Selenium Biofortification of Three Wild Species, Rumex acetosa L., Plantago coronopus L., and Portulaca oleracea L., Grown as Microgreens

Round 1

Reviewer 1 Report

This manuscript presents the investigation of Selenium biofortification of three wild species, grown as microgreens. Overall, the manuscript is an interesting contribution to scientific knowledge in the field of micronutrient biofortification of green vegetables to provide health benefits due to their nutraceutical value.

The manuscript fits within the scope of the journal Agronomy. Nevertheless, several problems/doubts should be solved before the manuscript is suitable to be published.

1. Introduction Section:

1.1. From Introduction it is not really clear whether there is a problem of selenium deficiency in th world (or in some regions) and whether it is important to increase its amount in food. How harmful to health is the high selenium content in vegetables caused by supplementation of plants with selenium? Please add the appropriate information.

1.2. Another important question, which the authors do not properly explain in the Introduction, is why these three wild species were chosen for the study? What nutritional properties do they have? What biologically active components do they contain? Have there been any previous studies on growing them as microgreens? Are they suitable for this? Please add the relevant information and references.

2. Materials and Methods Section:

2.1. Please describe more in details the seeds of Rumex acetosa, Plantago coronopus and Portulaca oleracea used in experiment. Were the seeds purchased or collected by authors? If the seeds were collected how they were stored and prepared for experiments? Was the germination test carried out before the experiments?

2.2. L99-101: Please add the name of analytical method used for selenium determination. This will allow the reader to assess the reliability of the analysis results without referring to the standard UNI EN ISO 17294-2:2016.

3. Results and discussion:

3.1. I suggest to change the names of Subsections 3.1 and 3.2 so that the reader would understand exactly what results are described in them.

3.2. L158-162: Since the figure shows the Se concentration in mg per kg dry weight, it is difficult to understand how the indicated EDI values were calculated. Please give also in the text of manuscript the concentration of selenium per kg (or g) fresh weight.

3.3. How was the health risk index (HRI) calculated? The tolerable upper intake level (UL) is 300 μg day^-1kg^-1. Did you calculate UL for average human weight?

3.4. L224-228, Figure 2: Why did authors use only three concentration of selenium (1.5, 5, and 10 mg L^-1)? Three points are not enough to make any conclusions about the relationship between selenium supplementation and Se accumulation in plant. With a larger number of points, including in the interval between 1.5 and 10, the equation might not be second-order polynomial quadratic. For more accurate interpretation of these data it will be also useful to calculate selenium content (μg Se per plant). On Figure 2 the selenium concentration in plants on Y-axis is given and not content. Additionally, the presented results about the regression of Se concentration in P. oleracea and Se concentration in the substrate are not discussed. Is it typical for micronutrients non-linear (second-order polynomial quadratic) regression between concentration in substrate and in plants? Or is it specific for selenium only? Or it depends on plant species? Please add appropriate information.

3.5. Please check the name of Y-axix on Figure 4a, e.g. μmol di Fe(II) g^-1 FW. What you mean under “di”?

Author Response

This manuscript presents the investigation of Selenium biofortification of three wild species, grown as microgreens. Overall, the manuscript is an interesting contribution to scientific knowledge in the field of micronutrient biofortification of green vegetables to provide health benefits due to their nutraceutical value.

The manuscript fits within the scope of the journal Agronomy. Nevertheless, several problems/doubts should be solved before the manuscript is suitable to be published.

1. Introduction Section:

1.1. From Introduction it is not really clear whether there is a problem of selenium deficiency in th world (or in some regions) and whether it is important to increase its amount in food. How harmful to health is the high selenium content in vegetables caused by supplementation of plants with selenium? Please add the appropriate information.

Text has been improved.

1.2. Another important question, which the authors do not properly explain in the Introduction, is why these three wild species were chosen for the study? What nutritional properties do they have? What biologically active components do they contain? Have there been any previous studies on growing them as microgreens? Are they suitable for this? Please add the relevant information and references.

Text has been improved.

2. Materials and Methods Section:

2.1. Please describe more in details the seeds of Rumex acetosa, Plantago coronopus and Portulaca oleracea used in experiment. Were the seeds purchased or collected by authors? If the seeds were collected how they were stored and prepared for experiments? Was the germination test carried out before the experiments?

Text has been improved.

The seed germinability was tested by the seed company.

2.2. L99-101: Please add the name of analytical method used for selenium determination. This will allow the reader to assess the reliability of the analysis results without referring to the standard UNI EN ISO 17294-2:2016.

The names of the methods have been added in the references.

3. Results and discussion:

3.1. I suggest to change the names of Subsections 3.1 and 3.2 so that the reader would understand exactly what results are described in them.

Text has been improved.

3.2. L158-162: Since the figure shows the Se concentration in mg per kg dry weight, it is difficult to understand how the indicated EDI values were calculated. Please give also in the text of manuscript the concentration of selenium per kg (or g) fresh weight.

Text has been improved.

3.3. How was the health risk index (HRI) calculated? The tolerable upper intake level (UL) is 300 μg day^-1kg^-1. Did you calculate UL for average human weight?

We erroneously wrote 300 µg day^-1kg^-1 rather than 300 µg day^-1. We apologize for the mistake. Text has been corrected.

3.4. L224-228, Figure 2: Why did authors use only three concentration of selenium (1.5, 5, and 10 mg L^-1)? Three points are not enough to make any conclusions about the relationship between selenium supplementation and Se accumulation in plant. With a larger number of points, including in the interval between 1.5 and 10, the equation might not be second-order polynomial quadratic. For more accurate interpretation of these data it will be also useful to calculate selenium content (μg Se per plant). On Figure 2 the selenium concentration in plants on Y-axis is given and not content.

The authors thank the reviewer for her/his observation. The study aimed to preliminarily investigate the possible relationship between the Se concentration applied and the maximum concentration suitable for the biofortification of microgreens. Further studies should be carried out using a higher number of Se-treatments and, possibly, different species of wild edible plants, to deeply investigate the mechanism of Se uptake in the microgreens of wild species. Our preliminary results indicated that 10 mg Se L^-1, and higher concentrations, are not suitable for microgreens of P. oleracea, and it would be interesting to test different concentrations in the range 1-2 mg Se L^-1.
The plant Se content (ng plant^-1) has been calculated and the results have been added to the text.

Additionally, the presented results about the regression of Se concentration in P. oleracea and Se concentration in the substrate are not discussed. Is it typical for micronutrients non-linear (second-order polynomial quadratic) regression between concentration in substrate and in plants? Or is it specific for selenium only? Or it depends on plant species? Please add appropriate information.

The discussion of the results about the regression has been improved.

3.5. Please check the name of Y-axix on Figure 4a, e.g. μmol di Fe(II) g^-1 FW. What you mean under “di”?

The figure has been corrected.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled „Selenium biofortification of three wild species, Rumex acetosa L., Plantago coronopus L. and Portulaca oleracea L., grown as  microgreens” shows the results of an interestingly planned experiments aimed into an assessment of the effect of selenium on the nutraceutical features of the microgreens based on the above three herb species. Additionally, the Authors of this work checked if there is a correlation between the increasing concentrations of Se added to the nutrient and the general content of Se in P. oleracea microgreen tissues. Next, they also assessed the potential impact of biofortified microgreens on Se demand in humans, as well as the possible health risks.

The Authors have obtained significant and important results which may be of interest to readers and other researchers involved into the issue of new vegetable products characterized by a high nutritional value, that at the same time ensure the sufficient consumption of selenium in the diet. It must be emphasized that in the Conclusions, the directions of the further studies were indicated.

For the above reasons I find this manuscript valuable and interesting, however I have a few small suggestions to it.

In the Introduction part, it would be recommended to supplement the information about the role of selenium in human health, and to refer not only to its positive role, but also to the negative effects resulting from either the deficiency or excess of the intake of this trace element. Also, please justify why between the three examined herb species, the Portulaca oleracea was chosen to search for the correlation between the increasing concentrations of Se added to the nutrient and the general content of Se in microgreen tissues.

Line 129-130: instead of `Taraxacus`, it should be `Taraxacum`

Line 381: it should be `Level on Vitamins`, it should be `Levels for Vitamins`  

Author Response

The manuscript entitled „Selenium biofortification of three wild species, Rumex acetosa L., Plantago coronopus L. and Portulaca oleracea L., grown as  microgreens” shows the results of an interestingly planned experiments aimed into an assessment of the effect of selenium on the nutraceutical features of the microgreens based on the above three herb species. Additionally, the Authors of this work checked if there is a correlation between the increasing concentrations of Se added to the nutrient and the general content of Se in P. oleracea microgreen tissues. Next, they also assessed the potential impact of biofortified microgreens on Se demand in humans, as well as the possible health risks.

The Authors have obtained significant and important results which may be of interest to readers and other researchers involved into the issue of new vegetable products characterized by a high nutritional value, that at the same time ensure the sufficient consumption of selenium in the diet. It must be emphasized that in the Conclusions, the directions of the further studies were indicated.

For the above reasons I find this manuscript valuable and interesting, however I have a few small suggestions to it.

In the Introduction part, it would be recommended to supplement the information about the role of selenium in human health, and to refer not only to its positive role, but also to the negative effects resulting from either the deficiency or excess of the intake of this trace element.

Test has been improved.

Also, please justify why between the three examined herb species, the Portulaca oleracea was chosen to search for the correlation between the increasing concentrations of Se added to the nutrient and the general content of Se in microgreen tissues.

Text has been improved.

Line 129-130: instead of `Taraxacus`, it should be `Taraxacum`

Text has been corrected.

Line 381: it should be `Level on Vitamins`, it should be `Levels for Vitamins`  

Text has been corrected.

Round 2

Reviewer 1 Report

The authors improved the manuscript well. I am satisfied with authors' responses to comments.