Safety and Protective Activities of Manufactured Alcohol-Free Beers
Round 1
Reviewer 1 Report
The work presented in this manuscript can consider for publication after addressing some minor mistakes, which I found.
- “simple compounds” is it right?
- Figures caption should be at the bottom of the figure.
- Figure 3 should be redrawn to make the results more visible.
Author Response
Authors thank all the reviewer's comments. Thanks of them, we consider that the manuscript has improved its quality.
Below we indicate the modifications and justifications asked for.
- “simple compounds” is it right?
Answer: Authors acknowledge the comment. The expression “simple compounds” has been changed by “single compounds”, being the last much more appropriate. Lines 107 and in Table 2 and Table 3.
- Figures caption should be at the bottom of the figure.
Answer: Figures captions have been moved to the bottom of the correspondent figure.
- Figure 3 should be redrawn to make the results more visible.
Answer: Figure 3 has been redrawn and amplified.
Reviewer 2 Report
General comments:
The topic looks interesting, but the manuscript failed to draw research gaps and novelties, and the experimental design did not support its conclusions. So the current version does not meet the general requirements for publishing in scientific journals.
Line 44. ECG is also commonly existed in tea, so why is it special in beers? Also, the most unique phytochemicals in beers are actually essential oils from hops, but why they were not even mentioned in the introduction?
Line 80 and 98. Yes, 'The available literature on comparisons of the two processes and their impact on the product quality and the economic aspects of brewing of LABs or AFBs is scarce.' But what is the research gap for the aim of this study on safety, protective and chemopreventive activities of three types of alcohol-free Lager beers'? The author wrote a lot of the text on conventional beers, ethanol-free beers, processes, etc.; and it is fine to provide a wide background, but the introduction failed to draw the research gaps that were filled by the topic of this study. Please clarify, 1) what is the scarcity of current studies on 'safety, protective and chemopreventive activities of beers'; 2) why it is novel to apply Drosophila animal model and human leukemia cells (HL-60) in vitro model for this study and are there previous articles used those models on beers or beverage evaluations, and what is the research gap, etc.
Line 108. The authors introduced 'alcohol-free (AFB) and low-alcohol beers (LAB)' as well as Lager and Ale...', but why only alcohol-free Lager beers were compared? Where are the Ale and low-alcohol beers? The title is 'alcohol-free beers ', so does only Larger represent all alcohol-free beers and Ale?
Line 113. What is the method for the determination of ECG? How did you know ECG was the dominant compound among other phytochemicals in the beers? Even if ECG was dominant, being the major component does not mean the antioxidant activities were attributed to ECG only, other compounds and phytochemicals such as hops essential oils, and other phenols may be also or the only active compounds that worked for the bioactivities.
The draft missed total phenol content and individual phenolic test by HPLC, LC-MASS, etc. The antioxidants profile is unclear, so the results on ECG were not supported by the experimental design. Other compounds may also contribute to antioxidant activities.
Author Response
Authors thank all the reviewer's comments. Thanks of them, we consider that the manuscript has improved its quality.
Below we indicate the modifications and justifications asked for.
Furthermore, reference numbers have been updated in the full manuscript as well as some parts of the text have been rewritten to eliminate the similarity percentage (in change form).
Line 44. ECG is also commonly existed in tea, so why is it special in beers?
Answer: authors added the following sentence in line 44:
“ECG is present in green tea and also in other plants such as grapes, strawberries, buckwheat,... This phenol was selected for this study due to [apart from the iso-Alpha-acids (iso-Cohumulone and iso-n-Humulone) and tyrosol] it is the most abundant phenolic in beer reaching up to 20 mg/L [6]. This value is in one order magnitude range of the green tea ECG content (8mg/g dw) [11]”.
- Gerhäuser, C. (2005). Beer constituents as potential cancer chemopreventive agents. European Journal of Cancer, 41(13), 1941-1954.
- Cui, L., Liu, Y., Liu, T., Yuan, Y., Yue, T., Cai, R., & Wang, Z. (2017). Extraction of Epigallocatechin Gallate and Epicatechin Gallate from Tea Leaves Using β-Cyclodextrin. Journal of food science, 82(2), 394-400.
Also, the most unique phytochemicals in beers are actually essential oils from hops, but why they were not even mentioned in the introduction?
Answer: we agree with the reviewer. Although the sentence "About 20–30% of beer polyphenols originate from hops, whereas 70–80% are malt derived (Gerhäuser, 2005)" is written in the introduction section in the original article, authors have added the following sentence in the introduction mentioning the unique phytochemicals from hops: "Beer is a complex beverage, containing a wide range of constituents. Also, some unique bioactive compounds are identified in beers as hop-derived prenylflavonoids: xanthohumol, isoxanthohumol, 8-prenylnaringenin [6], showing xanthohumol and iso-alpha humulone promising properties (safe, protective, cytotoxic and chemopreventive effects) [7,8].
- Gerhäuser, C. (2005). Beer constituents as potential cancer chemopreventive agents. European Journal of Cancer, 41(13), 1941-1954.
- Merinas-Amo, T., Tasset-Cuevas, I., Díaz-Carretero, A. M., Alonso-Moraga, Á., & Calahorro, F. (2016). In vivo and in vitro studies of the role of lyophilised blond Lager beer and some bioactive components in the modulation of degenerative processes. Journal of Functional Foods, 27, 274-294.
- Merinas-Amo, T., Merinas-Amo, R., Font, R., del Río Celestino, M., & Alonso-Moraga, Á. (2021). Toxicological and Epigenetic Studies of Two Types of Ale Beer, Tyrosol and Iso-Alpha Humulone. Processes, 9(3), 485.
Line 80 and 98. Yes, 'The available literature on comparisons of the two processes and their impact on the product quality and the economic aspects of brewing of LABs or AFBs is scarce.' But what is the research gap for the aim of this study on safety, protective and chemopreventive activities of three types of alcohol-free Lager beers'? The author wrote a lot of the text on conventional beers, ethanol-free beers, processes, etc.; and it is fine to provide a wide background, but the introduction failed to draw the research gaps that were filled by the topic of this study. Please clarify, 1) what is the scarcity of current studies on 'safety, protective and chemopreventive activities of beers'; 2) why it is novel to apply Drosophila animal model and human leukemia cells (HL-60) in vitro model for this study and are there previous articles used those models on beers or beverage evaluations, and what is the research gap, etc.
Answer: Following the recommendation of the reviewer, in the original lines 98-104, authors have added the next text:
“The final outcome of a specific beer is the result of a multifactorial entries (composition, alcohol content, brewing process) and, hence, the exhaustive study of beer become complicated. Nevertheless, it is known the increasing interest in health care and, consequently, a new lifestyle is emerging in the last times. For these reasons, we studied one of the most consumed beverages as it is believed to be healthier: the alcohol-free beer. A new data corpus is needed not only related to their brewing process, phenol content or flavour but related to their safety, genome protecting and longevity enhancing activities. [...] which contains more than 70% of human disease homologous genes [26,27], is a reliable system for in vivo screening assessing tests”
- Richardson HE, Willoughby L, Humbert PO: Screening for Anti-cancer Drugs in Drosophila. eLS 2015.
- Graf U, Wurgler FE, Katz AJ, Frei H, Juon H, Hall CB, Kale PG: Somatic mutation and recombination test in Drosophila melanogaster. Environ Mutagen 1984, 6:153-188.
Line 108. The authors introduced 'alcohol-free (AFB) and low-alcohol beers (LAB)' as well as Lager and Ale...', but why only alcohol-free Lager beers were compared? Where are the Ale and low-alcohol beers? The title is 'alcohol-free beers ', so does only Larger represent all alcohol-free beers and Ale?
Answer: Authors added the next paragraph following the reviewer comment:
“Only alcohol-free Lager beers were chosen for the present study due to its highest consumption over the world with respect to the alcohol-free Ale ones. It could be related with the economical impact that Ale beers may have in the dealcoholisation process, and also due to the brewing process (top-fermenting yeast at warm temperatures) and they have a much higher alcohol content than Lagers.”
Line 113. What is the method for the determination of ECG? How did you know ECG was the dominant compound among other phytochemicals in the beers? Even if ECG was dominant, being the major component does not mean the antioxidant activities were attributed to ECG only, other compounds and phytochemicals such as hops essential oils, and other phenols may be also or the only active compounds that worked for the bioactivities.
Answer: Authors agree with the reviewer. The biological activities assigned to beer consumption cannot be linked to one particular constituent due to the high variability and complexity in the polyphenolic profile; furthermore, brewing is influenced by many factors, such as the raw materials, yeast strains, wort composition and fermentation conditions. As authors have mentioned in the introduction section, we selected ECG due to it is the third most abundant phenol in alcohol-free beers after tyrosol and alpha-acid humulone and also because these two phenols were previously studied in our laboratory in other research project (Merinas-Amo et al. 2021).
- Merinas-Amo, T., Merinas-Amo, R., Font, R., del Río Celestino, M., & Alonso-Moraga, Á. (2021). Toxicological and Epigenetic Studies of Two Types of Ale Beer, Tyrosol and Iso-Alpha Humulone. Processes, 9(3), 485.
The next sentence is added in line 113 to clarify the selection of ECG:
"taking into account that alcohol-free beers have the same bioactive compounds (although in lower concentrations) than regular beer except for alcohol [28], the epicatechin gallate (ECG) was tested as single bioactive compound due to it is the third most abundant phenol in beers after tyrosol and iso-alpha humulone [6] not studied in our previous researches [8]"
The draft missed total phenol content and individual phenolic test by HPLC, LC-MASS, etc. The antioxidants profile is unclear, so the results on ECG were not supported by the experimental design. Other compounds may also contribute to antioxidant activities.
As authors mentioned in the 2.1 section, "taking into account that alcohol-free beers have the same bioactive compounds (although in lower concentrations) than regular beer except for alcohol [28], the epicatechin gallate (ECG) was tested as single bioactive compound due to it is the third most abundant phenol in beers after tyrosol and iso-alpha humulone [6] not studied in our previous researches [8]"
- Gerhäuser, C. (2005). Beer constituents as potential cancer chemopreventive agents. European Journal of Cancer, 41(13), 1941-1954.
- De Gaetano, G., Costanzo, S., Di Castelnuovo, A., Badimon, L., Bejko, D., Alkerwi, A. A., ... & Iacoviello, L. (2016). Effects of moderate beer consumption on health and disease: A consensus document. Nutrition, Metabolism and Cardiovascular Diseases, 26(6), 443-467.
Reviewer 3 Report
Review Journal; Processes (ISSN 2227-9717); Manuscript ID processes-1550073 Title: Safety and protecting activities of manufactured alcohol-free beers Authors: Tania Merinas-Amo, Mercedes Del Río Celestino, Rafael Font, Ángeles Alonso-Moraga Many people still wonder, "What is the purpose of non-alcoholic beer?" Others may not be aware of the many benefits of non-alcoholic beer - including its health benefits. One of the reasons why non-alcoholic beer is an effective substitute for alcoholic beverages is that the body associates taste and smell with beer of full strength. This leads to the production of dopamine, the same chemical that makes you feel good when you drink alcohol or eat chocolate with 80% cocoa. Your results suggest the safety properties of all compounds, although pale blue and solid beer showed genotoxic activity only at the lowest concentrations tested. The results obtained in this paper showed that non-alcoholic beers must be considered not only the cause of their reduced caloric, isotonic properties, but can also be recognized as potential nutrients due to safety, protective and chemopreventive results. Your research has shown excellent results and you should definitely continue in that direction.
Author Response
Authors thank all the reviewer's comments. Thanks of them, we consider that the manuscript has improved its quality.
Below we indicate the modifications and justifications asked for.
Answer: Authors highly appreciate to the reviewer for taking the time to review our manuscript and also for your valuable comments towards our research, specifically to the health benefits of non-alcoholic beer as an effective substitute for alcoholic beverages. In the past, we have carried out toxicological studies of Czech beers and their constituents with interesting results and we intend to continue in this direction. Thanks again for the encouragement.
Round 2
Reviewer 2 Report
Line 13 and others about ECG. Again, the most abundant phenol does not necessarily mean it is the active compound that contributed to the protection activities. It could be another small amount of compounds (i.e. other phenols and essential oils) that were the major contributors. It is also could be a synergistic effect of many compounds. Therefore, the unclear phytochemical profile is the major default of the experimental design, which results in the conclusion about ECG was not supported by the experiment. Total phenolic content should also be tested and provided.
Line 93. Yes. Limited literature reported the effects of processes on product quality, which indicated the research gap. But this study has yet to report and discuss any product quality content later. So this is an irreverent highlight of research gaps.
Line 132. There is no ECG determination method shown in the method section. A paragraph describes all chemicals and materials origination should be added. The total phenol content and individual phenolic profile of LBAFLB, LPBAFLB and LSAFLB must be determined by Folin-Ciocalteu assay and HPLC/HPLC-MS and provided in the draft.
Line 143. needs a reference.
Line 748. Figure 6. The significant markers are totally chaotic. How could the highest value and lowest value share the same marker 'b', when 'a' marked on values in the middle in Figure 6 B? Also in Figure 6 C, there is 'b' on both the highest and the lowest, while 'a' and 'c' marked in between? Obviously, the author does not have proper knowledge about statistics or did not understand or systematically learn some statistic classes.
Line 806. The conclusion is very general and bland, also does not provide useful information, key findings, and data.
In summary, the quality of the manuscript is low, the experiment does not properly design to support the conclusion. Figures did not carefully prepare. Phenol profiles of the tested beers are unclear. Many of the responses to reviews are irrelevant, plausible, ambiguous and evasive to the questions.
I am sorry that I have to reject the draft.
Author Response
Answer to Reviewer 2:
Line 13 and others about ECG. Again, the most abundant phenol does not necessarily mean it is the active compound that contributed to the protection activities. It could be another small amount of compounds (i.e. other phenols and essential oils) that were the major contributors. It is also could be a synergistic effect of many compounds. Therefore, the unclear phytochemical profile is the major default of the experimental design, which results in the conclusion about ECG was not supported by the experiment. Total phenolic content should also be tested and provided.
Dear Reviewer, thank you for your constructive comments. All of them will help us to improve the quality of the final manuscript. The final outcome of a specific beer is the result of multifactorial entries (composition, alcohol content, brewing process), so beer is a complex beverage, containing a wide range of constituents, hence, the exhaustive study of beer become complicated. Although several molecules contained in beer belonging to the phenol and vitamin groups are often claimed to be health promoters because they are involved in the reduction/remission of diseases age related, we focus our research in a phenolic (ECG) common in the three types of alcohol-free Lager beers (a blond, a pale-blond and a stout beer-variety), and also because it is in high concentration as several authors already determined [6, 13, 14].
[6] Gerhäuser, C. Beer constituents as potential cancer chemopreventive agents. European Journal of Cancer 2005, 41, 1941-1954.
[13] Bartolomé, B.; Pena-Neira, A.; Gómez-Cordovés, C. Phenolics and related substances in alcohol-free beers. European Food Research and Technology 2000, 210, 419-423.
[14] McMurrough, I.; Baert, T. Identification of proanthocyanidins in beer and their direct measurement with a dual electrode electrochemical detector. Journal of the Institute of Brewing 1994, 100, 409-416.
Line 93. Yes. Limited literature reported the effects of processes on product quality, which indicated the research gap. But this study has yet to report and discuss any product quality content later. So this is an irreverent highlight of research gaps.
Thanks again, may authors haven’t properly explained themselves. What we meant was that little attention has been paid to alcohol-free beers when compared to regular beers. In these last cases more literature can be found e.g. for lager beers [7, 8].
[7] Merinas-Amo, T.; Tasset-Cuevas, I.; Díaz-Carretero, A.M.; Alonso-Moraga, Á.; Calahorro, F. In vivo and in vitro studies of the role of lyophilised blond Lager beer and some bioactive components in the modulation of degenerative processes. Journal of Functional Foods 2016, 27, 274-294.
[8] Merinas-Amo, T.; Merinas-Amo, R.; Font, R.; del Río Celestino, M.; Alonso-Moraga, Á. Toxicological and Epigenetic Studies of Two Types of Ale Beer, Tyrosol and Iso-Alpha Humulone. Processes 2021, 9, 485.
Line 132. There is no ECG determination method shown in the method section. A paragraph describes all chemicals and materials origination should be added. The total phenol content and individual phenolic profile of LBAFLB, LPBAFLB and LSAFLB must be determined by Folin-Ciocalteu assay and HPLC/HPLC-MS and provided in the draft.
The tested concentrations cover a range that contains the values already described in literature for alcohol-free beer and that is why authors did no determined their amount in the three raw samples. Several published papers using other biological matrices (pepper, tomato, onion, wine, lemon, orange, vegetable oil, beta-carotene, capsaicin, lycopene, capsanthine, organosulfur, rutin, resveratrol, limonene, hesperidin, hydroxityrosol, sulforaphane, rosmarinic acid, sinigrin, food coloring and beer) have been published by our research group using for the calculation of concentrations from useful available data (Fernández-Bedmar, et al. 2019; Merinas-Amo, et al. 2019; Mateo-Fernández, et al. 2019; Mateo-Fernández, et al. 2021). So, for the concentration of ECG, authors apologize because we cannot satisfy the reviewer's request, since we have not got original samples to carry out the assays suggested.
- Fernández-Bedmar, Z.; Demyda-Peyrás, S.; Merinas-Amo, T.; del Río-Celestino, M. Nutraceutic Potential of Two Allium Species and Their Distinctive Organosulfur Compounds: A Multi-Assay Evaluation. Foods 2019, 8, 222. doi: 10.3390/foods8060222
- Merinas-Amo, R., Martínez-Jurado, M., Jurado-Güeto, S., Alonso-Moraga, Á., & Merinas-Amo, T. (2019). Biological effects of food coloring in in vivo and in vitro model systems. Foods 2019, 8, 176. doi: 10.3390/foods8050176
- Mateo-Fernández, M.; Alves-Martínez, P.; Del Río-Celestino, M.; Font, R.; Merinas-Amo, T.; Alonso-Moraga, Á. Food Safety and Nutraceutical Potential of Caramel Colour Class IV Using In Vivo and In Vitro Assays. Foods 2019, 8, 392. doi: 10.3390/foods8090392
- Mateo-Fernández, M.; Valenzuela-Gómez, F.; Font, R.; Del Río-Celestino, M.; Merinas-Amo, T.; Alonso-Moraga, Á. In Vivo and In Vitro Assays Evaluating the Biological Activity of Taurine, Glucose and Energetic Beverages. Molecules 2021, 26, 2198. doi: 10.3390/molecules26082198
Line 143. needs a reference.
A new reference has been added in the line 143 numbered as [29]. Consequently, references have been updated and English language has been checked.
[29] Pavsler, A., & Buiatti, S. (2009). Lager beer. In Beer in health and disease prevention (pp. 31-43). Academic Press.
Line 748. Figure 6. The significant markers are totally chaotic. How could the highest value and lowest value share the same marker 'b', when 'a' marked on values in the middle in Figure 6 B? Also in Figure 6 C, there is 'b' on both the highest and the lowest, while 'a' and 'c' marked in between? Obviously, the author does not have proper knowledge about statistics or did not understand or systematically learn some statistic classes.
We are sorry with this comment. In the Figures, statistical results are obtained by comparing the methylation status of HL-60 cells treated with each compound and, the same, for each repetitive element. The comparisons of interest are always with respect to their concurrent control (blue bar for each trial), with a type 1 error minor of 0.05 and irrespectively whether they mean hypo or hypermethylation as it is obvious when looking at the bars: if the bar is lower and significantly different to the control we can say that there is hypomethylating effect and if the bar is higher and significantly different to the control one can say that we have hypermethylating effect. No biological interest/meanning for the differences between hypo and hypermethylation levels.
According to the reviewer, the sentence “Different letters mean different values after one-way ANOVA and post hoc Tukey's test” has been deleted in the figure and a sentence has been added to clarify the significance markers in the Figures 6A,6B and 6C: “The different letters in each compound and each repetitive element mean differences respect to the negative control (ANOVA followed by post hoc Tukey’s test).
Line 806. The conclusion is very general and bland, also does not provide useful information, key findings, and data.
Authors added a new paragraph with results information. "Results suggested the safety properties of all compounds, although LPBAFLB and LSAFLB showed a genotoxic activity only at the lowest concentrations assayed. Moreover, all the studied alcohol-free beers and ECG were able to protect against H2O2 oxidative damage as well as to induce an increase of longevity with an improvement of quality of life in the in vivo animal model assayed. Promising results were obtained with the alcohol-free beers and ECG in the in vitro assays with human leukaemia cells as they inhibit the tumour cells growth, induce DNA damage and modify the methylation status of such a cancer cell line."
In summary, the quality of the manuscript is low, the experiment does not properly design to support the conclusion. Figures did not carefully prepare. Phenol profiles of the tested beers are unclear. Many of the responses to reviews are irrelevant, plausible, ambiguous and evasive to the questions. I am sorry that I have to reject the draft.
Round 3
Reviewer 2 Report
I appreciate the authors' efforts in revising the manuscript and improving the readability and soundness of the research. The phenol level provided from published papers is critical for the draft, but it would be much easier if the authors determined and reported at least the total phenol content by themself. All in all, the total phenol content is not a difficult job and can be tackled in 30 min to 1 hour, as compared to the long process of HPLC-MS/MS.
Anyway, I accept the current version.
Cheers,
