Identification of Seasonal Honey Based on Quantitative Detection of Typical Pollen DNA

Round 1

Reviewer 1 Report

Comments for author File: Comments.docx

Author Response

There was no question from reviewer 1

Reviewer 2 Report

The paper presents a methodology based on the quantification of DNA to authenticate some monofloral honey types produced in South Korea. It is understandable and it is well presented, however, some major concerns should be addressed before publication.

Main comments

In general, the document is excessively summarised and there is a lack of information that could be useful for a better understanding of the work. Many sentences drift to citations necessary information. Therefore, to improve the document I suggest to expand more some paragraphs including more details. Concretely,

Line 39-40: “However, this method must be conducted by a botanical specialist and is time-consuming”.

All the analytical procedures need specialists and are time-consuming, so the authors should justify this statement by indicating, for example, how much more time is needed to perform a pollen analysis using palynology in comparison to DNA quantification or, for example, whether DNA quantification can be performed by an untrained analyst. Also the cost of both types of analysis.

Line 41-43: “Detection of specific plant species based on the DNA that is isolated from the pollen in honey is considered as a reliable method for the accurate determination of honey’s origin”

Please explain a bit more the advantages to use this methodology instead of other methods. Also, it would very useful if the authors describe the main methods developed for DNA isolation from pollen in honey.

The paper is focused on four unifloral honey types produced in South Korea, due to the scarce international scientific literature about honey from this country, it would be very interesting for the readers to include a paragraph detailing principal honey types produced in the country or the main plant resources for honeybees. Also, a small description of the honey types used in this work, are they representative of the honey market in South Korea or why the authors choose these honey types, for example.

Line 50. “Selection of major nectar plants”, “The predominant seasonal nectar plants in South Korea were selected for their quantitative detection in the honey samples collected between April and July”

The sentence is a bit confusing as it does not explain how the species in the samples have been quantified. Presumably, the sampling period should be established before the quantification of DNA in samples (results of this paper). In one hand, if the samples have been collected in this period because it is the main period for honey production in the area, it should be clearly indicated. On the other hand, if the period has been established because it coincides with the flowering period of the target species, it should also be clearly indicated.

The main flowering plants important for honey production which blooms in the same period that target taxa can affect honey characteristics even DNA content. Therefore, a short description of these plants in the area where the honey samples were produced is recommended, as well as the predominant species of Prunus, Castanea and Kalopanax.

Line 56 and subsequent. Honey samples harvest were collected in each hive. It must be indicated how it has been done, pressing honey hives by centrifugation or how. It is important because it affects pollen content in honey samples.

Line 72, please explain a bit more the following steps.

Table 2. Explain or change the names of the second column.

Line 110, maybe 29.1, 2.83, 28.4 and 27.2 copies?

Line 122-125, This statement should be accompanied by information on the main flowering period of each species and it should be stated beforehand how this period has been determined.

Line 145-146, To confirm similarities and dissimilarities between samples the use of a statistical test is appreciated. In Aralia samples the quantity of Kalopanax seems to be less.

Regarding the last comment and considering that different honey types tend to have different pollen content, as the authors indicate in lines 181-182, this work should be accompanied by a palynological analysis to establish the pollen spectrum of the samples and quantitative relationships with the results found by DNA quantification.

Lines 168-169, this statement is not supported by the results as there is no information about the period of blooming.

Discussion should enlarge including information about the characteristics of these kinds of honey, the period of blooming of the main species, species that are in bloom together. Furthermore, the usefulness of the proposed methodology should justify to explain statements in lines 178-180: “Therefore, the older accumulated pollens could be remained in the hive for a long time. This accumulation leads to the difficulty of monofloral honey identification by melissopalynology method”

This is due to it is expected that this pollen content affects also DNA quantification.

I hope my comments help authors to improve their work.

Author Response

The answers to reviewer 2 were included the attached file.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

The presented manuscript aimed to develop a method to authenticate the origin of monofloral honey and the season during which it was produced. The quantity of DNA in the natural honey samples was determined and compared to identify the major source of nectar in each sample.

The research is conducted in the current trend – “food authentication”, so any effective action in the field of food quality and safety. Modern analytical methods can be used to the verify quality of food products based on their botanical sources, chemical composition, possible adulterations or specified geographical origin. The section  Materials and Methods is described  with great care. It is also worthy of noting that Authors used advanced testing methods.

This research is interesting as well as  has scientific value.  However, I have some suggestions for Authors to improve their study. These follow the text sequence:

• The Introduction needs to be improved:

Line 43: Please, briefly describe these methods.

A large proportion of the cited references  (in all article, not only in this section) is more than 10 years old. It should be replaced with newer ones.

• What is the innovation of the presented research carried out in relation to the achievements of other researchers in this topic?

• Are standard deviations presented in the Figure 1 and Figure 2? This information must be provided as  Explanatory notes under Figures.
• Section Conclusion: Please, complete the practical relevance of the obtained results.

From my standpoint, this manuscript will be  appropriate for publication in Journal – Applied Sciences  only after minor revision, given the above aspects.  

Author Response

The answers to reviewer 3 were included in the attached file.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Authors of this manuscript have done suggested changes by the reviewer and the manuscript has been improbed.