Dynamic Analysis of the Bacterial Community and Determination of Antioxidant Capacity during the Fermentation of Sour Tea
and Xiaoran Li 1,*,‡Round 1
Reviewer 1 Report
The manuscript describes the processing of sour tea and the organisms present in the fermentation and the changes in antioxidants present. The introduction covers the contribution of lactic acid bacteria to antioxidants and their presence in tea fermentation. Methods are described adequately. Overall the paper is well written and clear.
Particular points:
1. It would be good to include a comment on the approximate temperature of the fermentation over the time period as well as the pH, which is shown.
2. Figure 2 does not show very large changes in these components during fermentation.
3. Figure 3 shows a significant increase in antioxidant activity during the fermentation. In the top figure of figure 3 it would be better to explain the DPPH clearance and the orange and blue bars more completely in the legend for the reader to more easily be aware of the result shown.
4. In figure 4d, it is stated the microbial richness of the sour tea did not change much during the fermentation. This term “microbial richness” should be defined and further explanation of this point and its impact should be elaborated further.
5. Figure 5 the 60 and 70 time point samples seem to be quite different in composition than those of the surrounding time points. Some comment on this could be included.
6. Figure 6 is very interesting and well presented. It might be useful to include further comment on the proportions and sources of the component bacteria at zero time point, and compare that community to that community found in other tea samples or similar tea related samples in more detail.
Author Response
Response to Reviewer 1 Comments:
Dear Editor,
Thank you very much for handling our manuscript. The comments from you and referees are very helpful. According to your requests, we tried our best to improve the manuscript, and all of the points brought up by reviewer 1 were addressed and discussed point-by-point below. The changes performed in the modified version of the manuscript are highlighted. With these changes, we hope that we have sufficiently improved the manuscript for acceptance by fermentation.
Yours sincerely,
Xiao-Ran Li
College of Life Science and Technology
Kunming University of Science and Technology
Reviewer #1: The manuscript describes the processing of sour tea and the organisms present in the fermentation and the changes in antioxidants present. The introduction covers the contribution of lactic acid bacteria to antioxidants and their presence in tea fermentation. Methods are described adequately. Overall the paper is well written and clear.
In view of the authors very thorough responses to the prior criticisms, I would encourage several minor language revisions:
Point 1: It would be good to include a comment on the approximate temperature of the fermentation over the time period as well as the pH, which is shown.
Response 1: Thank you for your suggestion. I added a temperature change line chart in Figure 2. “The temperature of fermentation environment is detected during fermentation (Fig. 2a).”(Lines 188-189)
Point 2: Figure 2 does not show very large changes in these components during fermentation.
Response 2: Thank you for your suggestion. I can better show the changes in the fermentation process by narrowing the ordinate range.
Point 3: Figure 3 shows a significant increase in antioxidant activity during the fermentation. In the top figure of figure 3 it would be better to explain the DPPH clearance and the orange and blue bars more completely in the legend for the reader to more easily be aware of the result shown.
Response 3: Thank you for your suggestion. The legend in Figure 3 has been changed. (a) DPPH free radical scavenging rate of 24 strains of Lb. plantarum isolated from sour tea. Vc (0.8 mg/ml) was the positive control. Orange represents the bacterial total of 109 CFU, and blue represents the bacterial total of 1010 CFU. (b) Free radical scavenging rate of sour tea fermented at different times.(Lines 223-224)
Point 4: In figure 4d, it is stated the microbial richness of the sour tea did not change much during the fermentation. This term “microbial richness” should be defined and further explanation of this point and its impact should be elaborated further.
Response 4: Thank you for your suggestion. The microbial richness was expressed by number of OTUs. In addition, according to Figure 4d, the observed features index did not change significantly. There was no sig-nificant change in the observed features index, which may be due to the difference between the algorithm and other indexes (Lines 235-239)
- Hall, M.;Beiko, R. G. 16S rRNA Gene Analysis with QIIME2. Methods in molecular biology (Clifton, N.J.) 2018, 1849, 113-129, doi:10.1007/978-1-4939-8728-3_8.
Point 5. Figure 5 the 60 and 70 time point samples seem to be quite different in composition than those of the surrounding time points. Some comment on this could be included.
Response 5: Thank you for your suggestion. “On days 60 and 70 of fermentation,the abnormal proportion of Lactiplantibacillus may be due to the change of fermentation environment (take sour tea out of the pit for post-fermentation).” (Lines 258-260)
Point 6. Figure 6 is very interesting and well presented. It might be useful to include further comment on the proportions and sources of the component bacteria at zero time point, and compare that community to that community found in other tea samples or similar tea related samples in more detail.
Response 6: Thank you for your suggestion. “Although tea was pretreated before fermentation, some trace bacteria will still be retained. This may be one of the possible sources of Lb. plantarum in the fermentation process. ”(Lines 274-277) In the 0 day sour tea samples, roseomonas and sphingomonas account for the highest proportion. They are very common bacteria in the environment. This is the same as most fermented tea [33].
Li, Q.;Chai, S.;Li, Y. D.;Huang, J. A.;Luo, Y.;Xiao, L. Z. H. Biochemical Components Associated With Microbial Community Shift During the Pile-Fermentation of Primary Dark Tea. Front Microbiol 2018, 9, 1509.
Please see the attachment.
Author Response File: 
Author Response.doc
Reviewer 2 Report
Dear authors, in my opinion this study is interesting and different data were collected. However, the explanation of the methodology is not clear thus requiring a strong improvement to support results and discussion sections
Please check the method for catechin quantification in lines 111-116. Why there are two solvents in isocratic elution? Why organic acids if the evaluation was focused on catechins?
Please add references for polyphenols and caffeine detections
Please describe better the amminoacids analysis. What the sample is in this analysis (tea? extract?)
Please explain better what the water exrtact represents. Is it the difference between 2g and the dry residue?
Please add references for radical scavanging activity
Please add the statistical evaluation of all the results in the manuscript
Please add description of culture dependent microbiological characterization. How was Lb plantarum identified? Use Lactiplantibacillus plantarum istead of Latilactobacillus plantarum
Lb. plantarum was singly inoculated? In the introduction there is the description of the antioxidant properties of active LAB? why did you evaluate a cell free extract? Was the extract the tea or MRS broth?
In figure 7b please describe better the representation in the three graphs and the differences. What are 7b-1, 7b-2 and 7b-3 representing?
Author Response
Response to Reviewer 2 Comments:
Dear Editor,
Thank you very much for handling our manuscript. The comments from you and referees are very helpful. According to your requests, we tried our best to improve the manuscript, and all of the points brought up by reviewer 2 were addressed and discussed point-by-point below. The changes performed in the modified version of the manuscript are highlighted. With these changes, we hope that we have sufficiently improved the manuscript for acceptance by fermentation.
Yours sincerely,
Xiao-Ran Li
College of Life Science and Technology
Kunming University of Science and Technology
Reviewer #2: Dear authors, in my opinion this study is interesting and different data were collected. However, the explanation of the methodology is not clear thus requiring a strong improvement to support results and discussion sections:
Point 1: Please check the method for catechin quantification in lines 111-116. Why there are two solvents in isocratic elution? Why organic acids if the evaluation was focused on catechins?
Response 1: Thank you for your suggestion. I modified lines 111-116. The two solutions are used to ensure that all catechins are obtained “Catechin was monitored at 278 nm and its concentration was determined by integrating the calibration curve obtained from the standard.”
Point 2: Please add references for polyphenols and caffeine detections.
Response 2: Thank you for your suggestion. The identification method of tea polyphenols is from the Chinese national standard ISO 14502-1:2005. The caffeine identification method is from the Chinese national standard ISO 10727:1995.
Point 3: Please describe better the amminoacids analysis. What the sample is in this analysis (tea? extract?)
Response 3: Thank you for your suggestion. “The tea sample (3 g) was ground, soaked in 450 mL boiling water for 45 min, and then filtered. The sample was mixed with 0.5 mL phosphate buffer solution (pH 8) and 0.5 ml 2% ninhydrin solution. The mixture was heated in boiling water for 15 min and then cooled, and then the volume was stabilized to 25 mL with water. The absorbance of the mixed solution was measured at 570 nm after it was left at room temperature for 10 min.”(Lines 124-126)
Point 4: Please explain better what the water exrtact represents. Is it the difference between 2g and the dry residue?
Response 4: Thank you for your suggestion. Water extract refers to the substance that can be dissolved in water after tea is soaked in water. The weight of tea residue is the weight after removing the water extract.
Point 5 Please add references for radical scavanging activity.
Response 5: Thank you for your suggestion. I have added references on lines 171,177 and 184.
- Cao, Z. H.;Pan, H. B.;Li, S. J.;Shi, C. Y.;Wang, S. F.;Wang, F. Y.;Ye, P. F.;Jia, J. J.;Ge, C. R.;Lin, Q. Y.; et al. In Vitro Evaluation of Probiotic Potential of Lactic Acid Bacteria Isolated from Yunnan De'ang Pickled Tea. Probiotics and Antimicrobial Proteins 2019, 11, 103-112, doi:10.1007/s12602-018-9395-x.
- Smirnoff, N.;Cumbes, Q. J. Hyroxyl radical scavenging activity of compatible soluts. Phytochemistry 1989, 28(4): 1057-1060.
- Fernandez, C.;Kalpowitz, N.;Garcia, R. C.;Colell, A.;Miranda, M.;Mari, M.;Ardite, E.;Morales, A. GSH transport in mito-chondria: defense against TNF-induced oxidative stress and alcohol-induced defect. The American journal of physiology 1997, 273: 17-271
Point 6:Please add the statistical evaluation of all the results in the manuscript.
Response 6: Thank you for your suggestion. “The total amount of water extract gradually increased at the beginning of fermentation, and then decreased until Day 55 (P < 0.01). The contents of polyphenols and catechins increased significantly at the beginning of fermentation and gradually increased with the fermentation process (P < 0.01).” (Lines 191-193) “It was found that the highest DPPH clearance rate of Lb. plantarum 75-2-2 was 73.25% (73.25 ± 3.41), which was similar to that of Vc (0.8 mg/mL; 72.49% ± 1.34).” “It was found that the scavenging rate of hydroxyl radicals and superoxide anions increased with increasing fermentation time (P < 0.01).”(Lines 209-217)
Point 7:Please add description of culture dependent microbiological characterization. How was Lb plantarum identified? Use Lactiplantibacillus plantarum istead of Latilactobacillus plantarum.
Thank you for your suggestion. Lb plantarum strains were isolated and identified according to morphology, catalase test, Gram staining, and 16S rDNA sequences. I have replaced Lactiplantibacillus plantarum with Latilactobacillus plantarum. （Lines 22,53）
Point 8 Lb. plantarum was singly inoculated? In the introduction there is the description of the antioxidant properties of active LAB? why did you evaluate a cell free extract? Was the extract the tea or MRS broth?
Response 8: Thank you for your suggestion. Lb. plantarum was singly inoculated. According to Reference 11, the free radical scavenging rate of cell-free culture supernatant of Lb. plantarum is higher than that of cell-free extracts and intact cells. The cell-free culture supernatant of bacteria is to wash the bacteria with physiological saline, and then break them up and centrifuge.
Point 9 In figure 7b please describe better the representation in the three graphs and the differences. What are 7b-1, 7b-2 and 7b-3 representing?
Response 9: Thank you for your suggestion. “For PCoA, based on the unweighted UniFrac distances, the first principal components accounted for 47% of the total variance, which covered most of the variable information. As shown in Fig. 7b, The microbial communities of the three groups of samples were separated from each other, and different fermentation time had a major impact on the bacteria. The larger the difference in fermentation time of samples, the greater the difference in microbial communities.”(Lines 301-304)
