Improving Phenolic-Linked Antioxidant, Antihyperglycemic and Antibacterial Properties of Emmer and Conventional Wheat Using Beneficial Lactic Acid Bacteria
Round 1
Reviewer 1 Report
Overall, the manuscript is well written and organized. But some questions need to be considered:
Comments
- The different letters indicated statistically significant are very confusing. Please describe them in detail.
- The concentrations of extracts used for biological function assay are important for evaluating the feasibility of in vivo experiments. In the manuscript, the authors only provided the volumes of the compounds.
- There should be positive controls for antioxidant and anti-hyperglycemic properties assay.
Author Response
Reviewer 1:
Comment 1: Overall, the manuscript is well written and organized. But some questions need to be considered:
Response: We want to thank the reviewer for his encouraging comment and suggestions to further improve the manuscript. We have addressed all comments made by the reviewer and incorporated changes in the revised manuscript.
Comment 2: The different letters indicated statistically significant are very confusing. Please describe them in detail.
Response: Upper case letters indicating statistical significance in Figures 1, 2a and 2b has been changed to lowercase letters to maintain uniformity with Tables 1, 2 and 3. In Figure 2a, we have used brackets to show letters indicating statistically significant differences between fermentation time points (significant differences between main effect only for the ABTS based antioxidant activity, as interaction did not have statistically significant effect). For all other biochemical parameters, Tukey’s test revealed statistically significant effect of interactions between sample extract × fermentation time points. Therefore, in the figures and tables, we presented statistically significant differences between sample extract × fermentation time point interactions with different lowercase letters. We have explained the significance of statistical analysis and results in the Results and Discussion section of the revised manuscript.
Comment 3: The concentrations of extracts used for biological function assay are important for evaluating the feasibility of in vivo experiments. In the manuscript, the authors only provided the volumes of the compounds.
Response: In this study, we measured and presented results of the total phenolic content and concentration of the individual phenolic compounds based on total dry weight of the sample (wheat extracts) (Figure 1 & Table 1). The optimized concentration of the sample based on the phenolic content can be targeted in the future in vitro and in vivo experiments for antioxidant and anti-hyperglycemic benefits. The concentration of the wheat sample (20 g in 50 mL or 0.4 mg/mL), which was fermented with LAB was included in the “preparation of the wheat extracts” section of the Materials and Methods. We have included the concentration of wheat extracts in abstract and figure captions. The same concentration of wheat extracts following LAB fermentation (72 h) also showed inhibitory activity against Helicobacter pylori.
Comment 4: There should be positive controls for antioxidant and anti-hyperglycemic properties assay.
Response: We have included Trolox as positive control for antioxidant activity and Acarbose as positive control for α-amylase and α-glucosidase inhibitory activities. The results of the positive control closely matching the ABTS, DPPH, α-amylase and α-glucosidase inhibitory activities are included in respective figures and tables.
Reviewer 2 Report
Fermentation technology has been used as an effective bioprocessing strategy for many years, the author in the paper used LAB based fermentation and describe simple and effective to study various antioxidant, antimicrobial, prebiotic and carbohydrase’s like amylase, glucosidase activity, but authors need to revisit the study using different varieties Emmer .
The antimicrobial against H. Pylori is intriguing, and author need to discuss detail molecular mechanism of this bacterial inhibition.
It would be nice if FPLC profile of Phenolic compound were added to results section.
Please label the legend according to manuscript policy, and include statistical comparisons on all figures, it seems that some of the unfermented and pH adjusted procedure did not show much difference
Author Response
Reviewer 2:
Comment 1: Fermentation technology has been used as an effective bioprocessing strategy for many years, the author in the paper used LAB based fermentation and describe simple and effective to study various antioxidant, antimicrobial, prebiotic and carbohydrase’s like amylase, glucosidase activity, but authors need to revisit the study using different varieties Emmer.
Response: We appreciate the comments made by the reviewer. We agree with the reviewer that inclusion and comparison of different varieties/accessions of Emmer wheat would provide better and clear results. However, we targeted organically grown ND Common Emmer wheat based on our previous optimization, which was published and included in the reference of the current manuscript “Christopher, A.; Sarkar, D.; Zwinger, S.; Shetty, K. Ethnic food perspective of North Dakota common emmer wheat and relevance for health benefits targeting type 2 diabetes. J. Ethn. Foods 2018, 5, 66-74. https://doi.org/10.1016/j.jef.2018.01.002”.
We continued this fermentation study with same optimized emmer wheat source for consistency (different varieties and sources would lead to higher variability in bioactive content and profile) and to investigate potential improvement in phenolic bioactive-linked functionalities with LAB fermentation. We will definitely consider other Emmer wheat varieties for future investigations. This is an excellent suggestion of the reviewer as there will be regional and climate response variations of Emmer across the globe and we will follow this up for further studies.
Comment 2: The antimicrobial against H. pylori is intriguing, and author need to discuss detail molecular mechanism of this bacterial inhibition.
Response: We have revised the section on antimicrobial activity and have included information on the potential mechanism of antimicrobial activity of wheat phenolics and their possible synergistic action against Helicobacter pylori. We also explained the possible antibacterial activity of LAB fermented wheat extracts under low pH and due to the production of weak organic acid such as lactic acid.
Comment 3: It would be nice if HPLC profile of Phenolic compound were added to results section.
Response: We have included a figure (Figure 2) with the chromatogram of individual phenolic compounds detected in LAB fermented and unfermented wheat extracts. The concentration of individual phenolic compounds were also presented in the Table 1.
Comment 4: Please label the legend according to manuscript policy, and include statistical comparisons on all figures, it seems that some of the unfermented and pH adjusted procedure did not show much difference.
Response: We have revised the presentation of statistical significance results in the manuscript and improved the consistency of the description and presentation in all figures and tables. In Figure 2a, we have used brackets to show letters indicating statistically significant differences between fermentation time points (significant differences between main effect only for the ABTS based antioxidant activity, as interaction did not have any statistically significant effect). For all other biochemical parameters, Tukey’s test revealed statistically significant effect of interactions between sample extract × fermentation time points. Therefore, in the figures and tables, we presented statistically significant differences between sample extract × fermentation time point interactions with different lowercase letters. We have explained the significance of statistical analysis and results in the Results and Discussion section of the revised manuscript.
Round 2
Reviewer 1 Report
Thanks for the professional responses.
