A Novel Dairy–Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please find the review report in the attached file.

Comments for author File: Comments.pdf

Author Response

Manuscript ID: foods-4224179

 

Former title: Powdered beetroot dairy-drink microencapsulated: a cardioprotective, immunological and metabolic boosting product.

 

Actual title: A Novel Dairy-Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives Compounds

 

Authors: Lucileno Rodrigues da Trindade, Diego dos Santos Baião, Davi Vieira Teixeira da Silva, Fernanda Petzold Pauli, and Vania Margaret Flosi Paschoalin

 

GENERAL COMMENTS BY THE AUTHORS

 We believe that we have fully addressed all reviewer concerns and comments.  Several modifications were carried out in the revised manuscript.

Major modifications were carried out in the revised manuscript. The title was modified, the abstract was rewritten, including the reduction of some paragraphs, modifications in sentences were and two new paragraph addressing limitations were included, future directions and the best formulation of the present study were added in the manuscript. Two new paragraph addressing limitations were included, future directions and the best formulation of the present study were added in the manuscript. Minor corrections on grammar and syntax were performed for better reading and understanding. Nineteen references were included:

84. Labuza, T.P; Altunakar, B. Water Activity Prediction and Moisture Sorption Isotherms. In Water Activity in Foods (eds G.V. Barbosa-Cánovas, A.J. Fontana, S.J. Schmidt and T.P. Labuza), 2020.
85. Fan, F.; Liu, H.; Xu, Y.; Mou, T. Quantitative Analysis of Molecular Mobility in Amorphous Lactose Above Tg: A Novel Insight from Molecular Dynamic Simulation to Strength Parameter. Foods. 2025, 14, 928.
86. Saavedra-Leos, Z.; Leyva-Porras, C.; Araujo-Díaz, S.B.; Toxqui-Terán, A.; Borrás-Enríquez, A.J. Technological Application of Maltodextrins According to the Degree of Polymerization. Molecules. 2015, 20, 21067-21081.
87. Xiao, Z.; Xia, J.; Zhao, Q.; Niu, Y.; Zhao, D. Maltodextrin as wall material for microcapsules: A review. Carbohy. Poly. 2022, 298, 1-18.
88. Carvalho, G.R.; Fernandes, R.V.B.; de Castro, E.S.P.; Dessimoni, A.L.A.; Oliveira, C.R.; Borges, S.V.; Botrel, D.A. Influence of modified starches as wall materials on the properties of spray-dried lemongrass oil. J. Food Sci. Technol. 2019, 56, 4972-4981.
89. Bilenler Koc, T.; Fırat, I.; Karabulut, I.; Boztepe, C.; Topalcengiz, Z. Microencapsulation of Beetroot Anthocyanins: Inves-tigation of Degradation Kinetics and Modeling by Using Artificial Neural Networks. ACS Omega. 2025, 25, 2270-2284.
90. Shofinita, D.; Fawwaz, M.; Achmadi, A.B. Betalain extracts: Drying techniques, encapsulation, and application in food industry. Food Frontiers, 2023, 4, 576–623.
91. Ravichandran, K.; Palaniraj. R.; Saw, N.M.; Gabr, A.M.; Ahmed, A.R.; Knorr, D.; Smetanska, I. Effects of different encap-sulation agents and drying process on stability of betalains extract. J Food Sci Technol. 2014, 51, 2216-21.
92. Chakma, S.; Acharjee, M.R.; Tonni, I.J.; Tabassum, F.; Das, E.; Islam, S.; Faruk, S.A. Probiotic potential of yogurt: Explor-ing lactic acid bacteria for health-enhancing benefits. Appl Food Res. 2025, 5, 1-15.
93. Peng, Y.; Horne, D.S.; Lucey, J.A. Impact of preacidification of milk and fermentation time on the properties of yogurt. J. Dairy Sci. 2009, 92, 2977-2990.
94. Ren, Q.; Zhang, J.; Chen, X.; Pang, J.; Wichers, H.J.; Wen, Y.; Hettinga, K.A. Effects of fermentation on protein digestion and peptide release in an in vitro adult digestion model. Food Res. Inter. 2026, 230, 1-10.
95. Parvez, S.; Malik, K.A.; Ah Kang, S.; Kim, H.Y. Probiotics and their fermented food products are beneficial for health. J. Appl. Microbiol. 2006, 100, 1171–1185.
96. Makinen, O.E.; Wanhalinna, V.; Zannini, E.; Arendt, E.K. Foods for special dietary needs: Non-dairy plant-based milk substitutes and fermented dairy-type products. Crit. Rev. Food Sci. Nutr. 2016, 56, 339–349.
97. Guadalupe, H.S.D.; Berenice, O.R.L.; Elizabeth, C.G.A.; Guillermo, G.O.L.; Araceli, C.O. Probiotic fermented milk and type 2 diabetes mellitus: Mechanisms, benefits, and future directions. J. Funct. Foods. 2025, 134, 107068.
98. García-Burgos, M.; Moreno-Fernández, J.; Alférez, M.J.; Díaz-Castro, J.; López-Aliaga, I. New perspectives in fermented dairy products and their health relevance. J. Funct. Foods. 2020, 72, 104059.
99. Huligere, S.S.; Chandana Kumari, V.B.; Alqadi, T.; Kumar, S.; Cull, C.A.; Amachawadi, R.G.; Ramu, R. Isolation and characterization of lactic acid bacteria with potential probiotic activity and further investigation of their activity by α-amylase and α-glucosidase inhibitions of fermented batters. Front. Microbiol. 2023, 13, 1042263.
100. Hashemi Javaheri, F.S.; Nasiri Jounaghani. M.; Sahebkar, A.; Norouzzadeh, M.; Delgarm, P.; Shahinfar, H.; Mirdar Harijani, A. The effect of fermented dairy intake and abdominal obesity in adults: a systematic review and dose-response meta-analysis of cohort studies. Eat Weight Disord. 2025, 30, 23.
101. Mukarromah, T.A.; Rustanti, N.; Mahati, E.; Suparmi; Ayustaningwarno, F. The Impact of Fermented Milk Products on Gut Microbiota-Derived Metabolites in Obesity: A Narrative Review. J. Food Sci. 2025, 90, e70301.
102. Stachelska, M.A.; Karpiński, P.; Kruszewski, B. Health-Promoting and Functional Properties of Fermented Milk Bever-ages with Probiotic Bacteria in the Prevention of Civilization Diseases. Nutrients. 2025, 17, 9.

 

All modifications were highlighted in yellow.

After performing all the modifications suggested by the reviewers, the entire text was revised by an editing specialized company to improve English grammar and syntax. The authors have approved the final version of the manuscript.

We thank the Reviewer for his-her critical insightful of our manuscript.

We hope that in its revised form our manuscript will be found suitable for publication in this respectful journal.

 

Answers to Reviewer 1

Reviewer 1 comments preceding our responses.

 

Comments and Suggestions for Authors

Comments to the Author

Dear authors

The manuscript presents a practical and formulation strategy to overcome a key barrier in beetroot functional foods, aiming for delivering cardioactive compounds at meaningful levels without compromising sensory acceptance. The authors’ approach, namely freeze-dried, microencapsulated beetroot dairy drink using maltodextrin, cassava starch, and their combination, is clearly aligned with stability and consumer-acceptance goals. The characterization work supports the core claims about physicochemical performance and storage feasibility at room temperature. To strengthen the paper, I would encourage the authors to consider the following points for their manuscript revision. Clearer linkage between the formulation outcomes and the proposed physiological benefits: the discussion would benefit from more direct evidence (or careful qualification) regarding “bioaccessibility” and vascular/hemodynamic effects, plus more detail on sensory testing design, storage duration/conditions, and comparative performance across formulations (including nitrate and betalain retention over time). Overall, it is a promising and applicable contribution to functional beverage development, with minor-to-moderate revisions needed to better substantiate the health-impact framing and improve methodological transparency.

 

Title of the article:

1. The original article titled " Powdered beetroot dairy-drink microencapsulated: a cardioprotective, immunological and metabolic boosting product” immediately leads the readers’ expectation to see some results about cardioprotective, immunological activities and metabolic boosting of the experimented product. However, such activities were not experimented and reported in this article. It is suggested that the title is revised to conform to the key findings reported in this manuscript.

Answer: We agree and appreciate the reviewer`s suggestion and we refined the title to clarity and impact, choosing the title: "A Novel Dairy-Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotec-tive Bioactives" (page 1).

 

 

 

 

Abstract:

1. Line 24-26: mentions “…that achieve pharmacological concentration…”. Please consider if this statement was overstated since the pharmacological dose or clinical trial was not conducted.

Answer: We agree with the reviewer`s suggestion and the abstract was revised and modified to better understanding (page 1).

 

 

Introduction:

1. Line 53: Please check spelling “…dietary el able…”.

Answer: We agree with the reviewer`s suggestion and this sentence were modified.

 

 

2. Line 87: What was the type of beetroot juice reported there, fresh juice or processed juice?

Answer: We agree with the reviewer`s suggestion and we corrected it to fresh beetroot juice (page 2, line 78).

 

 

Methods:

1. Line 144: Please correct scientific writing of coli.

Answer: We agree with the reviewer`s suggestion and we fix it (page 4, line 151).

 

 

2. Line 173: Freeze-drying for 5 days sounds very long time for this type of product. Please confirm if this is correct.

Answer: Yes, we confirm this information and this time was based on previous freeze-drying tests that our group performed on beetroot milk beverage microencapsulated with different polysaccharide agents.

 

 

3. Line 179-180: Final preparation of the final microencapsulated powder involved grinding but approach to obtain uniform particle size, such as sieving at specific screen opening size, was not mentioned. Was the powder sieved after grinding? If not, please consider this when reporting the particle size distribution and solubility in the results and discussion.

 Answer: The powders were not sifted after grinding. When removing the products from the lyophilize vials, they have crumbled easily. Therefore, we only used a mortar and pestle to help crumble the samples. We revised the text to make it clear.

 

 

4. Line 185-188: SEM analysis of the microcapsule powder did not mention gold sputtering in sample preparation step. Since the product is not conductive, this is necessary. Please check. Also please provide the scanning conditions beside the kV.

Answer: We mention in page 4, lines 192-195 that the samples were covered with a thin layer of gold in Balzers metallizer, Union FL 9496 and were deposited on double-sided carbon adhesive tape, fixed on the surface of the microscope base, JEOL JSM 5310 at 15kV.

 

 

5. Line 196-197: Please provide the manufacturer information of FTIR instrument. Please specify the scanning wavelength range.

Answer: We agree with the reviewer`s suggestion and this sentence were modified (page 5, lines 202-206).

 

 

6. Line 204-208: Please include the solubility index calculation equation.

Answer: We agree with the reviewer`s suggestion and the solubility index calculation equation was added to the manuscript text (page 5, line 218).

 

 

7. Line 209: Please briefly describe the procedure of water absorption index analysis. Please also include the calculation equation for water absorption index.

Answer: We agree with the reviewer`s suggestion and we included a description  and added the equation for water absorption index calculation in the manuscript (page 5, lines 220-233).

 

 

8. Line 243: Please check the correctness of the unit for BT.

Answer: Yes, we  checked and confirmed this information.

 

 

9. Line 254: Please correct the EE(%) equation to EE(%)=((BT-BTS)/BT)100.

Answer: We agree with the reviewer`s suggestion and we corrected the equation (page 6, line 279).

 

 

10. Line 249-261 Section 2.5: The procedure used for extraction of the microcapsule would dissolve almost all the microcapsule structure. Hence, the betalains would be released into the supernatant almost 100%, meaning surface betalain and encapsulated betalains were released, not. The betalains content obtain here would be total betalains remaining both on surface and inside the microcapsule powder. The calculation equation would present % total betalains after freeze drying/total betalain added before freeze drying. I suggest that the authors clarify or redefine the term “Encapsulation Efficiency” for better alignment between the extraction and calculation, i.e. % betalains retention. Of course, the original definition of EE would be true if the BTS is only coming from the surface betalains.

Answer: The determination of encapsulation efficiency has been rewritten (page 6, lines 281-286), as suggested. First, the total betalains, betaxanthins, and betacyanins are quantified in the supernatant after dilution in water and centrifugation. The supernatant represents the betalains that the microparticle was unable to trap. Then, the ratio between supernatant values and pigment content employed in the microparticle powder after extraction. So, the EE (%) was determined after dilution of sample in water and centrifugation, followed by spectrophotometric quantification of betalains (betacyanins + betaxanthins) at 538 nm and 480 nm wavelengths. En-capsulation efficiency was determined by the ratio between betalain in the supernatant and betalain content added in the microparticle formulation.

 

11. Section 2.10: Please describe how the panelists prepare their samples before tasting.

Answer: We weighed and added a certain amount of the powdered samples to plastic cups and added a certain amount of ice-cold filtered water to the samples, homogenizing with a spoon until the product was completely dissolved. We agree with the reviewer`s suggestion and this information was included in the manuscript (page 7, lines 321-325).

 

 

 

Results:

1. Line 335: “…non-encapsulated powder dairy beetroot drink.” could read better as “…non-encapsulated dairy-beetroot powder.”

Answer: We agree with the reviewer`s suggestion and we modified to non-microencapsulated formulations as suggested by reviewer 2, along the whole manuscript.

 

 

2. Line 335-337: Please check the writing style of the units.

Answer: As suggested, we checked and confirm the units.

 

 

3. Table 1: Two numbers are reported for NO3- and NO2-, i.e. 15.970.92/990.14. What are they?

Answer: As shown on the side of the Table 1, for example NO₃^- and NO₂^- (mmol/mg), it was shown the contents in mmol and mg. Therefore, the first value before the / is in mmol and the second value after the / is in mg. This was clarified in Table 1. Thanks for the suggestion.

 

 

4. Line 440-441: “…the yield was reduced to 21.41%, demonstrating…in solid retention during and after freeze-drying method.” This may not be correct because the sample contains no extra added polysaccharide. Its initial solid content before drying was already lower than the sample with wall material added. Please consider revising this statement. To standardize the results in this section, the authors may wish to recalculate % yields based on the initial solids content before drying.

Answer: We agree with the reviewer`s suggestion and we rewritten the  sentence, as suggested. The idea was to show that the yield of samples without wall polysaccharide showed a yield lower than those entrapped into wall polysaccharides (page 12, lines 462-467).

 

 

5. Line 448-449: Please consider standardizing the water solubility index based on the initial solids content before drying.

Answer: At the beginning a liquid beetroot milk beverage was produced and the encapsulating agents were added to it and the mixture was subjected to freeze-dryer for microencapsulation, obtaining a powder of this beverage as the final product. So, the solubility index and the water absorption index were expressed in relation to the dried mass obtained, ensuring comparability between different batches and products. Furthermore, using the solids content of the liquid beverage before freeze-drying as a reference could be a way to standardize, but caution is necessary. Considering that freeze-drying removes virtually all water, it concentratated the solids. Thus, the resulting powder represents the total solids that were present in the original beverage. Furthemore, it is usual to calculate in relation to the dry mass of the powder, as this is the final product for commercialization.

 

 

Table 3: Please consider standardizing the % yield and water solubility index based on the initial solids content for better comparison of the results.

Answer: As explained above, we think more appropriate to consider the solids following the freeze-drying, in order to avoid losses during the processing, and would not be considered

 

 

Table 4: If the authors agreed to calculate EE% as the %betalains retained in microcapsule powder, please revise the results in this table.

Answer: The EE% is usually calculated by those material found in supernatant following the entrapment, that represents part of the initial material, that were not entrapped in the microparticles. To proceed that calculation the total betalains, betaxanthins, and betacyanins were quantified in the supernatant after dilution in water and centrifugation. Betalains in the supernatant were those unable to be entrapped in the microparticles. Then, finally, the ratio between supernatant contents and initial pigment content  in the microparticle powder after extraction.

 

 

Table 5: Molds and Yeats counts have number of 1.26101 CFU.g-1, for example. Does this mean 12.6 log CFU.g-1? Please clarify.

Answer: No, it doesn't mean that amount. When the count is presented as 1.26×10¹ CFU·g^-1, this corresponds to:

1.26x10¹ = 12.6 CFU·g^-1

This value is linear, meaning it directly expresses the number of colony-forming units per gram. The logarithm is always calculated from the absolute number of CFU·g^-1. Log₁₀(12.6) is approximately 1.10 log CFU·g^-1.

 

 

6. Line 532: Please check writing style of numbers and units, i.e. “50g” or “50 g”. Also check throughout the manuscript. Thank you.

Answer: We agree with the reviewer`s suggestion and we corrected the writing style of numbers and units in all manuscript.

 

 

7. Table 6: Please consider revising “Powdered beetroot dairy drink” to “Dairy-beetroot powder” for a better reading. This term may also be revise throughout the manuscript.

Answer: We agree with the reviewer`s suggestion and we modified this term in the entire manuscript.

 

 

 

 

Discussion:

1. Line 581-584: “During the encapsulation process, lipids may be partially retained or heterogeneously distributed in the encapsulating matrix, hindering their complete extraction, resulting in under-quantification.” This sentence may not be accurate since the procedure of Association of Official Analytical Chemists expects to detect all the lipid content in the sample. Please revise this statement.

Answer: Under the quantification conditions employed here, the complex polysaccharide matrix structure (polymeric ones) protects the lipid core, preventing the organic solvents used in standard methods, such as those of AOAC, from accessing and solubilizing the lipids. Heterogeneous retention or distribution happens for two main reasons:

• Physical Trapping: The matrix (e.g., maltodextrin, gum arabic, or protein isolates) forms a dense network. If the solvent cannot dissolve this network, the internal lipid remains untouched.
• Incomplete Encapsulation vs. Efficiency: The traditional AOAC method often quantifies only the surface lipid (free oil), ignoring the effectively encapsulated oil if there is no prior acid or base hydrolysis step.

This explanation and the reference below was added in the manuscript (page 17, lines 609-615).

 

Reference:

Bakry, A. M.; Abbas, S.; Ali, B.; Majeed, H.; Abouelwafa, M. Y.; Mousa, A.; Liang, L. Microencapsulation of Oils: A Com-prehensive Review of Benefits, Techniques, and Applications. Comprehensive reviews in food science and food safety. 2015, 15, 143-182.

 

 

2. Line 629: “Thus, the novel powdered beetroot dairy drink…” Please justify if the product can be claimed as “novel” product.

Answer: Indeed we believe that this is a novel formulation, joining beets, dairy product, obtaining from kefir fermentation. After searching the internet for this type of product, a powdered beetroot milk drink enriched with nitrate and probiotics (since eXact^® Kefir 1 cultures were added), we found no commercially available industrial product in the world, nor any published scientific articles that have produced a similar product.

 

 

3. Line 657-658: “…2015 mg.100 g-1 fresh weight…” Does the “fresh weight” mean the weight before or after freeze drying? Please clarify.

Answer: This refers to the weight after freeze-drying, and all calculations, and consequently the results, refer to the final product immediately after freeze-drying. In the dry weight basis, the water is completely excluded from the calculation, so the result represents the percentage of each nutrient considering the total solids. However, on the fresh weight basis, the calculation is based on the total weight of the sample, exactly as it is or comes out from the freeze-dryer, including water or residual moisture. It reflects the physical reality of the product. It is what the consumer will ingest when measuring a spoonful of the powder. Our objective was to express the product's results as soon as it came out of the equipment, and therefore, we chose the most accurate method, which is the Fresh Weight Basis. This explanation was added in the manuscript (page 7-8, lines 349-352).

 

 

4. Line 733-734: Discussion regarding water activity was focused on microbial safety aspect. How about the effects of water activity on the physical properties of the powder, i.e. its effects on powder stickiness/lumping/flowing and release of betalains during storage? Please extend the discussion to cover these important aspects of microcapsule product quality.

Answer: We agree with the reviewer`s suggestion and a discussion about effects of water activity on the physical properties of the powder were added in the manuscript (page 20-21, lines 771-820).

 

 

5. Line 760: Please check correctness of “…freeze-dried buriti oil..”.

Answer: We agree with the reviewer`s suggestion and corrected this term in the text (page 20, line 768).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

This is a well-executed and comprehensive study on the development of a functional powdered beetroot dairy drink. The research addresses a significant challenge in the field of functional foods: creating a palatable, stable, and effective delivery system for bioactive compounds like dietary nitrate, which are often associated with unpleasant sensory characteristics.

The authors have conducted a thorough characterization of their product, including physicochemical properties, bioactive compound retention, encapsulation efficiency, microbiological stability, and sensory analysis. The use of three different encapsulating agents (maltodextrin, cassava starch, and a combination) is a strong point, allowing for a comparative analysis that identifies the most effective formulation.

Overall Opinion: Minor to Moderate Revision

The manuscript is scientifically sound and presents valuable data. However, its impact and clarity can be significantly improved with revisions focused on contextualizing the findings, refining the language for precision, and strengthening the discussion.

Here are detailed revision suggestions to convey to the authors, categorized for clarity.

Revisions to Suggest to Authors

1. Major Conceptual and Structural Suggestions

• Refine the Title for Clarity and Impact: The current title is a bit of a "keyword salad." Consider a more focused title that clearly states the product and its key finding.

  • Suggested Alternative: "Development of a Microencapsulated Powdered Beetroot Dairy Drink: Enhanced Stability, Bioactive Retention, and Sensory Acceptance" or "A Novel Powdered Beetroot-Dairy Beverage: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives."

• Restructure the Abstract for Better Flow: The abstract is dense. Reorganize it to follow a standard structure: Background, Objective, Methods, Key Results, Conclusion. Move the detailed mechanism of NO action to the introduction. The abstract should focus on the what and what was found, not the exhaustive why.

• Strengthen the Introduction's Focus:

  • The introduction provides an excellent and detailed background on nitric oxide (NO) and its benefits (lines 45-92). This is a strength, but it is very long. The core challenge the paper addresses—formulating a palatable, high-nitrate product—gets somewhat lost. Consider shortening the detailed physiological mechanism of NO (lines 45-67) and focusing more sharply on the problem of poor patient adherence to current beetroot products.

  • The transition to the study's aim (lines 119-128) is good. Ensure the objectives are listed as clear, testable points that directly map to the Results section.

• Enhance the Discussion Section: The discussion is currently a point-by-point rehash of the results. It needs to be elevated to an argument about the significance of the findings.

  • Interpret, Don't Just Repeat: Instead of saying "The protein content was 19%," say "The high protein content (19%) meets regulatory standards and positions the product as a nutritionally complete option, surpassing typical protein levels in similar freeze-dried yogurts [53]." Do this for all key findings.

  • Create a Narrative: Structure the discussion around a central theme: "The combination of maltodextrin and cassava starch proved to be the optimal formulation." Use your data to build this case. Discuss why this combination works best (e.g., high EE from starch, high solubility from maltodextrin, leading to the best sensory scores).

  • Add a "Limitations and Future Directions" Paragraph: Acknowledge the study's limitations. For example, the shelf-life study was only 30 days, the sensory panel was untrained, and the actual bioavailability of the compounds in vivo was not tested. This directly leads to your final sentence in the conclusions, making it more impactful. Mention the need for clinical trials to prove the claimed "cardioprotective" effects.

2. Specific Technical and Writing Revisions

• Abstract (Lines 23-44):

  • Line 30-31: "higher than >90%" is redundant. Use "high solubility (>90%)".

  • Line 40: The mention of "probiotic microcapsules" is new. The kefir culture is mentioned in the methods, but its viability isn't a major focus of the results (only briefly in microbiology). Either present data on probiotic viability or remove the claim from the abstract.

• Introduction (Lines 80-92):

  • Line 86: "clinical tests concluded that large beetroot juice volumes... have limited the adherence" → This is the core problem. State it more strongly. "The requirement to consume large, unpalatable volumes of beetroot juice (500-700 mL) to achieve a clinical dose of nitrate has been a major barrier to patient adherence in long-term interventions [3,11,16-19]."

• Results (Lines 319-424):

  • General: The results are very clearly presented with excellent tables and figures.

  • Line 340 (Table 1): This table is information-dense and excellent. In the text, guide the reader to the most important comparisons. For example: "As expected, microencapsulated formulations showed significantly higher carbohydrate and energy content (p<0.05) due to the added polysaccharides, while protein and lipid content decreased proportionally. Critically, the concentration of the target bioactive, NO3-, remained high (>615 mg·100g-1) in all microencapsulated samples, although this was lower than the non-encapsulated control."

  • Line 353 (Table 1): The values for NO3- are given as mmol·mg-1 / mg·100g-1. The formatting is confusing. Clarify this in the table footnote. It should likely be mmol·100g-1 / mg·100g-1.

  • Line 392 (Table 2): The Span index is mentioned. Explain in the text what a "good" Span value is and why the high Span for the combination is not a problem if the d(0.5) values are small and solubility is high.

• Discussion (Lines 564-962):

  • Line 588: "lipids may be partially retained... resulting in under-quantification." This is a good point. Be more definitive: "The lower measured lipid content in microencapsulated samples is likely an artifact of the extraction method, as the polymeric wall material may hinder the solvent's ability to fully access the encapsulated lipids [49]."

  • Line 616-627: The paragraph on the clinical dose of nitrate is excellent. It perfectly links the product's composition to its intended purpose. This is the kind of impactful writing needed throughout the discussion.

  • Line 708-714: The explanation for the FTIR band at 1744 cm⁻¹ is good. State it more confidently: "The presence of the carbonyl band at 1744 cm⁻¹ exclusively in the encapsulated formulations, and its absence in the spectra of the pure encapsulating agents, serves as strong evidence that the core beetroot-dairy materials were successfully incorporated and preserved within the microcapsules [79,80]."

  • Line 863-893: The paragraphs on encapsulation efficiency are well-researched and explain the "why" behind the results. This is a model for the rest of the discussion.

  • Line 953-962: The conclusion is strong. It clearly states the best formulation and its key attributes. The mention of the patent is a nice touch.

• Materials and Methods (Lines 129-318):

  • This section is comprehensive and reproducible. No major revisions needed. Ensure all equations are correctly formatted (e.g., Equation 3 uses "BT" for both total and supernatant betalains—this is a typo that needs correction).

  • Line 253-255: Clarify the distinction between BT and BTS in Equation (3). It currently reads: EE(%) = (BT - BTS×100) / BT. This should likely be EE(%) = ((BT - BTS) / BT) × 100. Please verify.

3. Language and Presentation

• Minor Typos and Grammatical Errors:

  • Line 92: "already demonstrate very effective" → "already demonstrated to be very effective"

  • Line 101-102: The sentence "metabolic mediators, and flavors, has not yet been elucidated [22]." is repeated verbatim. (Lines 101 and 103).

  • Line 158: "maintained at room temperature in a cool, dry and dark place" → "maintained in a cool, dry, and dark place at room temperature".

  • Line 292: "an air- conditioned refrigerated and closed cabin" → "a temperature-controlled, closed sensory booth with white lighting".

  • Line 328: "betaxanins" is misspelled; it should be "betacyanins" in several places (e.g., lines 328, 344, 489, 492, 494, 496). A thorough spell-check for this term is recommended.

• Clarity and Concision:

  • Throughout the manuscript, sentences are often very long and contain multiple clauses. For example, lines 108-118. Breaking these into shorter, more direct sentences would improve readability.

  • Be consistent with terminology. "Powdered beetroot dairy drink microencapsulated" is a mouthful. After the first instance, you can refer to it as "the microencapsulated formulations" or "the powdered drinks" for brevity, as long as the context is clear.

By addressing these points,  can significantly enhance the manuscript's clarity, impact, and suitability for publication. The underlying science is excellent, and these revisions will help communicate its value more effectively.

Best regards

Author Response

Manuscript ID: foods-4224179

 

Former title: Powdered beetroot dairy-drink microencapsulated: a cardioprotective, immunological and metabolic boosting product.

 

Actual title: A Novel Dairy-Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives Compounds

 

Authors: Lucileno Rodrigues da Trindade, Diego dos Santos Baião, Davi Vieira Teixeira da Silva, Fernanda Petzold Pauli, and Vania Margaret Flosi Paschoalin

 

GENERAL COMMENTS BY THE AUTHORS

 We believe that we have fully addressed all reviewer concerns and comments.  Several modifications were carried out in the revised manuscript.

Major modifications were carried out in the revised manuscript. The title was modified, the abstract was rewritten, including the reduction of some paragraphs, modifications in sentences were and two new paragraph addressing limitations were included, future directions and the best formulation of the present study were added in the manuscript. Two new paragraph addressing limitations were included, future directions and the best formulation of the present study were added in the manuscript. Minor corrections on grammar and syntax were performed for better reading and understanding. Nineteen references were included:

84. Labuza, T.P; Altunakar, B. Water Activity Prediction and Moisture Sorption Isotherms. In Water Activity in Foods (eds G.V. Barbosa-Cánovas, A.J. Fontana, S.J. Schmidt and T.P. Labuza), 2020.
85. Fan, F.; Liu, H.; Xu, Y.; Mou, T. Quantitative Analysis of Molecular Mobility in Amorphous Lactose Above Tg: A Novel Insight from Molecular Dynamic Simulation to Strength Parameter. Foods. 2025, 14, 928.
86. Saavedra-Leos, Z.; Leyva-Porras, C.; Araujo-Díaz, S.B.; Toxqui-Terán, A.; Borrás-Enríquez, A.J. Technological Application of Maltodextrins According to the Degree of Polymerization. Molecules. 2015, 20, 21067-21081.
87. Xiao, Z.; Xia, J.; Zhao, Q.; Niu, Y.; Zhao, D. Maltodextrin as wall material for microcapsules: A review. Carbohy. Poly. 2022, 298, 1-18.
88. Carvalho, G.R.; Fernandes, R.V.B.; de Castro, E.S.P.; Dessimoni, A.L.A.; Oliveira, C.R.; Borges, S.V.; Botrel, D.A. Influence of modified starches as wall materials on the properties of spray-dried lemongrass oil. J. Food Sci. Technol. 2019, 56, 4972-4981.
89. Bilenler Koc, T.; Fırat, I.; Karabulut, I.; Boztepe, C.; Topalcengiz, Z. Microencapsulation of Beetroot Anthocyanins: Inves-tigation of Degradation Kinetics and Modeling by Using Artificial Neural Networks. ACS Omega. 2025, 25, 2270-2284.
90. Shofinita, D.; Fawwaz, M.; Achmadi, A.B. Betalain extracts: Drying techniques, encapsulation, and application in food industry. Food Frontiers, 2023, 4, 576–623.
91. Ravichandran, K.; Palaniraj. R.; Saw, N.M.; Gabr, A.M.; Ahmed, A.R.; Knorr, D.; Smetanska, I. Effects of different encap-sulation agents and drying process on stability of betalains extract. J Food Sci Technol. 2014, 51, 2216-21.
92. Chakma, S.; Acharjee, M.R.; Tonni, I.J.; Tabassum, F.; Das, E.; Islam, S.; Faruk, S.A. Probiotic potential of yogurt: Explor-ing lactic acid bacteria for health-enhancing benefits. Appl Food Res. 2025, 5, 1-15.
93. Peng, Y.; Horne, D.S.; Lucey, J.A. Impact of preacidification of milk and fermentation time on the properties of yogurt. J. Dairy Sci. 2009, 92, 2977-2990.
94. Ren, Q.; Zhang, J.; Chen, X.; Pang, J.; Wichers, H.J.; Wen, Y.; Hettinga, K.A. Effects of fermentation on protein digestion and peptide release in an in vitro adult digestion model. Food Res. Inter. 2026, 230, 1-10.
95. Parvez, S.; Malik, K.A.; Ah Kang, S.; Kim, H.Y. Probiotics and their fermented food products are beneficial for health. J. Appl. Microbiol. 2006, 100, 1171–1185.
96. Makinen, O.E.; Wanhalinna, V.; Zannini, E.; Arendt, E.K. Foods for special dietary needs: Non-dairy plant-based milk substitutes and fermented dairy-type products. Crit. Rev. Food Sci. Nutr. 2016, 56, 339–349.
97. Guadalupe, H.S.D.; Berenice, O.R.L.; Elizabeth, C.G.A.; Guillermo, G.O.L.; Araceli, C.O. Probiotic fermented milk and type 2 diabetes mellitus: Mechanisms, benefits, and future directions. J. Funct. Foods. 2025, 134, 107068.
98. García-Burgos, M.; Moreno-Fernández, J.; Alférez, M.J.; Díaz-Castro, J.; López-Aliaga, I. New perspectives in fermented dairy products and their health relevance. J. Funct. Foods. 2020, 72, 104059.
99. Huligere, S.S.; Chandana Kumari, V.B.; Alqadi, T.; Kumar, S.; Cull, C.A.; Amachawadi, R.G.; Ramu, R. Isolation and characterization of lactic acid bacteria with potential probiotic activity and further investigation of their activity by α-amylase and α-glucosidase inhibitions of fermented batters. Front. Microbiol. 2023, 13, 1042263.
100. Hashemi Javaheri, F.S.; Nasiri Jounaghani. M.; Sahebkar, A.; Norouzzadeh, M.; Delgarm, P.; Shahinfar, H.; Mirdar Harijani, A. The effect of fermented dairy intake and abdominal obesity in adults: a systematic review and dose-response meta-analysis of cohort studies. Eat Weight Disord. 2025, 30, 23.
101. Mukarromah, T.A.; Rustanti, N.; Mahati, E.; Suparmi; Ayustaningwarno, F. The Impact of Fermented Milk Products on Gut Microbiota-Derived Metabolites in Obesity: A Narrative Review. J. Food Sci. 2025, 90, e70301.
102. Stachelska, M.A.; Karpiński, P.; Kruszewski, B. Health-Promoting and Functional Properties of Fermented Milk Bever-ages with Probiotic Bacteria in the Prevention of Civilization Diseases. Nutrients. 2025, 17, 9.

 

All modifications were highlighted in yellow.

After performing all the modifications suggested by the reviewers, the entire text was revised by an editing specialized company to improve English grammar and syntax. The authors have approved the final version of the manuscript.

We thank the Reviewer for his-her critical insightful of our manuscript.

We hope that in its revised form our manuscript will be found suitable for publication in this respectful journal.

 

Answers to Reviewer 2

Reviewer 2 comments preceding our responses.

 

Comments and Suggestions for Authors

Comments to the Author

Dear authors

This is a well-executed and comprehensive study on the development of a functional powdered beetroot dairy drink. The research addresses a significant challenge in the field of functional foods: creating a palatable, stable, and effective delivery system for bioactive compounds like dietary nitrate, which are often associated with unpleasant sensory characteristics. The authors have conducted a thorough characterization of their product, including physicochemical properties, bioactive compound retention, encapsulation efficiency, microbiological stability, and sensory analysis. The use of three different encapsulating agents (maltodextrin, cassava starch, and a combination) is a strong point, allowing for a comparative analysis that identifies the most effective formulation.

 

Overall Opinion: Minor to Moderate Revision

The manuscript is scientifically sound and presents valuable data. However, its impact and clarity can be significantly improved with revisions focused on contextualizing the findings, refining the language for precision, and strengthening the discussion. Here are detailed revision suggestions to convey to the authors, categorized for clarity.

 

Revisions to Suggest to Authors

1. Major Conceptual and Structural Suggestions

Refine the Title for Clarity and Impact: The current title is a bit of a "keyword salad." Consider a more focused title that clearly states the product and its key finding. Suggested Alternative: "Development of a Microencapsulated Powdered Beetroot Dairy Drink: Enhanced Stability, Bioactive Retention, and Sensory Acceptance" or "A Novel Powdered Beetroot-Dairy Beverage: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives."

Answer: We agree and appreciate the reviewer`s suggestion and we refined the title for clarity and impact, choosing the title: "A Novel Dairy-Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactive Compounds" (page 1).

 

 

Restructure the Abstract for Better Flow: The abstract is dense. Reorganize it to follow a standard structure: Background, Objective, Methods, Key Results, Conclusion. Move the detailed mechanism of NO action to the introduction. The abstract should focus on the what and what was found, not the exhaustive why.

Answer: We agree with the reviewer`s suggestion and the abstract was revised and modified to better understanding (page 1, lines 24-44), as suggested we included standard structure such as Background, Objective, Key Results and Conclusion. Furthermore, we moved the detailed mechanism of NO action to introduction (page 2, lines 62-77).

 

 

Strengthen the Introduction's Focus:

The introduction provides an excellent and detailed background on nitric oxide (NO) and its benefits (lines 45-92). This is a strength, but it is very long. The core challenge the paper addresses—formulating a palatable, high-nitrate product—gets somewhat lost. Consider shortening the detailed physiological mechanism of NO (lines 45-67) and focusing more sharply on the problem of poor patient adherence to current beetroot products. The transition to the study's aim (lines 119-128) is good. Ensure the objectives are listed as clear, testable points that directly map to the Results section.

Answer: We agree with the reviewer`s suggestion and text about the detailed physiological mechanism of NO was reduced. Furthermore, the point of poor patient adherence to the ingestion of current beetroot products was improved, as suggested (page 2, lines 54-61 and 78-100). The objectives are listed as clear and testable points that directly map to the Results section.

 

 

Enhance the Discussion Section: The discussion is currently a point-by-point rehash of the results. It needs to be elevated to an argument about the significance of the findings. Interpret, Don't Just Repeat: Instead of saying "The protein content was 19%," say "The high protein content (19%) meets regulatory standards and positions the product as a nutritionally complete option, surpassing typical protein levels in similar freeze-dried yogurts [53]." Do this for all key findings.

Answer: We agree with the reviewer`s suggestion and we modified and corrected the 4. Discussion section according to reviewer 2 recommendations (page 18, lines 635-637).

 

 

Create a Narrative: Structure the discussion around a central theme: "The combination of maltodextrin and cassava starch proved to be the optimal formulation." Use your data to build this case. Discuss why this combination works best (e.g., high EE from starch, high solubility from maltodextrin, leading to the best sensory scores).

Answer: For a better understanding of the results presented in this manuscript, we discussed which formulation performed best in each analysis. However, we agreed with the reviewer`s suggestion, and at the end of the discussion, a new paragraph was written summarizing why and which formulation was the best (page 25, lines 1044-1054).

 

 

Add a "Limitations and Future Directions" Paragraph: Acknowledge the study's limitations. For example, the shelf-life study was only 30 days, the sensory panel was untrained, and the actual bioavailability of the compounds in vivo was not tested. This directly leads to your final sentence in the conclusions, making it more impactful. Mention the need for clinical trials to prove the claimed "cardioprotective" effects.

Answer: We agree with the reviewer`s suggestion and a paragraph addressing information about limitations and future directions has been added at the end of the discussion (page 26, lines 1055-1066).

 

 

2. Specific Technical and Writing Revisions

• Abstract (Lines 23-44):
• Line 30-31: "higher than >90%" is redundant. Use "high solubility (>90%)".

Answer: We agree with the reviewer`s suggestion and we fix it.

 

• Line 40: The mention of "probiotic microcapsules" is new. The kefir culture is mentioned in the methods, but its viability isn't a major focus of the results (only briefly in microbiology). Either present data on probiotic viability or remove the claim from the abstract.

Answer: We agree with the reviewer`s suggestion and we removed it.

 

• Introduction (Lines 80-92):
• Line 86: "clinical tests concluded that large beetroot juice volumes... have limited the adherence" → This is the core problem. State it more strongly. "The requirement to consume large, unpalatable volumes of beetroot juice (500-700 mL) to achieve a clinical dose of nitrate has been a major barrier to patient adherence in long-term interventions [3,11,16-19]."

Answer: We agree with the reviewer`s suggestion and this paragraph was rewritten (page 2 and 3, lines 78-100).

 

 

• Results (Lines 319-424):
• General: The results are very clearly presented with excellent tables and figures. Line 340 (Table 1): This table is information-dense and excellent. In the text, guide the reader to the most important comparisons. For example: "As expected, microencapsulated formulations showed significantly higher carbohydrate and energy content (p<0.05) due to the added polysaccharides, while protein and lipid content decreased proportionally. Critically, the concentration of the target bioactive, NO3-, remained high (>615 mg·100g-1) in all microencapsulated samples, although this was lower than the non-encapsulated control."

Answer: We agree with the reviewer`s suggestion and we draw the reader's attention more to the most important comparisons on 3. Results sections.

 

 

• Line 353 (Table 1): The values for NO3- are given as mmol·mg-1 / mg·100g-1. The formatting is confusing. Clarify this in the table footnote. It should likely be mmol·100g-1 / mg·100g-1.

Answer: We agree with the reviewer`s correction and the correct format is mmol/mg·100g^-1.  Table 1 was corrected as suggested (page 8).

 

 

• Line 392 (Table 2): The Span index is mentioned. Explain in the text what a "good" Span value is and why the high Span for the combination is not a problem if the d(0.5) values are small and solubility is high.

Answer: We agree with the reviewer`s suggestion and a new paragraph explained what good Span value is and why the high Span for the combination is not a problem with small d(0.5) values (page 19 and 20, lines 723-739).

 

 

• Discussion (Lines 564-962):
• Line 588: "lipids may be partially retained... resulting in under-quantification." This is a good point. Be more definitive: "The lower measured lipid content in microencapsulated samples is likely an artifact of the extraction method, as the polymeric wall material may hinder the solvent's ability to fully access the encapsulated lipids [49]."

Answer: We agree with the reviewer`s suggestion and this sentence was modified (page 17, lines 606-615).

 

 

• Line 616-627: The paragraph on the clinical dose of nitrate is excellent. It perfectly links the product's composition to its intended purpose. This is the kind of impactful writing needed throughout the discussion.

Answer: We appreciate the reviewer's comment.

 

 

• Line 708-714: The explanation for the FTIR band at 1744 cm⁻¹ is good. State it more confidently: "The presence of the carbonyl band at 1744 cm⁻¹ exclusively in the encapsulated formulations, and its absence in the spectra of the pure encapsulating agents, serves as strong evidence that the core beetroot-dairy materials were successfully incorporated and preserved within the microcapsules [79,80]."

Answer: We agree with the reviewer`s suggestion and this sentence was modified (page 20, 755-759).

 

 

• Line 863-893: The paragraphs on encapsulation efficiency are well-researched and explain the "why" behind the results. This is a model for the rest of the discussion.

Answer: We appreciate the reviewer's comment.

 

 

• Line 953-962: The conclusion is strong. It clearly states the best formulation and its key attributes. The mention of the patent is a nice touch.

Answer: We appreciate the reviewer's comment.

 

 

• Materials and Methods (Lines 129-318):
• This section is comprehensive and reproducible. No major revisions needed. Ensure all equations are correctly formatted (e.g., Equation 3 uses "BT" for both total and supernatant betalains—this is a typo that needs correction).

Answer: We agree with the reviewer`s correction and we correctly clarified each abbreviation included in the equation (page 6, lines 281-286).

 

 

• Line 253-255: Clarify the distinction between BT and BTS in Equation (3). It currently reads: EE(%) = (BT - BTS×100) / BT. This should likely be EE(%) = ((BT - BTS) / BT) × 100. Please verify.

Answer: We agree with the reviewer`s correction and we corrected the equation 3, as suggested (page 6, line 279).

 

 

3. Language and Presentation

• Minor Typos and Grammatical Errors:
• Line 92: "already demonstrate very effective" → "already demonstrated to be very effective"

Answer: We agree with the reviewer`s suggestion and we fix it (page 2-3, lines 97-98).

 

 

• Line 101-102: The sentence "metabolic mediators, and flavors, has not yet been elucidated [22]." is repeated verbatim. (Lines 101 and 103).

Answer: We agree with the reviewer`s suggestion and we fix it (page 3, lines 112).

 

 

• Line 158: "maintained at room temperature in a cool, dry and dark place" → "maintained in a cool, dry, and dark place at room temperature".

Answer: We agree with the reviewer`s suggestion and we fix it (page 4, lines 188-189).

 

 

• Line 292: "an air- conditioned refrigerated and closed cabin" → "a temperature-controlled, closed sensory booth with white lighting".

Answer: We agree with the reviewer`s suggestion and we fix it (page 7, lines 326).

 

 

• Line 328: "betaxanins" is misspelled; it should be "betacyanins" in several places (e.g., lines 328, 344, 489, 492, 494, 496). A thorough spell-check for this term is recommended.

Answer: We agree with the reviewer`s suggestion and we fix it.

 

• Clarity and Concision:
• Throughout the manuscript, sentences are often very long and contain multiple clauses. For example, lines 108-118. Breaking these into shorter, more direct sentences would improve readability.

Answer: We agree with the reviewer`s suggestion and we fix it.

 

• Be consistent with terminology. "Powdered beetroot dairy drink microencapsulated" is a mouthful. After the first instance, you can refer to it as "the microencapsulated formulations" or "the powdered drinks" for brevity, as long as the context is clear.

Answer: We agree with the reviewer`s suggestion and we fix it.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

Check for inconsistencies in the English language throughout the manuscript.

Revise abbreviations throughout the document and in figure legends (e.g., UHT, APHA).

Review the citation of references and remove the use of “by” where unnecessary.

Clarify how the product was presented during the sensory evaluation, liquid or purée.

Use consistent and appropriate terminology (molds, yeasts, fungi) throughout the manuscript and in Table 5. If possible, include the yeast species identified.

Include the lyophilization conditions.

In the Discussion section, address the relevance of the acidic pH of the new dairy drink in relation to potential health implications.

Section 3.3: verify units (line 391).

Consider updating the term “beneficial bacterial flora.” Microbiota is already included.

If Figure 4D shows the final appearance of the “dairy drink,” consider its texture when naming the final product, as it appears more similar to a custard or purée. Take this into account when revising the title.

 

The current title “Powdered beetroot dairy-drink microencapsulated: a cardioprotective, immunological and metabolic boosting product”, seems inappropriate since no experimental evaluation was performed”. Consider highlighting your results, like “Preservation of microencapsulated betalains in a powdered beetroot-based dairy drink with high sensory quality and consumer acceptance”.

Author Response

Manuscript ID: foods-4224179

 

Former title: Powdered beetroot dairy-drink microencapsulated: a cardioprotective, immunological and metabolic boosting product.

 

Actual title: A Novel Dairy-Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives Compounds

 

Authors: Lucileno Rodrigues da Trindade, Diego dos Santos Baião, Davi Vieira Teixeira da Silva, Fernanda Petzold Pauli, and Vania Margaret Flosi Paschoalin

 

GENERAL COMMENTS BY THE AUTHORS

 We believe that we have fully addressed all reviewer concerns and comments.  Several modifications were carried out in the revised manuscript.

Major modifications were carried out in the revised manuscript. The title was modified, the abstract was rewritten, including the reduction of some paragraphs, modifications in sentences were and two new paragraph addressing limitations were included, future directions and the best formulation of the present study were added in the manuscript. Two new paragraph addressing limitations were included, future directions and the best formulation of the present study were added in the manuscript. Minor corrections on grammar and syntax were performed for better reading and understanding. Nineteen references were included:

84. Labuza, T.P; Altunakar, B. Water Activity Prediction and Moisture Sorption Isotherms. In Water Activity in Foods (eds G.V. Barbosa-Cánovas, A.J. Fontana, S.J. Schmidt and T.P. Labuza), 2020.
85. Fan, F.; Liu, H.; Xu, Y.; Mou, T. Quantitative Analysis of Molecular Mobility in Amorphous Lactose Above Tg: A Novel Insight from Molecular Dynamic Simulation to Strength Parameter. Foods. 2025, 14, 928.
86. Saavedra-Leos, Z.; Leyva-Porras, C.; Araujo-Díaz, S.B.; Toxqui-Terán, A.; Borrás-Enríquez, A.J. Technological Application of Maltodextrins According to the Degree of Polymerization. Molecules. 2015, 20, 21067-21081.
87. Xiao, Z.; Xia, J.; Zhao, Q.; Niu, Y.; Zhao, D. Maltodextrin as wall material for microcapsules: A review. Carbohy. Poly. 2022, 298, 1-18.
88. Carvalho, G.R.; Fernandes, R.V.B.; de Castro, E.S.P.; Dessimoni, A.L.A.; Oliveira, C.R.; Borges, S.V.; Botrel, D.A. Influence of modified starches as wall materials on the properties of spray-dried lemongrass oil. J. Food Sci. Technol. 2019, 56, 4972-4981.
89. Bilenler Koc, T.; Fırat, I.; Karabulut, I.; Boztepe, C.; Topalcengiz, Z. Microencapsulation of Beetroot Anthocyanins: Inves-tigation of Degradation Kinetics and Modeling by Using Artificial Neural Networks. ACS Omega. 2025, 25, 2270-2284.
90. Shofinita, D.; Fawwaz, M.; Achmadi, A.B. Betalain extracts: Drying techniques, encapsulation, and application in food industry. Food Frontiers, 2023, 4, 576–623.
91. Ravichandran, K.; Palaniraj. R.; Saw, N.M.; Gabr, A.M.; Ahmed, A.R.; Knorr, D.; Smetanska, I. Effects of different encap-sulation agents and drying process on stability of betalains extract. J Food Sci Technol. 2014, 51, 2216-21.
92. Chakma, S.; Acharjee, M.R.; Tonni, I.J.; Tabassum, F.; Das, E.; Islam, S.; Faruk, S.A. Probiotic potential of yogurt: Explor-ing lactic acid bacteria for health-enhancing benefits. Appl Food Res. 2025, 5, 1-15.
93. Peng, Y.; Horne, D.S.; Lucey, J.A. Impact of preacidification of milk and fermentation time on the properties of yogurt. J. Dairy Sci. 2009, 92, 2977-2990.
94. Ren, Q.; Zhang, J.; Chen, X.; Pang, J.; Wichers, H.J.; Wen, Y.; Hettinga, K.A. Effects of fermentation on protein digestion and peptide release in an in vitro adult digestion model. Food Res. Inter. 2026, 230, 1-10.
95. Parvez, S.; Malik, K.A.; Ah Kang, S.; Kim, H.Y. Probiotics and their fermented food products are beneficial for health. J. Appl. Microbiol. 2006, 100, 1171–1185.
96. Makinen, O.E.; Wanhalinna, V.; Zannini, E.; Arendt, E.K. Foods for special dietary needs: Non-dairy plant-based milk substitutes and fermented dairy-type products. Crit. Rev. Food Sci. Nutr. 2016, 56, 339–349.
97. Guadalupe, H.S.D.; Berenice, O.R.L.; Elizabeth, C.G.A.; Guillermo, G.O.L.; Araceli, C.O. Probiotic fermented milk and type 2 diabetes mellitus: Mechanisms, benefits, and future directions. J. Funct. Foods. 2025, 134, 107068.
98. García-Burgos, M.; Moreno-Fernández, J.; Alférez, M.J.; Díaz-Castro, J.; López-Aliaga, I. New perspectives in fermented dairy products and their health relevance. J. Funct. Foods. 2020, 72, 104059.
99. Huligere, S.S.; Chandana Kumari, V.B.; Alqadi, T.; Kumar, S.; Cull, C.A.; Amachawadi, R.G.; Ramu, R. Isolation and characterization of lactic acid bacteria with potential probiotic activity and further investigation of their activity by α-amylase and α-glucosidase inhibitions of fermented batters. Front. Microbiol. 2023, 13, 1042263.
100. Hashemi Javaheri, F.S.; Nasiri Jounaghani. M.; Sahebkar, A.; Norouzzadeh, M.; Delgarm, P.; Shahinfar, H.; Mirdar Harijani, A. The effect of fermented dairy intake and abdominal obesity in adults: a systematic review and dose-response meta-analysis of cohort studies. Eat Weight Disord. 2025, 30, 23.
101. Mukarromah, T.A.; Rustanti, N.; Mahati, E.; Suparmi; Ayustaningwarno, F. The Impact of Fermented Milk Products on Gut Microbiota-Derived Metabolites in Obesity: A Narrative Review. J. Food Sci. 2025, 90, e70301.
102. Stachelska, M.A.; Karpiński, P.; Kruszewski, B. Health-Promoting and Functional Properties of Fermented Milk Bever-ages with Probiotic Bacteria in the Prevention of Civilization Diseases. Nutrients. 2025, 17, 9.

 

All modifications were highlighted in yellow.

After performing all the modifications suggested by the reviewers, the entire text was revised by an editing specialized company to improve English grammar and syntax. The authors have approved the final version of the manuscript.

We thank the Reviewer for his-her critical insightful of our manuscript.

We hope that in its revised form our manuscript will be found suitable for publication in this respectful journal.

 

Answers to Reviewer 3

Reviewer 3 comments preceding our responses.

 

Comments and Suggestions for Authors

Comments to the Author

Dear authors

Check for inconsistencies in the English language throughout the manuscript.

Answer: We agree and appreciate the reviewer`s suggestion and the inconsistencies were checked in the entire manuscript.

 

Revise abbreviations throughout the document and in figure legends (e.g., UHT, APHA).

Answer: We agree with the reviewer`s suggestion and all abbreviations were revised in the entire manuscript.

 

 

Review the citation of references and remove the use of “by” where unnecessary.

Answer: We agree with the reviewer`s suggestion and we removed the use of by before citation of references, as suggested.

 

 

Clarify how the product was presented during the sensory evaluation, liquid or purée.

Answer: After being reconstituted with ice water, the product had a smooth, homogeneous paste consistency, without any lumps. This information was added in the manuscript (page 16, lines 559-560), as suggested by the reviewer.

 

 

Use consistent and appropriate terminology (molds, yeasts, fungi) throughout the manuscript and in Table 5. If possible, include the yeast species identified.

Answer: We agree with the reviewer`s suggestion and we modified the terminology throughout the entire manuscript. Unfortunately, it was impossible to include the yeast species because the APHA methodology is focused solely on enumerating (counting) yeast colonies present in food or environmental samples, without providing detailed taxonomic information about which species are being tested. For identification, additional techniques such as biochemical tests, molecular sequencing, or specific isolation methods should be conducted.

 

 

 

 

Include the lyophilization conditions.

Answer: Information about the sublimation process performed by the freeze-dryer can be found on page 4, lines 180-182, as suggested.

 

 

In the Discussion section, address the relevance of the acidic pH of the new dairy drink in relation to potential health implications.

Answer: We agree with the reviewer`s suggestion and a discussion about the relevance of the acidic pH of the new dairy drink in relation to potential health implications were included in the manuscript (page 22, lines 848-883).

 

 

Section 3.3: verify units (line 391).

Answer: They are correct.

 

 

Consider updating the term “beneficial bacterial flora.” Microbiota is already included.

Answer: We agree with the reviewer`s suggestion and this term was modified to microbiota.

 

 

If Figure 4D shows the final appearance of the “dairy drink,” consider its texture when naming the final product, as it appears more similar to a custard or purée. Take this into account when revising the title.

Answer: The manuscript title is correct, as the product is in powder form and the consumer could choose how much ice water it would be added for product reconstitution. During the sensorial analysis, with one hundred volunteers, we have added a volume of water to obtain a pastier formulation like the natural yogurt.

 

 

The current title “Powdered beetroot dairy-drink microencapsulated: a cardioprotective, immunological and metabolic boosting product”, seems inappropriate since no experimental evaluation was performed”. Consider highlighting your results, like “Preservation of microencapsulated betalains in a powdered beetroot-based dairy drink with high sensory quality and consumer acceptance”.

Answer: We agree with the reviewer`s suggestion and in accordance with the suggestions of the other reviewers, we modified the title in the manuscript to: “A Novel Dairy-Beetroot Powder: Microencapsulation Improves Stability and Sensory Qualities While Preserving Cardioprotective Bioactives Compounds” (page 1).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Thank you for providing point by point answers to all requested revisions.

The maniscript can  now accepted.

Best regards

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have reviewed and addressed each of the observations of the manuscript, which has improved remarkably