Investigation of the Influence of Different Camellia sinensis Teas on Kombucha Fermentation and Development of Flavored Kombucha with Brazilian Fruits
, Amanda Lins Bispo Monteiro 2, Camila Macaúbas da Silva 2, Tania Maria Sarmento da Silva 2, Rodrigo Lira de Oliveira 3,*, Camila Souza Porto 4 and Tatiana Souza Porto 5,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Review report: beverages-3355093
Investigation of the influence of different Camellia sinensis teas on kombucha fermentation and development of flavored kombucha with Brazilian fruits
Brief summary of the content
The present work involves experimental studies on a small scale, which can be divided into 2 parts. In the first part, 10 individual tea fermentations were carried out, each with 30% of an unknown/non-characterized starter liquid, and individual chemical parameters were measured after 10 days. In the second part, a green tea fermented was mixed with 6 different fruit concentrates and incubated for a further 3 days. The total phenolic content and the flavonoid content of these batches were then analyzed.
General concept comments
It is an interesting topic and the manuscript is generally easy to read. However, if you take a closer look at the experimental procedure and try to reproduce the experiments, there are significant ambiguities and some deficiencies.
Article: Overall, it must be said that the studies are experiments that are unfortunately inadequately characterized and not sufficient defined from a scientific point of view. This makes it impossible to repeat the studies and an adequate evaluation of the data presented is also not possible due to the complete lack of analyses of the raw materials and starting points. When discussing the results, existing knowledge from other studies was mainly listed and the authors' own results were too rarely placed in a scientific context. Many fundamental connections regarding the processes during fermentation or the effects of the ingredients contained were too often merely speculated, which in turn can be traced back to the lack of own analyses. The following are concrete examples of the points just listed and other conspicuous features in the manuscript.
Form:
Unfortunately, the document has incorrect page numbering and no line numbering from page 12 onwards.
The only graphic (Figure 1) is illustrative and appealing, but the data is similar in content to the data in Table 1.
The significance of the qualitative analysis results in Table 3 is unfortunately limited, as the mere presence of the listed components does not bring any significant added value or new findings. From a scientific point of view, it would be appropriate to add reference measurements of the raw materials themselves and ideally to evaluate any changes during processing / fermentation.
Content:
Insufficient definition and characterization of the processes: Over 30% (30% liquid plus an undefined amount of bacterial cellulose) was used for the inoculation of the fermentations. This proportion is not addressed at all and the reference to a commercial product is insufficient. The scientific expert is aware that individual batches of such undefined "SCOOBY" including liquid can differ significantly in the quality and quantity of the microorganisms contained (variation in the composition, viability and vitality of the yeasts and AAB contained), the acid and sugar concentrations and possibly the ethanol concentrations. Manufacturers of such preparations do not normally provide precise specifications, as they cannot do so sufficiently due to the existing dynamics in the preparations, which is why a separate characterization is unavoidable. From the point of view of good scientific practice, it would have been necessary at least to characterize the starting liquids in terms of their comparability for the studies carried out here. This could be done, for example, by a profile of the chemical composition (sugar content, organic acids, EtOH) and ideally a microbial (concentration of yeasts and AAB, possibly LAB) evaluation.
Due to the situation described, the analysis of bacterial cellulose described under 2.3 and 3.3 is also useless, as measurements were only taken at one point and no statement can be made about the starting conditions, and thus no real statement about the cellulose production. The masses of the commercial SCOOBY can differ significantly. In order to evaluate this, a preliminary test with exemplary measurements of a certain number would have helped to evaluate the deviation.
In this context, the title under 2.3 is also inappropriate, as even with a statement about a real increase in BC, only one specific metabolic process of part of the microbiology (certain AAB) contained can be evaluated; other microbial activities are not addressed at all.
No possibility to evaluate the respective process and the repeatability: In the studies, none of the raw materials and starting materials were characterized (starter liquid, various teas, fruit components) or any statements on their content were made. This situation is incomprehensible. Also, no analysis was carried out at the start of the respective fermentation processes. At no point was there a microbiological analysis and concrete analyses of essential primary metabolic products (EtOH, gluconic acid, acetic acid, possibly lactic acid) and of the sugar content are not available, so that no indirect conclusions can be drawn about the dynamics of the complex microbiology contained. However, all of this would not only have been interesting from a technological point of view, but from the reviewer's opinion it would also have been necessary for this scientific work. It is also not clear to the reader why 10 days were chosen. As mentioned, in addition to the essential characterization of the starting conditions, a certain amount of process-related analysis would have been desirable. It can be assumed that the 10 days were not chosen arbitrarily, so information about the choice would be important. If this is the case for sensory reasons, then it would be necessary to explain why there is no sensory description in either part of the study.
Further points:
- Line 198: X days at X °C ??
- Line 276: The statement that the influence on metabolic components during fermentation was verified is, in the opinion of the reviewer, inappropriate. This would have to be proven concretely.
- Discussion: “…which impacts the presence and stability of bioactive compounds essential for the health benefits of kombucha” à Especially when statements are made about the “health benefits” of kombucha, this should not be done so superficially, as the available studies are still insufficient. A discussion here should not only be more specific, but should also try to better incorporate one's own results.
- In principle, an attempt should be made to make less superficial statements in the discussion. For example, comparisons such as the stated 3,206 g/L BC from another study are meaningless, since it is not mentioned at all whether and how the data are comparable (initial situation, process conditions, etc.).
- The statement made in the summary that green tea is the most suitable is not sufficiently clear from the results and the discussion. This would have to be critically evaluated, also taking into account the underlying and missing quality criteria.
Conclusion:
Based on the reasons described, this work is considered too incomplete to meet the level of good scientific practice that the reviewer sees for this journal. The snapshot of 10 days fermentation presented within the complex kombucha fermentations provide insufficient information to adequately evaluate the studies or even to repeat them. From a scientific point of view, no significant gain in knowledge for the topic or benefit for practice can therefore be identified.
Author Response
ANSWERS TO THE SUGGESTIONS OF REVIEWER 1
(All changes in the manuscript to satisfy the reviewers’ suggestions are written in red)
Review report: beverages-3355093
Investigation of the influence of different Camellia sinensis teas on kombucha fermentation and development of flavored kombucha with Brazilian fruits
Brief summary of the content
The present work involves experimental studies on a small scale, which can be divided into 2 parts. In the first part, 10 individual tea fermentations were carried out, each with 30% of an unknown/non-characterized starter liquid, and individual chemical parameters were measured after 10 days. In the second part, a green tea fermented was mixed with 6 different fruit concentrates and incubated for a further 3 days. The total phenolic content and the flavonoid content of these batches were then analyzed.
General concept comments
It is an interesting topic and the manuscript is generally easy to read. However, if you take a closer look at the experimental procedure and try to reproduce the experiments, there are significant ambiguities and some deficiencies.
Article: Overall, it must be said that the studies are experiments that are unfortunately inadequately characterized and not sufficient defined from a scientific point of view. This makes it impossible to repeat the studies and an adequate evaluation of the data presented is also not possible due to the complete lack of analyses of the raw materials and starting points. When discussing the results, existing knowledge from other studies was mainly listed and the authors' own results were too rarely placed in a scientific context. Many fundamental connections regarding the processes during fermentation or the effects of the ingredients contained were too often merely speculated, which in turn can be traced back to the lack of own analyses. The following are concrete examples of the points just listed and other conspicuous features in the manuscript.
Form:
Unfortunately, the document has incorrect page numbering and no line numbering from page 12 onwards.
Answer: We apologize for the mistake in page numbering.
The only graphic (Figure 1) is illustrative and appealing, but the data is similar in content to the data in Table 1.
Answer: Dear reviewer, the Figure 1 corresponds to the contour plots obtained from the data presented in Table 1 for each dependent variable studied. Therefore, they are not the same data. Table 1 shows the experimental data and Figure 1 shows the models calculated with the data obtained experimentally. The mentioned Figure contributes directly from the suitable visualization of the influence of mixtures of different C. sinensis teas, in particular the optimum regions, on the experimental design responses facilitating the reader's understanding.
The significance of the qualitative analysis results in Table 3 is unfortunately limited, as the mere presence of the listed components does not bring any significant added value or new findings. From a scientific point of view, it would be appropriate to add reference measurements of the raw materials themselves and ideally to evaluate any changes during processing / fermentation.
Answer: This research has a more than scientific nature, as it is research applied to real products that are commercialized. Kombucha goes through two fermentation processes, an initial one that was studied and presented in this manuscript. After this process, fruit extracts were added for the second fermentation, after which the kombucha is suitable for consumption. In this way, the addition of fruit extracts is commonly used in commercial Kombuchas, highlighting that in Brazil fruits are widely used due to the culture of drinking fruit juices. The qualitative analysis of flavored kombuchas is relevant to identifying the bioactive compounds present. The analysis of the compounds in the fruits is already reported in the literature, but which of these compounds are present in the final composition of the kombuchas after two fermentation processes. Moreover, no changes were observed in the profile of phenolic compounds in flavored kombucha beverages as observed in Table 3.
Content:
Insufficient definition and characterization of the processes: Over 30% (30% liquid plus an undefined amount of bacterial cellulose) was used for the inoculation of the fermentations. This proportion is not addressed at all and the reference to a commercial product is insufficient. The scientific expert is aware that individual batches of such undefined "SCOOBY" including liquid can differ significantly in the quality and quantity of the microorganisms contained (variation in the composition, viability and vitality of the yeasts and AAB contained), the acid and sugar concentrations and possibly the ethanol concentrations. Manufacturers of such preparations do not normally provide precise specifications, as they cannot do so sufficiently due to the existing dynamics in the preparations, which is why a separate characterization is unavoidable. From the point of view of good scientific practice, it would have been necessary at least to characterize the starting liquids in terms of their comparability for the studies carried out here. This could be done, for example, by a profile of the chemical composition (sugar content, organic acids, EtOH) and ideally a microbial (concentration of yeasts and AAB, possibly LAB) evaluation.
Answer: Kombucha fermentation was carried out numerous times. Sugar, pH, EtOH and organic acid analyses were carried out on all occasions. In this way, it was defined through previously performed experiments that 30% would be the ideal amount of fermented liquid to inoculum the fermentation process. It was also defined prior to this study in partnership with the industry that there is no need to add SCOBY during fermentation. All kombuchas were evaluated for ethanol content, as according to the legislation this drink is non-alcoholic and must have an ethanol content of <0.05%. Both unflavored and flavored kombuchas comply with the legislation. Microbiological analysis of the complex (SCOBY) is not often performed. However, this step will be performed in a partner laboratory next year and these results will be part of a new publication. Moreover, the microbiological analysis of kombucha was not the objective of this study.
Due to the situation described, the analysis of bacterial cellulose described under 2.3 and 3.3 is also useless, as measurements were only taken at one point and no statement can be made about the starting conditions, and thus no real statement about the cellulose production. The masses of the commercial SCOOBY can differ significantly. In order to evaluate this, a preliminary test with exemplary measurements of a certain number would have helped to evaluate the deviation.
Answer: Dear reviewer, during the production of the kombucha beverage (first fermentation), the SCOBY was not used, so the process was carried out without SCOBY, only a previously prepared kombucha as inoculum for fermentation, this information was added to manuscript (Lines 97 to 99). The SCOBY mass evaluated was only that of the SCOBYs that form naturally after each fermentation., In this case, the SCOBYs formed in each mixture design condition were evaluated in terms of cellulosic biomass produced and its relationship with the types of teas.
In this context, the title under 2.3 is also inappropriate, as even with a statement about a real increase in BC, only one specific metabolic process of part of the microbiology (certain AAB) contained can be evaluated; other microbial activities are not addressed at all.
Answer: As suggested by the reviewer, the title of item 2.3 has been changed to a more appropriate title that encompasses the analyses performed, given that the aim of this item was to verify the changes in the weight of bacterial cellulose formed during the fermentation of kombucha by different C. sinensis teas (Lines 128 to 133).
No possibility to evaluate the respective process and the repeatability: In the studies, none of the raw materials and starting materials were characterized (starter liquid, various teas, fruit components) or any statements on their content were made. This situation is incomprehensible. Also, no analysis was carried out at the start of the respective fermentation processes. At no point was there a microbiological analysis and concrete analyses of essential primary metabolic products (EtOH, gluconic acid, acetic acid, possibly lactic acid) and of the sugar content are not available, so that no indirect conclusions can be drawn about the dynamics of the complex microbiology contained. However, all of this would not only have been interesting from a technological point of view, but from the reviewer's opinion it would also have been necessary for this scientific work.
Answer: Dear reviewer, as answered in the previous question, the fermented liquid was evaluated in previous studies (pH, ethanol, acidity, etc.). The raw materials are well described in the literature and were not the objective of this study. In industrial practice, numerous raw materials are used for fermentation as well as flavoring. In this study, we do not intend to explain the metabolism of the different processes of the SCOBY complex during the fermentation of Kombucha. The objective was to select raw materials and improve the process based on the presence of bioactive molecules. We can cite some articles in the literature that characterized the compounds present C. sinensis teas and some fruits used for kombucha flavoring (Prasanth et al, 2019. DOI:10.3390/nu11020474; Aboulwafa et al, 2019. DOI:10.3390/antiox8100455; Kuzu et al, 2023. DOI:10.20944/preprints202306.0850.v1; Júnior et al, 2022. DOI:10.1016/j.crfs.2022.01.023).
It is also not clear to the reader why 10 days were chosen. As mentioned, in addition to the essential characterization of the starting conditions, a certain amount of process-related analysis would have been desirable. It can be assumed that the 10 days were not chosen arbitrarily, so information about the choice would be important. If this is the case for sensory reasons, then it would be necessary to explain why there is no sensory description in either part of the study.
Answer: Dear reviewer, the fermentation time can vary between one week and two months, however the FDA recommends a fermentation period of 10 days, prolonging the fermentation time can cause excessive acidification of the kombucha drink which can reduce the health-promoting properties or have adverse effects on consumers, therefore the 10-day fermentation period of kombucha in the present work was chosen based on scientific articles found in the literature (Kitwetcharoen et al; 2023. DOI: 10.3390/fermentation9010048; Amarasinghe et al, 2017. DOI: 10.1002/fsn3.605; Malbaša et al; 2011. DOI:10.1016/j.foodchem.2011.02.048). Concerning sensory analysis, the results presented in this paper are part of a larger research, where also the sensory evaluation of flavored kombuchas was carried out using the CATA and RATA techniques. From the obtained data, an article focused exclusively on sensory analysis is in the final stage of writing.
Further points:
Line 198: X days at X °C ??
Answer: This information are concerning the time (days) and temperature used in the green tea kombucha fermentative processes and were added to the caption of Table 1.
Line 276: The statement that the influence on metabolic components during fermentation was verified is, in the opinion of the reviewer, inappropriate. This would have to be proven concretely.
Answer: Dear reviewer, this observation was not verified in any place of manuscript. Being the detailed evaluation of metabolic pathways of fermentative process and the formation of compounds with biological activity are not objectives of the present study.
Discussion: “…which impacts the presence and stability of bioactive compounds essential for the health benefits of kombucha” à Especially when statements are made about the “health benefits” of kombucha, this should not be done so superficially, as the available studies are still insufficient. A discussion here should not only be more specific, but should also try to better incorporate one's own results.
Answer: Changes have been made to the text to clarify the discussion about the influence of different teas on kombucha production, where the aim of the present work focused on observing the physicochemical behavior of kombucha samples (Lines 288 to 294).
In principle, an attempt should be made to make less superficial statements in the discussion. For example, comparisons such as the stated 3,206 g/L BC from another study are meaningless, since it is not mentioned at all whether and how the data are comparable (initial situation, process conditions, etc.).
Answer: Dear reviewer, with regard to the evaluation of the influence of different C. sinensis teas on the bacterial cellulose formed, the results obtained in the present work were better presented, reporting both the formulations of the trials that obtained the highest values and also these values, in addition to comparisons with other studies and justifications for this increase or decrease in the production of bacterial cellulose (Lines 401 to 412).
The statement made in the summary that green tea is the most suitable is not sufficiently clear from the results and the discussion. This would have to be critically evaluated, also taking into account the underlying and missing quality criteria.
Answer: The manuscript presents results regarding the influence of C. sinensis teas on the variables total phenolic content and antioxidant capacity against the radicals DPPH and ABTS, where it was observed that for these highlighted variables the formulation that has only green tea presented higher values for these, thus highlighting in the text the choice of green tea to continue to the kombucha flavoring stage (lines 319 to 329).
Conclusion:
Based on the reasons described, this work is considered too incomplete to meet the level of good scientific practice that the reviewer sees for this journal. The snapshot of 10 days fermentation presented within the complex kombucha fermentations provide insufficient information to adequately evaluate the studies or even to repeat them. From a scientific point of view, no significant gain in knowledge for the topic or benefit for practice can therefore be identified.
Answer: Dear reviewer, we appreciate your contributions to improving the manuscript. Views on quality also depend on experiences in the areas of professional practice. We have made the changes suggested by all reviewers and respect all points of view raised by the reviewers.
Reviewer 2 Report
Comments and Suggestions for Authors
The manuscript addresses the optimization of Kombucha beverages production in a double approach. The first one is the use of the statistical mixture designs to study the interactions between different components of the raw materials (teas) mixture to maximize the response. In addition, local Brazilian fruits were tested for their influence on the final product, which brings a certain level of novelty and may lead to the development of new commercial products. Generally, the methodology was presented in sufficient detail and was appropriate and the conclusions are well supported by the results.
One problem is, that, starting from page 10 the lines are not anymore counted, so, it is difficult to make observations/comments on the manuscript.
Here are some observations to be taken into account:
Introduction: please provide some more information on other plants/fruits used to obtain flavored kombucha
Line 138: please be more specific on what does it means “extracts from various fruits”. What kind of extracts? How the fruits were prepared? What was the origin of the fruits?
Table 2: try to keep the equation in the same line
Line 248: you wrote “with increasing pH, there is a decrease in acidity”. Please add a note that this is normal, nothing surprising.
Line 250: you stated “positively favors the production of kombucha”; can you explain on which side/parameter is positive?
Lines 251-269: you have dedicated many explanation on how the interpret the blend counter plots, which is good; however, when you come to the interpretation itself, your inputs are scarce. Please provide more details on the results.
Table 3: the tile is unclear, because A, B…G letters representing the variant can not be found inside the table. Which compound correspond to which variant?
Discussions section (which has no counted lines):
- For the pH evolution you emphasize that several studies reported similar results, but you provide only one reference [24]; please complete
- Regarding the BC (Bacterial Cellulose) please explain why it is important for the development of kombucha commercial beverages
- “In plants they are known as UV protectants”: what do you mean by “they”?
- Please provide clear examples of other fruits used in combination with kombucha and compare with your results.
Author Response
ANSWERS TO THE SUGGESTIONS OF REVIEWER 2
(All changes in the manuscript to satisfy the reviewers’ suggestions are written in red)
The manuscript addresses the optimization of Kombucha beverages production in a double approach. The first one is the use of the statistical mixture designs to study the interactions between different components of the raw materials (teas) mixture to maximize the response. In addition, local Brazilian fruits were tested for their influence on the final product, which brings a certain level of novelty and may lead to the development of new commercial products. Generally, the methodology was presented in sufficient detail and was appropriate and the conclusions are well supported by the results.
One problem is, that, starting from page 10 the lines are not anymore counted, so, it is difficult to make observations/comments on the manuscript.
Answer: We apologize for the mistake in page numbering.
Here are some observations to be taken into account:
Introduction: please provide some more information on other plants/fruits used to obtain flavored kombucha
Answer: As requested by the reviewer, more information and examples of kombucha flavored beverages are added to the Introduction section (Lines 81 to 82).
Line 138: please be more specific on what does it means “extracts from various fruits”. What kind of extracts? How the fruits were prepared? What was the origin of the fruits?
Answer: The fruits used in this study were purchased from a cooperative of producers, who normally sell high-quality fruits. To formulate the fruit extracts, a proportion of 1 Kg of seedless fresh fruit pulp and 0.5 L of water was used, and the fruit was cooked at 100°C (土 2°C) for 1 hour. After cooling the concentrated fruit extracts, they were incorporated into the fermented tea. This information was added to the Section 2.6 of manuscript (Lines 155-162).
Table 2: try to keep the equation in the same line
Answer: Dear reviewer, due to the journal template was not possible to keep the equation in the same line.
Line 248: you wrote “with increasing pH, there is a decrease in acidity”. Please add a note that this is normal, nothing surprising.
Answer: As suggested by the reviewer, it was stated in the article text that this is expected behavior (Line 296).
Line 250: you stated “positively favors the production of kombucha”; can you explain on which side/parameter is positive?
Answer: This indicates that the increase in the proportion of these teas in the mixture is associated with the increase in the values of these response variables. Thus, it is possible to understand how the different types of tea and their combinations positively influence the studied variables, especially in the phenolic compounds and antioxidant capacity, considering that these variables were used as parameters to characterize the quality of kombucha as these variables are related to several health benefits indicated in the literature. Furthermore, defining optimal kombucha production conditions as well as which type of mixture is richest in bioactive compounds favors the development of optimized processes, as well as improvements for kombucha producers.
Lines 251-269: you have dedicated many explanations on how to interpret the blend contour plots, which is good; however, when you come to the interpretation itself, your inputs are scarce. Please provide more details on the results.
Answer: More details about the results obtained in the work were added to the text of the article, such as which compositions of the tests studied presented higher values for the DPPH and ABTS radicals, and what these values were. (Lines 288 to 294).
Table 3: the tile is unclear, because A, B…G letters representing the variant can not be found inside the table. Which compound corresponds to which variant?
Answer: All identified compounds can be found within the table, the letters were removed.
Discussions section (which has no counted lines):
For the pH evolution you emphasize that several studies reported similar results, but you provide only one reference [24]; please complete
Answer: More references have been added to reinforce the study of pH (Line 386 to 387).
Regarding the BC (Bacterial Cellulose) please explain why it is important for the development of kombucha commercial beverages
Answer: A more detailed explanation the relationship between cellulosic biofilm and the development of commercial kombucha beverages were add to manuscript (Lines 411 to 418).
“In plants they are known as UV protectants”: what do you mean by “they”?
Answer: The mentioned paragraph deals with flavonoids, where it was explained what the benefits are for plants and humans. So, when we talk about UV protectors, it is related to the fact that in plants, flavonoids have this function as well (Lines 491 to 497).
Please provide clear examples of other fruits used in combination with kombucha and compare with your results.
Answer: Other examples were added to the manuscript, more specifically kombucha flavored with Bacuri and Umbu-caja compared with the formation of phenolic compounds (Line 508 to 515).
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript entited: “Investigation of the influence of different Camellia sinensis teas on kombucha fermentation and development of flavored kombucha with Brazilian fruits”, evaluates the properties of teas from Camellia sinensis in kombucha fermentation, and kombuchas flavored with fruits. The topic has been also to evaluate the potential and production optimization with attention to kombucha beneficial effects of flavored beverages. The manuscript fits the scope and aims of the Journal. Some remarks to be addressed are reported in the following. The symbiotic culture of bacteria and yeast (SCOBY) used origin should be better specified. In paragraph 2.2 and 2.3, please give more details on the experimental procedures used. Please explain why the 30% extracts from the mentioned fruits were used: which is the rational behind this choice and the choice of three days (paragraph 2.6)? Please specify the origin of the used extracts and how they have been obtained. Is there are specific reason for choosing the mentioned fruits in the tea blend? At line 150 and elsewhere please specify the apparatus used. Table 1: please include the esd. The LOD and LOQ should be mentioned. While the overall experimental approach and results are properly substantiated, the rationale behind the proposed study should be better assessed as well as the possible impact in the area of interest. A sensory evaluation could be useful to be added to better assess the impact of the flavored preparations compared with the non flavored teas. The overall novelty and possible impact in the area of interst should be better exploited.
Author Response
ANSWERS TO THE SUGGESTIONS OF REVIEWER 3
(All changes in the manuscript to satisfy the reviewers’ suggestions are written in red)
The manuscript entited: “Investigation of the influence of different Camellia sinensis teas on kombucha fermentation and development of flavored kombucha with Brazilian fruits”, evaluates the properties of teas from Camellia sinensis in kombucha fermentation, and kombuchas flavored with fruits. The topic has been also to evaluate the potential and production optimization with attention to kombucha beneficial effects of flavored beverages. The manuscript fits the scope and aims of the Journal. Some remarks to be addressed are reported in the following.
The symbiotic culture of bacteria and yeast (SCOBY) used origin should be better specified.
Answer: Regarding the origin of the colony used for kombucha fermentation, the culture starter was purchased from Indupropil Indústria e Comércio Ltda. (Ijuí, RS, Brazil), this information was added to the article (Line 96 to 97).
In paragraph 2.2 and 2.3, please give more details on the experimental procedures used.
Answer: As requested by the reviewer, more information was added, such as procedures and equations used (Lines 119 to 127 and 128 to 133).
Please explain why the 30% extracts from the mentioned fruits were used: which is the rational behind this choice and the choice of three days (paragraph 2.6)?
Answer: This research is being developed in partnership with a company that produces kombucha beverages, with aspects related to formulations, highlighting the choice of using 30% fruit extract based on the internal production already defined by the company, and scientific articles suggest that between 10% and 30% of fruit extracts can be used for flavoring (Crispino, et al, 2024. DOI: 10.1590/0001-3765202420230356; Júnior et al. 2021. DOI: 10.1016/j.fbio.2021.101380; Aziz et al, 2023. DOI: 10.26538/tjnpr/v7i5.10). Regarding the choice of 3 days of fermentation for flavoring kombucha, the Kombucha Brewers International (https://kombuchabrewers.org/) recommends that when performing kombucha secondary fermentation (flavoring), make sure it is brief (2-3 days) and then refrigerate to stop further fermentation, limiting additional alcohol production.
Please specify the origin of the used extracts and how they have been obtained. Is there are specific reason for choosing the mentioned fruits in the tea blend?
Answer: The fruits used were purchased from a cooperative of producers and were chosen for flavoring because they are typical Brazilian fruits and widely consumed, and the grape was selected considering that the region where the study was carried out has many wineries, thus attracting consumers and favoring the consumption and sale of kombuchas with fruits that are already known and consumed.
At line 150 and elsewhere please specify the apparatus used.
Answer: As requested by the reviewer, the apparatus used to keep the samples sheltered from light was added (Line 177).
Table 1: Please include the esd.
Answer: As requested by the reviewer, the standard deviation values were added for each studied variable, except to the bacterial cellulose content, since in mixture designs the number of experiments are reduced and not was possible calculate the standard deviation for this dependent variable.
The LOD and LOQ should be mentioned.
Answer: Unfortunately, we did not perform the quantification of the substances in this study; therefore, we cannot include the LOD and LOQ values. In this regard, we kindly request that the publication of the article be considered without this data.
While the overall experimental approach and results are properly substantiated, the rationale behind the proposed study should be better assessed as well as the possible impact in the area of interest.
Answer: More information has been added about the relevance of the study, highlighting how the fruits used add value to green tea kombucha are presented in Conclusion section (Lines 547 to 553).
A sensory evaluation could be useful to be added to better assess the impact of the flavored preparations compared with the non flavored teas. The overall novelty and possible impact in the area of interest should be better exploited.
Answer: Dear reviewer, the results presented in this manuscript are part of a larger research, where also the sensory evaluation of flavored kombuchas was carried out using the CATA and RATA techniques. From the obtained data, an article focused exclusively on sensory analysis is in the final stage of writing.
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
The reviewer appreciates that the revision of the manuscript has clarified some previously unclear points and has thus led to an improvement.
The reviewer, however, still believes that the processes used here are unfortunately not sufficiently characterized in order to 1. understand the processes sufficiently, 2. draw important conclusions for further control options and 3. (most importantly) give the possibility to reproduce the processes.
The reviewer doubts that the alcohol content remains/is below 0.05% permanently or at this particular time when fruit components are added. How do the authors come to the conclusion that the alcohol content is below this without any analyses being available? That makes no sense and is not self-evident, since the addition of the fruit components in particular has the potential to promote yeast activity and thus alcohol formation. If there are analysis results, these must be shown.
Any argument that the alcohol content was measured several times in previous studies/productions or product analyses and that the process is standardized so that these values ​​can also be applied to the products produced here would not be sufficient from the reviewer's point of view, since the studies carried out here did not take place under standardized production conditions.
The nature of the results shown here therefore corresponds, from the reviewer's point of view, more to endpoint determinations of different products (which cannot be reproduced by other scientists), with the focus on the qualitative analyses of the antioxidant components.
The reviewer does not agree with the argument that the ingredients of the fruit components can be obtained from the literature and therefore no reference measurements are necessary. In fact, the analyses carried out here only make it possible to state what the qualitative composition is at the end of the fermentations. The majority of the fermentation process remains a black box, and it is not possible to make adequate statements about whether ingredients or chemical/physical parameters are influenced in any way by the complex fermentation processes. Even if the focus was not on the microbiological side, this is a fundamental aspect of kombucha and all relevant product parameters.
The situation still remains that 30% of the beverage matrix examined here, namely the starter culture liquid, is undefined in this work. It is good to hear that only the liquid phase was used as a starter, an important fact to mention. This of course also solves the points about the bacterial cellulose increase. Nevertheless, all the points mentioned that the production processes were comprehensively characterized and analyzed in the past are useless for this work if the data are not provided here or this fact is not even explained in detail.
In principle, it should also be ensured that discussions and comparisons with other works in which kombucha was produced must always be very specific, since no kombucha production process and no kombucha product is the same. Therefore, arguments such as a selected process time of 10 days based on an FDA recommendation are very questionable, especially when it comes to practical/commercial products. Normally, the taste in combination with the chemical parameters would determine the process time. Are there really no statements on this? It is even mentioned that there were sensory tests.
The reviewer considers the described character/focus of the studies in combination with insufficient reproducibility to be insufficient in terms of scientific quality for a scientific journal.
From the reviewer's point of view, the transparency of the manuscript for publication would have to be increased. The minimum here would be that more detailed information on the starter culture liquid is given and that this is also discussed critically. This starter liquid will certainly contain tea as a base and perhaps even other nutrient-rich ingredients? This must be taken into account when presenting the design and the final composition, as this will change. In addition, the alcohol content and ideally the characteristic organic acids (acetic and gluconic acid) should also be discussed critically.
Author Response
ANSWERS TO THE SUGGESTIONS OF REVIEWER 1
(All changes in the manuscript to satisfy the reviewers’ suggestions are written in red)
The reviewer appreciates that the revision of the manuscript has clarified some previously unclear points and has thus led to an improvement.
The reviewer, however, still believes that the processes used here are unfortunately not sufficiently characterized in order to 1. understand the processes sufficiently, 2. draw important conclusions for further control options and 3. (most importantly) give the possibility to reproduce the processes.
The reviewer doubts that the alcohol content remains/is below 0.05% permanently or at this particular time when fruit components are added. How do the authors come to the conclusion that the alcohol content is below this without any analyses being available? That makes no sense and is not self-evident, since the addition of the fruit components in particular has the potential to promote yeast activity and thus alcohol formation. If there are analysis results, these must be shown. Any argument that the alcohol content was measured several times in previous studies/productions or product analyses and that the process is standardized so that these values can also be applied to the products produced here would not be sufficient from the reviewer's point of view, since the studies carried out here did not take place under standardized production conditions.
Answer: As observed by the reviewer, there were a little mistake in the previous answer about ethanol content. In fact, all drinks had an ethanol content of less than 0.5%, not 0.05%. We apologize for the error. As requested by the reviewer, results concerning the alcohol content on kombucha flavored beverages were added to Table 4 and in the corresponding section (Lines 373 to 377), their discussion (Lines 550 to 558) and the method used for ethanol quantification by HLPC (Lines 181 to 192).
The nature of the results shown here therefore corresponds, from the reviewer's point of view, more to endpoint determinations of different products (which cannot be reproduced by other scientists), with the focus on the qualitative analyses of the antioxidant components. The reviewer does not agree with the argument that the ingredients of the fruit components can be obtained from the literature and therefore no reference measurements are necessary. In fact, the analyses carried out here only make it possible to state what the qualitative composition is at the end of the fermentations. The majority of the fermentation process remains a black box, and it is not possible to make adequate statements about whether ingredients or chemical/physical parameters are influenced in any way by the complex fermentation processes. Even if the focus was not on the microbiological side, this is a fundamental aspect of kombucha and all relevant product parameters.
Answer: Dear reviewer, the fruit extracts were added in the second fermentation, that have main aim the carbonation and the development of characteristic flavours. In this stage, the main contribution of the fruits is as a carbon source for the action of yeasts on the production of ethanol and carbon dioxide. Moreover, we recognize that there may be an impact of the addition of fruits on changes in the chemical composition of the kombucha flavored beverages, so much so that in this article the specific class of phenolic compounds has already been evaluated. The reviewer's suggestion to characterize the raw materials and compare the changes in the chemical composition is very valid and will serve as a basis for seeking partnerships to evaluate more compounds present in the beverage, such as volatile compounds and amino acids, which could form part of a future study.
The situation still remains that 30% of the beverage matrix examined here, namely the starter culture liquid, is undefined in this work. It is good to hear that only the liquid phase was used as a starter, an important fact to mention. This of course also solves the points about the bacterial cellulose increase. Nevertheless, all the points mentioned that the production processes were comprehensively characterized and analyzed in the past are useless for this work if the data are not provided here or this fact is not even explained in detail.
Answer: As indicated by the reviewer, more information concerning to the chemical composition of previously used fermented kombucha used as starter were added to the manuscript (Lines 98 to 99).
In principle, it should also be ensured that discussions and comparisons with other works in which kombucha was produced must always be very specific, since no kombucha production process and no kombucha product is the same. Therefore, arguments such as a selected process time of 10 days based on an FDA recommendation are very questionable, especially when it comes to practical/commercial products. Normally, the taste in combination with the chemical parameters would determine the process time. Are there really no statements on this? It is even mentioned that there were sensory tests.
Answer: Dear reviewer, in addition to the FDA’s recommendation and the fermentation time choiced were present in the range used in various published studies (Neffe-SkociÅ„ska et al, 2017. DOI:10.1080/19476337.2017.1321588; Prasanth et al, 2019. DOI:10.3390/nu11020474; Aboulwafa et al, 2019. DOI:10.3390/antiox8100455; Kuzu et al, 2023. DOI:10.20944/preprints202306.0850.v1; Júnior et al, 2022. DOI:10.1016/j.crfs.2022.01.023) this research was performed in partnership with a Brazilian kombucha manufacturer which defined this time based on the sensory aspects of the product. We recognized that the fermentation time was a direct influence on sensory aspects of kombucha beverages, with longer times can result in excessive acidity and increase the fermented characteristic odor, which can directly impact consumer choice. However, the expertise of our industrial partnership can contour these undesired sensory aspects. We would like to highlight that an article entirely focused on the study of the sensory aspects of kombucha flavored beverages, using the CATA and RATA methods using semi-trained panels and consumers, is in the final stage of writing, the results of which will contribute to, if necessary, changes in the production process.
The reviewer considers the described character/focus of the studies in combination with insufficient reproducibility to be insufficient in terms of scientific quality for a scientific journal. From the reviewer's point of view, the transparency of the manuscript for publication would have to be increased. The minimum here would be that more detailed information on the starter culture liquid is given and that this is also discussed critically. This starter liquid will certainly contain tea as a base and perhaps even other nutrient-rich ingredients? This must be taken into account when presenting the design and the final composition, as this will change. In addition, the alcohol content and ideally the characteristic organic acids (acetic and gluconic acid) should also be discussed critically.
Answer: Dear reviewer, as known the reproducibility of studies involving kombucha beverage production are conditioned to the use of the same SCOBY, since the microbial composition is very variable and can be changed with the time and due influence of environmental conditions. Concerning the starter liquid, as mentioned in Item 2.1 , the starter culture used in the present study were prevenient from a previous kombucha fermentation, other than green tea and sucrose, no other ingredients were added. For better understanding, information about the chemical composition, such as pH, total titratable acidity and the total reducing sugar content present in the starter culture has been added to the corresponding section (Lines 98 to 99). Regardingwas alcohol content, as mentioned in a previous answer, the results obtained for each flavored kombucha beverage were added to Table 4 and a correspondent discussion was added (Lines 373 to 377 and 550 to 558). The identification and quantification of organic acids present in kombucha beverages was not the aim of the present study, therefore we do not have this data. We recognize that the presentation of these data provides valuable information, specifically concerning gluconic acid, and we are willing to develop future studies aimed to evaluate the profile of organic acids in kombucha beverages. We emphasize that the focus of this article is the development of new flavored kombucha beverages using Brazilian fruits and their bioactive properties, specifically the antioxidant capacity.
Reviewer 2 Report
Comments and Suggestions for Authors
No further comments. The authors have addressed all issues.
Author Response
We are grateful for the reviewer's comments, all of which were very pertinent to improving the article.
