Evaluation of the Thermal Processes on Changing the Phenotypic Characteristics of Escherichia coli Strains from Ice Cream Compared to Non-Pasteurized Milk
Round 1
Reviewer 1 Report (Previous Reviewer 2)
The authors responded to most of the reviewer's concerns. The manuscript can be now accepted for publication.
Author Response
Dear Dear Ms. Irene Yi,
Thank you very much for your letter of 29 November 2022 and for the constructive comments made by the reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compared to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised again according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The green, and yellow highlight in the revised-unclean-manuscript related to the final reviewer 1, and 2 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(fermentation-1999649)
Reviewer #1:
The authors responded to most of the reviewer's concerns. The manuscript can be now accepted for publication.
Reply: Thank you very much for your very wise judgment at this stage as well as the previous stages. We really implemented all your suggested comments one by one and thank you for your comment about accepting the article. Hopefully, I will be able to submit better articles to the scientific community in the future with your comments, dear referee.
Author Response File: 
Author Response.pdf
Reviewer 2 Report (Previous Reviewer 3)
The authors of "Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compared to non-pasteurized milk" have presented an improved version of their initial manuscript. However, minor English language and style errors need to be corrected.
- Check in all the text that 16S rDNA/16S rRNA is not in Italics.
- Line 28: substitute "which" with "that".
- Line 53: rephrase, the subject of the sentence, "adults", seems inappropriate and not correct. The prhase needs to be corrected.
- Line 85: "antimicrobial" not clear.
- Line 108: substitute "Bacteroids" with "Bacteroides".
- Line 151-152: change “Following that, 0.5 mL of sodium hydroxide (NaOH), 5M was added” to “Subsequently, 0.5 mL of 5 M sodium hydroxide (NaOH) was added”.
- Line 174: “Following purification” is not clear.
- Line 175: Specify each acronym, for example, “EC (E. coli)“, the first time it appears.
- Line 189: replace “2-Mercaptoethanol” with “2-mercaptoethanol”.
- Line 196: check the repetition and correct it.
- Line 240: Modify "There were 82 samples tested"; I suggest: “Among 82 tested samples”.
- Still not clear the caption of Figure 1.
- Line 373: "Gen bank" should be correct into "Genbank".
- I suggest an improvement of the second part of the discussion from line 433 in terms of the relevance of the content with respect to the general topic and the aim of the study.
Author Response
Dear Dear Ms. Irene Yi,
Thank you very much for your letter of 29 November 2022 and for the constructive comments made by the reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compared to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised again according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The green, and yellow highlight in the revised-unclean-manuscript related to the final reviewer 1, and 2 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(fermentation-1999649)
Reviewer #1:
The authors responded to most of the reviewer's concerns. The manuscript can be now accepted for publication.
Reply: Thank you very much for your very wise judgment at this stage as well as the previous stages. We really implemented all your suggested comments one by one and thank you for your comment about accepting the article. Hopefully, I will be able to submit better articles to the scientific community in the future with your comments, dear referee.
Reviewer #2:
The authors of "Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compared to non-pasteurized milk" have presented an improved version of their initial manuscript. However, minor English language and style errors need to be corrected.
Reply: Thank you very much for your very wise judgment at this stage as well as the previous stages. We really implemented all your suggested comments one by one.
Check in all the text that 16S rDNA/16S rRNA is not in Italics.
Reply: It was done in throughout the manuscript.
- Line 28: substitute "which" with "that".
Reply: It was done. Thank you so much for your suggestion.
- Line 53: rephrase, the subject of the sentence, "adults", seems inappropriate and not correct. The prhase needs to be corrected.
Reply: The exact sentence was contradictory and caused confusion for the reader. By removing this sentence, the continuity of the content in the text was observed.
- Line 85: "antimicrobial" not clear.
Reply: Thank you so much for the wise and scientific comment. It was amended as the following text:
In nature, bacteria are subjected to a variety of harmful and severe stresses, including nutrient deficiency, osmotic pressure, extremely high or low temperatures, acid, and antimicrobial compounds.
- Line 108: substitute "Bacteroids" with "Bacteroides".
Reply: It was done according your intelligent comment.
- Line 151-152: change “Following that, 0.5 mL of sodium hydroxide (NaOH), 5M was added” to “Subsequently, 0.5 mL of 5 M sodium hydroxide (NaOH) was added”.
Reply: It was done according to the scientific comment as the follow:
Subsequently, 0.5 mL of 5 M sodium hydroxide (NaOH) was added to each tube to induce blue fluorescence using a long-wave UV light source (366 nm).
- Line 174: “Following purification” is not clear.
Reply: Thank you so much for your attention in studying and improving the article as much as possible. This phrase was deleted. Please consider the following text:
Biochemical assays were used to compare each isolate to the reference strain of E. coli ATCC 25922. Further biochemical tests were performed on the isolates to provide a more precise diagnosis. Table 2 provides a list of the tests performed to identify the E. coli species isolated from ice cream.
- Line 175: Specify each acronym, for example, “EC (E. coli)“, the first time it appears.
Reply: It was done according to your suggestion as the following:
In EC (Escherichia coli) broth, LMX broth, and lauryl sulfate tryptose broth medium, gas might be generated from lactose by the production of acid from a variety of substrates.
- Line 189: replace “2-Mercaptoethanol” with “2-mercaptoethanol”.
Reply: it was done and replaced as you proposed it. Thank you so much for your scientific comment.
- Line 196: check the repetition and correct it.
Reply: Thank you so much for your attention in studying and improving the article as much as possible. Please notice to the revise text as the following:
The difference in protein profile of the isolates produced from infected specimens was examined by reference strain, and the degree of conformity between the isolates with the reference strain and with each other was calculated after determining the similarity.
- Line 240: Modify "There were 82 samples tested"; I suggest: “Among 82 tested samples”.
Reply: Thank you so much for accuracy of your opinion. Your wise comment was performed as the following:
Among 82 tested samples, 48 isolates were obtained.
- Still not clear the caption of Figure 1.
Reply: It was changed as the following:
Fig. 1. Dendrogram for phenotypic characteristics of 48 isolates among ice cream samples based on UPGMA algorithm of utilized carbon sources. Phenon 1 (1–16), Phenon 2 (22–29), Phenon 3 (39–42), Phenon 4 (2), Phenon 5 (6), Phenon 6 (32), Phenon 7 (21–35), and Phenon 8 (46).
- Line 373: "Gen bank" should be correct into "Genbank".
Reply: It was done according to your scientific suggestion. Thank you so much.
- I suggest an improvement of the second part of the discussion from line 433 in terms of the relevance of the content with respect to the general topic and the aim of the study.
Reply: With your scholarly judgment, dear reviewer, this section was not relevant to the title and objectives of the study, and we removed these few lines.
Thank you so much for scientific comment. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you dear reviewer. Please, if you still have valuable suggestions, do not hesitate to submit them to us.
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This manuscript is mainly about the detecting of E. coli strains from ice cream and milk samples by using several different methods (in this sense the title seems inappropriate). Although the conclusion is not surprising, the manuscript provides a relatively systematic comparison of these methods.
Other comments:
1. The introduction and discussion parts should include recent studies about the approaches used for E. coli detection.
2. Most of the references are old and not closely related with the main topic of the manuscript.
3. The Latin names of the microorganisms should be in italic.
Author Response
Dear dear Ms. Irene Yi,
Thank you very much for your letter of 25 October 2022 and for the constructive comments made by the Reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream by whole-cell protein profile, 16s rDNA and protein pattern compare to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The yellow, green, and turquoise highlight in the revised-unclean-manuscript related to the final reviewer 1, 2, and 3 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(Fermentation-1999649)
Reviewer #1: This manuscript is mainly about the detecting of E. coli strains from ice cream and milk samples by using several different methods (in this sense the title seems inappropriate). Although the conclusion is not surprising, the manuscript provides a relatively systematic comparison of these methods.
Reply: In agreement with this valuable recommendation, The title and conclusion of the article were modified. Our aim in this article is to investigate temperature stress in causing phenotypic changes on Escherichia coli bacteria as discussed in the article. We wanted to show that these temperature stresses can affect the diagnostic characteristics of bacteria. According to the ambiguity created in the article, the abstract was also modified.
Other comments:
- The introduction and discussion parts should include recent studies about the approaches used for E. coli detection.
Reply: In agreement with this valuable recommendation, we performed. Our aim was to investigate the effects of thermal shock on the changes in the phenotypic characteristics of E.coli as a health indicator. Our aim was to investigate the effects of temperature shock on the changes in the phenotypic characteristics of Escherichia coli as a health indicator. Therefore, related references were added to the text, which are highlighted in turquoise and yellow.
- Most of the references are old and not closely related with the main topic of the manuscript.
Reply: In agreement with this valuable recommendation, we revised it, as requested. Therefore, related references were added to the text, which are highlighted in turquoise and yellow.
- The Latin names of the microorganisms should be in italic.
Reply: In agreement with this valuable recommendation, we revised it, as requested. The corrected items are highlighted in yellow in the text.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by the Reviewers.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript, Ranjbar, Goli & Nedaeinia present a comparative study on how thermal processing (heat shock) can influence the phenotypic characteristics of Escherichia coli strains. For this purpose, the authors isolated strains from ice cream and non-pasteurized milk, and compared them with standardized E. coli strains (ATCC) in terms of phenotypic tests, whole-cell protein profiles, and 16s rDNA sequencing.
There are some issues that the authors need to address before the manuscript may be considered for publication.
· Genus and species names should be italicized all throughout the manuscript (the authors are not consistent in this regard).
· It is not clear on what criteria the isolates were grouped, classified or named.
· Section 3.2 is very confusing. For example, the first sentence, i. e. „The regression equation was used to evaluate the molecular weight of a substance from bacteria” is not clear. Regression equation of what? To evaluate the molecular weight of what „substance from bacteria”?
· Figures 1-3: the legends are not informative, as it is not clear what each lane represents. For instance, in Figure 1, lane 3 corresponds to 126, but what „126” means is difficult to understand. And the same applies to each lane. Also, it is hard to understand what is really compared.
· Figure 2: 11 lanes are enumerated in the legend, but there are only 10 lanes on the electrophoregram.
· Figure 4: bands are not discrete. What are lanes 1-5? Why do the authors skip from lane 5 to lanes 9,10?
· Legends of Figures 5 and 6 are also confusing: for example, what is „1 - 286 92, 93 - 184, 185 – 276 and 277 – 368”? Also, is there any alignment with ATCC strain?
· Figures 5 and 6 should be included as supplementary material, and only the sequence identities/differences must be discussed within the text.
· Lines 241-247 must be shifted to Materials and Methods section.
· Lines 160-162: it is not clear where equation (1) was used and what data were used for comparison (there is no reference to this equation in the Results section).
Author Response
Dear dear Ms. Irene Yi,
Thank you very much for your letter of 25 October 2022 and for the constructive comments made by the Reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream by whole-cell protein profile, 16s rDNA and protein pattern compare to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The yellow, green, and turquoise highlight in the revised-unclean-manuscript related to the final reviewer 1, 2, and 3 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(Fermentation-1999649)
Reviewer #2: In this manuscript, Ranjbar, Goli & Nedaeinia present a comparative study on how thermal processing (heat shock) can influence the phenotypic characteristics of Escherichia coli strains. For this purpose, the authors isolated strains from ice cream and non-pasteurized milk, and compared them with standardized E. coli strains (ATCC) in terms of phenotypic tests, whole-cell protein profiles, and 16s rDNA sequencing.
There are some issues that the authors need to address before the manuscript may be considered for publication.
- Genus and species names should be italicized all throughout the manuscript (the authors are not consistent in this regard).
Reply: In agreement with this valuable recommendation, we revised it, as requested.
- It is not clear on what criteria the isolates were grouped, classified or named.
Reply: In agreement with this valuable recommendation, the dendrogram of the isolates, which shows their grouping, was added to the results section of paragraph 3-1, which is highlighted in green in the text of the article.
Section 3.2 is very confusing. For example, the first sentence, i. e. „The regression equation was used to evaluate the molecular weight of a substance from bacteria” is not clear. Regression equation of what? To evaluate the molecular weight of what „substance from bacteria”?
Reply: In agreement with this valuable recommendation, Figure 2 was added to clear the ambiguity. We needed this graph to determine the molecular weight of some protein bands so that we could calculate the differences between the isolates obtained from ice cream compared to the standard strain and the isolates obtained from raw milk.
- Figures 1-3: the legends are not informative, as it is not clear what each lane represents. For instance, in Figure 1, lane 3 corresponds to 126, but what „126” means is difficult to understand. And the same applies to each lane. Also, it is hard to understand what is really compared.
Reply: In agreement with this valuable recommendation, According to the addition of the dendrogram as Figure 1, all the isolates mentioned in section 2-3 can be seen by number in the grouped diagram of the dendrogram. Therefore, it resolves the ambiguity created.
- Figure 2: 11 lanes are enumerated in the legend, but there are only 10 lanes on the electrophoregram.
Reply: In agreement with this valuable recommendation, Figure 2, which is Figure 4 according to the figures added above, was wrongly uploaded and replaced with the correct figure.
- Figure 4: bands are not discrete. What are lanes 1-5? Why do the authors skip from lane 5 to lanes 9,10?
Reply: The mentioned numbers are the numbers of the isolates based on the sample number and because it is mentioned in the dendrogram with this number, we could not change the name. In order to solve this ambiguity, the explanation below Figure 4, which is now Figure 6, was added as a turquoise highlight.
- Legends of Figures 5 and 6 are also confusing: for example, what is „1 - 286 92, 93 - 184, 185 – 276 and 277 – 368”? Also, is there any alignment with ATCC strain?
Reply: In agreement with this valuable recommendation, The mentioned figure was removed from the article and its modified version was added to the article under the title of Supplementary File S1.
- Figures 5 and 6 should be included as supplementary material, and only the sequence identities/differences must be discussed within the text.
Reply: In agreement with this valuable recommendation, we performed.
- Lines 241-247 must be shifted to Materials and Methods section.
Reply: In agreement with this valuable recommendation, we performed. Section “2.3. Heat shock in bacteria isolated from raw milk “was added to the method section, which is highlighted in green.
- Lines 160-162: it is not clear where equation (1) was used and what data were used for comparison (there is no reference to this equation in the Results section).
Reply: This formula was mathematically designed by a statistician to check the percentage of similarity, which we used in two parts of the article, but due to the large number of protein bands of each bacterium and the differences in the intensity of the bands, it was only used for estimation.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by the Reviewers.
Author Response File: Author Response.pdf
Reviewer 3 Report
Major comments:
The paper " Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream by whole-cell protein profile, 16s rDNA and protein pattern compare to non-pasteurized milk " has serious flaws that must be reviewed before being considered for publication.
First, I miss a clear explanation what was the exact aim of the study. Highlight more specifically the purpose of the work showing the importance of the results obtained. For example, which is the functional role of each phenon of Escherichia coli strains from ice cream in relation to the thermal processes? Which is the relationship between all the characteristics of the isolates and their protein assets? And after thermal treatments? What does provide the comparison of the total protein profile with E. coli strains isolated from milk?
The Introduction section should be improved with updated References and I suggest including References depicting the actual background in terms of molecular characterization of bacteria submitted to thermal processes.
The results on the number of isolates, their features, and the number of isolates whose profile has been characterized should be clearly explained.
One of the two main experimental parts of the work (Electrophoresis of bacteria proteins) doesn't seem to be conclusive. The comparison of the bands with the presented method is not convincing in relation to the results obtained "Due to differences in the severity or position of the bands, accurate calculation of the percentage of similarity was not possible."
The Reference of the applied method for determining the band similarity should be reported.
The Result section can be improved. Moreover, the description of the phenon should be addressed in the Result section and further commented and explored in the Discussion section.
Amplification of 16s rDNA and its sequencing appeared to be only a validation of the genera of the isolates. It does not add any new information on the changes deriving from the thermal processes on the phenotypic characteristics of Escherichia coli strains from ice cream.
In the Discussion, hsp genes are mentioned. Would have been interesting the identification of their pattern before/after thermal processes to better characterize E. coli profiling.
Minor comments:
- Correct in italics all the genera and species of bacterial strains
- English grammar needs extensive correction.
- Check for missing space in the text.
- The content of Table 1 could be integrated into the text
- Clarify all the contents of Table 3, e.g., what do the numbers represent, such as 50 and 50 for Lysine decarboxylase? Clarify in the text the number of isolates considered that were included in Table 3.
- Figure 1-3: not clear which strain belongs to which profile and it is not clearly presented why these profiles were chosen with respect to others. Are the meaningful profiles? The pictures are not clear, they should be improved.
- Figures 4, 5, and 6 do not add any important conclusion, they can be removed.
- Lines 398-402 were copied and pasted in the Conclusion section.
Author Response
Dear dear Ms. Irene Yi,
Thank you very much for your letter of 25 October 2022 and for the constructive comments made by the Reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream by whole-cell protein profile, 16s rDNA and protein pattern compare to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The yellow, green, and turquoise highlight in the revised-unclean-manuscript related to the final reviewer 1, 2, and 3 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(Fermentation-1999649)
Reviewer #3: Major comments:
The paper "Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream by whole-cell protein profile, 16s rDNA and protein pattern compare to non-pasteurized milk " has serious flaws that must be reviewed before being considered for publication.
First, I miss a clear explanation what was the exact aim of the study. Highlight more specifically the purpose of the work showing the importance of the results obtained. For example, which is the functional role of each phenon of Escherichia coli strains from ice cream in relation to the thermal processes? Which is the relationship between all the characteristics of the isolates and their protein assets? And after thermal treatments? What does provide the comparison of the total protein profile with E. coli strains isolated from milk?
Reply: In agreement with this valuable recommendation, The title and conclusion of the article were modified. Our aim in this article is to investigate temperature stress in causing phenotypic changes on Escherichia coli bacteria as discussed in the article. We wanted to show that these temperature stresses can affect the diagnostic characteristics of bacteria. According to the ambiguity created in the article, the abstract was also modified. The added parts in the abstract, introduction and conclusion were highlighted in turquoise. In order to specify the grouping, the dendrogram was added in section 1-3 (green highlight). Regarding each phenon and its differences with the standard strain, it is explained in the discussion section. In the case of the protein pattern, the differences were mostly qualitative, and in the cases where the protein bands were important, the molecular weight was calculated using the regression added in section 2-3 (green highlight).
The Introduction section should be improved with updated References and I suggest including References depicting the actual background in terms of molecular characterization of bacteria submitted to thermal processes.
Reply: In agreement with this valuable recommendation, we revised it, as requested.
The results on the number of isolates, their features, and the number of isolates whose profile has been characterized should be clearly explained.
Reply: Regarding the number of samples, necessary explanations are given in section 2.1 (turquoise highlight). A dendrogram was added to identify the groups in section 3.1 (green highlight) of Figure 1. Regarding the explanation of each phenon, the necessary information is given in the discussion section, which is highlighted in turquoise.
One of the two main experimental parts of the work (Electrophoresis of bacteria proteins) doesn't seem to be conclusive. The comparison of the bands with the presented method is not convincing in relation to the results obtained "Due to differences in the severity or position of the bands, accurate calculation of the percentage of similarity was not possible."
Reply: We agree with the referee's opinion and we have mentioned this point. Because the nature of protein electrophoresis pattern by SDS-PAGE method is qualitative. In some places, we noted the prominent protein bands that differed from the standard strain in the ice cream samples. Regarding the protein pattern obtained from milk, we only wanted to show the difference between the isolates obtained before and after the temperature shock. The main comparison of differences with biochemical tests has already been given in Table 2.
The Reference of the applied method for determining the band similarity should be reported.
Reply: This method was designed by a statistical consultant and its correctness has been proven mathematically.
The Result section can be improved. Moreover, the description of the phenon should be addressed in the Result section and further commented and explored in the Discussion section.
Reply: In agreement with this valuable recommendation, we revised it, as requested. Modified items are highlighted in turquoise and green.
Amplification of 16s rDNA and its sequencing appeared to be only a validation of the genera of the isolates. It does not add any new information on the changes deriving from the thermal processes on the phenotypic characteristics of Escherichia coli strains from ice cream.
Reply: Our aim was the effects of temperature shocks on the biochemical and structural characteristics of bacteria. Finally, we showed that because those characteristics can be changed, it is better to use genomic methods for special cases. In addition, we mentioned the phenotypic characteristics that change less or are more stable in Table 2, which seems to be useful for more definite diagnosis.
In the Discussion, hsp genes are mentioned. Would have been interesting the identification of their pattern before/after thermal processes to better characterize E. coli profiling.
Reply: We agree with the referee's opinion. It will definitely be desired by the research team in future projects.
Minor comments:
- Correct in italics all the genera and species of bacterial strains
Reply: Modified as requested
- English grammar needs extensive correction.
Reply: the English grammar was corrected as possible as that you want.
- Check for missing space in the text.
Reply: Modified as requested
- The content of Table 1 could be integrated into the text
Reply: Because the text of the article will be long. In addition, we preferred to keep the table as it will help the reader to understand correctly.
- Clarify all the contents of Table 3, e.g., what do the numbers represent, such as 50 and 50 for Lysine decarboxylase? Clarify in the text the number of isolates considered that were included in Table 3.
Reply: All groups are explained in the discussion section. The requested item was added as a turquoise highlight under the text.
- Figure 1-3: not clear which strain belongs to which profile and it is not clearly presented why these profiles were chosen with respect to others. Are the meaningful profiles? The pictures are not clear, they should be improved.
Reply: The dendrogram of the isolates, which shows their grouping, was added to the results section of paragraph 3-1, which is highlighted in green in the text of the article. which specifies the role of the groups below. This figure is used to qualitatively compare the protein profile of the isolates obtained from ice cream and the effect of temperature shock on the isolates obtained from raw milk before and after the shock.
- Figures 4, 5, and 6 do not add any important conclusion, they can be removed.
Reply: The requested items were transferred to the supplementary file 1 and removed from the text of the article.
- Lines 398-402 were copied and pasted in the Conclusion section.
Reply: In agreement with this valuable recommendation, we revised it, as requested. Modified items are highlighted in turquoise in conclusion.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by the Reviewers.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors did not respond to reviewer's concerns, and they did not improve the presentation of the data.
For example: lines 285-286 "The regression equation was used to evaluate the molecular weight of a substance from bacteria" do not make sense... The answer to this concern was the introduction of the superfluous Figure 2, and not a clarification on what "substance" was actually investigated.
Besides, the numbers associated to the gel lanes in Figures 3-5 are still chaotic, Figure 6 included gels of poor quality, there is no correlation between data, etc.
Author Response
Dear Dear Ms. Irene Yi,
Thank you very much for your letter of 16 November 2022 and for the constructive comments made by the reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compared to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised again according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The green, and turquoise highlight in the revised-unclean-manuscript related to the final reviewer 2, and 3 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(fermentation-1999649)
Reviewer #2:
*The authors did not respond to reviewer's concerns, and they did not improve the presentation of the data.
For example: lines 285-286 "The regression equation was used to evaluate the molecular weight of a substance from bacteria" do not make sense... The answer to this concern was the introduction of the superfluous Figure 2, and not a clarification on what "substance" was actually investigated.
Reply: Thank you so much for wise comment. We tried as much as possible to address the referee's concerns. Nevertheless, if there is still a problem, we tried to fix it in the new version of the article. In agreement with this valuable recommendation, we revised it, as requested. Therefore, in the article section 3.2. Electrophoresis of bacteria proteins in the first line "Substance" was replaced with "unknown proteins" in order to clear the ambiguity with a green highlight. Figure 2 and its description were removed based on the reviewer's comments.
**Besides, the numbers associated to the gel lanes in Figures 3-5 are still chaotic, Figure 6 included gels of poor quality; there is no correlation between data, etc.
Reply: Thank you so much for intelligent suggestion. In agreement with this valuable recommendation, we revised it. In fig. 3, next to each number; we mentioned the name of the Phenon, which were clustered according to Figure 1 (Dendrogram for phenotypic characteristics). We showed with a green highlight. In Figure 3, when we wanted to compare the difference in the protein bands of the isolates obtained from ice creams, which we were absolutely sure E.coli, with the reference strain; we used (phenon 1) isolates and showed the differences. For figures 4 and 5, next to each number, we mentioned the name of the E. coli strain (From milk), which we showed with green highlights.
***Figure 6 actually shows that despite the differences in biochemical and protein characteristics. The PCR test was to confirm our diagnosis method, after which we performed the sequencing test. Sequencing (Supplementary file 2) confirmed our results. According to the referee's comment, figure 6 was transferred to the supplementary file 1.
Reply: It was done according to your scientific suggestion.
Thank you so much for scientific comment. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you dear reviewer. Please, if you still have valuable suggestions, do not hesitate to submit them to us.
It is worth noting, dear referee, that the entire text was completely revised in the native language.
Reviewer #3:
*The authors of "Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compare to non-pasteurized milk " have presented a revised version of their manuscript.
Although the authors have made significant improvements, English errors are still present (also in the title "compare" should be "compared"), and some sentences are obscure, especially the newly added parts of the Abstract. Moreover, some changed parts are not evidenced or not reported correctly in the point-by-point generating difficulties in reading.
Overall, some issues still need to be addressed and improved, instead of evidencing some not changed sentences of the manuscript.
They have not answered some of my comments, e.g., regarding the italic of genera and species of the strains. For example in the Introduction (L 58-60): e.g., Lactococcus, Lactobacillus, Streptococcus, Propionibacterium, and fungal populations), causing spoilage (e.g., Pseudomonas, Clostridium, Bacillus, and other spore-forming...
Reply: Thank you so much for comprehensive and intelligent comment. We tried as much as possible to address the referee's concerns. Nevertheless, if there is still a problem, we tried to fix it in the new version of the article. In agreement with this valuable recommendation, we performed.
**About the comment "The Reference of the applied method for determining the band similarity should be reported." The reply: "This method was designed by a statistical consultant and its correctness has been proven mathematically." is not exhaustive. If it has been used, the method should be reported.
Reply: Thank you so much for kind and wise suggestion. Reference related to the text of the article section “2.6. Determination of the percentage of similarity” was added and the highlight became turquoise.
***Also, the improvement of the Result section with the description of the phenon is still missing. The authors preferred maintaining the intact text of the description of the phenons within the Discussion.
Reply: I would like to thank you, dear judges, for your careful comments. In agreement with this valuable recommendation, we revised it with the highlight became turquoise.
****About my comment "The results on the number of isolates, their features, and the number of isolates whose profile has been characterized should be clearly explained." They answered that the necessary explanation is in section 2.2 but it is the same as before and the newly added Fig. 1 is not readable. Regarding Fig 1, the numbers are too close to each other and it is not easy to detect the groups that are reported in the caption in a not clear way (e.g., Phenon 7 (21-35) should be maybe Phenon 7 (21, 33-35)).
Reply: I would like to thank you, dear judges, for your careful comments. In agreement with this valuable recommendation, the description of each phenon is at the end of section “3.1. Phenotypic tests of isolated coliforms” were added and highlighted with turquoise. We increased the quality of Figure 1. By the way, the caption of Figure 1 is correct.
*****Captions of Fig. 4 and Fig. 5 are not clear even with the new dendrogram. The numbers in the caption are not present in the new Fig. 1. They should be further explained.
Reply: I am especially grateful for your very valuable suggestions to improve and increase the scientific value of the article. The caption of figure 2, 3 and 4 was modified and highlighted with green. It should be noted that figure 1 is only for isolates obtained from ice cream, which is related to figure 2, and is not used for figures 3 and 5, which are isolates obtained from milk.
******Figure 6 should be included in supplementary material or even not necessary. The sequence comparison can be reported in the text.
Reply: I am especially grateful for your very valuable suggestions to improve and increase the scientific value of the article. Figure 6 was transferred to the supplementary file 1.
*******The discussion still needs improvements.
Reply: Thank you very much for your concern and importance to make the article more scientific. In agreement with this valuable recommendation, we revised it with the highlight became turquoise. The results related to phenons were moved to the results section.
Thank you so much for scientific comment. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you dear reviewer. Please, if you still have valuable suggestions, do not hesitate to submit them to us.
It is worth noting, dear referee, that the entire text was completely revised in the native language.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors of "Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compare to non-pasteurized milk " have presented a revised version of their manuscript.
Although the authors have made significant improvements, English errors are still present (also in the title "compare" should be "compared"), and some sentences are obscure, especially the newly added parts of the Abstract. Moreover, some changed parts are not evidenced or not reported correctly in the point-by-point generating difficulties in reading.
Overall, some issues still need to be addressed and improved, instead of evidencing some not changed sentences of the manuscript.
They have not answered some of my comments, e.g., regarding the italic of genera and species of the strains. For example in the Introduction (L 58-60): e.g., Lactococcus, Lactobacillus, Streptococcus, Propionibacterium, and fungal populations), causing spoilage (e.g., Pseudomonas, Clostridium, Bacillus, and other spore-forming...
About the comment "The Reference of the applied method for determining the band similarity should be reported." The reply: "This method was designed by a statistical consultant and its correctness has been proven mathematically." is not exhaustive. If it has been used, the method should be reported.
Also, the improvement of the Result section with the description of the phenon is still missing. The authors preferred maintaining the intact text of the description of the phenons within the Discussion.
About my comment "The results on the number of isolates, their features, and the number of isolates whose profile has been characterized should be clearly explained." They answered that the necessary explanation is in section 2.2 but it is the same as before and the newly added Fig. 1 is not readable. Regarding Fig 1, the numbers are too close to each other and it is not easy to detect the groups that are reported in the caption in a not clear way (e.g., Phenon 7 (21-35) should be maybe Phenon 7 (21, 33-35)).
Captions of Fig. 4 and Fig. 5 are not clear even with the new dendrogram. The numbers in the caption are not present in the new Fig. 1. They should be further explained.
Figure 6 should be included in supplementary material or even not necessary. The sequence comparison can be reported in the text.
The discussion still needs improvements.
Author Response
Dear Dear Ms. Irene Yi,
Thank you very much for your letter of 16 November 2022 and for the constructive comments made by the reviewers on our manuscript, entitled “Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compared to non-pasteurized milk” (fermentation-1999649), submitted for consideration to be published as a research article in Fermentation.
We are pleased to send you the manuscript carefully revised again according to the comments of the Reviewers. Concerning the format, a more concise writing style has been adopted. Concerning the specific points raised by the Reviewers, we have carefully revised our manuscript according to the comments and suggestions of the Reviewers. The point-to-point reply to the Reviewers is enclosed; in order to help you and the Reviewers in the identification of the modifications in the revised manuscript, we indicate in the reply letter the pages where the changes to the main text have been made. Moreover, we uploaded, as a supplementary file, a version of the revised manuscript with changes highlighted. All authors are aware of and agree to the content of the manuscript.
We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you and the Reviewers and meets the quality standard for publication in the Journal of the Fermentation. Thank you very much for your kind consideration.
The green, and turquoise highlight in the revised-unclean-manuscript related to the final reviewer 2, and 3 proposed amendments, respectively.
Yours sincerely,
Mohammad Goli (Ph.D.), Associate Professor
Department of Food Science & Technology, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
Email: [email protected]
REPLIES TO REVIEWERS’ COMMENTS
(fermentation-1999649)
Reviewer #2:
*The authors did not respond to reviewer's concerns, and they did not improve the presentation of the data.
For example: lines 285-286 "The regression equation was used to evaluate the molecular weight of a substance from bacteria" do not make sense... The answer to this concern was the introduction of the superfluous Figure 2, and not a clarification on what "substance" was actually investigated.
Reply: Thank you so much for wise comment. We tried as much as possible to address the referee's concerns. Nevertheless, if there is still a problem, we tried to fix it in the new version of the article. In agreement with this valuable recommendation, we revised it, as requested. Therefore, in the article section 3.2. Electrophoresis of bacteria proteins in the first line "Substance" was replaced with "unknown proteins" in order to clear the ambiguity with a green highlight. Figure 2 and its description were removed based on the reviewer's comments.
**Besides, the numbers associated to the gel lanes in Figures 3-5 are still chaotic, Figure 6 included gels of poor quality; there is no correlation between data, etc.
Reply: Thank you so much for intelligent suggestion. In agreement with this valuable recommendation, we revised it. In fig. 3, next to each number; we mentioned the name of the Phenon, which were clustered according to Figure 1 (Dendrogram for phenotypic characteristics). We showed with a green highlight. In Figure 3, when we wanted to compare the difference in the protein bands of the isolates obtained from ice creams, which we were absolutely sure E.coli, with the reference strain; we used (phenon 1) isolates and showed the differences. For figures 4 and 5, next to each number, we mentioned the name of the E. coli strain (From milk), which we showed with green highlights.
***Figure 6 actually shows that despite the differences in biochemical and protein characteristics. The PCR test was to confirm our diagnosis method, after which we performed the sequencing test. Sequencing (Supplementary file 2) confirmed our results. According to the referee's comment, figure 6 was transferred to the supplementary file 1.
Reply: It was done according to your scientific suggestion.
Thank you so much for scientific comment. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you dear reviewer. Please, if you still have valuable suggestions, do not hesitate to submit them to us.
It is worth noting, dear referee, that the entire text was completely revised in the native language.
Reviewer #3:
*The authors of "Evaluation of the thermal processes on changing the phenotypic characteristics of Escherichia coli strains from ice cream compare to non-pasteurized milk " have presented a revised version of their manuscript.
Although the authors have made significant improvements, English errors are still present (also in the title "compare" should be "compared"), and some sentences are obscure, especially the newly added parts of the Abstract. Moreover, some changed parts are not evidenced or not reported correctly in the point-by-point generating difficulties in reading.
Overall, some issues still need to be addressed and improved, instead of evidencing some not changed sentences of the manuscript.
They have not answered some of my comments, e.g., regarding the italic of genera and species of the strains. For example in the Introduction (L 58-60): e.g., Lactococcus, Lactobacillus, Streptococcus, Propionibacterium, and fungal populations), causing spoilage (e.g., Pseudomonas, Clostridium, Bacillus, and other spore-forming...
Reply: Thank you so much for comprehensive and intelligent comment. We tried as much as possible to address the referee's concerns. Nevertheless, if there is still a problem, we tried to fix it in the new version of the article. In agreement with this valuable recommendation, we performed.
**About the comment "The Reference of the applied method for determining the band similarity should be reported." The reply: "This method was designed by a statistical consultant and its correctness has been proven mathematically." is not exhaustive. If it has been used, the method should be reported.
Reply: Thank you so much for kind and wise suggestion. Reference related to the text of the article section “2.6. Determination of the percentage of similarity” was added and the highlight became turquoise.
***Also, the improvement of the Result section with the description of the phenon is still missing. The authors preferred maintaining the intact text of the description of the phenons within the Discussion.
Reply: I would like to thank you, dear judges, for your careful comments. In agreement with this valuable recommendation, we revised it with the highlight became turquoise.
****About my comment "The results on the number of isolates, their features, and the number of isolates whose profile has been characterized should be clearly explained." They answered that the necessary explanation is in section 2.2 but it is the same as before and the newly added Fig. 1 is not readable. Regarding Fig 1, the numbers are too close to each other and it is not easy to detect the groups that are reported in the caption in a not clear way (e.g., Phenon 7 (21-35) should be maybe Phenon 7 (21, 33-35)).
Reply: I would like to thank you, dear judges, for your careful comments. In agreement with this valuable recommendation, the description of each phenon is at the end of section “3.1. Phenotypic tests of isolated coliforms” were added and highlighted with turquoise. We increased the quality of Figure 1. By the way, the caption of Figure 1 is correct.
*****Captions of Fig. 4 and Fig. 5 are not clear even with the new dendrogram. The numbers in the caption are not present in the new Fig. 1. They should be further explained.
Reply: I am especially grateful for your very valuable suggestions to improve and increase the scientific value of the article. The caption of figure 2, 3 and 4 was modified and highlighted with green. It should be noted that figure 1 is only for isolates obtained from ice cream, which is related to figure 2, and is not used for figures 3 and 5, which are isolates obtained from milk.
******Figure 6 should be included in supplementary material or even not necessary. The sequence comparison can be reported in the text.
Reply: I am especially grateful for your very valuable suggestions to improve and increase the scientific value of the article. Figure 6 was transferred to the supplementary file 1.
*******The discussion still needs improvements.
Reply: Thank you very much for your concern and importance to make the article more scientific. In agreement with this valuable recommendation, we revised it with the highlight became turquoise. The results related to phenons were moved to the results section.
Thank you so much for scientific comment. We very much hope that the present version of the manuscript satisfactorily addresses all the observations made by you dear reviewer. Please, if you still have valuable suggestions, do not hesitate to submit them to us.
It is worth noting, dear referee, that the entire text was completely revised in the native language.
Author Response File: Author Response.docx
