Changes in Microstructure, Germination, Sprout Growth, Phytochemical and Microbial Quality of Ultrasonication Treated Adzuki Bean Seeds

Round 1

Reviewer 1 Report

This manuscript entitled: "Changes in microstructure, germination, sprout growth, phytochemical and microbial quality of ultrasonication treated adzuki bean seeds" is an article that contributes to the investigation of the improvement of germination and microbial control of adzuki bean sprouts, using an ultrasonication treatment.  The following considerations need to be taken into account:

 

• Throughout the manuscript but especially during the abstract on several occasions there is redundant information, specifically with the ultrasonication treatment. The values that have been used in this treatment are detailed on several occasions during the abstract, including successive sentences. It would be convenient to restructure the abstract to avoid this and have a clearer and more direct message.

 

• It is necessary to correct a formatting problem that exists throughout the manuscript. Each period is preceded by a "\t" instead of space.

 

• Line 34: The phrase “Sprouts are the product…” would be convenient to refer to the sprouts of the species that is being studied in this article. Since not specifying it leads to understanding that all sprouts are edible.

 

• It would be interesting to introduce the species' scientific name in their first appearance in the manuscript, as in the case of mungbean, pea, etc.

 

• Line 84: fix the formatting error causing the paragraphs to be joined.

 

• To avoid possible confusion, I would recommend unifying the nomenclature used for α-amylase / alpha-amylase.

 

• From lines 113 to 119 it would be necessary to order the references.

 

• Line 268: remove the color from “(Table 2)”.

 

• Specify in the figure what the 4 arrows show on the data from the time scale.

 

• Correct the underlines and colors of references number 9 and 21.

Author Response

Thanks for your recommendations.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript describes the potential of ultrasonication treatments to improve water uptake and germination speed from adzuki beans and analyses the mechanisms limiting water uptake in comparing tow varieties with different seed coat structure. These data are interesting to be published! Next to that the author describes results of measurements of alpha-amylase activity, which can purely be explained by the faster water uptake and germination, although the author reason differently. Subsequently the author reports on measurements of phenolic compounds, flavonoid, saponin and anti-oxidant levels, without any indication of a relation between the different treatments, nor any suggestion why the treatments should influence this. The effect on microbial load has been reported before by the author.

I suggest to rewrite the manuscript (a short communication?), limiting to describing the effect on the speed of water uptake, germination and earlier yield, supported by the electron microscopy images. This will give a much better and interesting publication. However before that the manuscript needs also a thorough editing step with correction of a large number of grammatical and typing errors.

Here more detailed comments comments.

• Line 77 seed are stored at 19% MC, that is unusually high to maintain seed quality. Calculating the equilibrium relative humidity (http://data.kew.org/sid/viability/mc1.jsp?oil=0.40&drytemp=5&equib=81&constid=24128) shows that this is storage at around 81% RH, which are poor seed storage conditions. Nothing is mentioned how long the seeds were stored under those conditions. Seed viability data (frequency of living seeds) are lacking.
• Germination curves are lacking in the figures, but this may be compensated by the MGT data
• Line 85. How many replicates were used? Three as mentioned on line 128?
• Line 88. Why using a self-citation for the calculation of mean germination time (MGT). Mean Emergence Time and the formula was introduced by Ellis & Roberts in 1980 (Ellis, R.H. and Roberts, E.H. (1980). Towards a rational basis for testing seed quality. In Seed Production, (ed. P.D. Hebblethwaite), pp. 605-635, Butterworths, London) and Matthews & Hosseini used that formula principle in 2006 to calculate Mean Germination Time (Matthews, S., and Mohammad Khajeh Hosseini. "Mean germination time as an indicator of emergence performance in soil of seed lots of maize (Zea mays)." Seed Science and Technology 34.2: 339-347).
• Line 110. How were the seeds dried? Freeze dried? Under nitrogen gas? This seems to me essential for determination of residual anti-oxidant capacity (line 113).
• Line 128. Does the randomised block design also hold for the sonication treatments themselves? Meaning three series of treatments in a randomised order? Was in the germination, water uptake and other tests each replicate derived from such an independent treatment?
• Line 140. A sigmoidal water uptake curve is very typical for all seeds with a water permeable seed coat. See for instance the seed biology handbook: Bewley JD, Bradford KJ, Hilhorst HWM, Nonogaki H (2013) Seeds: Physiology of development, germination and dormancy. 3rd Edition edn. Springer, New York.
• Line 145. Compliments for this combined observation, explaining the differences in water uptake speed by the testa structure.
• Figure 1. It is not mentioned in the legend what the arrows point at.
• Figure 2. The magnification will depend on the editing of the publication and on screen display with the electronic version. At the bottom it is written 100 µm but it is not clear if that refers to the distance between the single lines/dots. Better to add a size bar in the picture. It is a pity that picture B is rather dark, can the exposure and contrast be improved in post-processing. I suggest to indicate in the figure with names from the different morphological layers.
• Line 200. The sonication results in a faster water uptake and faster mean germination time. As a consequence of the latter, it is very logic that the germination related processes as alfa-amylase activity are also initiated at a shorter time. Therefore I do not see the ratio for measuring this. Can the author explain the reason?
  Moreover, the author suggest that the faster alfa-amylase activity might be “caused by the ultrasound-induced starch granules damages”. It is common biological knowledge that alpha-amylase activity cleaves starch and aids in ‘digesting’ the starch granules. Earlier and higher activity will result in faster digestion.
• Line 209. Isn’t it too logic that 2 days faster germination results in faster sprout growth, of which the hypocotyl and roots form the major parts (line 210). However, I understand that the faster sprout growth and faster yield are interesting for practical application.
• Line 224 – 235. To me there is no clear effect on the polyphenols and flavonoids. Why did the author expect that? Why is it interesting to mention? I think only if the author can explain a potential relation with the treatments. Moreover, I disagree with the author about significant effects. Indeed there are significant differences between samples, but there seems no relation with the different sonication treatments. Yes significant differences, but no significant effects. I’m no convinced that these results can consequently be reproduced when repeated several times.
• The same holds for the saponins and for the anti-oxidant levels
• Table 3 and line 274-293. How much has this not already been reported in the previous publication by the author [#7]?
• What is more relevant for the reader and the application is that there seems an optimum in the frequency of the sonication treatment. I expect also in the duration, but that has not been analysed apparently. Is there is some damaging effect when the frequency is too high or duration too long? Why has this not been tested?

 

Author Response

I appreciated the valuable suggestions made by reviewer, and some suggestions were added to the manuscript. However, I still hope the said manuscript can be submitted for publishing as a full length article.  Therefore I did not re-write it.

Author Response File: Author Response.docx