1. Introduction
Constipation is not simply defined as a small number of bowel movements. Treatment is required because patients with constipation have a significantly lower survival rate [1] and more impediments to their daily activities [2] than in those with other gastrointestinal diseases. Constipation can be either organic (associated with morphological changes in the large bowel; e.g., inflammatory bowel disease, intestinal obstruction) or functional (not associated with large bowel morphological changes; e.g., irritable bowel syndrome, drug-induced constipation) [1] [3] . Functional constipation is a health problem that negatively impacts the quality of life (QOL) [4] , according to systematic review and meta-analysis results, its global prevalence among adults is between 14%, and its prevalence increases modestly with increasing age [5] [6] . Therefore, constipation is a health problem that must be addressed. Typically, various agents, such as laxatives and fibers, are used to treat constipation, and probiotics have also been reported to be effective [7] . The mechanism of action by which probiotics such as Bifidobacterium improve constipation includes normalizing the intestinal environment [8] [9] [10] , decreasing methanogenic bacteria [11] , and increasing serotonin production [12] [13] . However, the effectiveness of probiotics varies significantly among individuals because the human intestinal environment is diverse.
Our previous double-blind study of healthy volunteers prone to constipation revealed that heat-killed B. longum CLA8013 effectively increased the frequency of defecation when ingested continuously over a 2-week period at a dose of 25 billion cells/day [14] . The present trial comprised two studies aimed at evaluating the safety of B. longum CLA8013 in healthy volunteers to verify the potential of B. longum CLA8013 as a food or supplement: a long-term intake safety study and an excessive intake safety study.
2. Participants and Methods
The long-term intake and excessive intake safety studies were approved by the Ethics Review Committee of the Kobuna Orthopedic Clinic (approval date: November 10, 2021; approval numbers: MK-2211-03 and MK-2211-02, respectively) and were registered and published in the UMIN clinical trial registration system (clinical trial registration numbers: UMIN000049596 and UMIN000049771, respectively). The trial was conducted in accordance with the principles of the Declaration of Helsinki and the Ethical Guidelines for Medical Research Involving Human Participants. The principal investigator adequately explained what the study entailed to the participants who voluntarily provided written consent to participate.
The study was conducted at a clinic in Tokyo, Japan. Medical interviews and examinations were conducted by physicians and nurses at the Shinagawa Season Terrace Health Care Clinic. The studies were conducted in collaboration with the KSO Corporation, a contract research organization that was responsible to recruit participants.
2.1. Participants
Healthy adult male and female volunteers (age: 20 - 65 years old) were recruited by the KSO Corporation’s participants panel. The recruitment criteria is subjective symptoms that prone to constipation with 3 - 5 spontaneous bowel movements per week or normal defecation exceeding 5 times per week. All participants were provided with adequate explanation of the study, including its purpose, contents, methods, and potential adverse events. Written informed consent was obtained from all participants before study initiation.
The inclusion and exclusion criteria are presented in Table 1. These criteria were set up to recruit for healthyorprone to constipation participants, three–five spontaneous bowel movements per week was set because one of the criteria for functional constipation is fewer than three bowel movements per week [3] . Discontinuation criteria are listed in Table S1.
2.2. Study Design
The two safety studies were placebo-controlled, randomized, double-blind, and parallel. Ingestion of the study substance began after selecting the participants
![]()
Table 1. Inclusion and exclusion criteria.
via screening and after a 2-week pre-observation period. The intervention periods were 12 and 4 weeks for the long-term and excessive intake safety studies, respectively. Post-study observation of the participants was conducted for 2 weeks after the conclusion of both studies (Figure 1). Participants were randomly assigned to one of the two groups at the time of screening to preclude biases based on age, sex, body mass index, or defecation frequency. In the long-term intake safety study, participants ingested one capsule once per day, whereas in the excessive intake safety study, participants ingested five capsules once per day. The participants were instructed to drink large amounts of water while ingesting the capsules. If the participants forgot to take the capsules at the designated time, they were instructed to take the capsules as soon as they became aware of the missed dose, rather than doubling the dose the following day.
The study substance comprised capsules containing heat-killed B. longum CLA8013 at a dose of 25 billion cells, in a formulation containing crystalline cellulose, calcium stearate, and silicon dioxide. The placebo group received capsules that did not contain heat-killed B. longum CLA8013. Both sets of capsules were manufactured by API Co., Ltd. (Gifu, Japan; Table 2). In the long-term intake safety study, participants in the active group ingested 25 billion cells/day for 12 weeks, while in the excessive intake safety study, participants in the active group ingested 125 billion cells/day for 4 weeks.
In the long-term intake safety study, we measured the parameters indicated below at the baseline; at ingestion weeks 4, 8, and 12; and subsequently at 2 weeks following the end of ingestion. In the excessive intake safety study, we
![]()
Figure 1. Intervention schedule. (a) The long-term intake safety study was conducted from November 2022 to March 2023; (b) The excessive intake safety study was conducted from January 2023 to March 2023.
![]()
Table 2. Composition, nutrients, duration and dosage of capsules.
measured the same parameters at baseline; at ingestion weeks 2 and 4; and at 2 weeks after the end of ingestion.
In addition, we instructed the participants to maintain diaries containing data on their physical condition, meal content, and defecation status. We confirmed the safety of participants using this data. The parameters examined were as follows: 1) a medical interview conducted by a physician, 2) physical examination (body weight, body mass index [BMI], systolic blood pressure, diastolic blood pressure, and pulse rate), 3) hematological examination (white blood cell count, red blood cell count, hemoglobin (Hb), hematocrit (Ht), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differential leucocyte count [neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EOSINO), and basophils (BASO)], 4) blood biochemical examination (total protein, albumin, aspartate aminotransferase (AST (GOT)), alanine aminotransferase (ALT (GPT)), lactic dehydrogenase (LD (IFCC)), total bilirubin, alkaline phosphatase (ALP (IFCC)), gamma-glutamyl transferase (gamma-GT), urea nitrogen (UN), creatinine, uric acid (UA), sodium (Na), chloride (Cl), potassium (K), calcium (Ca), total cholesterol, low density lipoprotein (LDL)-cholesterol, high density lipoprotein (HDL)-cholesterol, triglyceride (TG), glucose, and HbA1c (NGSP value)), 5) urinalysis (urine protein, urine glucose, urobilinogen, urine bilirubin, urine pH, urine specific gravity, urine ketone body, and occult blood reaction) 6) defection frequency (defecation frequency, number of defecation days, stool volume, and stool consistency (Bristol Stool Form Scale)).
2.3. Statistical Analysis
The means and standard deviations were calculated for all the baseline parameters measured during the screening period before intake. Statistical analysis was performed using one-way analysis of variance (ANOVA), and the measured values were tabulated.
The full analysis set (FAS) at the start of the ingestion of the study substance was used to analyze all the safety evaluation parameters. If a participant had ingested the study substance, tests were performed at the time of dropout as necessary, and the results of the parameters related to safety were included in our evaluation. All data were presented as mean values and standard deviations. The study data that were believed to be outliers were also included in our evaluation. Statistical analysis was performed using the unpaired t-test to compare the active treatment groups with the placebo groups, and the Wilcoxon rank-sum test was performed on the qualitative evaluation parameters. Statistical significance was set at 5%. Statistical analyses were performed using SPSS ver. 27 (IBM Corp., Armonk, NY, USA).
3. Results
3.1. Long-Term Intake Safety Study
1) Participant characteristics
A Consolidated Standards of Reporting Trials (CONSORT) flow chart for this study is shown in Figure 2, and participant characteristics are listed in Table 3. Of the 30 participants, there was one discontinuation/dropout from the active group. Therefore, 29 participants (placebo group: 15; active group: 14) completed the study. One participant withdrew consent to participate in the study for personal reasons.
2) Physical, Hematological, Urinalysis, and Biochemical Examinations
No significant changes were observed in the physical, (Table S2), hematological (Table S3), or urine examination parameters (Table S5). The biochemical examinations revealed that the active group exhibited significantly higher glucose levels and HbA1C than the placebo group. No significant differences were observed in other parameters (Table S4). Although significant changes were observed in several parameters during the study, these were within the range of physiological fluctuations and were deemed clinically irrelevant. Based on a comprehensive analysis of the examination results, the principal investigator determined that there were no safety concerns regarding the long-term or excessive intake of heat-killed B. longum CLA8013.
3) Defecation frequency
Compared with the placebo group, the active group exhibited significant changes in the following parameters: higher defecation frequency at week 14 and higher stool volume at weeks 1 and 14. No significant changes were observed in other parameters (Table S6). Based on the examination results, the principal investigator concluded that there were no safety concerns.
![]()
Figure 2. Flow diagram of the long-term intake safety study.
4) Adverse events
Twelve adverse events (active group: seven events in five participants, placebo group: five events in four participants) were observed during the study period (Table 4). The principal investigator concluded that none of the adverse events were related to the study capsules because the events were accidental or occurred outside the intervention period.
3.2. Excessive Intake Safety Study
1) Participant characteristics
A CONSORT flow diagram for this trial is shown in Figure 3, and the participant characteristics are listed in Table 5. None of the participants met the predefined discontinuance criteria during the study period. Therefore, data obtained from all 30 participants were included in the analysis.
2) Physical, Hematological, Urinalysis, and Biochemical Examinations
No significant changes were observed in the physical (Table S7) or hematological examination parameters (Table S8). However, biochemical examinations revealed significant changes in the active group compared with the placebo group in the following parameters: higher uric acid level at week 2, lower sodium level at week 6, lower chloride level at week 6, and higher potassium level at week 6. No significant changes were observed in any other parameters (Table S9). Urinalysis revealed that the active group had significantly lower urine pH at weeks 2 and 6 than that in the placebo group. No significant changes were observed in other parameters (Table S10). Although significant changes were
![]()
![]()
(a)p value: ANOVA.
(b)p value: Wilcoxon’s rank sum test (asymptotic significance probability).
(a)
Table 3. (a) Baseline parameters of physical, hematological, and biochemical examinations (Long-term intake safety study). (b) Baseline parameters of urine examinations (Long-term intake safety study).
observed in uric acid, sodium, chloride, potassium, and urine pH levels during the study, these changes were within the range of physiological fluctuations and were deemed clinically irrelevant. Based on the examination results, the principal investigator concluded that there were no safety concerns.
3) Defecation frequency
The active group showed a significantly lower difference in stool volume per week at week 2 than that of the placebo group. No significant changes were observed in other parameters (Table S11).
4) Adverse events
Three adverse events (active group: 1, placebo group: 2) were observed during the study period (Table 6). The principal investigator concluded that none of the adverse events were related to the study capsules because the events were accidental or occurred outside the intervention period.
4. Discussion
To develop a food with a functional claim that would be effective for constipation, we focused on hard stools as a cause of difficulty with bowel movements and conducted screening to evaluate the effectiveness of the intervention in
![]()
Table 4. Adverse events (Long-term intake safety study).
![]()
![]()
(a)p value: ANOVA.
(b)p value: Wilcoxon’s rank sum test (asymptotic significance probability).
(a)
Table 5. (a) Baseline parameters of physical, hematological, and biochemical examinations (Excessive intake safety study). (b) Baseline parameters of urine examinations (Excessive intake safety study).
promoting fluid secretion within the intestinal tract. This allowed us to identify the human-derived Bifidobacterium known as Bifidobacterium longum. B. longum is a species listed on the European Food Safety Authority (EFSA) “list of qualified presumption of safety (QPS)-recommended biological agents intentionally added to food or feed as notified to EFSA” [15] . In vivo efficacy evaluation showed that heat-killed B. longum CLA8013 improved the moisture content of feces and promoted bowel movements (unpublished data). An in-house safety evaluation was performed using approximately 2.1 billion units of heat-killed B. longum CLA8013 as the study substance. Single-dose toxicity testing using
![]()
Table 6. Adverse events (Excessive intake safety study).
![]()
Figure 3. Flow diagram of the excessive intake safety study. A total of 75 individuals were registered and assessed for eligibility; thirty participants participated in the study.
Sprague–Dawley rats revealed that, at a maximum dose of 2000 mg/kg, the general condition, body weight, and autopsy results were normal. Repeated oral dosage toxicity testing at doses of 0, 500, 1000, and 2000 mg/kg showed no toxic changes in any of the following: general condition, body weight, amount of intake, ophthalmological tests, urinalysis, hematological tests, blood chemical tests, organ weight, autopsy results, or histopathologic tests. Based on these results, the no observed adverse effect level (NOAEL) for both men and women was determined to be 2000 mg/kg. The results of chromosome aberration and Ames tests of Salmonella typhimurium and Escherichia coli using the Chinese hamster lung cell line (CHL/IU) were negative.
The effectiveness of heat-killed B. longum CLA8013 was previously evaluated in a double-blind, placebo-controlled study of healthy volunteers prone to constipation, which was conducted after confirming its safety in animals. The study showed that ingesting heat-killed B. longum CLA8013 at a dose of 25 billion cells/day for 2 weeks effectively improved bowel movements [14] . After confirming the usefulness of heat-killed B. longum CLA8013, we conducted the two safety studies: long-term intake safety study and excessive intake safety study. Based on our study findings, we conclude that heat-killed B. longum CLA8013 is not associated with safety-related issues.
5. Conclusion
Our randomized, double-blind, placebo-controlled, parallel-group study confirmed the safety of long-term (25 billion cells/day for 12 weeks) and excessive (125 billion cells/day for 4 weeks) intake of heat-killed B. longum CLA8013.
Acknowledgements
Data acquisition was performed by the staff of the KSO Corporation. We gratefully acknowledge the contributions of all the staff involved in this study.
Funding
The funding for this study was provided by Biofermin Pharmaceutical Co., Ltd. Daisuke Takami, Keisuke Okada, Yutaka Makizaki, Yoshiki Tanaka, and Hiroshi Ohno are employees of Biofermin Pharmaceutical Co. Ltd.
Appendixs (Tables S1-S11)
https://zenodo.org/records/10016356/files/Supplemental%20Tables.xlsx?download=1