In vitro and in silico studies for the identification of anti-cancer and antibacterial peptides from camel milk protein hydrolysates

Mohammad Javad Taghipour; Hamid Ezzatpanah; Mohammad Ghahderijani

doi:10.1371/journal.pone.0288260

Peer Review History

Original SubmissionFebruary 23, 2023
19 Apr 2023 Decision Letter - Ghulam Mustafa, Editor PONE-D-23-05337In vitro and in silico studies for identification bioactive peptides from camel milk protein hydrolysatesPLOS ONE Dear Dr. Ezzatpanah, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 03 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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: No ****** 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: Minor modifications required: 1. Indicate the bioactivities that are being investigated in the study in your title - i.e. you can add anti-cancer and anti-bacterial bioactivities in the title. 2. Line 43: Remove the link and reference it according to the guidelines. 3. Line 80: State the storage conditions between the period of purchase and analysis. 4. Line 90: Avoid using ddH2O, instead use distilled water/double distilled water accordingly. 5. Table 4 in the reference "" ext-link-type="uri" xlink:type="simple">https://doi.org/10.1016/j.foodchem.2021.131444" has a list of camel milk-derived bioactive peptides with potential antibacterial properties. Check it out and use it in your discussion to see if there are any similar trends. Reviewer #2: The manuscript “In vitro and in silico studies for identification bioactive peptides from camel milk protein hydrolysates” is a manuscript that investigates isolating the protein from camel milk and hydrolyzing it using two different enzymes to achieve the hydrolysate fractions with biological activities. In my opinion, the study has been well-designed, and the manuscript covers the objectives. However, some points should be carefully addressed by the authors. I believe this manuscript needs MAJOR Modification to be suitable for publishing in the journal. Comments: 1. Title: Add “the” and “of” to the title. In vitro and in silico studies for the identification of bioactive peptides from camel milk protein hydrolysates 2. Introduction- Page 9, Line 43: use the references according to the journal requirement for web pages. 3. Materials and Methods- Page 11, Line 93: Why did the authors select 12 h for hydrolysis of the proteins? 4. Did the authors measure the DH of the parent protein as a control? If not, how do they justify that the DH of protein hydrolysates is higher or lower than the parent protein? 5. The above question is important about the bioactivities of the protein hydrolysates. How do they claim that the anticancer activity of protein hydrolysates is higher than the parent proteins? Please justify. 6. Why do authors use only 4 bacteria for the antibacterial activities? For antibacterial studies, usually, a combination of gram-negative and gram-positive bacteria should be used. Justify please. ****** 6. 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? 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Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0288260.r001
Revision 1
27 May 2023 Author Response Reviewer #1: Minor modifications required: 1. Indicate the bioactivities that are being investigated in the study in your title - i.e. you can add anti-cancer and anti-bacterial bioactivities in the title. Response: Dear Reviewer, we would like to thank you for your time and for helping improve our manuscript with your valuable comments. Title was revised as you wished. 2. Line 43: Remove the link and reference it according to the guidelines. Response: we revised it according to the journal guideline. 3. Line 80: State the storage conditions between the period of purchase and analysis. Response: we added it in the main text (line 81). 4. Line 90: Avoid using ddH2O, instead use distilled water/double distilled water accordingly. Response: we revised it as you wished. 5. Table 4 in the reference "https://doi.org/10.1016/j.foodchem.2021.131444" has a list of camel milk-derived bioactive peptides with potential antibacterial properties. Check it out and use it in your discussion to see if there are any similar trends. Response: Thank you for your good suggestion. We included it in the discussion (lines 221-228). Reviewer #2: The manuscript "In vitro and in silico studies for identification bioactive peptides from camel milk protein hydrolysates" is a manuscript that investigates isolating the protein from camel milk and hydrolyzing it using two different enzymes to achieve the hydrolysate fractions with biological activities. In my opinion, the study has been well-designed, and the manuscript covers the objectives. However, some points should be carefully addressed by the authors. I believe this manuscript needs MAJOR Modification to be suitable for publishing in the journal. Comments: 1. Title: Add "the" and "of" to the title. In vitro and in silico studies for the identification of bioactive peptides from camel milk protein hydrolysates Response: Dear Reviewer, we would like to thank you for your time and for helping improve our manuscript with your valuable comments. Title was edited as you wished. 2. Introduction- Page 9, Line 43: use the references according to the journal requirement for web pages. Response: we revised it according to the journal guideline. 3. Materials and Methods- Page 11, Line 93: Why did the authors select 12 h for hydrolysis of the proteins? Response: This time was chosen due to (i) Ensure the complete hydrolysis of the protein by the enzyme, that is, the enzyme has the opportunity to recognize and cut all its recognition sites (ii) Since our aim was to evaluate the antibacterial and anticancer activities, therefore we wanted to ensure the production of 1-7 kD peptides because the peptides with these sizes usually exhibit these activities (https://doi.org/10.1098/rsob.200004; https://doi.org/10.1128/AAC.02369-13). Also, in a study by Wang et al. (https://doi.org/10.3390/ani10020337), they stated that hydrolysis of camel milk proteins for 12h could produce peptides for killing their desired bacteria (lines 228-232). 4. Did the authors measure the DH of the parent protein as a control? If not, how do they justify that the DH of protein hydrolysates is higher or lower than the parent protein? Response: Thank you for your precious question. At time 0, no hydrolysis has taken place, so this in turn represents the degree of hydrolysis of the original proteins initially. However, we added the following sentence: “At time 0, no hydrolysis has taken place, so that the degree of hydrolysis of the parent proteins varied from 1.9 to 2.8% and increased over time with the addition of enzymes (Fig. 1)" in lines 195-197 to avoid any misunderstanding. 5. The above question is important about the bioactivities of the protein hydrolysates. How do they claim that the anticancer activity of protein hydrolysates is higher than the parent proteins? Please justify. Response: According to the Fig. 2, we measured anticancer activity of parent proteins and hydrolysates, therefore whey and casein proteins (parent proteins that used as control) respectively killed approximately 30% and 10% of total cancerous cells, which is lower than anticancer activity of hydrolysates (Fig. 2). Also, it is better to say that since we boiled the mixture after hydrolysis to inactivate the enzyme, all of proteins, including enzyme and parent proteins were removed using centrifugation. Therefore, there are only peptides in the mixture. But if there are parent proteins in the mixture, they are inactive. However, we clarified it by adding a sentence in lines 250-252. 6. Why do authors use only 4 bacteria for the antibacterial activities? For antibacterial studies, usually, a combination of gram-negative and gram-positive bacteria should be used. Justify please. Response: Thank you so much for your good question. As you said we also used a combination of gram-negative and gram-positive bacteria (3 gram-negatives bacteria, including Klebsiella pneumoniae (PTCC 1053), Pseudomonas aeruginosa (PTCC 1074), and Escherichia coli (PTCC 1330); and 1 gram-positive bacteria, including Staphylococcus aureus (PTCC 1112)). They are from different families and commonly use in antibacterial studies, because they cause deadly diseases in humans. Therefore, we decided to choose them for evaluating antibacterial tests. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0288260.r002
22 Jun 2023 Decision Letter - Ghulam Mustafa, Editor In vitro and in silico studies for the identification of anti-cancer and antibacterial peptides from camel milk protein hydrolysates PONE-D-23-05337R1 Dear Dr. Ezzatpanah, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ghulam Mustafa, PhD 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: All comments have been addressed Reviewer #2: All comments have been addressed ******** 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Yes Reviewer #2: Yes ****** 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: Yes Reviewer #2: Yes ****** 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: All comments have been addressed. The manuscript can be accepted in its current form. Thanks for the revision. Reviewer #2: (No Response) ****** 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 ******** https://doi.org/10.1371/journal.pone.0288260.r003
Formally Accepted
3 Jul 2023 Acceptance Letter - Ghulam Mustafa, Editor PONE-D-23-05337R1 In vitro and in silico studies for the identification of anti-cancer and antibacterial peptides from camel milk protein hydrolysates Dear Dr. Ezzatpanah: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ghulam Mustafa Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0288260.r004

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