PONE-D-25-19776Optimization of lyophilization process for long-term storage of probiotic Bacillus and Staphylococcus: Effects on survival and functionalityPLOS ONE

Dear Dr. Habib,

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Academic Editor

PLOS ONE

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Additional Editor Comments :

Dear respected Authors,

Your manuscript entitled" Optimization of lyophilization process for long-term storage of probiotic Bacillus and Staphylococcus: Effects on survival and functionality was evaluated and based on our reviewers

suggestions needs revisions. So, please follows the reviewers comments and prepare point by point responses for all details. Due to use different Bacillus Spp. , staphylococcus Spp. and one strain of Lactobacillus salivarius, indicate in the title in a correct way.

Best regards,

M.Faezi (PhD)

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The data presented here are interesting. However, there are several shortcomings reduce the value of the manuscript. The language definitely requires attention, there are several typos and grammatical errors. Additionally, some parts are poorly described or confused (see also below).

Introduction:

-The manuscript requires improvement in the Introduction.

- What is the novelty of your study?

Material and Methods:

- Please add references for all protocols.

- Add name and reference of the mediums and materials.

- Antimicrobial activity section misses important data and assays may not have been conducted as recommended by antibiotic susceptibility testing guidelines. According to Clinical and Laboratory Standard Institute (CLSI) and European committee for antibiotic susceptibility testing (EUCAST) the determination of MICs with the method must use bacterial inoculum standardized at 5 x 105 CFU/ml that does not seem to be the case here. Did you control the number of CFU/ml in your inoculum? Which OD cut-off was considered positive growth? If the antimicrobial testing was not performed according to guidelines, it must be discussed in the manuscript.

- What is test bacterial strain??

Results

- The figures 1, 2, and 3 are very illegible, the quality of the charts should be improved.

- I strongly advise authors to revise the explanation of the figures. Legends of many figures has to be increased.

- What does RD mean in the tables legends?

Discussion

- Some of the content presented here are repetition of the results.

- I feel that the Discussion is too long and can be shorted by 25% or so easily without losing important information.

-Some of the content presented here are subject to the Introduction.

- I also would recommend a better organization of ideas in the Discussion.

Reviewer #2: - Strengthen the introduction by highlighting the probiotic species of Bacillus and Staphylococcus.

-Line 111, Bacterial cells washed twice with sterile distilled water. Won't washing bacteria with distilled water lead to their osmotic lysis?

-In Table one, mention the used concentration of each compound in preparation of storage conditions.

-In Table1 (Suspended cells), what are the washed cells suspended in?

-In2.4 A 1 mL sample is mixed with 9 mL of SGF. Mention the CFU of bacteria in 1 mL of sample.

-According to 2.4, the text mentions that following gastric exposure, the surviving sample is transferred into 9 mL of SIF to replicate the intestinal phase. As the population of bacteria is important in their resistance rate, in related results, mention the population of survived bacteria for second test.

- Please include reference for each section of methods.

-The effect of different excipient concentrations (2–10%) on the viability of various bacterial strains was not assessed in methods. The related results are presented in 3.1.

-In Line 202, how do you find Skim Milk Powder is the most effective excipient for probiotic strains?

-For 3.6, authors need to substantiate pathogenic bacterial growth inhibition with plate photographs.

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Reviewer #1: No

Reviewer #2: No

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Optimization of cryoprotectants and storage temperatures for preserving viability and probiotic properties of lyophilized bacterial strains from chicken gut

PONE-D-25-19776R1

Dear Dr. Habib,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mohammad Faezi Ghasemi, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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