing the administered strain from indigenous lactobacilli. Both fresh and fermented milk were effective as vehicles in terms of delivery of the probiotic, as measured by isolated faecal numbers, although fresh milk was marginally better than fermented product. A possible reason for this may be more rapid gastric emptying for milk than fermented product (29), with less exposure to gastric acid and enzymes. In contrast, consumption of fermented milk containing the L. salivarius U CC118 Rif strain was associated with signi cant increases in faecal excretion of enterococci and enhanced faecal numbers of total lactobacilli not accounted for by consumption of the U CC118Rif Lacto bacillus alone. These changes were not as evident when the probiotic bacteria were consumed in fresh milk and did not occur in either of the control groups. It is likely that the fermentative process undertaken by U CC118Rif lactobacilli generates metabolic bio-active products that stimulate speci c microbial populations. Indeed, some of the health bene ts of consumption of certain lactobacilli are
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Fig. 4. F aeca l excretion of total lactobacilli associated with consumption of L. salivarius UCC118R if. Total lactobacilli number s isolated from faeces (CF U:g wet weight) before (T0), immediately after (T1) and 21 days after (T2) consumptio n of Lactobacillus UCC118 Rif-containing fermented milk (* ¾ Statistically signi cant difference; p B0.05; means 9SD).
Fig. 5. The effect of L. salivarius UCC118 Rif consumption on faeca l excretion of enterococci. Enterococci number s isolated from faeces (CF U :g wet weight) before (T0), immediately after (T1) and 21 days after (T2) consump tion of UCC118Rif-containing fermented milk (* ¾ Statistically signi cant difference; p B0.05).
Table I Changes in salivary IgA reactive a against L. salivarius UCC118 Rif associated with consumption of fermented milk -borne and fresh milk-borne probiotic Products administered b
Initiation of probiotic consumption
F ermented milk-borne U CC118Rif (n¾ 20) Probiotic-free fermented milk (n ¾ 20) F resh milk-borne U CC118Rif (n¾ 20) Probiotic-free fresh milk (n ¾ 20)
3.42 4.02 3.12 3.57
a
(2.70) c (4.89) (6.51) (3.52)
Mean levels of salivary IgA antibod y concentration (g:l). UCC118 Rif was administered at a concentration of 1½10 8 CF U :ml fermented or fresh milk. c Standard deviations are given in parentheses. b
Day 21 of probiotic consumption 9.11 3.05 3.92 5.03
(14.62) (3.12) (6.67) (6.82)
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Table II Changes in peripheral granulocyte phagocytic activity
a
associated with consumption of fermented milk-borne and fresh milk-borne L. salivarius UCC118 Rif
Products administered b
Initiation of probiotic consumption
F ermented milk-borne U CC118Rif (n¾ 20) Probiotic-free fermented milk (n ¾ 20) F resh milk-borne U CC118Rif (n¾ 20) Probiotic-free fresh milk (n ¾ 20)
70.05 72.50 68.75 64.84
(9.51) c (8.38) (8.55) (17.13)
Day 21 of probiotic consumption 76.37 69.56 71.06 70.05
(7.68)d (8.45)d (11.02) (10.18)
a
Phagocytic activity is expressed as a percentage of cells with ingested uorescen t bacteria, as determined by ow cytometry. UCC118 Rif was administered at a concentration of 1½10 8 CF U :ml fermented or fresh milk. c Standard deviations are given in parentheses. d There was a statistically signi cant difference in the change from baselin e phagocytic activity for those subject s who received fermented milk-borne UCC118Rif versus probiotic-free fermented milk for the 21 day period (p ¾ 0.0088). b
though t to be indirect and due to stimulation of the bi dobacteria population and inhibition of anaerobes, although in this study signi cant bi dogenic activity was not observed in vivo with L. salivarius U CC118 Rif. There has been unresolved speculation as to whether the health bene ts associated with probiotic consumption are dependent on the micro-organism becoming a long-term resident in the gut. F ew feeding studies in humans have demonstrated persistent long-term colonisation by probiotic lactic acid bacteria (LAB) (30). In this study, we found that ve of the 40 subjects (12.5%) consuming L. salivarius U CC118 Rif displayed signi cant faecal concentrations of the strain up to 3 weeks after feeding was discontinued and one individual was excreting the probiotic strain 6 months later. This was not correlated with local IgA responses and colonisation was not an absolute requirement for probiotic-associated alterations in faecal ora. Although optimal dose and duration of consumption have not been well correlated with impact on enteric ora for any probiotic (31) continual feeding will probably be required for most individuals if probiotics in their current form are to be exploited for health bene ts. This should not be taken to imply that the probiotic is in simple transit within the lumen of the digestive tract without any direct engagement with the host. Several lines of evidence in vivo and in vitro indicate that probiotics engage and interact with host immune and other mucosal cells (12, 32, 33). In this study, consumption of L. salivarius UCC118 Rif was associated with a speci c secretary IgA anti-UCC118Rif response as detected in saliva by a dilution-controlled, protein-standardised ow cytometric detection system. In contrast, a systemic serologic immune response against the strain was not evident. Although increased granulocyte (but not monocyte) phagocytic activity was observed in subjects fed the U CC118Rif strain in fermented but not in fresh milk, and a similar nding has previously been reported after consumption of L. acidophilus (34, 35), the biological signi cance of this is unclear. It does, however, indicate an increased immune
status, which is a concept frequently, associated with, and promoted by, the marketing of probiotics. M odi cation of the host- ora interface with probiotics promises much. Gastrointestinal disorders where probiotics might be anticipated to have a therapeutic application include antibiotic-associated diarrhoea; bacterial overgrowth and other diarrhoeal syndromes; in ammatory bowel disease, and colonic cancer (1, 12, 36). Probiotics also offer the very real potential to deliver therapeutically relevant proteins including enzymes, vaccines and cytokines (37). H owever, the scienti c credibility of the functional food and probiotic industry suffers from an excess of soft claims, anecdotal reports, and insuf cient data on probiotic performance or mechanisms of action. It seems reasonable to propose that putative probiotic strains be subjected to rigorous study with current molecular technology in the context of enteric microbial ecology, cellular physiology and human immunology before launching expensive human clinical trials in speci c diseases.
ACKNOWLEDGEMENTS The authors are supported in part by grants from the Irish Higher Education Authority; the Irish Health R esearch Board; BioR esearch Ireland; and the Europea n Commission (PR OBDEM O: F AIR-CT96-1028, LABVAC: BIO4-CT96-0542 and PROGID : QLKI-2000-00563) .
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