Seaweed (Laminaria digitata) and Honey Kombucha: A Fermented Antioxidant-Rich Beverage

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in yellow in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

The subject "kombucha" is an interesting food due to its potential health benefits.

However, the hypothesis of substitution of sugar with honey and inclusion of seaweed as well as other adjuncts should be reconstructed, with optimisation data. The statistical evaluation is missing; and valuable analysis techniques such as PCA, HCA or their heatmap should be conducted to improve the quality of the research. (Main comments in attached file).

Response: More relevant information around statistics has been incorporated where applicable.

Comments

1.     The study seems to be conducted to fulfil the gap of product formulation; but needs optimisation studies (i.e. RSM)

Response 1: Indeed, we acknowledge the need for employing optimization tools such as RSM to understand the interplay of various factors such as batch time, level of adjuncts, etc. In this study, we only looked at one factor at a time and hope to standardize the formulation with more complex tools in the future.

2.     If the aim is to evaluate the effect of seaweed and honey addition on quality parameters of kombucha beverage, why ginger and lavender are also included in the formulation.. since seaweed, ginger and lavender are adjuncts - sugar and honey are sweetening agents.

Response 2:  The objective of this study has been clarified to include formulating a novel kombucha beverage that incorporates adjuncts such as seaweed (Laminaria digitata), cinnamon, ginger, lavender, as well as an alternate sugar source in the form of local honey and further evaluating its antioxidant capacities and sensory qualities against traditional kombucha formulations. This has been edited in the draft. Since Ireland has locally harvested seaweed and is known for phytochemical-rich local honey, these were prioritized in the discussions over other adjuncts.

3.     these parameters are basic determinants for fermentation ongoing.. however to support the hypothesis of the present research there needs further analysis; ie aminoacid profile, fatty acid composition,  organic acids, bioavailability or phenolic profile.It is ineviatable that all these mentioned parameters will change according to additives included in formulation

Response 3: The presented research was more focused on processing parameters and their impact on overall antioxidant activity screening. We acknowledge the importance of compositional differences and are conducting further studies involving an integrated omics approach to thoroughly understand microbial composition and post-fermentation metabolites.

4.     Preparation of kombucha is well described, however in further tables and results section plain kombucha and commercial kombucha (V / V shot) has been mentioned. They must be control samples. Why they are not mentioned here.

Response 4: V/V shots are commercial kombucha samples, and due to a confidentiality agreement, details cannot be disclosed. These samples displayed low antioxidant activity and were not included in further investigations.

5.     why ginger, cinnamon and lavender are added. how did you decide the addition ratio for each adjunct

Response 5: Ginger, cinnamon and lavender are phytochemical rich adjuncts known to possess anti-inflammatory and anti-oxidant potential. Also lavender is a locally grown and available just as seaweed and honey chosen in the study. The excipients were used as follows; Ginger and seaweed were added at 0.15% while lavender and cinnamon were added at 0.05%. The addition ratios were determined based on pre-trials with sensory testing, ensuring kombucha's aroma and flavor were acceptable. Information on similar adjuncts by other researchers has been added, supporting the chosen concentrations. (DOI 10.2298/CICEQ120205048V), added to the manuscript.

6.     here statistical analysis has been given; however in tables/text the statistical evaluation is not mentioned

Response 6: Statistical analysis results have been documented more clearly, with significant results highlighted while comparing antioxidant activity, as shown in Table 4, where significant differences are indicated by different letters according to Tukey's test

7.     explain the sensory properties in detail.. the effect of ginger, cinnamon, lavender and seaweed addition.. why only mention honey and sugar substitution

Response 7: , HCK, SCK, SLK were also analyzed for sensorial preferences, and this has been added to the text.

8.     Why it is mentioned ? Did you conduct any shelflife analysis in your current research ? (Line 300)

Response 8: Yes, preliminary sensory studies were conducted on the kombucha samples. The stored bottles at 4 ℃ were checked weekly for signs of spoilage, such as mold or off odors as well as checked via FRAP assay for antioxidant activity. This study found that kombucha maintained its quality and antioxidant properties for three months.

9.     Although there are many studies on kombucha from different substrates and with different additives, the discussion section is quite weak due to unsatisfactory experiments

Response 9: Further references have been added to strengthen the discussion section.

10.  explain the relevance of seaweed? Is it true to conclude this without considering bioavailability?

Response 10: Many bioactive compounds are found in Laminaria digitata, including polyphenols such as flavonoids. Bioactive peptides, sulphated and non sulphated carbohydrates as well as pigments have been reported to present in this seaweed. While bioavailability is an important component, often there is little correlation between invitro and invivo models for bioavailability. Quantifying postbiotics and bioavailability using validated animal models will strengthen scientific evidence and will be pursued in future studies.

11.  explain and discuss please the other additives used (Line 318)

Response 11: Details on the role and impact of other additives has been expanded in the materials & methods section.

12.  ginger, cinnamon and lavender have antimicrobial activity, aside serving as flavouring agent. did you take this into consideration

Response 12: Since the SCOBY was able to ferment the sugar containing Kombucha, we did not delve into the reported antimicrobial activity. We acknowledge that it might impact some microbes more than the others which in turn might impact the final product. However, at the concentrations used, our culture didn’t get significantly impacted (weight and diameter of SCOBY consistently increased during the length of fermentation).

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in yellow in the resubmitted file.

2. Point-by-point response to Comments and Suggestions for Authors

This paper reviews the mechanisms of food packaging monomers’ migration into food matrix and the implications on human health. It has certain significance and value. This paper studies the antioxidant properties and sensory qualities of kelp (Laminaria digitata) and honey-fermented kombucha, which is innovative. Kombucha, as a functional beverage with potential health benefits, has received a lot of attention in recent years. By introducing ingredients such as kelp and honey, it is of great significance to study their effects on the antioxidant capacity and flavor of kombucha, which is of great significance for the development of new health drinks. However, this paper need to be revised. The specific comments are addressed as follows:

Comments

1. INTRODUCTION: Mechanistic hypotheses on the synergistic effects of seaweed and honey in kombucha (e.g., polyphenols interacting with probiotics) need to be added to enhance the elaboration of the research motivation.

  Response 1: This has been added via the following lines in introduction: Polyphenol-rich fermented foods (PFFs) such as Kombucha are reported to have better bioavailability and bioactivity than polyphenol-rich foods such as tea, honey or seaweed by itself. This is because polyphenolic substrates are hydrolyzed into smaller more bioavailable and bioactive phenolic compounds (such as quercetin, kaempferol, gallic acid, ellagic acid, etc.) formed during food fermentation by polyphenol-associated enzymes [10].

1. Addition of excipients: It is not clear whether the proportion of excipients (e.g. seaweed, cinnamon) added is based on literature or pre-tests, and additional evidence is needed.

Response 2: The excipients were used as follows; Ginger and seaweed were added at 0.15% while lavender and cinnamon were added at 0.05%. This was primarily done using pre-trials where sensory testing was done to ensure aroma and flavour of the kombucha was acceptable. Other researchers who have used adjuncts such as stinging nettle for example have used excipient at 0.25% (DOI 10.2298/CICEQ120205048V).

1. Sensory evaluation: the sample size of only 10 people may affect the representativeness of the results, it is recommended to expand the sample size or explain the limitations.

Response 3: The authors acknowledge the small sample size and its limitations. Although expanding is not feasible for this study, future evaluations will consider a larger, more diverse panel. Our study included males and females between the age group of 18 -55 years at the school of food science & environmental health at TU Dublin.

1. DISCUSSION: Some of the results (e.g., low sensory scores of HKG) were shallowly analyzed and need to be further interpreted in the context of specific flavor compounds (e.g., volatiles of honey versus pungent components of ginger).

Response 4: Though ginger sugar fermented Kombucha is widely available in the marketplace, our study showed that the combination of honey and ginger led to less preferable profiles. This could also be a result of biotransformation of ginger volatile compounds during fermentation that altered the flavor of Kombucha unfavorably. Modification or transformation of compounds by bacteria and yeast during fermentation is a known phenomenon (https://doi.org/10.3390/foods13121947 )

More in-depth metabolomics studies will be carried out as an extension of this work.

1. Line 11, "synbiotic" is misspelled.

Response 5: In line 11 we want to say that synbiotic effects of Kombucha made using adjuncts i.e combination of pre and probiotics will be present in the beverage.

1. Line 17 "DPPH", "TE" appears for the first time, please write the full name.

Response 6: 2,2-Diphenyl-1-picrylhydrazyl (DPPH) inhibition, Trolox Equivalent and Ferric reducing antioxidant power (FRAP) full forms have been added in the abstract.

1. Picture 1 Please change the background to the same color.

Response 7: All figure backgrounds have been standardized in color.

1. Line 156 "FRAP", line 160 "TPTZ" first appear, please write the full name.

Response 8: Full form 2,4,6-Tripyridyl-s-triazine, has been added.

1. Line 160 and line 162, "H2O" writing method is not standard, please correct.

Response 9: Chemical notations have been standardized.

The data in Table 3 ，please deal with significant differences to make the data more intuitive.

Response 10: Table 3 has been enhanced with significance markers and an additional table highlighting these.

The number of references in the paper is not enough, please increase the number of references.

Response 11: More references have been incorporated.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted in yellow in the resubmitted manuscript.

2. Point-by-point response to Comments and Suggestions for Authors

The objective of this study is to formulate a novel kombucha beverage that incorporates both Laminaria digitata and honey, evaluating its antioxidant capacities and sensory qualities against traditional kombucha formulations. There are several issues in this paper.

Comments

(1) lines 71-88 It should be a paragraph.

Response 1: The sentences have been merged into a single coherent paragraph.

(2) Recent trends in kombucha production have seen innovative approaches that incorporate a variety of ingredients beyond traditional tea and sugar. Add references.

Response 2: In the material and methods section other adjuncts such as food byproducts (pecan shells; soapberry; sugar cane syrup; hydrolysate of pomace, pineapple, and pear peel; pomace; sweet sorghum; cellar residues) and stinging nettle have been reported to be used by other researchers.

(3) The integration of Laminaria digitata, a type of brown seaweed, into kombucha production has been a recent focus of many product formulation studies. Add more discussion and references.

Response 3: Discussions reflecting the rich micronutrient profile of Laminaria digitata and its contribution to antioxidant properties have been expanded as follows: This significant antioxidant potential can be associated with the rich micronutrient profile of honey as well as the bio-actives present in Laminaria digitata. According to one study [29], the incorporation of honey enhances the phenolic content of the final beverage, contributing to its antioxidant profile. Additionally, the bioactive compounds found in Laminaria digitata, including polyphenols such as flavonoids [30], further enhance overall antioxidant composition and support the health-promoting attributes of the kombucha.

(4) Figure 1. Delete

Response 4: Since the other reviewers have mentioned editing the legends of this figure and keeping it, it hasn’t been deleted. It could be added as a supplementary file and the authors would wait for the editor suggestion on this.

(5) Figure 2. Where is its discussion? Redraw with the professional drawing software. 

Response 5: Figure 2 has been reconstructed in excel and the scale has been changed for better visualisation

(6) 3.8. Shelf-life Analysis No data for this study was found in this section. Do you have experimental data?

Response 6: The stored bottles at 4 ℃ were checked weekly for signs of spoilage, such as mold or off odors. This study found that kombucha maintained its quality and antioxidant properties for three months when stored in glass bottles under refrigerated conditions in agreement with reported studies.