Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes the impact of administering the probiotic bacterium H. coagulans through sugar-free chewing gum on dental biofilm. The authors conducted a double-blind randomised controlled study, including two groups: one that consumed the probiotic and one that consumed placebo gum. They investigated the fate of the probiotic in the dental plaque using qPCR and analysed its effects on the microbiome using amplicon sequencing.

This topic will be of interest to readers of Nutrients and to scientists working in oral microbiology and dentistry. The manuscript is well written. However, the following major points raised by the reviewer must be addressed:

1) When were the dental plaque samples taken? How can the authors be sure that they took enough plaque material for their experiments?

2) The alpha- and beta-diversity diagrams for all time points should be removed from the supplementary materials and included in the results section. Unfortunately, access to the supplementary materials was not possible. Nevertheless, these results form a key part of this study and must be included in the main manuscript.

3) The authors mentioned an earlier study with the same probiotic bacterium that described the fate of the probiotic in the saliva of consumers. Could this bacterium be recultured from the salivary samples over a longer period of time after consumption? Detecting this bacterium by qPCR does not provide information about its viability. Hence, it is a pity that the authors did not attempt to culture this bacterium from the plaque samples alongside the detection using qPCR.

4) The conclusion regarding the effects on microbiome composition should be weakened, as the results from the small cohort included in this study do not justify strong conclusions.

Author Response

Dear Reviewer,

We sincerely thank you for the positive appraisal of our manuscript and for the valuable suggestions provided to improve its quality. We will address each comment point by point below and you will find in evidence in the revised version of the manuscript.

1) When were the dental plaque samples taken? How can the authors be sure that they took enough plaque material for their experiments?

Dental plaque sampling was performed in the morning, at least 24 hours after the last oral hygiene procedure and at least 2 hours after food or drink intake, in order to standardize plaque accumulation conditions. Sampling was conducted before any clinical manipulation and prior to chewing gum intake at each time point.

To ensure adequate biomass for downstream molecular analyses, plaque was collected systematically from both buccal and lingual surfaces of all teeth. DNA yield and concentration were quantified fluorometrically prior to qPCR and 16S analysis.

2) The alpha- and beta-diversity diagrams for all time points should be removed from the supplementary materials and included in the results section. Unfortunately, access to the supplementary materials was not possible. Nevertheless, these results form a key part of this study and must be included in the main manuscript.

As suggested, we have incorporated tables and figures related to alpha-diversity and beta-diversity into the revised manuscript.

3) The authors mentioned an earlier study with the same probiotic bacterium that described the fate of the probiotic in the saliva of consumers. Could this bacterium be recultured from the salivary samples over a longer period of time after consumption? Detecting this bacterium by qPCR does not provide information about its viability. Hence, it is a pity that the authors did not attempt to culture this bacterium from the plaque samples alongside the detection using qPCR.

We thank the reviewer for this comment. We agree that distinguishing between viable cells and DNA presence is a critical distinction in probiotic research. Our decision to prioritize qPCR over culture-based methods was based on several factors. First, the primary scope of this study was to monitor the overall dental plaque microbial composition following chewing gum consumption. Consequently, the detection of H. coagulans was performed mainly to assess volunteer compliance with the intervention. Second, due to the limited biomass of the plaque samples, we opted for qPCR to ensure we could perform both this compliance check and the microbial profiling from the same DNA extract. Furthermore, our previous findings on H. coagulans in saliva showed a rapid decline in concentration within the first two hours post-consumption (Cirio S, Salerno C, Guglielmetti SD, Mezzasalma V, Sarrica A, Kirika N, Campus G, Cagetti MG. In Vivo Study on the Salivary Kinetics of Two Probiotic Strains Delivered via Chewing Gum. Microorganisms. 2025 Mar 24;13(4):721. do: 10.3390/microorganisms13040721). In the present study, the target was detected seven days post-intervention in only 2 out of 21 subjects. This low frequency of detection via molecular assay suggests that its persistence in the oral cavity is transient, which aligns with the rapid loss of viability previously observed in saliva. While we acknowledge that culturing would have provided additional information regarding metabolic activity, we believe the qPCR data effectively confirms the "wash-out" trend and serves its purpose as a marker of adherence to the study protocol.

4) The conclusion regarding the effects on microbiome composition should be weakened, as the results from the small cohort included in this study do not justify strong conclusions.

In accordance with your suggestion, we have revised the manuscript to moderate the conclusions regarding the effects on microbiome composition, ensuring that they accurately reflect the limited sample size and the exploratory nature of the study

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

My comments and suggestions are provided in the attached review.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We sincerely thank you for the positive appraisal of our manuscript and for the valuable suggestions provided to improve its quality. We will address each comment point by point below and you will find in evidence in the revised version of the manuscript.

Abstract

The study evaluates microbiome composition and strain detection but does not include clinical oral health parameters. It would be helpful to clarify in the Discussion and Conclusions that the findings reflect ecological shifts in dental biofilm rather than demonstrated clinical improvement.

The observed effects were modest and reversible, and qPCR detection does not distinguish viable bacteria from free DNA; therefore, the findings should not be interpreted as true colonization. The authors may also briefly acknowledge the potential influence of chewing gum use itself (e.g., increased salivary flow and mechanical plaque disruption).

In accordance with your suggestion, we have revised the abstract to moderate the conclusions regarding the effects on microbiome and and emphasized the absence of clinical outcomes.

Introduction

The introduction could be streamlined by shortening the general discussion of the oral microbiome and moving more directly to probiotic modulation and its relevance to dental biofilm.

The clinical relevance should be stated more explicitly by briefly explaining how modulation of dental biofilm ecology may be relevant for preventive or adjunctive oral care.

The knowledge gap would benefit from clearer definition, particularly the limited evidence on delivery of probiotic strains into dental plaque and their ecological impact within the oral biofilm, as well as the rationale for chewing gum as a delivery system.

In accordance with the comments, we have streamlined the Introduction by shortening the general discussion on the oral microbiome and moving more directly to probiotic modulation and its relevance to dental biofilm. Furthermore, we have more explicitly clarified the clinical relevance by briefly explaining how modulation of dental biofilm ecology may contribute to preventive strategies and adjunctive oral care. Finally, we have better defined the existing knowledge gap, particularly emphasizing the limited evidence regarding the delivery of probiotic strains into dental plaque, their ecological impact within the oral biofilm, and the rationale for selecting chewing gum as a delivery system.

 Materials and Methods

The study is exploratory in nature; therefore, describing it explicitly as a pilot or hypothesis-generating study would help clarify the rationale for the chosen sample size.

Because chewing itself may influence salivary flow and mechanical biofilm disruption, it would be helpful to explicitly state that placebo gum was used to control for these effects.

We have now more clearly specified the exploratory nature of the study, explicitly describing it as a pilot and hypothesis-generating investigation in Section 2.2 of the manuscript.

Additionally, as suggested, we have clarified in Section 2.3 that a placebo chewing gum was used in order to control for the potential effects of chewing itself, including increased salivary flow and mechanical biofilm disruption.

Discussion

The discussion clearly describes the ecological and transient nature of the findings. It may be useful to briefly emphasize that microbiome modulation does not necessarily translate into clinical benefit and that variability among participants may reflect individual host factors and the need for continued use to sustain the observed effects.

We have carefully considered the suggestions and have accordingly integrated the Discussion section to further emphasize that microbiome modulation does not necessarily translate into a direct clinical benefit. In addition, we have highlighted that the variability observed among participants may reflect individual host-related factors and that sustained use may be necessary to maintain the ecological effects detected in this exploratory study.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All points have been addressed sufficiently