Lactobacillus acidophilus 74-2 and Bifidobacterium animalis ... - Nature

European Journal of Clinical Nutrition (2008) 62, 584–593

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ORIGINAL ARTICLE

Lactobacillus acidophilus 74-2 and Bifidobacterium animalis subsp lactis DGCC 420 modulate unspecific cellular immune response in healthy adults A Klein1, U Friedrich2, H Vogelsang3 and G Jahreis1 1 Institute of Nutrition, Department of Nutritional Physiology, Friedrich Schiller University, Jena, Germany; 2Research & Development, Danisco Deutschland GmbH, Busch-Johannsen-Str. 1, Niebu¨ll, Germany and 3Institute of Clinical Chemistry and Laboratory Diagnostics, Friedrich Schiller University, Jena, Germany

Objective: It was determined whether a combination of Lactobacillus acidophilus (L. acidophilus) 74-2 and Bifidobacterium animalis subsp lactis DGCC 420 (B. lactis 420) affect the faecal microbiota as well as immunological parameters and blood lipids in healthy adults. Design: A placebo-controlled, double-blinded, randomized crossover trial was conducted. Subjects: Twenty-six healthy volunteers (mean age 25 years) were recruited by advertising in academical buildings. All of them completed the study. Methods: After 3-week run-in period, half of the volunteers consumed 300 g/day of yoghurt supplement containing probiotic strains L. acidophilus 74-2 and B. lactis 420, and the other half received the placebo product for a period of 5 weeks. The two groups were crossed during the following 5-week period. Blood and faecal samples were collected at the end of each period. The faecal content of probiotic bacteria, faecal short-chain fatty acids (SCFA), serum lipids and plasma immune system biomarkers were evaluated. Results: Faecal proportions of L. acidophilus and of B. lactis increased significantly from 0.02 to 0.19 and 0.4 to 1.4% (Po0.05), respectively. Percentages of granulocytes and monocytes showing phagocytic activity were significantly elevated from 92 to 95% during probiotic intervention, whereas their oxidative burst activity and specific immune parameters remained unaffected. Fecal SCFA and serum cholesterol levels were not influenced by the probiotics. However, serum concentrations of triacylglyceroles decreased significantly by 11.6% (Po0.05) in the probiotic supplementation period. Conclusions: L. acidophilus and B. lactis were recovered in faeces in significantly elevated numbers after supplementation. They are able to modulate unspecific cellular immune response indicated by the increased phagocytic activity.

European Journal of Clinical Nutrition (2008) 62, 584–593; doi:10.1038/sj.ejcn.1602761; published online 18 April 2007 Keywords: probiotic; Lactobacillus; Bifidobacterium; immune system; cholesterol; faecal recovery

Introduction Correspondence: Professor G Jahreis, Department of Nutritional Physiology, Institute of Nutrition, Friedrich Schiller University, Dornburger Str. 24, Jena D-07743, Germany. E-mail: [email protected] Guarantor: G Jahreis. Contributors: AK and GJ were responsible for the study design. AK conducted the study and was responsible for data acquisition and analysis. AK performed the statistical analysis and drafted the manuscript. UF established the FISH methodology and validated the oligonucleotide probes. HV performed the immunological analysis. GJ was responsible for obtaining funding and acted as adviser throughout the study. UF and GJ were responsible for the critical review of the manuscript. None of the authors had any personal or financial conflict of interest. Received 21 November 2006; revised 31 January 2007; accepted 8 March 2007; published online 18 April 2007

The interest in probiotics has substantially increased over the last decade. Numerous studies support the beneficial effect of living microbial food supplements in health and disease (Marcos et al., 2004; Valeur et al., 2004; De Vrese et al., 2005; Viljanen et al., 2005; Weston et al., 2005; Ewaschuk and Dieleman, 2006; Olivares et al., 2006; Suomalainen et al., 2006). The functional properties of probiotics are strainspecific. Therefore, validation of individual properties is required by controlled intervention studies. Balanced blood cholesterol levels and immune functions are desirable to decrease the risk of disease. The ability of

Physiological effects of probiotic yoghurt A Klein et al

585 lactic acid bacteria to deconjugate bile salts is discussed as the underlying mechanism to affect cholesterol metabolism (Gilliland et al., 1985; Klaver and Van der Meer, 1993). The resulting free bile acids are less water soluble and are hardly reabsorbed. The deficit of bile acids leads to a compensatory synthesis of new bile acids from cholesterol in the liver (Brashears et al., 1998; De Smet et al., 1998; Ditscheid et al., 2005). However, there is little evidence that probiotic bacteria are able to reduce serum cholesterol in vivo. Most studies investigated the cholesterol-lowering effect in hypercholesterolaemic patients or after a high-fat diet (Bertolami et al., 1999; Kawase et al., 2001; Doncheva et al., 2002; Abd El-Gawad et al., 2005). There are few reports documenting the influence on normocholesterolaemic subjects (De Roos et al., 1999; Jahreis et al., 2002). As a precondition for the effect of probiotic bacteria on the immune system, the interaction between the microbiota and intestinal immune cells such as Peyer’s patches and intraepithelial lymphocytes are required. Adhesion to the intestinal mucosa and bacterial–epithelial–crosstalk are important criteria for exerting effects (Lu and Walker, 2001; Ouwehand et al., 2003). Numerous studies have shown an influence of probiotics on markers for the unspecific cellular immune response (Chiang et al., 2000; Gill et al., 2001; Sheih et al., 2001; Jahreis et al., 2002). Concerning specific cellular markers for immunity, the results are inconsistent. However, most of these studies revealing such immunological effects did not consider the detection of administered probiotics in faeces (Chiang et al., 2000; Gill et al., 2001; Sheih et al., 2001). Some multispecies probiotic products were already shown ¨ ck and Gebbers, to exert useful effects on human health (Glu 2003; Wang et al., 2004; Wildt et al., 2006). For this study Lactobacillus acidophilus 74-2 (L. acidophilus 74-2) and Bifidobacterium animalis subsp lactis DGCC 420 (B. lactis 420) were chosen for various reasons. They showed good antimicrobial activity against Staphylococcus aureus and Escherichia coli in vitro (Kristensen 2002; unpublished results). Furthermore, they were able to increase the intestinal production of short-chain fatty acids in vitro and in vivo, respectively (Gmeiner et al., 2000; Ouwehand et al., 2004). For B. lactis 420, good adhesion to human mucus was demonstrated in subjects of different age groups (Ouwehand et al., 1999). Thus, a combination of these two probiotic strains could be more effective in exerting health effects than a single strain. To the best of our knowledge, this is the first human intervention study evaluating broad physiological effects of L. acidophilus 74-2 and B. lactis 420. The aim of the study was to investigate the effects of the administered probiotics especially on the immune system and on markers of intestinal health. For this purpose, various plasma parameters of the specific and unspecific immune response and serum cholesterol levels were determined. Furthermore, L. acidophilus and B. lactis were detected in faecal samples and faecal concentrations of short-chain fatty acids were evaluated.

Methods Subjects Twenty-six normocholesterolemic volunteers, 13 men and 13 women, were recruited for this study. Before commencement of the trial, the selection criteria included general good health, no record of milk product intolerance, no intake of pharmaceuticals and nutritional or vitamin supplements. The study was approved by the Ethical Committee of the Medical Faculty of the Friedrich Schiller University of Jena. Informed consent was obtained from all volunteers.

Study design The study was designed as placebo-controlled and crossover study to ensure that each volunteer served as his or her own control. At the beginning of the study, each subject had to record his or her individual intake of food and drink over 1 week to estimate the individual requirements. Data were interpreted using PC-software PRODI 4.4 expert (NutriScience GmbH, Hausach, Germany). After the first yoghurtfree period (3 weeks), volunteers were randomly assigned to two groups: group A (7 female and 6 male) and group B (6 female and 7 male). The group A subjects consumed first 300 g placebo yoghurt daily, and the group B volunteers received first 300 g/day probiotic yoghurt over 5 weeks. In the following 5-week period, intervention changed between the two groups. During the last 7 days of each period all participants had to consume a defined diet. During this time, all volunteers were given the same quality and similar quantities of food and drink depending on their individual requirements. The first 2 days were used for the transition from individual to defined diet. From the 3rd to 7th day the faeces were collected completely in plastic boxes and immediately frozen at 201C. Fresh faecal samples were prepared for fluorescence in situ hybridization (FISH) analysis within 1 h of defecation. At the end of each period, all faecal samples of each subject were defrosted, homogenized and aliquots were stored at 801C. On the last day of each period, venous blood samples were obtained from subjects after overnight fasting for immunological and blood lipid analyses. At the end of the study, volunteers had to document their compliance with the dietary regimen in an anonymous questionnaire.

Yoghurt and diet The placebo product contained the standard yoghurt starters L. bulgaricus and Streptococcus thermophilus. The probiotic yoghurt was supplemented with B. lactis 420 (3.0 106 colony-forming units (cfu)/g) and L. acidophilus 74-2 (9.3 108 cfu/g; Danisco Deutschland GmbH, Germany). These strains were chosen because of their resistance to hydrochloric acid and bile acids (confirmed by in vitro assays; Danisco Deutschland GmbH). Yoghurt was produced in a specialized dairy once a week for immediate consumption. European Journal of Clinical Nutrition


586 Table 1 Data on oligonucleotide probes used in the present study Probe

Target

Sequence (50 -30 )

EUB 338

Bacteria 16S rRNA Negative control L. acidophilus 23S rRNA B. animalis subsp. lactis 16S rRNA

GCTGCCTCCCGTAGGAGT

20

Amann et al. (1990)

ACTCCTACGGGAGGCAGC TCTTTCGATGCATCCACA

20 20

Wallner et al. (1993) Pot et al. (1993)

CGATGGAGCGGAGCATCC

35

GGTATTACCACCCGTTTC

35

Complementary to PCR primer of Kok et al. (1996) This study

ATTTCCCACCCCACCATG

35

This study

NON 338 Lba LW420ca

H159 H195

B. animalis subsp. lactis 16S rRNA B. animalis subsp. lactis 16S rRNA

Formamide concentration in the hybridization buffer (%)

Reference

a

Used in combination with helperoligonucleotides H159 und H195.

Contents of water, fat, protein and minerals (Ca, Mg, P and Fe) were comparable in all batches of yoghurt products (data not shown). The defined diet was adapted to individual requirements resulting from the dietary protocols. All foodstuffs were bought in batches, weighed accurately to the nearest gram and prepacked for home consumption. Lunch was prepared freshly at the refectory of the Friedrich Schiller University Jena, portioned and immediately frozen at 201C until required. Tea, water, milk and apple juice were provided. Other beverages were permitted with the exception of alcoholic drinks. Volunteers had to record additional intakes of drinks and collect an aliquot. Aliquots were taken and frozen from all supplied food and drinks at 201C for subsequent analyses.

Analytical techniques Nutrient analyses. The nutrient content of food and their concentration in faeces was analyzed as described by Ditscheid et al. (2005). Fluorescence in situ hybridization. Preparation of faecal samples for FISH was performed by the method of Langendijk et al. (1995). For hybridization, 5 ml of fixed samples were applied to a precleaned (washed in 1% HCl and rinsed with 70% ethanol) and gelatin-coated [0.075% gelatine and 0.01% CrK(SO4)2] glass slide. After drying for 10 min, slides were dehydrated in 50, 80 and 100% ethanol each for 3 min. To optimize test conditions for the Lba probe, samples were incubated with 20 ml of lysozyme (10 mg ml1) at 41C for 10 min as described previously (Beimfohr et al., 1993). Hybridization was carried out as described by Manz et al. (1992). Unlabeled helper probes (Fuchs et al., 2000) were designed for probe LW420c (complementary to the primer of Kok et al., 1996) to increase the otherwise low probe conferred fluorescence. A formamide gradient between 0 and 80% in European Journal of Clinical Nutrition

the hybridization buffer was used to assess the optimal stringency for the newly designed helper probes in combination with LW420c. The optimal stringency for the helper probes in combination with LW 420c was achieved with a formamide concentration of 35%. Oligonucleotides were synthesized and fluorescently labeled with sulphoindocyanine dye (Cy3) at the 50 end by MWG Biotech (Ebersberg, Germany). A list of applied oligonucleotides is shown in Table 1. Slides were incubated in a buffer-saturated hybridization chamber at 461C for 15 h. After hybridization, slides were washed for 15 min in 50 ml of wash buffer (20 mM Tris—HCl, pH 8, 215 mM NaCl in the case of 20% formamide and 70 mM NaCl in the case of 35% formamide, 5 mM ethylenediaminetetraacetic acid, addition of distilled water to a final volume of 50 ml and 0.01% sodium dodecyl sulphate) at 481C for 15 min, rinsed with distilled water and air-dried. Staining with 4,6-diamidino-2-phenylindole (DAPI; 1 mg ml1) was carried out for 10 min at 41C. Slides were rinsed briefly, air-dried and mounted in Vectashield (Alexis ¨ nberg, Germany). Deutschland GmbH, Gru Samples were evaluated by the use of a Zeiss Axiovert 25 CA microscope (Carl Zeiss Jena GmbH, Germany) equipped with a HBO 50 mercury lamp, an objective Plan-Neofluar ( 100/1.3) and specific filter sets (no. 01 and MF 20 Cy3; Carl Zeiss Jena GmbH, Germany). At least 500 DAPI-stained cells were counted visually by examining a minimum of 15 randomly chosen microscopic fields analyzing probe Eubacteria (EUB). For evaluation of the specific probes Lba and LW420, at least 1000 DAPI-stained cells in a minimum of 15 randomly chosen microscopic fields were enumerated. Probe-positive cells were determined relative to DAPI counts. Each sample was analyzed in duplicate.

Immunological parameters For determination of immunological parameters, 100 ml heparinized blood was incubated with 20 ml fluorescein


587 isothiocyanate and phycoerthrine conjugated monoclonal antibodies at room temperature for 20 min. Erythrocytes were lysed after incubation with 2 ml of fluorescent-activated cell sorting (FACS) lysing solution (Becton Dickinson GmbH, Heidelberg, Germany) for 10 min. After two washing, steps cells were resuspended in 0.5 ml Cellwash (Becton Dickinson GmbH, Heidelberg, Germany) and analyzed on a flowcytometer FACScan using the blue-green excitation light (488 nm argon-ion laser) with SimulSET- and CellQuest software (Becton Dickinson GmbH, Heidelberg, Germany). Two thousand lymphocytes were measured using the following antibodies: CD3, CD19, CD4, CD8, CD16&CD56, CD57, HLA-DR (Becton Dickinson GmbH, Heidelberg, Germany), CD25, CD122, and CD54 (BD Biosciences Pharmingen, Heidelberg, Germany). The results were expressed as the percentage of mononuclear cells that stained positively for each cell surface marker. Phagocytic activity of granulocytes and monocytes was determined using the commercial test kit Phagotest (Orpegen, Heidelberg, Germany). Heparinized blood was incubated with fluorescein-labelled opsonized E. coli at 371C for 10 min. The reaction was stopped by the addition of icecold quenching solution. After two washing steps, addition of lysing solution and DNA staining, the percentage of granulocytes that ingested fluorescein isothiocyanatelabelled opsonized E. coli was detected by flow cytometry (FACScan, CellQuest software Becton Dickinson GmbH, Heidelberg, Germany). Oxidative burst activity was analyzed by the commercial test kit Phagoburst (Orpegen, Heidelberg, Germany). Heparinized blood was incubated with different stimuli unlabelled opsonized E. coli, protein kinase C ligand (phorbol 12myristate 13-acetate) and the chemotactic peptide N-formylMetLeuPhe at 371C for 10 min. Addition and oxidation of fluorogenic substrate dihydrorhodamine 123 results in the formation of reactive oxidants. After stopping the reaction by the addition of lysing solution, washing and DNA staining, the percentage of cells that produced reactive oxygen radicals was measured with the same flow cytometer as described above.

Blood lipids Venous blood samples were taken from subjects after overnight fasting using serum monovettes. For lipid analyses blood was centrifuged for 15 min at 1400 g. High-density lipoprotein (HDL) cholesterol was separated from the lowdensity lipoprotein (LDL) and the very-low-density lipoprotein (VLDL) fraction by precipitation performed with magnesium and phosphorwolframate. Total cholesterol and HDL cholesterol were measured after an enzymatic cholesterol oxidase–peroxidase treatment using a commercially available kit (Beckman, Krefeld, Germany) and a Synchron LX 20 (Beckman, Krefeld, Germany). Determination of triacylglyceroles was performed by a full enzymatic glycerophosphate oxidase–peroxidase technique with Beckman test

kits and a CX 7 Synchron Beckman (Beckman, Krefeld, Germany). LDL cholesterol was precipitated and measured with an electrophoretic technique using a commercially available test kit (Immuno AG, Heidelberg, Germany).

Short-chain fatty acids To detect short-chain fatty acids on two different days, two times 1 g of fresh faeces were weighted into 4 ml cryo-tubes and 2 ml distilled water was added. Samples were stored at 201C. For analyses, samples were defrosted, homogenized and centrifuged at 6000 g for 15 min. Subsequently, 500 ml of the supernatant were added to 50 ml of internal standard (i-caproic acid), vortexed and centrifuged once more. For gas-chromatographic measurement, (on a Shimadzu model GC 17A, Shimadzu, Kyoto, Japan) 1 ml was injected. The following operating conditions were used for separation and detection of short-chain fatty acids: a flame-ionization detector, a 15 m FFAP-column (15 m 0.25 mm 0.25 mm), hydrogen as carrier gas, flow rate 0.8 ml/min and a temperature program with start temperature 1051C, an increase of 351C/min and a final temperature of 1701C.

Statistical analysis A power analysis was performed using PASS 6.0 (NCSS Statistical Software, Kaysville, UT, USA) based on data from the literature and of our study group to evaluate sample size. It resulted in a power of 90% for this study. All data was checked for normal distribution applying the Kolmogorov– Smirnov-Test. Statistical analyses were conducted by SPSS version 11.5 (SPSS Inc., Chicago, USA). To evaluate data in terms of statistical significance, analysis of variance with general linear model and repeated measurements was applied. This model considers both individual and interindividual changes over time. The influence of the sex on parameters was analyzed by pasting the sex as intersubject. Furthermore, the intervention sequence as a consequence of the crossover design was considered by pasting the sequence as covariate. No influence of sex and sequence was determined for the analyzed parameters except for nutrient intake and excretion. Relationships between variables were examined by calculating Pearson’s correlation coefficients and linear regressions. For each comparison, Po0.05 was considered as significant. Significant differences between periods are indicated by unequal superscripts. Results are expressed as the mean7s.d.

Results Nutrient intake and excretion All 26 subjects completed the study in good health. Dry matter, fat and protein intake were comparable in both study periods (Table 2). As expected, the intake of nutrients differed significantly between men and women (dry matter: European Journal of Clinical Nutrition


588 639775 vs 462761 g/day (placebo), 635763 vs 462790 (probiotic); fat: 119720 vs 75715 g/day (placebo), 117717 vs 75719 (probiotic); protein 94710 vs 67714 g/day (placebo), 95712 vs 63711 (probiotic); values are means7s.d., men vs women). The daily faecal excretion of nutrients, measured during defined diet, remained unaffected by the probiotic intervention (Table 3), but faecal excretion was significantly different between men and women (dry matter: 30.678.1 vs 22.578.7 g/d (placebo), 29.478.8 vs 22.877.9 (probiotic); fat: 4.571.3 vs 3.171.6 g/day (placebo), 5.072.7 vs 3.471.5 (probiotic); protein 10.572.8 vs 7.772.9 g/day (placebo), 9.972.6 vs 7.772.6 (probiotic); values are means7s.d., men vs women). Although not being significant, a tendency of increase in relative fat excretion (P ¼ 0.052) from 3.2/100 g faeces in the placebo period to 3.8/100 g faeces in the probiotic period, independent of the sex, was observed.

Faecal microbiota Total bacteria and administered probiotics were detected in faecal samples. Percentages of total bacteria determined as percentage of EUB-positive cells versus DAPI-stained cells accounted for 83.2 and 79.2% in the placebo and probiotic period, respectively (P ¼ 0.087). Significant changes in faecal samples were determined in terms of the applied probiotic bacteria. Cell counts of L. acidophilus increased significantly

Table 2 Characteristics of all volunteers and averaged intake of nutrients during the defined diet Placebo period Age (years) Body mass index (kg/m2) Dry matter (g/day) Beverage (l/day) Protein (g/day) Fat (g/day)

Probiotic period

(P ¼ 0.027) in the probiotic period. Also the FISH-based quantification of B. lactis showed a highly significant increase (P ¼ 0.003) after 5 weeks of probiotic intake (Figure 1). A high interindividual variation of the FISH-based proportions was found for L. acidophilus and B. lactis.

Immunological measurements Most of the investigated immunological parameters were not influenced by the probiotic intervention. Cell counts of lymphocytes, monocytes, granulocytes and the expression of CD3, CD19, CD4, CD8, CD16&CD56, CD57, CD3 þ HLA-DR, CD25, CD122 and CD54 did not change throughout the study. However, percentages of monocytes and granulocytes showing phagocytic activity as a marker for the unspecific cellular immune response increased significantly. After 5week of placebo intake, percentages of active cells were 9276% and increased to 9574% in the probiotic period (P ¼ 0.044). The oxidative burst activity remained unaffected by probiotic cultures (placebo period 9174% vs probiotic period 9276%).

Blood lipids Total cholesterol, HDL and LDL cholesterol levels were not significantly influenced by the probiotic intervention. However, concentration of triacylglyceroles decreased significantly by 11.6% (P ¼ 0.045) in the probiotic period (Table 4). All values were within the normal range.

P-value

2573 21.572.0 5517112 2.270.5 80.9718.1 97.2728.0

21.372.1 5607108 2.270.5 79.0719.7 96.0727.9

0.068 0.456 0.618 0.415 0.603

Values are means7s.d., n ¼ 26. No significant differences were observed between the probiotic and the placebo period.

Table 3 Daily faecal excretion of dry matter, fat and protein during the defined diet

Fresh matter (g/day) Dry matter (g/day) Protein (g/day) Fat (g/day)

Placebo period

Probiotic period

P-value

132763 26.679.2 9.173.1 3.871.6

122751 26.178.8 8.872.7 4.272.3

0.313 0.813 0.627 0.400

Values are means7s.d., n ¼ 26. No significant differences were observed between probiotic and placebo period.

European Journal of Clinical Nutrition

Figure 1 Percentage changes of L. acidophilus and B. lactis in faecal samples after 5-week intervention with 300 g/day probiotic yoghurt containing L. acidophilus 74-2 and B. lactis 420 and a placebo yoghurt. Results were obtained by FISH. Probe-positive cells are determined relative to DAPI counts. Values are means7s.d., n ¼ 26. a,b Significant differences between mean values of unequal superscripts, Po0.05.


589 Short-chain fatty acids The concentration of total short-chain fatty acids did not differ significantly between the placebo and the probiotic period. Concentrations of acetate, propionate, butyrate, valerate and caproate were not influenced by probiotic cultures (Table 5). The pH value of fresh faeces remained stable in both intervention periods. Additionally, there was no influence on the distribution (mol%) of the short-chain fatty acids (data not shown).

Discussion Faecal microbiota mainly represents the composition of the luminal microbiota in the distal colon. Luminal microbiota is responsible for the activation of the innate and, in particular, of the adaptive immunity (Ouwehand et al., 2004). The administration of L. acidophilus 74-2 at a dose of 2.8 109 cfu/day and B. lactis 420 at a dose of 9.0 108 cfu/day over 5 weeks was well tolerated by the subjects and resulted in a significant increase in faecal bacterial counts of both species. Studies that investigated biopsy specimens from different parts of the gastrointestinal tract and faecal samples showed a clear correlation between the appearance

Table 4 Concentrations of blood lipids during the intervention study

Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL/HDL ratio Triacylglycerol (mmol/l)

Placebo period

Probiotic period

P-value

4.470.9 2.570.8 1.570.3 1.770.5 1.070.42a

4.570.9 2.570.8 1.570.3 1.770.6 0.870.4b

0.219 0.812 0.402 0.755 0.045

Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein. Values are means7s.d., n ¼ 26. a,b Significant differences between mean values of unequal superscripts, Po0.05.

Table 5 Total concentration of short-chain fatty acids, their distribution (mmol/g) in faeces and pH of faeces after 5 weeks intervention with probiotic and placebo yoghurt

Total short-chain fatty acids (mmol/g) Acetate (mmol/g) Propionate (mmol/g) i-Butyrate (mmol/g) n-Butyrate (mmol/g) i-Valerate (mmol/g) n-Valerate (mmol/g) n-Caproate (mmol/g) pH of faeces

Placebo period

Probiotic period

P-value

88.5719.3

85.0725.6

0.251

49.5710.0 16.977.0 2.070.8 15.174.7 2.470.9 2.070.8 0.670.7 7.170.5

46.7712.6 16.778.7 1.970.6 14.676.4 2.370.7 2.170.8 0.770.7 7.270.6

0.085 0.779 0.603 0.528 0.346 0.682 0.520 0.100

Values are means7s.d., n ¼ 26. No significant differences were observed between the probiotic and the placebo period.

of the bacteria in the biopsy samples and their presence in faeces (Ouwehand et al., 2004; Valeur et al., 2004; Van der Waaij et al., 2005). These observations allow the assumption that the significant elevated faecal counts of both administered probiotic bacteria indicate transient colonization. Furthermore, Valeur et al. (2004) stated that an increase in faecal bacterial counts is a traditional indication of colonization. Interestingly, L. acidophilus was detected in 30.8% (eight out of 26 volunteers), whereas B. lactis was found in 88.5% (23 out of 26 volunteers) after probiotic intake. The relative low recovery of L. acidophilus in only eight subjects might be caused by several factors. It is known that each subject harbours its individual intestinal microbiota (Tannock et al., 2000; Mai et al., 2004). Analyses of the microbiota (before and after administration of probiotics) demonstrated varying counts of administered bacteria in faeces indicating the need for differentiation between responders and nonresponders (Johansson et al., 1993; Tannock et al., 2000; ¨ et al., 2006). The interaction with Jahreis et al., 2002; Ma¨tto the carbohydrate moieties of the mucus is responsible for a successful binding of bacteria (Ouwehand et al., 1999). Possibly, there were differences in the expression of epithelial binding sites, which serve as receptors (Salminen et al., 1998). Tannock et al. (2000) monitored the influence of a probiotic intervention on the composition of the faecal microbiota over 15 months. They detected low numbers of probiotic L. rhamnosus in all subjects harbouring just one or two predominant Lactobacillus strains, whereas in subjects with a fluctuating Lactobacillus population the probiotic fraction was predominant during intervention. Thus, the establishment of administered bacteria may depend on the persistence of autochthonal strains. Secondly, the detection limit of FISH is approximately 0.1% (equal to 1 target cell if 1000 DAPI-stained cells are counted). Harmsen et al. (1999, 2002) and Mueller et al. (2006) detected between 0.01 and 0.2% of total DAPI counts in faeces using the group-specific probe LAB158. In consideration of the species specificity of the Lba probe used in this study (Pot et al., 1993) relatively high values were observed. For two individuals, members of L. acidophilus were also detected during the placebo period (0.25 and 0.24%). The same two subjects showed the highest proportion of members of the species L. acidophilus during the probiotic period (0.64 and 1.75%). Thus, it appears that L. acidophilus is one of the predominant species of the genus Lactobacillus in these two volunteers. In the probiotic period, a higher incidence of L. acidophilus can be assumed. However, in 69.2% (18 out of 26 volunteers) members of this species were not detectable. Numbers of bifidobacteria in the colon change during lifetime. Group-specific numbers of bifidobacteria varied between 3.5 and 4.2% of the total microbiota in healthy adults (Franks et al., 1998; Welling et al., 2000, 2002). In this study, the detection of B. lactis 420 was carried out using B. lactis-specific probe LW420c (complementary to PCR primer LW420c of Kok et al., 1996). Since members of B. lactis were also detected during the placebo period (in 46% of European Journal of Clinical Nutrition


590 all subjects), this taxon may represent a component of the resident microbiota in these subjects. In recent human studies, B. lactis was detected in biopsy and faecal samples ¨ et al., 2006). The increased (Bartosch et al., 2005; Ma¨tto numbers of B. lactis after 5 weeks of probiotic consumption may be a consequence of the good adhesion properties of this probiotic bacteria that have been shown in vitro by Ouwehand et al. (1999). The significant increase of probiotic bacteria in faeces suggests an influence on the immune system because of the known interaction between the microbiota and the gutassociated lymphoid tissue (GALT) (Salminen et al., 1998). This interaction is based on the ability of bacteria to induce expression of glycoconjugates by host epithelial cells. This oligosaccharide unit can serve as a receptor for the attachment of bacteria (Lu and Walker, 2001). Phagocytic activity and oxidative burst activity of monocytes and granulocytes are qualified markers to determine the influence on the unspecific cellular immune response (Cummings et al., 2004). A positive correlation between the percentages of monocytes and granulocytes showing phagocytic and oxidative burst activity was observed (Po0.001), although the oxidative burst activity remained unaffected by the probiotic intervention. Numerous studies dealing with the influence of probiotics on phagocytically active cells found corresponding results. For the strains L. rhamosus HN001, B. lactis HN019 and L. paracasei LTH 2579, it was shown to enhance the mononuclear leukocyte phagocytosis (Arunachalam et al., 2000; Chiang et al., 2000; Gill et al., 2001; Sheih et al., 2001; Jahreis et al., 2002). Indicating a stimulation of the immune system, these outcomes can be important for several population groups, for example elderly, stressed workers and children that are susceptible for infections. An enhancement of the immune system induced by probiotic bacteria can reduce duration and severity of infectious diseases (Schiffrin et al., 1997; Hatakka et al., 2001; Turchet et al., 2003). The expression of the specific cellular surface markers (CDs) remained unaffected through this study. Thus, the administered probiotic bacteria affect the unspecific immune response, but did not change specific immune parameters. One reason for these findings may be that healthy volunteers were chosen for this study. It is always somewhat problematic to recruit healthy young volunteers and to expect overall effects on the immune system. They normally have an optimal functioning immune system so that extensive effects on specific immune parameters may not be expected. However, to evaluate efficacy of a new probiotic combination, it is always safer to do this first in healthy individuals. Although in vitro experiments demonstrate the ability of L. acidophilus to decrease the concentration of cholesterol (Gilliland et al., 1985), blood cholesterol levels remained unaffected in this study. Influences of dietary habits were excluded by the administration of a standardized diet 1 week before collecting blood samples. Fat, protein and dry matter intake did not differ significantly between the placebo and European Journal of Clinical Nutrition

the probiotic periods (Table 2). Earlier studies dealing with the influence of probiotics on cholesterol metabolism have shown contradictory results (Schaafsma et al., 1998; AgerholmLarsen et al., 2000; Jahreis et al., 2002; Kieling et al., 2002; Lewis and Burmeister, 2005). These contradictions might be caused by different strains and amounts of administered probiotic cultures, no standardized application forms (milk products, sausage and capsules) and varying lengths of treatment periods. Moreover, a cholesterol-lowering effect of probiotics was more obviously, when hypercholesterolaemic individuals were chosen as subjects or when a cholesterol-enriched diet was given (Kieling et al., 2002; Xiao et al., 2003; Abd El-Gawad et al. 2005). There is no precise indication of the mechanism concerning a decrease in cholesterol levels in human. However, we observed a significant decrease of the concentration of triacylglyceroles after probiotic consumption (Table 4). This was also found by Taranto et al. (1998) and Kikuchi-Hayakawa et al. (2000), who studied the effect of lactobacilli in mice and hamsters, respectively. These authors supposed a lower intestinal absorption of lipids or a higher lipid catabolism because of a modified activity of lipogenic enzymes. In this study, an increase in relative fat excretion (per 100 g faeces) by 18.8% was measured in the probiotic period. This confirms the observation of Taranto et al. (1998) and Kikuchi-Hayakawa et al. (2000). Probiotic bacteria are able to ferment dietary fibres to produce short-chain fatty acids in the gut that may result in a decrease in the systemic levels of blood lipids by inhibiting hepatic cholesterol synthesis (Pereira and Gibson, 2002). Although the yoghurt contained two probiotic cultures, no significant change in the concentration and ratio of shortchain fatty acids in faeces were observed. The identical pH value after probiotic and placebo period supports this finding. The relative proportions of acetate, propionate and butyrate were about 50:17:17 (Table 5). In other studies, this ratio was about 60:20:15 and 60:16:16 (St-Onge et al., 2000; Kieling et al., 2002). Relative proportions are variable depending on the fermented substrates, but acetate represents quantitatively the main part followed by propionate and butyrate. The molar ratio of acetate to propionate is an intermediate marker for the influence on the cholesterol metabolism. It is known that a high concentration of acetate when entering the hepatocytes results in an increase of cholesterogenesis and lipogenesis in the liver (Delzenne and Williams, 2002). Conversely, a high concentration of propionate leads to a competitive inhibition of the activity of the acetyl-coenzyme A synthetase that is responsible for the entry of acetate into the liver (Delzenne and Williams, 2002; Rochet et al., 2006). Consequently, this mechanism of affecting the profile of short-chain fatty acids may be an explanation for the discussed cholesterol-lowering effect of pre- and probiotics. However, in this study, no effects of L. acidophilus 74-2 and B. lactis 420 on short-chain fatty acid concentrations in faeces and on cholesterol levels in serum were observed. These results are confirmed by recent human


591 studies (Lewis and Burmeister, 2005; Rochet et al., 2006; Yamano et al., 2006). In conclusion, this study shows that L. acidophilus and B. lactis administered at a daily dose of about 109 cfus resulted in a significant increase in faeces indicating transient colonization. The increment of the phagocytic activity suggests an enhancement of the immune function. The administered probiotics did not influence parameters of the specific immune response, total cholesterol, LDL and HDL cholesterol in serum and faecal concentrations of shortchain fatty acids. To detect clearer changes on the immune response or on blood lipids, further studies should be conducted in a subject group with suboptimal health status, like elderly or hypercholesterolaemic.

Acknowledgements ¨ ckel for technical assistance and We thank U Helms and P Mo R Schubert for advise on statistical analysis. In addition, we thank all volunteers for providing serum and faecal samples.

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