Valorization of Organic Third-Category Fruits Through Vinegar Fermentation: A Laboratory-Scale Evaluation of Apples, Peaches, and Clementines

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Introduction: The descriptions of the three fruit vinegar raw materials—apple, peach, and clementine—are insufficiently detailed. Among them, there area considerable amount of researches on vinegar production using apple and peach as the raw material. Please supplement with relevant advances in research.
2. Materials and Methods: Why are different culture media used in Sections 2.2 and 2.5？
3. There is no indication of relative error in Fig. 2. 2 is suggested to be divided into four separate graphs. As described in the text, the results can be presented separately for °Brix, ethanol concentration, yeast counts, and TMA counts, with each graph comparing the differences among the three fruit types.
4.  3 exhibits the same problems as Fig. 2.
5. Section 3.4: The substantial standard deviations in the aroma measurements raise concerns about data precision. For instance, the value reported for 2,4-bis(1,1-dimethylethyl)-phenol(4.25 ± 3.69) has a standard deviation that is 87% of the mean, indicating extreme variability. How do the authors explain this and justify the robustness of their conclusions?"
6. 7: FRAP values were determined by measuring the samplesin absorbance at 593 nm, not 700nm.
7. Line 437-438: Peach show better performance in acetic fermentation, possibly because of its higher initial ethanol level, offering more substrate for acetic acid bacteria.
8. Line 515-520:The high vitamin C content in clementine may partly account for its strong antioxidant activity. The authors may consider conducting additional experiments to verify this observation or support it with references from the literature.

Author Response

Comments and Suggestions for Authors

1. Introduction: The descriptions of the three fruit vinegar raw materials—apple, peach, and clementine—are insufficiently detailed. Among them, there area considerable amount of researches on vinegar production using apple and peach as the raw material. Please supplement with relevant advances in research.

• CA: Ok, thank you for this comment. We have accordingly amended the relevant information in the introduction part (LL. 80-91).

1. Materials and Methods: Why are different culture media used in Sections 2.2 and 2.5?

• CA: We followed the suggested media provided by DSMZ for reviving freeze-dried forms of S. cerevisiae DSM 1848 and G. oxydans DSM 7145, as well as for stocking them for their use in laboratory. We did not use the same media (Yeast Malt for yeasts, Yeast Peptone Mannitol for acetic acid bacteria) when estimating cell density of the inoculated microorganisms during fermentation. For that aim, we instead preferred agar media that are more commonly used, namely Sabouraud Dextrose and Glucose Yeast Carbonate, respectively for yeasts and acetic acid bacteria.

1. There is no indication of relative error in Fig. 2. 2 is suggested to be divided into four separate graphs. As described in the text, the results can be presented separately for °Brix, ethanol concentration, yeast counts, and TMA counts, with each graph comparing the differences among the three fruit types.

• CA: OK. Thank you for your recommendation. Figure 2 has been revised (now it shows relative error) and reformatted as suggested in 4 separate graphs.

1.  3 exhibits the same problems as Fig. 2.

• CA: OK. Thank you for your recommendation. Figure 3 has been revised (now it shows relative error) and reformatted as suggested in 4 separate graphs.

1. Section 3.4: The substantial standard deviations in the aroma measurements raise concerns about data precision. For instance, the value reported for 2,4-bis(1,1-dimethylethyl)-phenol(4.25 ± 3.69) has a standard deviation that is 87% of the mean, indicating extreme variability. How do the authors explain this and justify the robustness of their conclusions?"

CA: Ok, Thank you for your comment. The analyses were performed on biological replicates, and because this was an untargeted analysis of VOCs, the relatively wide standard deviations can be explained by differences in volatility as well as variability among biological replicates. Nevertheless, the robustness of the conclusions is supported by the statistical analysis performed (two-way ANOVA with Tukey’s post hoc test).

1. 7: FRAP values were determined by measuring the samplesin absorbance at 593 nm, not 700nm.

• CA: Ok, thank you. Although we acknowledge that ferrous ion absorbs best at 593 nm, other authors (https://doi.org/10.1016/j.lwt.2014.11.013; https://doi.org/10.1080/02773813.2018.1488873) expressed FRAP in terms of absorbance at 700 nm.

1. Line 437-438: Peach show better performance in acetic fermentation, possibly because of its higher initial ethanol level, offering more substrate for acetic acid bacteria.

• CA: Ok, thank you for this suggestion. The sentence has been modified accordingly (ll. 634-635).

1. Line 515-520:The high vitamin C content in clementine may partly account for its strong antioxidant activity. The authors may consider conducting additional experiments to verify this observation or support it with references from the literature.

• CA: OK, thank you. We have supported the text with additional references from the literature (Ll 726-728).

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, fruit vinegars (from organic apples, peaches, and clementines) were produced at a laboratory scale. Sensory analysis, color, volatile organic compounds, and antioxidant activity were analyzed.

The basic idea of this study is of interest for the production of vinegars from so-called “third-category” fruits and valorize the wastes discarded for aesthetic reasons, but several mistakes and explanations need to be included:

Keywords, lines 139, 150…: The name of microorganisms, in italics. Correct throughout the manuscript.

Line 161: …a ratio of 1:50 (v/v) using…”. Please, to add (v/v).                   

Line 198: Please, delete “(2024)”.

Line 220: I don't understand why the authors express the concentration of volatile compounds in ug/g. The concentration of the internal standard is 33 mg/L and the vinegars are liquid samples. It would be logical to express it as ug/L or mg/L. Regardless of the unit used, Table 2 (supplementary) should indicate that they are expressed as 4-methyl-2-pentanol.

Line 227: Please, delete “(2018)”.

Figure 2: The total mesophilic aerobi microorganisms graph does not appear. The axis is there, but not the trend line (which should have triangles, as indicated in the figure's legend).

Line 292: It is not a subsection title, it is a result or a conclusion. Please write another one.

Line 318: It is not a subsection title, it is a result or a conclusion. Please write another one.

Line 330: It is not a subsection title, it is a result or a conclusion. Please write another one.

The supplementary tables cannot be accessed.

Line 342: Which terpenol are you referring to in particular? because, for example, linalool is a terpenol !!!

Line 343: The identification of the 2,4-bis(1,1-dimethylethyl)-phenol is very very unusual. Are the authors sure about this identification?

Line 344: butylated hydroxytoluene: Are the authors sure about this identification? BHT is a synthetic antioxidant!!

Lines 344-345: “These volatiles, which contribute to citrus and floral aromatic characteristics” only true for linalool.

It does not appear that the identification of compounds has been correct for the four compounds listed in lines 342-344. I also seem to read “styrene” in graph 4, and I do not believe this identification to be correct. The identification of Benzenedicarboxylic acid, bis(2-methylpropyl) ester and Dibutyl phthalate is also incorrect. If the authors did not inject any sample into the GC-MS, they would verify that these two compounds appear in the chromatogram.

Line 369: It is butanol, not buthanol.

Figure 7.B: The results of the FRAP method are expressed as absorbance (at 700 nm)???? The authors should have prepared a calibration curve with a standard (e.g., ascorbic acid). In addition, absorbance readings to ensure compliance with Lambert-Beer's Law should not exceed unity. If exceeded, the sample should be diluted and the dilution factor taken into account.

Lines 463-464: “…exhibited darker, warmer, and more saturated tones, associated with natural pigments…” And don't the authors think that pasteurization at 100 °C (line 138) will have an influence?

Lines 482-487: Some of these studies performed sensory analysis, allowing the authors to compare their data with that obtained in those studies. The authors criticize the fact that these studies did not compare experimental vinegars with commercial samples. In this study, the authors analyzed a commercial sample, but it was apple vinegar... therefore, it will only be valid to compare it with their experimental apple vinegar, because it does not make much sense to compare commercial apple vinegar with peach or clementine vinegar.

Lines 503 and 519: Terpinol ?????

Lines 504-506. “These compounds, along with others (e.g., linalool and phenylethyl alcohol), probably contributed to citrus and floral aromatic characteristics…” I repeat that this sentence is incorrect.

Lies 510-511 “..furfural, associated with floral and fruity aromas.” this sentence is incorrect.

Line 553: “high-quality vinegars” ???? Based on the sensory results obtained, it does not appear to be of high quality. These vinegars may be acceptable to consumers and allow for the revaluation of discarded fruit.

 

The idea seems promising from a revaluation point of view, but the identification of volatile compounds has not been correct and must be revised in order for the article to be published. In addition, authors should compare their results with those obtained by other authors.

 

Author Response

Please, find our replies in the attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have corrected several of the suggestions proposed to improve their manuscript. There are several of my criticisms with which I still disagree, such as:
* The compounds benzenedicarboxylic acid, bis(2-methylpropyl) ester, and dibutyl phthalate are NOT in their samples. Even if they provide me with the spectra from the mass spectrometer library. These compounds appear in any type of sample injected into a GC-MS because they are “column fragments”; they are not volatile compounds present in the samples.
* I do not understand why the conversion of VOCs from ug/g matches ug/mL.
* The FRAP results are expressed in absorbance units.
* My suggestion that the darkening of the color of the vinegars could be influenced by pasteurization at 100 °C...could be...and the authors have considered it as the cause.

In any case, you can publish this article because I know that these issues will not be modified.