PONE-D-22-08028Polyphenol and flavonoid content in major Teff [ Eragrostis tef (Zuccagni) Trotte r] varieties in Amhara Region, EthiopiaPLOS ONE

Dear Dr. Atlabachew,

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The paper needs careful revision. Authors are requested to respond to the individual comments of the reviewers. Please pay special attention to the statistical analysis

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper “Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region, Ethiopia” provided interesting information about the polyphenol content of different Teff samples and varieties in relation to their region of provenience. For this aim a suitable number of samples was collected and analyzed.

I found the paper very interesting, however I have also identified some limitations that will be listed below:

- In the abstract the following sentence was reported “Such a difference is also evident in consumers' preference for teff grains from one location”. Report and explain better this consideration in “Results and discussions” or “Conclusions” paragraphs.

- In the introduction could be interesting to better explain the nutritional characteristics of Teef compared to other cereals: “Teff is gluten-free, abundant in minerals, vital amino acids, protein, and carbohydrates than other grains.”

- Explain the meaning of TTeff the first time that appeared

- Three different varieties of Teff were cited (and also analyzed). If the literature are reported differences, in term of phenolic compounds, between the Teff varieties (white, brown, and red) you could report them in the Introduction

- At the end of the introduction are cited, for the study, only two white Teff varieties

“Thus, this study was intended to quantify the level of total phenolic and total flavonoid contents in Kuncho teff (DZ-CR-387) and Tseday teff (DZ-CR-37) varieties collected from 21 sub-districts of three zones (East Gojjam, West Gojjam, and Awi)”

However, in Sample Collection and Pre-treatment paragraph were reported four different varieties:

“Four varieties of TTeff, such as Kuncho (white teff), Tseday (white teff), Brown (mixed), and Red teff were collected from model farmers in January 2020.”

Correct this difference and better explain the meaning of mixed for Brown Teff.

- In this study the determination of total flavonoid content was conducted expressing the results as catechin or quercetin equivalents.

In materials and methods was specify that some flavonoids such as quercetin, rutin, and galangin prefer to form a complex with aluminium chloride in an acid medium, therefore this class of flavonoids, was estimated in acidic conditions. However, in the description of method the used of acid medium was not reported. Pękal and Pyrzynska (2014) reported that acid or acetate solution could be used but, in some cases, only methanol or water was added. Correct or better explain the incongruity between the theory and the method applied.

Pękal A, Pyrzynska K. Evaluation of aluminium complexation reaction for flavonoid content assay. Food Anal Methods. 2014;7(9):1776-82.

- Some contradictions were observed between the text (in the results paragraph) and Tables 1 and 2; for example:

“On the other hand, the phenolic content recorded in samples from the West Gojjam and East Gojjam zones were ranged between 46.2 -92.2 mg/100 g and 45.95-128.5 mg/100 g, respectively.” The two zones were inverted and the data “45.95” was not reported in the Table 1.

“Comparing the mean value of TPC in samples from the three administrative zones, the highest average value of polyphenols was noted in the teff samples from the West Gojjam zone (95.04 mg G.A.E./100 g), followed by the Awi zone (79.37 mg G.A.E./100 g), and East Gojjam zone (71.39 mg G.A.E./100 g) (Table 2).” The data 79.37 and 71.39 don’t correspond to values reported in table 2

Carefully check all results.

- The sentence: “The lowest and highest mean concentrations of TFC were found in teff samples from East Gojjam (15.45 mg Q.E./100g) and the Awi zone (113.12 mg Q.E./100g), respectively, based on the mean value of total flavonoids at each sampling zone.” The results reported are not the mean concentrations listed in Table 2.

- Explain better how authors have conclude that:

“This implies that brown or mixed teff variety (from Awi zone) contained a higher total phenolic content than Kuncho teff variety (from East Gojjam zone) and Tseday teff variety (from West Gojjam zone). “(paragraph of Results and Discussion)

“the phenolic content in different teff samples was obtained in Brown teff > Kuncho teff > Tseday teff > Red Teff.” (paragraph of conclusions)

Overall, the manuscript is relatively well written however there are some typing and punctuation errors. For example, in Table 2, the Standard Error of the mean (indicated as SEM in the text), was reported as SE in the table and ESE in the legend.

Reviewer #2: Ref. No.: PONE-D-22-08028

Title: Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter]

varieties in Amhara Region, Ethiopia

The manuscript deals with a topic that could be of interest limited to the comparison of the polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region (Ethiopia).

The manuscript in some parts lacks clarity and an accurate revision is mandatory.

I have some questions and comments listed below:

Abstract:

the abstract is well organized.

In the line 3: ‘ ‘ region’s, please remove symbols.

Makeup: make-up

Last sentence of the abstract could be improved: “ These two dimensions (PC1 and PC2) explained the 87.2% of the total variance of the dataset”.

Introduction:

the introduction section is very rich but sometimes not clear and not presented in a logical way. Please revise this part.

“Vital” amino acids: please replace with essential.

“Its flour is mainly used to make "injera," Ethiopians' favourite national dish”: this has been said before, it is a repetition. Please modify in the manuscript.

“light to dark brown”: please modify “light brown to dark brown”.

“Reports indicate that total flavonoids are determined using two approaches: flavonols and flavones are selectively determined at 410–430 nm by reacting the sample solutions with AlCl3 in acidic or acetate solution. In contrast, flavan-3-ols (catechins and others), luteolin, and rutin could selectively react with AlCl3 solution in sodium nitrite and alkaline conditions, and measurement was done at 510 nm [24].

However, to the best of our knowledge, we did not find reports comparing the level of flavonoids determined by the two approaches simultaneously, either in teff or other products. Instead, the former method is most frequently reported to determine the total flavonoid content in various products”: this part should not be included in the introduction. Please move it to the discussion section.

“As information obtained from Amhara Region's Bureau of Agriculture, about 42 improved varieties of teff have been registered in the country, but only a few are widely cultivated in the region. Different types of teff are produced in all region zones”: In my opinion, this sentence is not so clear. Please better explain in the text.

Materials and Methods:

Please improve the resolution of Figure 1. In the map shown at the top left the names are not clearly legible. The sampling administration districts highlight in green in the maps are not visible.

In “Chemicals, Reagents, and Instruments”: ”Cary 60” Agilent, not Carry.

The Wetted sample: please remove the capital letter.

(porta centrifuge, Japan): please add the specifications of the centrifuge used in the previous paragraph.

and reported as mg gallic acid equivalent (mg G.A.E./100 g) of samples.

In “ Determination of total phenolic content by Folin–Ciocalteu assay”: please modify last sentence: “and reported as mg gallic acid equivalent for 100 g of samples (mg G.A.E./100 g).

Before reference [24], please remove the round bracket.

In “Determination of total flavonoid content as catechin equivalent“: “All measurements were performed in triplicate readings”. Were no analytical replicas of the spectrophotometric test made?

In “Determination of total flavonoid content as quercetin equivalent“: “All measurements were performed in triplicate readings”. Were no analytical replicas of the spectrophotometric test made?

Were quartz cuvettes used for the spectrophotometric readings? with which optical path? Please detail in the manuscript.

Statistical data analysis: Please express the acronyms TPC and TFC, TFC-QE first in the text.

Results and Discussion:

Figure 3: Concerning the calibration curve of catechin, the range of linearity was too short (abs from 0.00 to 0.2). Why were not considered standard solutions with higher concentration?

“The total phenolic content (TPC) is recorded as a milligram of gallic acid equivalent per 100 g of teff sample (mg of G.A.E./100 g teff). In addition, the catechin-like and quercetin-like total flavonoid contents are reported as milligram of catechin equivalent per 100 g of teff sample (mg of C.E./100 g) and milligram of quercetin equivalent per 100 g of teff sample (mg of Q.E./100 g teff) respectively”: this has already been said in materials and methods. Please remove it from Results and Discussion section.

Table1: please improve the formatting of table 1.

Figure 5 and Table 2 gives us the same information. Please remove Table 2 from the text (it could be added as supplementary materials). In Figure 5 please add error bars.

In the Discussion, Authors said that “The ANOVA test confirmed significant variations (P < 0.05) in its phenolic content among the 72 teff samples”. Where are reported the ANOVA results?

Table 3 is not clear. Please better explain in the text. Moreover, Principal component analysis has been applied but is not explain (in material and method section) and is not adequately discussed. This part needs to be improved.

Please check all the manuscript because there are several formatting errors. Please proofread the English, too.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-08028R1Polyphenol and flavonoid content in major Teff [ Eragrostis tef (Zuccagni) Trotte r] varieties in Amhara Region, EthiopiaPLOS ONE

Dear Dr. Minaleshewa Atlabachew

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by July 15, 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Patrizia Restani, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

The paper has improved and now requires only minor changes of the text.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper “Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region, Ethiopia” was improve by authors and all the suggestions were amended.

The paper is very interesting; however, some little corrections could be done:

- some little typing and punctuation errors were still observed (e.g. in the tile and in the test be lacking spaces);

- In the Introduction replace “It's a chasmogamous C4 annual cereal with a fibrous…” with “Teff is a chasmogamous C4 annual…”;

- In the Results and Discussion, the Figure 1 on gallic acid results is the Figure 2;

- In the Results and Discussion, I suggest to modify the following sentence “This implies that brown or mixed teff variety (from Awi zone) contained a higher total phenolic content than Kuncho teff variety (from East Gojjam and West Gojjam zone) and Tseday teff variety (from Awi zone).” Some samples from West Gojjam zone have an high TPC, indeed the mean value of TPC and TFC-Q.E were found to be the highest in West Gojjam teff samples compared to the samples from other zones.

Among varieties from Awi zone, the brown samples showed the highest TPC. Similar comments could be also arranged for the TFC.

- In the Results and Discussion, in the sentence “On the other hand, relatively comparable TPC values with the present study were reported by [14] for white and brown teff varieties collected from the Tigray region of Ethiopia.” could be interest to specify the values reported by the reference authors.

- Table 3 must be insert in the text as a Figure or as a Table?

Reviewer #2: Authors responded to comments by significantly improving the quality of the manuscript. In my opinion, the work in this formcan be accepted for its publication in Plos One.

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Reviewer #1: No

Reviewer #2: No

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Polyphenol and flavonoid content in major Teff [ Eragrostis tef (Zuccagni) Trotte r] varieties in Amhara Region, Ethiopia

PONE-D-22-08028R2

Dear Dr. Atlabachew

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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