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American Journal of Clinical Nutrition, Vol. 88, No. 5, 1438-1446, November 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers1,2,3

Jelena Vulevic, Alexandra Drakoularakou, Parveen Yaqoob, George Tzortzis and Glenn R Gibson

1 From the Department of Food Biosciences, The University of Reading, Reading, United Kingdom (JV, AD, PY, and GRG), and Clasado Ltd, Milton Keynes, United Kingdom (GT)

2 Supported by a grant from Clasado Ltd.

3 Address reprint requests to J Vulevic, Department of Food Biosciences, The University of Reading, SCFP, PO Box 226, Reading RG6 6AP, United Kingdom. E-mail: j.vulevic{at}reading.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Aging is associated with reduced numbers of beneficial colonic bifidobacteria and impaired immunity. Galactooligosaccharides (GOSs) stimulate the growth of bifidobacteria in younger adults, but little is known about their effects in the elderly and their immunomodulatory capacity.

Objective: We assessed the effect of a prebiotic GOS mixture (B-GOS) on immune function and fecal microflora composition in healthy elderly subjects.

Design: In a double-blind, placebo-controlled, crossover study, 44 elderly subjects were randomly assigned to receive either a placebo or the B-GOS treatment (5.5 g/d). Subjects consumed the treatments for 10 wk, and then went through a 4-wk washout period, before switching to the other treatment for the final 10 wk. Blood and fecal samples were collected at the beginning, middle (5 wk), and end of the test period. Predominant bacterial groups were quantified, and phagocytosis, natural killer (NK) cell activity, cytokine production, plasma cholesterol, and HDL cholesterol were measured.

Results: B-GOS significantly increased the numbers of beneficial bacteria, especially bifidobacteria, at the expense of less beneficial groups compared with the baseline and placebo. Significant increases in phagocytosis, NK cell activity, and the production of antiinflammatory cytokine interleukin-10 (IL-10) and significant reduction in the production of proinflammatory cytokines (IL-6, IL-1β, and tumor necrosis factor-{alpha}) were also observed. B-GOS exerted no effects on total cholesterol or HDL-cholesterol production, however.

Conclusions: B-GOS administration to healthy elderly persons resulted in positive effects on both the microflora composition and the immune response. Therefore, B-GOS may be a useful dietary candidate for the enhancement of gastrointestinal health and immune function in elderly persons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A progressive increase in the proportion and number of persons >60 y old has led to heightened attention to their physiologic and health needs, as well as to increased costs to the health care system. Aging is associated with changes in the function of many organs and tissues, including the gastrointestinal tract (GIT). The composition of the elderly intestinal microflora differs from that of younger adults. Studies investigating these differences report increases in putrefactive bacteria, such as clostridia (especially Clostridium perfringens) and enterobacteria at the expense of more beneficial groups such as Eubacterium spp., and more importantly, bifidobacteria (1-4). The aging process also leads to a marked decline in immune function (immunosenescence), which can promote hyporesponsiveness to vaccination and a predisposition to infectious and noninfectious diseases (5). Immunosenescence is characterized by decreased numbers of circulating CD3+ T cells, increased production of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-{alpha} (TNF-{alpha}) by peripheral blood mononuclear cells (PBMCs), as well as diminished phagocytic and natural killer (NK) cell activities (6-8).

Dietary substrates reaching the large intestine are able to influence the composition and activities of bacteria present through their fermentation capacities. Metabolic products from colonic bacteria may affect the immune system. Modulation of the intestinal microflora by dietary means is the basis for the probiotic (9) and prebiotic (10) concepts. Various strains of bifidobacteria and lactobacilli as probiotics were also shown to exert immunostimulatory properties (11, 12).

It is well known that prebiotics, namely inulin-type fructooligosaccharides (FOSs) and galactooligosaccharides (GOSs), support the growth of beneficial bacteria in younger adult populations. In general, studies report increased numbers of lactobacilli, bifidobacteria, or both and decreased enterobacteria after FOS supplements (13, 14). However, studies have not fully determined their effect on the gut microflora of elderly persons.

The immunomodulatory potential of prebiotics has not been adequately investigated, and most data originate from animal models. For elderly human subjects, data are scarce and contradictory for modulation of the gut microflora. In one study, FOS administration to frail elderly subjects in a nursing home for 3 wk resulted in increased bifidobacteria, reduced phagocytic activity, and reduced expression of IL-6 mRNA in PBMCs (15). However, in another study, the same researchers reported that 12 wk of FOS administration to malnourished elderly subjects diminished IL-6 and TNF-{alpha} mRNA without any effect on the fecal microflora (16).

The effects of GOSs as potential modulators of both the elderly gut microflora and the immune system have not been reported thus far, although some reports suggest that these prebiotics have a good prebiotic effect in younger adults (17). In the present study, we have investigated the effect of administering a GOS mixture (B-GOS, Bi2muno) on immune function and fecal microflora composition of healthy elderly subjects in a double-blind, randomized, placebo-controlled crossover study.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Unless otherwise stated all chemicals and reagents were obtained from Sigma-Aldrich Co Ltd (Poole, United Kingdom) or BDH Chemicals Ltd (Poole, United Kingdom). Fluorescent probes, the K562 cell line and cell culture media, and supplements were also obtained from Sigma-Aldrich Co Ltd.

Subjects and study design
Forty-four, free-living, elderly volunteers [28 women and 16 men; average (±SD) age: 69.3 ± 4.0 y; range: 64–79 y; body mass index (BMI; in kg/m2) range: 22–31] were enrolled into the study to provide 99% statistical power (MGH BIOSTATISTICS SOFTWARE, Massachusetts General Hospital, Boston, MA) for the studied parameters on the basis of the data reviewed in the literature. Of these, 3 volunteers did not complete the study (1 fatality was unrelated to the study, 1 volunteer was reluctant to provide fecal samples regularly and was withdrawn, and 1 volunteer experienced tolerance problems and withdrew after 3 wk). Written consent was obtained from all volunteers, and they were assessed before the start of the trial for good health and selected according to certain exclusion and inclusion criteria. The exclusion criteria included use of antibiotics 3 mo before the study; regular use of prebiotic, probiotic, or symbiotic supplements 2 wk before the study; use of immunosuppressive or antiinflammatory drugs or any drugs affecting intestinal mobility; former participation in another study involving prebiotic, probiotic, or symbiotic preparations or other investigational drugs 6 mo before the study; and history or evidence of disorder of the GIT and malnutrition defined on the basis of BMI (minimal BMI required was 20 for women and 22 for men). The trial protocol was reviewed and approved by the University of Reading Ethics and Research Committee.

Volunteers were randomly assigned into 2 groups; one starting with a placebo (Maltodextrin) and the other with a prebiotic GOS mixture (B-GOS; Bi2muno), both provided in powder form (5.5 g) and supplied by Clasado Ltd, Milton Keynes, United Kingdom. The composition of the B-GOS mixture, in terms of degree of polymerization and saccharide linkages, is shown in Table 1Go. Subjects were asked to reconstitute sachets immediately before consumption by mixing the powder with water and to consume it every day at approximately the same time of day. They consumed the products for 10 wk, followed by a 4-wk washout period, before switching to the other treatment for the final 10 wk. Volunteers were required to visit the University of Reading on 7 separate occasions during this period and to provide a fecal sample at each visit (2 wk before the start; on day 0; and after 5, 10, 14, 19, and 24 wk). During all (except 2 wk before the start) visits, blood was sampled, and blood pressure, weight, use of any medication (including vitamin-mineral supplements), and adverse events were also recorded. In addition, subjects were asked to confirm their compliance with, or deviation from, the study protocol and to keep daily diaries recording bowel movements, stool consistency, and incidences of abdominal pain, flatulence, and bloating (data not presented).


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  		TABLE 1. Composition of the prebiotic galactooligosaccharide mixture (B-GOS) used in the study, for degree of polymerization (DP) and saccharide linkages, as percentage of the galactooligosaccharide content1

 
Preparation and collection of fecal samples
Fecal samples were collected in plastic pots lined with a sterile plastic bag at the University of Reading, and they were immediately diluted in anaerobic sterile phosphate buffer [phosphate-buffered saline (PBS), 0.1 mol/L, pH 7.0, (1:10, wt:vol), Oxoid Ltd, Basingstoke, United Kingdom], homogenized in a Stomacher 400 (Seward, Norfolk, United Kingdom), and used for fluorescent in situ hybridization (FISH). A portion of each fecal sample was removed before dilution and used to freeze-dry and measure the dry weight.

FISH analysis
Differences in bacterial populations were assessed with the use of FISH analysis with oligonucleotide probes designed to target specific diagnostic regions of 16S rRNA. The probes were synthesized commercially and labeled with the fluorescent dye Cy3. The probes used were Bif164 (18), Bac303 (19), Lab158 (20), Erec482 and His150 (21), Ec1531 (22), and Srb687 (23), specific for Bifidobacterium spp., Bacteroides spp., Lactobacillus-Enterococcus spp., the Clostridium coccoides–Eubacterium rectale group, the Clostridium histolyticum group, Escherichia coli, and Desulfovibrio spp., respectively. For total bacterial counts, the nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI) was used. Fecal homogenates from the volunteers were diluted in 4% (wt:vol) paraformaldehyde and fixed overnight at 4 °C. Cells were then centrifuged at 1500 x g for 5 min, washed twice with PBS (0.1 mol/L, pH 7.0), resuspended in a mixture of PBS and 99% ethanol (1:1, wt:vol), and stored at –20 °C for at least 1 h. Hybridization of cell suspensions was performed with the use of the modified well method described by Daims et al (24). Briefly, the cell suspension was diluted appropriately for each probe and added to the well of a Teflon- and poly L-lysine-coated 6-well slide (Tekdon Inc, Myakka City, FL). Slides were dried in a desktop plate incubator, dehydrated in ethanol (50%, 80%, and 96%, by vol), and dried again. For the Lab158 probe, samples were treated in a lysozyme solution to increase cell permeability before dehydration in ethanol. The hybridization mixture was then added to each well, and slides were placed onto the slide tray, which was sealed and left for 4 h in the hybridization oven set at the appropriate temperature for each probe (37 °C for Ec1531; 46 °C for Bac303 and Srb687; 50 °C for Bif164, Lab158, Erec482, and His150). Once the hybridization was complete, slides were removed from the slide tray and transferred into the wash buffer (warmed at the appropriate temperature for each probe), where they were left for a further 15 min. Slides were then dipped into ice-cold distilled water for 2–3 s and dried with a stream of compressed air. Antifade solution (ProLong Gold; Invitrogen Ltd, Paisley, United Kingdom) was then added to each well, and slides were examined with the use of a fluorescent microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan). The DAPI-stained cells were examined under ultraviolet light, and hybridized cells were viewed with the use of a DM510 filter. For each slide, at least 15 random different fields of view were counted. The following equation was used to calculate the numbers of bacteria: log10 (450 x A1 x 6732.42 x S1), where 450 is the dilution factor, A1 is the average count derived from 15 fields of view, 6732.42 is area of the well divided by the area of the field of view, and S1 is the sample size expressed in µL. The number of cells obtained is expressed as grams of feces (dry weight).

Analysis of plasma total cholesterol and HDL cholesterol
Blood was collected into 10-mL EDTA-coated tubes and centrifuged for 10 min at 1600 x g and 4 °C. Plasma was removed and stored at –20 °C until analysis. Samples were measured with the use of an ILab 600 biochemical analyser and enzymatic colorimetric kits (Instrumentation Laboratories Ltd, Warrington, United Kingdom).

Preparation of PBMCs
Blood samples (40 mL) were collected into sodium heparin-coated tubes and diluted 1:0.75 with PBS. The diluted blood was layered onto Polymorphprep (Axis-Shield, Kimbolton, United Kingdom) and centrifuged for 30 min at 600 x g and 20 °C. Plasma was collected for cytokine analysis; PBMCs were removed and washed with sterile PBS. Cells were then resuspended in 2.5 mL PBS, layered onto 5 mL Lympholyte (Cedarlane Laboratories, Burlington, NC), centrifuged, and washed once again, to ensure a lower degree of erythrocyte contamination, before final resuspension in the appropriate medium.

Measurement of NK cell activity
The NK cell activity of PBMC preparations against the K562 cell line was determined with the use of a commercially available kit (MorphoSys Ltd, Oxford, United Kingdom), which measures the release of cellular lactate dehydrogenase (LDH) after target cell lysis. PBMCs were prepared as described earlier and resuspended in RPMI medium supplemented with glutamine (2 mmol/L) and antibiotics (1% by vol). K562 cells were washed 3 times and resuspended in RPMI medium supplemented with bovine serum albumin (1% by vol) and antibiotics (1% by vol). PBMCs (1 x 107 cells/mL) and K562 cells (2 x 105 cells/mL) were added to each well of a 96-well microtiter plate to give ratios of PBMC to K562 cells of 100:1, 50:1, or 25:1. Spontaneous release of LDH by PBMCs was determined by incubating the PBMCs in the absence of target cells, and spontaneous release of LDH by K562 cells was determined by incubating the K562 cells in the absence of effector cells. Maximal target cell lysis was determined by incubating K562 cells with the lysing reagent supplied in the cytotoxicity kit. Plates were centrifuged for 3 min at 100 x g and then incubated at 37 °C in an atmosphere of air to carbon dioxide (19:1) for 4 h. After incubation, 50 µL of ice-cold RPMI-bovine serum albumin-antibiotics medium was added to each well, and the plates were centrifuged at 400 x g for 5 min. Supernatant fluid (100 µL) from each well was removed and transferred onto the wells of a flat-bottomed 96-well plate. The activity of LDH in these supernatant fluids was assayed according to the instructions provided by the manufacturer of the kit. The following equation was used to calculate the percentage of cytolysis: % Cytolysis = (LDH release –PBMC spontaneous LDH release –K562 cell spontaneous LDH release)/(maximum K562 cell LDH release –K562 cell spontaneous LDH release) x 100.

Measurement of cytokine production by PBMC cultures
PBMCs (2 x 106 cells/mL) were resuspended in RPMI-glutamine-antibiotics medium supplemented with autologous plasma (5% by vol). Cells were then plated onto 24-well plates in the presence of lipopolysaccharide (10 µg/mL) and incubated at 37 °C in an air to carbon dioxide (19:1) atmosphere for 24 h. After incubation, the plates were centrifuged at 400 x g for 5–6 min, and the supernatant fluids were collected and frozen in aliquots (200 µL). The concentration of cytokines (IL-6, IL-10, IL-1β, TNF-{alpha}, and IL-8) was measured by enzyme-linked immunoabsorbent assay with the use of commercially available kits (R&D Systems Inc, Minneapolis, MN) and the instructions provided by the manufacturer. The limits of detection for these assays were as follows: IL-6, 2.5 ng/mL; IL-10, 3.3 ng/mL; IL-1β, 7.2 ng/mL; TNF-{alpha}, 3.7 ng/mL; and IL-8, 3.6 ng/mL (data were supplied by the manufacturer of the kits).

Measurement of phagocytic activity
Phagocytosis was determined with the use of Phagotest kits (Orpegen Pharma, Heidelberg, Germany). Before use, blood was cooled on ice for 10 min and mixed by vortexing. Aliquots (100 µL) of blood were incubated on ice (control) or at 37 °C in a preheated water bath for 10 min with opsonized fluorescein isothiocyanate-labeled E. coli (20 µL). The reaction was stopped by adding ice-cold quenching solution (100 µL) and vortexing. Samples were then washed several times in solution provided by the manufacturer, erythrocytes were lysed, leukocytes were fixed, and the DNA was stained. Cell preparations (200 µL) were transferred onto glass slides containing wells and covered with a cover slip. Slides were protected from light and examined with the use of phase-contrast and fluorescence microscopy within 1 h. At least 10–15 random fields of view were examined, and the number of leukocytes (monocytes and neutrophils), number of leukocytes that had internalized bacteria, and the number of bacteria engulfed by these cells were recorded.

Statistical analysis
All statistical tests were performed with the use of SPSS version 15.0 (SPSS Inc, Chicago, IL), and a value of P < 0.05 was taken to indicate statistical significance. All data were analyzed by an analysis of variance model with repeated measurements taking into account the crossover design. In the analysis of variance model, treatment period (0, 5, and 10 wk) and sequence of treatments were introduced as fixed effects and subject measurements as random effect. Significant differences were determined by Tukey's honestly significant difference test. Reported P values are 2-sided, and no statistically significant interaction between sequence and treatment or between period and treatment was found for any of the measurements taken. When it existed, the linear relation between the variables measured in this study, at the end of the trial period (10 wk), was analyzed by bivariate correlation and presented with the use of Pearson's correlation coefficient with significant levels.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of B-GOS on bacterial populations
The fecal microflora composition of the elderly volunteers who participated in this study was determined by the use of FISH and probes targeting bacterial groups of interest. No differences were observed in bacterial populations in the 2 treatment groups at baseline (Table 2Go). The 2 treatments administered did not appear to have an effect (P > 0.14) on the total bacterial count during the study. After 5 wk, the placebo treatment did not alter the numbers of Bifidobacterium spp., the C. coccoides–E. rectale group, the C. histolyticum group, or Desulfovibrio spp., whereas those of Bacteroides spp. (P < 0.001) and E. coli (P < 0.01) increased and Lactobacillus-Enterococcus spp. (P < 0.05) decreased (Table 2Go). At the same time, B-GOS had a significant effect on all bacterial groups measured compared with the baseline and placebo groups. Higher numbers of Bifidobacterium spp., Lactobacillus-Enterococcus spp., and the C. coccoides–E. rectale group were observed compared with both baseline and the placebo treatment (Table 2Go). In contrast, numbers of Bacteroides spp., the C. histolyticum group, E. coli, and Desulfovibrio spp. decreased compared with both baseline and the placebo treatment (Table 2Go).


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  		TABLE 2. Bacterial populations of the monitored fecal microflora components for the placebo and prebiotic galactooligosaccharide mixture (B-GOS) treatments used in elderly volunteers during the study period as determined by fluorescent in situ hybridization1

 
By the end of the trial period (10 wk), both treatments had exerted a significant effect on all bacterial groups monitored in this study compared with baseline (Table 2Go). The placebo treatment resulted in increased numbers of Bifidobacterium spp. (P < 0.001), Bacteroides spp. (P < 0.001), the C. histolyticum group (P < 0.01), E. coli (P < 0.001), and Desulfovibrio spp. (P < 0.05), and decreased numbers of Lactobacillus-Enterococcus spp. (P < 0.05) and the C. coccoides–E. rectale group (P < 0.05; Table 2Go). Furthermore, numbers of Bifidobacterium spp., Bacteroides spp., the C. histolyticum group, and Desulfovibrio spp. were significantly higher than after 5 wk of the placebo treatment (P < 0.01, P < 0.05, P < 0.01, and P < 0.001 for Bifidobacterium spp., Bacteroides spp., the C. histolyticum group, and Desulfovibrio spp., respectively; Table 2Go). The effect of B-GOS treatment observed at 5 wk was also further enhanced by 10 wk. There were higher numbers of Bifidobacterium spp. (P < 0.001 for all), Lactobacillus-Enterococcus spp. (P < 0.001 for all), and the C. coccoides–E. rectale group (P < 0.001 for all) and lower numbers of Bacteroides spp. (P < 0.001 for all), the C. histolyticum group (P < 0.001 for all), E. coli (P < 0.001 for all), and Desulfovibrio spp. (P < 0.001 for all) compared with baseline, the 5-wk B-GOS treatment, and the 10-wk placebo treatment (Table 2Go).

Plasma total cholesterol and HDL cholesterol
Plasma concentrations of total cholesterol and HDL cholesterol did not differ significantly at the beginning of each treatment (Table 3Go). In addition, concentrations of both remained unchanged regardless of the treatment or time during the trial period (Table 3Go).


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  		TABLE 3. Effect of the placebo and prebiotic galactooligosaccharide mixture (B-GOS) treatments on total cholesterol and HDL cholesterol during the study period1

 
Effect of B-GOS on NK cell activity
NK cell activity measured at effector-to-target cell ratios (E:T) of 100:1 and 25:1 did not differ significantly (P > 0.06) at baseline between the 2 treatments, whereas the activity measured at an E:T of 50:1 tended to be lower (P = 0.06) before the administration of B-GOS compared with the placebo baseline (Table 4Go). After 5 wk of placebo supplement, NK cell activity at an E:T of 25:1 was higher (P < 0.05) than that observed at the baseline, whereas the B-GOS supplement resulted in increased activity at all E:T values (P < 0.001 for all) compared with baseline (Table 4Go). At E:T values of 100:1 and 50:1 NK cell activity was significantly higher (P < 0.05 for both) after B-GOS than after the placebo treatment at 5 wk (Table 4Go).


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  		TABLE 4. Effect of placebo and prebiotic galactooligosaccharide mixture (B-GOS) treatments on natural killer cell activity of peripheral blood mononuclear cells1

 
By 10 wk, both treatments resulted in highly significant increases in NK cell activity compared with the baseline (P < 0.001 for both treatments and all ratios) and compared with the 5-wk time point for the same treatment (P < 0.001 for both treatments and all ratios, except at E:T value of 25:1 for the placebo, P < 0.01; Table 4Go). However, the effect of B-GOS on the NK cell activity was significantly higher (P < 0.001 at E:T values of 100:1 and 50:1 and P < 0.01 at E:T value of 25:1) than that observed with placebo (Table 4Go).

The association between the effect of placebo and B-GOS, at the end of the trial period, on bacterial groups measured in this study and NK cell activity was determined by bivariate correlation analysis (Table 5Go). This analysis showed that the numbers of Bifidobacterium spp., Lactobacillus-Enterococcus spp., and the C. coccoides–E. rectale group were significantly positively (P < 0.01) correlated, and the numbers of Bacteroides spp. were significantly negatively (P < 0.01) correlated with NK cell activity (Table 5Go).


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  		TABLE 5. Relation between bacterial groups measured and natural killer (NK) cell activity and phagocytosis at 10 wk of the treatments as determined by bivariate correlation analysis1

 
Effect of B-GOS on cytokine production by PBMCs
The effects of the 2 treatments on the production of cytokines (IL-10, IL-1β, TNF-{alpha}, IL-6, and IL-8) are shown in Table 5Go. Baseline TNF-{alpha} concentration was significantly higher (P < 0.05) before B-GOS supplementation (Table 6Go). No significant effects of time or treatment were observed on the production of IL-8 (Table 6Go). However, differences were observed in the production of other cytokines. At 5 wk, the placebo treatment resulted in increased production of IL-1β (P < 0.05), TNF-{alpha} (P < 0.001), and IL-6 (P < 0.05), whereas production of IL-10 remained unchanged compared with baseline (Table 6Go). B-GOS supplementation, however, resulted in decreased production of TNF-{alpha} (P < 0.01) and IL-6 (P < 0.05), increased production of IL-10 (P < 0.05), and no change in the concentration of IL-1β compared with the baseline at this time point (Table 6Go). TNF-{alpha} and IL-6 production after B-GOS treatment was significantly lower (P < 0.05 for both cytokines) at this time point than that observed after placebo treatment, whereas IL-10 and IL-1β concentrations did not differ significantly from placebo (Table 6Go). After 10 wk of the placebo treatment, no further increases were observed in the production of IL-1β, TNF-{alpha}, and IL-6, but the values were still significantly higher than those observed at baseline (P < 0.05, P < 0.001, and P < 0.05 for IL-1β, TNF-{alpha}, and IL-6, respectively). In the case of IL-10 production, the placebo treatment resulted in a further reduction at 10 wk, which was significant when compared with baseline (P < 0.01) and 5 wk (P < 0.05). At the same time, B-GOS treatment resulted in further decreases in the production of IL-1β (P < 0.01), TNF-{alpha} (P < 0.001), and IL-6 (P < 0.001) compared with both baseline (P < 0.05, P < 0.05, and P < 0.001 for IL-1β, TNF-{alpha}, and IL-6, respectively) and 5 wk (Table 6Go). Production of IL-10 was further significantly increased compared with both baseline (P < 0.01) and 5 wk (P < 0.01) after the B-GOS treatment (Table 6Go). Furthermore, production of IL-1β (P < 0.05), TNF-{alpha} (P < 0.01), and IL-6 (P < 0.001) was significantly lower and production of IL-10 (P < 0.01) was significantly higher after 10 wk of B-GOS treatment than was the placebo treatment at the same time (Table 6Go).


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  		TABLE 6. Production of cytokines by peripheral blood mononuclear cells with placebo and prebiotic galactooligosaccharide mixture (B-GOS) treatments during the study period1

 
The change in the production of IL-10, IL-1β, TNF-{alpha}, and IL-6 at 10 wk of placebo and B-GOS treatments from baseline is shown in Figure 1Go. This figure summarizes clearly opposite and significantly different effects of the 2 treatments on the production of these cytokines.


Figure 1
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  		FIGURE 1.. Mean ± SD (n = 41) change, at 10 wk from baseline, in a prebiotic mixture of galactooligosaccharide (B-GOS) and placebo treatments in elderly volunteers in the production of interleukin-10 (IL-10), IL-1β, tumor necrosis factor-{alpha} (TNF-{alpha}), and IL-6. The mean for the B-GOS group was significantly different from that for the placebo group for all cytokines (IL-6: P < 0.001; IL-10 and TNF-{alpha}: P < 0.01; IL-1β: P < 0.05). Values were analyzed by ANOVA with 3 fixed effects (treatment, period of treatment, and sequence of treatment). No significant interactions were observed between sequence and treatment or between period and treatment.

 
Effect of B-GOS on phagocytosis
Total phagocytic activity of leukocytes (neutrophils and monocytes) is expressed as the percentage of cells able to internalize fluorescently labeled E. coli (number of cells involved in phagocytosis) and as the quantity of internalized bacteria per active cell (level of activity). As shown in Figure 2Go, the baseline samples did not differ significantly (P > 0.8) between the 2 treatments. The placebo treatment had no effect on either the percentage of cells engaged in phagocytosis or the amount of phagocytic activity, regardless of time (Figure 2Go). B-GOS, however, resulted in a significant increase in the percentage of cells engaged in phagocytosis of E. coli after 5 wk of treatment (P < 0.001 compared with baseline and placebo), and this effect was further enhanced after 10 wk (P < 0.001 compared with baseline, placebo, and 5 wk; Figure 2Go). Furthermore, B-GOS treatment also increased the phagocytic capacity, as assessed by the average number of phagocytosed bacteria per active cell (Figure 2Go). This increase was significant after 5 wk of treatment compared with baseline (P < 0.001) and with the placebo treatment (P < 0.05) at the same time point and was further enhanced after 10 wk of the B-GOS treatment (P < 0.001 compared with baseline, 5 wk, and placebo).


Figure 2
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  		FIGURE 2.. Phagocytic activity of the placebo and prebiotic mixture of galactooligosaccharide (B-GOS) treatments during the study period in response to Escherichia coli. Data are expressed as mean ± SD (n = 41). The percentage of cells engaged in phagocytosis and the amount of phagocytic capacity are shown. {ddagger}Significantly different from baseline (P < 0.001) and placebo (P < 0.001); {dagger}significantly different from baseline (P < 0.001) and placebo (P < 0.05); *significantly different from baseline (P < 0.001), placebo (P < 0.001), and 5 wk (P < 0.001). Values were analyzed by ANOVA with 3 fixed effects (treatment, period of treatment, and sequence of treatment). No significant interactions were observed between sequence and treatment or between period and treatment.

 
The association between the effect of placebo and B-GOS on bacterial groups measured in this study and phagocytosis as determined by bivariate correlation analysis is shown in Table 5Go. The numbers of Bifidobacterium spp., Lactobacillus-Enterococcus spp., and the C. coccoides–E. rectale group were significantly positively (P < 0.01) correlated, and the numbers of Bacteroides spp. and E. coli were significantly negatively (P < 0.01) correlated with both the percentage of cells involved in phagocytosis and the amount of phagocytic activity. In addition, the numbers of the C. histolyticum group and Desulfovibrio spp. were also significantly negatively (P < 0.01) correlated with the percentage of cells involved in phagocytosis (Table 5Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The intestinal microflora can be considered as an organ composed of a large diversity of bacterial cells that can perform different functions for the host. Specific components of this microflora, including bifidobacteria and lactobacilli, are associated with beneficial effects for the host, such as promotion of gut maturation and integrity, antagonism against pathogens, and immune modulation (9, 10, 12, 17). The principal aim of this study was to assess the effect of a prebiotic, B-GOS, compared with placebo on the major bacterial groups found in feces of elderly persons and to assess the effect of this change in the microflora components on immune function of the host.

Age-related changes in GIT physiology and function, such as greater permeability of mucosal membrane, reduced transit times, and secretion of acids by the gastric mucosa, result in a significant change in the composition of the intestinal microflora, marked by a decline in bifidobacterial numbers and an increase in putatively detrimental populations such as clostridia and enterobacteria (2, 3, 25). These bacteriologic and other physiologic changes may result in increased putrefaction in the colon and greater susceptibility to disease. In this sense, any dietary supplement, such as prebiotics, that could counteract these changes is of value. However, the effect of prebiotics on the fecal microflora of elderly has not been extensively investigated. GOSs are generally recognized prebiotics (17, 26), and the specific GOS ingredient used in this study, B-GOS, produced with the use of bifidobacterial enzymes (27), was shown to promote the selective growth of bifidobacteria in in vitro mixed culture systems (28) and in vivo in younger healthy adults (29). Furthermore, B-GOS was shown in ex vivo experiments to selectively promote the growth of Bifidobacterium bifidum and Bifidobacterium longum (29), 2 species reported to have good immunomodulatory properties (30, 31).

In the present study, the placebo treatment increased fecal populations of bacteroides and E. coli and decreased lactobacilli, but it had no effect on the other bacterial groups tested at 5 wk. B-GOS, however, reduced numbers of less beneficial bacteria (bacteroides, the C. histolyticum group, E. coli, and Desulfovibrio spp.) and increased numbers of beneficial bacteria (bifidobacteria, lactobacilli, and the C. coccoides-E. rectale group). These effects became statistically significant by 5 wk of treatment (P < 0.05), but they were further enhanced at 10 wk (P < 0.001), showing that the bifidogenic effect of B-GOS was not lost or reduced over this time course. Although there were some increases in bifidobacteria after 10 wk of placebo treatment compared with the baseline sample, this was offset by significant increases in less beneficial bacterial groups and decreases in lactobacilli and the C. coccoides-E. rectale group. The effect of B-GOS treatment on host immune function was assessed with the use of a range of biomarkers of the innate and adaptive immune system. Probiotic bacteria, such as lactobacilli and bifidobacteria, were shown in vivo to interact with mouse epithelial cells of the small intestine and be internalized with the use of distinct pathways into the epithelial cells of the large intestine by the follicle-associated epithelium cells (32). They make contact with underlying immune tissues, and, through these interactions, they were suggested to bring about strain-specific immunomodulation (33), such as differentially induced cytokine production by macrophages in a concentration-dependent manner (34). Probiotics were also shown to regulate the balance between necessary and excessive immune response to support an adequate immune response to infection by enhancing phagocytosis, NK cell activity, and TH1 responses associated with the release of proinflammatory cytokines, but, at the same time, almost all probiotics induce antiinflammatory cytokines (IL-10 and transforming growth factor-β) through the regulatory Tr1 and TH3 cells (33).

Orally applied prebiotics were also associated with immune-modulating effects encompassing both innate and specific immunity, although evidence was mostly derived from animal models or in vitro experiments (35, 36). Because prebiotics are fermented to varying degrees by the beneficial intestinal bacteria (bifidobacteria, lactobacilli), the immunomodulatory properties were mainly attributed to microflora-dependent effects. However, some prebiotics are also reported to bind to specific receptors on cells of the immune system, suggesting that a direct interaction between prebiotics and the host is feasible (37, 38).

Both phagocytosis and NK cell activity are depressed in the elderly in comparison to younger counterparts (6). These activities are a first line of defense for the elimination of invading pathogens, infected cells, or (in the case of NK activity) tumor cells. In the present study, both the placebo and B-GOS treatments significantly increased NK cell activity. For the B-GOS treatment, the effect was already significant after 5 wk of treatment and was further enhanced after 10 wk, whereas, for the placebo treatment, the effect only became apparent at 10 wk with the effect of the B-GOS treatment being significantly greater than that after the placebo treatment (P < 0.05).

B-GOS unequivocally enhanced phagocytic activity in a time-dependent manner, whereas the placebo treatment had no such effect. Although, mechanisms by which prebiotics are able to influence these activities are not understood, we have shown a clear positive correlation between numbers of beneficial bacteria and both NK cell activity and phagocytosis, whereas some less beneficial bacteria (ie, bacteroides) were negatively correlated. This suggests that, at least in part, the observed modulating effects of the 2 treatments on the microflora were responsible for the changes in NK and phagocytic activities.

Cytokines have a range of important roles in the regulation of immunity. Elevated concentrations of proinflammatory cytokines are associated with both aging (7, 8, 39) and chronic inflammatory disease (40). In the present study, there was considerable intersubject variation in production of the cytokines studied, some of which could be attributed to factors such as sex, genetic polymorphisms, smoking status, BMI, and dietary habits, as well as early life events, hormonal status, and gut microflora (41, 42). Nevertheless, B-GOS significantly decreased the production of both TNF-{alpha} and IL-6 by 5 wk, whereas the placebo treatment significantly increased production of the 2 cytokines. B-GOS also decreased the production of IL-1β at 10 wk, whereas the placebo treatment resulted in an initial increase at 5 wk and no further effect at the end of the treatment. In the case of IL-10, the antiinflammatory cytokine, B-GOS significantly increased production by 5 wk and further production by 10 wk, whereas the placebo treatment decreased production of this cytokine at 10 wk. These data clearly show opposite effects of the 2 treatments on the proinflammatory and antiinflammatory cytokine profiles, with the B-GOS treatment resulting in an overall antiinflammatory effect and the placebo treatment showing a tendency to produce a proinflammatory effect.

The antiinflammatory effect of IL-10 is partly achieved through suppression in the production of macrophage inflammatory proteins IL-1, IL-6, IL-12, and TNF-{alpha} (43). In vivo evidence for the importance of IL-10 as a significant immunoregulator with potent antiinflammatory and immunosuppressive activities comes from the observation that IL-10–deficient mice develop chronic enterocolitis similar to inflammatory bowel disease (44). Many of the proinflammatory cytokines showed to be suppressed by IL-10 are known to be regulated by the nuclear transcription factor {kappa}B (45), which also has a role in IL-10 gene expression (46, 47). In this study we have shown that B-GOS treatment resulted in a clearly less inflammatory profile, and it could be speculated that the mechanism of action involves the nuclear transcription factor {kappa}B pathway.

To date, the immunomodulatory potential of GOS has not been directly shown. Two studies looked at the effect of combined application of probiotics with GOS on gut health and immunity (48, 49). In the first study, combination of GOS with Bifidobacterium lactis (HN019) enhanced NK cell activity compared with probiotic alone (48), and, in the other, the effect of GOS in combination with Lactobacillus rhamnosus (HN001) was not significantly different from the probiotic alone (49).

Several animal studies have shown that administration of probiotics, prebiotics, or fermented milk products is effective in lowering blood cholesterol concentrations. However, in vivo results are variable, with some studies reporting lowering effects (50) and increasing HDL cholesterol (51, 52) and others without any effect (53, 54). Most studies reporting decreased concentrations of serum cholesterol or increased HDL cholesterol or both, after either probiotic or prebiotic administration, have used subjects with elevated serum cholesterol concentrations. Results from our study showed that B-GOS had no effect on total serum cholesterol and HDL cholesterol concentrations. The reason for this might be because our subjects had normal serum cholesterol concentrations before the start of the trial.

In conclusion, this study showed that B-GOS administration to healthy elderly subjects led to a significant decrease in less beneficial bacteria and a significant increase in beneficial bacteria, especially bifidobacteria. We also found significant positive effects on the immune response, evidenced by an improvement in NK cell activity and phagocytosis, increased secretion of the antiinflammatory cytokine IL-10, and decreased secretion of proinflammatory cytokines (IL-6, IL-1β, and TNF-{alpha}) by stimulated PBMCs. Therefore, dietary intervention that uses B-GOS is an attractive option for enhancement of both the GIT and immune system, which could be of particular importance for the elderly.


   ACKNOWLEDGMENTS
 
The author's responsibilities were as follows—JV, GT, and GRG: designed the study; PY: helped with a choice of immune methods; JV: prepared protocols, selected and recruited volunteers, analyzed data, and wrote the manuscript; AD: collected and processed all the samples; JV and AD: performed the experimental work; PY, GT, and GRG: reviewed the manuscript. GT is employed by Clasado Ltd. None of the other authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication April 2, 2008. Accepted for publication July 29, 2008.





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