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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Background : Probiotics are efficacious for treating and maintaining remission of ulcerative colitis.

Aim : To conduct a randomized placebo-controlled trial of bifidobacteria-fermented milk supplementation as a dietary adjunct in treating active ulcerative colitis.

Methods : Twenty patients with mild to moderate, active, ulcerative colitis randomly received 100 mL/day of bifidobacteria-fermented milk or placebo for 12 weeks with conventional treatment.

Results : Clinical and endoscopic activity indices and histological scores were similar in the two groups before treatment. Although improvements were significant in both groups, the clinical activity index was significantly lower in the bifidobacteria-fermented milk than in the placebo group after treatment. The post-treatment endoscopic activity index and histological score were significantly reduced in the bifidobacteria-fermented milk, but not the placebo group. Increases in faecal butyrate, propionate and short-chain fatty acid concentrations were significant in the bifidobacteria-fermented milk, but not the placebo group. No adverse effects were observed in either group.

Conclusion : Supplementation with this bifidobacteria-fermented milk product is safe and more effective than conventional treatment alone, suggesting possible beneficial effects in managing active ulcerative colitis. This is a pilot study and further larger studies are required to confirm the result these preliminary results.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

The widely accepted theory of inflammatory bowel disease (IBD) pathogenesis is that chronic intestinal inflammation is the consequence of an overly aggressive cell-mediated immune response to commensal enteric bacteria in a genetically susceptible host.1, 2 Recently, the pathogenic role of luminal bacteria in IBD has been a source of controversy but the leading hypothesis is that resident bacterial flora are an essential cofactor in driving the inflammatory process in IBD.3 Ulcerative colitis (UC) is a chronic inflammatory condition and appears to be the result of defective regulation of mucosal interactions with the enteric microflora. A causative role for Bacteroidesvulgatus or Fusobacterium varium in experimental UC has been suggested.4, 5 In addition, decreases in Lactobacillus and bifidobacteria concentrations in colonic biopsy specimens from patients with active UC and reduced faecal concentrations of lactobacilli and bifidobacteria in patients with active pouchitis have been reported.1, 6, 7

Probiotics are defined as living organisms that improve the bacterial balance in the gut.8 Theoretically, probiotics can modify the composition and some metabolic activities of microflora by preventing overgrowth of potentially pathogenic bacteria and by maintaining the integrity of the gut mucosal barrier.9, 10 Reconditioning of the flora through direct supplementation with protective bacteria or by indirect stimulation with a prebiotic compound could play a protective role in the inflammatory process. Accordingly, probiotics and/or prebiotics, which have the potential to enhance the growth of indigenous bifidobacteria, are expected to be a treatment and/or prophylaxis for UC. The potential of probiotic therapy in IBD is based on convincing evidence of the effectiveness of this treatment strategy in several animal models of experimental colitis.11, 12Lactobacillus prevented the development of spontaneous colitis in interleukin-10-deficient mice.13 It has been suggested that some probiotics can help maintain remission of UC.14, 15 In addition, the administration of a highly concentrated probiotic preparation (VSL #3), containing bifidobacteria and lactobacilli, to chronic pouchitis patients was recently reported to maintain remission of the disease.16, 17 Furthermore, Gionchetti et al. reported the efficacy of VSL #3 for prophylaxis of pouchitis in a double-blind randomized placebo-controlled study.18 The use of fermented milk products to treat gastrointestinal complaints dates from antiquity. Ishikawa et al. demonstrated the effectiveness of supplementation with bifidobacteria-fermented milk (BFM), which contains live bifidobacteria, Yakult strains of Bifidobacterium breve, Bifidobacterium bifidum and Lactobacillus acidophillus, in maintaining remission of UC.19 Therefore, although the efficacy of probiotics in maintaining UC remission has been demonstrated, whether probiotic therapy is effective in the active stage of UC has not as yet been investigated in a randomized placebo-controlled study.

We designed and conducted a randomized placebo-controlled clinical trial to determine whether the remission of active mild to moderate UC could be achieved by altering the intestinal flora through supplementation with BFM as a dietary adjunct in combination with a standard medication such as mesalazine (5-ASA) or salazosulphapyridine (SASP).

Study subjects

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Subjects were recruited from among out-patients and hospitalized patients, seen at our facility and other hospitals, who had been diagnosed with active UC. The diagnosis of active UC was confirmed by characteristic clinical, endoscopic and histological features. Patients receiving topical or systemic steroids or antibiotics within the preceding 4 weeks were excluded. Approval for the trial was granted by our University Hospital's ethics committee and all participants gave informed written consent before enrolment. All subjects were enrolled consecutively between September 2001 and March 2003. Of the 21 patients invited to participate, informed consent was obtained from 20. The reason for the refusal of one was an unwillingness to participate in a clinical trial. The 20 subjects were then randomly assigned to either the group (n = 10) receiving BFM supplementation (BFM group), or the control group (n = 10) receiving a placebo (placebo group), using a random number table. Blood was collected at the time of recruitment and after 3 months, for blood counts and biochemistry.

Clinical characteristics of the BFM and placebo groups are presented in Table 1. The age distribution and mean disease duration were similar in the two groups. All 20 patients had active mild to moderate UC (eight mild and 12 moderate) according to the criteria of Truelove and Witts.20 No significant differences were found between the two groups in age, gender, extent of disease, disease severity, disease duration or treatment. Four patients in the BFM and three in the placebo group with recurrence had taken low-dose prednisolone and had then discontinued them at least 1 year before starting this trial.

Table 1.  Baseline characteristics of the patients
 BFM group (n = 10)Placebo group (n = 10)P
  1. BFM, bifidobacteria-fermented milk; UC, ulcerative colitis; SASP, salazosulphapyridine; N.S., not significant.

Mean age (year)30.233.7N.S.
Sex (male:female)5:55:5N.S.
Clinical pattern of UCFirst: 4, relapsing: 5, chronic: 1First: 5, relapsing: 4, chronic: 1N.S.
Disease extent (n)Total: 5, left-sided: 3, proctitis: 2Total: 3, left-sided: 4, proctitis: 3N.S.
Disease severity (n)Mild: 4; moderate: 6Mild: 4; moderate: 6N.S.
Disease duration (year, mean) (range)3.4 (0.3–12)3.8 (0.3–12)N.S.
Treatment5-ASA: 9; SASP 15-ASA: 9; SASP 1N.S.
Previous steroid use43N.S.

Medications

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Treatment for UC was provided on the usual clinical grounds, using SASP (500 mg tablet, 3–4 g/day) or 5-ASA (250 mg tablet, 2250–3000 mg/day). Standard doses of these medications had been given for at least months before enrolment in the study. The dose and delivery of these agents subsequently remained stable throughout the study period. No patients in either the BFM or the control group were treated with steroids during the study period. All patients in both groups were instructed not to ingest any other fermented milk products during this time. No dietary alterations were made once the patients entered the study. The commercially available BFM product was provided by Yakult Co., Ltd. (Tokyo, Japan) and contained live bacteria of the Bifidobacterium breve strain Yakult, Bifidobacterium bifidum strain Yakult and a Lactobacillus acidophillus strain, numbering at least 10 billion per 100 mL bottle. This product was delivered to the homes of BFM group members, who were instructed to drink 100 mL each day for 12 weeks. The BFM placebo product without the above live bacteria, which was also manufactured by Yakult Co., Ltd., was delivered to the homes of placebo group members. There was no difference in either taste or appearance between the two preparations.

Study evaluation

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

At out-patient appointments every 4 weeks, compliance with medications and BFM and placebo products in both groups were checked by questioning by doctors and through questionnaires. Subjects were examined as to items relevant to the clinical activity index (CAI). All patients confirmed good compliance with ingestion of the BFM product throughout the test period (12 weeks). Good compliance with ingestion was defined as >95% consumption of BFM products.

Clinical activity index scores, which have been shown to reflect changes in the clinical status of patients with UC, are based on a sum of the following: (i) number of episodes of diarrhoea (0–4); (ii) presence of nocturnal diarrhoea (0–1); (iii) degree of visible blood in stool (0–3); (iv) presence of faecal incontinence (0–1); (v) degree of abdominal pain or cramping (0–3); (vi) general well-being (0–5); (vii) degree of abdominal tenderness (0–3); and (viii) need for anti-diarrhoeal drugs (0–1).21 The overall CAI score can range from 0 to 21. The score rises as symptoms worsen. This index was determined at baseline and thereafter by attending physicians. The primary efficacy endpoint was clinical improvement at 12 weeks, as indicated by a decrease in the CAI score of at least three points.

Colonoscopic examinations were performed before and after the treatment period and endoscopic mucosal biopsies were taken from several areas including those appearing to have the most severe inflammation. Each of five variables, erythema, oedema, friability, granularity and erosions, was scored as absent (grade 0), mild (1) or severe (2). The sum of the scores was taken as the endoscopic activity index score with a range of 0–10.22, 23 The endoscopic activity index score was first determined at the time of the procedure, based on a review of endoscopic photographs taken by experienced endoscopists, blinded to the mode of treatment. All endoscopists had been certified by the Japanese Society of Gastroenterological Endoscopy.

Histopathological inflammatory activity was assessed before and 12 weeks after starting treatment with at least four biopsy specimens taken from actively inflamed mucosa, including a rectal biopsy, with the most severe inflammation being documented by pathologists who had no knowledge of the treatment. The histopathological appearance was, as previously described by Matts, scored as follows: l, normal appearance, 2, some infiltration of the mucosa or lamina propria with either round cells or polymorphs, 3, much cellular infiltration of the mucosa, lamina propria and submucosa, 4, presence of crypt abscesses, with much infiltration of all layers of the mucosa and 5, ulceration, erosion, or necrosis of the mucosa, with cellular infiltration of some or all of its layers.24

Remission was defined as absence of rectal bleeding, a rectal mucosa without erythema, granularity, or friability and normal or near-normal sigmoidoscopic findings.15

Analysis of organic acid in faeces

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Short-chain fatty acids (SCFAs) such as butyrate, propionic and acetic acids are the main products of microbial breakdown of carbohydrates in the large intestine and provide energy to colonocytes. SCFA metabolism is necessary for salt and water absorption by the colonic mucosa. Topical butyrate has been shown to be an effective treatment for UC. Previous studies has shown that prebiotic, germinated barley food stuff (GBF) increased stool butyrate concentrations.25 Thus, in order to evaluate the effect of BFM supplementation in luminal SCFA production, we measured faecal SCFA contents.

Stool specimens were collected from the subjects immediately after recruitment and again after 12 weeks. SCFAs were assayed, as described previously.19 Briefly, subjects were asked to put faeces into a plastic tube upon defecation, then immediately place this in an ice-cooled sealed container. A portion of the stool collected was deproteinated with perchloric acid and used for analysis of SCFAs. The faecal SCFA content was measured as follows: the sample was deproteinized and then centrifuged and the supernatant was analysed using high-performance liquid chromatography. The column used was a Shodex KC-811 (x2) (Showa Electronics, Tokyo, Japan) with an internal standard.

Microbiological analysis of faeces

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Recovery of the probiotic strains B. breve and B. bifidum was estimated by culturing on T-CBPC (transglycosylated oligosaccharide agar supplemented with 5 mg/mL of streptomycin sulphate and 1 µg/mL carbenicillin) and T-LCM (transglycosylated oligosaccharide agar supplemented with 5 mg/mL of streptomycin sulphate and 2 µg/mL lincomycin), respectively, and confirmed with monoclonal antibodies to each strain. Numbers of Bacteroides and Bifidobacterium species were estimated based on bacterial DNA in the faeces as described previously.26–28 In brief, the bacterial suspension was obtained by centrifugation of the faeces after repeated washing with 10 volumes of phosphate-buffered saline (PBS) using glass beads (diameter 2.5 mm) (Biospec Products Inc., Bartlesville, OK, USA) within 24 h after sample collection. The washed bacterial suspension was kept under −75°C until DNA extraction. DNA was extracted from the bacterial suspension with phenol by shaking in FastPrep FP120 (Qbiogene Inc., Carlsbad, CA, USA) for 30 s with the aid of glass beads (diameter 0.1 mm) (Biospec Products Inc). Then, the supernatant containing bacterial DNA was partially purified with isopropanol and ethanol. The numbers of Bacteroides and Bifidobacterium species were estimated by the real-time polymerase chain reaction (PCR) method using species-specific primer sets and a standard strain of each species as described elsewhere.26–28

Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Results were expressed as mean ± S.E.M. The Wilcoxon signed rank test for paired samples was used to compare differences within treatment groups and the Mann–Whitney U-test was used to compare differences between groups. The Friedman non-parametric repeated-measures test was used for comparisons among the three groups, as appropriate. For all studies, an associated probability (P-value) of <0.05 was considered statistically significant.

Participation

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

All 10 patients in the BFM group and nine of 10 in the placebo group completed the 12-week study. These patients were all able to continue consuming the BFM and placebo drinks with 5-ASA or SASP. One member of the placebo group withdrew, because of worsening symptoms 2 days after the start of the trial. Compliance was satisfactory overall. Administrations of BFM and the placebo were well tolerated and no subjects reported adverse events that might have been related to BFM or placebo supplementation. There were no significant changes in routine biochemical data or urine analyses after treatment in either the BFM or the placebo supplementation group. There was a slight improvement in the inflammatory reaction [decreased C-reactive protein and erythrocyte sedimentation rate (ESR)], but it did not reach statistical significance.

Response to therapy and clinical activity index score

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

The mean CAI score was 7.9 ± 0.8 in the BFM and 7.9 ± 0.6 in the placebo group at the start of the study and not significantly different.

A response-to-treatment was defined as a decrease in the CAI score of at least three points. Seven of 10 patients (70%) in the BFM group responded and clinical remission was achieved in 40% (4/10). In contrast, three of nine (33%) patients in the placebo group responded, as defined by the treatment regimen and clinical remission was achieved in 33% (3/9).

The average CAI score in the BFM group had decreased to 5.1 ± 0.5 by 4 weeks, and had significantly decreased to 4.1 ± 0.4 by 8 weeks (P < 0.05) and to 3.7 ± 0.4 by 12 weeks (P < 0.001) after starting BFM supplementation (Figure 1a). In the placebo group, the CAI score had decreased to 6.1 ± 0.4 by 4 weeks, had significantly decreased to 5.7 ± 0.7 by 8 weeks (P < 0.05) and was still significantly decreased with a value of 5.8 ± 0.8 at 12 weeks (P < 0.05) (Figure 1b). The CAI score in the BFM group was significantly lower than that of the placebo group at 12 weeks (P < 0.05).

image

Figure 1. Clinical activity index scores at 0, 4, 8 and 12 weeks of treatment with mesalazine/salazosulphapyridine (5-ASA/SASP) and bifidobacteria-fermented milk (a) or 5-ASA/SASP and placebo supplementation (b). The mean clinical activity index score (CAI) of patients receiving standard treatment and bifidobacteria-fermented milk (BFM) for 8 and 12 weeks was significantly decreased when compared with before treatment (n = 10, *P < 0.05, **P < 0.001 respectively). The mean CAI score of patients receiving standard treatment and the placebo for 8 and 12 weeks was also significantly decreased when compared with before treatment (n = 9, P < 0.05*). *Friedman non-parametric repeated-measures test.

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Endoscopic and histological results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Figure 2a shows changes in the endoscopic activity index score demonstrating a mild but statistically significant reduction in the BFM group. The average score was significantly decreased from 5.1 ± 0.4 to 3.0 ± 0.5 after 12 weeks of treatment with BFM (P < 0.01). In contrast, there was no significant difference between the values before vs. 12 weeks after starting treatment in the placebo group, although the endoscopic activity index score did decrease from 4.9 ± 0.4 to 3.6 ± 0.7 (Figure 2b). The endoscopic activity index score in the BFM group was not significantly different from that of the placebo group at 12 weeks.

image

Figure 2. Endoscopic activity index scores before and 12 weeks after treatment with mesalazine/salazosulphapyridine (5-ASA/SASP) and bifidobacteria-fermented milk (a) or 5-ASA/SASP and placebo supplementation (b). The mean endoscopic activity index score of patients receiving standard treatment and bifidobacteria-fermented milk for 12 weeks was significantly decreased as compared with before treatment (n = 10, P < 0.01*). There was no significant difference before vs. after 12 weeks of treatment in the placebo group (n = 9). *Wilcoxon signed rank test.

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The histological score in the BFM group was also significantly reduced from 4.4 ± 0.3 to 3.1 ± 0.3 after 12 weeks of treatment (P < 0.01). In contrast, there was no significant difference between the values before vs. 12 weeks after starting treatment in the placebo group, although the score did decrease from 3.6 ± 0.3 to 3.0 ± 0.4.

Organic acid constituents of the faeces

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Relative proportions of faecal concentrations of SCFAs in 18 UC patients, whose faeces could be collected at the commencement and completion of the study, are shown in Table 2. One patient in the placebo group was excluded from this evaluation because faeces could not be satisfactorily obtained. There were no differences in SCFA concentrations except for propionate between the placebo and BFM groups at commencement. A significant increase in total SCFAs was seen in the BFM group after 12 weeks of BFM supplementation (P < 0.05), with marked increases in butyrate and propionate (P < 0.05). There were no changes in either individual concentrations or total SCFAs in the placebo group.

Table 2.  Faecal concentrations of organic acids in ulcerative colitis patients before and after bifidobacteria-fermented milk or placebo supplementation for 12 weeks
Organic acid (mm)BFMPlacebo
BeforeAfterBeforeAfter
  1. BFM, bifidobacteria-fermented milk; SCFA, short chain fatty acid.

  2. * Values indicate the average (±S.E.) in BFM (n = 10) or placebo group (n = 8).

  3. † Significantly different, P < 0.05 by Wilcoxon signed rank test.

Succinic10.2 ± 5.9*0.2 ± 0.220.8 ± 14.74.1 ± 2.3
Lactic1.4 ± 1.00.4 ± 0.30.9 ± 0.63.2 ± 2.2
Acetic50.1 ± 8.173.3 ± 8.077.6 ± 14.782.6 ± 11.4
Propionic10.3 ± 2.319.8 ± 2.4†23.8 ± 4.320.7 ± 3.1
Butyric5.4 ± 2.115.1 ± 3.6†9.8 ± 3.49.3 ± 3.0
Total SCFA65.5 ± 11.1108.1 ± 12.2†111.2 ± 22.4112.5 ± 15.4

In addition, there were no significant differences in butyrate and propionate concentrations at 12 weeks between the two groups, although the butyrate concentration in the BFM group was higher than that in the placebo group.

Bacterial analysis of faeces

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

Recoveries of probiotic strains, B. breve and B. bifidum, after daily intake of a 100 mL bottle of BFM were 6.8 ± 1.1 colony-forming units (cfu)/g faeces and 7.9 ± 0.7 cfu/g faeces respectively. The faecal numbers of B. breve and B. pseudocatenulatum among Bifidobacterial species were significantly increased in the BFM group (P < 0.05), but not in the placebo group. The faecal numbers of other Bifidobacterial species such as B. longum were not significantly changed in either group (Figure 3). While the number of B. fragilis species among Bacteroidaceae tended to be reduced in the BFM group, the change was not statistically significant (Figure 4). There were no significant differences in faecal numbers of other Bacteroides species, such as B. ovatus, B.vulgatus, B. eggerthii, B. thetaiotaomicron and B.uniformis.

image

Figure 3. Bacterial numbers for each Bifidobacterium species before (open bar) and after (closed bar) bifidobacteria-fermented milk (BFM) or placebo supplementation. Bacterial number was determined by the real-time polymerase chain reaction method. The logarithmic value of the average of the BFM (n = 10) or placebo (n = 8) group is presented. Bifidobacteriumbreve and B. pseudocatenulatum were significantly increased in the BFM group after 12 weeks of BFM ingestion (*P < 0.05; Wilcoxon signed rank test).

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image

Figure 4. Bacterial numbers for each Bacteroides species before (open bar) and after (closed bar) bifidobacteria-fermented milk (BFM) or placebo supplementation. Bacterial number was determined by the real-time polymerase chain reaction method as described in the text. The logarithmic value of the average of the BFM (n = 10) or placebo (n = 8) group is presented.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

The results of our present randomized placebo-controlled clinical trial suggest that BFM supplementation is effective, well tolerated and safe for the treatment of active UC at a mild to moderate stage. BFM supplementation with 5-ASA/SASP treatment resulted in significant improvement of CAI, the endoscopic activity index and the histological score. While the endoscopic activity index and histological score were not significantly decreased in the placebo group, a slight but significant improvement in the CAI score was noted. A previous randomized controlled study using BFM as maintenance treatment in the remission stage of UC indicated its possible preventive effects against relapse of UC.19 The results of the present randomized placebo-controlled study strongly suggest that BFM supplementation also effectively ameliorates symptoms in patients with active UC, although the clinical remission ratio as that defined by remission of symptoms such as rectal bleeding and endoscopic findings did not differ between the BFM and placebo groups at 12 weeks.

The efficacy of this probiotic preparation may be related to the increased concentrations of faecal (luminal) SCFAs. Probiotic bacteria can improve epithelial function via production of SCFA.29 In this study, total SCFA concentrations, and butyric and propionic acids in faeces were significantly increased after BFM supplementation, the observations being consistent with those of a study on prebiotics published previously.25 SCFAs, particularly butyrate, are the major energy source for colonocytes and appear to function in immunological regulation including the suppression of proinflammatory cytokines through inhibition of NF-κB activation.30 Colonocytes isolated from patients with UC were shown to have impaired butyrate utilization when compared with healthy controls.31 Butyrate or SCFA enemas are reportedly effective in the treatment of refractory distal UC.32 Therefore, enhancement of butyrate production via BFM supplementation is considered to be efficacious and may be related to the significant improvements in CAI, the endoscopic activity index and histological scores. However, as the concentrations of SCFAs including that of propionic acid at 12 weeks were similar in the two groups, the results do not fully support this explanation. A larger number of subjects are required to assess this possibility.

The efficacy of this probiotic preparation may also be related to increased concentrations of some species of protective bacteria such as B. breve and B. pseudocatenulatum, as shown by the faecal microbiologic data of the BFM group following BFM consumption. Other successful clinical trials have shown normalization of intestinal flora with administration of B.breve preparations, giving the same strain as in our BFM to preterm infants or B. bifidum and Streptococcus thermophilus preparations to patients with diarrhoea.33, 34 On the contrary, Bacteroides species such as B. vulgatus in the colonic mucosa or blood levels of antibodies are often reportedly increased in patients with UC.35,6 Although no significant changes in Bacteroidaceae were identified in the present study, a previous study in which BFM supplementation was provided for 1 year showed a significant decrease in the relative percentage of B. vulgatus among the Bacteroidaceae.19 These observations raise the possibility that one of the mechanisms underlying the efficacy of this BFM supplement is a reduction in B. vulgatus, although it may be necessary that BFM be consumed for a more than 3 months to siginificantly reduce B. vulgatus. In dextran sulphate-treated gnotobiotic mice carrying Bacteroides strains isolated from UC patients, Bifidobacterium strains appear to suppress exacerbation of intestinal inflammation by inhibiting growth of the B. vulgatus strain.36

An important advantage of BFM supplementation in combination with 5-ASA or SASP is the lack of side-effects and toxicity. We have observed no significant changes in routine blood chemistry or urinalysis results and no allergic symptoms. As described above, a previous study using this BFM for 1 year in addition to conventional treatment also demonstrated efficacy and safety. These data reflect the fact that BFM has long been used clinically in Japan as a probiotic product. Therefore, BFM supplementation in addition to a conventional treatment such as 5-ASA appears to be well tolerated by patients, regardless of whether UC is in the active or the remission stage.

In conclusion, BFM significantly improved CAI, the endoscopic activity index and histological scores in patients with mild to moderate active UC when compared with those receiving a BFM placebo. In addition, we observed increases in probiotic strains and butyrate in the faeces. These preliminary results encourage us to conduct a multi-centred double-blind randomized clinical trial with more subjects to determine which ingredients of this BFM product are effective and their mechanisms of action.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References

The authors thank Ms H. Setoyama and Ms T. Hara for the analysis of intestinal flora. We thank Ms S. Komuro, Ms E. Murata and Mr K. Takahashi for dealing with patients and Dr B. Barfod for assistance in preparation of the manuscript. Mr T. Kimura, Mr M. Hayasaka and Ms M. Yagoshi are also appreciated for their assistance with the randomized controlled trial.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Study subjects
  6. Medications
  7. Study evaluation
  8. Analysis of organic acid in faeces
  9. Microbiological analysis of faeces
  10. Statistical analysis
  11. Results
  12. Participation
  13. Response to therapy and clinical activity index score
  14. Endoscopic and histological results
  15. Organic acid constituents of the faeces
  16. Bacterial analysis of faeces
  17. Discussion
  18. Acknowledgements
  19. References
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