Round 1

Reviewer 1 Report

1. The informations aout Ligilactobacillus salivarius and Limosilactobacillus fermentum have to provie more detail.
2. The strains must be deposited in the public Type Culture Collection and have the strain numbers before publication. 
3. The contents or compositions of the cell-free supernatants from probiotics have to evaluate clearly. 
4. The data of HAS1 mRNA have to show in the manuscript.
5. Hyaluronan (HA)-degrading enzymes, HYAL-1, -2, -3 and -4, have to investigate in this manuscript.
6.  Reference 25 is not proper in the academic paper. Please revised!

Author Response

Authors’ response (in blue) to the Reviewer#1’s comments (in italic black):

We thank the anonymous reviewer for thoroughly reading our manuscript and providing helpful comments and suggestions. The detailed responses to comments are listed below:

 

Q1. The information about Ligilactobacillus salivarius and Limosilactobacillus fermentum have to prove more detail.
Response :

Thank you for your comment, we have added more information about Ligilactobacillus salivarius and Limosilactobacillus fermentum in line 43-47.

P1, L43-47 – “Among Lactobacillaceae Family, Ligilactobacillus salivarius (Lactobacillus salivarius, L. salivarius) and Limosilactobacillus fermentum (Lactobacillus fermentum, L. fermentum) have the basic properties of Lactobacilli, such as inhibition of pathogen growth in the intestine, immunomodulatory activity, and intestinal barrier regulation, and are also known to have superior antibacterial effects against S. aureus compared to other species [7].”

Q2. The strains must be deposited in the public Type Culture Collection and have the strain numbers before publication. 

Response :

Thank you for your comment, we have mentioned about NCBI accession number in line 66-70.

P2, L66-70 – “All lactic acid bacteria (LAB), containing L. salivarius MG242 (NCBI accession num-ber, MN055708.1), MG4265 (NCBI accession number, MN060992.1), MG4227 (NCBI ac-cession number, MF597747.1) and L. fermentum MG901 (NCBI accession number, MN055709.1), MG4231 (NCBI accession number, MW947163.1), MG4244 (NCBI accession number, MW947154.1)…”

Q3. The contents or compositions of the cell-free supernatants from probiotics have to evaluate clearly. 

Response :

Thank you for your comment. As previously mentioned in our study, the cell-free supernatants from L. fermentum and L. salivarius contained D-, and L-lactic acid, acetic acid, propionic acid, butyric acid analyzed by GC/MS [13,14]. we have added that content in line 50-52.

P2, L50-52 – “Especially, the cell-free supernatants from L. fermentum and L. salivarius contained SCFA such as D-, and L-lactic acid, acetic acid, propionic acid, butyric acid [12,13].”

Q4. The data of HAS1 mRNA have to show in the manuscript.

Response:

Thank you for your comment. In the HaCaT keratinocytes used in this study, HAS2 was most expressed and reported to promote HA production, so HAS2 has been evaluated (Ref.: Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan, 2001, JBC). In addition, as mentioned in the text, HAS3 is known to synthesize a shorter form of HA, so it has been confirmed. However, we will make sure to check the expression of HAS1 by reflecting on your opinion in further studies.

 

Q5. Hyaluronan (HA)-degrading enzymes, HYAL-1, -2, -3 and -4, have to investigate in this manuscript.

Response:

Thank you for your comment. In this study, only HAS expression been checked to confirm the improvement of the moisturizing effect. We will make sure to check the expression of HA-degrading enzymes by reflecting on your opinion in further studies.

Q6. Reference 25 is not proper in the academic paper. Please revised!

Response:

Thank you for your comment. Reference 25 is a website citation, and it is stated in the Instructions for Authors of the Fermentation Journal that it can be used as a reference. Please check it here: https://www.mdpi.com/journal/fermentation/instructions.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is very well written; clear, precise, and easy to understand. However, some comments should be addressed. 

- Please add more quantitative data in the abstract. For example, how many times/folds increases HA.

- Scientific names and latin phrases it should be in Italics. For example, references 4,8, 9,10,11,14,16,18,22. Please double-check.

- The last reference is showed as 1. Please double-check.

My main concern is about the uses of the selected strains to evaluate the antimicrobial activity of CFS. Are those strains of skin importance? Please specify

 

Author Response

Authors’ response (in blue) to the Reviewer#2’s comments (in italic black):

The manuscript is very well written; clear, precise, and easy to understand. However, some comments should be addressed. 

We thank the anonymous reviewer for thoroughly reading our manuscript and providing helpful comments and suggestions. The detailed responses to comments are listed below:

Q1. Please add more quantitative data in the abstract. For example, how many times/folds increases HA.

Response :
Thank you for your comments, we added folds of HA in line 20.

P1, L18-20 – “The results showed that CFS from L. salivarius MG242 and L. fermentum MG901 increased the expression of these genes along with the production of HA (2.40, and 1.95-fold of control).

Q2. Scientific names and latin phrases it should be in Italics. For example, references 4,8, 9,10,11,14,16,18,22. Please double-check.

Response :
Thank you for your comments, we have double-checked and modified references.

Q3. The last reference is showed as 1. Please double-check.

Response :
Thank you for your comments, we have double-checked and modified the last reference.

Q4. My main concern is about the uses of the selected strains to evaluate the antimicrobial activity of CFS. Are those strains of skin importance? Please specify

Response :
As we mentioned in manuscript, S. aureus is closely related to skin infections (in line 174). L. fermentum and L. salivarius have been reported to have an antibacterial effect against S. aureus, especially compared to other species, suggesting that the species may play an important role in the skin. We have additionally written about the antibacterial effect of L. fermentum and L. salivarius against S. aureus in line 43-47.

P1, L43-47 – “Among Lactobacillaceae Family, Ligilactobacillus salivarius (Lactobacillus salivarius, L. salivarius) and Limosilactobacillus fermentum (Lactobacillus fermentum, L. fermentum) have the basic properties of Lactobacilli, such as inhibition of pathogen growth in the intestine, immunomodulatory activity, and intestinal barrier regulation, and are also known to have superior antibacterial effects against S. aureus compared to other species [7].”

 

Author Response File: Author Response.docx

Reviewer 3 Report

The study focuses on investigating the skin barrier-supporting properties of several strains of L. salivarius and L. fermentum in vitro. It has been reported that the skin may profit from oral ingestion of probiotic bacteria (reduced skin sensitivity, support of the skin´s immune function). In addition, probiotic metabolites can be incorporated into cosmetic products and directly applied to the skin to support barrier function of the skin and maintain a healthy skin microbiome. From this point of view, the study provides valuable data for the further selection of effective probiotic strains with medical potential to support skin properties. A weakness of the manuscript is that the authors did not analyse the composition of CFS, so it is not clear which component has beneficial effects on skin cells. Although the manuscript is clear and presented in a well-structured manner, authors should respond to the following comments and include corrections or additions to the manuscript.

 

1.       Line 43: I recommend putting the former names of the tested strains in brackets to make it clear to readers who are not yet familiar with the changes in the nomenclature of the genus Lactobacillus.

2.       Ref. 7 should be deleted.

3.       Please specify the methods section in more detail.

-        How was CFS prepared? I am referring to the length of time the bacteria are cultivated before the supernatant is collected. If this information is added, citation 17 can be removed, since the entire CFS preparation will already be mentioned in the manuscript.

-        This section should list the strains that have been tested.

-        References 18 and 19 are redundant and should be deleted. (Ref. 18: The MTT test is a well-known and widely used method for testing the cytotoxic effect of various molecules, including CFS. Ref. 19: ELISA was performed according to the manufacturer's instructions. “After incubation, the absorbance of 81 the supernatants was measured at 450 nm according to....“ I propose to correct the sentence because it appears that the authors measured the absorbance of the cell supernatants immediately after sampling, and this is not true.)

-        Line 117 - state how the cells were handled after the adherence test (washed, scraped, etc.) before plating the bacteria

4.        Ref. 32 is marked as n. 1 in the list of references.

Author Response

Authors’ response (in blue) to the Reviewer#3’s comments (in italic black):

The study focuses on investigating the skin barrier-supporting properties of several strains of L. salivarius and L. fermentum in vitro. It has been reported that the skin may profit from oral ingestion of probiotic bacteria (reduced skin sensitivity, support of the skin´s immune function). In addition, probiotic metabolites can be incorporated into cosmetic products and directly applied to the skin to support barrier function of the skin and maintain a healthy skin microbiome. From this point of view, the study provides valuable data for the further selection of effective probiotic strains with medical potential to support skin properties. A weakness of the manuscript is that the authors did not analyse the composition of CFS, so it is not clear which component has beneficial effects on skin cells. Although the manuscript is clear and presented in a well-structured manner, authors should respond to the following comments and include corrections or additions to the manuscript.

 We thank the anonymous reviewer for thoroughly reading our manuscript and providing helpful comments and suggestions. The detailed responses to comments are listed below:

Q1.       Line 43: I recommend putting the former names of the tested strains in brackets to make it clear to readers who are not yet familiar with the changes in the nomenclature of the genus Lactobacillus.

Response :
Thank you for your comments, we have put the former names of strains in brackets in line 43-44.

P2, L43-44 – “Among Lactobacillaceae Family, Ligilactobacillus salivarius (Lactobacillus salivarius, L. salivarius) and Limosilactobacillus fermentum (Lactobacillus fermentum, L. fermentum)…”

Q2.       Ref. 7 should be deleted.

Response :
Thank you for your comments, we have deleted reference 7.

Q3.       Please specify the methods section in more detail.

-        How was CFS prepared? I am referring to the length of time the bacteria are cultivated before the supernatant is collected. If this information is added, citation 17 can be removed, since the entire CFS preparation will already be mentioned in the manuscript.

Response :
Thank you for your comments, We did not specifically mention the bacterial incubation time for preparing CFS as it was performed in the same manner as reference 17, the method we had previously carried out. The process of preparing for CFS is as follows: The probiotic strains grown in MRS broth were diluted to an optical density of 0.9–1.0 (10⁸–10⁹ CFU/mL) at 600 nm and then inoculated at 2% (v/v) in fresh MRS broth. After 24 h, the probiotic strains were centrifuged at 4000× g at 4 °C for 10 min.

-        This section should list the strains that have been tested.

Response :
Thank you for your comments, we have listed all strains used in this study in line 66-70.

P2, L66-70 – “All lactic acid bacteria (LAB), containing L. salivarius MG242 (NCBI accession num-ber, MN055708.1), MG4265 (NCBI accession number, MN060992.1), MG4227 (NCBI ac-cession number, MF597747.1) and L. fermetum MG901 (NCBI accession number, MN055709.1), MG4231 (NCBI accession number, MW947163.1), MG4244 (NCBI accession number, MW947154.1)…”

-        References 18 and 19 are redundant and should be deleted. (Ref. 18: The MTT test is a well-known and widely used method for testing the cytotoxic effect of various molecules, including CFS. Ref. 19: ELISA was performed according to the manufacturer's instructions. “After incubation, the absorbance of the supernatants was measured at 450 nm according to....“ I propose to correct the sentence because it appears that the authors measured the absorbance of the cell supernatants immediately after sampling, and this is not true.)

Response:
Thank you for your comments, we have deleted references 18 and 19.

-        Line 117 - state how the cells were handled after the adherence test (washed, scraped, etc.) before plating the bacteria

Response:
Thank you for your comments, we have stated the adhesion test in more detail in lines 116-118.

P3, L116-118 – “After 2 h, the non-adherent LAB was discarded, washed twice with PBS, and scrapping of LAB adherents to colorectal cells. The LAB attached to the cells were counted using the plate count method on MRS agar.”

Q4.        Ref. 32 is marked as n. 1 in the list of references.

Response:
Thank you for your comments, we have double-checked and modified the last reference.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

1. NCBI is not a Type Culture Collection Center. If the strain is not deposited in the public centre, the reader has no chance to confirm the results. 

2. The authors do not answer the effects of HAS-1 and HA-degrading enzymes. I think they are essential for this topic.

Author Response

Authors’ response (in blue) to the Reviewer#1’s comments (in italic black):

We thank the anonymous reviewer for thoroughly reading our manuscript and providing helpful comments and suggestions. The detailed responses to comments are listed below:

 

Q1. NCBI is not a Type Culture Collection Center. If the strain is not deposited in the public center, the reader has no chance to confirm the results.
Response :

Thank you for your comment, we did not purchase the strain from Type Culture Collection Center and used the strain isolated from MEDIOGEN Co., Ltd. as in our previously study(Antioxidant activity and short‑chain fatty acid production of lactic acid bacteria isolated from Korean individuals and fermented foods, 2021, 3 Biotch). Therefore, the NCBI number registered separately from MEDIOGEN Co., Ltd. was written, not the Type Culture Collection Center registration number, as in other reports (line 70).

Q2. The authors do not answer the effects of HAS-1 and HA-degrading enzymes. I think they are essential for this topic.

Response :

Thank you for your comment, HAS-1 expression is low in keratinocytes, and HAS2 is mainly involved in HA production, as mentioned in the previous revision. Therefore, since many studies mainly observed the expression of HAS2 in the keratinocytes, the study focused on confirming the expression of HAS2. In the case of HA-degrading enzymes, a longer study period is required to add the expression level, and in the present paper, HAS expression been focused on the study. In addition, this paper is a communication, not an article, that can suggest future research directions. When we proceed with this study and extend it to animal testing, we will make sure to include your suggestions in the further study.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.

Author Response

Authors’ response (in blue) to the Reviewer#2’s comments (in italic black):

Q1. The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.

Respose: We thank the anonymous reviewer for thoroughly reading our manuscript.