Lactobacillus helveticus UA881 Improves Body Composition, Lipid Profiles, and Gut Microbiota in Overweight Adults: A Randomized Double-Blind Placebo-Controlled Trial

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article entitled Lactobacillus helveticus UA881 improves body composition, lipid profiles, and gut microbiota in overweight adults: a randomized double-blind placebo-controlled trial addresses a topic of great interest to the international scientific community: how modification of the microbiome can positively influence body recomposition, improving the health status of obese and overweight individuals. However, several aspects need to be improved before publication.

Below are the comments organized by section:

Abstract

• The name of the microorganism must be written in italics, and this should be reviewed and corrected throughout the document whenever a microorganism is

Introduction

• The full name of the microorganism Lactobacillus helveticus UA881 must be stated in italics, followed by the abbreviation in parentheses (UA881), which should be used consistently throughout the

Materials and Methods

Materials and Methods

• In the subsection “Participants Enrollment,” the clinical trial registration where the study was recorded must be indicated, as well as a reference to the Helsinki Declaration.
• The exclusion criteria do not clarify whether diabetic individuals were excluded or whether they could be included in the study.
• The CONSORT diagram detailing participant distribution must be included, with proper reference to this figure as required in clinical trials. The figure should also be adapted according to CONSORT guidelines. The method of randomization employed in the study is not described.
• In the subsection “UA881 Probiotics and Placebo Capsules Administration,” it is not specified under what conditions participants took the capsules (e.g., fasting or with meals).
• In “Body Characteristics Assessment,” the instrument used to measure bioimpedance characteristics is not mentioned, nor are the conditions (e.g., whether measurements were taken by the same person or by multiple evaluators). It is also unclear whether participants engaged in physical activity during the study, which should be included in the analysis.

• In “Blood Sampling and Serum Separation,” the method of blood collection should be specified (e.g., venipuncture of the cubital vein). If all biochemical parameters were measured in serum, why is plasma collection also mentioned?
• In “Statistics,” several aspects must be clarified: a. The method used to calculate sample size, or the rationale for the chosen sample size. b. The methods used for analyzing clinical biochemistry parameters and microbiome data. c. The design of the measured variables must be specified: what is the primary variable of the study? What are the secondary variables? This information should also be reflected in the public trial registry where the study is registered.

Results

• Figure 1 must comply with CONSORT criteria, be referenced accordingly, and be cited in the section describing participant distribution in Materials and Methods.
• Table 2 should include information on treatments received by participants, for example, antihypertensive therapy, since hypertension is common among obese or overweight individuals. Additionally, the total number of participants per group should be shown in the column rather than the row.
• In Table 3, “Changes in body characteristics before and after UA881 intervention,” the legend does not clearly explain the analyses represented by the p-values. It should specify whether the analysis was within each group or between groups, and distinguish them as p1, p2, etc.

Discussion

• Among the study limitations, it should be noted that HbA1c and insulin measurements should have been performed, as these parameters may be altered in obesity.
• It should be verified whether the observed effects were maintained over time, and whether participants engaged in physical activity during the study.
• What happened to the placebo group participants? Were they offered the treatment at the end of the study?

Comments for author File: Comments.pdf

Author Response

Reviewer1

The article entitled Lactobacillus helveticus UA881 improves body composition, lipid profiles, and gut microbiota in overweight adults: a randomized double-blind placebo-controlled trial addresses a topic of great interest to the international scientific community: how modification of the microbiome can positively influence body recomposition, improving the health status of obese and overweight individuals. However, several aspects need to be improved before publication.

Response: We sincerely thank the reviewer for their positive and thoughtful feedback on our manuscript. We are pleased to hear that the topic of microbiome modification and its potential impact on body recomposition is of interest. We appreciate the reviewer’s constructive suggestions and have addressed each of the points raised in the revised manuscript. Detailed responses to the reviewer’s comments are provided below.

Below are the comments organized by section:

Abstract

• The name of the microorganism must be written in italics, and this should be reviewed and corrected throughout the document whenever a microorganism is

Response: We thank the reviewer for this comment. We have carefully reviewed the entire manuscript and corrected the formatting of all microorganism names to ensure that genus and species names are consistently written in italics, in accordance with standard scientific nomenclature. (Please see the revised version we highlighted in yellow. E.g. Line 24)

Introduction

• The full name of the microorganism Lactobacillus helveticusUA881 must be stated in italics, followed by the abbreviation in parentheses (UA881), which should be used consistently throughout the

Response: We thank the reviewer for this comment. The full name Lactobacillus helveticus UA881 has been formatted in italics at first mention, and the abbreviation (UA881) is used consistently throughout the manuscript. (Please see the revised version we highlighted in yellow. E.g. Line 63)

Materials and Methods

Materials and Methods

• In the subsection “Participants Enrollment,” the clinical trial registration where the study was recorded must be indicated, as well as a reference to the Helsinki Declaration.

Response: We thank the reviewer for this important comment. The Participants Enrollment subsection has been revised to clearly state that the study protocol was reviewed and approved by the Institutional Review Board of Chung Shan Medical University Hospital (IRB No. CS2-23191) and conducted in accordance with the principles of the Declaration of Helsinki. In addition, the clinical trial registration information has been added, indicating that the study was registered at ClinicalTrials.gov (Identifier: NCT06554314). These revisions are highlighted in yellow in the revised manuscript. (Lines 117-121)

• The exclusion criteria do not clarify whether diabetic individuals were excluded or whether they could be included in the study.

Response: We thank the reviewer for this comment. Diabetes was not listed as an explicit exclusion criterion. However, participants using medications with pharmacological effects that could interfere with the study outcomes or potentially confound the intervention effects were excluded. As most individuals with diabetes require pharmacological treatment, such participants were therefore not eligible for enrollment. In practice, no participants with diagnosed diabetes were included in this study. This clarification has been added to the revised manuscript. (Line 129-144)

• The CONSORT diagram detailing participant distribution must be included, with proper reference to this figure as required in clinical trials. The figure should also be adapted according to CONSORT guidelines. The method of randomization employed in the study is not described.

Response: We thank the reviewer for this important comment. In accordance with CONSORT guidelines, we have revised and included a CONSORT flow diagram (Figure 1) to clearly illustrate participant enrollment, allocation, follow-up, and analysis. The diagram now details the number of participants assessed for eligibility, excluded with reasons, randomized, allocated to each intervention, lost to follow-up, and included in the final analysis.

In addition, we have revised the Participants Enrollment subsection to clearly describe the randomization procedure. Participants were assigned to the UA881 or placebo group via computer-generated covariate-adaptive randomization. A computer-generated randomization sequence was used, with stratification by gender, age, and body mass index (BMI), to minimize potential confounding effects on study outcomes. Allocation was performed by an independent study staff member who was not involved in participant recruitment, intervention administration, or outcome assessment, thereby ensuring allocation concealment. Both participants and investigators were blinded to group assignment throughout the study period.

These revisions have been incorporated into the Methods section, and Figure 1 has been revised to fully comply with CONSORT reporting standards. (Lines 300-301)

• In the subsection “UA881 Probiotics and Placebo Capsules Administration,” it is not specified under what conditions participants took the capsules (e.g., fasting or with meals).

Response: We thank the reviewer for this important comment. We have revised the subsection “UA881 Probiotics and Placebo Capsules Administration” to clearly specify the administration conditions. Participants were instructed to take one capsule daily in a fasting state every morning during the intervention period, without concurrent food intake. This information has now been added to the revised manuscript. (Lines 151-152)

• In “Body Characteristics Assessment,” the instrument used to measure bioimpedance characteristics is not mentioned, nor are the conditions (e.g., whether measurements were taken by the same person or by multiple evaluators). It is also unclear whether participants engaged in physical activity during the study, which should be included in the analysis.

Response: We thank the reviewer for these constructive comments. We have revised the Body Characteristics Assessment subsection to provide additional methodological details as follows.

First, body composition parameters were measured by using a bioelectrical impedance analyzer (InBody 270, Biospace Co., Ltd., Seoul, Korea), and this information has now been stated in the revised manuscript. Second, all anthropometric and bioimpedance measurements were performed by the same trained evaluator throughout the study to minimize inter-observer variability.

Regarding physical activity, participants were allowed to maintain their usual daily activities during the intervention period. To reduce potential confounding effects, participants were instructed to keep their habitual lifestyle patterns unchanged; those who engaged in regular exercise prior to enrollment were asked to maintain the same exercise frequency and intensity throughout the study. Physical activity was not specifically restricted or quantitatively analyzed, but lifestyle consistency was emphasized and monitored through participant instructions. These clarifications have been incorporated into the revised Methods section. (Lines 176-179)

• In “Blood Sampling and Serum Separation,” the method of blood collection should be specified (e.g., venipuncture of the cubital vein). If all biochemical parameters were measured in serum, why is plasma collection also mentioned?

Response: We thank the reviewer for this important comment. Blood samples were collected by venipuncture of the cubital vein, and this information has now been stated in the revised manuscript. We also clarify that only whole blood and serum samples were collected and used for complete cell counts (CBC) and biochemical analyses in this study. The previous mention of plasma collection was inaccurate and has been removed. We has been revised this subsection content accordingly to avoid confusion. (Lines 182-189)

• In “Statistics,” several aspects must be clarified: a. The method used to calculate sample size, or the rationale for the chosen sample size. b. The methods used for analyzing clinical biochemistry parameters and microbiome data. c. The design of the measured variables must be specified: what is the primary variable of the study? What are the secondary variables? This information should also be reflected in the public trial registry where the study is registered.

Response: We thank the reviewer for these very important comments. We have revised the Statistics subsection accordingly.

(a) Sample size rationale:

This study was designed as an exploratory, proof-of-concept randomized controlled trial to evaluate the potential metabolic effects of UA881 probiotic supplementation in overweight adults. As limited prior human data were available for this specific strain, a formal a priori sample size calculation was not performed. The sample size was determined based on feasibility considerations and consistency with previous exploratory probiotic intervention studies of similar duration. This rationale has now been clarified in the revised manuscript. (Lines 274-278)

(b) Statistical analysis methods:

The statistical methods used for analyzing clinical biochemical parameters, anthropometric data, inflammatory markers, fecal short-chain fatty acids, and microbiome outcomes have been clearly described in the original manuscript and are summarized in the revised Statistics subsection. Briefly, within-group comparisons were performed using paired tests, and between-group intervention effects were further evaluated using analysis of covariance (ANCOVA) to adjust for baseline variability. Microbiome data were analyzed using alpha diversity indices and relative abundance measures, with appropriate statistical comparisons as specified. (Lines 283-292)

(c) Primary and secondary outcomes:

We have clarified that serum triglyceride (TG) level was defined as the primary endpoint of the study, in accordance with the publicly registered trial protocol. All other anthropometric parameters, serum biochemical markers, inflammatory cytokines, fecal SCFAs, and gut microbiota outcomes were designated as secondary endpoints. This information has been explicitly stated in the revised manuscript and is consistent with the study registration at ClinicalTrials.gov. (Lines 279-282)

Results

• Figure 1 must comply with CONSORT criteria, be referenced accordingly, and be cited in the section describing participant distribution in Materials and Methods.

Response: We thank the reviewer for this comment. Figure 1 has been revised to comply with the CONSORT flow diagram guidelines, including detailed information on participant enrollment, allocation, follow-up, and analysis. The figure is now appropriately referenced and cited in the Materials and Methods section describing participant enrollment and distribution. The revised CONSORT flow diagram clearly illustrates the number of participants assessed for eligibility, excluded with reasons, randomized, allocated to each group, and included in the final analysis. (Please see the revised Fig.1)

• Table 2 should include information on treatments received by participants, for example, antihypertensive therapy, since hypertension is common among obese or overweight individuals. Additionally, the total number of participants per group should be shown in the column rather than the row.

Response: We thank the reviewer for this helpful suggestion.

1. Regarding concomitant medications (e.g., antihypertensive therapy):
   Participants who were receiving medications with potential metabolic effects were excluded according to the predefined exclusion criteria. Specifically, individuals using antihypertensive, antidiabetic, lipid-lowering, or other metabolism-related medications were not eligible for enrollment. Therefore, no participants with diagnosed metabolic comorbidities requiring pharmacological treatment were included in this study. This information has been clarified in the Participants Enrollment subsection. (Lines 129-134)
2. Regarding the presentation of participant numbers in Table 2:
   In accordance with the reviewer’s recommendation, we have revised Table 2 so that the total number of participants in each group is now clearly indicated in the column rather than in the rows. This modification improves clarity and consistency with common reporting practices in clinical trials. (Please see the revised Table2)

• In Table 3, “Changes in body characteristics before and after UA881 intervention,” the legend does not clearly explain the analyses represented by the p-values. It should specify whether the analysis was within each group or between groups, and distinguish them as p1, p2, etc.

Response: We thank the reviewer for this valuable comment. We agree that the original legend of Table 3 did not sufficiently clarify the statistical meaning of the reported p-values. In the revised manuscript, we have updated the table legend to explicitly specify the statistical analyses represented by each p-value. Specifically, p₁ now denotes within-group comparisons between baseline (day 0) and day 28 assessed by paired two-tailed t-tests, while p₂ denotes between-group comparisons assessed by the Brown–Forsythe test for homogeneity of variance. These clarifications have been added to the legend of Table 2 and Table 3 to improve transparency and reader comprehension. (Please see the revised Table 2 and Table 3)

Discussion

• Among the study limitations, it should be noted that HbA1c and insulin measurements should have been performed, as these parameters may be altered in obesity.

Response: We thank the reviewer for this helpful suggestion. HbA1c and insulin measurements were not included in the present study, which primarily focused on lipid metabolism and gut microbiota modulation. This limitation has been added in the Discussion section, and we note that future studies should incorporate these parameters to further evaluate glucose metabolism and insulin sensitivity. (Lines 588-591)

• It should be verified whether the observed effects were maintained over time, and whether participants engaged in physical activity during the study.

Response: We sincerely thank the reviewer for this important comment. The present study evaluated the effects of UA881 supplementation over a 28-day intervention period, and no follow-up assessments beyond the intervention were conducted. Therefore, whether the observed effects were maintained over time could not be determined in this study. This point has now been acknowledged as a limitation in the Discussion section. (Lines 585-587)

Regarding physical activity, participants were allowed to maintain their usual daily activities during the study. To minimize potential confounding effects, participants were instructed to keep their habitual lifestyle patterns unchanged, including exercise frequency and intensity if they had pre-existing exercise routines. Physical activity was not quantitatively assessed or included as a covariate in the analysis, and this has been clarified in the revised manuscript. (Lines 176-179 and 595-598)

• What happened to the placebo group participants? Were they offered the treatment at the end of the study?

Response: We thank the reviewer for this question. Participants in the placebo group did not receive UA881 supplementation after completion of the trial, as no post-trial intervention was planned in the study protocol. However, participants who were identified as having potential metabolic health risks based on study assessments were advised to seek further medical evaluation and follow-up with healthcare professionals as appropriate. This approach was consistent with ethical standards for clinical research and participant welfare. We have further stated in the revised version, please see the Lines 142-144.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors in this manuscript aimed to evaluate the metabolic and gut microbiota-modulating effects of Lactobacillus helveticus UA881 in a 28-day randomized controlled trial with 50 overweight adults. They found that UA881 supplementation significantly improved body composition, reduced serum triglycerides and total cholesterol, and beneficially altered the gut microbiota. The authors concluded that UA881 shows potential as a probiotic for improving metabolic health, warranting further investigation. While the study demonstrates promising findings regarding improvements in body composition, lipid profiles, and microbiota modulation, several critical methodological and reporting issues require revision. Below are several comments, suggestions, and questions that need to be considered.

• The abstract states participants were "overweight or obese adults (BMI 25–27 kg/m²)." A BMI of 25-27 kg/m² typically defines overweight, not obesity (which is generally BMI ≥ 30 kg/m²). The authors should clarify this or revise the terminology to "overweight" for consistency and accuracy.
• The introduction refers to UA881 as a "novel probiotic formulation" but provides no background on this specific strain. Why was L. helveticus UA881 selected for this study? Was it based on pre-clinical evidence?
• The authors do not justify why n=25 per group was chosen. Was a power analysis conducted based on an expected effect size for the primary outcome?
• The criteria are clearly defined. However, as noted previously, the BMI inclusion criterion of 25-27 kg/m² is very narrow and does not include obese individuals. This limits the generalizability of the findings and should be explicitly acknowledged. The title and abstract should be revised to reflect "mildly overweight" or "overweight" rather than "overweight and obese."
• Some details are missing in the method section that would be necessary for full replication:
  • The specific model of the "calibrated bioelectrical analyzer" used for body composition is not mentioned.
  • The commercial ELISA kits used for IL-6 and TNF-α analysis (manufacturer, catalog number) should be specified.
• The authors state they used one-tailed paired t-tests (line 213). This requires strong justification, as it assumes an effect in only one direction. A two-tailed test is more conservative and generally standard. The authors should either justify the use of a one-tailed test or re-analyze with a two-tailed test.
• The description of normality testing appears twice (line 210 and 213-214), which is redundant.
• Body Characteristics (Table 3): The significant reduction in weight and BMI in both groups is an important finding. While the authors suggest a Hawthorne effect, this should be discussed more deeply.
• The key finding that only the probiotic group showed significant reductions in Body Fat Mass (BFM) and Visceral Fat Level (VFL) is well-highlighted.
• The Results section (line 294) states that in the ANCOVA, "the slopes differed significantly between the probiotics and placebo groups." This implies a significant interaction between the treatment and the baseline value. If so, where are the p-values for this interaction test reported?
• The legend for Figure 2 is inconsistent. It states that ANCOVA demonstrated effects on "TG, TCHO, and UA," but the figure only displays plots for TG, TCHO, and LDL-C. Please correct the figure legend or the figure itself.
• The number of participants for SCFA and microbiota analysis was reduced to 20 per group. Was this sub-sampling pre-specified in the protocol? The authors should state this and acknowledge if it has any implications for statistical power.
• The placebo group showed a significant increase in propanoic acid. Do the authors have any biological explanation for this observation?
• The discussion attributes weight loss in the placebo group solely to the Hawthorne effect. Could the placebo itself (maltodextrin) have had a mild prebiotic effect, contributing to some changes? This possibility should be considered.
• The significant worsening of several metabolic markers in the placebo group over just 28 days is quite pronounced. Is this expected fluctuation in an overweight population, or could there be another reason?
• Could the authors elaborate on how a change in butyrate levels might specifically lead to a reduction in serum triglycerides and cholesterol, citing relevant mechanistic pathways?

8. Minor Issues

• Line 213-214: The sentence "Normality of continuous variables was assessed using the Shapiro-Wilk test to determine the use of parametric or non-parametric statistical methods" is a repetition of the sentence on line 210.
• Section "Gut Microbiota Composition at the Genus Level" is numbered 3.7, the same as the preceding section. It should be 3.8.
• Line 420: The phrase "more obvious" could be replaced with more scientific language, such as "more pronounced" or "of greater magnitude."
• Line 439: "our data suggest that UA881 may protect against metabolic dysregulation." Consider rephrasing to "may attenuate" or "mitigate" metabolic dysregulation.
• Conflicts of Interest: The authors declare no conflicts of interest. However, the manuscript states it was funded by Nutrarex Bioceutical, and two authors (P.-C.H. and S.-Y.L.) are affiliated with Percheron Bioceutical Research Center / NUTRAREX BIOTECH, the company that also produced the supplements. The statement "The funders had no role in the design... collection, analyses, or interpretation of data..." is therefore questionable

Author Response

Reviewer2

The authors in this manuscript aimed to evaluate the metabolic and gut microbiota-modulating effects of Lactobacillus helveticus UA881 in a 28-day randomized controlled trial with 50 overweight adults. They found that UA881 supplementation significantly improved body composition, reduced serum triglycerides and total cholesterol, and beneficially altered the gut microbiota. The authors concluded that UA881 shows potential as a probiotic for improving metabolic health, warranting further investigation. While the study demonstrates promising findings regarding improvements in body composition, lipid profiles, and microbiota modulation, several critical methodological and reporting issues require revision. Below are several comments, suggestions, and questions that need to be considered.

Response: We sincerely thank the reviewer for their positive and thoughtful assessment of our study. We are grateful for the recognition of the promising findings regarding the metabolic and gut microbiota-modulating effects of Lactobacillus helveticus (UA881). We also appreciate the reviewer’s constructive suggestions, which we have carefully addressed in the revised manuscript. Detailed responses to each comment, suggestion, and question are provided below.

• The abstract states participants were "overweight or obese adults (BMI 25–27 kg/m²)." A BMI of 25-27 kg/m² typically defines overweight, not obesity (which is generally BMI ≥ 30 kg/m²). The authors should clarify this or revise the terminology to "overweight" for consistency and accuracy.

Response: We thank the reviewer for this comment. We agree that a BMI of 25–27 kg/m² corresponds to overweight rather than obesity. Accordingly, we have revised the terminology throughout the manuscript, including the Abstract and main text, to refer to the study population as “overweight adults” for accuracy and consistency. (Lines 122-124)

• The introduction refers to UA881 as a "novel probiotic formulation" but provides no background on this specific strain. Why was L. helveticus UA881 selected for this study? Was it based on pre-clinical evidence?

Response: We thank the reviewer for this important comment. Lactobacillus helveticus (UA881) was selected based on pre-clinical screening studies demonstrating its potential metabolic relevance. Specifically, UA881 exhibited favorable in vitro properties related to purine and uric acid metabolism, antioxidant capacity, and intestinal barrier support. Although these pre-clinical findings are part of ongoing and unpublished work, they provided the biological rationale for selecting UA881 for the present exploratory human study. We have added a brief justification in the Introduction to clarify the rationale for strain selection, without overinterpretation. (Lines 89-94)

• The authors do not justify why n=25 per group was chosen. Was a power analysis conducted based on an expected effect size for the primary outcome?

Response: We thank the reviewer for these very important comments. This study was designed as an exploratory, proof-of-concept randomized controlled trial to evaluate the potential metabolic effects of UA881 probiotic supplementation in overweight adults. As limited prior human data were available for this specific strain, a formal a priori sample size calculation was not performed. The sample size was determined based on feasibility considerations and consistency with previous exploratory probiotic intervention studies of similar duration. This rationale has now been clarified in the revised manuscript. (Lines 274-278)

• The criteria are clearly defined. However, as noted previously, the BMI inclusion criterion of 25-27 kg/m² is very narrow and does not include obese individuals. This limits the generalizability of the findings and should be explicitly acknowledged. The title and abstract should be revised to reflect "mildly overweight" or "overweight" rather than "overweight and obese."

Response: We thank the reviewer for this important comment. We agree that the BMI inclusion range of 25–27 kg/m² represents a narrowly defined overweight population and does not include individuals with obesity. Accordingly, the generalizability of the findings to obese populations is limited. This limitation has now been explicitly acknowledged in the Discussion section. (Lines 599-604)

In addition, we have revised the title, abstract, and relevant sections of the manuscript to refer to the study population as overweight adults, rather than obese, to ensure accuracy and consistency with the inclusion criteria. (Please see the revised version highlighted in yellow. E.g. Line 21)

• Some details are missing in the method section that would be necessary for full replication:
  • The specific model of the "calibrated bioelectrical analyzer" used for body composition is not mentioned.

Response: We thank the reviewer for this comment. The specific model of the bioelectrical impedance analyzer has now been specified in the revised manuscript. Body composition was measured using the InBody 270 system (Biospace Co., Ltd., Seoul, Korea). (Lines 170-171)

• The commercial ELISA kits used for IL-6 and TNF-α analysis (manufacturer, catalog number) should be specified.

Response: We thank the reviewer for this comment. The manufacturer and catalog numbers of the commercial ELISA kits used for IL-6 and TNF-α measurements have now been specified in the revised manuscript. (Lines 199-202)

• The authors state they used one-tailed paired t-tests (line 213). This requires strong justification, as it assumes an effect in only one direction. A two-tailed test is more conservative and generally standard. The authors should either justify the use of a one-tailed test or re-analyze with a two-tailed test.

Response: We thank the reviewer for this important comment. We agree that the use of one-tailed paired t-tests requires strong a priori justification and that two-tailed tests represent a more conservative and widely accepted approach. In response to this concern, we have reanalyzed the relevant data using two-tailed paired t-tests. The Results section and corresponding figures and tables have been revised accordingly to reflect the updated statistical analyses. Importantly, the main conclusions of the study remained unchanged after reanalysis using two-tailed tests. (Please see the revised Table 3 and Table 4)

• The description of normality testing appears twice (line 210 and 213-214), which is redundant.

Response: Thanks for your reminding. We have revised the duplicated description of normality testing. (Lines 270-271)

• Body Characteristics (Table 3): The significant reduction in weight and BMI in both groups is an important finding. While the authors suggest a Hawthorne effect, this should be discussed more deeply.

Response: We thank the reviewer for this insightful comment. We agree that the significant reductions in body weight and BMI observed in both groups warrant further discussion. Accordingly, we have expanded the Discussion to more thoroughly address the potential contribution of the Hawthorne effect, including increased health awareness, study participation–related behavioral changes, and improved adherence to lifestyle recommendations during the trial period. At the same time, we emphasize that the magnitude of reduction was greater in the UA881 group, suggesting an additional intervention-related effect beyond behavioral influences. The Discussion section has been revised accordingly. (Lines 513-526)

• The key finding that only the probiotic group showed significant reductions in Body Fat Mass (BFM) and Visceral Fat Level (VFL) is well-highlighted.

Response: We thank the reviewer for highlighting this important point. Following the reviewer’s recommendation, we reanalyzed the data using two-tailed statistical tests. After reanalysis, the reduction in body fat mass (BFM) in the probiotic group remained statistically significant, whereas the change in visceral fat level (VFL) no longer reached statistical significance. We have revised the Results section accordingly to accurately reflect these updated findings and now emphasize BFM as the primary body composition parameter showing a significant improvement after UA881 supplementation. (Lines 317-322)

• The Results section (line 294) states that in the ANCOVA, "the slopes differed significantly between the probiotics and placebo groups." This implies a significant interaction between the treatment and the baseline value. If so, where are the p-values for this interaction test reported?

Response: We thank the reviewer for this important issue. We agree that a statement regarding significant differences in regression slopes implies a significant treatment × baseline interaction and that the corresponding p-values should be clearly reported in the manuscript.

Accordingly, we have revised the Results section to clearly report the p-values for the interaction terms derived from the ANCOVA models. Specifically, significant treatment × baseline interactions were observed for serum uric acid (slope p = 0.036) and triglycerides (slope p < 0.001), indicating baseline-dependent intervention effects of UA881 supplementation. For total cholesterol, the interaction term was not significant (slope p = 0.658); however, a significant difference in intercepts was detected (p = 0.013), reflecting a baseline-adjusted group difference independent of slope effects. These p-values are now clearly stated in the revised version in the Results section and correspond to the data presented in Figure 2. (Lines 386-396)

• The legend for Figure 2 is inconsistent. It states that ANCOVA demonstrated effects on "TG, TCHO, and UA," but the figure only displays plots for TG, TCHO, and LDL-C. Please correct the figure legend or the figure itself.

Response: We thank the reviewer for identifying this inconsistency. To address this issue, we have revised the Results section to ensure that the description of the ANCOVA findings is fully consistent with the data presented in Figure 2 and its legend. The revised text now accurately reflects the parameters shown in the figure, and the inconsistency between the Results and the figure legend has been corrected. (Lines 367-378)

• The number of participants for SCFA and microbiota analysis was reduced to 20 per group. Was this sub-sampling pre-specified in the protocol? The authors should state this and acknowledge if it has any implications for statistical power.

Response: We thank the reviewer for this comment. Fecal sample collection for short-chain fatty acid (SCFA) and gut microbiota analyses was conducted on a voluntary basis. Among the participants who completed the intervention, complete paired fecal samples at both baseline and post-intervention time points were obtained from 20 participants per group, which constituted the final dataset used for these analyses.

This sample size reflects participant willingness to provide stool samples and the availability of complete paired specimens rather than a predefined sub-sampling strategy. We have clarified this point in the Materials and Methods section. The SCFA and microbiota analyses were conducted as secondary and exploratory outcomes, and the corresponding methods and results have been reported accordingly. (Lines 204-207 and 223-226)

• The placebo group showed a significant increase in propanoic acid. Do the authors have any biological explanation for this observation?

Response: We thank the reviewer for this comment. We agree that propanoic acid increased significantly within the placebo group over the 28-day period. Because the placebo capsule contained no active probiotic strain, this within-group change is unlikely to represent a direct intervention effect. Instead, fecal propionate can vary with short-term fluctuations in habitual diet (particularly fermentable carbohydrate and fiber intake), day-to-day bowel habits, and inter-individual microbiota dynamics. In this study, participants were asked to maintain their usual lifestyle rather than follow a standardized diet; therefore, spontaneous temporal variation within a group is possible. We have revised the Discussion to clearly describe this finding as a within-placebo change and avoided overinterpretation regarding a specific placebo bio-effects. (Lines 546-555)

• The discussion attributes weight loss in the placebo group solely to the Hawthorne effect. Could the placebo itself (maltodextrin) have had a mild prebiotic effect, contributing to some changes? This possibility should be considered.

Response: We thank the reviewer for this insightful comment. We agree that maltodextrin, although commonly used as an inert placebo, is a digestible carbohydrate and cannot be regarded as completely biologically inactive under all conditions. A mild fermentative or caloric contribution cannot be entirely excluded. To address this point, we have revised the Discussion to acknowledge that both behavioral factors (e.g., increased health awareness during trial participation) and the potential metabolic influence of the placebo matrix may have contributed to the modest within-group changes observed in the placebo arm. Importantly, the key findings of this study are based on between-group comparisons and baseline-adjusted analyses, which demonstrated more pronounced metabolic and microbiota-related effects in the UA881 group, supporting a treatment-specific effect beyond placebo-related influences. (Lines 513-526)

• The significant worsening of several metabolic markers in the placebo group over just 28 days is quite pronounced. Is this expected fluctuation in an overweight population, or could there be another reason?

Response: We thank the reviewer for this comment. Participants in this study were instructed to maintain their habitual diet, physical activity, and daily lifestyle throughout the intervention period. Therefore, short-term fluctuations in metabolic markers observed in the placebo group may reflect normal inter-individual physiological variability commonly seen in overweight populations under non-interventional conditions, rather than a true deterioration induced by the study protocol. Importantly, our conclusions rely on between-group and baseline-adjusted analyses, which consistently demonstrated that UA881 supplementation mitigated these unfavorable trends compared with placebo. (Lines 530-541)

• Could the authors elaborate on how a change in butyrate levels might specifically lead to a reduction in serum triglycerides and cholesterol, citing relevant mechanistic pathways?

Response: We appreciate the reviewer’s insightful question regarding the potential mechanistic link between butyrate and lipid metabolism. Following the reviewer’s recommendation, we reanalyzed the SCFA data using two-tailed statistical tests. Under this more conservative approach, the increase in fecal butyrate levels in the UA881 group did not reach statistical significance, although an upward trend was consistently observed. This finding suggests a potential, but not definitive, effect of UA881 on butyrate production.

From a mechanistic perspective, butyrate is well recognized as a key microbial metabolite involved in host energy and lipid metabolism. Previous studies have demonstrated that butyrate can activate AMP-activated protein kinase (AMPK) signaling, enhance fatty acid oxidation, suppress hepatic lipogenesis, and regulate cholesterol homeostasis through modulation of genes such as ACC, FAS, and HMG-CoA reductase. In addition, butyrate has been reported to influence intestinal lipid absorption and bile acid metabolism, which may collectively contribute to reductions in circulating triglycerides and cholesterol.

Although causality cannot be established in the present study, the concurrent trend toward increased butyrate levels and the observed improvements in serum triglycerides and total cholesterol support a biologically plausible link between UA881-induced microbial metabolic activity and host lipid regulation. Further studies with larger sample sizes and longer intervention periods are warranted to clarify this relationship. (Lines 556-569)

1. Minor Issues

• Line 213-214: The sentence "Normality of continuous variables was assessed using the Shapiro-Wilk test to determine the use of parametric or non-parametric statistical methods" is a repetition of the sentence on line 210.

Response: Thank you for pointing this out. We have revised this repetition. (Lines 270-273)

• Section "Gut Microbiota Composition at the Genus Level" is numbered 3.7, the same as the preceding section. It should be 3.8.

Response: Thank you for pointing this out. We have revised this typo. (Line 479)

• Line 420: The phrase "more obvious" could be replaced with more scientific language, such as "more pronounced" or "of greater magnitude."

Response: Thanks for your suggestion. We have revised as your comment. (Line 500)

• Line 439: "our data suggest that UA881 may protect against metabolic dysregulation." Consider rephrasing to "may attenuate" or "mitigate" metabolic dysregulation.

Response: Thanks for your suggestion. We have revised as your comment. (Line 529)

• Conflicts of Interest: The authors declare no conflicts of interest. However, the manuscript states it was funded by Nutrarex Bioceutical, and two authors (P.-C.H. and S.-Y.L.) are affiliated with Percheron Bioceutical Research Center / NUTRAREX BIOTECH, the company that also produced the supplements. The statement "The funders had no role in the design... collection, analyses, or interpretation of data..." is therefore questionable

Response: We thank the reviewer for raising this important point. We agree that the original Conflict of Interest statement did not adequately reflect the role of Nutrarex Bioceutical in this study. Nutrarex Bioceutical not only provided research funding, but also supplied the study products (UA881 probiotic and placebo) and contributed to specific experimental procedures and data analysis. We have therefore revised the Conflict of Interest statements to transparently disclose these contributions and clarify the respective roles of the academic investigators and the company-affiliated authors. (Lines 635-641)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors emphasize the crucial role of gut microbiota dysbiosis in the development of obesity and metabolic disorders and highlight the potential of probiotic intervention as a safe dietary strategy. Additionally, the beneficial impacts of Lactobacillus helveticus UA881 on improving body composition, lipid metabolism, and gut microbiota structure in overweight adults are demonstrated. However, there are certain aspects that the authors should take into account, and the detailed comments are listed as follows:

Major Comments:

1. The manuscript only mentions the effects of butyrate on intestinal barrier function and anti-inflammation. It is suggested to supplement the potential molecular mechanisms by which butyrate regulates host lipid metabolism and energy homeostasis.
2. A statistical power analysis should be added to justify that the sample size of 25 participants is sufficient to detect inter-group differences.
3. What is the rationale for the selected probiotic dose (5×10⁹ CFU/capsule)?
4. Why was SCFA analysis performed on only 20 (rather than all 50) participants? Are the selection criteria clearly defined?
5. The colonization status of the probiotic was not assessed, which prevents confirmation of whether the probiotic successfully colonized and exerted its effects.

Minor Comments:

6. For Figures 2 and 3 specifically, to improve readability, it is recommended to clearly label which indicators exhibit significant inter-group differences either within the figures or their legends.
7. The reference format needs standardization, as the abbreviations of some journal names are inconsistent.

Author Response

Reviewer3

In this study, the authors emphasize the crucial role of gut microbiota dysbiosis in the development of obesity and metabolic disorders and highlight the potential of probiotic intervention as a safe dietary strategy. Additionally, the beneficial impacts of Lactobacillus helveticus UA881 on improving body composition, lipid metabolism, and gut microbiota structure in overweight adults are demonstrated. However, there are certain aspects that the authors should take into account, and the detailed comments are listed as follows:

Response: We sincerely thank the reviewer for the thoughtful and positive evaluation of our study, as well as for recognizing the potential metabolic and microbiota-modulating effects of Lactobacillus helveticus (UA881). We appreciate the reviewer’s constructive suggestions and have carefully revised the manuscript accordingly. Detailed responses to each comment are provided below.

Major Comments:

1. The manuscript only mentions the effects of butyrate on intestinal barrier function and anti-inflammation. It is suggested to supplement the potential molecular mechanisms by which butyrate regulates host lipid metabolism and energy homeostasis.

Response: We appreciate the reviewer’s insightful question regarding the potential mechanistic link between butyrate and lipid metabolism. Following the reviewer’s recommendation, we reanalyzed the SCFA data using two-tailed statistical tests. Under this more conservative approach, the increase in fecal butyrate levels in the UA881 group did not reach statistical significance, although an upward trend was consistently observed. This finding suggests a potential, but not definitive, effect of UA881 on butyrate production.

From a mechanistic perspective, butyrate is well recognized as a key microbial metabolite involved in host energy and lipid metabolism. Previous studies have demonstrated that butyrate can activate AMP-activated protein kinase (AMPK) signaling, enhance fatty acid oxidation, suppress hepatic lipogenesis, and regulate cholesterol homeostasis through modulation of genes such as ACC, FAS, and HMG-CoA reductase. In addition, butyrate has been reported to influence intestinal lipid absorption and bile acid metabolism, which may collectively contribute to reductions in circulating triglycerides and cholesterol.

Although causality cannot be established in the present study, the concurrent trend toward increased butyrate levels and the observed improvements in serum triglycerides and total cholesterol support a biologically plausible link between UA881-induced microbial metabolic activity and host lipid regulation. Further studies with larger sample sizes and longer intervention periods are warranted to clarify this relationship. (Lines 556-569)

1. A statistical power analysis should be added to justify that the sample size of 25 participants is sufficient to detect inter-group differences.

Response: We thank the reviewer for these very important comments. This study was designed as an exploratory, proof-of-concept randomized controlled trial to evaluate the potential metabolic effects of UA881 probiotic supplementation in overweight adults. As limited prior human data were available for this specific strain, a formal a priori sample size calculation was not performed. The sample size was determined based on feasibility considerations and consistency with previous exploratory probiotic intervention studies of similar duration. This rationale has now been clarified in the revised manuscript. (Lines 274-278)

1. What is the rationale for the selected probiotic dose (5×10⁹ CFU/capsule)?

Response: We thank the reviewer for this question regarding the rationale for the selected probiotic dose. The dosage of 5 × 10⁹ CFU per capsule was selected based on commonly reported effective doses for functional probiotics in clinical studies, which generally range from 10⁹ to 10¹⁰ CFU / day. As this study was designed as an exploratory clinical trial, the chosen dose represents a practical and safe level expected to elicit biological effects while maintaining good tolerability. We have added the rationale in the Materials and Methods section. (Lines 160-162)

1. Why was SCFA analysis performed on only 20 (rather than all 50) participants? Are the selection criteria clearly defined?

Response: We thank the reviewer for this comment. Fecal sample collection for short-chain fatty acid (SCFA) and gut microbiota analyses was conducted on a voluntary basis. Among the participants who completed the intervention, complete paired fecal samples at both baseline and post-intervention time points were obtained from 20 participants per group, which constituted the final dataset used for these analyses.

This sample size reflects participant willingness to provide stool samples and the availability of complete paired specimens rather than a predefined sub-sampling strategy. We have clarified this point in the Materials and Methods section. The SCFA and microbiota analyses were conducted as secondary and exploratory outcomes, and the corresponding methods and results have been reported accordingly. (Lines 204-207 and 223-226)

1. The colonization status of the probiotic was not assessed, which prevents confirmation of whether the probiotic successfully colonized and exerted its effects.

Response: We thank the reviewer for this valuable comment. We agree that assessment of probiotic colonization would provide additional insight into the mechanisms underlying the observed effects. In the present study, in vivo colonization of UA881 in participants was not directly evaluated. However, we have conducted complementary in vitro experiments demonstrating that UA881 is capable of adhering to and colonizing intestinal epithelial cells (Caco-2 model), supporting its potential for gastrointestinal persistence. These mechanistic studies are part of our ongoing work and will be reported separately in a future publication. We have therefore focused the current manuscript on the clinical and metabolic outcomes observed following UA881 supplementation.

Minor Comments:

1. For Figures 2 and 3 specifically, to improve readability, it is recommended to clearly label which indicators exhibit significant inter-group differences either within the figures or their legends.

Response: We thank the reviewer for this helpful suggestion. To improve readability, we have revised Figures 2 and 3 by adding asterisks (*) within the figures to clearly indicate indicators showing statistically significant inter-group differences. (Please see the revised Fig.2 and Fig.3)

1. The reference format needs standardization, as the abbreviations of some journal names are inconsistent.

Response: Thank you for your effective suggestion. We have revised as your comment. (Please see the revised Reference section)

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This study is a randomized, double-blind, placebo-controlled trial evaluating Lactobacillus helveticus UA881 for 28 days in adults with BMI 25–27 kg/m², assessing anthropometrics, serum markers, fecal SCFAs, and 16S-based microbiota. The subject aligns with the journal's focus, and the multi-omics-style outcome set is a notable advantage. However, several design/reporting and interpretation issues (population definition, statistical approach, and microbiome methods reporting) currently limit confidence in the conclusions.

Major issues

The manuscript repeatedly refers to “obese” participants and “obesity”, including in the Abstract and framing, but the eligibility criterion is BMI 25–27 kg/m² (overweight range), and baseline BMI values are in the overweight range. This is a substantive misrepresentation of the studied population and must be corrected throughout (title/abstract/main text), or the inclusion criteria must be clarified if obesity was truly included.

Statistical approach: extensive within-group testing + one-tailed paired t-tests + multiple outcomes: The use of one-tailed paired t-tests and many endpoints without a clear primary outcome or multiplicity strategy increases false-positive risk. Please justify one-tailed testing (directionality prespecified) or switch to two-tailed tests. Also define primary/secondary endpoints and consider correction for multiple comparisons (or at minimum, report adjusted p-values/sensitivity analyses).

Primary endpoint and trial reporting standards are not clearly specified: The manuscript does not clearly state the primary outcome, power/sample-size rationale, or whether the trial was prospectively registered. Please add CONSORT-style details (allocation concealment, randomization procedure, registration ID, and sample size calculation) to support internal validity.

Diet/physical activity control is insufficient for body composition outcomes: The trial is “free-living” and both groups show weight/BMI reduction. Without robust monitoring/control (dietary intake, physical activity logs, energy intake), attributing changes to UA881 is challenging. This should be strengthened in Methods (what was recorded and how) and discussed more cautiously.

Microbiome analysis: insufficient methodological detail for reproducibility: Key details are missing/limited: bioinformatics pipeline (e.g., OTU clustering vs ASVs), database used for taxonomy, filtering thresholds, rarefaction/normalization strategy, batch effects, and handling of compositionality. These are essential for microbiome claims.

Subsampling for SCFA and microbiota endpoints (n=20) needs justification and transparency: Selecting only 20 participants per group for SCFAs/microbiota reduces power and may introduce selection bias unless the randomization/subsampling procedure is clearly described and baseline comparability of the subsample is shown.

Potential conflicts of interest / sponsor involvement require careful transparency: The study is funded by an industry entity; some authors appear affiliated with related organizations. The statement that funders had “no role” should be reconciled with affiliations and any sponsor involvement in product manufacture/quality control and data handling.

 

Minor issues 

Lines 20-39, esp. 23-30: Abstract should specify microorganism, dose, and timing of effectiveness more explicitly. While the strain is mentioned, the dose and a clear statement that effects were observed after 28 days should be explicitly included in the Abstract for clarity and clinical interpretability.

Lines 25-26: BMI range 25–27 kg/m² indicates overweight, not obesity—remove/replace “obese/obesity” wording. The Abstract states “overweight or obese adults (BMI 25–27 kg/m²)”, which is incorrect classification and internally inconsistent. Please remove “obese/obesity” terminology unless obesity participants were actually included. Lines

Lines: 25–29, 123–129, 136: Abstract mentions “for 28 days” but includes “for 28 days” and “day 28” assessment; ensure consistent phrasing as “28 days” or “4 weeks” and match across Abstract/Methods/Results. 

Lines 209–215: Shapiro–Wilk normality assessment is described twice; please remove redundancy and clarify exact decision rules for parametric vs non-parametric tests

Lines 212–214: One-tailed paired t-tests should be stated with rationale. If retained, explicitly justify pre-specified directionality; otherwise revise to two-tailed.

Lines 358–373: OTU terminology vs modern ASV approaches: the Results discuss “observed OTUs” (alpha diversity). If ASVs were used, revise terms; if OTUs were used, specify clustering threshold and pipeline.

Line 490: Double period at end of “manuscript.”

Table 1: “Dosage regiment” should be “Dosage regimen”; “administrated capsule” should be “administered capsule”.

Minor formatting artifacts in tables (e.g., “Homogeneity”, broken hyphenation) likely from template/PDF conversion; please clean prior to final submission.

Figure captions could be more specific about statistical tests. Captions for Figs. 2–5 would benefit from naming the test used for group comparisons and whether correction for multiple testing was applied.

 

Author Response

Reviewer4

This study is a randomized, double-blind, placebo-controlled trial evaluating Lactobacillus helveticus UA881 for 28 days in adults with BMI 25–27 kg/m², assessing anthropometrics, serum markers, fecal SCFAs, and 16S-based microbiota. The subject aligns with the journal's focus, and the multi-omics-style outcome set is a notable advantage. However, several design/reporting and interpretation issues (population definition, statistical approach, and microbiome methods reporting) currently limit confidence in the conclusions.

>>Response: We thank the reviewer for the constructive and balanced assessment of our study. We appreciate the recognition of the study design and the multi-dimensional outcome approach. In response to the reviewer’s valuable comments, we have carefully revised the manuscript to address issues related to population definition, statistical methodology, and microbiome analysis reporting, as detailed below.

Major issues

The manuscript repeatedly refers to “obese” participants and “obesity”, including in the Abstract and framing, but the eligibility criterion is BMI 25–27 kg/m² (overweight range), and baseline BMI values are in the overweight range. This is a substantive misrepresentation of the studied population and must be corrected throughout (title/abstract/main text), or the inclusion criteria must be clarified if obesity was truly included.

>>Response: We thank the reviewer for this important clarification. We agree that the original terminology did not accurately reflect the studied population. According to the inclusion criteria and baseline characteristics, participants in this study were within the overweight range (BMI 25–27 kg/m²), rather than obese. We have therefore revised all obesity-related terminology throughout the manuscript, including the title, abstract, and main text, to consistently refer to overweight individuals, ensuring alignment with the BMI definition and the actual study population. (Please see the revised version we highlighted in yellow, such as Lines 122-124)

Statistical approach: extensive within-group testing + one-tailed paired t-tests + multiple outcomes: The use of one-tailed paired t-tests and many endpoints without a clear primary outcome or multiplicity strategy increases false-positive risk. Please justify one-tailed testing (directionality prespecified) or switch to two-tailed tests. Also define primary/secondary endpoints and consider correction for multiple comparisons (or at minimum, report adjusted p-values/sensitivity analyses).

>>Response: We thank the reviewer for this important comment regarding the statistical approach. In response, we have revised the statistical analyses by replacing the one-tailed paired t-tests with two-tailed tests throughout the manuscript, in accordance with standard and more conservative practice. In addition, we have clearly defined the primary and secondary endpoints of the study: serum triglycerides (TG) were prespecified as the primary outcome, while all other anthropometric, biochemical, inflammatory, and microbiota-related parameters were designated as secondary outcomes, consistent with the trial registration. Given the exploratory nature of this trial, analyses of secondary outcomes were interpreted descriptively. The Methods and Results sections have been revised accordingly to reduce the risk of false-positive findings associated with multiple outcomes. (Please see the revised text in Lines 279-282, and the corresponding data presented in Table 4.)

Primary endpoint and trial reporting standards are not clearly specified: The manuscript does not clearly state the primary outcome, power/sample-size rationale, or whether the trial was prospectively registered. Please add CONSORT-style details (allocation concealment, randomization procedure, registration ID, and sample size calculation) to support internal validity.

>>Response: We thank the reviewer for this important comment regarding trial reporting standards. We have revised the manuscript to clearly specify the primary and secondary endpoints: serum triglycerides (TG) were prespecified as the primary outcome, while all other anthropometric, biochemical, inflammatory, and microbiota-related parameters were defined as secondary outcomes, consistent with the public trial registration. The study was prospectively registered at ClinicalTrials.gov (Identifier: NCT06554314), and this information has been clearly stated in the revised manuscript. (Lines 117-121)

With regard to trial design, participants were assigned to the UA881 or placebo group using a computer-generated covariate-adaptive randomization procedure, controlling for gender, age, and BMI to minimize potential confounding effects. Allocation concealment and the double-blind design have been clarified in the Methods section. (Lines 134-141)

Regarding sample size, this study was designed as an exploratory clinical trial, and a formal a priori power calculation was therefore not performed. The chosen sample size was based on feasibility considerations and on commonly used group sizes in exploratory probiotic intervention studies. These CONSORT-related details have now been added or clarified in the Materials and Methods section to strengthen the internal validity and transparency of the trial reporting. (Please see revised Figure 1 in Lines 300-301)

Diet/physical activity control is insufficient for body composition outcomes: The trial is “free-living” and both groups show weight/BMI reduction. Without robust monitoring/control (dietary intake, physical activity logs, energy intake), attributing changes to UA881 is challenging. This should be strengthened in Methods (what was recorded and how) and discussed more cautiously.

Response: We thank the reviewer for this important comment. We acknowledge that this study was conducted under free-living conditions and that dietary intake and physical activity were not strictly controlled or quantitatively monitored throughout the intervention. As described in the Methods section, participants were instructed to maintain their habitual diet and physical activity patterns during the study period, and no structured lifestyle intervention was implemented. We have revised the Methods section to clarify this aspect of the study design. (Lines 176-179)

We agree that such free-living conditions may contribute to within-group changes in body composition observed in both groups. Accordingly, we have revised the Discussion to interpret these findings more cautiously, emphasizing that our primary conclusions are based on between-group and baseline-adjusted analyses rather than within-group changes alone. These revisions are intended to provide a balanced interpretation while acknowledging the inherent variability associated with real-world clinical settings. (Lines 530-541)

Microbiome analysis: insufficient methodological detail for reproducibility: Key details are missing/limited: bioinformatics pipeline (e.g., OTU clustering vs ASVs), database used for taxonomy, filtering thresholds, rarefaction/normalization strategy, batch effects, and handling of compositionality. These are essential for microbiome claims.

>>Response: We thank the reviewer for this important comment regarding the microbiome methodology. We agree that detailed reporting of the bioinformatics pipeline is essential for reproducibility. Accordingly, we have expanded the Materials and Methods section to provide additional details on the microbiome analysis workflow.

Briefly, 16S rRNA gene sequencing data (V3–V4 regions) were processed using a standard OTU-based pipeline. Quality filtering was performed to remove low-quality reads and chimeric sequences prior to downstream analysis. Operational taxonomic units (OTUs) were clustered at 97% sequence similarity, and taxonomic assignment was conducted using SILVA reference database. OTUs with extremely low abundance or prevalence across samples were filtered to reduce noise.

For diversity analyses, data were normalized by rarefaction to an even sequencing depth to minimize bias due to sequencing depth variation. Alpha diversity indices (Observed OTUs, ACE, Chao1, and Shannon index) and beta diversity metrics were then calculated based on the normalized data. Relative abundance data were used for taxonomic comparisons and visualizations at the phylum and genus levels, acknowledging the compositional nature of microbiome data. As all samples were processed and sequenced in the same laboratory using identical protocols, batch effects were minimized. These methodological details have now been clarified in the revised manuscript to improve reproducibility of the microbiome analyses. Please see the subsection entitled “2.7.2. Library Construction and Sequencing” (Lines 238–266)

Subsampling for SCFA and microbiota endpoints (n=20) needs justification and transparency: Selecting only 20 participants per group for SCFAs/microbiota reduces power and may introduce selection bias unless the randomization/subsampling procedure is clearly described and baseline comparability of the subsample is shown.

>>Response: We thank the reviewer for this comment. Fecal sample collection for short-chain fatty acid (SCFA) and gut microbiota analyses was conducted on a voluntary basis. Among the participants who completed the intervention, complete paired fecal samples at both baseline and post-intervention time points were obtained from 20 participants per group, which constituted the final dataset used for these analyses.

This sample size reflects participant willingness to provide stool samples and the availability of complete paired specimens rather than a predefined sub-sampling strategy. We have clarified this point in the Materials and Methods section. The SCFA and microbiota analyses were conducted as secondary and exploratory outcomes, and the corresponding methods and results have been reported accordingly. (Lines 204-207 and 223-226)

Potential conflicts of interest / sponsor involvement require careful transparency: The study is funded by an industry entity; some authors appear affiliated with related organizations. The statement that funders had “no role” should be reconciled with affiliations and any sponsor involvement in product manufacture/quality control and data handling.

>>Response: We thank the reviewer for raising this important point. We agree that the original Conflict of Interest statement did not adequately reflect the role of Nutrarex Bioceutical in this study. Nutrarex Bioceutical not only provided research funding, but also supplied the study products (UA881 probiotic and placebo) and contributed to specific experimental procedures and data analysis. We have therefore revised the Conflict of Interest statements to transparently disclose these contributions and clarify the respective roles of the academic investigators and the company-affiliated authors. (Lines 635-641)

Minor issues 

Lines 20-39, esp. 23-30: Abstract should specify microorganism, dose, and timing of effectiveness more explicitly. While the strain is mentioned, the dose and a clear statement that effects were observed after 28 days should be explicitly included in the Abstract for clarity and clinical interpretability.

>>Response: We thank the reviewer for this valuable comment. We agree that clearer specification of the probiotic strain, dosage, and timing of observed effects would improve the clinical interpretability of the Abstract. Accordingly, we have revised the Abstract to explicitly state the administered dose of Lactobacillus helveticus UA881 (5 × 10⁹ CFU/day) and to clarify that the reported effects were observed after a 28-day intervention period. These revisions have been incorporated throughout the Abstract. (Lines 25-30)

Lines 25-26: BMI range 25–27 kg/m² indicates overweight, not obesity—remove/replace “obese/obesity” wording. The Abstract states “overweight or obese adults (BMI 25–27 kg/m²)”, which is incorrect classification and internally inconsistent. Please remove “obese/obesity” terminology unless obesity participants were actually included. Lines

>>Response: Thanks for your effective comment. As the response to your first Major Comment, we have revised all obese-related wording as overweight-related terminology. Please see the revised version we highlighted in yellow. (Lines 122-124)

Lines: 25–29, 123–129, 136: Abstract mentions “for 28 days” but includes “for 28 days” and “day 28” assessment; ensure consistent phrasing as “28 days” or “4 weeks” and match across Abstract/Methods/Results. 

>>Response: We thank the reviewer for this comment. To ensure consistency, we have revised the manuscript to use the phrasing “28 days” uniformly throughout the Abstract, Methods, and Results sections, replacing mixed usage of “4 weeks” or “28 days”. (Please see the revised version we highlighted in yellow, such as Line 148.)

Lines 209–215: Shapiro–Wilk normality assessment is described twice; please remove redundancy and clarify exact decision rules for parametric vs non-parametric tests

>>Response: We thank the reviewer for pointing out this redundancy. The repeated description of the Shapiro–Wilk normality test has been removed. In addition, we have clarified the decision rules in the revised Methods section. (Lines 270-273)

Lines 212–214: One-tailed paired t-tests should be stated with rationale. If retained, explicitly justify pre-specified directionality; otherwise revise to two-tailed.

>>Response: We thank the reviewer for this important comment. We agree that the use of one-tailed paired t-tests requires strong a priori justification and that two-tailed tests represent a more conservative and widely accepted approach. In response to this concern, we have reanalyzed the relevant data using two-tailed paired t-tests. The Results section and corresponding figures and tables have been revised accordingly to reflect the updated statistical analyses. Importantly, the main conclusions of the study remained unchanged after reanalysis using two-tailed tests. (Please see the revised Table 3 and Table 4)

Lines 358–373: OTU terminology vs modern ASV approaches: the Results discuss “observed OTUs” (alpha diversity). If ASVs were used, revise terms; if OTUs were used, specify clustering threshold and pipeline.

>>Response: We thank the reviewer for this important comment regarding microbiome terminology. In the present study, operational taxonomic units (OTUs) were generated using a conventional OTU-based pipeline rather than an ASV-based approach. OTUs were clustered at 97% sequence similarity, which is consistent with widely used practices in 16S rRNA gene-based microbiome studies. We have revised the Methods section to clearly specify the OTU clustering strategy, similarity threshold, and taxonomic assignment pipeline. (Lines 248-257)

Line 490: Double period at end of “manuscript.”

>>Response: Thanks for the kind reminder. The double period has been removed. (Line 623)

Table 1: “Dosage regiment” should be “Dosage regimen”; “administrated capsule” should be “administered capsule”.

>>Response: Thanks for the kind reminder. We have revised these typos. (Lines 159-160)

Minor formatting artifacts in tables (e.g., “Homogeneity”, broken hyphenation) likely from template/PDF conversion; please clean prior to final submission.

>>Response: We appreciate the reviewer’s careful attention to formatting details. These minor artifacts arose from the manuscript template, and all tables and text will be thoroughly checked and corrected to ensure a clean and consistent presentation in the final version. (Please see the revised Table 3 and Table 4)

Figure captions could be more specific about statistical tests. Captions for Figs. 2–5 would benefit from naming the test used for group comparisons and whether correction for multiple testing was applied.

>>Response: We thank the reviewer for this helpful suggestion. We have revised the figure captions for Figures 2–5 to explicitly specify the statistical tests used for group comparisons (e.g., paired two-tailed t-tests or ANCOVA, as appropriate). In addition, we clarified in the figure legends that no formal correction for multiple testing was applied, consistent with the exploratory nature of the secondary and microbiome-related analyses. (Please see revised legends of Fig. 2-5)

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to express my appreciation for the thorough and thoughtful manner in which you have addressed the comments and suggestions provided during the review process. The revisions made to the manuscript demonstrate a clear commitment to scientific rigor and clarity, and they have significantly strengthened the overall quality of the work. The responses to the reviewers’ queries were comprehensive and well-supported, reflecting careful consideration of the feedback received.
I am satisfied with the adjustments implemented, which have enhanced the presentation of the study’s methodology, results, and discussion. The manuscript now provides a more coherent and persuasive account of the clinical relevance of Lactobacillus helveticus UA881 in improving body composition, lipid profiles, and gut microbiota in overweight adults. I commend the authors for their diligence and responsiveness in refining the paper to meet high academic standards.

Author Response

Reviewer1

I would like to express my appreciation for the thorough and thoughtful manner in which you have addressed the comments and suggestions provided during the review process. The revisions made to the manuscript demonstrate a clear commitment to scientific rigor and clarity, and they have significantly strengthened the overall quality of the work. The responses to the reviewers’ queries were comprehensive and well-supported, reflecting careful consideration of the feedback received.

I am satisfied with the adjustments implemented, which have enhanced the presentation of the study’s methodology, results, and discussion. The manuscript now provides a more coherent and persuasive account of the clinical relevance of Lactobacillus helveticus UA881 in improving body composition, lipid profiles, and gut microbiota in overweight adults. I commend the authors for their diligence and responsiveness in refining the paper to meet high academic standards.

>>Response: We sincerely thank the reviewer for the kind and encouraging comments. We appreciate the reviewer’s recognition of our revisions and are pleased that the manuscript has been improved accordingly.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all points raised in the previous round of review. I appreciate their efforts to improve the work and I have no further concerns.

Author Response

Reviewer2

The authors have addressed all points raised in the previous round of review. I appreciate their efforts to improve the work and I have no further concerns.

>>Response: We sincerely thank the reviewer for the positive evaluation and encouraging comments. We greatly appreciate the time and effort devoted to reviewing our manuscript, and we are grateful for the valuable feedback that has helped improve the quality of our work.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

no

Author Response

Reviewer3

No further comment.

>>Response: We thank the reviewer for the evaluation of our manuscript and for confirming that no further comments are raised.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Most comments addressed; however, please correct remaining inconsistencies:

Minor issues

Line 96: please remove „and obese”

Line 204-205: please correct from „complete paired fecal samples at baseline and day 0” to: complete paired fecal samples at baseline (day 0) and day 28.”

Author Response

Reviewer4

Most comments addressed; however, please correct remaining inconsistencies:

Minor issues

Line 96: please remove „and obese”

>>Response: We thank the reviewer for this comment. The wording has been revised, and the phrase “and obese” has been removed to ensure consistency with the study population definition. (Please see the revision we highlighted in Red in Line 96)

Line 204-205: please correct from „complete paired fecal samples at baseline and day 0” to: complete paired fecal samples at baseline (day 0) and day 28.”

>>Response: We thank the reviewer for pointing out this inconsistency. The text has been corrected to read “complete paired fecal samples at baseline (day 0) and day 28,” as suggested. (Line 205)

Author Response File: Author Response.docx