‘Kombucha’-like Beverage of Broccoli By-Products: A New Dietary Source of Bioactive Sulforaphane
Round 1
Reviewer 1 Report
1. INTRODUCTION:
A) - In line 60, a bibliographic reference is missing.
B) – The authors did not provide any bibliographic references from studies that have used alternative substrates for the production of kombucha analogs. I believe it is important to include this information since that was the aim of this study.
2. RESULTS:
A) - Why was titratable acidity calculated in citric acid? The predominant acid in kombucha is acetic acid, not citric acid. Furthermore, in lines 186 and 187, the results are presented as % titratable acidity in citric acid, not % citric acid.
B) - In line 189, to which parameter are the authors referring? This paragraph is unclear. The authors should provide clarification.
C) - Figure 2 can be subdivided into a, b, and c, with Figure 2a (°Brix), Figure 2b (pH), and Figure 2c (titratable acidity). This way, it will be clearer in the text which graph each section refers to.
D) - In Figure 3, I recommend the same subdivision as previously suggested for Figure 2. Label them as Figure 3a and Figure 3b.
3. CONCLUSIONS:
A) - Which bibliographic references did you use to conclude that "...the development process of traditional 'kombucha' is restricted to five days..."? It is known that most studies report fermentation periods of 7 to 14 days, so this statement in the conclusion should be corrected.
Author Response
POINT-BY-POINT RESPONSE
(Beverages-26472116)
Reviewer 1
INTRODUCTION:
In line 60, a bibliographic reference is missing.
Following the reviewer’s suggestion, on page 2, line 60, of the reviewed version of the manuscript (MS), the statement was properly referenced.
The authors did not provide any bibliographic references from studies that have used alternative substrates for the production of kombucha analogs. I believe it is important to include this information since that was the aim of this study.
According to the reviewer’s suggestion, the reviewed version of the MS included a citation covering the use of alternative vegetable materials to develop kombucha-like beverages (page 2, line 71).
Emiljanowicz, K.E.; Malinowska-Pańczyk, E. Kombucha from Alternative Raw Materials – The Review. Crit Rev Food Sci Nutr 2020, 60, 3185–3194, doi:10.1080/10408398.2019.1679714.
RESULTS:
Why was titratable acidity calculated in citric acid? The predominant acid in kombucha is acetic acid, not citric acid. Furthermore, in lines 186 and 187, the results are presented as % titratable acidity in citric acid, not % citric acid.
The use of the term titratable acidity instead of percentage of citric acid was mistaken during the article drafting. This has been double-checked and corrected throughout the entire MS. Beyond this, according to the reviewer’s comment, the use of the percentage of citric acid as an indicator of titratable acidity was replaced by the percentage of acetic acid for which calculation was applied a conversion factor 0.9377 according to sectorial literature. Accordingly, Figure 2 was modified to express titratable acidity as the percentage of acetic acid.
In line 189, to which parameter are the authors referring? This paragraph is unclear. The authors should provide clarification.
Following the reviewer’s suggestion, the paragraph on page 6, lines 194-202, has been redrafted to clearly indicate that the information provided is concerning total soluble solids.
Figure 2 can be subdivided into a, b, and c, with Figure 2a (°Brix), Figure 2b (pH), and Figure 2c (titratable acidity). This way, it will be clearer in the text which graph each section refers to.
According to the reviewer’s suggestion, Figure 2 was modified by changing the scale of the line plot corresponding to titratable acidity to express it by the % of acetic acid. Also, each separate plot is referred to as A (ºBrix), B (pH), and C (percentage of acetic acid) in the reviewed version of the MS.
In Figure 3, I recommend the same subdivision as previously suggested for Figure 2. Label them as Figure 3a and Figure 3b.
Following the reviewer’s suggestion, each separate plot is referred to as A (sulforaphane) and B (sulforaphane-N-acetylcysteine) in the reviewed version of the MS.
CONCLUSIONS:
Which bibliographic references did you use to conclude that "...the development process of traditional 'kombucha' is restricted to five days..."? It is known that most studies report fermentation periods of 7 to 14 days, so this statement in the conclusion should be corrected.
According to the reviewer’s suggestion, in the conclusions subsection, the statement “Concerning the evolution of the quality and compositional parameters considered in the present characterization, the development process of traditional ‘kombucha’ is restricted to five days, which coincides with the higher concentration of bioactive compounds generated as a result of the microbial metabolism of the different GSL” was corrected as follows “Concerning the evolution of the quality and compositional parameters considered in the present characterization, the development process of traditional ‘kombucha’ is enclosed to fermentation of 7 to 14 days, which coincides with the higher concentration of bioactive compounds generated as a result of the microbial metabolism of the different GSL” on page 9, lines 324-328 of the reviewed version of the MS.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors,
Your Manuscript aims to promote the valorisation of vegetable by-products and their further applications for the production of different functional products (beverages). In this your study, the kombucha like beverage was produced and characterized in detail using powerful chromatographic techniques, as the first step in the formulation of a functional product (beverage). However, in future research, it is necessary to evaluate the sensory properties of this beverage, as well as its in vitro and in vivo functional properties/activities. Finally, this your study gives very interesting approach for valorisation of broccoli by-product, which fits into the concept of green economy and environmental sustainability.
However, there are some corrections/improvements that must be done before any further Manuscript processing.
I will enumerate different requirements to improve the manuscript. All my comments are listed below and separated as General and specific comments.
General comments:
1. The abstract should contain the objective and all the novelties that make this study unique, the most relevant results, and the conclusion (whether the obtained product is in accordance with the set hypotheses). I suggest to authors that meaningfully shorten the existing version of the abstract and to point out the specifics of this research. This part in Abstract (line 8-14) is more appropriate for Introduction section.
2. Kombucha or kombucha-like beverages have been studied a lot until now, which is not indicated in the introduction section. So, I suggest to authors that in the introduction section add some previous research which analyzed kombucha or kombucha-like beverages, and only then to indicate the specifics of your research.
3. (a) In Material and methods (section 2.4.), you described only the preparation of dried broccoli by-products for chromatographic analysis. How did you prepare kombucha-like beverage for chromatographic analysis?
(b) Describe in detail both chromatographic techniques (HPLC-PDA-ESI/MSn and UHPLC-ESI-QqQ-MS) used for identification and quantification of glucosinolates and their metabolites.
(c) In which ionization mode, did you analyse isothiocyanates and indoles (section 2.5)?
(d) How did you determine the content of sulforaphane-N-acetylcysteine, when you did not have this standard?
If you have expressed the content of this compound in equivalents of another standard, this should be emphasized in the Material and methods section.
(e) Add parameters of quantification (linear regression, r2, LOD, LOQ, and linear range) for used standards of glucosinolates, isothiocyanates and indoles.
4. A deeper expertise and additional explanations of the obtained results are necessary in the Results and discussion section.
(a) Explain why the content of total soluble solids, pH values and titratable acidity of kombucha beverage changes with time of fermentation, as well as in comparison to control sample (section 3.1).
(b) Did you identified/quantified of glucosinolates in kombucha-like beverages?
If you did the identification/quantification of glucosinolates in beverage, then in that case make a comparison between dried broccoli and its beverage? (Additional explanations are necessary in section 3.2.)?
(c) Why are isothiocyanates and indoles dominant in kombucha beverage?
Did you identified/quantified only sulforaphane and sulforaphane-N-acetylcysteine in a kombucha-like beverage?
If you identified/quantified other isothiocyanates and indoles in your beverage (presented in Figure 1), complete section 3.3. with these results?
Specific comments:
Line 22-25: This sentence “Therefore, these results… functional beverages.” Is too long and confused. I suggest to authors that meaningfully rewrite the mentioned sentence.
Line 32: “..., Due to…” replace with “…, due to…” (small letter)
Line 34-36: This sentence “These positive effects … and postbiotic compounds” should be meaningfully rewritten. Moreover, this sentence (claim) should be further supported with appropriate reference/s.
Line 54-58: This sentence “In the search … broccoli consumption.” Is too long and confused. I suggest to authors that that meaningfully rewrite the mentioned sentence.
Line 63-66: These sentences “The use of … and functional profiles (7).” must be meaningfully rewritten.
Line 82: “Acetic acid and ammonium acetate were obtained/purchased from …”
Line 83-84: “Methanol used for extractions and acetonitrile (analytical grade) were purchased from …”
Line 85: “For preparation of reagents and mobile phases were used milliQ water (Milli-Q system, Millipore, Bedford…).”
Line 88. “Plant material and beverage development” (without point)
Line 117: This part (“newly developed”) delete from sentence.
Line 122: Term (“obtained”) delete from sentence. “The extracts were centrifuged…”
Line 137-143: This sentence (“The chromatographic separation … chromatographic column), is too long and confused. I suggest to authors that meaningfully rewrite the mentioned sentence.
Line 147-148: This sentence (“The concentration … day of analysis.”) is confused and must be meaningfully explained and rewritten.
Line 154-155: This sentence (“The results are… performed (n=3).”) must be meaningfully rewritten.
Line 164: “While Brix remained…” replace with “Values for TSS were not significantly change during first 6 days….”
Line 183-188: These sentences (“Finally, and following … 1.3% citric acid (Figure 2)”) are confused and must be meaningfully rewritten.
Line 189-194. Whole this part (“The most … fermentative activity”) must be meaningfully rewritten.
Line 197: “… pre-cursor…” replace with “…precursor…”
I have no comments.
Author Response
POINT-BY-POINT RESPONSE
(Beverages-26472116)
Reviewer 2
GENERAL COMMENTS
The abstract should contain the objective and all the novelties that make this study unique, the most relevant results, and the conclusion (whether the obtained product is in accordance with the set hypotheses). I suggest to authors that meaningfully shorten the existing version of the abstract and to point out the specifics of this research. This part in Abstract (line 8-14) is more appropriate for Introduction section.
Following the reviewer’s suggestion, the introductory statements in the abstract were removed. This information has been included in the introduction subsection taking care to avoid duplications.
Kombucha or kombucha-like beverages have been studied a lot until now, which is not indicated in the introduction section. So, I suggest to authors that in the introduction section add some previous research which analyzed kombucha or kombucha-like beverages, and only then to indicate the specifics of your research.
According to the reviewer’s suggestion, an additional statement and references on the research focused on ‘kombucha’ development and characterization has been included on page 1, lines 36-37.
(a) In Material and methods (section 2.4.), you described only the preparation of dried broccoli by-products for chromatographic analysis. How did you prepare kombucha-like beverage for chromatographic analysis?
Following the reviewer’s suggestion, the beverages’ processing conditions for quality properties and organosulfur compounds content was described on page 3, lines 113-115.
(b) Describe in detail both chromatographic techniques (HPLC-PDA-ESI/MSn and UHPLC-ESI-QqQ-MS) used for identification and quantification of glucosinolates and their metabolites.
The chromatographic techniques used in the present work have been developed and validated in our laboratory and they have been described comprehensively in the references provided in the main text (Domínguez-Perles et al., 2014; Baenas et al., 2017; Abellán et al., 2021). This form of presenting the methodology is totally accepted in the scientific literature and avoids plagiarism issues. Therefore, in our opinion, providing additional information in the present MS is not necessary at all.
(c) In which ionization mode, did you analyse isothiocyanates and indoles (section 2.5)?
Following the reviewer’s suggestion, the ionization mode for isothiocyanates and indoles has been described on page 4, lines 150-151, and in the figure caption of Figure 1. Also, this information was provided for glucosinolates on page 3, line 13, as well as in the headline of Table 1.
(d) How did you determine the content of sulforaphane-N-acetylcysteine, when you did not have this standard? If you have expressed the content of this compound in equivalents of another standard, this should be emphasized in the Material and methods section.
Indeed, we account for the standard of sulforaphane-N-acetylcysteine as you can see in the references that provide further details on the analytical methodology (Domínguez-Perles et al., 2014). The gap concerning the description of this standard in the reagents subsections has been corrected in the reviewed version of the MS (page 2, lines 82-83).
(e) Add parameters of quantification (linear regression, r2, LOD, LOQ, and linear range) for used standards of glucosinolates, isothiocyanates and indoles.
In our opinion, this information is provided when reporting the validation of a new method (concerning the limit of detection and quantification) that is included in the references providing the details of the analytical method (Domínguez-Perles et al., 2014; Baenas et al., 2017). In the current version of the MS, the quality of the regression coefficient is provided for glucosinolates and breakdown products on page 4 lines 140-142 and lines 156-157, correspondingly.
A deeper expertise and additional explanations of the obtained results are necessary in the Results and discussion section.
(a) Explain why the content of total soluble solids, pH values and titratable acidity of kombucha beverage changes with time of fermentation, as well as in comparison to control sample (section 3.1).
Following the reviewer’s comments, additional discussion on the causes responsible for the modification of the total soluble solids, pH, and titratable acidity values has been included on pages 6-7, lines 194-221 of the reviewed version of the MS.
(b) Did you identified/quantified of glucosinolates in kombucha-like beverages?
If you did the identification/quantification of glucosinolates in beverage, then in that case make a comparison between dried broccoli and its beverage? (Additional explanations are necessary in section 3.2.)?
Subsection 3.2 is referred to the organosulfur content of the plant material. Therefore, in our opinion, the requested information should be included in subsection 3.3 (Bioactive organosulfur compounds content of broccoli kombucha). In this concern, no quantifiable concentration of glucosinolate was recorded as explained in the first paragraph of the subsection (modified for a better understanding on page 8, lines 257-262).
(c) Why are isothiocyanates and indoles dominant in kombucha beverage?
Following the reviewer’s comment, additional discussion was included on pages 8-9, lines 285-297 on the mechanisms responsible for the formation of ITC and indoles from their GSL precursors as a result of the enzymatic activity during the fermentation process. Actually, this is the rationale for the preponderance of ITC and indoles in fermented beverages.
Did you identified/quantified only sulforaphane and sulforaphane-N-acetylcysteine in a kombucha-like beverage?
Yes. Despite the diversity of organosulfur compounds monitored in the present work, which are detailed in subsection 2.1 and Figure 1, only SFN and SFN-NAC were found in quantifiable concentrations. Thereby, these are the only compounds described relative to the quantitative profile ITC according.
If you identified/quantified other isothiocyanates and indoles in your beverage (presented in Figure 1), complete section 3.3. with these results?
Figure 1 presents the diversity of ITC and indoles included in the analytical method. Nonetheless, when analyzing the experimental samples, only SFN and SFN-NAC were found in concentrations higher than LOD and LOQ.
SPECIFIC COMMENTS
Line 22-25: This sentence “Therefore, these results… functional beverages.” Is too long and confused. I suggest to authors that meaningfully rewrite the mentioned sentence.
Following the reviewer's suggestion, the statement “Therefore, these results demonstrate that this process increases the concentration of the bioactive compounds of interest, which will have a key impact on their bioavailability, and thereby, on health, especially in relation to anti-inflammatory activity, thus boosting the development of new functional beverages.” Was re-drafted towards “Therefore, these results demonstrate that the increase of the concentration of the bioactive compounds would provide higher bioavailability and health benefits. This is especially relevant concerning anti-inflammatory activity. Reporting additional proof of enhanced biological benefits will boost the development of new functional beverages.” On page 1, lines 16-20 of the reviewed version of the MS for a better understanding.
Line 32: “..., Due to…” replace with “…, due to…” (small letter)
According to the reviewer’s suggestion, “Due to…” was replaced by “due to…” on page 1, line 27 of the reviewed version of the MS.
Line 34-36: This sentence “These positive effects … and postbiotic compounds” should be meaningfully rewritten. Moreover, this sentence (claim) should be further supported with appropriate reference/s.
Following the reviewer’s suggestion, the statement referred to was re-drafted to “These positive effects are due, to a high extent, to the presence of functional microorganisms with the metabolic capacity to transform the chemical compounds present in the food matrices, giving rise to highly bioavailable prebiotic and postbiotic compounds [4].” For a better understanding on page 1, lines 29-32. Also, a valid reference “Dahiya, D.; Nigam, P.S. Probiotics, Prebiotics, Synbiotics, and Fermented Foods as Potential Biotics in Nutrition Improving Health via Microbiome-Gut-Brain Axis. Fermentation 2022, Vol. 8, Page 303 2022, 8, 303, doi:10.3390/FERMENTATION8070303.”, was included to support the sentence (claim).
Line 54-58: This sentence “In the search … broccoli consumption.” Is too long and confused. I suggest to authors that that meaningfully rewrite the mentioned sentence.
According to the reviewer’s suggestion, the sentence referred to has been rewritten in the reviewed version of the MS as follows: “In the search for alternative plant materials with the potential to be used as ingredients for the development of new fermented beverages, broccoli by-products have been pointed out as good sources of bioactive phytochemicals (e.g., glucosinolates (GSL), isothiocyanates (ITC), and indoles). These compounds have been demonstrated on a valuable capacity to modulate several pathophysiological situations [12], thus contributing to the diversity of biological benefits associated with broccoli consumption [13].” (page 2, lines 51-57).
Line 63-66: These sentences “The use of … and functional profiles (7).” must be meaningfully rewritten.
According to the reviewer’s suggestion, the statement referred to was modified towards “Despite this potential as a source of bioactive compounds, broccoli by-products are currently affected by a gap in consolidated and sustainable methods of valorization [15]. Achieving this objective has provided stabilized material with preserved phytochemical content that could constitute the basis for the development of new fermented, “3S”, beverages. The valorization of broccoli by-products in this way will increase the competitiveness of broccoli production and boost the panel of marketable fermented beverages by incorporating new nutritional and functional profiles [10].” For a better understanding on page 2, lines 61-67 of the reviewed version of the MS.
Line 82: “Acetic acid and ammonium acetate were obtained/purchased from …”
Following the reviewer’s suggestion, the grammatic mistake was replaced in the subsection 2.1 (page 2, line 85) of the reviewed version of the MS.
Line 83-84: “Methanol used for extractions and acetonitrile (analytical grade) were purchased from …”
The modifications suggested by the reviewer were included on page 2, lines 86-87 of the reviewed version of the MS (Methanol used for extractions and acetonitrile (analytical grade) were purchased from J.T. Baker (Philipsburg, NJ, USA).)
Line 85: “For preparation of reagents and mobile phases were used milliQ water (Milli-Q system, Millipore, Bedford…).”
The modifications suggested by the reviewer were included on page 2, lines 87-88 of the reviewed version of the MS (For the preparation of reagents and mobile phases Milli-Q water was used (Milli-Q system (Millipore, Bedford, MA, USA).).
Line 88. “Plant material and beverage development” (without point)
Following the reviewer’s suggestion, the point was removed from the head of the subsection 2.2 (page 2, line 90 of the reviewed version of the MS).
Line 117: This part (“newly developed”) delete from sentence.
According to the reviewer’s suggestion, “newly developed” was removed from the sentence on page 3, line 119 of the reviewed version of the MS.
Line 122: Term (“obtained”) delete from sentence. “The extracts were centrifuged…”
Following the reviewer’s suggestion, “obtained” was removed from the statement referred to (page 3, line 127 of the reviewed version of the MS).
Line 137-143: This sentence (“The chromatographic separation … chromatographic column), is too long and confused. I suggest to authors that meaningfully rewrite the mentioned sentence.
According to the reviewer’s suggestion, the sentence referred to was modified for a better understanding as follows “The chromatographic separation of the ITC and indoles present in the analytical extracts and in the broccoli-based fermented beverage developed was performed according to the methodology described by Domínguez-Perles et al. (2014) and Baenas et al. (2017) [17,20]. Briefly, it was used a Zorbax Eclipse Plus C18 chromatographic column (2.1 × 50.0 mm, 1.7 µm) integrated into a UHPLC coupled with a triple-quadrupole-MS/MS detector model 6460 (Agilent Technologies, Waldbronn, Germany), operated in positive ionization mode.” on page 4, lines 145-151 of the reviewed version of the MS.
Line 147-148: This sentence (“The concentration … day of analysis.”) is confused and must be meaningfully explained and rewritten.
Following the reviewer’s suggestion, the referred statement was re-drafted on page 4, lines 155-157 of the reviewed version of the MS.
Line 154-155: This sentence (“The results are… performed (n=3).”) must be meaningfully rewritten.
Following the reviewer’s suggestion, the referred statement was re-drafted on page 6, line 172 of the reviewed version of the MS.
Line 164: “While Brix remained…” replace with “Values for TSS were not significantly change during first 6 days….”
Following the reviewer’s suggestion, the referred statement was re-drafted on page 6, line 183 of the reviewed version of the MS.
Line 183-188: These sentences (“Finally, and following … 1.3% citric acid (Figure 2)”) are confused and must be meaningfully rewritten.
Following the reviewer’s suggestion and also according to the previous comment, the paragraph was deeply modified. The new draft is included on pages 6, lines 209-214 of the reviewed version of the MS.
Line 189-194. Whole this part (“The most … fermentative activity”) must be meaningfully rewritten.
Following the reviewer’s suggestion, on page 7, lines 215-221, the referred paragraph was redrafted for a more understandable version.
Line 197: “… pre-cursor…” replace with “…precursor…”
Following the reviewer’s suggestion, on page 7, line 225, “pre-cursor” has been replaced by “precursor).
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
I have no additional suggestion/comments.
