Promising Immobilization of Industrial-Class Phospholipase A₁ to Attain High-Yield Phospholipids Hydrolysis and Repeated Use with Optimal Water Content in Water-in-Oil Microemulsion Phase

Round 1

Reviewer 1 Report

The manuscript reported by Hayakawa et al.,  showed some optimization to attain high yield hydrolysis of phospholipids by inductrial-class immobilized of phospholipase A1 with optimal water content in water-in-oil (W/O) microemulsion phase. Although the author showed some condition that may help to increase the yield of hydrolysis reaction of phospholipase A1, however, the manuscript still requires a lot to improve as below:

1. Abstract: I cannot identify from where it was actually their finding/conclusion or it was from beginning. I request the authors revise it so that readers can follow the order: general background, biological questions, and results and conclusion in the abstract.
2. The introduction was not written well about why did they conduct this study? What kind of problems in previous study in this field? How did they solve this problem? How significant of the work to industrial application as they mentioned in the title.
3. Most of results were not clearly concluded. This is because they did not explain well the data of every single figure.
4. The background of all conditions: for example GA concentration, water content and so on, was not mentioned in the introduction so readers find hard to follow. What happens if the GA concentration is higher than 10%?
5. Fig. 2 can be Fig. 1 and Fig. 1 can be changed to Fig. 2.
6. 3 and 4 can be combined.
7. All legend of all figures must be revised so that reviewers understand what the authors are trying to show/explain?
8. The “Discussion” part was just a summary of their results, they were not discussing about their results and the previous works of this topic.
9. The manuscript must go to English language editting.

Author Response

The reply from authors to reviewer#1
Thank you very much for your kind review of our manuscript. Your comments are valuable for improving the paper, and the authors have revised the text accordingly.

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Hayakawa et al proposed the article “Promising immobilization of industrial-class immobilized phospholipase A1 to attain high yield hydrolysis of phospholipids for repeated use involved with optimal water content in W/O microemulsion phase “.

I think the article was well written. I liked the clarity of the methods section.

Here are my comments:

Introduction:

I think that the introductory part is too concise and must necessarily be integrated with notions that cannot be considered as a mere outline.

For example, it is necessary to indicate how for lipases the use on an industrial level can concern not only the canonical mechanism of hydrolysis but also the equally important mechanism of esterification. The immobilized lipase CALB is used all over the world, which proves to be flexible in different esterification contexts, not limited only to W / O systems but also to solventless systems. For example, Zappaterra et al (Zappaterra, F.; Summa, D.; Semeraro, B.; Buzzi, R.; Trapella, C.; Ladero, M.; Costa, S.; Tamburini, E. Enzymatic Esterification as Potential Strategy to Enhance the Sorbic Acid Behavior as Food and Beverage Preservative. Fermentation 2020.) recently reported the enzymatic esterification of sorbic acid for industrialization in the agri-food field.

line 39, it would be interesting to briefly indicate some practical examples of the emulsifiers used in the food and pharma industries.

line 41, is necessary to indicate how many times, generally, a catalyst can be reused without losing its effectiveness to be able to compare the literature data to those provided by this paper. Where we talk about challenges in the industrial application of hydrophobic reactions it would be better to indicate what are the main limitations of this application.

Materials and Methods:

line 72: the sentence "is made of a polypropylene-based hydrophobic granular support [22]" must be moved to the introductory part at line 66.

line 73: Aspergillus oryzae must be written in italics.

line 91: please specify rpm used for stirring.

line 111: it would be correct to indicate the quantity (s) or concentrations used instead of using the word desired.

My biggest concern is that the experiments were carried out in triplicate.

Results and Discussion:

My criticism concerns all the graphs shown; first of all the trend line is reported and not the actual trend of the various samples, furthermore there are no errors (standard deviations or standard error), this could suggest that the tests have been carried out in single so that the result obtained could be completely random. I advise authors to review these aspects.

Figure 3: it is not clear to me what is the difference between the initial adsorbed phospholipase A1 before glutaraldehyde treatment and the concentration of C_GA0%?

Line 184: for greater clarity I would use the abbreviations used in the graphs in the text for example: The initial reaction rate (Vi), this applies to all the abbreviations used in the graphs.
Going to resume the abbreviations at the end of the article does not facilitate reading.

Lines 194-195: "The volumetric amount of the supplemental acetate buffer solution (Hb) was changed as follows; 0 (★), 0.05 (●), 0.075 (○), 0.1 (▼), and 0.2 (■) mL." This is a repetition of the caption of figure 6, it would be useful to remove it.

Author Response

The reply from authors to reviewer#2
Thank you very much for your kind review of our manuscript. Your comments are valuable for improving the paper, and the authors have revised the text accordingly.

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised manuscript by Hayakawa et al.,  showed significant improvement compared to the previous version. I have only a few minor comment as follows:

1. The authors could provide the method of SEM in Figure 4(a) if the SEM image was obtained from their experiment, if not, please provide the original source of the data.
2. The authors could perform statistical analysis of their data to show significant differences between different conditions.

Author Response

The reply from authors to reviewer#1:

  Thank you very much for your kind review of our manuscript. Your comments are valuable for improving the paper, and the authors have revised the text accordingly.

Reviewer#1’s comment 1:

The authors could provide the method of SEM in Figure 4(a) if the SEM image was obtained from their experiment, if not, please provide the original source of the data.

Our comments:

Thank you for your comments. We provided the method of SEM in Figure 4(a), and revised the text as follows.

Revised text: P.4, L.129

2.3. Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) was employed to examine the surface of Accurel. The sample was coated with gold and viewed under a high vacuum. A surface of Accurel was carried out using a JSM-6701F scanning electron microscope (JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 10.0 kV.

Reviewer#1’s comment 2:

The authors could perform statistical analysis of their data to show significant differences between different conditions.

Our comments:

Authors agree your comment. The experiments were performed in triplicates and the data deviation was expressed by error bars. To clear presentation on data for readers, the error bars were eliminated reluctantly in Figure 2, 5, 7, 9, and 11. In this study, the statistical analysis was examined that was results valid statistically significant if p≦0.05.

Revised text: P.4, L.164

2. 6 Statistical analysis

The experiments were performed in triplicates and the data deviation was expressed by error bars. To clear presentation on data for readers, the error bars were eliminated reluctantly in Figure 2, 5, 7, 9, and 11. In this study, the statistical analysis was examined that was results valid statistically significant if p≦0.05.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

all changes have been made to the manuscript, only an error remains in table 1: The term Esterificatoin should be corrected.

Author Response

Please see the attachment

Author Response File: Author Response.docx