Analysis of the Antioxidant Mechanism of Ozone Treatment to Extend the Shelf Life and Storage Quality of ‘Korla’ Fragrant Pears Based on Label-Free Proteomics

Round 1

Reviewer 1 Report

In the Abstract there are many abbreviations without the correspondent meaning. Only very few examples: MDA, ROS, SOD, CAT….). So, they need to be clarified when they are cited for the first time.

The same for the Introduction section. Only very few examples: D-SDS-PAGE, ASA-GSH.

Line 68, Ioannis et al. This reference lacks the respective number.

Line 74: Zhang et al., which is the reference?

Why is the range time on the x-axis in Figure 2b 5 days, whereas the range in the other figures is 4 days?

In the section Antioxidant index would be possible to correlate the trend of superoxide and hydrogen peroxide with SOD, POD and CAT activities?

Pyrus ussuriensis must be in italic

Would be possible to find an explanation for the generation rate of O2-, H2O2 content, and changes in respiration rate showed a clear positive correlation (P<0.05), and the MDA content showed a clear positive correlation with the weight loss rate and a clear negative correlation with firmness: Through metabolic explanation or, if possible, with a schema? Although this is not required, I think this strategy could be better utilized and would make it easier for readers, especially students, to comprehend the concepts.

Author Response

Dear reviewer,

Thank you for the comments concerning our manuscript entitled “Analysis of the antioxidant mechanism of ozone treatment to extend the shelf life and storage quality of ‘Korla’ fragrant pears based on label-free proteomics” (ID: horticulturae-2939548). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied and revised the comments carefully. The responses to the comments are as flowing:

1.In the Abstract there are many abbreviations without the correspondent meaning. Only very few examples: MDA, ROS, SOD, CAT….). So, they need to be clarified when they are cited for the first time.

 The same for the Introduction section. Only very few examples: D-SDS-PAGE, ASA-GSH.

Answer: Thank you for your comment, and we modified this in the Abstract and Introduction.

2.Line 68, Ioannis et al. This reference lacks the respective number.

Answer: Thank you for your comment. We added the reference number.

3.Line 74: Zhang et al., which is the reference?

Answer: Thank you for your comment. We modified it and added the regerence number.

4.Why is the range time on the x-axis in Figure 2b 5 days, whereas the range in the other figures is 4 days?

Answer: Thank you for your comment. We modified the Figure 2b.

5.In the section Antioxidant index would be possible to correlate the trend of superoxide and hydrogen peroxide with SOD, POD and CAT activities?

Answer: Thank you for your comment, and we added this in the part Results and Discussion, “In general, the superoxide anion generation rate of postharvest fragrant pears gradually increased with storage time, which stimulated the increase of SOD enzyme activity, and the hydrogen peroxide produced by the metabolism of superoxide anion induced the ac-tivities of POD and CAT. The antioxidant enzyme activity of fragrant pears was promoted by ozone to improve the antioxidant capacity of the tissue and maintain fruit quality. This finding is in agreement with previous work on Acibenzolar-S-methyl treated pears[25,26], Zhao et al. also concluded that the antioxidant enzyme activities of postharvest pears were stimulated by ozone at appropriate concentrations, especially POD enzyme[18]”

[18] Zhao, Z.; Xu, G.; Han, Z.; Li, Q.; Chen, Y.; Li, D. Effect of ozone on the antioxidant capacity of "qiushui" pear (pyrus pyrifolia nakai cv. Qiushui) during postharvest storage. J. Food Qual. 2013, 36, 190-197.

6.Pyrus ussuriensis must be in italic

Answer: Thank you for your comment, and we modified it.

7.Would be possible to find an explanation for the generation rate of O2-, H2O2 content, and changes in respiration rate showed a clear positive correlation (P<0.05), and the MDA content showed a clear positive correlation with the weight loss rate and a clear negative correlation with firmness: Through metabolic explanation or, if possible, with a schema? Although this is not required, I think this strategy could be better utilized and would make it easier for readers, especially students, to comprehend the concepts.

Answer: Thank you for your comment, and we added the explanation.

“The generation rate of O2-, H2O2 content, and changes in respiration rate showed a clear positive correlation (P<0.05), some studies have found that the increase in respiration rate and the accumulation of ROS were signs of aging in harvested fruits and vegetables[31,32]. The MDA content showed a clear positive correlation with the weight loss rate and a clear negative correlation with firmness (P < 0.05), permeability of harvested fruit and vegetable cells was induced by peroxidation of cell membranes, resulting in a decrease in hardness and an increase in weight loss rate[33].”

[31] Lin, Y.; Lin, H.; Chen, Y.; Wang, H.; Lin, M.; Ritenour, M.A.; Lin, Y. The role of ros-induced change of respiratory metab-olism in pulp breakdown development of longan fruit during storage. Food Chem. 2020, 305, 125439, doi: https://0-doi-org.brum.beds.ac.uk/10.1016/j.foodchem.2019.125439.

[32] Meitha, K.; Pramesti, Y.; Suhandono, S. Reactive oxygen species and antioxidants in postharvest vegetables and fruits. Int. J. Food Sci. 2020, 2020, 1-11, doi: 10.1155/2020/8817778.

[33] Song, L.; Yang, H.; Cheng, S.; Zhang, Z.; Zhang, L.; Su, R.; Li, Y.; Zhan, X.; Yang, B.; Lin, L.; et al. Combination effects of ultrasound and citral nanoemulsion against shigella flexneri and the preservation effect on fresh-cut carrots. Food Control 2023, 155, 110069, doi: https://0-doi-org.brum.beds.ac.uk/10.1016/j.foodcont.2023.110069.

We tried our best to improve the manuscript and made some changes in the manuscript. We appreciate your warm work earnestly and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present work titled “  Analysis of the Antioxidant Mechanism of Ozone Treatment to 2 Extend the Shelf Life and Storage Quality of ‘Korla’ Fragrant 3 Pears Based on Label-Free Proteomics” deals with effects of ozone as a posthavest treatment and analyses the expression of certain enzymatic ROS processing system in pears.

The overall manuscript shows some new information about the ozone treatment in postharvest, but there are several important issues that should be discussed. There is also another work that must be added to the references and compare the main results. The present version needs some amendments, and more information should be added to clarify some issues.

Line 64: There is a previous work in other species of pear that explores the same objectives in postharvest. Please add this reference of Zhao and coworkers (2013): 10.1111/jfq.12021

Line 95-97: It is very important to detail the procedure and justify the chosen concentration. Please add supplementary information that documents the chosen concentration of 1ppm of ozone.

Line 99: It is not clear if the time 0 of the experiments starts after the 4 d ozone treatment or starts for both groups (treated and untreated) without ozone in the chamber. Please clarify this in the M&M section.

Line 230: It is also interesting to discuss the effects of ozone at exactly 16 d after harvest. At this specific timepoint the is a raise in the respiration rate, coupled to the increase of ROS and the activity of the enzymatic antioxidant system (Figure 3) coinciding with the peak of certain SOD, POD and CAT (Figure 3).

Line 257: I encourage the authors to add the work of Zhao et al (2013). Similar results in POD, CAT and SOD activities were found in another species of pear.

Line 267: If we look at the activities of the principal enzymes, in the upper left corner there is a strong correlation between groups, but these correlations are less important when compared to specific enzymes. Only POD seams to correlate with other PODs and CAT2 and POD10 with other SODs. This effect can be also seen in Figure 2 at 16 d after postharvest in the fold change of these enzymes.

Author Response

Dear reviewer,

Thank you for the comments concerning our manuscript entitled “Analysis of the antioxidant mechanism of ozone treatment to extend the shelf life and storage quality of ‘Korla’ fragrant pears based on label-free proteomics” (ID: horticulturae-2939548). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied and revised the comments carefully. The responses to the comments are as flowing:

1.Line 64: There is a previous work in other species of pear that explores the same objectives in postharvest. Please add this reference of Zhao and coworkers (2013): 10.1111/jfq.12021IF: 3.3 Q2

Answer: Thank you for your comment, the previous work is very meaningful and helpful for our studies, and we added the references in the section Introduction and Results and discussion.

[18] Zhao, Z.; Xu, G.; Han, Z.; Li, Q.; Chen, Y.; Li, D. Effect of ozone on the antioxidant capacity of "qiushui" pear (pyrus pyrifolia nakai cv. Qiushui) during postharvest storage. J. Food Qual. 2013, 36, 190-197.

2.Line 95-97: It is very important to detail the procedure and justify the chosen concentration. Please add supplementary information that documents the chosen concentration of 1ppm of ozone.

Answer: Thank you for your comment. The effects of different concentrations of ozone on the shelf life of postharvest fragrant pears. In another of our studies (under submission). For example, the influence of different concentrations of ozone treatment on the decay rate of postharvest fragrant pears is shown in the figure.

Figure 1. The effect of ozone treatment on rot
ting rate

3.Line 99: It is not clear if the time 0 of the experiments starts after the 4 d ozone treatment or starts for both groups (treated and untreated) without ozone in the chamber. Please clarify this in the M&M section.

Answer: Thank you for your comment. We added the details in the Material and Method.

4.Line 230: It is also interesting to discuss the effects of ozone at exactly 16 d after harvest. At this specific time point the is a raise in the respiration rate, coupled to the increase of ROS and the activity of the enzymatic antioxidant system (Figure 3) coinciding with the peak of certain SOD, POD and CAT (Figure 3).

Answer: Thank you for your comment. We added these results in the part Results and Discussion.

 “the superoxide anion generation rate of postharvest fragrant pears gradually increased with storage time, which stimulated the increase of SOD enzyme activity, and the hydrogen peroxide produced by the metabolism of superoxide anion induced the activities of POD and CAT. ”

On the 16th day of shelf life, the respiration rate, superoxide anion production rate and hydrogen peroxide content of postharvest fragrant pears in the ozone treatment group reached their peaks, which were consistent with the peaks of SOD, POD and CAT enzyme activities, while the difference in protein expression levels of SOD2, SOD6, CAT2, and POD2 also reached the maximum fold. Li et al. also concluded that the decrease in postharvest ‘Yali’ pears quality was related to the decrease in the enzyme activities of SOD, CAT, and POD[29]. The gene expression of POD was stimulated in the postharvest ‘Zaosu’ and ‘Nanguo’ pears to maintain high quality during storage time[25,29].

5.Line 257: I encourage the authors to add the work of Zhao et al (2013). Similar results in POD, CAT and SOD activities were found in another species of pear.

Answer: Thank you for your comment, and we added this in the part Results and Discussion, “Zhao et al. also concluded that the antioxidant enzyme activities of postharvest pears were stimulated by ozone at appropriate concentrations, especially POD enzyme.”

[18] Zhao, Z.; Xu, G.; Han, Z.; Li, Q.; Chen, Y.; Li, D. Effect of ozone on the antioxidant capacity of "qiushui" pear (pyrus pyrifolia nakai cv. Qiushui) during postharvest storage. J. Food Qual. 2013, 36, 190-197.

6.Line 267: If we look at the activities of the principal enzymes, in the upper left corner there is a strong correlation between groups, but these correlations are less important when compared to specific enzymes. Only POD seams to correlate with other PODs and CAT2 and POD10 with other SODs. This effect can be also seen in Figure 2 at 16 d after postharvest in the fold change of these enzymes.

Answer: Thank you for your comment, and we modified the Results and Discussion. “the superoxide anion generation rate of postharvest fragrant pears gradually increased with storage time, which stimulated the increase of SOD enzyme activity, and the hydrogen peroxide produced by the metabolism of superoxide anion induced the activities of POD and CAT. ”

On the 16th day of shelf life, the respiration rate, superoxide anion production rate and hydrogen peroxide content of postharvest fragrant pears in the ozone treatment group reached their peaks, which were consistent with the peaks of SOD, POD and CAT enzyme activities, while the difference in protein expression levels of SOD2, SOD6, CAT2, and POD2 also reached the maximum fold. Li et al. also concluded that the decrease in postharvest ‘Yali’ pears quality was related to the decrease in the enzyme activities of SOD, CAT, and POD[29]. The gene expression of POD was stimulated in the postharvest ‘Zaosu’ and ‘Nanguo’ pears to maintain high quality during storage time[26,30].

We tried our best to improve the manuscript and made some changes in the manuscript. We appreciate your warm work earnestly and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The work was enhanced and the authors addressed the queries.

Author Response

Dear review,

It's very kind of you for the comments that are helpful for our work.

Chen Cunkun

[email protected]

Reviewer 2 Report

The present version of the manuscript has been improved and I consider that it is appropriate for its publication in Horticulturae. All the mayor amendments were answered, but there is still one detail that should be corrected. Please add in the M&M section that the final concentration of 1 ppm of ozone was previously tasted and declare "data not shown". 

Author Response

Dear reviewer,

Thank you for your comment, and we added the details in Line 101-102.

Chen Cunkun

[email protected]