Screening and Evaluation In Vitro of Bacillus-Based Probiotics for Feed Additives
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
“Screening and Evaluation in vitro of Bacillus-based Probiotics for Feed Additives”
The manuscript presents the initial preclinical characterization of two promising Bacillus strains, FJAT-10508 and FJAT-13563, selected from a collection of 394 Bacillus isolates. The screening encompasses the evaluation of extracellular enzyme production (cellulase, protease, and amylase) and antibacterial activity against Escherichia coli, Staphylococcus aureus, and Salmonella enterica, employing standard microbiological and biochemical methodologies. Subsequent characterization of the selected strains includes safety assessment through genome sequencing and investigation of probiotic traits such as auto-aggregation, endospore formation efficiency, tolerance to simulated gastrointestinal conditions, antibiotic susceptibility, and hemolytic activity.
While the identification of novel Bacillus strains for feed additive applications is not an entirely new research topic, the manuscript is well organized, and the results are clearly presented. Overall, the study provides a meaningful contribution to the current understanding of Bacillus-based probiotics and their potential use as feed additives.
Nevertheless, prior to publication, the authors are encouraged to address the following points:
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Introduction – Nomenclature of pathogens:
The use of abbreviations for pathogenic species (e.g., Staphylococcus aureus as St. aureus and Salmonella enterica as Sa. enterica) should be consistent throughout the manuscript (see line 98). -
Statistical Analysis:
Please clarify whether a normality test was performed on the analyzed data to ensure the validity of subsequent statistical evaluations. -
Primary Screening (Line 126):
The manuscript states that “Each obtained culture was adjusted to OD₆₀₀ nm = 0.5.” The authors are requested to convert this optical density value to colony-forming units per milliliter (CFU/mL). Expressing the inoculum concentration in CFU/mL provides greater accuracy, as OD₆₀₀ readings can vary substantially among different Bacillus strains. -
General/Results:
It would strengthen the study to include in vitro assays using a benchmark Bacillus strain for comparative purposes. Such data would allow a clearer evaluation of the relative probiotic potential of the newly identified strains. -
Antibacterial Screening and Co-aggregation Assay:
Considering the results obtained from the antibacterial and co-aggregation assays, the authors should discuss their interpretation of the predominant mechanism of action against S. aureus, S. enterica, and E. coli. Is the inhibition primarily due to growth suppression, co-aggregation, or a combination of both? This discussion should be incorporated into the relevant section of the manuscript. -
Safety of Bacillus Strains – EFSA Guidelines:
According to the European Food Safety Authority (EFSA), the safety evaluation of Bacillus strains intended for food and feed applications should include assessment of the cytotoxicity of Bacillus supernatants on epithelial cell lines. This step is essential for identifying strains capable of producing harmful toxins, including non-ribosomally synthesized peptides, which cannot be detected solely through genomic analysis. The authors should clarify whether the presence of non-ribosomal peptide synthesis pathways has been evaluated in the selected strains. This analysis is considered mandatory for new Bacillus probiotic candidates. If such evaluation could not be performed, this limitation should be explicitly acknowledged in the manuscript.
Author Response
Please see the attachment.
Author Response File:
Reviewer 2 Report
Comments and Suggestions for Authors
The article is a detailed, consistent description of the extensive work to identify strains with valuable probiotic properties. The logic of the work and the methods (the authors used standard methods generally accepted for this type of research) are described clearly and in sufficient detail. The design of the work corresponds to the goals, the results obtained are presented clearly, the conclusions drawn are justified and correspond to the results obtained. No excessive or inappropriate citations were found. There were no ethical issues.
There are some minor comments that do not affect the scientific value of the article.
In section 2.6, please clarify how the bacterial suspension was obtained for measuring aggregation and autoaggregation. Is the Bacillus suspension used (~108 CFU ml-1) a stationary-phase growth sample?
It is advisable to rewrite the Results and Discussion sections.
In particular, move lines 522-542 from the Discussion section to the Introduction section.
In the Results section, leave only the results of the experiments performed, moving the references and discussion to the Discussion section.
Author Response
Please see the attachment.
Author Response File:
Reviewer 3 Report
Comments and Suggestions for Authors
This study addresses a topic of great relevance to sustainable animal production, highlighting the importance of Bacillus velezensis as a promising probiotic candidate due to its enzymatic activity, antibacterial potential, stress tolerance, and safety profile. The methodology is clear, comprehensive, and well-aligned with current international guidelines, combining extensive in vitro evaluations with genomic analyses, which significantly strengthens the scientific value of the work. Overall, the manuscript is well-written, logically structured, and based on accurate experimental data. However, some minor revisions are recommended to further enhance clarity and coherence. It is necessary to explicitly state the study's objective at the beginning of the abstract to improve clarity and ensure better alignment between the methodology, results, and the overall purpose of the study. Furthermore, while the methodology is technically sound, it is excessively detailed and could be made more concise by condensing repetitive descriptions. The introduction would also benefit from minor adjustments, mainly by simplifying some sections to improve focus and flow without altering the scientific content. All the suggestions in the article are in the corrections marked in yellow.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File:
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
The authors have satisfactorily addressed all reviewer comments. The manuscript is therefore recommended for publication.
