Effects of a Probiotic Fermented Milk on Functional Constipation

A Randomized, Double-blind, Placebo-Controlled Study

Mena Mustapha Mazlyn; Lee Hun-Leong Nagarajahl Arshad Fatimah; A Karim Norimah; Khean-Lee Goh

J Gastroenterol Hepatol. 2013;28(7):1141-1147.

Methods

Study Design

A randomized, double-blind, placebo-controlled, parallel group study involving 100 adults with functional constipation was conducted in the Klang Valley area of Peninsular Malaysia between March and June 2010.

A third-party controller assigned color codes to probiotic and placebo drinks, respectively. Subjects were stratified based on age and severity of constipation measured using Chinese Constipation Questionnaire (CCQ)^[19] score and were allocated to two groups with 1:1 ratio using randomization software. Both researchers and subjects were blinded to the allocation.

A painstaking and stringent selection process was undertaken in the recruitment of subjects. A database was obtained and broadly screened to identify a large pool of potential subjects. The database had been collected via surveys at public health awareness events organized by medical societies that were not pharmaceutical marketing events. From among the pool of potential subjects, all those who agreed to participate in the screening were interviewed by the researchers and underwent medical examination by a physician. Only those who met the strict eligibility requirements were invited to participate in the trial.

After a 2-week baseline, subjects received once-daily doses of either fermented milk with LcS or placebo nutrient drink without LcS for 4 weeks. Study drinks were delivered to subjects once every 3 days. Cold chain was maintained below 10°C during storage, transportation, and distribution. Final assessment was done 1 week postintervention. Subjects recorded information regarding their bowel movements in diaries and constipation severity was assessed weekly via CCQ^[19] throughout the 7 weeks. For use in ethnic Malay subjects, the CCQ^[19] was translated to Malay by expert panel forward-back translation and pre-tested. Subjects were required to maintain their usual diets and lifestyle throughout the study.

Ethical Considerations

The study was conducted in accordance with Malaysian Guidelines for Good Clinical Practice, the Declaration of Helsinki, and local laws and regulations. The protocol was approved by the joint research and ethics committee of International Medical University, Kuala Lumpur on December 17, 2009 (Research project identification no. IMU R052 2009). All subjects gave written informed consent. The study was registered at ClinicalTrials.Gov (#NCT01161784).

Safety Considerations

Adverse events (AEs) that occurred during the study were recorded. For safety assessment, only AEs that took place during intervention phase were analyzed. Stimulant laxative Dulcolax (Boehringer Ingelheim, Ridgefield, CT, USA) was assigned as rescue therapy in the event of absence of bowel movement for 4 consecutive days.

Study Agents

The test beverages consisted of fermented milk containing viable LcS ("probiotic drink") and fermented milk without LcS (placebo "nutrient drink"). The beverages were packaged in unmarked individual plastic bottles in doses of 80 mL. The ingredients were sugar, skimmed milk powder, glucose, calcium, vitamins, and permitted flavorings. Both test beverages contained approximately 1-g lactose per bottle and did not contain dietary fiber. The probiotic drink contained at least 3.0 × 10¹⁰ colony-forming unit (CFU) of live LcS per serving, while the placebo did not contain LcS. Every effort was made to ensure similarity between the probiotic and placebo drinks in terms of nutritional content, taste, color, texture, and smell, with the only difference being the presence or absence of LcS.

Eligibility

Subjects were of either gender aged between 18 and 60 years diagnosed with Rome II-defined functional constipation^[2] with CCQ^[19] score of at least 5, satisfying the CCQ^[19] cut-off point for constipation. Subjects were otherwise-healthy, free of cardiovascular-related diseases, diabetes, cancer, neurological diseases, or other serious illnesses, and not physically or mentally handicapped. Subjects lived in the Klang Valley, with no predetermined plans to be out of town for longer than 6 days during the intervention. Subjects were excluded if they had a body mass index (BMI) of below 16 (severe thinness), or 30 and above (obese); were pregnant; consumed probiotic products more than once a week in the month preceding screening or at any point during intervention; regularly used laxatives (more than once a week); used anticholinergics, anti-diarrheals, or antibiotics in the month preceding screening or at any point during intervention; had milk protein allergy; had constipation of organic or neurological origin; had alarm features indicative of colorectal cancer; failed to either confirm intake of study drink or provide data records for 6 days or more during intervention; or experienced AEs that necessitated discontinuation of intervention.

Efficacy Measurements

Primary efficacy outputs: CCQ^[19] severity score and stool frequency.

Secondary efficacy outputs: Stool consistency assessed on a 5-point scale modified from the Bristol stool form chart^[20] and stool quantity estimations based on visual comparison of stool volume with size of 40-mm diameter standard unit (ping-pong ball).^[15]

CCQ^[19] score was calculated using a questionnaire consisting of six items that has been found to be satisfactory in terms of validity and reliability as a self-report measure for constipation severity by a review.^[21] The six items are: (i) severity of false alarm (having urge but unable to pass stool); (ii) frequency of < 3 weekly defecations; (iii) severity of incomplete evacuation; (iv) severity of lumpy or hard stools; (v) number of laxatives used; and (vi) severity of abdominal bloating.^[19] Items (i), (ii), (iii), (iv), and (vi) are rated using a 5-point Likert scale.

Sample Size

Sample size was determined based on primary end-point measures, CCQ^[19] score, and stool frequency. According to priori power analysis for two-tailed t-test,^[22] with α error 0.05 and β error 0.20, assuming mean change in CCQ score and weekly stool frequency of −3.5 and +0.95, and standard deviation of 5.0 and 1.5, respectively, a total of 80 subjects (40 per group) were needed. However, if α level was set at 0.10, while other parameters remained unchanged, the minimum sample size required would be 26 (13 subjects per group).

Statistical Analysis Plan

Analysis was conducted on intent-to-treat population (i.e. all randomized subjects exposed to at least one dose of study drink, with at least one post-baseline efficacy assessment, and excluding outliers with major eligibility violation—possible misdiagnosis). Student's t-test or non-parametric equivalent (α = 0.05 level) was employed to compare the mean primary efficacy outcomes of the probiotics and control at weekly intervals. Baseline values are the mean of 2-week pre-intervention period. Magnitude of change was computed using Cohen's effect size.

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Table 1. Subjects' demographic and anthropometric data at screening
Parameter	Probiotics	Control
Total no. of subjects	47	43
Males	5	7
Females	42	36
Age (years)a	31.8 ± 9.4	31.7 ± 9.4
Ethnicity (no. of subjects)
Malays	22	17
Chinese	20	24
Indian	5	2
Body weight (kg)^a	56.8 ± 9.3	58.1 ± 9.5
Height (m)^a	1.60 ± 0.06	1.59 ± 0.09
Body mass index (kg/m²)^a	22.3 ± 3.4	22.9 ± 3.2
Waist-to-hip ratio^a	0.81 ± 0.06	0.82 ± 0.08

^aMean ± standard deviation.
Discrete and continuous data were analyzed using χ² test and Mann–Whitney U-test, respectively.

Table 2. Subjects' constipation-related data at screening
Parameters	Probiotics	Control
Severity of constipation (CCQ score)^a	10.5 ± 3.4	10.1 ± 3.1
Frequency of constipation symptoms^bc (%)
Hard or lumpy stools	89.4	74.4
Feeling of incomplete emptying after defecation	85.1	86.0
Straining during defecation	85.1	74.4
Fewer than three defecations a week	68.1	69.8
Sensation of blockage when defecating	83.0	69.8
Abdominal fullness, bloating	63.8	55.8
Need to press on or around anus or vagina to try to remove stool in order to complete defecation	46.8	44.2
Duration of constipation^bd
Less than 5 years	25.5	18.6
Between 5 and 25 years	42.6	48.8
Others (unaware, etc)	31.9	32.6

^aMean ± standard deviation, assessment period was the 3 months preceding screening.
^bPercentage of subjects.
^cSymptom was present at least one-fourth of the time in the preceding 3 months.
^dSelf perceived based on subjects' own understanding of constipation.
Discrete and continuous data were analyzed using χ² test and Mann–Whitney U-test, respectively.
CCQ, Chinese Constipation Questionnaire.¹⁹

Table 3. Mean comparisons of end-point measures between probiotics and control groups and their effect sizes
End-points	Probioticsa	Controla	P value	Effect sized (95% CI)
Primary end-point measures
Severity of constipation (CCQ score)
Baseline	5.06 ± 2.75	5.21 ± 2.82	0.955	–
Week 2 of treatment	3.83 ± 3.29*	3.96 ± 3.40*	0.856	0.04 (−1.00–0.98)
Week 4 of treatment	2.33 ± 2.62,*	3.38 ± 2.78*	0.058	0.39 (−0.47–1.18)
Withdrawal period	3.78 ± 3.46*	3.73 ± 3.04*	0.966	−0.02 (−0.96–1.04)
Weekly stool frequency
Baseline	3.84 ± 1.78	3.91 ± 1.94	0.919	–
Week 2 of treatment	4.21 ± 1.78	5.22 ± 2.52,*	0.035	−0.46 (−0.98–0.30)
Week 4 of treatment	4.56 ± 1.99*	4.83 ± 2.24*	0.737	−0.13 (−0.72–0.57)
Withdrawal period	4.20 ± 2.18	4.55 ± 2.32*	0.528	−0.16 (−0.83–0.56)
Secondary end-point measures
Stool consistencyb
Baseline	2.61 ± 0.60	2.63 ± 0.62	0.842	–
Week 2 of treatment	2.62 ± 0.72	2.67 ± 0.72	0.594	−0.06 (−0.27–0.16)
Week 4 of treatment	2.97 ± 0.64*	2.86 ± 0.51	0.483	0.19 (0.00–0.35)***
Withdrawal period	2.92 ± 0.61*	2.73 ± 0.73	0.188	0.29 (0.11–0.52)***
Weekly stool quantityc
Baseline	10.09 ± 5.33	11.90 ± 7.11	0.340	–
Week 2 of treatment	11.82 ± 6.02*	14.90 ± 9.43*	0.163	−0.39 (−2.12–2.49)
Week 4 of treatment	13.36 ± 6.54*	14.80 ± 9.74	0.830	−0.17 (−2.13–2.84)
Withdrawal period	12.43 ± 7.38	13.60 ± 9.57	0.737	−0.14 (−2.40–2.83)

Student's t-test (paired samples) or Wilcoxon signed-rank test; *P < 0.05, versus baseline.
Student's t-test (independent samples) or Mann–Whitney U-test **P < 0.10, versus control or versus treatment.
***P < 0.05, versus control.
^aMean ± standard deviation.
^bAssessed by a 5-point scale modified according to Bristol stool form scale:²⁰ 1 indicates hard lumps; 2 lumpy, hard sausage; 3 normal sausage; 4 soft blobs; and 5 watery or mushy.
^cExpressed as number of balls (40-mm diameter) to the first decimal place using ping-pong ball as reference.
^dCohen's d.
CCQ, Chinese Constipation Questionnaire;¹⁹ –, not applicable.

Table 4. Post-hoc exploratory analysis of CCQ component symptoms
CCQ component scores^a	Baseline	Week 4 of treatment	P value
i) False alarm^a
Probiotics	0.89 ± 0.87	0.28 ± 0.63	0.518
Control	0.71 ± 0.70	0.35 ± 0.66	0.518
ii) Less than three defecations per week^b
Probiotics	0.89 ± 1.31	0.65 ± 1.49	0.883
Control	1.12 ± 1.53	0.70 ± 1.54	0.883
iii) Incomplete defecation^b
Probiotics	1.19 ± 0.74	0.40 ± 0.58	0.003*
Control	1.28 ± 0.91	1.05 ± 1.11	0.003*
iv) Lumpy/hard stoolsb
Probiotics	1.29 ± 0.89	0.67 ± 0.94	0.8
Control	1.22 ± 0.84	0.83 ± 0.81	0.8
v) Frequency of laxative usagec
Probiotics	0.02 ± 0.15	0.00 ± 0.00	1.000
Control	0.02 ± 0.15	0.00 ± 0.00	1.000
vi) Abdominal bloatingb
Probiotics	0.78 ± 0.90	0.33 ± 0.64	0.362
Control	0.86 ± 0.90	0.45 ± 0.71	0.362

Student's t-test (independent samples) or Mann–Whitney U-test; *P < 0.05 versus control.
^aMean ± standard deviation.
^bAssessed with 5-point ordinal scale; for (i), (iii), (iv) and (vi): 0 indicates none, 1 mild, 2 moderate, 3 severe, and 4 very severe; for (ii): 0 indicates never, 1 less than half the time, 2 half the time, 3 more than half the time, and 4 all the time.
^cCalculated based on number of times used averaged over a 2-week period.
CCQ, Chinese Constipation Questionnaire.¹⁹