Formulation and Functional Properties of Whey Protein-Based Tissue Adhesive Using Totarol as an Antimicrobial Agent
Round 1
Reviewer 1 Report
Manuscript Ref. Ref. No.: processes-771753
The present manuscript describes the formulations of the protein-based tissue adhesive with tutarol as an antimicrobial agent. The article is interesting, in fact finding more applications for a by-product closing a cycle for circular economy. However, the manuscript lacks in discussion of the presented results; the authors never showed critical view when analyzing and discussing the results. Throughout the article they only show some results, but do not discuss them, and any connection between proteins theoretical approaches and the obtained results is presented.
Why does tutarol give better results between 2 and 3%? The authors show that this happens, but never explain why? Why is the formulation so stable over the time? In the SEM results, why does the (C) WPI-tutarol mixture after crosslinking with GTA image looks so different?
In the conclusions section is not made any reference to future studies. Authors are very succinct and only present the main results they showed throughout the article. But is there anything else to explore?
In short, I think the article requires a better presentation of a scientific component and a deep improvement of the discussion of the results shown.
Comments for author File: 
Comments.pdf
Author Response
Point 1: Why does totarol give better results between 2 and 3%?
Response 1: Previous research suggests that totarol disrupts bacterial energy metabolism at high concentrations. However, the mode of antimicrobial action of totarol is not well-established yet. It is discussed in the introduction part. There are studies searching for the explanation of the mechanism of totarol’s antimicrobial activity. Theories are totarol could alter the cytoplasmic membrane’s integrity and permeability, which led to the leakage of cellular materials, or compromise the functional integrity of cell membranes. Totarol could also act by inhibiting several NADH-related enzymes and affecting the oxidation of NADH in membrane preparation. It is also argued that totarol was a potent efflux pump inhibitor in S. aureus and could reduce the biofilm formation.
Point 2: Why is the formulation so stable over the time?
Response 2: The testing period of this study is one month, in which the samples remain stable. However, longer storage time could lead to change of bonding strength and gelation of the samples. So a longer testing period may be needed in future studies. This is also added in the conclusion section addressing future explorations. (Line 388-389)
Point 3: In the SEM results, why does the (C) WPI-tutarol mixture after crosslinking with GTA image looks so different?
Response 3: The SEM result (c) is a comparison to (a) and (b). The bonding strength of WPI/GTA tissue adhesive is provided by the crosslinking of amino groups (provided by whey protein and tissue protein) and aldehyde groups (provided by GTA). Graph (c) showed a continuous structure compared to (a) and (b), which is the evidence of the reaction (crosslinking) between the protein and GTA.
Point 4: In the conclusions section is not made any reference to future studies. Authors are very succinct and only present the main results they showed throughout the article. But is there anything else to explore?
Response 4: A few points that address future studies are added in the conclusion section. (Line 385-389)
Author Response File: Author Response.pdf
Reviewer 2 Report
- Why was the totarol concentration of the WPI-totarol solutions set at 0.1; 0.2 and 0.3%?
- In page 3, the authors mentioned that they used ASTM F2255-05 to assess the shear strength of the joints - the used standard should be explained in detail.
- In page 3, authors giving dimensions of porcine skin elements and joint dimensions use different units of measurement (millimeters and centimeters) - units should be unified.
- The gluing area in the sample is not given correctly.
- The number of samples in each series should be given in the article.
- The presentation of the test results in Figure 1 is misleading - the connection of average results with a line suggests that for exaple for 0.15% WPI-totarol solution the Aerobic Count can be about 10,000 CFU/ml what was not be proved in experiment.
- References to Figures 1 and 2 should be placed in the text.
- The results of the preliminary experimental studies of lap joints strength given in numbers are difficult to interpret, it would be better to present them in the form of a graph.
- The results given in row 239 should be removed from the text and transferred to the chart in Figure 4.
Author Response
Point 1: Why was the totarol concentration of the WPI-totarol solutions set at 0.1; 0.2 and 0.3%?
Response 1: According to previous study [1], the minimum inhibitory concentration (MIC) value of totarol against Staphylococcus aureus strains was 2µg/mL, which was 0.2% by percentage, and this gave totarol a potential to act as an antimicrobial agent. So in our study, in order to determine the content of totarol in the final formulation of tissue adhesive, the concentration of totarol were set at 0.1, 0.2, and 0.3% to see the antimicrobial effect of totarol at these concentrations. (Line 66-67)
Point 2: In page 3, the authors mentioned that they used ASTM F2255-05 to assess the shear strength of the joints - the used standard should be explained in detail.
Response 2: The ASTM standard [2] details are added in the text. (Line 116-122)
Point 3: In page 3, authors giving dimensions of porcine skin elements and joint dimensions use different units of measurement (millimeters and centimeters) - units should be unified.
Response 3: The units are unified as millimeters. (line 137)
Point 4: The gluing area in the sample is not given correctly.
Response 4: The WPI-totarol solution and GTA solution were mixed on the porcine skin. The mixture was spread evenly in the gluing area (50mm×20mm).
Point 5: The number of samples in each series should be given in the article.
Response 5: There were 3 replicates for each sample. (Line 101-102, 135-136).
Point 6: The presentation of the test results in Figure 1 is misleading - the connection of average results with a line suggests that for exaple for 0.15% WPI-totarol solution the Aerobic Count can be about 10,000 CFU/ml what was not be proved in experiment.
Response 6: The line connecting the average results was removed so the graph is now a scatter plot. The same change was also made to Figure 2. (Line 197-234)
Point 7: References to Figures 1 and 2 should be placed in the text.
Response 7: The results in Figures 1 and 2 were explained at the beginning of section 3.1.
Point 8: The results of the preliminary experimental studies of lap joints strength given in numbers are difficult to interpret, it would be better to present them in the form of a graph.
Response 8 : Two more figures (Figure 4 and 5) are added under the paragraph explaining the preliminary results of lap-shear bonding strength. The results in the text were removed so that the text could be better interpreted. (Line 267-316)
Point 9: The results given in row 239 should be removed from the text and transferred to the chart in Figure 4.
Response 9: The results in the text are removed and transferred into Figure 4. (Line 326-347)
Added references:
- Shi, C.; Zhao, X.C.; Li, W.L.; Meng, R.Z.; Liu, Z.H.; Liu, M.Y.; Guo, N.; Yu, L. Inhibitory effect of totarol on exotoxin proteins hemolysin and enterotoxins secreted by Staphylococcus aureus. World J. Urol 2015 31, 1565-1573. (Line 431-433)
- ASTM. Standard test method for strength properties of tissue adhesives in lap-shear by tension loading. ASTM International: West Conshohocken, PA, USA, 2015. (Line 447-448)
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Manuscript can be accepted in present form.
Reviewer 2 Report
I am satisfied with the corrections made.
