Encapsulation of Combi-CLEAs of Glycosidases in Alginate Beads and Polyvinyl Alcohol for Wine Aroma Enhancement
Round 1
Reviewer 1 Report
The manuscript catalysts-1301569 presents biochemical data to support the effectiveness of a new methodology for wine aroma enhancement.
The topic is original, and the information contained is relevant for the research in this field. The methodological approach is correct and adequate to the research. The manuscript is well organized and well written.
The discussion is sufficiently argued. I consider that this manuscript is appropriate for publication in Catalysts after little implementation and a rereading by the Authors, to resolve some typos.
The evidence provided is convincing, but a statistical support is needed to validate the differences obtained. The Authors stated that they have performed a statistical analysis and reported the related results in the Supplementary Information (sections 1.2 and 2.2). But I don't see this part in the manuscript. A description of how the statistical analysis was performed should be included in the materials and methods section.
Also, in the Supplementary Materials section it is written that this material (figures and tables) is available online, but I don't see any trace of it.
In any case, in this part, reference is made to an analysis of variance with numerosity n = 2. I imagine that this sample size is the same as the graphs in Figures 1 and 2. If so, it is my opinion that this is a bit too limited. Authors should add at least one more replica to arrive at n = 3, and report statistically significant differences in the graphs.
As for the graphs of Figure 3, I don't see any error bars. Does this mean that the measurement was only performed once? If so, also in this case the Authors should add a number of replicates to arrive at least n = 3, perform the statistical analysis, and report the statistically significant differences in the graphs.
Finally, I suggest moving the materials and methods section either after the introduction or after the conclusions.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
The article provides data on the use of combi-СLEAs immobilized in alginate or polyvinyl alcohol beads for the aromatic expression of wine. The results are interesting and may be important for the wine industry.
I believe the paper should be published after some corrections:
- Was the release of enzymes from the beads into the liquid phase observed? Especially in the case of using alginate balls, which turned out to be unstable.
- The study showed that with a decrease in the average diameter of the beads, the specific activity increases. Is there a reasonable limit on the minimum diameter of the beads?
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript entitled “Encapsulation of combi-CLEAs of glycosidases in alginate beads and polyvinyl alcohol for wine aroma enhancement”, presented a study to produce combined glycosidases cross-linked and encapsulated in two different materials (alginate and polyvinyl alcohol). The novelty of the study is based on the selected materials for encapsulated the combi-CLEAs. Finally, the physical integrity of the materials was tested in a white wine. The study is well structured and written. Different concerns about this manuscript are:
- A description of the “residual activity” calculus should be provided in the materials and methods section.
- The authors should provide an explanation of why choose these concentrations (1 g/L / 1 g/L / 50 g/L) used in operational stability assays. According to the supplier instructions (Rapidase Revelation Aroma commercial preparation) should be added in wine between 1 to 4 g/hL.
- There is too much information not essential or marginal results provided as supplementary material, taking into account that this is not a characterization or an optimization study of materials.
The following considerations may be taken into consideration by the authors:
- Title: In my opinion, the title of the paper could be improved. As a suggestion: “Encapsulation of glycosidases by combined cross-linked enzyme aggregates in alginate beads and polyvinyl alcohol for white wine aroma enhancement”.
- Page 1, line 20: Please complete this sentence as “Afterwards, the stability of the biocatalysts was tested in a white wine”.
- Page 2, line 52: Please correct this sentence “…(CLEAs or combi-CLEAs in the case of the co-immobilization of all the enzymes enzymes)[9, 10]”.
- Page 2, line 75: “…used as carrier for enzyme immobilization in numerous applications…” Please clarify if this material (PVA) can be used for food applications.
- Page 2, lines 79-82: This paragraph has not been properly justified with a reference, for instance [4-6]
- Page 3, lines 96-97: Please change the caption of figure 1. “Effect of combi-CLEAs loading in alginate beans on the expressed specific activity of βG (a) and ARA (b), in beans of two sizes: 2.8 mm average diameter (▲) and 4.2 mm average diameter (●)”.
- Page 4, lines 136-137: Please change the caption of figure 2. “Effect of combi-CLEAs loading in polyvinyl alcohol hydrogel particles (PVA) on the expressed specific activity of βG (a) and ARA (b), in PVA particles of two sizes: 2.6 mm average diameter (▲) and 6.0 mm average diameter (●)”.
- Page 5, line 192: Please change this subtitle “3.1. Hydrolytic activity assay” by “3.1. Specific activity analysis (SA)”.
- Page 6, line 251: Please complete this sentence as “Samples were taken periodically measuring βG and ARA residual activities”.
- Supplementary information, page 2: It is confusing to use “lentikats” instead of polyvinyl alcohol in this section, select only one, and should be used consistently throughout all the text.
- Supplementary information, pages 2 and 3: Please, move the tables S1, S2, S3, and S4 to the results section.
- Supplementary information, pages 5 to 7: Please, provide p-values consistently in the tables. Change values as “1.51 × 10-4” by “< 0.0001”.
Author Response
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Author Response File: Author Response.pdf
