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Peer-Review Record

Rhizopus oryzae Inulinase Production and Characterization with Application in Chicory Root Saccharification

Microbiol. Res. 2023, 14(1), 297-315; https://doi.org/10.3390/microbiolres14010024

by Asmaa Abdella^1,*, Mahmoud Al-Saman¹

, Fatma I. Abou-Elazm² and Shaymaa Wagdy El-Far³

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Microbiol. Res. 2023, 14(1), 297-315; https://doi.org/10.3390/microbiolres14010024

Submission received: 31 December 2022 / Revised: 7 February 2023 / Accepted: 17 February 2023 / Published: 20 February 2023

Round 1

Reviewer 1 Report

Dear Authors,

Article entitled "Rhizopus oryzae inulinase production, and characterization with application in chicory root saccharification". I read with interest. I believe that the conducted research has a great cognitive value as well as utilitarian significance. The applied screening methods made it possible to achieve the set goals. Notes and comments are included below. Authors should take them into account when preparing the manuscript for publication. I would like to point out that in order to standardize the notation of cited literature, it should comply with the Journal's requirements. Currently, the authors sometimes give the full name of the journals, other times their abbreviated notation.

Abstract

Line 16 - is "from" - should be for (for Rhizopus oryzae NRRL 3563)

Introduction

In this part of the article, the style of some sentences should be corrected, e.g. lines 56-59, and the presented content should be organized so that it constitutes a logical whole.

Lines 60-61 - the sentence "Paecilomyces variotii is the asexual state of Byssochlamys

spectabilis, a member of the Phylum Ascomycota (Family Trichocomaceae) [11]" - is detached, it should be preceded by the sentence from line 61 - "A potential source of thermotolerant enzymes is thermophilic fungus."

Material and methods

This chapter requires additions and corrections.

Lines 90-93 - should be before the sentence " Oat, and wheat bran were ...." (line 85)

Line 102 - 2.3. Effect of different carbon sources - requires a more detailed description (how the experiment was performed, how much substrate was used ...)

Line 106 - 2.4. Multifactorial experiments for optimizing inulinase production.

Please provide the components tested in the first phase and in the second phase - the most significant concentrations

Line 127 - it is known that "(-1) and (+1) are coded levels in Plackett-Burman design", but what exactly does -1 and +1 mean?

Line 141 – please list these two factors (…….) and give three levels (….)

Line 173 - give the meaning of "SDS-PAGE", briefly what this protein analysis technique is and what it uses

Results and discussion

This part of the article should have been better presented, the results should have been interpreted with greater insight.

You can't start discussing research results with "Table 3 shows..." from the construction of the table it is obvious what it contains. This also applies to Lines: 269; 280; 296; 442; 469; 470.

The methodology (2.1.) shows that the experiment was also carried out on Rhizopus oryzae NRRL 3562 - and was not included in the results?

- In table 3 there is "Cell dry weight (g)" – „cel” ... or maybe „fungus” ...

Lines 235-238 - please write something more about the research results of the cited authors (this information is very general)

Line 247 - remove the dot before [29]

Table 5 - "Term"? or Factor?

Line 282 - Please explain if "In contrast, chicory root, KCl, and pH had a negative impact on inulinase production, indicating that as their concentrations increased, inulinase production was suppressed. That's why in Lines 291-294 "The concentrations of chicory root and NaNO3 were determined to be the two major significant factors studied on the synthesis of fungal inulinase based on the Plackett-Burman design results, and they were chosen for further optimization using the Box - Behnken design”.

In 3.3.2. there is no interpretation for table 6.

Line 298 - is Table 5 should be Table 7

Table 6 - consistently should be Chicory root (capital first letter)

Table 7 - "Term"? or Factor?

Line 316 - explain RSM (appears for the first time in the article). Write more precisely how changes in carbon and nitrogen content affected the production of inulinase

Line 330 and 332 is Rhizopus oryzaei NRRL 3563 should be Rhizopus oryzae NRRL 3563

Lines 339 and 343 is "cultivated on 1% chicory root"; "on 3% chicory root" - it will be better on "a medium with 1% (3%) of chicory root pulp

Figures 3-6 - please standardize the notation of all values: for example in Fig. 3.4 is "30,000.0" and in Fig. 5.6 "30000"

Table 11 - Should be Temperature (°C)

Tables 12 and 14 - "Term" or Factor

In 3.8.2. Optimization of medium components using Box-Behnken design- A more accurate interpretation of tables 13 and 14 would be useful.

Author Response

Comments and Suggestions for Authors

Dear Authors,

Thanks for all valuable comments of reviwer

All changes were made in red text in the revised manuscript.

Abstract

Line 16 - is "from" - should be for (for Rhizopus oryzae NRRL 3563)

Thanks for your valuable comment.

From was changed into for in the revised manuscript.

Introduction

In this part of the article, the style of some sentences should be corrected, e.g. lines 56-59, and the presented content should be organized so that it constitutes a logical whole.

Thanks for your valuable comment.

Sentences in lines 56-59 have been rephrased to be logical.

Lines 60-61 - the sentence "Paecilomyces variotii is the asexual state of Byssochlamys

Thanks for your valuable comment.

Sentences have been rearranged in the revised manuscript.

Material and methods

This chapter requires additions and corrections.

Lines 90-93 - should be before the sentence " Oat, and wheat bran were ...." (line 85)

Thanks for your valuable comment.

Lines 90-93 have been placed before line 85 in the revised manuscript

Line 102 - 2.3. Effect of different carbon sources - requires a more detailed description (how the experiment was performed, how much substrate was used ...)

Thanks for your valuable comment.

More details were added to the revised manuscript.

Line 106 - 2.4. Multifactorial experiments for optimizing inulinase production.

Please provide the components tested in the first phase and in the second phase - the most significant concentrations.

Thanks for your valuable comment.

The components tested in the first phase were mentioned in section 2.5 and significant components tested in the second phase were mentioned in section 3.3.2

Line 127 - it is known that "(-1) and (+1) are coded levels in Plackett-Burman design", but what exactly does -1 and +1 mean?

Thanks for your valuable comment.

"(-1) and (+1) are coded levels of low concentration and high concentration, respectively.

Line 141 – please list these two factors (…….) and give three levels (….)

Thanks for your valuable comment.

Sorry for that I wrote two factors by mistake, they were three factors (KCI, NaNO3, and chicory roots)) and three different levels (-1, 0, and 1)

It was corrected in the revised manuscript.

Line 173 - give the meaning of "SDS-PAGE", briefly what this protein analysis technique is and what it uses

Thanks for your valuable comment.

SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight.

Results and discussion

This part of the article should have been better presented, the results should have been interpreted with greater insight.

You can't start discussing research results with "Table 3 shows..." from the construction of the table it is obvious what it contains. This also applies to Lines: 269; 280; 296; 442; 469; 470.

Thanks for your valuable comment.

All sentences have been rephrased, so they don’t begin with Table No

The methodology (2.1.) shows that the experiment was also carried out on Rhizopus oryzae NRRL 3562 - and was not included in the results?

Thanks for your valuable comment.

Rhizopus oryzae NRRL 3562 was written by mistake, it was removed from revised manuscript

- In table 3 there is "Cell dry weight (g)" – „cel” ... or maybe „fungus” ...