Alimentary Pharmacology & Therapeutics

Clinical trial: the effects of a fermented milk containing three probiotic bacteria in patients with irritable bowel syndrome – a randomized, double-blind, controlled study M. SIMRE´ N*, L. O¨ HMAN*, J. OLSSON , U. SVENSSONà, K. OHLSONà, I. POSSERU D* & H. STRID*

*Department of Internal Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden;  KPL Good Food Practice, Uppsala, Sweden; àArla Foods Innovation, Stockholm, Sweden Correspondence to: Dr M. Simre´n, Department of Internal Medicine, Sahlgrenska University Hospital, S-41345 Gothenburg, Sweden. E-mail: [email protected]

Publication data Submitted 23 September 2009 First decision 4 October 2009 Resubmitted 14 October 2009 Accepted 22 October 2009 Epub Accepted Article 26 October 2009

SUMMARY Background The effects of probiotic bacteria in IBS remain controversial. Aim To study the effects of a probiotic product on IBS symptoms. Methods We randomized 74 IBS patients to receive 8 weeks of daily treatment with 400 mL milk fermented with the yoghurt bacteria and containing Lactobacillus paracasei, ssp. paracasei F19, Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 (Cultura; active) or acidified milk without these bacteria (control). The primary endpoint was the proportion of subjects reporting adequate relief of their IBS symptoms at least 50% of the weeks. IBS symptom severity, psychological symptoms and quality of life were assessed. Results The proportion of responders was 38% (14 ⁄ 37 patients) in the active group and 27% (10 ⁄ 37 patients) in the control group (P = 0.3). IBS symptom severity improved significantly in both groups during the treatment period. This change was greater in the active group during the first 2 weeks, but thereafter, no significant group differences were seen. Conclusions We could not detect a clearly positive effect of fermented milk containing three probiotic bacteria on GI symptoms in IBS patients compared with the control treatment. However, a trend towards a more favourable effect during the first weeks was seen in the active group. Aliment Pharmacol Ther 31, 218–227

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ª 2010 Blackwell Publishing Ltd doi:10.1111/j.1365-2036.2009.04183.x

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BACKGROUND Irritable bowel syndrome (IBS) is characterized by pain and abnormal bowel habits (diarrhoea and ⁄ or constipation).1 IBS is common in the western world and affects 10–20% of the population, but the causes of the disorder are not well known. The symptoms are often severe and no effective treatment treating all the IBS symptoms exists.2 Different pathophysiological alterations have been proposed to be of relevance for IBS, such as visceral hypersensitivity, disturbed gastrointestinal (GI) motility and secretion, altered central processing of signals emanating from the gut and psychosocial factors.3 Recent data also suggest the presence of low-grade inflammation within the GI tract of IBS patients, as well as alterations in the immune system, both in the gut and in the blood.4 Also, abnormal colonic fermentation5 because of altered bacterial flora in the colon might be of relevance to the symptoms of IBS patients, as well as minor alterations in the bacterial flora of the small intestine.6 Recently, several studies have implicated a positive effect of treatment with probiotic bacteria in IBS patients. For instance, a 4 week treatment with Lactobacillus plantarum demonstrated a positive effect on overall symptoms as well as gas-related symptoms in a mixed group of IBS patients7 and a positive effect on GI symptoms was also demonstrated in another trial using Bifidobacterium infantis.8 However, in this study, no effect could be detected after treatment with Lactobacillus salivarius. Moreover, there are also other more or less negative trials, where the probiotic used has had minor or no effect on symptoms.9, 10 Therefore, it is fair to say that at this stage, the role of probiotic treatment for patients with IBS remains unclear. In the present study, we examined the symptomatic effect in patients with IBS of milk fermented with the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus and containing at least 5 · 107 cfu ⁄ mL of three different probiotic bacteria, Lactobacillus paracasei, ssp. paracasei F19, Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 (Cultura; active treatment) or acidified milk without these bacteria (control treatment), comparable in colour, texture and taste.

METHODS We included patients with IBS according to the Rome II criteria11 recruited from a pool of IBS patients at the Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

Table 1. Inclusion and exclusion criteria Inclusion criteria 1 Signed written informed consent 2 Age ‡18 and £70 years at baseline visit 3 IBS according to the Rome II criteria 4 Ability to understand and willingness to comply to the study procedures Exclusion criteria 1 Participation in another clinical study 1 month prior to screening visit and throughout the study 2 Abnormal results on the screening laboratory tests clinically relevant for study participation 3 Other gastrointestinal disease(s) explaining the patient’s symptoms, as judged by the investigator 4 Other severe disease(s) such as malignancy, severe heart disease, kidney disease or neurological disease 5 Symptoms indicating other severe disease(s) such as gastrointestinal bleeding, weight loss or fever 6 Severe psychiatric disease 7 Previous history of drug or alcohol abuse 6 months prior to screening 8 Intolerance or allergy against milk products or gluten 9 Use of other probiotic products (according to sponsor’s list) 2 weeks prior to the study and throughout the study 10 Consumption of antibiotics 1 month prior to screening and throughout the study 11 Consumption of cortisone, NSAID or other anti-inflammatory drugs on a regular basis 2 weeks prior to screening and throughout the study 12 Pregnant or lactating or wish to become pregnant during the period of the study

unit, as well as from consecutive referrals from primary care physicians. The first patient entered the trial in September 2005 and the study was completed in October 2006. The patients were evaluated by a gastroenterologist (IP, HS, MS) at a screening visit for eligibility in the study based on inclusion and exclusion criteria (Table 1). Thereafter, the patients entered a two-weeks screening period (Figure 1). At the screenRandomization Active treatment Follow up

Screening and Run-in

Placebo treatment –2

0

2

4

6

8

16

V1

V2

V3

V4

V5

V6

V7

Time (weeks)

Figure 1. Schematic presentation of the study design.

220 M . S I M R E´ N et al.

ing visit, the patients received questionnaires to complete during the screening period to assess GI and psychological symptom severity, as well as quality of life (see below) and blood samples for biochemistry and haematology tests were taken. After the screening period, the patients returned for a second visit and entered the treatment period if they fulfilled the randomization criteria (see below) and still fulfilled all inclusion criteria and had no exclusion criteria (Table 1). Patients were then randomized to receive either active treatment with milk fermented with the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus and containing at least 5 · 107 cfu ⁄ mL of three different probiotic bacteria (Lactobacillus paracasei, ssp. paracasei F19, Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 (Cultura; active), or a control treatment with acidified milk without these bacteria, comparable in colour, texture and taste (as assessed by the study team before the start of the study). Hundred grams of the products (approximately 100 mL) contains 200 kJ ⁄ 50 kcal (protein 4%, carbohydrates 4%, fat 1.5%). The patients were instructed to ingest 200 mL of the investigational product twice daily, preferably in the morning and in the evening. The treatment period lasted 8 weeks during which symptoms were evaluated using self-administered questionnaires. During this period, the patients made short visits to our study unit every 2 weeks to assess compliance with the investigational product, hand in the evaluation forms for the previous 2 weeks and to receive new evaluation forms and the investigational product for the coming 2 weeks. Following the treatment period, the patients were followed up for another 8 weeks regarding symptom evolution, by completing symptom questionnaires and was contacted again 8 weeks after the treatment period over the phone to remind the patients to return the questionnaires completed during the follow-up period by mail and to check for adverse events. Signed informed consent was obtained from each subject before entering the study. The study protocol was approved by the Ethics Committee of the University of Gothenburg.

Questionnaires Gastrointestinal and extraintestinal symptoms. The patients were instructed to complete a questionnaire daily during the study, assessing the severity of five

different symptoms (bloating, flatulence, abdominal discomfort, nausea and abdominal pain) on a scale ranging from 0 (no symptoms) to 3 (severe symptoms) (GI symptom questionnaire), as well as the stool form using the Bristol Stool Form Scale.12 On a weekly basis, the patients also completed the IBS Symptom Severity Index (IBS SSI),13 which is a widely used questionnaire divided into two categories measuring IBS related (pain frequency, severity of pain, bloating, bowel habit dissatisfaction and interference with daily life) and extra-colonic symptoms on a visual analogue scale. For both these categories, scores ranging from 0 (no symptoms) to 500 (maximum severity) can be obtained. The patients also responded to a weekly question whether they had had adequate relief of their IBS symptoms during the preceding week or not.14

Psychological symptoms. At the randomization visit and at the last day of the treatment phase, the patients completed the Hospital Anxiety and Depression (HAD) scale, which is a self-assessment mood scale developed for use in a clinical setting to identify patients with psychiatric distress.15 It has been extensively validated and is one of the most widely used brief inventories for assessment of anxiety and depression.16 There are seven items each for anxiety and depression. It uses a 4-point Likert scale (0–3) which provides a minimum score of 0 and a maximum score of 21 on each sub-scale.

Quality of life. At the randomization visit and at the last day of the treatment phase, the patients also completed the Irritable Bowel Syndrome Quality of Life Questionnaire (IBSQOL). The IBSQOL is a recently developed disease-specific QOL instrument designed to measure nine domains found to be of relevance for patients with IBS: emotional health, mental health, sleep, energy, physical functioning, food ⁄ diet, social functioning, physical role and sexual relations.17 It has been shown to be valid,17, 18 reliable18 and responsive to change.19 The questionnaire was scored according to recommendations from the developers17 and each scale score was transformed to a scale of 0–100, with 100 representing the best possible QOL.

Diet registration. The subjects recorded all the food they had eaten, except the test products, during two weekdays and one weekend day in the Menybok, a standardized form developed and evaluated by the Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

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National Food Administration in Sweden (Livsmedelverket). This was performed once during the screening period and during the end of the treatment period. Nutrients were calculated using dietary analysis software (Stor MATs Rudans La¨ttdata, Va¨stera˚s, Sweden), which is based on the Swedish national nutrient composition database.

Randomization and blinding At the randomization visit following the 2-weeks screening period, inclusion and exclusion criteria were again reviewed by one of the three gastroenterologists involved in the study (HS, IP, MS). Based on the IBS SSI form and the response to the adequate relief question, it was decided by a study nurse if the patient fulfilled the criteria for randomization. The patient was judged to be eligible for randomization, if he or she had VAS >40 on at least one of the five questions of IBS SSI and had not reported adequate relief of their IBS symptoms the week preceding the randomization visit. If so, the patient then received one of the two treatment options according to a randomization list delivered by the statistical department of Uppsala Clinical Research, Uppsala University, Sweden. Concealed allocation was assured by the distribution of investigational products through an organization within Arla Foods, otherwise not involved in the study. The randomization schedule was kept at Arla Foods until the completion of the trial and all the data were entered into a computer data base. Patient data and results from questionnaires were entered into the database by dedicated people at KPL Good Food Practice, otherwise not involved in the conduct of the study. Thus, investigators and participants were blinded to treatment arm assignments.

Compliance The subject reported the daily consumption of the study product in a study diary. The compliance was calculated as percentage of planned ingestion of the study product, and a compliance rate above 80% was set as a minimum requirement to be regarded as acceptable. Moreover, as a second evaluation of compliance with the study product, the presence and number of Lactobacillus paracasei, ssp. paracasei F19 in faecal samples were analysed at the end of the treatment period on Rogos (Merck Ltd). Colonies were isolated and the presence of the probiotic strain was confirmed by randomly amplified polymorphic DNA (RAPD)-PCR using the primers LBC -19 for Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

screening and OPA-02 for confirmation of Lactobacillus paracasei, ssp. paracasei F19.

Statistical analyses The primary endpoint of this study was the proportion of patients reporting adequate relief of their IBS symptoms at least 50% of the weeks during the treatment period (‘responders’). The analysis for the primary endpoint was based on the intention-to-treat paradigm with drop-outs counted as nonresponders. As secondary endpoints, the effect of the investigational products on GI and extraintestinal symptoms assessed with the weekly IBS SSI questionnaire and the scores from the daily questions of the GI symptom questionnaire (average weekly score),20 as well as the effect on psychological symptoms (HAD scale) and quality of life (IBS-QOL) were analysed. For these variables, both between- and within-group comparisons were made for exploratory reasons. We also evaluated the proportion of responders on the basis of adequate relief question weeks 1–4 and 5–8 during the treatment period to see if there was a time effect of the product. The data are presented as mean  s.d. unless otherwise stated. Categorical variables were compared with the Chi squared test, whereas continuous variables were compared with non-parametric tests, the Wilcoxon signed rank test for paired comparisons and the Wilcoxon rank sum test for unpaired comparisons. All the hypotheses tested were two-sided and statistical significance was accepted at the 5% level. No adjustments for multiple comparisons were made. Our planned sample size calculated for this study (n = 70) was based on the intention to be able to detect a 30% difference in the proportion of responders in the treatment arms with 80% power at a = 0.05, using a two-sided chi-square test, assuming a 15% placebo response. Knowing that this was a very optimisitc calculation based on differences between placebo and active treatment arms in previous clinical trials, we also planned to perform sample size calculations for future studies based on the proportion of responders in our treatment groups.

RESULTS Subjects One hundred eighteen patients entered the screening phase of the study (mean age 44 years; 80 females)

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118 screened

44 screening failures 74 randomized

37 Active

37 Control

4 drop-outs

33 completed the study

3 drop-outs

34 completed the study

(Figure 2). Of these, 74 subjects (mean age 42  16 years; 52 females) were randomized and entered the treatment phase. The main reasons for not being randomized were that the patient reported adequate relief of their IBS symptoms during the last week of the screening phase or had too mild symptoms, i.e. not VAS >40 on any of the five GI questions of IBS SSI (n = 34). A small number of subjects also withdrew their informed consent before the treatment phase (n = 10) for reasons of factors unrelated to the study. There were no significant differences between screening failures and the patients that were randomized regarding age, gender or IBS subgroup, but the patients who were randomized reported somewhat more severe IBS symptoms (IBS-SSI score: 270  96 vs. 209  109; P < 0.05). Thirty-seven patients were randomized to receive the active treatment and 37 to the control group. There were no significant differences in baseline characteristics between the groups (Table 2). Thirty-three patients in the active group and 34 in the control group completed the trial and seven dropped out prematurely during the treatment phase because of lack of effect of the treatment (n = 5) or factors unrelated to the study (n = 2) (Figure 2). The yoghurt was well tolerated and no adverse events were reported by the patients. No clinically significant effects on biochemistry or haematology were noted. The compliance was >95% in both groups as analysed from the study diaries. The high compliance was confirmed by the faecal analyses, where Lactobacillus paracasei, ssp. paracasei F19 could be detected in 88% of the participants in the active treatment group. The results from the background diet registration showed that patients in the two groups consumed the

Figure 2. Flow chart demonstrating the number of patients in the different phases of the study.

Table 2. Baseline characteristics of the randomized patients Active (n = 37) Females, n (%) Age, mean  s.d. Predominant bowel habit Diarrhoea, n (%) Constipation, n (%) Mixed, n (%) IBS symptom severity (IBS SSI), mean  s.d. IBS SSI extracolonic symptoms, mean  s.d. HAD anxiety score, mean  s.d. HAD depression score, mean  s.d.

Control (n = 37)

26 (70) 42  15

26 (70) 44  16

15 (41) 5 (13) 17 (46) 256  79

11 (30) 6 (16) 20 (54) 298  96

138  71

127  77

6.5  3.7

7.9  2.9

3.6  3.1

4.0  3.1

same amount of energy and macro nutrients such as fat, carbohydrate and proteins during the screening period (data not shown). Both groups decreased their energy intake with 10–15% during the study which corresponded to the energy intake from the study products.

Gastrointestinal and extraintestinal symptoms The number of patients meeting the primary endpoint, i.e. adequate relief at least 50% of the weeks during the treatment phase was numerically higher in the active vs. the placebo group, but the difference was not statistically significant [14 ⁄ 37 (38%) vs. 10 ⁄ 37 Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

45

450

40

400

35

350

IBS SSI score

Proportion with adequate relief (%)

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30 25 20

*

**

*

**

*

**

**

**

**

**

**

**

**

**

**

300 250 200 150

15

100

10

**

50

5

0 Baseline W1 W2 W3 W4 W5 W6 W7 W8 FU Active (n = 37) Control (n = 37)

0 W2 W3 W4 Active (n = 37)

W5 W6 W7 Control (n = 37)

W8

Figure 3. Proportion of subjects reporting adequate relief of IBS symptoms each week during the treatment period.

(27%) patients; P = 0.3]. As can be seen from Figure 3, this numerical difference seems to relate primarily to a larger proportion of responders in the active group during the first weeks of the treatment period (N.S.). A power calculation, using these responder data, demonstrated that it would have been necessary to include 284 patients in each treatment group to detect a difference of 11% in the proportion of responders with 80% power at a = 0.05. Compared with baseline, both groups demonstrated significant reductions of their IBS SSI scores during the entire treatment period, as well as at the follow-up visit in the control group, but not in the active treatment group (Figure 4). The change in GI symptom severity assessed with IBS SSI was similar in both groups except for a larger improvement in the active group during the first treatment week (P < 0.05). At the end of the treatment, the reduction in IBS SSI did not differ statistically between the groups (P = 0.24). (Figure 5). When evaluating the five different GI questions of the IBS SSI separately (pain frequency, pain severity, bloating severity, satisfaction with bowel habits, interference with daily life) no significant differences between the groups could be detected, even though a positive treatment effect at the end of the treatment period compared with baseline could be detected in both groups (Table 3). The extracolonic score of IBS SSI, assessing symptoms such as backache, headache, palpitations etc., was reduced at the end of the treatment period compared with baseline, but did not differ between the groups (Table 3). Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

Figure 4. The overall GI symptom severity, assessed by IBS-SSI. FU = follow-up 8 weeks after the end of the treatment period. Mean, s.d. *P < 0.05 **P < 0.01 vs. baseline.

0 IBS SSI Change from baseline

W1

W1

W2

W3

W4

W5

W6

W7

W8

FU

–20 –40 –60 –80 –100 –120

*

–140 –160 –180 Active (n = 37)

Control (n = 37)

Figure 5. Change in IBS SSI from baseline. *P < 0.05 vs. the control group.

The symptom scores for the five GI symptoms of the GI symptom questionnaire were added and a weekly total score was obtained.20 The baseline data were similar in both groups. The active treatment group had a prompt response and demonstrated a significant improvement already at week 1 and this improvement was sustained for the remainder of the study, including the follow-up visit. In the control treatment group, a significant improvement of the symptom severity was obtained from week 4 and onwards (Figure 6). The change in GI symptoms relative to baseline was significantly greater in the active treatment group

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Table 3. IBS symptom severity index – IBS related and extracolonic symptoms – at baseline and at the end of the treatment period (mean  s.d.) Active (n = 37) End of treatment period

Baseline Pain severity Pain Frequency Bloating severity Satisfaction with bowel habits Interference with daily life Extracolonic symptoms

36 50 51 64 58 133

     

Control (n = 37)

30 40 30 24 25 73

28 40 41 48 48 111

     

End of treatment period

Baseline

29 40 33* 28* 29 70

34 60 57 72 66 124

*

5

     

30 40 25 25 27 85

31 50 43 56 30 111

     

P-value – between groups

31* 40* 30* 29* 32* 86

0.27 0.15 0.68 0.72 0.17 0.78

* P < 0.05 vs. baseline.

W1 *

*

GI symptom severity

50

*

*

*

40 30 20 * 10

*

*

*

*

*

*

*

*

0 Baseline W1 W2 W3 W4 W5 W6 W7 W8 FU Active (n = 37) Control (n = 37)

Figure 6. The weekly total scores of the GI symptom questionnaire. *P < 0.05 vs. Baseline. FU = Follow-up.

GI symptom change from baseline

60

W2

W3

W4

W5

W6

W7

W8

FU

0 –5 –10 –15 ** –20 * –25 –30 Active (n = 37)

Control (n = 37)

Figure 7. The change in GI symptoms assessed with the GI symptom questionnaire. FU = follow-up 8 weeks after the end of the treatment period. *P < 0.05 **P < 0.01 vs. control.

during weeks 1 and 2 of the treatment period, but thereafter, no significant group differences could be detected (Figure 7).

in both groups, but none of the changes observed differed significantly between the groups (Table 4).

Quality of life and psychological symptoms

DISCUSSION

There was a significant reduction after the treatment period vs. baseline in the HAD anxiety score in the control group (8  3 vs. 6  3; P = 0.001), but not in the active treatment group (6  4 vs. 6  4; N.S.), but this change did not differ significantly between the groups (N.S.). No significant changes in HAD depression scores were detected in any of the groups (both groups: 4  3 vs. 3  3; N.S.). Some of the domains of the disease-specific IBSQOL questionnaire improved

On the basis of our primary endpoint, i.e. the proportion of patients with adequate relief at least half of the weeks during the treatment period (=responders), we could not detect a significant difference between milk fermented with the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus and containing three probiotic bacteria (Lactobacillus paracasei, ssp. paracasei F19, Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12) and acidified milk without Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

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Table 4. Irritable bowel syndrome quality of life questionnaire Active (n = 37)

Baseline Emotional Mental Health Sleep Energy Physical functioning Food Social functioning Role physical Sexual relations

57 81 73 69 77 67 65 65 67

        

20 14 20 22 21 22 23 31 24

Control (n = 37) End of treatment period 64 81 77 71 84 68 71 69 75

        

18 16 24 20 19 * 18 22 * 28 23

Baseline 52 81 74 58 81 72 64 67 67

        

19 16 22 23 22 18 22 26 28

End of treatment period 64 88 77 64 78 74 70 73 67

        

20* 14* 20 29 20 17 26 25 35

P-value – between groups 0.39 0.12 0.82 0.41 0.07 0.79 0.84 0.96 0.50

* P < 0.05 vs. baseline.

any added probiotic bacteria. However, in both groups, the severity of IBS symptoms was gradually reduced during the eight-week treatment period, with a more pronounced effect in the active treatment group during the first week ⁄ weeks. The clinical relevance of this subtle difference deserves further investigations. Moreover, a numerically higher proportion of subjects who received the active treatment compared with the control treatment were responders according to our primary endpoint definition (38% vs. 27%), but our sample size was too small to detect such minor differences. Larger studies are needed to evaluate if there is a small, but significant, superiority for the probiotic product used in this study compared with acidified milk without bacteria. During recent years, alterations in the gut microflora in patients with IBS have been reported by several independent research groups.6, 21, 22 On this basis, treatment options directed towards normalizing the intestinal microflora seem logical. Both anti- and probiotics have been tested with somewhat divergent results. Some IBS patients report worsening of their IBS symptoms following treatment with systemic antibiotics,23 whereas non-absorbed antibiotics, especially rifaximin, have been found to have favourable effects on IBS symptoms.24–26 There are also several trials evaluating the effects of various probiotic products on IBS-related symptoms with both positive7, 8, 27–29 and negative10, 30 results. However, a recent meta-analysis, including 19 randomized controlled trials, demonstrated that probiotics appear to be efficacious in IBS, but the magnitude of benefit and the most effective Aliment Pharmacol Ther 31, 218–227 ª 2010 Blackwell Publishing Ltd

species and strain, as well as optimal doses remain uncertain.31 In this study, milk fermented with the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus and containing Lactobacillus paracasei, ssp. paracsei F19 together with Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 was evaluated. Lactobacillus paracasei, ssp. paracsei F19 has a documented ability to survive during passage through the GI tract and can transiently colonize both the colonic lumen and mucosa.32–34 Moreover, it also has properties making it suitable for consumer products35 and can prevent antibiotic-associated ecological disturbances in the intestine.36 The yoghurt used in this study also contained two other probiotic bacteria, i.e. Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12, resulting in a combination not tested before in IBS, even though previous studies have evaluated probiotic products containing Lactobacillus acidophilus and ⁄ or Bifidobacterium lactis.27, 37–39 Although we failed to detect a significant group difference on the basis of our primary endpoint, we believe that the positive trends for some symptom variables in our relatively small sample size, provide support for further assessment of this product in patients with functional bowel disorders. The responder definition used in this study, i.e. the proportion of patients with adequate relief at least half of the weeks, is in line with what has been used in drug trials in IBS.40, 41 Even though we failed to detect a significant difference in the proportion of responders

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between our active treatment and the control treatment, the numerical difference was 11% more responders in the active treatment group. A power calculation showed that we would have needed almost 600 patients to demonstrate such a small difference between the treatment groups with reasonable certainty, which is also in agreement with the size of the majority of drug trials in IBS. There are several trials assessing the effect of probiotics in IBS where an effect on the primary endpoint was not evident, but benefits on different secondary endpoints were seen,9, 28 which can be as a result of too small sample sizes, as might be the case also in our study. The recently published meta-analyses showing benefit of probiotics in IBS supports this assumption.31, 42 However, larger studies assessing the effect of our product containing Lactobacillus paracasei, ssp. paracasei F19 together with Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 are needed to prove this. It could also be questioned whether an 11% difference in the proportion of responders between active and control treatment is clinically significant. However, a real effect of the control product in our study, i.e. the acidified milk, should also be considered, as it is possible that bioactive products in the milk can affect the gut microbiota,43 when 400 mL ⁄ day is consumed during 8 weeks. An interesting finding in the present study was the rapid onset of a positive treatment effect in the active group as compared with the control group that received acidified milk without probiotic bacteria, where the positive effect came later. One could argue that a rapid onset of a positive effect is needed to motivate the patients to continue with the treatment beyond the first week ⁄ weeks, which speaks in favour of the yoghurt used in this study. However, one should

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be aware of the fact that the IBS symptom severity assessments with the two questionnaires were secondary endpoints and mainly included for exploratory purposes generating hypotheses for future studies. To conclude, in this study, we could not detect a clearly positive effect of milk fermented with the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus and containing three probiotic bacteria, Lactobacillus paracasei, ssp. paracasei F19, Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12, on IBS symptoms compared with control treatment. However, some positive trends, including a more favourable effect during the first treatment weeks in the group receiving probiotics, emphasize that this product should be included in future trials with different study design and larger sample sizes to explore further its potential usefulness in IBS.

ACKNOWLEDGEMENTS Disclosure of personal interests: Magnus Simre´n has served as a speaker for AstraZeneca, Novartis, Albireo, Danone, Movetis Addex and Succampo, and has received research funding from AstraZeneca and Danone. Johan Olsson is an employee of KPL Good Food Practice. Ulla Svensson and Kajsa Ohlson are employees of Arla Foods Innovation. Disclosure of funding interests: This study was supported by an unrestricted grant from Arla Foods Innovation, Stockholm, Sweden to the department of Internal Medicine, Sahlgrenska University Hospital, Sweden. This study was supported by the Swedish Medical Research Council (grant 13409), and by the Faculty of Medicine, University of Go¨teborg.

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croflora is associated with reduction in abdominal bloating and pain in patients with irritable bowel syndrome. Am J Gastroenterol 2000; 95: 1231–8. 8 O’Mahony L, McCarthy J, Kelly P, et al. Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 2005; 128: 541–51. 9 Kim HJ, Camilleri M, McKinzie S, et al. A randomized controlled trial of a probiotic, VSL#3, on gut transit and symptoms in diarrhoea-predominant irritable bowel

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