Anethole Stability in Aniseed Spirits: Storage Condition Repercussions on Commercial Products

Round 1

Reviewer 1 Report

Very good and interesting work! Just a few grammar notes below.

line 14: 'result'

line 68: 'but they showed results considerably slower'?

line 79: units on the 25

line 145: units

line 333: crystallization misspelled

line 338: 'no crystals formation'

line 339: 'could depends'

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Materials and methods section

2.1. Samples and solutions.

The Sambuca and Mistrà samples were provided by the spirits maker, as can be deduced from the Acknowledgments section. However, the Mistrà sample does not meet the legal requirements regarding the minimum concentration of t-anethole (Regulation EU 2019/787).

If the purpose of the current study is to provide spirit makers with information on the best conditions for storage and protection of the characteristics of the product, it would be important to start from recently prepared samples. Thus, some questions should be answered:

Do you have any information on the previous storage conditions for both samples?

Have the samples of Sambuca and Mistrà been made in close proximity to each other?

An attempt should be made to explain the low concentration of t-anethole in the Mistrà samples.

2.2. HPLC and GC methods.

The concentration of the internal standard is missing.

2.3., 2.4. and 2.5: Assays of stability with UV-light and temperature.

It seems that all the variations in the t-anethole concentration due to the different light and thermal treatments have been made against the initially measured t-anethole concentration in both products. Please specify.

3. Results and Discussion

3.1. HPLC method validation

For the analysis of the aniseed liquors by HPLC, a 1/10 dilution is made with a methanol / water mixture. In view of the chromatogram shown in Figure 1, the peak corresponding to t-anethole appears distorted, perhaps due to excess signal. Has the influence of dilutions on the accuracy of t-anethole values been verified? Would it be feasible to carry out a higher dilution of the samples? What is the linearity range of this HPLC method?

3.2. Effects of light

Lines 214-217: This paragraph is confusing. If the Pyrex tube in which the samples are placed to establish the effect of two wavelengths on the concentration of t-anethole shows some selectivity for certain wavelengths, how is the influence of ultraviolet light deduced?

3.4. Effects of VIS light exposure

Lines 277-278: The paragraph is confusing. Sucrose does not absorb in the 400-500 nm range. What should be highlighted here is that the observed differences in absorbance of the different treatments are not affected by possible interferences caused by the absorption of sucrose.

4. Conclusions

Lines 355-358: The study evaluates the influence of extreme conditions on the stability of t-anethol, however it is concluded that the degradation of this compound

Minor details:

Figure 1 captions: make proper identifications of the chromatographic peaks. The reference “HPLC chromatogram of Sambuca (black)” is not correct. Identify peaks with numbers on the same chromatogram.

The wavelength of the treatment referred to as “UVB” changes throughtout the entire manuscript. In the Materials and Methods section "it is 312 nm; in section 3.2 (line 201) it is 321nm; at the bottom of Figure 2 it is 313nm … and so on.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The paper entitled ”Anethole stability in aniseed spirits: storage condition repercussions on commercial products” offers a novel perspective about the risks present in aniseed spirits and justly recommend the revision of the Regulation for this category of beverages.

The manuscript is well structured, clearly written and the quality of methodology used and results presentation offered are high.

I recommend minor language editing.

Author Response

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Reviewer 4 Report

The present work is focused on the evaluation trans-anethole degradation on alcoholic beverages due to the action of the light (UV and visible) and temperature emulating the storage conditions of some aniseed spirits. The obtained results are interesting for researchers and for the industry of these types of alcoholic beverages, however, some details about the experiments carried out must be clarified. In addition, maybe the addition of new figures of chromatograms could improve the quality of the manuscript.

Specific comments:

In the Introduction section:

Page 2-Lines 47-55. Maybe it is not necessary to explain so much about aspects related to healthy and non-healthy effects. Only as a suggestion, this part could be summarized.

In the material and methods section:

Page 3. Lines 119-128: Please add the GC method conditions: column oven temperature program, injector temperature, volume injected.

Page 3: Lines 130-141. Which is the intensity supplied by the visible light instrument? Why 20 minutes of light exposition in both cases?

Page 4. Lines 143-146: Which sample volume is used in these experiments? Why selected 5 hours?

In general, related to the preparation of samples for each set of experiments, please explain if the test tubes have an air headspace or not. How many samples are prepared for each experiment (4? this information is presented in some figure captions)? Why different sample volumes for UV, visible and temperature assays?

In the results and discussion section:

In figure 1 caption (or in figure 1): Please indicate retention times and peak name.

Page 6. Lines 223-239. Is it possible to add a figure with the GC and HPLC chromatograms?

Page 8. Lines 275-276. Are comparable the experiments with UV and visible light? (Different volume, radiation intensity probably different).  

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript is now clearer. It would have been very interesting to have been able to carry out the stability study of the Mistrà under the same conditions as those of the Sambucca, that is, starting from a recently prepared spirit. This is relevant to establish optimal storage conditions or periods of preferential consumption.

Author Response

Reviewer 2

The manuscript is now clearer. It would have been very interesting to have been able to carry out the stability study of the Mistrà under the same conditions as those of the Sambucca, that is, starting from a recently prepared spirit. This is relevant to establish optimal storage conditions or periods of preferential consumption.

Authors: of course we agree with the reviewer, but as explained in our first response the main goal of our project was to study the shelf life of Sambuca (starting from a fresh prepared sample), while the Mistrà sample was added later. Even though the Mistrà sample was not a fresh one, we decided to add the data in the paper as they help in understanding the role of sugars on the t-anethole stability.

I hope the reviewer can understand that starting with a new freshly prepared Mistrà sample would mean rewriting a large part of the paper.

Reviewer 4 Report

Minor comments:
line 218. Isocratic is not commonly used in GC analysis. "Isothermal" or "at constant temperature" is probably more adequate.

Author Response

Reviewer 4

line 218. Isocratic is not commonly used in GC analysis. "Isothermal" or "at constant temperature" is probably more adequate.

Authors: of course, the reviewer is right, we used the wrong term. We substituted isocratic with isothermal.