Abstract
Fermented milk containing Lacticaseibacillus paracasei strain Shirota (LcS) has been shown to improve stool consistency in various countries; however, its effect on hard or lumpy stools (HLS) in the Vietnamese population remains unclear. We investigated the effects of LcS-fermented milk on constipated Vietnamese adults with a high prevalence of HLS. In a single-centre, open-label trial, 51 participants with HLS were randomised to receive one bottle per day of fermented milk containing â¥6.5 à 109 cfu of LcS for 4 weeks (probiotic group) or no intervention (control group), followed by a 2-week washout. The primary endpoint was the proportion of participants with HLS (Bristol Stool Form Scale score of 1 or 2) in â¥25% of bowel movements over 4 weeks. Secondary endpoints included total stool frequency, HLS frequency, ideal stool form, and defecation-related symptoms â all recorded in daily diaries. The Chinese Constipation Questionnaire (CCQ) and gut microbiota composition (16S rRNA sequencing) were assessed every 2 weeks. During the intervention, the probiotic group had a significantly lower proportion of participants with HLS in â¥25% of bowel movements compared to controls (odds ratio: 0.00; 95% confidence interval: 0.00-0.16;
The study was registered at ClinicalTrials.gov (ID: NCT05982743).
1 Introduction
Constipation is a common symptom-based gastrointestinal disorder characterised by infrequent bowel movements, often accompanied by pain and a sense of incomplete evacuation, affecting 2.6 to 26.9% of the adult population worldwide (Forootan et al., 2018; Schmidt and Santos, 2014). Acute constipation can lead to intestinal obstruction, potentially requiring surgical intervention, whereas the chronic form significantly impairs patientsâ quality of life and places a substantial burden on the healthcare system (Belsey et al., 2010; Benninga et al., 2005). The production of hard or lumpy stools (HLS) is strongly associated with the development of constipation and is considered a core symptom (Patimah et al., 2017). HLS is frequently observed in individuals with chronic constipation and in healthy individuals. Therefore, reducing the incidence of HLS may benefit a broad range of individuals by reducing their overall risk of constipation.
Among various treatment options, probiotics have emerged as a promising intervention for managing constipation. Recent systematic reviews and meta-analyses have shown that probiotics can significantly improve bowel movement frequency and stool consistency in adults with constipation, although the magnitude of the effect varies depending on the strain and formulation (Ding et al., 2024; Garzon Mora and Jaramillo, 2024). Lactobacillus casei strain Shirota (reclassified as Lacticaseibacillus paracasei strain Shirota in April 2020) (LcS) is a well-characterised probiotic, first isolated and cultured by Dr Minoru Shirota in 1930. LcS reaches the intestines alive and confers various health benefits to the host (Qi et al., 2020; Yan et al., 2022; Zheng et al., 2020). Several studies have investigated the effects of fermented milk beverages containing LcS (Yakult®) on constipation symptoms, including HLS. Randomised controlled trials conducted in constipated populations in Europe (Koebnick et al., 2003; Sakai et al., 2011; Tilley et al., 2014; Tsujibe et al., 2024), the United States (Cook et al., 2025), and Japan (Matsumoto et al., 2006) have demonstrated that daily intake of LcS-containing fermented milk alleviates gastrointestinal symptoms and reduces the prevalence of hard stools. In one such study, Matsumoto et al. observed an increase in beneficial microbes (e.g. Bifidobacterium) along with elevated concentrations of organic acids, suggesting a favourable shift in the intestinal environment (Matsumoto et al., 2006). Additionally, Chen et al. examined the effects of LcS-containing fermented milk in Chinese patients with functional constipation, stratified by baseline stool consistency (Chen et al., 2019). Notably, in individuals with hard stools (the HS group), 4 weeks of daily LcS supplementation significantly alleviated constipation symptoms and increased stool frequency. These clinical improvements were accompanied by distinct alterations in the gut microbiota, including increased abundances of Pseudobutyrivibrio and Roseburia, and elevated concentrations of organic acids. These findings suggest that the anti-constipation effects of LcS, particularly in individuals with hard stools, may be mediated through the modulation of the intestinal microbiota and its metabolic environment. However, the specific patterns of modulation may vary across populations.
The promising findings about the anti-constipation effect of LcS-containing fermented milk have not been evaluated in the Vietnamese population. To address this gap, the present pilot study was conducted as a single-centre randomised controlled trial to investigate the efficacy of this beverage in healthy Vietnamese adults with HLS. Additionally, we evaluated whether the observed clinical outcomes were associated with gut microbiome shifts.
2 Materials and methods
Ethical considerations
This study was reviewed and approved by the IRB of Bach Mai Hospital (approval ID: 2138/QD-BVBM, dated 2 August 2021) and conducted in accordance with the principles of the Declaration of Helsinki, the International Council for Harmonisation-Good Clinical Practices (ICH-GCP E6), and Vietnamese regulatory laws governing biomedical research involving human participants. The study was registered at ClinicalTrials.gov (ID: NCT05982743).
Study design and setting
This single-centre, open-label, randomised controlled trial was conducted at Bach Mai Hospital and Vietnam National University (Hanoi, Vietnam), from April 2022 to March 2023. Participants with HLS were identified from individuals attending routine health check-ups at the Outpatient Department of Bach Mai Hospital, based on the Bristol Stool Form Scale (BSFS) score. Block randomisation stratified by sex was performed using randomly permuted blocks of 2 or 4 to allocate participants to either the probiotic group or the control group in a 1:1 ratio through a centralised, web-based randomisation system. The system automatically generated the randomisation number and treatment assignment in real time, ensuring blinded group allocation. The probiotic group consumed one bottle of LcS-containing fermented milk daily for 4 weeks, and the control group received no interventions. Subsequently, both groups underwent a 2-week washout period without interventions. CRO Smart Research (https://www.smartresearch.com.vn/) supported the study by drafting the study documents, generating and concealing the randomisation sequence, managing the site, monitoring data, and performing biostatistics.
Eligibility criteria
The inclusion criteria were: age between 18 and 60 years, awareness of frequently producing HLS (BSFS score of 1 or 2), production of HLS in â¥25% of bowel movements during a 2-week screening period and provision of written informed consent.
The exclusion criteria were primary organic disease of the colon or pelvic floor or use of medication causing constipation, presence of significant concomitant diseases including neurological or metabolic disorders, known allergy to milk ingredients, pregnancy or breastfeeding, inability to refrain from or anticipated use of medications including diuretics, laxatives, prescription and non-prescription drugs, vitamins, and herbal supplements, except for paracetamol, oral contraceptives, or hormonal replacement therapy from 2 weeks before screening until study completion, inability to refrain from or anticipated use of probiotics, prebiotics, or yogurts during the same period, participation in another investigational study within 2 months before this study and drug or alcohol abuse.
Investigational product
LcS-fermented milk (Yakult®), each 65 ml, containing at least 6.5 à 109 cfu of LcS at the end of shelf life, was supplied by Yakult Vietnam Co., Ltd. (Ho Chi Minh City, Vietnam). The viable microbial count was guaranteed by the manufacturer using the same quality specifications applied to the commercially available product. The investigated product (IP) was stored at 2-10 °C. Although the manufacturerâs recommended storage temperature in Vietnam is 5-10 °C, a broader range was predefined to account for potential operational challenges in maintaining a narrow temperature band. Continuous temperature monitoring with a data logger ensured compliance with this range. Shipments were delivered biweekly, with expiry dates managed to ensure at least 21 days of remaining shelf life upon arrival at the study site. Participants consumed one bottle daily for 4 weeks, preferably after breakfast, reflecting real-world consumer behaviour. Adherence was defined as consumption of at least 80% of the IP. Participants could withdraw at any time.
Study endpoints
The primary efficacy endpoint was the proportion of participants producing HLS in â¥25% of bowel movements. Secondary efficacy endpoints included total stool frequency; frequency of HLS; ideal stool form (BSFS score 4); and defecation-related symptoms, including straining during evacuation, sensation of remaining stool in the rectum, sensation of anorectal blockage, and use of manual manoeuvres, and the Chinese Constipation Questionnaire (CCQ) score. A cut-off score of â¥5 on the CCQ, with higher scores indicating functional constipation, was established to distinguish healthy participants from patients with functional constipation, with 91% sensitivity and 91% specificity (Chan et al., 2005). Gut microbiota measures were assessed using faecal samples, including alpha-diversity, beta-diversity, and relative abundance of Bifidobacterium and other bacterial taxa. Adverse events (AEs) were recorded and followed up for safety throughout the study period.
Data collection
Data collection schedules are presented in Figure 1 and Supplementary Table S1. Demographic and clinical data were collected during the study visits. Stool consistency (BSFS) and bowel habits were recorded in a daily diary per bowel movement. Participants provided stool samples for stool microbiota analysis and completed the CCQ every 2 weeks after the start of the intervention.
Analysis of stool microbiota
Stool microbiota were analysed using next-generation sequencing on samples collected at the end of a 2-week baseline period (Week 0), on Weeks 2 and 4 after the intervention, and following a subsequent 2-week washout period (Week 6). Genomic DNA was extracted from stool samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany), following a previously published protocol, with slight modifications (Matsuki et al., 2016). The V1-V2 regions of the 16S rRNA gene were amplified using the forward primer 5â²-AGAGTTTGATCMTGGCTCAG-3â² and the reverse primer 5â²-TGCTGCCTCCCGTAGGAGT-3â² (Kim et al., 2013) in PCR reactions with a total volume of 15 μl, comprising Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 2 μM of each primer, and 10 ng of template DNA. The thermal cycling protocol included an initial denaturation at 96 °C for 1 min, followed by 30 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, with a final extension step at 72 °C for 5 min. PCR products were quantified and qualified by mixing with an equal volume of 1X loading buffer containing SYBR Green, followed by electrophoresis on a 2% agarose gel. The PCR products were then combined in equidensity ratios and purified using a Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturerâs instructions, with index codes added. Library quantity and quality were assessed using the Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). Libraries were then sequenced on an Illumina NovaSeq platform, producing 250 bp paired-end reads.
Microbiota analysis was performed based on 16S rRNA gene V1-V2 sequencing using the open-source software QIIME 2 (Version Qiime2-amplicon-2024.2) (Bolyen et al., 2019). The resulting sequences were assigned to amplicon sequence variants (ASVs) using the USEARCH algorithm (Edgar, 2010), with a 99% identity threshold. Taxonomy for each representative ASV was assigned using an RDP-naïve Bayesian classifier with a minimum bootstrap threshold of 50% (Wang et al., 2007). A single representative from each OTU was aligned using the MUSCLE alignment tool (Edgar, 2004), and a phylogenetic tree was constructed using FastTree (Price et al., 2009). Alpha-diversity (observed ASVs, Chao1 index, Shannon index, and Faithâs phylogenetic diversity) and beta-diversity (Bray-Curtis, Jaccard, unweighted UniFrac, and weighted UniFrac) were estimated for 57,571 randomly selected sequences (the minimum sample depth across samples) to account for differences in sampling effort.
Changes to trial protocol
Although dietary information was originally planned to be collected using a Food Frequency Questionnaire (FFQ), valid data could not be obtained due to issues during the data-collection process. As a result, dietary intake could not be analysed as initially intended.
Sample size
The sample size calculation was based on the primary endpoint â the proportion of individuals producing HLS in â¥25% of bowel movements. The expected proportion in the control arm (not consuming the IP) was 85%, versus 36.8% in the probiotic arm. This expectation was based on data from a study conducted in Belgium that applied similar eligibility criteria and used fermented milk containing the same dose of LcS (Sakai et al., 2011). Accordingly, a sample size of 22 participants per group was required. This study aimed to recruit 50 participants to account for potential losses during follow-up. Assuming a screening failure rate of approximately 50%, 100 individuals were screened.
Statistical methods
Efficacy analyses followed the intention-to-treat principle and included all randomised individuals with available efficacy data (Full Analysis Set). Statistical analyses were performed using R (version 4.3.1), unless otherwise specified. Between-group comparisons were performed using Fisherâs exact test for categorical variables and the Mann-Whitney U test for continuous variables. Mixed-effects logistic regression with a random intercept, adjusting for baseline values and sex, was used to estimate the overall treatment effect on the risk of producing HLS in â¥25% of bowel movements and exceeding the CCQ score â¥5 throughout the intervention. For microbiota outcomes, differential abundance analyses at the phylum, class, order, family, and genus levels were performed at each time point using ANCOM-BC, applying the default settings of the ancombc function with Holm-adjusted P-values (Lin and Peddada, 2020). Alpha- and beta-diversity metrics were compared between the groups using the Wilcoxon-Mann-Whitney U test and PERMANOVA on the QIIME 2 platform. P-values less than 0.05 were considered statistically significant.
3 Results
Patient disposition, demographic, and baseline characteristics
A total of 51 eligible participants were randomised in a 1:1 ratio to the probiotic (n = 24) and control (n = 27) groups. The flow of participants throughout the study, from enrolment to analysis, is shown in Figure 2. Demographic and baseline characteristics are presented in Table 1. Two participants were excluded from the efficacy analysis because of missing efficacy data, resulting in 49 participants in the Full Analysis Set.
All 24 randomised individuals in the probiotic arm were considered treatment compliant, having consumed at least 80% of the dispensed product (Supplementary Table S2).
Analysis of efficacy â primary endpoint
The proportion of individuals with HLS (BSFS score of 1 or 2) in â¥25% of bowel movements from Week 0 to Week 6 is shown in Table 2. Overall, this proportion decreased in the probiotic group but remained stable in the control group, resulting in significant differences between the groups at Weeks 2, 4, and 6 (
Demographics and baseline characteristics1
Citation: Beneficial Microbes 2026; 10.1163/18762891-bja00116
Proportion of participants who produced hard or lumpy stools (HLS) in â¥25% of bowel movements
Citation: Beneficial Microbes 2026; 10.1163/18762891-bja00116
Mixed-effects logistic regression parameters for hard or lumpy stools (HLS) in â¥25% of bowel movements
Citation: Beneficial Microbes 2026; 10.1163/18762891-bja00116
Secondary endpoint (clinical outcomes)
Changes in defaecation status (based on stool diary) from Week 0 to Week 6.1
Citation: Beneficial Microbes 2026; 10.1163/18762891-bja00116
Changes in defaecation status based on stool diary data are shown in Table 4 and Supplementary Table S3. AÂ significant increase in total stool frequency was observed in the probiotic group compared with the control group after 4 weeks of probiotic consumption (
Changes in constipation-related symptom scores (based on the CCQ) from Week 0 to Week 6 are presented in Supplementary Table S4 and Supplementary Figure S2. In the probiotic group, the proportion of individuals with a CCQ score â¥5, indicative of constipation (Chan et al., 2005), declined from 100% at baseline to 45.8% at the end of the intervention period (Week 4), and then rose slightly to 62.5% after the follow-up period (Week 6). In contrast, the control group remained relatively stable (88â96%). Significant between-group differences were evident at Weeks 4 (
Secondary outcome (gut microbiota)
The baseline bacterial composition at the phylum level is shown in Figure 3. Participantsâ gut microbiota was dominated by Firmicutes and Bacteroidetes, followed by Proteobacteria and Actinobacteria (including Bifidobacterium). This composition aligns with previous findings in healthy, non-constipated Vietnamese individuals (Pereira-Dias et al., 2021).
Alpha-diversity metrics showed no significant differences between the groups at any time point (Supplementary Table S6). Significant differences in beta-diversity were observed at 2 weeks post-intervention, based on BrayâCurtis dissimilarity (
Summary of adverse events
Four AEs were reported in four participants across both study arms. In the probiotic group, two AEs (eye pain and headache) occurred in two participants (8.3%), whereas in the control group, two AEs (bleeding haemorrhoids and leg joint pain) occurred in two participants (7.4%) (Supplementary Tables S13 and S14). Bleeding haemorrhoids in the control group were classified as a serious AE (SAE), resulting in study withdrawal. Except for the SAE, all AEs were mild, unrelated to the IP or study procedures, and did not require medical intervention (Supplementary Tables S13 and S14). All AEs were resolved by the end of the study period.
4 Discussion and conclusions
The results of this open-label, randomised controlled trial conducted among constipated adults in Vietnam demonstrated that daily consumption of fermented milk containing LcS significantly reduced the proportion of participants experiencing HLS in â¥25% of bowel movements after 2 and 4 weeks of intervention. The proportion remained largely unchanged in the control group (88%) but decreased to 45.8% in the LcS group after 4 weeks. These findings align with previous studies that investigated the effects of LcS-containing fermented milk on individuals with constipation, particularly those conducted in comparable settings in Belgium (Sakai et al., 2011) and the United States (Cook et al., 2025). In the Belgian study, 3 weeks of LcS consumption reduced the proportion of participants with â¥25% HLS to 36.8% in the treatment group, compared to 85% in the control group. Similarly, in a US study, the proportion decreased to 28% in the treatment group versus 64% in the control group after 4 weeks. Additionally, despite differences in age groups, a controlled trial in Vietnamese children demonstrated that a 12-week intake of fermented milk containing LcS significantly reduced the incidence of constipation and helped prevent acute respiratory infections compared to the control group (Mai et al., 2021). These findings suggest that the efficacy of LcS-containing fermented milk can be generalised across different populations and geographical regions.
In this study, we also observed a notable reduction in clinically meaningful constipation-related symptoms (defined as CCQ score â¥5) in participants consuming fermented milk containing LcS. The proportion of participants reporting such symptoms decreased substantially in the LcS group â from 100% at baseline to 45.8% after 4 weeks of consumption â whereas this proportion increased in the control group from 88 to 96% over the same period. Similar improvements were reported in previous clinical trials. For instance, in the aforementioned study in the United States, daily intake of LcS-containing fermented milk led to improvements in Patient Assessment of Constipation Quality of Life (PAC-QOL) scores (Cook et al., 2025). In a placebo-controlled randomised trial among Belgian women during the puerperium period, significant improvements were reported in both overall Patient Assessment of Constipation Symptom scores and the satisfaction subscale of the PAC-QOL in the LcS group compared to placebo (Sakai et al., 2015). Finally, a double-blind, placebo-controlled trial in Germany among individuals with functional constipation demonstrated that consumption of LcS-containing fermented milk alleviated constipation-related symptoms by reducing the prevalence of hard stools (Koebnick et al., 2003). Although the specific symptom profile may vary depending on population characteristics and study context, the collective evidence from these trials supports the reliability and generalisability of our findings.
Baseline gut microbiota composition at the phylum level. The stacked bar chart shows relative abundances of bacterial compositions at the phylum level at baseline in the faecal microbiome of individuals suffering from hard stools.
Citation: Beneficial Microbes 2026; 10.1163/18762891-bja00116
The gut microbiome plays multifaceted roles in the maintenance and restoration of human health. Our study revealed the impact of LcS-fermented milk on the beta-diversity of the gut microbiota after 2 weeks, followed by changes in specific taxa in the subsequent study period. The three bacterial groups associated with the beta-diversity shift at 2 weeks post-intervention â Peptococcaceae, Clostridium_methylpentosum, and Clostridia (unclassified at the order level) â have been linked to an unfavourable gut environment for constipation. Clostridium_methylpentosum_group and Clostridia (unclassified at the order level) have been shown to positively correlate with inflammatory markers tumour necrosis factor-α, interleukin-1β, and prostaglandin E2 in the colon of loperamide-induced functional constipation mice (Gao et al., 2023). Additionally, an increased abundance of these bacterial groups is significantly linked to motilin suppression and somatostatin elevation, both of which are key gastrointestinal hormones that together result in decreased intestinal motility and the progression of constipation within this model (Gao et al., 2023). Peptococcaceae is associated with slower gastrointestinal motility, as non-dietary induction of gastrointestinal transit leads to a decreased abundance of this bacterium (Kashyap et al., 2013). The reduction of these taxa by LcS-fermented milk may provide objective support for our studyâs findings that probiotic drinks decrease the prevalence of HLS and improve overall constipation. This microbial community change in the probiotic group was followed by an orderly increase in Lachnospiraceae_UCG-004 and Lachnospiraceae_ND3007 group at 4 weeks post-intervention and at the end of the follow-up period, respectively. These two bacterial groups are key contributors to short-chain fatty acid production and are significantly depleted in constipated individuals (Gao et al., 2022; Johnson-MartıÌnez et al., 2024). Their abundance is also reduced in patients with Parkinsonâs disease, where constipation is prevalent (Drobny et al., 2021; Nishiwaki et al., 2020; Wallen et al., 2020). A distinct effect of LcS-containing fermented milk on Lachnospiraceae_UCG-004 was previously reported in an intervention study involving patients with constipation who met the Rome III criteria (Chen et al., 2019). In that study, the consumption of a probiotic beverage led to an increase in Lachnospiraceae_UCG-004 among individuals with harder stools (BSFS score <3), whereas a decrease was observed in those with softer stools (BSFS score 4â5). The reproducibility of this finding, particularly in Southeast Asian populations, suggests a consistent and potentially region-specific modulatory effect of LcS-fermented milk on this genus.
Summary of differential abundance analysis using ANCOM-BC1
Citation: Beneficial Microbes 2026; 10.1163/18762891-bja00116
In contrast to this reproducible finding, we did not observe an increase in the abundance of Bifidobacterium after daily consumption of LcS-containing fermented milk, as reported in previous studies conducted in Japan (Matsumoto et al., 2006; Nagata et al., 2011). This discrepancy is likely multifactorial. First, population-level differences in gut microbial ecology may contribute. International metagenomic surveys indicate that the Japanese gut microbiome is enriched in Actinobacteria, particularly Bifidobacterium, whereas Vietnamese adults harbour distinct community structures with different dominant taxa (Nishijima et al., 2016). In our cohort, the median relative abundance of Bifidobacterium remained below 0.025% throughout the study â orders of magnitude lower than the 2â3% reported in healthy Japanese individuals (Watanabe et al., 2021) and the 4â5% observed in constipation-prone individuals (Matsumoto et al., 2006). Under such Bifidobacterium-poor conditions, the same probiotic intervention may not translate into a detectable expansion of this niche. Moreover, the extremely low baseline abundance reduces the dynamic range for detecting changes with 16S rRNA geneâbased profiling and, together with the sample size, may have limited statistical power. The modest, non-significant upward trend observed during the intervention may therefore reflect a small effect that could become detectable in larger studies or in populations with higher baseline Bifidobacterium levels. Second, dietary patterns and host genetic background may further modulate the bifidogenic response to LcS. Bifidobacterium species preferentially utilise complex glycans, including milk-derived carbohydrates (Xiao et al., 2024). In Japan, dairy consumption is relatively common and has been positively associated with Bifidobacterium abundance, potentially reflecting the availability of undigested lactose as a substrate in the colon (Kato et al., 2018). In contrast, traditional Vietnamese diets have historically contained little dairy and rely more heavily on rice and plant-based carbohydrates (Van et al., 2022). These differences in carbohydrate sources and lactose availability may alter microbial resource utilisation and cross-feeding interactions, potentially limiting the extent to which LcS intake promotes Bifidobacterium expansion in this population.
Collectively, LcS-fermented milk induced a favourable shift in the microbiota of constipated adults with a high prevalence of HLS in Vietnam, supporting improvements in clinical outcomes. However, the specific mechanisms linking these microbial changes to constipation remain unclear. Evidence from animal models suggests that LcS supplementation promotes intestinal homeostasis via multiple pathways to alleviate gastrointestinal motility disorders (Cheng et al., 2023). For example, in constipated mice, LcS was suggested to accelerate small intestinal transit and increase faecal pellet output, accompanied by elevated levels of pipecolinic acid in the gut (Ou et al., 2019). Furthermore, in a randomised, double-blind, placebo-controlled trial, LcS intake significantly improved colonic transit in individuals with slow-transit constipation (Krammer et al., 2011). These findings underscore the need for further investigation of the complex interactions between LcS, the host gut environment, and functional outcomes to better elucidate the mechanisms underlying its beneficial effects on constipation.
One limitation of this study is its open-label design. As the study endpoints relied on self-reported measures (BSFS, Stool Diary, and CCQ), participantsâ awareness of the intervention may have influenced their response, potentially introducing bias. To address this limitation, stool samples were collected from all participants, and the gut microbiota composition was analysed. The observed favourable modulation of constipation-associated microbiota in the probiotic group provides objective support for the efficacy of the intervention and strengthens the clinical findings. Another limitation relates to dietary assessment. Although dietary intake was originally intended to be evaluated using an FFQ, technical issues during data collection prevented the acquisition of valid dietary information. Consequently, we were unable to explore potential interactions between habitual diet and the intervention. Nevertheless, given the randomised controlled design of the study, any unmeasured dietary variation is expected to have been evenly distributed between groups. Therefore, while this limitation constrains secondary exploratory analyses, it is unlikely to compromise the internal validity of the between-group comparisons or alter the conclusions regarding the effect of LcS consumption.
In conclusion, daily consumption of fermented milk containing LcS over 4 weeks significantly reduced the incidence of HLS in constipated adults in Vietnam compared with no intervention. Constipation-related symptoms improved concurrently. These clinical benefits were accompanied by favourable shifts in the gut microbiota, supporting the biological plausibility of the observed effects. Although the physiological significance of individual microbial changes requires further investigation, the overall microbiota modulation observed in this study is consistent with the clinically meaningful improvement in constipation-related symptoms. These results suggest that daily intake of LcS-containing fermented milk is a beneficial and well-tolerated approach for managing constipation in this population, with no new safety concerns identified.
Corresponding author; e-mail:Â takuya-akiyama@yakult.co.jp
Acknowledgements
We acknowledge Smart Research for its professional support in conducting this study. We also appreciate the support provided by Editage including medical writing, assembling tables, creating high-resolution images based on the authorsâ detailed directions, collating author comments, copyediting, fact-checking, and referencing.
Authorsâ contribution
Conceptualisation, VHV and TA; data curation, VHV, TDV, NNB, UNQ, and CNTL; formal analysis, VHV, UNQ, CNTL, and TA; investigation, VHV, TDV, NNB, UNQ, and CNTL; methodology, VHV, UNQ, CNTL, and TA; project administration, TDV, NNB, CDX, and TA; resources, VHV, CDX, and TA; supervision, TA; validation, VHV and TA; visualisation, VHV, UNQ, CNTL, and TA; writing â original draft, VHV and TA; writing â review and editing, VHV, TDV, NNB, UNQ, CNTL, CDX, and TA.
Conflict of interest
Takuya Akiyama is affiliated with Yakult Honsha Co., Ltd. â the sponsor of this study. The investigational product was manufactured and distributed in Vietnam by its subsidiary. The article reflects the views of the authors and not necessarily those of the funder. All other authors declare no potential conflicts of interest.
Funding
This study was funded by Yakult Honsha Co., Ltd.
References
Belsey, J., Greenfield, S., Candy, D. and Geraint, M., 2010. Systematic review: impact of constipation on quality of life in adults and children. Alimentary Pharmacology & Therapeutics 31: 938-949. https://doi.org/10.1111/j.1365-2036.2010.04273.x
Benninga, M., Candy, D.C., Catto-Smith, A.G., Clayden, G., Loening-Baucke, V., Di Lorenzo, C., Nurko, S. and Staiano, A., 2005. The Paris Consensus on Childhood Constipation Terminology (PACCT) group. Journal of Pediatric Gastroenterology & Nutrition 40: 273-275. https://doi.org/10.1097/01.mpg.0000158071.24327.88
Bolyen, E., Rideout, J.R., Dillon, M.R., Bokulich, N.A., Abnet, C.C., Al-Ghalith, G.A., Alexander, H., Alm, E.J., Arumugam, M., Asnicar, F., Bai, Y., Bisanz, J.E., Bittinger, K., Brejnrod, A., Brislawn, C.J., Brown, C.T., Callahan, B.J., Caraballo-RodrıÌguez, A.M., Chase, J., Cope, E.K., Da Silva, R., Diener, C., Dorrestein, P.C., Douglas, G.M., Durall, D.M., Duvallet, C., Edwardson, C.F., Ernst, M., Estaki, M., Fouquier, J., Gauglitz, J.M., Gibbons, S.M., Gibson, D.L., Gonzalez, A., Gorlick, K., Guo, J., Hillmann, B., Holmes, S., Holste, H., Huttenhower, C., Huttley, G.A., Janssen, S., Jarmusch, A.K., Jiang, L., Kaehler, B.D., Kang, K.B., Keefe, C.R., Keim, P., Kelley, S.T., Knights, D., Koester, I., Kosciolek, T., Kreps, J., Langille, M.G.I., Lee, J., Ley, R., Liu, Y., Loftfield, E., Lozupone, C., Maher, M., Marotz, C., Martin, B.D., McDonald, D., McIver, L.J., Melnik, A.V., Metcalf, J.L., Morgan, S.C., Morton, J.T., Naimey, A.T., Navas-Molina, J.A., Nothias, L.F., Orchanian, S.B., Pearson, T., Peoples, S.L., Petras, D., Preuss, M.L., Pruesse, E., Rasmussen, L.B., Rivers, A., Robeson, M.S., Rosenthal, P., Segata, N., Shaffer, M., Shiffer, A., Sinha, R., Song, S.J., Spear, J.R., Swafford, A.D., Thompson, L.R., Torres, P.J., Trinh, P., Tripathi, A., Turnbaugh, P.J., Ul-Hasan, S., van der Hooft, J.J.J., Vargas, F., VaÌzquez-Baeza, Y., Vogtmann, E., von Hippel, M., Walters, W., Wan, Y., Wang, M., Warren, J., Weber, K.C., Williamson, C.H.D., Willis, A.D., Xu, Z.Z., Zaneveld, J.R., Zhang, Y., Zhu, Q., Knight, R. and Caporaso, J.G., 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology 37: 852-857. https://doi.org/10.1038/s41587-019-0209-9
Chan, A.O.O., Lam, K.F., Hui, W.M., Hu, W.H., Li, J., Lai, K.C., Chan, C.K., Yuen, M.F., Lam, S.K. and Wong, B.C., 2005. Validated questionnaire on diagnosis and symptom severity for functional constipation in the Chinese population. Alimentary Pharmacology & Therapeutics 22: 483-488. https://doi.org/10.1111/j.1365-2036.2005.02621.x
Chen, S., Ou, Y., Zhao, L., Li, Y., Qiao, Z., Hao, Y. and Ren, F., 2019. Differential effects of Lactobacillus casei Strain Shirota on patients with constipation regarding stool consistency in China. Journal of Neurogastroenterology & Motility 25: 148-158. https://doi.org/10.5056/jnm17085
Cheng, S., Li, H., Ding, Y., Huo, J., Zheng, Y., Jiang, Y., Zhang, Y. and Man, C., 2023. The probiotic combination of Lacticaseibacillus paracasei JY062 and Lactobacillus gasseri JM1 alleviates gastrointestinal motility disorder via Improving gut microbiota. Nutrients 15: 839. https://doi.org/10.3390/nu15040839
Cook, CM., Akiyama, T., Blonquist, T., Mah, E., Derrig, L. and Shibata, H., 2025. Effect of daily consumption of a fermented milk containing Lacticaseibacillus paracasei Strain Shirota (LcS) on stool consistency in United States adults with hard or lumpy stools: a randomized controlled trial. Journal of Nutrition 155: 1183-1192. https://doi.org/10.1016/j.tjnut.2025.02.021
Ding, F., Hu, M., Ding, Y., Meng, Y. and Zhao, Y., 2024. Efficacy in bowel movement and change of gut microbiota on adult functional constipation patients treated with probiotics-containing products: a systematic review and meta-analysis. BMJ Open 14: e074557. https://doi.org/10.1136/bmjopen-2023-074557
Drobny, A., Ngo, P.A., Neurath, M.F., Zunke, F. and LoÌpez-Posadas, R., 2021. Molecular communication between neuronal networks and intestinal epithelial cells in gut inflammation and Parkinsonâs disease. Frontiers in Medicine 8: 655123. https://doi.org/10.3389/fmed.2021.655123
Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792-1797. https://doi.org/10.1093/nar/gkh340
Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460-2461. https://doi.org/10.1093/bioinformatics/btq461
Forootan, M., Bagheri, N. and Darvishi, M., 2018. Chronic constipation: a review of literature. Medicine 97: e10631. https://doi.org/10.1097/MD.0000000000010631
Gao, X., Yang, W., Li, S., Liu, S., Yang, W., Song, S., Sheng, J., Zhao, Y. and Tian, Y., 2023. Moringa oleifera leaf alleviates functional constipation via regulating the gut microbiota and the enteric nervous system in mice. Frontiers in Microbiology 14: 1315402. https://doi.org/10.3389/fmicb.2023.1315402
Gao, Y., Hou, L., Hu, M., Li, D., Tian, Z., Wen, W., Fan, B., Li, S. and Wang, F., 2022. Effects of Bacillus subtilis BSNK-5-fermented soymilk on the gut microbiota by in vitro fecal fermentation. Foods 11: 3501. https://doi.org/10.3390/foods11213501
Garzon Mora, N. and Jaramillo, A.P., 2024. Effectiveness of probiotics in patients with constipation: a systematic review and meta-analysis. Cureus 16: e52013. https://doi.org/10.7759/cureus.52013
Johnson-MartıÌnez, J.P., Diener, C., Levine, A.E., Wilmanski, T., Suskind, D.L., Ralevski, A., Hadlock, J., Magis, A.T., Hood, L., Rappaport, N. and Gibbons, S.M., 2024. Aberrant bowel movement frequencies coincide with increased microbe-derived blood metabolites associated with reduced organ function. Cell Reports Medicine 5: 101646. https://doi.org/10.1016/j.xcrm.2024.101646
Kashyap, P.C., Marcobal, A., Ursell, L.K., Larauche, M., Duboc, H., Earle, K.A., Sonnenburg, E.D., Ferreyra, J.A., Higginbottom, S.K., Million, M., Tache, Y., Pasricha, P.J., Knight, R., Farrugia, G. and Sonnenburg, J.L., 2013. Complex interactions among diet, gastrointestinal transit, and gut microbiota in humanized mice. Gastroenterology 144: 967-977. https://doi.org/10.1053/j.gastro.2013.01.047
Kato, K., Ishida, S., Tanaka, M., Mitsuyama, E., Xiao, J.Z. and Odamaki, T., 2018. Association between functional lactase variants and a high abundance of Bifidobacterium in the gut of healthy Japanese people. PLoS One 13: e0206189. https://doi.org/10.1371/journal.pone.0206189
Kim, S., Suda, W., Kim, S., Oshima, K., Fukuda, S., Ohno, H., Morita, H. and Hattori, M., 2013. Robustness of gut microbiota of healthy adults in response to probiotic intervention revealed by high-throughput pyrosequencing. DNA Research 20: 241-253. https://doi.org/10.1093/dnares/dst006
Koebnick, C., Wagner, I., Leitzmann, P., Stern, U. and Zunft, H.J.F., 2003. Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation. Canadian Journal of Gastroenterology 17: 655-659. https://doi.org/10.1155/2003/654907
Krammer, H.J., von Seggern, H., Schaumburg, J. and Neumer, F., 2011. Effect of Lactobacillus casei Shirota on colonic transit time in patients with chronic constipation. Coloproctology 33: 109-113. https://doi.org/10.1007/s00053-011-0177-0
Lin, H. and Peddada, S.D., 2020. Analysis of compositions of microbiomes with bias correction. Nature Communications 11: 3514. https://doi.org/10.1038/s41467-020-17041-7
Mai, T.T., Thu, P.T., Hang, H.T., Trang, T.T.T., Yui, S., Shigehisa, A., Tien, V.T., Dung, T.V., Nga, P.B., Hung, N.T. and Tuyen, L.D., 2021. Efficacy of probiotics on digestive disorders and acute respiratory infections: a controlled clinical trial in young Vietnamese children. European Journal of Clinical Nutrition 75: 513-520. https://doi.org/10.1038/s41430-020-00754-9
Matsuki, T., Yahagi, K., Mori, H., Matsumoto, H., Hara, T., Tajima, S., Ogawa, E., Kodama, H., Yamamoto, K., Yamada, T., Matsumoto, S. and Kurokawa, K., 2016. A key genetic factor for fucosyllactose utilization affects infant gut microbiota development. Nature Communications 7: 11939. https://doi.org/10.1038/ncomms11939
Matsumoto, K., Takada, T., Shimizu, K., Kado, Y., Kawakami, K., Makino, I., Yamaoka, Y., Hirano, K., Nishimura, A., Kajimoto, O. and Nomoto, K., 2006. The effects of a probiotic milk product containing Lactobacillus casei Strain Shirota on the defecation frequency and the intestinal microflora of sub-optimal health state volunteers: a randomized placebo controlled cross-over study. Bioscience & Microflora 25: 39-48. https://doi.org/10.12938/bifidus.25.39
Nagata, S., Asahara, T., Ohta, T., Yamada, T., Kondo, S., Bian, L., Wang, C., Yamashiro, Y. and Nomoto, K., 2011. Effect of the continuous intake of probiotic-fermented milk containing Lactobacillus casei strain Shirota on fever in a mass outbreak of Norovirus gastroenteritis and the faecal microflora in a health service facility for the aged. British Journal of Nutrition 106: 549-556. https://doi.org/10.1017/S000711451100064X
Nishijima, S., Suda, W., Oshima, K., Kim, S.W., Hirose, Y., Morita, H. and Hattori, M., 2016. The gut microbiome of healthy Japanese and its microbial and functional uniqueness. DNA Research 23: 125-133. https://doi.org/10.1093/dnares/dsw002
Nishiwaki, H., Hamaguchi, T., Ito, M., Ishida, T., Maeda, T., Kashihara, K., Tsuboi, Y., Ueyama, J., Shimamura, T., Mori, H., Kurokawa, K., Katsuno, M., Hirayama, M. and Ohno, K., 2020. Short-chain fatty acid-producing gut microbiota is decreased in Parkinsonâs disease but not in rapid-eye-movement sleep behavior disorder. mSystems 5: e00797-20. https://doi.org/10.1128/mSystems.00797-20
Ou, Y., Chen, S., Ren, F., Zhang, M., Ge, S., Guo, H., Zhang, H. and Zhao, L., 2019. Lactobacillus casei Strain Shirota alleviates constipation in adults by increasing the pipecolinic acid level in the gut. Frontiers in Microbiology 10: 324. https://doi.org/10.3389/fmicb.2019.00324
Patimah, A.W., Lee, Y.Y. and Dariah, M.Y., 2017. Frequency patterns of core constipation symptoms among the Asian adults: a systematic review. BMC Gastroenterology 17: 115. https://doi.org/10.1186/s12876-017-0672-z
Pereira-Dias, J., Minh, C.N.N., Hong, C.T.T., Nguyen, T.N.T., Thanh, T.H., Zellmer, C., The, H.C., Pike, L., Higginson, E.E. and Baker, S., 2021. The gut microbiome of healthy Vietnamese adults and children is a major reservoir for resistance genes against critical antimicrobials. Journal of Infectious Diseases 224: S840-S847. https://doi.org/10.1093/infdis/jiab398
Price, M.N., Dehal, P.S. and Arkin, A.P., 2009. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Molecular Biology and Evolution 26: 1641-1650. https://doi.org/10.1093/molbev/msp077
Qi, X., Fan, G. and Jia, H., 2020. The probiotic Lactobacillus casei Shirota attenuates symptoms of vestibular migraine: a randomised placebo-controlled double-blind clinical trial. Beneficial Microbes 11: 469-476. https://doi.org/10.3920/BM2020.0058
Sakai, T., Kubota, H., Gawad, A., Gheyle, L., Ramael, S. and Oishi, K., 2015. Effect of fermented milk containing Lactobacillus casei strain Shirota on constipation-related symptoms and haemorrhoids in women during puerperium. Beneficial Microbes 6: 253-262. https://doi.org/10.3920/BM2014.0076
Sakai, T., Makino, H., Ishikawa, E., Oishi, K. and Kushiro, A., 2011. Fermented milk containing Lactobacillus casei strain Shirota reduces incidence of hard or lumpy stools in healthy population. International Journal of Food Sciences & Nutrition 62: 423-430. https://doi.org/10.3109/09637486.2010.542408
Schmidt, F.M.Q. and Santos, V.L., 2014. Prevalence of constipation in the general adult population: an integrative review. Journal of Wound, Ostomy, & Continence Nursing 41: 70-76, quiz E1. https://doi.org/10.1097/01.WON.0000438019.21229.b7
Tilley, L., Keppens, K., Kushiro, A., Takada, T., Sakai, T., Vaneechoutte, M. and Degeest, B., 2014. A probiotic fermented milk drink containing Lactobacillus casei strain Shirota improves stool consistency of subjects with hard stools. International Journal of Probiotics & Prebiotics 9: 23-29.
Tsujibe, S., Gawad, A., Shigehisa, A., Matsuda, K., Fujimoto, J. and Takahashi, T., 2024. Evaluating the effect of Lacticaseibacillus paracasei Strain Shirota on the physical consistency of stool in healthy participants with hard or lumpy stools: a double-blind, randomized, placebo-controlled study. Nutrients 16: 2469. https://doi.org/10.3390/nu16152469
Van, D.T.T., Trijsburg, L., Do, H.T.P., Kurotani, K., Feskens, E.J.M. and Talsma, E.F., 2022. Development of the Vietnamese Healthy Eating Index. Journal of Nutritional Science 11: e45. https://doi.org/10.1017/jns.2022.44
Wallen, Z.D., Appah, M., Dean, M.N., Sesler, C.L., Factor, S.A., Molho, E., Zabetian, C.P., Standaert, D.G. and Payami, H., 2020. Characterizing dysbiosis of gut microbiome in PD: evidence for overabundance of opportunistic pathogens. NPJ Parkinsonâs Disease 6: 11. https://doi.org/10.1038/s41531-020-0112-6
Wang, Q., Garrity, G.M., Tiedje, J.M. and Cole, J.R., 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied & Environmental Microbiology 73: 5261-5267. https://doi.org/10.1128/AEM.00062-07
Watanabe, S., Kameoka, S., Shinozaki, N.O., Kubo, R., Nishida, A., Kuriyama, M. and Takeda, A.K., 2021. A cross-sectional analysis from the mykinso cohort study: establishing reference ranges for japanese gut microbial indices. Bioscience of Microbiota, Food & Health 40: 123-134. https://doi.org/10.12938/bmfh.2020-038
Xiao, M., Zhang, C., Duan, H., Narbad, A., Zhao, J., Chen, W., Zhai, Q., Yu, L. and Tian, F., 2024. Cross-feeding of bifidobacteria promotes intestinal homeostasis: a lifelong perspective on the host health. NPJ Biofilms Microbiomes 10: 47. https://doi.org/10.1038/s41522-024-00524-6
Yan, R., Wang, K., Wang, Q., Jiang, H., Lu, Y., Chen, X., Zhang, H., Su, X., Du, Y., Chen, L., Li, L. and Lv, L., 2022. Probiotic Lactobacillus casei Shirota prevents acute liver injury by reshaping the gut microbiota to alleviate excessive inflammation and metabolic disorders. Microbial Biotechnology 15: 247-261. https://doi.org/10.1111/1751-7915.13750
Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P., OâToole, P.W., Pot, B., Vandamme, P., Walter, J., Watanabe, K., Wuyts, S., Felis, G.E., Gänzle, M.G. and Lebeer, S., 2020. A taxonomic note on the genus lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and Union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology 70: 2782-2858. https://doi.org/10.1099/ijsem.0.004107
