Review Reports - Fermentation-Driven Valorization of a Carrot Juice By-Product into an Exopolysaccharide-Enriched Beverage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

*Please highlight whether the novelty is primarily EPS in situ synthesis, beverage formulation, or sensory enhancement. Add a concise “novelty statement” at the end of the introduction (1–2 sentences).

*Viscosity measurement lacks shear rate or spindle specification, which may affect reproducibility.

*Sensory panel size (n=10) is acceptable, but it is close to the limit. The justification could be improved.

*Authors should add a comment on the relation between sugar metabolism, EPS synthesis, and sensory outcomes.

*Clarify the rationale for sucrose level (5% w/v) in relation to EPS synthesis.

*Explicitly emphasize microbiological safety (low Enterobacteriaceae) in one sentence.

*Molecular characterization of EPS is mentioned but not done. This limitation should be clearly acknowledged.

*Authors should add a brief paragraph that clearly outlines the study's limitations. These include the lack of molecular EPS characterization, the absence of a storage study, and no biological validation.

*Please briefly mention industrial scalability or regulatory considerations as future perspectives.

Author Response

Reviewer 1

The novelty of this study lies primarily in the repurposing strategy, which considers carob pomace as a whole matrix rather than a minor ingredient added to an entirely different product. Secondly, the fermentation process is noteworthy for its ability to facilitate in situ EPS synthesis, an essential aspect for ensuring the uniformity and complexity of the sensory profile of the beverage. The introduction and the conclusion were updated with a clearer statement (Lines 75-81 and 443-448).

*Viscosity measurement lacks shear rate or spindle specification, which may affect reproducibility.

As suggested the material and methods section for the viscosity measurement was updated.

*Sensory panel size (n=10) is acceptable, but it is close to the limit. The justification could be improved.

We agree with the reviewer, although acceptable, panel size (n=10) is close to the limit. Nevertheless, in this study, the sensory analysis was a preliminary assessment to discriminate between samples and of course will be done with a higher panel size once the product will be intended for industrial scale-up. Moreover, to make sure the analysis was performed in the absence of fundamental errors, the panellists enrolled were trained, possessed demonstrated abilities and prior expertise in plant-based product assessment. These aspects were reported in the text.

*Authors should add a comment on the relation between sugar metabolism, EPS synthesis, and sensory outcomes.

The relation of sugar metabolism, EPS synthesis, and sensory outcomes was further discussed in lines 346-411)

*Clarify the rationale for sucrose level (5% w/v) in relation to EPS synthesis.

Usually, the amount of sucrose added to facilitate in situ EPS synthesis ranges between 2 and 20% (Koirala et al., 2025. Microbial Biotechnology, 18(4), e70116; Montemurro et al., 2025. Food Research International, 201, 115537; Woo et al., 2025. Food Research International, 117523.). For this study, preliminary tests were conducted at 2.5, 5, and 10% of sucrose, and 5% was chosen because it represented the best compromise between viscosity reached and residual sugar content. This aspect was included in the discussion (Lines 325-329).

*Explicitly emphasize microbiological safety (low Enterobacteriaceae) in one sentence.

As suggested, a sentence emphasizing the microbiological significance of low Enterobacteriaceae was added (Lines 332-335).

*Molecular characterization of EPS is mentioned but not done. This limitation should be clearly acknowledged.

We thank the Reviewer for the comment. We fully agree that a complete structural characterization of the EPS, including molecular weight distribution, monomer composition, and detailed rheological behaviour, would strengthen structure–function interpretations. However, the primary goal of the study, was to evaluate the technological feasibility of in situ EPS enrichment of a carrot by-product beverage, rather than to provide an exhaustive physicochemical characterization of the polymers produced. Accordingly, EPS quantification was intentionally limited to a gravimetric approach, which is widely used in comparative fermentation studies to assess EPS production levels and their macroscopic technological effects (e.g., viscosity enhancement and syneresis reduction).

Moreover, although direct EPS structural analyses (FTIR, NMR, GPC/SEC) were not performed, the nature of the polymers can be reasonably inferred from strain-specific literature. Leuconostoc pseudomesenteroides DSM20193 is a well-established dextran producer via dextransucrase activity, yielding predominantly α-(1→6)-linked glucans with low branching, while Levilactobacillus brevis AM7 has been reported to synthesize heteropolysaccharides composed of rhamnose, mannose, glucose, and glucosamine. These strain-dependent EPS profiles and their rheological implications have been previously demonstrated in comparable cereal- and legume-based matrices and are explicitly cited and discussed (Lines 346-411).

Ok. The limitations were clearly stated in the conclusion.

*Please briefly mention industrial scalability or regulatory considerations as future perspectives.

The fermentation process described in this study is technically feasible for industrial scale-up, as it relies on well-established food fermentation principles and industrially compatible lactic acid bacteria since they have the QPS status. However, challenges related to raw material variability and consumer acceptance may occur. Hence, further pilot-scale studies will be required to optimize process parameters and validate product stability and functionality under industrial conditions. This aspect was also mentioned in the conclusion (Lines 449-456).

Reviewer 2 Report

Comments and Suggestions for Authors

EPS characterization was evaluated solely based on gravimetric amount (g/100 g d.m.). However, molecular weight, monomer composition, and homopolymer/heteropolymer differentiation were not performed. The increase in viscosity is associated with the EPS amount, but there is no analysis of rheological behavior (shear-rate, flow behavior index).
The main claim of the study is based on EPS enrichment. However, structure-function interpretations made without showing what type of EPS it is remain speculative.
Therefore, structural verification by FTIR or ¹H-NMR, molecular weight distribution by GPC/SEC, or at least a clear “limitations” paragraph based on the literature is required.

2. The “prebiotic” and ‘functional’ claims are insufficient in experimental terms. This is because the Abstract and Discussion include statements such as “potential prebiotic properties,” “functional beverages,” and “health-promoting effects.” There is no in vitro prebiotic test (colon fermentation, selective growth, etc.). Or the bioavailability of EPS or phenolics has not been measured. If these analyses are not feasible, these statements should be softened from “potential” to ‘hypothesized’ or “technologically functional,” and clinical/biological claims should be clearly limited in the Conclusions section.

3. EPS Quantities Should Be Discussed in Terms of Realism. DSM reports 16.8 g/100 g d.m. EPS for EPS⁺. This value is at the upper limit of many plant fermentation studies. Missing here are:
• EPS recovery yield (% recovery)
• Protein/oligosaccharide contamination control
• Purity discussion after TCA-ethanol method
Recommendation:
• The risk of overestimation of the EPS isolation method should be explicitly discussed
• A quantitative comparison with the literature should be added
4. Rheological Analysis is Insufficient. Only a single viscosity value (Pa·s) is reported. Flow behavior (Newtonian/pseudoplastic) is not specified. Measurement speed (rpm/shear rate), repeatability (SD), and limitations of the EPS-viscosity relationship should be explained.

Number of panelists: n = 10. Radar charts are present, but: Multiple comparison correction and panelist variation are not specified. Furthermore, standard deviations are not present in the radar chart. Perhaps changing the chart type could present these standard deviations in a way that is more accessible to the reader.
Sugar concentrations are reported on a volumetric basis (g/L), which is highly dependent on dilution and solid loading. For a more meaningful and comparable interpretation, the authors are requested to additionally express sugar contents normalized to dry matter (e.g., g/kg d.w.), or clearly justify the use of volumetric units in relation to the constant solid content of the system.
The DPPH assay is reported as percent radical scavenging activity; however, the manuscript does not specify the sample preparation/extraction, the concentration (or dilution) of the tested extract, the DPPH solution concentration, reaction volumes, and incubation conditions. Please provide these methodological details (and ideally report the activity normalized to sample concentration, e.g., IC₅₀ or µmol Trolox equivalents/g d.w., to enable comparability).
Total polyphenol content (TPC) was determined using the Folin–Ciocalteu spectrophotometric method; however, essential analytical validation details are missing. Please provide full information on the calibration curve, including the number of calibration points, the calibration equation, and the regression coefficient (R²). In addition, clarify whether the reported variability (± SD) reflects analytical replicates or sample replicates, and report the associated measurement uncertainty. Without these details, the reliability and comparability of the TPC data cannot be adequately assessed.

Author Response

Reviewer 2

EPS characterization was evaluated solely based on gravimetric amount (g/100 g d.m.). However, molecular weight, monomer composition, and homopolymer/heteropolymer differentiation were not performed. The increase in viscosity is associated with the EPS amount, but there is no analysis of rheological behavior (shear-rate, flow behavior index).
The main claim of the study is based on EPS enrichment. However, structure-function interpretations made without showing what type of EPS it is remain speculative.
Therefore, structural verification by FTIR or ¹H-NMR, molecular weight distribution by GPC/SEC, or at least a clear “limitations” paragraph based on the literature is required.

We thank the Reviewer for the comment. We fully agree that a complete structural characterization of the EPS, including molecular weight distribution, monomer composition, and detailed rheological behaviour, would strengthen structure–function interpretations. However, the primary aim of the study, was to evaluate the technological feasibility of in situ EPS enrichment of a carrot by-product beverage, rather than to provide an exhaustive physicochemical characterization of the polymers produced. Accordingly, EPS quantification was intentionally limited to a gravimetric approach, which is widely used in comparative fermentation studies to assess EPS production levels and their macroscopic technological effects (e.g., viscosity enhancement and syneresis reduction).

Regarding EPS type, although direct structural analyses (FTIR, NMR, GPC/SEC) were not performed, the nature of the polymers can be reasonably inferred from strain-specific literature. Leuconostoc pseudomesenteroides DSM20193 is a well-established dextran producer via dextransucrase activity, yielding predominantly α-(1→6)-linked glucans with low branching, while Levilactobacillus brevis AM7 has been reported to synthesize heteropolysaccharides composed of rhamnose, mannose, glucose, and glucosamine. These strain-dependent EPS profiles and their rheological implications have been previously demonstrated in comparable cereal- and legume-based matrices and are explicitly cited and discussed (Lines 346-411).

The “prebiotic” and ‘functional’ claims are insufficient in experimental terms. This is because the Abstract and Discussion include statements such as “potential prebiotic properties,” “functional beverages,” and “health-promoting effects.” There is no in vitro prebiotic test (colon fermentation, selective growth, etc.). Or the bioavailability of EPS or phenolics has not been measured. If these analyses are not feasible, these statements should be softened from “potential” to ‘hypothesized’ or “technologically functional,” and clinical/biological claims should be clearly limited in the Conclusions section.

We thank the reviewer for the comment and fully agree with the concern raised. The statements were softened to avoid unverified claims.
3. EPS Quantities Should Be Discussed in Terms of Realism. DSM reports 16.8 g/100 g d.m. EPS for EPS⁺. This value is at the upper limit of many plant fermentation studies. Missing here are:
• EPS recovery yield (% recovery)
• Protein/oligosaccharide contamination control
• Purity discussion after TCA-ethanol method
Recommendation:
• The risk of overestimation of the EPS isolation method should be explicitly discussed
• A quantitative comparison with the literature should be added

The reviewer makes an interesting point, and we acknowledge that the value of 16.8 g EPS/100 g dry matter (d.m.) may appear high when viewed in isolation. However, it is important to emphasize that this value is expressed relative to the dry matter of the beverage, not to the wet product or fermentation substrate. Approximately 100 g of dry matter correspond to 1.4 L of fermented beverage, owing to the high water content of the system. When normalized to beverage volume, the reported EPS concentration therefore corresponds to roughly 1.2 g EPS per 100 mL, which falls within the upper but still realistic range reported for EPS-producing strains in sucrose-supplemented plant-based fermentations.

This aspect was included in the discussion (Lines 388-394).

Rheological Analysis is Insufficient. Only a single viscosity value (Pa·s) is reported. Flow behavior (Newtonian/pseudoplastic) is not specified. Measurement speed (rpm/shear rate), repeatability (SD), and limitations of the EPS-viscosity relationship should be explained.
We acknowledge that viscosity was measured at a single shear condition and that full flow curves and modelling (e.g., flow behavior index, shear-thinning behavior) were not determined. In the revised manuscript, we have therefore restricted viscosity-related claims to comparative technological effects and avoided over-interpretation of rheological mechanisms. Moreover, this aspect was included in the limitations of the study.

Number of panelists: n = 10. Radar charts are present, but: Multiple comparison correction and panelist variation are not specified. Furthermore, standard deviations are not present in the radar chart. Perhaps changing the chart type could present these standard deviations in a way that is more accessible to the reader.

As suggested the chart type was modified so that it could better display standard deviations and be more accessible to the reader.

Sugar concentrations are reported on a volumetric basis (g/L), which is highly dependent on dilution and solid loading. For a more meaningful and comparable interpretation, the authors are requested to additionally express sugar contents normalized to dry matter (e.g., g/kg d.w.), or clearly justify the use of volumetric units in relation to the constant solid content of the system.

The reviewer makes an interesting point and we would agree with it if the volumetric unit corresponded to an extract or some diluted sample. Instead, the volumetric unit was used because the samples are liquid beverages, and all data were expressed on L or mL, hence no conversion is required.
The DPPH assay is reported as percent radical scavenging activity; however, the manuscript does not specify the sample preparation/extraction, the concentration (or dilution) of the tested extract, the DPPH solution concentration, reaction volumes, and incubation conditions. Please provide these methodological details (and ideally report the activity normalized to sample concentration, e.g., IC₅₀ or µmol Trolox equivalents/g d.w., to enable comparability).

Ok. The material and method section was updated with all the necessary information.
Total polyphenol content (TPC) was determined using the Folin–Ciocalteu spectrophotometric method; however, essential analytical validation details are missing. Please provide full information on the calibration curve, including the number of calibration points, the calibration equation, and the regression coefficient (R²). In addition, clarify whether the reported variability (± SD) reflects analytical replicates or sample replicates, and report the associated measurement uncertainty. Without these details, the reliability and comparability of the TPC data cannot be adequately assessed.

Ok. Details on the TPC methodology were added to better ensure reproducibility. Also, as reported in the statistical analysis paragraph, standard deviations reflect both biological and technical replicates since analyses were carried out on samples obtained in three separate replicates and each sample was analysed in duplicate (Lines 144-158).

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents research with clear innovation and applied value, aligning with trends in circular bioeconomy and functional foods. The experimental design is reasonable, the data are comprehensive, and the conclusions largely support the research objectives.
1. Introduction
The review of existing valorization routes for "carrot by-product" is somewhat general and does not sufficiently highlight the distinctiveness of the present study. It is suggested to add a short paragraph stating that research on using lactic acid bacteria (LAB) to ferment carrot by-products for EPS-enriched beverages is currently scarce, thereby underscoring the gap this work fills.
2. Materials and Methods
Some method descriptions are too brief. It should include a concise summary of key steps or cite a more detailed methodological reference to enhance methodological reproducibility.
3. Discussion
3.1 The explanation for the differences in EPS synthesis between the two strains could be deepened, for example by linking it to their metabolic pathways (e.g., dextransucrase activity).
3.2 Further discuss the quantitative relationship between sensory attributes and EPS content/viscosity, rather than relying solely on qualitative descriptions.
3.3 The mention of "release of phenolic compounds" could be supported by citing relevant literature on the impact of LAB fermentation on the bioaccessibility of phenolics.
4. Conclusions
The conclusions are somewhat general and do not highlight the practical application value and industrial potential of the research. It is suggested to add a sentence regarding the preliminary feasibility assessment of this process at pilot or industrial scale.
5.It is suggested to clearly state the three key innovative aspects of this study compared to existing research, either at the end of the Introduction or the beginning of the Discussion: Utilization of carrot by-product as a sole substrate for EPS-enrichment fermentation. Comparative analysis of EPS synthesis capability of two LAB strains with and without sucrose supplementation. Development of a clean-label beverage combining high fiber, antioxidant properties, and favorable sensory attributes.

Author Response

Reviewer 3

The authors thank the reviewer for the comments. All suggestions were taken into consideration, and a point-by-point revision was provided.

Introduction
The review of existing valorization routes for "carrot by-product" is somewhat general and does not sufficiently highlight the distinctiveness of the present study. It is suggested to add a short paragraph stating that research on using lactic acid bacteria (LAB) to ferment carrot by-products for EPS-enriched beverages is currently scarce, thereby underscoring the gap this work fills.

Ok. As suggested, this aspect was included in the introduction (Lines 67-81).
2. Materials and Methods
Some method descriptions are too brief. It should include a concise summary of key steps or cite a more detailed methodological reference to enhance methodological reproducibility.

As suggested, the material and methods section was expanded in several paragraphs to ensure clarity and reproducibility.
3. Discussion
3.1 The explanation for the differences in EPS synthesis between the two strains could be deepened, for example by linking it to their metabolic pathways (e.g., dextransucrase activity).

The differences between the EPS produced by the two strains, as well as their metabolic pathways was reported in the discussion (Lines 346-411).
3.2 Further discuss the quantitative relationship between sensory attributes and EPS content/viscosity, rather than relying solely on qualitative descriptions.

Ok, as suggested, the discussion was expanded to more explicitly address the quantitative associations between EPS concentration, viscosity, and selected sensory attributes, rather than limiting the interpretation to qualitative descriptors (Lines 397-411).
3.3 The mention of "release of phenolic compounds" could be supported by citing relevant literature on the impact of LAB fermentation on the bioaccessibility of phenolics.

As suggested, the impact of LAB fermentation on the bioaccessibility of phenolic compounds was deepened in the discussion (Lines 418-428).
4. Conclusions
The conclusions are somewhat general and do not highlight the practical application value and industrial potential of the research. It is suggested to add a sentence regarding the preliminary feasibility assessment of this process at pilot or industrial scale.

The conclusion was modified highlighting the practical application and industrial potential of the research, as well as with limitation and future perspectives (Lines 443-456).
5.It is suggested to clearly state the three key innovative aspects of this study compared to existing research, either at the end of the Introduction or the beginning of the Discussion: Utilization of carrot by-product as a sole substrate for EPS-enrichment fermentation. Comparative analysis of EPS synthesis capability of two LAB strains with and without sucrose supplementation. Development of a clean-label beverage combining high fiber, antioxidant properties, and favorable sensory attributes.

These aspects were reported throughout the text (lines 75-81, 429-435, 443-452)

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for compiling this manuscript.

I have the following recommendations and comments to improve its quality.

There's a mix of American and UK English. Please check this
The abstract is missing critical empirical data, lines 21-31
Lines 65-73 of the introduction need additional references
Change deriving in line 76 to derived
Please provide a formulation table for the beverages
Line 112, the broth was diluted by which factor?
Be consistent when expressing centrifugation speed; sometimes it is in x g and sometimes in rpm
Sometimes time is expressed as min, sometimes minutes (be consistent)
Add an equation for DPPH scavenging, and label it as equation 1
Add the manufacturer, brand, and country of origin for the freeze dryer, line 144
Was an ethical clearance obtained for the sensory evaluation of the beverages from an institutional body?
Table 1, carbohydrates for Ct, AM7 EPS-, and DSM EPS- should not be less than the fibre, which is part of the carbohydrates
TTA, line 212 is not described in the methodology
Figure 2, there's not much difference between DSM EPS- and AM7 EPS+ could the monosacharide composition of the carrot pomace and AM7 EPS be similar, this needs to be reflected in the discussion
A recommendation to study the monosaccharide composition of the EPS if resources and time allow
Line 345, was synerisis studied over time?
The conclusion doesn't highlight the implications of the findings; it just lists what happened in the study.

Comments on the Quality of English Language

There's a mix of American and UK English. Please check this

Author Response

Reviewer 4

Dear Authors,

Thank you for compiling this manuscript.

The authors thank the reviewer for the comments. All suggestions were taken into consideration, and a point-by-point revision was provided.

I have the following recommendations and comments to improve its quality.

There's a mix of American and UK English. Please check this

Ok. The manuscript was checked for typos and inconsistencies in the English language.

The abstract is missing critical empirical data, lines 21-31

As suggested, the abstract was revised to explicitly include quantitative microbiological data, EPS, viscosity, and sensory-related results.

Lines 65-73 of the introduction need additional references

As suggested the relevant references to the literature were added to the introduction (Lines 67-81).

Change deriving in line 76 to derived

Ok.

Please provide a formulation table for the beverages

As suggested, a formulation table was provided.

Line 112, the broth was diluted by which factor?

Serial decimal dilutions were performed. This was added to the text.

Be consistent when expressing centrifugation speed; sometimes it is in x g and sometimes in rpm

Done.

Sometimes time is expressed as min, sometimes minutes (be consistent)

Done.

Add an equation for DPPH scavenging, and label it as equation 1

As suggested, the equation was added.

Add the manufacturer, brand, and country of origin for the freeze dryer, line 144

Done.

Was an ethical clearance obtained for the sensory evaluation of the beverages from an institutional body?

Yes, it was. the sensory analysis received ethical approval from the local health board. This was reported in the Institutional Review Board Statement.

Table 1, carbohydrates for Ct, AM7 EPS-, and DSM EPS- should not be less than the fibre, which is part of the carbohydrates

Table 1 was modified adding data for total carbohydrates, sugars and fibres as separate rows.

TTA, line 212 is not described in the methodology

The reviewer is correct, the TTA method was missing from the previous version, it was an oversight. It was included in the revised version (Lines 129-132).

Figure 2, there's not much difference between DSM EPS- and AM7 EPS+ could the monosacharide composition of the carrot pomace and AM7 EPS be similar, this needs to be reflected in the discussion

Although in smaller amount, EPS are produced even without sucrose addition if the monosaccharide composition of the substrate used for fermentation is ideal for their synthesis. Indeed, in this study, carrot pomace contained roughly 15 g of sucrose per 100g of dry matter and in samples fermented with both strains, EPS synthesis was observed even if sucrose wasn’t added (DSM EPS- and AM7 EPS-). Nevertheless, the amount was significantly lower than the respective beverage fermented with sucrose supplementation and most of all, viscosity was almost ca. 7 times lower in DSM EPS- and AM7 EPS- compared to DSM EPS+ and AM7 EPS+. This suggests a different conformation of the synthesized EPS which in tourn had different structural properties. Indeed, it was recently (Woo et al., 2025. Food Research International, 117523) observed the different sucrose concentrations induce structural modifications of EPS, particularly changes in branching patterns and molecular, which modifies the tecno-functional properties of the EPS. This aspect was included in the discussion (Lines 388-393).

A recommendation to study the monosaccharide composition of the EPS if resources and time allow

We thank the Reviewer for the comment. We fully agree that a complete structural characterization of the EPS, including molecular weight distribution, monomer composition, and detailed rheological behaviour, would strengthen structure–function interpretations. However, the primary aim of the study, was to evaluate the technological feasibility of in situ EPS enrichment of a carrot by-product beverage, rather than to provide an exhaustive physicochemical characterization of the polymers produced. Accordingly, EPS quantification was intentionally limited to a gravimetric approach, which is widely used in comparative fermentation studies to assess EPS production levels and their macroscopic technological effects (e.g., viscosity enhancement and syneresis reduction).

Line 345, was synerisis studied over time?

The reviewer makes an interesting point, but unfortunately syneresis was not studied overtime. It was however visually observed in the control and in samples fermented without the addition of sucrose. This aspect was reported in the text.

The conclusion doesn't highlight the implications of the findings; it just lists what happened in the study.

The conclusion was modified highlighting the practical application and industrial potential of the research, as well as with limitations and future perspectives (Lines 443-456).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the points raised in the previous review. Although some aspects (e.g., EPS structural characterization and advanced rheological analysis) are discussed based on strain-specific literature rather than direct experimental verification, these limitations are now clearly acknowledged and appropriately framed within the technological scope of the study.

The revision successfully avoids overinterpretation, softens unsupported functional claims, and improves methodological transparency and data presentation. In its revised form, the manuscript meets the scientific and editorial standards of the journal and is acceptable for publication.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for addressing the comments and improving the manuscritpt.