Arch. Pharm. Res. DOI 10.1007/s12272-014-0522-2
RESEARCH ARTICLE
Effect of a double-coated probiotic formulation on functional constipation in the elderly: a randomized, double blind, controlled study Youngran Yeun • Jaejin Lee
Received: 29 July 2014 / Accepted: 21 November 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract A randomized, double blind, controlled study was conducted to evaluate a multi-species probiotic product, DuolacÒ Care, for the efficacy in functional constipation of elderly nursing home residents. Forty participants were randomly assigned to receive either one capsule containing six bacterial strains (2.5 9 108 viable cells/ capsule) doubly coated with proteins and polysaccharides (DC group) or one capsule containing the same bacterial strains without the coating layers outside the cell (NC group) twice a day (5 9 108 viable cells/day). After consumption of 2 weeks, NC group showed a significant improvement in a symptom of Rome III criteria for constipation, ‘effort to evacuate’, but failed to show a significant improvement in weekly defecation frequency. By contrast, DC group showed significant improvements in symptoms of ‘effort to evacuate’ and ‘sensation of anorectal obstruction or blockage’ as well as being improved in ‘defecation frequency’. The quantitative analysis of the fecal bacteria revealed that the levels of all the bacterial species consumed were significantly increased after consumption in both groups. However DC group showed larger increases in the bacterial levels than NC group, and the overall bacterial levels in DC group were about 100-fold or greater than those in NC group. Therefore the improvements in the symptoms appeared to be closely associated with large increases of the bacterial species consumed. The
Y. Yeun J. Lee (&) Sahmyook Medical Center, Seoul, Republic of Korea e-mail: [email protected] Y. Yeun Department of Nursing, Kangwon National University, Samcheok, Republic of Korea
double-coating layers of proteins and polysaccharides ensured increase in the levels of bacteria, indicating that the layers function to protect the bacteria from the digestive action in the GI tract. Therefore, DuolacÒ Care could be a treatment option in functional constipation. Keywords Probiotics Functional constipation Doublecoated bacteria Non-coated bacteria Defecation frequency
Introduction Constipation is a common digestive complaint, characterized by straining during defecation, lumpy or hard stools, sensation of incomplete evacuation or anorectal obstruction, manual maneuvers to facilitate defecation, and/or less than three defecations per week (Longstreth et al. 2006). It is prevalent among people of all ages and both sexes, but its occurrence increases with age. It was reported that constipation has affected about 26 % of men and 34 % of women over 65 years of age but less than 2 % of the nonelderly people (Johanson et al. 1989; Whitehead et al. 1989). In particular, at11 least 75 % of elderly hospitalized patients and nursing home residents are known to suffer from constipation (Primrose et al. 1987). Possible factors for the development of functional constipation in the elderly include dietary changes, increased intestinal transit times, lack of physical activity, and frequent use of medications. Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO/WHO 2001). Their beneficial roles in many gastrointestinal disorders such as irritable bowel syndrome (IBS) (Hoveyda et al. 2009),
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inflammatory bowel disorder (IBD) (Meijer and Dieleman 2011), and Clostridium difficile-associated diarrhea (CDAD) (Fitzpatrick 2013) have been reviewed. The rationale for the use of probiotics is they may have potential to modulate the gut function through interactions between microbes and/or between microbes and host cells. In this regard, probiotics have been introduced as a treatment option for constipation, and some probiotic strains were evaluated in randomized, controlled trials, revealing significant improvement of the symptoms of constipation (Koebnick et al. 2003; Del Piano et al. 2010). However, studies of probiotic intervention in constipation of the elderly have been limited. There is a controversy whether multi-species probiotics have greater health-promoting effects over single strain probiotics. A review examining this issue concluded that multi-species probiotics appear to have superior efficacy to single strain probiotics (Chapman et al. 2011). A recent clinical trial showed that a multispecies mixture consisting of three Bifidobacterium species and three Lactobacillus species was effective in constipation of pregnant women, where the primary outcome, defecation frequency, and some secondary outcomes were significantly improved compared to baseline (de Milliano et al. 2012). The superior efficacy of multi-species probiotics may come from the different spatial distribution of bifidobacteria and lactobacilli along the gastrointestinal tract where lactobacilli are highly abundant in the small intestine and bifidobacteria are mostly found in the large intestine (Reuter 2001). DuolacÒ Care has been formulated based on the above speculation and contains three Bifidobacterium species (Bifidobacterium bifidum, B. lactis, and B. longum), two Lactobacillus species (Lactobacillus acidophilus and L. rhamnosus), and Streptococcus thermophilus at a total of 2.5 9 108 viable cells in a capsule. In a previous randomized, double blind, placebo controlled study, a daily dosage of 1 9 1010 viable cells of the strains of DuolacÒ Care for 4 weeks was found to improve the symptoms of IBS compared to the placebo control (Yoon et al. 2014). The probiotic strains are doubly coated with proteins and polysaccharides during the process of production. Thanks to the double coating layers, the coated bacteria showed even higher resistance to acids and bile salts than noncoated bacteria (Kang et al. 2013). Therefore the doublecoated bacteria are expected to better survive during their passage through the GI tract and thus to have greater impact on the consumer than the non-coated counterparts. In this study, we investigated efficacy of DuolacÒ Care in constipation of elderly nursing home residents and evaluated the effect of the double coating layers on the viability of the consumed bacteria by quantifying fecal bacteria.
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Materials and methods Study participants Residents who were 60 years of age or older and fulfilled Rome III criteria for functional constipation (Longstreth et al. 2006) were eligible for this study. The criteria are (1) straining during at least 25 % of defecations, (2) lumpy or hard stools in at least 25 % of defecations, (3) sensation of incomplete evacuation for at least 25 % of defecations, (4) sensation of anorectal obstruction/blockage for at least 25 % of defecations, (5) manual maneuvers to facilitate at least 25 % of defecations, and (6) fewer than three defecations per week. Those who have two or more of the six criteria in at least 25 % of the evacuations during the past 6 months were included for the study. Informed consent was obtained from participants or their next of kin. People who were sensitive to probiotic products and were using medications that may affect the study result (e.g. probiotics, intestinal drugs) were excluded. Study design and procedures The study was conducted in the nursing home called Ujawon of Sahmyook Medical Center in Seoul, Korea between March 2014 and April 2014 and was approved by the Clinical Research Ethics Committee of Sahmyook Medical Center (SYMC IRB 1401-02). The enrolled participants were randomly assigned to receive either one capsule (350 mg) containing six bacterial strains doubly coated with proteins and polysaccharides (DC group) or one capsule (350 mg) containing the same bacterial strains without the coating layers outside the cell (NC group) twice a day, 30 min after breakfast and dinner, for 2 weeks. DuolacÒ Care (Cell Biotech, Co. Ltd., Gimpo, Korea) is a capsule form product containing six species of live bacteria: Bifidobacterium bifidum (KCTC 12199BP), B. lactis (KCTC 11904BP), B. longum (KCTC 12200BP), Lactobacillus acidophilus (KCTC 11906BP), L. rhamnosus (KCTC 12202BP), and Streptococcus thermophilus (KCTC 11870BP). A total of 2.5 9 108 viable cells in lyophilized powder form were included in each capsule. Participants were allocated to DC group or NC group using a computer-generated randomization schedule with a 1:1 allocation ratio. Before the start of the study, participants were instructed on the information of the study product and the way of consumption. They were also highly recommended to keep their lifestyle, e.g. eating habits, physical activity, and fluid intake, during the study period. Drug compliance was defined as the percentage of the number of capsules taken to the number of capsules prescribed, and consumption of more than 80 % of the capsules was considered to be the minimum level of compliance. All adverse reactions were reported.
A multi-species probiotic product improved symptoms of constipation
Measurements
Table 1 Questionnaire for assessing the symptoms of functional constipation based on Rome III criteria
Study outcomes
Effort to evacuate
The primary study outcome was the change of defecation frequency per week before and after treatment. The secondary outcome was the changes in symptoms of Rome III criteria. A questionnaire to assess defecation frequency and the symptoms of Rome III criteria was previously described (Longstreth et al. 2006; Favretto et al. 2013) and was recorded at the beginning and end of the study (Table 1).
In the past six months, how often have you had to force or make an effort to defecate?
(1) Sometimes (2) Often (3) Most of the times (4) Always Hard or lumpy stools In the past six months, how often did you have hard stools, hardened or as if they were marbles?
(2) Often (3) Most of the times (4) Always Sensation of incomplete evacuation In the past six months, how often have you had the sensation of incomplete evacuation, i.e. even after evacuation, the need remained?
Results
(0) Never or rarely (1) Sometimes (2) Often
Statistics The sample size was determined based on the assumptions of 30 % difference in the primary endpoint (Yoon et al. 2014), statistical power at 80 %, two-sided a risk at 5 %, and drop-out rate at 20 %. Thus we concluded that 40 people in total were needed to enroll. Efficacy and safety parameters and bacterial numbers were assessed by intention-to-treat (ITT) analysis. Descriptive statistics were reported as median with inter-quartile range or mean ± SD, unless otherwise stated. Statistical comparative analysis was performed using Prism 5 (GraphPad Software, USA). The Chi square test for categorical variables and t test (paired t test for intra-group comparison and unpaired t test for inter-group comparison) for continuous variables were used. The Wilcoxon signed rank test and the Mann–Whitney test were used to evaluate the Rome III questionnaire before and after the intervention. P \ 0.05 was considered significant.
(0) Never or rarely (1) Sometimes
Change of fecal bacteria Fecal specimens were collected at the beginning and end of the study and were used for quantification of fecal bacteria. Preparation of genomic DNA from fecal samples, quantitative real time PCR, and PCR primers were previously described (Yoon et al. 2014).
(0) Never or rarely
(3) Most of the times (4) Always Sensation of anorectal blockage or obstruction In the past six months, how often have you felt that the stool fails to pass, that it is locked or trapped in the anus?
(0) Never or rarely (1) Sometimes (2) Often (3) Most of the times (4) Always
Manual maneuvers to facilitate defecation In the past six months, how often have you had to use your finger to help in the passage of stool?
(0) Never or rarely (1) Sometimes (2) Often (3) Most of the times (4) Always
Defecation frequency per week How many times per week do you usually evacuate?________ times/ week
Participants and baseline characteristics Symptoms of constipation The forty participants were randomly assigned to receive either non-coated probiotic product (NC group) or doublecoated product (DC group) and completed the study. As shown in Table 2, there were no significant differences in the baseline characteristics (age, gender, weight, height, and BMI) between two groups.
The effects of the non-coated or double-coated bacteria on the symptoms of constipation were assessed (Table 3). Defecation frequency per week, the primary outcome, was little changed in NC group where median was determined to be 3.0 (1.5–5.3) at baseline and 3.0 (1.5–4.0) at week 2.
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assessed were found to be significantly improved in DC group after intervention (P \ 0.05) (Table 3).
Table 2 Baseline characteristics Non-coated probioticsa (n = 20)
Double coated probioticsa (n = 20)
P
Age (years)
81.10 ± 9.12
82.45 ± 9.36
NSb
Gender (male/ female)
3/20
7/20
NSc
Weight (kg)
45.92 ± 13.31
44.50 ± 7.09
NSb
Height (cm)
154.8 ± 8.70
158.5 ± 11.14
NSb
2
19.05 ± 4.89
18.20 ± 3.01
NSb
BMI (kg/m )
Quantitative changes in fecal bacteria Five hundred million of probiotic bacteria, non-coated or double-coated with proteins and polysaccharides, were consumed daily for 2 weeks. In order to investigate how the consumed bacteria impacted the gut microbiota, we examined the quantitative changes in the levels of the bacteria species consumed using quantitative real time PCR. As shown in Table 4, all the bacterial species consumed were found to be significantly increased in both groups compared to the levels at baseline. However the degree of increase in the bacterial numbers was greatly different between groups. In NC group, the numbers of the bacteria were increased by from 3.4-fold (S. thermophilus) to 14-fold (B. bifidum and L. rhamnosus). By contrast, the bacterial numbers in DC group were increased hundreds of times compared to the numbers at baseline. The increases compared to the baseline levels were from 213-fold (S. thermophilus) to 2519-fold (L. rhamnosus). Therefore this result indicates that the double-coating layers play a protective role, making the consumed bacteria more resistant to the harsh conditions due to, e.g. gastric acid and bile acids encountered in the GI tract. On the other hand, the differences in the bacterial levels between the groups after consumption were statistically significant (P \ 0.01 for all except for L. acidophilus (P \ 0.05)). Compared to the bacterial levels of NC group, the overall bacterial levels after consumption in DC group were about 100-fold or greater.
NS not significant a
Values are expressed as mean ± SD
b
Two-sided unpaired t test
c
Chi square test
By contrast, the consumption of the double-coated bacteria significantly improved stool frequency, median of which was increased from 3.0 (1.5–7.8) at baseline to 5.0 (3.1–8.8) at week 2 (P \ 0.01). It should be noted that nearly half of the participants evacuated from 4 to 20 times per week during the study but the amount was so little at baseline that it only wetted underwear or diapers. However there were some participants in both groups who reported an increase in stool amount. Meanwhile, NC group showed a significant improvement only in ‘effort to evacuate’ (P \ 0.05). By contrast, DC group showed significant improvements in ‘effort to evacuate’ (P \ 0.05) as well as ‘sensation of anorectal obstruction or blockage’ (P \ 0.05). When comparison was made between groups before and after intervention, there were no significant differences in the criteria at baseline. However, ‘hard or lumpy stools’, ‘manual maneuvers to facilitate defecation’, and ‘defecation frequency per week’ among the six criteria
Table 3 Changes in symptoms of Rome III criteria before and after intervention Symptoms
Non-coated probiotics (n = 20) Before
a
After
a
Double coated probiotics (n = 20) P
b
Before
a
After
a
P
b
Intergroup comparison Pc Before
After
Effort to evacuate
1.0 (1.0–2.0)
1.0 (0.4–1.0) \0.05
1.0 (0.2–1.0)
0.0 (0.0–1.0)
\0.05
NS
NS
Hard or lumpy stools
1.0 (0.0–1.0)
1.0 (0.4–1.0)
1.0 (0.0–1.0)
0.0 (0.0–1.0)
NS
NS
\0.05
NS
Sensation of incomplete evacuation
2.0 (2.0–2.0)
2.0 (2.0–2.0)
NS
2.0 (2.0–2.0)
2.0 (1.0–2.0)
NS
NS
NS
Sensation of anorectal obstruction or blockage
1.0 (1.0–1.0)
1.0 (1.0–1.0)
NS
1.0 (0.2–1.0)
0.0 (0.0–1.0)
\0.05
NS
NS
Manual maneuvers to facilitate defecation
1.0 (0.0–1.0)
1.0 (0.0–1.0)
NS
1.0 (0.0–1.0)
0.0 (0.0–0.0)
NS
NS
\0.05
Defecation frequency per week
3.0 (1.5–5.3)
3.0 (1.5–4.0)
NS
3.0 (1.5–7.8)
5.0 (3.1–8.8)
\0.01
NS
\0.05
NS not significant a
values are expressed as median and inter-quartile range (25, 75 %)
b
Wilcoxon signed rank test
c
Mann–Whitney test
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A multi-species probiotic product improved symptoms of constipation Table 4 Quantitative changes of fecal bacteria assessed before and after intervention Non-coated probiotics (n = 20) log10 cells/g feces Before
a
Double coated probiotics (n = 20) P
b
After
log10 cells/g feces Before
a
P
Between groups after treatment b
Fold change
Pc
After
B. bifidum
5.28 ± 5.55
6.42 ± 6.72
\0.05
5.87 ± 6.23
8.45 ± 8.64
\0.01
108
\0.01
B. lactis
5.46 ± 5.72
6.13 ± 6.41
\0.05
5.65 ± 5.79
8.26 ± 8.39
\0.01
137
\0.01
B. longum L. acidophilus
6.17 ± 6.34 5.44 ± 5.52
7.09 ± 7.16 6.26 ± 6.43
\0.001 \0.01
6.19 ± 6.55 5.46 ± 5.62
9.08 ± 9.08 8.22 ± 8.48
\0.001 \0.05
98 92
\0.001 \0.05
L. rhamnosus
1.98 ± 1.91
3.06 ± 3.02
\0.001
2.01 ± 2.06
5.41 ± 5.59
\0.01
221
\0.01
S. thermophilus
4.80 ± 4.88
5.33 ± 5.40
\0.01
5.03 ± 5.12
7.36 ± 7.38
\0.001
105
\0.001
a
Values are expressed as mean ± SD
b
One-sided paired t test
c
Two-sided unpaired t test
Safety The treatments were well tolerated, and the drug compliance rates were [80 % in all patients. No adverse events or serious adverse events were reported in both groups during and after consumption.
Discussion The definition of probiotics by FAO/WHO (2001) indicates that the consumed microbes must reach the intestine alive to exert their health-promoting activities. The rationale for the use of probiotics in treatment of constipation is disturbed state of the intestinal microbiota reported in patient with constipation (Zoppi et al. 1998) and their potential to improve intestinal motility (Salminen and Salminen 1997). DuolacÒ Care is a multi-species probiotic product produced with double coating layers of proteins and polysaccharides, and the coating technology has been patented in Korea (patent no. 0429495), Japan (patent no. 3720780), and Europe (patent no. 1514553B). Since the bacterial strains with the coating layers showed higher viability under a simulated gastrointestinal condition compared to the non-coated counterparts (Kang et al. 2013), it has been assumed that the double-coated bacteria better survive in the gastrointestinal tract upon ingestion, giving the host health benefits. In this study, we compared an efficacy of a double-coated multi-species probiotic product, DuolacÒ Care, in functional constipation in 40 elderly nursing home residents with its non-coated counterparts. Based on the higher viability observed in the previous study (Kang et al. 2013), it was expected that the double-coated bacteria would have greater effects in this study. When the symptoms of Rome III criteria before and after intervention were assessed, the consumption of the
no-coated bacteria appeared to be significantly effective in certain symptoms, ‘effort to evacuate’ and ‘hard or lump stools’. By contrast, DC group showed significant improvements in all the symptoms assessed. Thus the result shows that the consumption of the double-coated bacteria has greater effects in constipation than the non-coated counterparts. Since both groups consumed the same bacteria, the presence of the coating layers likely affected viable cell numbers reaching the intestine and in turn contributed to improve the symptoms of constipation. This speculation was supported by the quantitative analysis of the fecal bacteria before and after consumption. Although the bacterial levels examined were significantly increased after consumption in both groups, the increases in DC group were even larger than those in NC group. When compared to the bacterial levels after consumption in NC group, those in DC group were even 100-fold or greater. Species of Bifidobacterium and Lactobacillus have been found to be effective in the management of constipation (Favretto et al. 2013; Lahtinen et al. 2009; Koebnick et al. 2003). The proposed mechanisms are changes in the composition of the gut flora (e.g. increase in the levels of bifidobacteria), increase in the production of short chain fatty acids which stimulate bowel movement, and consequently decrease of transit time (Salminen and Salminen 1997). Thus it is reasonable to think that the larger increase in the levels of all the bacterial species was closely associated with the greater improvements in the symptoms of constipation in DC group. On the other hand, the improvements of the symptoms in the DC group seem not to be large considering that the bacterial levels were increased 100-fold or greater. This may be primarily due to the short consumption period (2 weeks) or/and old age of the participants ([81 years of age). As aforementioned, functional constipation in the elderly can be developed by many factors such as dietary changes, increased intestinal
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transit times, lack of physical activity, and frequent use of medications. Thus the consumption period of 2 weeks may have not been long enough to highly elicit changes in the physiology and function of the GI tract, and longer period of consumption (e.g. [1 month) may be needed for the elderly. In conclusion, the consumption of DuolacÒ Care for 2 weeks improved defecation frequency and other symptoms of constipation in elderly nursing home residents. The improvements appeared to be closely associated with large increases of the bacterial species consumed. The doublecoating layers of proteins and polysaccharides ensured increase in the levels of bacteria, indicating that the layers function to protect the bacteria from the digestive action in the GI tract. Acknowledgments We would like to thank Dr. Seo from Cell Biotech for technical assistance. This study was funded by Cell Biotech. Conflict of interest The authors declare no conflict of interest that could inappropriately influence their work.
References Chapman, C.M., G.R. Gibson, and I. Rowland. 2011. Health benefits of probiotics: are mixtures more effective than single strains? European Journal of Nutrition 50: 1–17. de Milliano, I., M.M. Tabbers, J.A. van der Post, and M.A. Benninga. 2012. Is a multispecies probiotic mixture effective in constipation during pregnancy? ‘A pilot study’. Nutrition Journal 11: 80. Del Piano, M., S. Carmagnola, A. Anderloni, S. Andorno, M. Ballare`, M. Balzarini, F. Montino, M. Orsello, M. Pagliarulo, M. Sartori, R. Tari, F. Sforza, and L. Capurso. 2010. The use of probiotics in healthy volunteers with evacuation disorders and hard stools: A double-blind, randomized, placebo-controlled study. Journal of Clinical Gastroenterology 44(1): S30–S34. FAO/WHO (2001) Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria. Cordoba, Argentina: Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation report 1–34. Favretto, D.C., B. Pontin, and T.R. Moreira. 2013. Effect of the consumption of a cheese enriched with probiotic organisms (Bifidobacterium lactis bi-07) in improving symptoms of constipation. Arquivos de Gastroenterologia 50: 196–201.
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Fitzpatrick, L.R. 2013. Probiotics for the treatment of Clostridium difficile associated disease. World Journal Gastrointestinal Pathophysiology 4: 47–52. Hoveyda, N., C. Heneghan, K.R. Mahtani, R. Perera, N. Roberts, and P. Glasziou. 2009. A systematic review and meta-analysis: probiotics in the treatment of irritable bowel syndrome. BMC Gastroenterology 16: 9–15. Johanson, J.F., A. Sonnenberg, and T.R. Koch. 1989. Clinical epidemiology of chronic constipation. Journal of Clinical Gastroenterology 11: 525–536. Kang, J.-H., D.-K. Lee, J.-E. Park, M.-J. Kim, J.S. Lee, J.-G. Seo, M.J. Chung, H.-S. Shin, and N.-J. Ha. 2013. Dual coating improves the survival of probiotic Bifidobacterium strains during exposure to simulated gastrointestinal conditions. Kor. Journal of Microbiology 49: 275–281. Koebnick, C., I. Wagner, P. Leitzmann, U. Stern, and H.J. Zunft. 2003. Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation. Canadian Journal of Gastroenterology 17: 655–659. Lahtinen, S.J., L. Tammela, J. Korpela, R. Parhiala, H. Ahokoski, H. Mykka¨nen, and S.J. Salminen. 2009. Probiotics modulate the Bifidobacterium microbiota of elderly nursing home residents. Age (Dordr) 31: 59–66. Longstreth, G.F., W.G. Thompson, W.D. Chey, L.A. Houghton, F. Mearin, and R.C. Spiller. 2006. Functional bowel disorders. Gastroenterology 130: 1480–1491. Meijer, B.J., and L.A. Dieleman. 2011. Probiotics in the treatment of human inflammatory bowel diseases: update. Journal of Clinical Gastroenterology 45: S139–S144. Primrose, W.R., A.E. Capewell, G.K. Simpson, and R.G. Smith. 1987. Prescribing patterns observed in registered nursing homes and long-stay geriatric wards. Age and Ageing 16: 25–28. Reuter, G. 2001. The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession. Current Issues in Intestinal Microbiology 2: 43–53. Salminen, S., and E. Salminen. 1997. Lactulose, lactic acid bacteria, intestinal microecology and mucosal protection. Scandinavian Journal of Gastroenterology 32: 45–48. Whitehead, W.E., D. Drinkwater, L.J. Cheskin, B.R. Heller, and M.M. Schuster. 1989. Constipation in the elderly living at home. Definition, prevalence, and relationship to lifestyle and health status. Journal of the American Geriatrics Society 37: 423–429. Yoon, J.-S., W. Sohn, O.-Y. Lee, S.-P. Lee, K.-N. Lee, D.-W. Jun, H.L. Lee, B.-C. Yoon, H.-S. Choi, W.-S. Chung, and J.-G. Seo. 2014. Effect of multispecies probiotics on irritable bowel syndrome: a randomized, double-blind, placebo-controlled trial. Journal of Gastroenterology and Hepatology 29: 52–59. Zoppi, G., M. Cinquetti, A. Luciano, A. Benini, A. Muner, and M.E. Bertazzoni. 1998. The intestinal ecosystem in chronic functional constipation. Acta Paediatrica 87: 836–841.
RESEARCH ARTICLE
Effect of a double-coated probiotic formulation on functional constipation in the elderly: a randomized, double blind, controlled study Youngran Yeun • Jaejin Lee
Received: 29 July 2014 / Accepted: 21 November 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract A randomized, double blind, controlled study was conducted to evaluate a multi-species probiotic product, DuolacÒ Care, for the efficacy in functional constipation of elderly nursing home residents. Forty participants were randomly assigned to receive either one capsule containing six bacterial strains (2.5 9 108 viable cells/ capsule) doubly coated with proteins and polysaccharides (DC group) or one capsule containing the same bacterial strains without the coating layers outside the cell (NC group) twice a day (5 9 108 viable cells/day). After consumption of 2 weeks, NC group showed a significant improvement in a symptom of Rome III criteria for constipation, ‘effort to evacuate’, but failed to show a significant improvement in weekly defecation frequency. By contrast, DC group showed significant improvements in symptoms of ‘effort to evacuate’ and ‘sensation of anorectal obstruction or blockage’ as well as being improved in ‘defecation frequency’. The quantitative analysis of the fecal bacteria revealed that the levels of all the bacterial species consumed were significantly increased after consumption in both groups. However DC group showed larger increases in the bacterial levels than NC group, and the overall bacterial levels in DC group were about 100-fold or greater than those in NC group. Therefore the improvements in the symptoms appeared to be closely associated with large increases of the bacterial species consumed. The
Y. Yeun J. Lee (&) Sahmyook Medical Center, Seoul, Republic of Korea e-mail: [email protected] Y. Yeun Department of Nursing, Kangwon National University, Samcheok, Republic of Korea
double-coating layers of proteins and polysaccharides ensured increase in the levels of bacteria, indicating that the layers function to protect the bacteria from the digestive action in the GI tract. Therefore, DuolacÒ Care could be a treatment option in functional constipation. Keywords Probiotics Functional constipation Doublecoated bacteria Non-coated bacteria Defecation frequency
Introduction Constipation is a common digestive complaint, characterized by straining during defecation, lumpy or hard stools, sensation of incomplete evacuation or anorectal obstruction, manual maneuvers to facilitate defecation, and/or less than three defecations per week (Longstreth et al. 2006). It is prevalent among people of all ages and both sexes, but its occurrence increases with age. It was reported that constipation has affected about 26 % of men and 34 % of women over 65 years of age but less than 2 % of the nonelderly people (Johanson et al. 1989; Whitehead et al. 1989). In particular, at11 least 75 % of elderly hospitalized patients and nursing home residents are known to suffer from constipation (Primrose et al. 1987). Possible factors for the development of functional constipation in the elderly include dietary changes, increased intestinal transit times, lack of physical activity, and frequent use of medications. Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (FAO/WHO 2001). Their beneficial roles in many gastrointestinal disorders such as irritable bowel syndrome (IBS) (Hoveyda et al. 2009),
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inflammatory bowel disorder (IBD) (Meijer and Dieleman 2011), and Clostridium difficile-associated diarrhea (CDAD) (Fitzpatrick 2013) have been reviewed. The rationale for the use of probiotics is they may have potential to modulate the gut function through interactions between microbes and/or between microbes and host cells. In this regard, probiotics have been introduced as a treatment option for constipation, and some probiotic strains were evaluated in randomized, controlled trials, revealing significant improvement of the symptoms of constipation (Koebnick et al. 2003; Del Piano et al. 2010). However, studies of probiotic intervention in constipation of the elderly have been limited. There is a controversy whether multi-species probiotics have greater health-promoting effects over single strain probiotics. A review examining this issue concluded that multi-species probiotics appear to have superior efficacy to single strain probiotics (Chapman et al. 2011). A recent clinical trial showed that a multispecies mixture consisting of three Bifidobacterium species and three Lactobacillus species was effective in constipation of pregnant women, where the primary outcome, defecation frequency, and some secondary outcomes were significantly improved compared to baseline (de Milliano et al. 2012). The superior efficacy of multi-species probiotics may come from the different spatial distribution of bifidobacteria and lactobacilli along the gastrointestinal tract where lactobacilli are highly abundant in the small intestine and bifidobacteria are mostly found in the large intestine (Reuter 2001). DuolacÒ Care has been formulated based on the above speculation and contains three Bifidobacterium species (Bifidobacterium bifidum, B. lactis, and B. longum), two Lactobacillus species (Lactobacillus acidophilus and L. rhamnosus), and Streptococcus thermophilus at a total of 2.5 9 108 viable cells in a capsule. In a previous randomized, double blind, placebo controlled study, a daily dosage of 1 9 1010 viable cells of the strains of DuolacÒ Care for 4 weeks was found to improve the symptoms of IBS compared to the placebo control (Yoon et al. 2014). The probiotic strains are doubly coated with proteins and polysaccharides during the process of production. Thanks to the double coating layers, the coated bacteria showed even higher resistance to acids and bile salts than noncoated bacteria (Kang et al. 2013). Therefore the doublecoated bacteria are expected to better survive during their passage through the GI tract and thus to have greater impact on the consumer than the non-coated counterparts. In this study, we investigated efficacy of DuolacÒ Care in constipation of elderly nursing home residents and evaluated the effect of the double coating layers on the viability of the consumed bacteria by quantifying fecal bacteria.
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Materials and methods Study participants Residents who were 60 years of age or older and fulfilled Rome III criteria for functional constipation (Longstreth et al. 2006) were eligible for this study. The criteria are (1) straining during at least 25 % of defecations, (2) lumpy or hard stools in at least 25 % of defecations, (3) sensation of incomplete evacuation for at least 25 % of defecations, (4) sensation of anorectal obstruction/blockage for at least 25 % of defecations, (5) manual maneuvers to facilitate at least 25 % of defecations, and (6) fewer than three defecations per week. Those who have two or more of the six criteria in at least 25 % of the evacuations during the past 6 months were included for the study. Informed consent was obtained from participants or their next of kin. People who were sensitive to probiotic products and were using medications that may affect the study result (e.g. probiotics, intestinal drugs) were excluded. Study design and procedures The study was conducted in the nursing home called Ujawon of Sahmyook Medical Center in Seoul, Korea between March 2014 and April 2014 and was approved by the Clinical Research Ethics Committee of Sahmyook Medical Center (SYMC IRB 1401-02). The enrolled participants were randomly assigned to receive either one capsule (350 mg) containing six bacterial strains doubly coated with proteins and polysaccharides (DC group) or one capsule (350 mg) containing the same bacterial strains without the coating layers outside the cell (NC group) twice a day, 30 min after breakfast and dinner, for 2 weeks. DuolacÒ Care (Cell Biotech, Co. Ltd., Gimpo, Korea) is a capsule form product containing six species of live bacteria: Bifidobacterium bifidum (KCTC 12199BP), B. lactis (KCTC 11904BP), B. longum (KCTC 12200BP), Lactobacillus acidophilus (KCTC 11906BP), L. rhamnosus (KCTC 12202BP), and Streptococcus thermophilus (KCTC 11870BP). A total of 2.5 9 108 viable cells in lyophilized powder form were included in each capsule. Participants were allocated to DC group or NC group using a computer-generated randomization schedule with a 1:1 allocation ratio. Before the start of the study, participants were instructed on the information of the study product and the way of consumption. They were also highly recommended to keep their lifestyle, e.g. eating habits, physical activity, and fluid intake, during the study period. Drug compliance was defined as the percentage of the number of capsules taken to the number of capsules prescribed, and consumption of more than 80 % of the capsules was considered to be the minimum level of compliance. All adverse reactions were reported.
A multi-species probiotic product improved symptoms of constipation
Measurements
Table 1 Questionnaire for assessing the symptoms of functional constipation based on Rome III criteria
Study outcomes
Effort to evacuate
The primary study outcome was the change of defecation frequency per week before and after treatment. The secondary outcome was the changes in symptoms of Rome III criteria. A questionnaire to assess defecation frequency and the symptoms of Rome III criteria was previously described (Longstreth et al. 2006; Favretto et al. 2013) and was recorded at the beginning and end of the study (Table 1).
In the past six months, how often have you had to force or make an effort to defecate?
(1) Sometimes (2) Often (3) Most of the times (4) Always Hard or lumpy stools In the past six months, how often did you have hard stools, hardened or as if they were marbles?
(2) Often (3) Most of the times (4) Always Sensation of incomplete evacuation In the past six months, how often have you had the sensation of incomplete evacuation, i.e. even after evacuation, the need remained?
Results
(0) Never or rarely (1) Sometimes (2) Often
Statistics The sample size was determined based on the assumptions of 30 % difference in the primary endpoint (Yoon et al. 2014), statistical power at 80 %, two-sided a risk at 5 %, and drop-out rate at 20 %. Thus we concluded that 40 people in total were needed to enroll. Efficacy and safety parameters and bacterial numbers were assessed by intention-to-treat (ITT) analysis. Descriptive statistics were reported as median with inter-quartile range or mean ± SD, unless otherwise stated. Statistical comparative analysis was performed using Prism 5 (GraphPad Software, USA). The Chi square test for categorical variables and t test (paired t test for intra-group comparison and unpaired t test for inter-group comparison) for continuous variables were used. The Wilcoxon signed rank test and the Mann–Whitney test were used to evaluate the Rome III questionnaire before and after the intervention. P \ 0.05 was considered significant.
(0) Never or rarely (1) Sometimes
Change of fecal bacteria Fecal specimens were collected at the beginning and end of the study and were used for quantification of fecal bacteria. Preparation of genomic DNA from fecal samples, quantitative real time PCR, and PCR primers were previously described (Yoon et al. 2014).
(0) Never or rarely
(3) Most of the times (4) Always Sensation of anorectal blockage or obstruction In the past six months, how often have you felt that the stool fails to pass, that it is locked or trapped in the anus?
(0) Never or rarely (1) Sometimes (2) Often (3) Most of the times (4) Always
Manual maneuvers to facilitate defecation In the past six months, how often have you had to use your finger to help in the passage of stool?
(0) Never or rarely (1) Sometimes (2) Often (3) Most of the times (4) Always
Defecation frequency per week How many times per week do you usually evacuate?________ times/ week
Participants and baseline characteristics Symptoms of constipation The forty participants were randomly assigned to receive either non-coated probiotic product (NC group) or doublecoated product (DC group) and completed the study. As shown in Table 2, there were no significant differences in the baseline characteristics (age, gender, weight, height, and BMI) between two groups.
The effects of the non-coated or double-coated bacteria on the symptoms of constipation were assessed (Table 3). Defecation frequency per week, the primary outcome, was little changed in NC group where median was determined to be 3.0 (1.5–5.3) at baseline and 3.0 (1.5–4.0) at week 2.
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assessed were found to be significantly improved in DC group after intervention (P \ 0.05) (Table 3).
Table 2 Baseline characteristics Non-coated probioticsa (n = 20)
Double coated probioticsa (n = 20)
P
Age (years)
81.10 ± 9.12
82.45 ± 9.36
NSb
Gender (male/ female)
3/20
7/20
NSc
Weight (kg)
45.92 ± 13.31
44.50 ± 7.09
NSb
Height (cm)
154.8 ± 8.70
158.5 ± 11.14
NSb
2
19.05 ± 4.89
18.20 ± 3.01
NSb
BMI (kg/m )
Quantitative changes in fecal bacteria Five hundred million of probiotic bacteria, non-coated or double-coated with proteins and polysaccharides, were consumed daily for 2 weeks. In order to investigate how the consumed bacteria impacted the gut microbiota, we examined the quantitative changes in the levels of the bacteria species consumed using quantitative real time PCR. As shown in Table 4, all the bacterial species consumed were found to be significantly increased in both groups compared to the levels at baseline. However the degree of increase in the bacterial numbers was greatly different between groups. In NC group, the numbers of the bacteria were increased by from 3.4-fold (S. thermophilus) to 14-fold (B. bifidum and L. rhamnosus). By contrast, the bacterial numbers in DC group were increased hundreds of times compared to the numbers at baseline. The increases compared to the baseline levels were from 213-fold (S. thermophilus) to 2519-fold (L. rhamnosus). Therefore this result indicates that the double-coating layers play a protective role, making the consumed bacteria more resistant to the harsh conditions due to, e.g. gastric acid and bile acids encountered in the GI tract. On the other hand, the differences in the bacterial levels between the groups after consumption were statistically significant (P \ 0.01 for all except for L. acidophilus (P \ 0.05)). Compared to the bacterial levels of NC group, the overall bacterial levels after consumption in DC group were about 100-fold or greater.
NS not significant a
Values are expressed as mean ± SD
b
Two-sided unpaired t test
c
Chi square test
By contrast, the consumption of the double-coated bacteria significantly improved stool frequency, median of which was increased from 3.0 (1.5–7.8) at baseline to 5.0 (3.1–8.8) at week 2 (P \ 0.01). It should be noted that nearly half of the participants evacuated from 4 to 20 times per week during the study but the amount was so little at baseline that it only wetted underwear or diapers. However there were some participants in both groups who reported an increase in stool amount. Meanwhile, NC group showed a significant improvement only in ‘effort to evacuate’ (P \ 0.05). By contrast, DC group showed significant improvements in ‘effort to evacuate’ (P \ 0.05) as well as ‘sensation of anorectal obstruction or blockage’ (P \ 0.05). When comparison was made between groups before and after intervention, there were no significant differences in the criteria at baseline. However, ‘hard or lumpy stools’, ‘manual maneuvers to facilitate defecation’, and ‘defecation frequency per week’ among the six criteria
Table 3 Changes in symptoms of Rome III criteria before and after intervention Symptoms
Non-coated probiotics (n = 20) Before
a
After
a
Double coated probiotics (n = 20) P
b
Before
a
After
a
P
b
Intergroup comparison Pc Before
After
Effort to evacuate
1.0 (1.0–2.0)
1.0 (0.4–1.0) \0.05
1.0 (0.2–1.0)
0.0 (0.0–1.0)
\0.05
NS
NS
Hard or lumpy stools
1.0 (0.0–1.0)
1.0 (0.4–1.0)
1.0 (0.0–1.0)
0.0 (0.0–1.0)
NS
NS
\0.05
NS
Sensation of incomplete evacuation
2.0 (2.0–2.0)
2.0 (2.0–2.0)
NS
2.0 (2.0–2.0)
2.0 (1.0–2.0)
NS
NS
NS
Sensation of anorectal obstruction or blockage
1.0 (1.0–1.0)
1.0 (1.0–1.0)
NS
1.0 (0.2–1.0)
0.0 (0.0–1.0)
\0.05
NS
NS
Manual maneuvers to facilitate defecation
1.0 (0.0–1.0)
1.0 (0.0–1.0)
NS
1.0 (0.0–1.0)
0.0 (0.0–0.0)
NS
NS
\0.05
Defecation frequency per week
3.0 (1.5–5.3)
3.0 (1.5–4.0)
NS
3.0 (1.5–7.8)
5.0 (3.1–8.8)
\0.01
NS
\0.05
NS not significant a
values are expressed as median and inter-quartile range (25, 75 %)
b
Wilcoxon signed rank test
c
Mann–Whitney test
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A multi-species probiotic product improved symptoms of constipation Table 4 Quantitative changes of fecal bacteria assessed before and after intervention Non-coated probiotics (n = 20) log10 cells/g feces Before
a
Double coated probiotics (n = 20) P
b
After
log10 cells/g feces Before
a
P
Between groups after treatment b
Fold change
Pc
After
B. bifidum
5.28 ± 5.55
6.42 ± 6.72
\0.05
5.87 ± 6.23
8.45 ± 8.64
\0.01
108
\0.01
B. lactis
5.46 ± 5.72
6.13 ± 6.41
\0.05
5.65 ± 5.79
8.26 ± 8.39
\0.01
137
\0.01
B. longum L. acidophilus
6.17 ± 6.34 5.44 ± 5.52
7.09 ± 7.16 6.26 ± 6.43
\0.001 \0.01
6.19 ± 6.55 5.46 ± 5.62
9.08 ± 9.08 8.22 ± 8.48
\0.001 \0.05
98 92
\0.001 \0.05
L. rhamnosus
1.98 ± 1.91
3.06 ± 3.02
\0.001
2.01 ± 2.06
5.41 ± 5.59
\0.01
221
\0.01
S. thermophilus
4.80 ± 4.88
5.33 ± 5.40
\0.01
5.03 ± 5.12
7.36 ± 7.38
\0.001
105
\0.001
a
Values are expressed as mean ± SD
b
One-sided paired t test
c
Two-sided unpaired t test
Safety The treatments were well tolerated, and the drug compliance rates were [80 % in all patients. No adverse events or serious adverse events were reported in both groups during and after consumption.
Discussion The definition of probiotics by FAO/WHO (2001) indicates that the consumed microbes must reach the intestine alive to exert their health-promoting activities. The rationale for the use of probiotics in treatment of constipation is disturbed state of the intestinal microbiota reported in patient with constipation (Zoppi et al. 1998) and their potential to improve intestinal motility (Salminen and Salminen 1997). DuolacÒ Care is a multi-species probiotic product produced with double coating layers of proteins and polysaccharides, and the coating technology has been patented in Korea (patent no. 0429495), Japan (patent no. 3720780), and Europe (patent no. 1514553B). Since the bacterial strains with the coating layers showed higher viability under a simulated gastrointestinal condition compared to the non-coated counterparts (Kang et al. 2013), it has been assumed that the double-coated bacteria better survive in the gastrointestinal tract upon ingestion, giving the host health benefits. In this study, we compared an efficacy of a double-coated multi-species probiotic product, DuolacÒ Care, in functional constipation in 40 elderly nursing home residents with its non-coated counterparts. Based on the higher viability observed in the previous study (Kang et al. 2013), it was expected that the double-coated bacteria would have greater effects in this study. When the symptoms of Rome III criteria before and after intervention were assessed, the consumption of the
no-coated bacteria appeared to be significantly effective in certain symptoms, ‘effort to evacuate’ and ‘hard or lump stools’. By contrast, DC group showed significant improvements in all the symptoms assessed. Thus the result shows that the consumption of the double-coated bacteria has greater effects in constipation than the non-coated counterparts. Since both groups consumed the same bacteria, the presence of the coating layers likely affected viable cell numbers reaching the intestine and in turn contributed to improve the symptoms of constipation. This speculation was supported by the quantitative analysis of the fecal bacteria before and after consumption. Although the bacterial levels examined were significantly increased after consumption in both groups, the increases in DC group were even larger than those in NC group. When compared to the bacterial levels after consumption in NC group, those in DC group were even 100-fold or greater. Species of Bifidobacterium and Lactobacillus have been found to be effective in the management of constipation (Favretto et al. 2013; Lahtinen et al. 2009; Koebnick et al. 2003). The proposed mechanisms are changes in the composition of the gut flora (e.g. increase in the levels of bifidobacteria), increase in the production of short chain fatty acids which stimulate bowel movement, and consequently decrease of transit time (Salminen and Salminen 1997). Thus it is reasonable to think that the larger increase in the levels of all the bacterial species was closely associated with the greater improvements in the symptoms of constipation in DC group. On the other hand, the improvements of the symptoms in the DC group seem not to be large considering that the bacterial levels were increased 100-fold or greater. This may be primarily due to the short consumption period (2 weeks) or/and old age of the participants ([81 years of age). As aforementioned, functional constipation in the elderly can be developed by many factors such as dietary changes, increased intestinal
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transit times, lack of physical activity, and frequent use of medications. Thus the consumption period of 2 weeks may have not been long enough to highly elicit changes in the physiology and function of the GI tract, and longer period of consumption (e.g. [1 month) may be needed for the elderly. In conclusion, the consumption of DuolacÒ Care for 2 weeks improved defecation frequency and other symptoms of constipation in elderly nursing home residents. The improvements appeared to be closely associated with large increases of the bacterial species consumed. The doublecoating layers of proteins and polysaccharides ensured increase in the levels of bacteria, indicating that the layers function to protect the bacteria from the digestive action in the GI tract. Acknowledgments We would like to thank Dr. Seo from Cell Biotech for technical assistance. This study was funded by Cell Biotech. Conflict of interest The authors declare no conflict of interest that could inappropriately influence their work.
References Chapman, C.M., G.R. Gibson, and I. Rowland. 2011. Health benefits of probiotics: are mixtures more effective than single strains? European Journal of Nutrition 50: 1–17. de Milliano, I., M.M. Tabbers, J.A. van der Post, and M.A. Benninga. 2012. Is a multispecies probiotic mixture effective in constipation during pregnancy? ‘A pilot study’. Nutrition Journal 11: 80. Del Piano, M., S. Carmagnola, A. Anderloni, S. Andorno, M. Ballare`, M. Balzarini, F. Montino, M. Orsello, M. Pagliarulo, M. Sartori, R. Tari, F. Sforza, and L. Capurso. 2010. The use of probiotics in healthy volunteers with evacuation disorders and hard stools: A double-blind, randomized, placebo-controlled study. Journal of Clinical Gastroenterology 44(1): S30–S34. FAO/WHO (2001) Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria. Cordoba, Argentina: Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation report 1–34. Favretto, D.C., B. Pontin, and T.R. Moreira. 2013. Effect of the consumption of a cheese enriched with probiotic organisms (Bifidobacterium lactis bi-07) in improving symptoms of constipation. Arquivos de Gastroenterologia 50: 196–201.
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Fitzpatrick, L.R. 2013. Probiotics for the treatment of Clostridium difficile associated disease. World Journal Gastrointestinal Pathophysiology 4: 47–52. Hoveyda, N., C. Heneghan, K.R. Mahtani, R. Perera, N. Roberts, and P. Glasziou. 2009. A systematic review and meta-analysis: probiotics in the treatment of irritable bowel syndrome. BMC Gastroenterology 16: 9–15. Johanson, J.F., A. Sonnenberg, and T.R. Koch. 1989. Clinical epidemiology of chronic constipation. Journal of Clinical Gastroenterology 11: 525–536. Kang, J.-H., D.-K. Lee, J.-E. Park, M.-J. Kim, J.S. Lee, J.-G. Seo, M.J. Chung, H.-S. Shin, and N.-J. Ha. 2013. Dual coating improves the survival of probiotic Bifidobacterium strains during exposure to simulated gastrointestinal conditions. Kor. Journal of Microbiology 49: 275–281. Koebnick, C., I. Wagner, P. Leitzmann, U. Stern, and H.J. Zunft. 2003. Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation. Canadian Journal of Gastroenterology 17: 655–659. Lahtinen, S.J., L. Tammela, J. Korpela, R. Parhiala, H. Ahokoski, H. Mykka¨nen, and S.J. Salminen. 2009. Probiotics modulate the Bifidobacterium microbiota of elderly nursing home residents. Age (Dordr) 31: 59–66. Longstreth, G.F., W.G. Thompson, W.D. Chey, L.A. Houghton, F. Mearin, and R.C. Spiller. 2006. Functional bowel disorders. Gastroenterology 130: 1480–1491. Meijer, B.J., and L.A. Dieleman. 2011. Probiotics in the treatment of human inflammatory bowel diseases: update. Journal of Clinical Gastroenterology 45: S139–S144. Primrose, W.R., A.E. Capewell, G.K. Simpson, and R.G. Smith. 1987. Prescribing patterns observed in registered nursing homes and long-stay geriatric wards. Age and Ageing 16: 25–28. Reuter, G. 2001. The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession. Current Issues in Intestinal Microbiology 2: 43–53. Salminen, S., and E. Salminen. 1997. Lactulose, lactic acid bacteria, intestinal microecology and mucosal protection. Scandinavian Journal of Gastroenterology 32: 45–48. Whitehead, W.E., D. Drinkwater, L.J. Cheskin, B.R. Heller, and M.M. Schuster. 1989. Constipation in the elderly living at home. Definition, prevalence, and relationship to lifestyle and health status. Journal of the American Geriatrics Society 37: 423–429. Yoon, J.-S., W. Sohn, O.-Y. Lee, S.-P. Lee, K.-N. Lee, D.-W. Jun, H.L. Lee, B.-C. Yoon, H.-S. Choi, W.-S. Chung, and J.-G. Seo. 2014. Effect of multispecies probiotics on irritable bowel syndrome: a randomized, double-blind, placebo-controlled trial. Journal of Gastroenterology and Hepatology 29: 52–59. Zoppi, G., M. Cinquetti, A. Luciano, A. Benini, A. Muner, and M.E. Bertazzoni. 1998. The intestinal ecosystem in chronic functional constipation. Acta Paediatrica 87: 836–841.
