Heat-Killed Lactobacillus paracasei SMB092 Reduces Halitosis by Stimulating the Expression of β-Defensins in Oral Keratinocytes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this article the authors have investigated mucin binding capacity of Lactobacillus paracasei SMB092, its role in host oral immunity and the effects on P. gingivalis and its production of sulfur compound (H2S). The manuscript is well written and structured, the subject relevant to the field and the research design is scientifically sound. The cited references are suitable and recent, and the figures and tables are appropriate and informative. The results are well presented and the conclusions supported by the obtained data.

Author Response

We thank you to the reviewer for your valuable time and effort in reviewing the manuscript.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is on an interesting subject however it can be improved.

Comments for author File: Comments.pdf

Author Response

We thank the Reviewer for your constructive critiques that enabled us to bring substantial improvements to the initial manuscript. Our responses to these critiques are presented below in a point-by-point manner:

 

Comments 1: The title is too long, try to shorten it

Response 1: We apologize for confusion caused by too long a title. We agree with this comment. Therefore, we have shortened “Heat-Killed Lactobacillus paracasei SMB092 Reduces P. gingi-valis and Halitosis by Stimulating the Expression of β-Defensins in Normal Human Oral Keratinocytes” to “Heat-Killed Lactobacillus paracasei SMB092 Reduces Halitosis by Stimulating the Expression of β-Defensins in Oral Keratinocytes” on page 1, lines 2 to 4.

 

Comments 2: The abstract must be reformulated for improved clarity

Response 2: We apologize for the lack of clarity. We have reconstructed the abstract as below and have incorporated it to the section of abstract on page 1, lines 14 to 27.

“The purpose of this study is to evaluate Lactobacillus paracasei SMB092 as a prophylactic agent for oral pathogens. We examined the physical interaction of SMB092 with a host by identifying the presence of mucus binding (MuB) protein domains and the capacity of the mucin binding. We de-termined the role of heat-killed SMB092 in host oral immunity by quantifying the mRNA levels of β-defensins (BDs), Toll-like receptors (TLRs) and their cofactors (CD14/CD36) in normal human oral keratinocytes (HOK-16B cells). To assess the clinically relevant oral health effects of heat-killed SMB092, the growth of Porphyromonas (P.) gingivalis and the production of a volatile sulfur compound (H2S) were also measured in the filtered condition media (FCM) obtained from its cultures with HOK-16B cells. SMB092 possessed 14 putative MuB protein domains and was attached to mucin. Significant amounts of hBD1/2 and TLR2/6 were expressed in heat-killed-SMB092-treated HOK-16B cells. The specific neutralization of TLR2 attenuated the expression of hBD1/2 and CD14/CD36. The FCM inhibited the growth of P. gingivalis and the production of H2S. Our data indicate that the heat-killed SMB092 may contribute to a healthy oral microbiome as an immune stimulant in the production of BDs via the activation of the TLR2/6 signaling pathway” on page 1, lines 14 to 27.

 

Comments 3: The authors should state the null hypothesis

Response 3: We agree with this comment. Therefore, we have added the following statement on page 2, lines 62 to 64. “Therefore, our null hypothesis is that heat-killed Lactobacillus (L.) paracasei SMB092 (SMB092) stimulates oral epithelial cells to produce BDs that prevents the growth of oral pathogens and the development of halitosis.”

 

Comments 4: Please reformulate the purpose of this study so it is more clear

Response 4: We apologize for the unclear. We have reorganized the paragraphs as below and added them on page 2, lines 67 to 77.

“The aim of this study is to find the potential of heat-killed SMB092 as a prophylactic ingredient for oral health maintenance. Thus, to confirm the physical interaction of SMB092 with a host’s oral cavity, we identified whether SMB092 has putative cell adhesion genes targeting mucus binding (MuB) proteins and is capable of binding to mucus. We also evaluated the effects of heat-killed SMB092 on host oral immunity by measuring the production of antimicrobial peptides (β-defensins: BDs), microbial membrane-bound receptors (Toll-like receptors: TLRs), and their cofactors (CD14/CD36) in primary normal human oral keratinocytes (HOK-16B cells). Additionally, we examined whether the filtered conditioned media (FCM) obtained from HOK-16B cell cultures with heat-killed SMB092 exhibited antimicrobial activity against oral pathogens related to halitosis and periodontal disease.”

 

Comments 5: The images are not clear, please increase in size.

Response 5: We apologize that the images are not big enough to understand. Based on your comments, we have increased the size of the images and inserted them in figure 1 on page 5 to 6.

Comments 6: The authors should also mention the importance of saliva in the oral homeostasis.

I suggest: Nicolae V, Neamtu B, Picu O, Stefanache MA, Cioranu VS. The comparative evaluation of salivary biomarkers (Calcium, phosphate, Salivary pH) in cario-resistance versus cario-activity. Rev. Clim 2016 Apr 1, 67:821-4.

 

Response 6: Thank you for your insightful comments. We have added the statements related to the importance of saliva in the oral homeostasis as below and incoporated them on page 10, lines 275 to 280.

“The oral cavity harbors extensive microbiota including commensal and pathogenic bacteria. It is constantly covered with saliva containing antimicrobial peptides that produced by oral epithelial tissues. Saliva plays a major role to maintain the homeo-stasis between the oral microbiota and the host [Carpenter, G.H. The secretion, components, and properties of saliva. Annu. Rev. Food Sci. Technol. 2013, 4, 267-276]. It is important strategy to strength the host’s oral mucosal barrier by stimulating the expression of the defense factors such as BDs in the saliva.”

 

Comments 7: The authors should also mention how excipients influence the effectiveness of medication. I suggest: Chiriac AP, Diaconu A, Nita LE, Tudorachi N, Mititelu-Tartar L, Creteanu A, Dragostin O, Rusu D, Popa G. The influence of excipients on physical and pharmaceutical properties of oral lyophilizates containing pregabalin-acetaminophen combination. Expert Opinion on Drug Delivery 2017May 4: 14(5)

Response 7: Thank you for your comments. However, the purpose of this study is not related to excipients. Therefore, we gently declined not to add the information about “how excipients influence the effectiveness of mediation” in this section of discussion.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been improved