Isolation and Characterization of Bacteriocin-like-Producing Companilactobacillus farciminis YLR-1 and the Inhibitory Activity of Bacteriocin Against Staphylococcus aureus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting study on the bacteriocin like compounds produced by Companilactobacillus farciminis and their inhibitory activity against S. aureus. However, I would like to draw your attention to the following:

Abstract: Since you studied only one pathogen the word should not appearin plural. 

At certain parts of the MS (L119, L 122) species names appear inlarger fonts.

 Introduction

L84 the selected strain Lactobacillus... this is the first mention of Companilactobacillus farciminis as Lactobacillus. Either explain the use of older and more recent taxonomy (Companilactobacillus farciminis, previously Lactobacillus farciminis) or try to be consistent.

Materials and Methods

L. 124 Since you are working woth two bacteria (one probiotic and one patogen) ou might consider reporting species name at each supernatant mention for best clarity. e.g. L124, C. farciminis cell free. Also, what was the duration of S. aureus incubation? Please state that you checked various time pointsof C. farciminis culture (ie production of antibacterials as a function of culture time).

2.5. This part has to be rewritten with more clarity. Please state bacterial species( L129...cells.... which cells?) . 1 ml extracted by centrifugation....do you keep the sediment and resuspend in what or the supernatant?

2.6. L142 Please state that MRS was previously autoclaved , prior to the addition of the bacteria

2.7. You state that you used a rotary evaporator at 40 C to downsize the volume of an aqueous sample! I cannot see how this is possible. 

2.8. Purification implies that only one compound is isolated and purified. However all compounds with MW lower than 500D are isolated in your study.Hence, purification is an overstatement.

2.10. L. 174 Please state that all samples were run (SDS) in duplicate and half the gel was stained

2.11. Please provide a more detailed description of the procedure and mention electron microscope and fluorescence microscope (2.12) type and operating conditions.

Results

You state that "Growth data for C. farciminis YLR-1 were collected through 18 samplings over 36 206 h". Does this mean  two samples per time point? How many independent samples (n) did you employ?

Figures 2, 3

 Please state control survival in legend

 discussion

 L. 326. You state that BLIS may function as secondary metabolite . Please consider that production of bacteriocins is not only strain specific but also phase specific

L 327 Probiotic potential; A probiotic has to meet other criteria as well ..ie autoaggregation, biofilm , no toxicity against host, to be characterized as such. Hence,you might consider replacing probiotic with potential probiotic. 

L 358 You state that BLIS might have a proteinaceous nature due to the action of proteinake K. Did you quantify protein in your isolation steps? 

L377These properties suggest it likely belongs to Class I bacteriocins[41, 42]. Such classification requires more evidence . The presence of unusual peptides might strengthen your hypothesis

 conclusions : You state " In conclusion, C. farciminis YLR-1, a bacteriocin-producing strain, preliminary probiotic properties indicated the potential of the strain for direct use in the gut". Ι consider this conclusion risky since hemolytic and cytotoxic activity against human or other hosts has not been checked. 

 

Author Response

Dear editor and reviewers,

We are very grateful for your constructive comments and suggestions for our manuscript entitled “Isolation and characterization of bacteriocin-like-producing Companilactobacillus farciminis YLR-1, and the inhibitory activity of bacteriocin against Staphylococcus aureus” (fermentation-3743773). Your comments are very valuable and helpful for improving our manuscript. The revisions made to the manuscript are highlighted in yellow text. The comments of three reviewers are provided in italics and bold below, and specific questions have been numbered. Our responses are presented in normal font. Please refer to the attached Response Letter for detailed corrections. We have studied comments and modified our manuscript carefully. We sincerely hope to get your approval.

Reviewer: 1

1. Abstract: Since you studied only one pathogen the word should not appearin plural.

The author’s answer： Thank you for the suggestion. This sentence was revised according to the comment (Line 21-23).

-- This study aimed to identify a probiotic bacterium with antagonistic activity against foodborne pathogen Staphylococcus aureus （S. aureus） and investigate the mechanism of its antibacterial components.

 

2. At certain parts of the MS (L119, L 122) species names appear inlarger fonts.

The author’s answer： Thank you for your suggestions. We have verified the species names throughout the manuscript and standardized their formatting (Line 121, Line 124).

-- a 1 mL aliquot of the C. farciminis YLR-1 overnight culture was centrifuged,

-- Antimicrobial activity against S. aureus ATCC25923 was evaluated using the agar well diffusion method^[18].

 

3. L84 the selected strain Lactobacillus... this is the first mention of Companilactobacillus farciminis as Lactobacillus. Either explain the use of older and more recent taxonomy (Companilactobacillus farciminis, previously Lactobacillus farciminis) or try to be consistent.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised to ensure consistency with the rest of the text, based on the given comment (Line 82).

-- Notably, the selected strain, C. farciminis YLR-1, exhibited significant inhibitory activity against S. aureus.

 

4. 124 Since you are working woth two bacteria (one probiotic and one patogen) ou might consider reporting species name at each supernatant mention for best clarity. e.g. L124, C. farciminis cell free. Also, what was the duration of S. aureus incubation? Please state that you checked various time pointsof C. farciminis culture (ie production of antibacterials as a function of culture time).

The author’s answer： Thank you for the suggestion. Based on the given annotations, the content related to the supernatant mentioned in this article has been revised, and consistency throughout the entire text has been ensured (Line 127, Line164, Line280). The incubation duration of Staphylococcus aureus has been revised (Line 125-127). As the bacteriocin measured is non-purified, exact yield quantification is unattainable. Consequently, we cannot establish a time-production relationship and instead infer approximate bacteriocin yield changes through inhibition zone diameters in time-course supernatants.

-- Aliquots (200 μL) of C. farciminis YLR-1 cell free supernatants were loaded into preformed wells.

-- C. farciminis YLR-1 cell free supernatants served as controls.

-- Inhibitory zones on the in situ antibacterial activity assay after sodium dodecyl SDS-PAGE of C. farciminis YLR-1 cell free supernatants.

-- Briefly, adjust an overnight culture (12 h) of Staphylococcus aureus ATCC 25923 to 10⁸ CFU/mL, then inoculate 100 μL of this suspension into 20 mL of soft agar medium.

 

5. This part has to be rewritten with more clarity. Please state bacterial species( L129...cells.... which cells?) . 1 ml extracted by centrifugation....do you keep the sediment and resuspend in what or the supernatant?

The author’s answer： Thank you for the question. We have added the information required as explained above (Line 121-123).

-- To analyze the growth kinetics of C. farciminis YLR-1, a 1 mL aliquot of the C. farciminis YLR-1 overnight culture was centrifuged, resuspended in phosphate-buffered saline (PBS), inoculated into 300 mL of autoclaved MRS Liquid medium, and cultured at 37°C.

 

6. L142 Please state that MRS was previously autoclaved , prior to the addition of the bacteria.

The author’s answer： Thank you for the question. We have added the information required as explained above (Line 135).

-- Viable cells were enumerated on autoclaved MRS agar plates to assess the acid tolerance of C. farciminis YLR-1.

 

7. You state that you used a rotary evaporator at 40 C to downsize the volume of an aqueous sample! I cannot see how this is possible.

The author’s answer： Thank you for the question. We regret an error in the Methods section regarding rotary evaporation temperature during bacteriocin concentration. The submitted temperature of 40°C is incorrect; the accurate value is 50°C. To prevent BLIS property alterations or thermal degradation from excessive heat during concentration, we employed 50℃ processing. This lower-temperature approach required extended duration but preserved sample integrity. We have corrected the temperature in the main text (Line 151-152).

-- The filtrate was concentrated tenfold using a rotary evaporator (50°C),

 

8. Purification implies that only one compound is isolated and purified. However all compounds with MW lower than 500D are isolated in your study.Hence, purification is an overstatement.

The author’s answer： Thank you for the question. We have replaced the term "purification" with "preliminary extraction" in the text (Line 154, Line 345).

-- 2.8 Preliminary extraction of YLR-1 BLIS

-- In this study, preliminary extraction was carried out using the dialysis membrane method as described in the previous study^[23],

 

9. 174 Please state that all samples were run (SDS) in duplicate and half the gel was stained

The author’s answer： Thank you for the question. We have added the information required as explained above (Line 177-179).

-- The gels for Coomassie Brilliant Blue staining and the in situ activity assay were prepared simultaneously under identical conditions on the same apparatus.

 

10. Please provide a more detailed description of the procedure and mention electron microscope and fluorescence microscope (2.12) type and operating conditions.

The author’s answer： Thank you for the suggestion. We have supplemented the main text with corresponding instrument models, detailed protocol descriptions, and operating conditions (Line 182-184, Line 186-187 , Line 198-199).

-- The effect of YLR-1 BLIS on S. aureus ATCC25923 membrane integrity was analyzed via SEM (hitachi SU8100). Adjust an overnight culture (12 h) of Staphylococcus aureus ATCC 25923 to 10⁸ CFU/mL,

-- Samples were collected, fixed, and dried, then mounted on carbon-coated conductive tape. Following sputter-coating with gold, specimens were imaged using SEM. 

-- The status of the bacteria was examined under dark-field conditions using a fluorescence microscope (Nikon).

 

11. You state that "Growth data for C. farciminis YLR-1 were collected through 18 samplings over 36 206 h". Does this mean two samples per time point? How many independent samples (n) did you employ?

The author’s answer： Thank you for the question. As explained in the paper, in the process of the experiment, we collected three samples at each time point and did the experiment and took the average value for the drawing of the graph (Line 223-224).

 

12. Figures 2, 3Please state control survival in legend

The author’s answer： Thank you for the question. In both experiments, we assessed C. farciminis YLR-1 survival following pH and bile salt exposure using 0h viability as the baseline control.

 

13. 13. 326. You state that BLIS may function as secondary metabolite . Please consider that production of bacteriocins is not only strain specific but also phase specific.

The author’s answer： Thank you for your question. We have added the required information and the corresponding references as described above, with a slight change in the order of the original references due to the addition of the references 31-32, 32-33, 33-34, 34-35, 35-31 (Line 327-328).

-- The growth-phase-dependent production and subsequent decline of BLIS antibacterial activity suggest this bacteriocin may function as a secondary metabolite^{[17, 31]}.

 

14. L 327 Probiotic potential; A probiotic has to meet other criteria as well ..ie autoaggregation, biofilm , no toxicity against host, to be characterized as such. Hence,you might consider replacing probiotic with potential probiotic.

The author’s answer： Thank you for the suggestion. We have added the information required as explained above (Line 345).

-- These results indicate that YLR-1 has promising Probiotic potential.

 

15. L 358You state that BLIS might have a proteinaceous nature due to the action of proteinake K. Did you quantify protein in your isolation steps?

The author’s answer： Thank you for the question. The bacteriocin referenced is a crude preparation rather than a purified product;  therefore, specific protein quantification cannot be performed. The protease dosage employed follows established protocols in existing literature.

 

16. L377These properties suggest it likely belongs to Class I bacteriocins[41, 42]. Such classification requires more evidence . The presence of unusual peptides might strengthen your hypothesis

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 370-371). Following content removal, we have concurrently deleted references 41 and 42.

-- These findings suggest YLR-1 BLIS may be an unconventional bacteriocin.

 

17. conclusions : You state " In conclusion, C. farciminis YLR-1, a bacteriocin-producing strain, preliminary probiotic properties indicated the potential of the strain for direct use in the gut". Ι consider this conclusion risky since hemolytic and cytotoxic activity against human or other hosts has not been checked.

The author’s answer： Thank you for the suggestion. This sentence was revised according to the comment (Line 374-375).

-- In conclusion, C. farciminis YLR-1—a bacteriocin-producing strain—demonstrates promising potential for development as a probiotic.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the isolation of new potential probiotic from fermented vegetables, the characterisation of its bacteriocin-like inhibitory metabolite, and the evaluation of its anti-Staphylococcus activity. The study addresses a timely topic and presents a substantial amount of experimental work. Nevertheless, significant editorial polishing and several clarifications are needed.

Major Comments

1)  The Introduction lacks explicitly stated research aim. Please add a concise closing paragraph that identifies the problem and/or knowledge gap and states the main objective and/or hypothesis.

2) Given that the complete genome sequence of strain YLR-1 was obtained, why not use it for the in silico search for bacteriocin gene clusters? Would be great to verify whether the genome encodes class I lantibiotic, class II, or other bacteriocin loci, and discuss concordance with the < 2.7 kDa peptide observed experimentally. Otherwise, using whole genome sequencing seems to be unjustified for this research. Bioinfomatic tools such as BAGEL 4, antiSMASH 7 or RAST can accomplish this within minutes. Please consider performing and reporting it, or justify its absence.

Comments and Corrections by lines:

The font size in Figure 5 is too small to read when printed at journal column width.

Line 19 - Extra spaces before “pathogens”

Line 24 - “pH 3 and pH 4” – maybe write as “pH 3–4”.

Line 25, 27 - Genus–species not italicised

Throughout the texst, check the consistency of italics and remove unintended italics in plain text.

Line 20- “0.03%, 0.3%, and 0.05%” Likely “0.03%, 0.3%, and 0.5%”. Please verify experimental design.

Line 130 “PBS solution with an optical density of 0.5 at 600 nm (OD600)” -  “PBS (OD600 ≈ 0.5)”.

Line 382 Sentence appears inconsistent and seems to lack something.

Overall, the study is promising but requires major revisions to address the clarity of objectives, integrate genome mining, correct formatting and typographical errors, and improve figure quality.

Author Response

Dear editor and reviewers,

We are very grateful for your constructive comments and suggestions for our manuscript entitled “Isolation and characterization of bacteriocin-like-producing Companilactobacillus farciminis YLR-1, and the inhibitory activity of bacteriocin against Staphylococcus aureus” (fermentation-3743773). Your comments are very valuable and helpful for improving our manuscript. The revisions made to the manuscript are highlighted in yellow text. The comments of three reviewers are provided in italics and bold below, and specific questions have been numbered. Our responses are presented in normal font. Please refer to the attached Response Letter for detailed corrections. We have studied comments and modified our manuscript carefully. We sincerely hope to get your approval.

Reviewer: 2

1. The Introduction lacks explicitly stated research aim. Please add a concise closing paragraph that identifies the problem and/or knowledge gap and states the main objective and/or hypothesis.

The author’s answer： Thank you for your suggestion. We have added the content to the main text (Line 84-86).

-- Therefore, in response to the prevailing food safety risks in the current social environment, this study aims to identify a food preservative alternative that effectively inhibits foodborne pathogens while minimizing potential side effects.

 

2. Given that the complete genome sequence of strain YLR-1 was obtained, why not use it for thein silico search for bacteriocin gene clusters? Would be great to verify whether the genome encodes class I lantibiotic, class II, or other bacteriocin loci, and discuss concordance with the < 2.7 kDa peptide observed experimentally. Otherwise, using whole genome sequencing seems to be unjustified for this research. Bioinfomatic tools such as BAGEL 4, antiSMASH 7 or RAST can accomplish this within minutes. Please consider performing and reporting it, or justify its absence.

The author’s answer： Thank you for your suggestion. With regard to the identification of bacteriocin gene clusters, we employed BAGEL 4 and antiSMASH 7 for gene cluster prediction at the early stage of the experiment. However, the analysis revealed no matching entries in the existing gene cluster databases. Therefore, we speculate that there might be the following reasons:

(1) Incomplete genome annotation or limitations of prediction tools

-- The gene cluster was not correctly identified

-- Bacteriocin genes are typically small (e.g., class II bacteriocins often encode just 30–60 amino acids) and may reside in non-coding genomic regions or on mobile genetic elements such as plasmids or prophages. They are thus prone to oversight by standard annotation pipelines. Common prediction tools like BAGEL and antiSMASH rely on known bacteriocin databases. Consequently, bacteriocins with sequences highly divergent from characterized types may escape detection.

• Bacteriocins are encoded by non-classical genes

-- Certain bacteriocins are biosynthesized through non-classular pathways (e.g., nisin in Lactococcus lactis requires precursor peptide modification). Consequently, their genetic determinants may not reside within dedicated bacteriocin gene clusters.

• Via horizontal gene transfer or plasmid-encoding

-- Bacteriocin genes may reside on plasmids or within prophages.  These elements risk incomplete capture during sequencing if plasmids are inadequately extracted or lost during assembly.

• novel or uncharacterized bacteriocins

-- This bacteriocin may constitute an entirely novel structural type, requiring validation of its genetic origin through mass spectrometric sequencing or heterologous expression.

 

3. The font size in Figure 5 is too small to read when printed at journal column width.

The author’s answer： Thank you for the suggestion. We have increased the font size and applied bold formatting to Figure 5. The original and modified figures, along with their corresponding in-text versions, will be submitted as supplementary files with the revised manuscript.

 

4. Line 19 - Extra spaces before “pathogens”

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 22).

-- This study aimed to identify a probiotic bacterium with antagonistic activity against foodborne pathogen Staphylococcus aureus （S. aureus） and investigate the mechanism of its antibacterial components.

 

5. Line 24 - “pH 3 and pH 4” – maybe write as “pH 3–4”.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 23).

-- but also has a high survival rate at pH 3–4.

 

6. Line 25, 27 - Genus–species not italicised.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 27, Line 29).

-- The bacteriocin-like inhibitory substance (BLIS) isolated from C. farciminis YLR-1 was dialyzed using a membrane with a molecular weight cut-off (MWCO) of 500 Da,

-- The results indicated that the BLIS produced by C. farciminis YLR-1 is a small-molecule peptide.

 

7. Throughout the texst, check the consistency of italics and remove unintended italics in plain text.

The author’s answer： Thank you for your suggestion. We have italicized the names of the bacteria that were previously not italicized in the text and have corrected the formatting of non-bacterial terms that were inadvertently italicized. The revisions to the font are highlighted in blue text

 

8. Line 20- “0.03%, 0.3%, and 0.05%” Likely “0.03%, 0.3%, and 0.5%”. Please verify experimental design.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 24-26).

-- The results indicated that Companilactobacillus farciminis (C. farciminis) YLR-1 not only had high tolerance to salt conditions (0.03%, 0.3%, and 0.5%）,

 

9. Line 130 “PBS solution with an optical density of 0.5 at 600 nm (OD600)” - “PBS (OD600 ≈ 0.5)”.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 132).

-- The cells were suspended in PBS (OD₆₀₀ ≈ 0.5) after 1 mL of the overnight culture was extracted by centrifugation at 10,000 × g for 5 min.

 

10. Line 382 Sentence appears inconsistent and seems to lack something.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 374-375).

-- In conclusion, C. farciminis YLR-1—a bacteriocin-producing strain—demonstrates promising potential for development as a probiotic.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments to authors

Thank you for giving me the second opportunity to participate in the review of the manuscript entitled “Isolation and Characterization of Bacteriocin-Like-Producing Companilactobacillus farciminis YLR-1, and the Inhibitory Activity of Bacteriocin Against Staphylococcus aureus.” In the present study, the authors explain the isolation of lactic acid bacteria from pickles and probiotic characteristics of C. farciminis YLR-1. The antimicrobial properties of BLIS of LAB strain YLR-1 were also determined against pathogenic bacteria. The work is devoted to the important problem of protecting the food product. The work described in the present article is basic, but it is essential from a practical point of view. Therefore, after resolving the following comments, it is suggested to publish it in Fermentation.

The author should write the results of the manuscript in the abstract and avoid writing the methods.

Line 94: “Inoculated” or “spread”. Please correct.

Line 96: The authors should write “……LAB strain YLR-1…”.

Line 97: Please replace “target strain” with “LAB strain”.

Line 100: “….the isolate…..” which isolate or strain used for the extraction of genomic DNA. Please write.

Line 120: “…..autoclaved medium ….” Please write the name of medium used for the growth.

Line 121: “….regular intervals…” Please write the time.

Line 291: The untreated S aureus ATCC25923 images (A1-A3) are similar. The authors should select one SEM image for the untreated S. aureus ATCC 25923.

The author should italicize the name of the bacterial strain throughout the manuscript.

Author Response

Dear editor and reviewers,

We are very grateful for your constructive comments and suggestions for our manuscript entitled “Isolation and characterization of bacteriocin-like-producing Companilactobacillus farciminis YLR-1, and the inhibitory activity of bacteriocin against Staphylococcus aureus” (fermentation-3743773). Your comments are very valuable and helpful for improving our manuscript. The revisions made to the manuscript are highlighted in yellow text. The comments of three reviewers are provided in italics and bold below, and specific questions have been numbered. Our responses are presented in normal font. Please refer to the attached Response Letter for detailed corrections. We have studied comments and modified our manuscript carefully. We sincerely hope to get your approval.

Reviewer: 3

1. The author should write the results of the manuscript in the abstract and avoid writing the methods.

The author’s answer： Thank you for your suggestion. We have appropriately removed the experimental methods from the main text (Line 23-26, Line 34-36).

-- Acid and bile salt tolerance are vital indicators for evaluating probiotic survival in the gastrointestinal tract. The results indicated that Companilactobacillus farciminis (C. farciminis) YLR-1 not only had high tolerance to salt conditions (0.03%, 0.3%, and 0.5%）,

-- Scanning electron microscopy (SEM) and fluorescence imaging revealed that BLIS induced membrane damage in S. aureus ATCC25923, causing cytoplasmic leakage and cell death.

 

2. Line 94: “Inoculated” or “spread”. Please correct.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 94).

-- a 2 g sample of each pickle was spread onto de Man-Rogosa-Sharpe (MRS) agar culture medium and incubated at 37°C for 24 h.

 

3. Line 96: The authors should write “……LAB strain YLR-1…”.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 95-96).

-- Among the isolated strains, the supernatant produced by LAB strain YLR-1 exhibited the highest antibacterial activity against S. aureus ATCC25923 and was selected as the subject of further research.

 

4. Line 97: Please replace “target strain” with “LAB strain”.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 97).

-- The isolated LAB strain was preserved in MRS broth containing glycerol (25%, v/v) at -80°C.

 

5. Line 100: “….the isolate…..” which isolate or strain used for the extraction of genomic DNA. Please write.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 101).

-- The total genomic DNA of the isolated LAB strain YLR-1 was extracted using the rapid extraction kit.

 

6. Line 120: “…..autoclaved medium ….” Please write the name of medium used for the growth.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 123).

-- inoculated into 300 mL of autoclaved MRS Liquid medium, and cultured at 37°C.

 

7. Line 121: “….regular intervals…” Please write the time.

The author’s answer： Thank you for the suggestion. The content of this sentence has been revised (Line 123-124).

-- Samples were collected at regular 2-h intervals to monitor bacterial growth and antimicrobial activity

 

8. Line 291: The untreatedS aureus ATCC25923 images (A1-A3) are similar. The authors should select one SEM image for the untreated  aureus ATCC 25923.

The author’s answer：Thank you for your suggestion. We have replaced the similar images in the unprocessed Staphylococcus aureus ATCC 25923 panels (A1-A3). The revised original files and corresponding in-text figures will be submitted as supplementary materials alongside the amended manuscript (Line 293).

 

9. The author should italicize the name of the bacterial strain throughout the manuscript.

The author’s answer： Thank you for your suggestion. We have italicized the names of the bacteria that were previously not italicized in the text and have corrected the formatting of non-bacterial terms that were inadvertently italicized. The revisions to the font are highlighted in blue text.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All points raised were addressed and the relevant corrections were performed.