Cheese Whey Valorization via Microbial Fermentation (Lactic Acid Bacteria, Yeasts/Fungi, and Microalgae), Postbiotic Production, and Whey-Based Encapsulation Strategies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is positioned as a comprehensive review spanning from whey fermentation by LAB/yeasts-fungi/microalgae, to postbiotics, and whey-based encapsulation. The scope is extremely broad relative to the depth of analysis delivered, resulting in a largely descriptive catalogue of studies (tables + narrative summaries) with limited critical synthesis. Moreover, the manuscript does not report any review methodology (search strategy, databases, timeframe, inclusion/exclusion criteria, screening process), which makes coverage, potential bias, and “recent advances” claims difficult to evaluate.

In fact, from the Tables i noted many important missing, like the massive utilization of whey and dairy side-streams for sustainable traustochytrids cultivation (like Aurantiochytrium sp.). Also, the utilizazion of cheese whey for the oomycete Pythium irregulare is missing (https://www.mdpi.com/2071-1050/15/2/1147). I suggest to check better for the literature.

The review notes whey variability (type, pretreatment, supplementation), yet does not convert this into actionable guidance (like a decision tree: whey type → pretreatment → organism choice). The composition section lists typical fractions and BOD/COD ranges, but does not discuss how these ranges translate into inhibition thresholds (salts/low pH) or contamination risk and sterilization strategy

Since the manuscript tries to cover fermentation platforms, postbiotics, and encapsulation in one piece, the consequence is that each area receives largely “study-by-study” reporting with few cross-cutting conclusions. In fact i sensed a limited discussion of trade-offs (yield vs inhibition vs contamination risk vs downstream cost) despite claiming “performance trade-offs relevant to industrial scale-up"

Tables list outcomes using mixed endpoints (CFU/mL, g/L, % removal etc.), which prevents meaningful comparison. For a valorization review, minimum harmonized metrics should be discussed consistently.

The paper repeatedly frames whey bioprocessing as circular bioeconomy/low-cost/scale-up relevant, and even calls for pilot-scale/TEA/LCA in the conclusion, but it does not synthesize any actual quantitative TEA/LCA evidence (even at high level) nor does it identify the dominant cost/environmental drivers by process family. I suggest to include some insights from TEA on traustochytrids/microalgae case studies.

Given repeated reference to “food-grade” systems and “probiotics/postbiotics,” the review should address the regulatory status differences across regions (GRAS/QPS constraints, novel food considerations) and general safety issues.

The conclusion lists fashionable directions (precision fermentation, AI/ML, multi-omics, microfluidics, LCA) but these appear as “aspirational add-ons” rather than being derived from rigorously identified gaps and bottlenecks mapped earlier in the manuscript.

Comments on the Quality of English Language

Occasional grammar/typos

Author Response

This is positioned as a comprehensive review spanning from whey fermentation by LAB/yeasts-fungi/microalgae, to postbiotics, and whey-based encapsulation. The scope is extremely broad relative to the depth of analysis delivered, resulting in a largely descriptive catalogue of studies (tables + narrative summaries) with limited critical synthesis. Moreover, the manuscript does not report any review methodology (search strategy, databases, timeframe, inclusion/exclusion criteria, screening process), which makes coverage, potential bias, and “recent advances” claims difficult to evaluate.

Response: We thank you for this thorough and constructive comment. About your concern, we clarify that the manuscript was conceived as a structured narrative review, rather than a systematic review, with the objective of integrating recent advances in whey valorization across fermentation platforms. To improve transparency, we have now explicitly incorporated a brief review methodology section, detailing the literature search strategy, timeframe, database, keywords, and inclusion criteria (lines 77–82.

 

In fact, from the Tables i noted many important missing, like the massive utilization of whey and dairy side-streams for sustainable traustochytrids cultivation (like Aurantiochytrium sp.). Also, the utilizazion of cheese whey for the oomycete Pythium irregulare is missing (https://www.mdpi.com/2071-1050/15/2/1147). I suggest to check better for the literature.

Response: We thank the reviewer for pointing out the omission of thraustochytrids and oomycetes. We note that these organisms fall outside the primary scope of this section, which is focused on photosynthetic microalgae and cyanobacteria cultivated in whey-supported systems. Nevertheless, given their phylogenetic proximity to microalgae and their emerging relevance in dairy side-stream valorization, we have incorporated a dedicated subsection (Section 5.4) to briefly address their use (lines 481-503).

 

The review notes whey variability (type, pretreatment, supplementation), yet does not convert this into actionable guidance (like a decision tree: whey type → pretreatment → organism choice). The composition section lists typical fractions and BOD/COD ranges, but does not discuss how these ranges translate into inhibition thresholds (salts/low pH) or contamination risk and sterilization strategy

Response: We agree that whey variability should be more explicitly translated into practical guidance. In response, we have revised the manuscript to strengthen the link between whey composition, pretreatment requirements, and microorganism selection. Additionally, we also expanded the discussion of how parameters and strategies may influence bioprocess. Changes have been added in the new section 2.2. (lines 122-142).

 

Since the manuscript tries to cover fermentation platforms, postbiotics, and encapsulation in one piece, the consequence is that each area receives largely “study-by-study” reporting with few cross-cutting conclusions. In fact i sensed a limited discussion of trade-offs (yield vs inhibition vs contamination risk vs downstream cost) despite claiming “performance trade-offs relevant to industrial scale-up"

Response: We acknowledge the reviewer’s concern. We would like to clarify that trade-offs relevant to scale-up were already addressed through platform-specific discussion sections, particularly within the “limitations and considerations” subsections of each fermentation platform. These discussions are further integrated in the Conclusions, where cross-platform insights are summarized.

 

Tables list outcomes using mixed endpoints (CFU/mL, g/L, % removal etc.), which prevents meaningful comparison. For a valorization review, minimum harmonized metrics should be discussed consistently.

Response: We thank the reviewer for this observation. As discussed in the new Section 2.3 (lines 144–153), the use of mixed performance endpoints reflects the fundamentally different objectives and evaluation frameworks across whey valorization platforms, which currently prevents full metric harmonization. This limitation is explicitly acknowledged and discussed as an inherent challenge in cross-platform comparison.

 

The paper repeatedly frames whey bioprocessing as circular bioeconomy/low-cost/scale-up relevant, and even calls for pilot-scale/TEA/LCA in the conclusion, but it does not synthesize any actual quantitative TEA/LCA evidence (even at high level) nor does it identify the dominant cost/environmental drivers by process family. I suggest to include some insights from TEA on traustochytrids/microalgae case studies.

Response: We thank the reviewer for this suggestion. Available TEA evidence from thraustochytrid-based systems has been incorporated in Section 5.4, where a techno-economic assessment of Aurantiochytrium spp. identifies enzymatic hydrolysis and dilution as key cost drivers in whey-based processes (lines 481-503).

 

Given repeated reference to “food-grade” systems and “probiotics/postbiotics,” the review should address the regulatory status differences across regions (GRAS/QPS constraints, novel food considerations) and general safety issues.

Response: We thank the reviewer for this important comment. To address regulatory and safety considerations, we have added Section 8, which outlines regional regulatory frameworks and general safety requirements relevant to whey-derived probiotics, postbiotics, and encapsulated ingredients (lines 776-800).

 

The conclusion lists fashionable directions (precision fermentation, AI/ML, multi-omics, microfluidics, LCA) but these appear as “aspirational add-ons” rather than being derived from rigorously identified gaps and bottlenecks mapped earlier in the manuscript.

Response: We thank the reviewer for this comment. The Conclusions have been revised to explicitly derive future research directions from the technical and economic bottlenecks identified in the review (810-820).

Finally, The manuscript has been carefully revised to correct minor grammatical issues and typographical errors throughout the text.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present an important and up-to-date review on the application of cheese whey in the food industry. Although whey is already widely used in the production of foods such as dairy beverages, cheese whey is still understudied, especially with regard to fermentation.

Although whey is used worldwide for fermentation with lactic acid bacteria, the authors presented an updated perspective on fermentation with fungi/yeasts and, especially, microgases. Furthermore, the main positive and negative aspects for its application in postbiotic products were addressed. The authors also provide relevant information on the use of more traditional technologies, such as spray drying, and more recent encapsulation techniques for these compounds.

Therefore, the review offers an interesting conclusion to all points addressed in the text, including data organized in tables that facilitate the interpretation of the mentioned articles and a complete and up-to-date understanding of the subject.

Author Response

The authors present an important and up-to-date review on the application of cheese whey in the food industry. Although whey is already widely used in the production of foods such as dairy beverages, cheese whey is still understudied, especially with regard to fermentation.

 

Although whey is used worldwide for fermentation with lactic acid bacteria, the authors presented an updated perspective on fermentation with fungi/yeasts and, especially, microgases. Furthermore, the main positive and negative aspects for its application in postbiotic products were addressed. The authors also provide relevant information on the use of more traditional technologies, such as spray drying, and more recent encapsulation techniques for these compounds.

 

Therefore, the review offers an interesting conclusion to all points addressed in the text, including data organized in tables that facilitate the interpretation of the mentioned articles and a complete and up-to-date understanding of the subject.

 Response: We thank the reviewer for the positive and encouraging evaluation of our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript title was “Cheese whey valorization via microbial fermentation (lactic acid bacteria, yeasts/fungi, and microalgae), postbiotic production, and whey-based encapsulation strategies” This review summarizes a lot of content, but my idea is that the introduction should focus on one aspect.

Author Response

The manuscript title was “Cheese whey valorization via microbial fermentation (lactic acid bacteria, yeasts/fungi, and microalgae), postbiotic production, and whey-based encapsulation strategies” This review summarizes a lot of content, but my idea is that the introduction should focus on one aspect.

Response: We thank the reviewer for the positive and encouraging evaluation of our manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

thanks for the detailed response, i have no further requests. Take time to make a carefull proof review for grammar and typos.

Reviewer 3 Report

Comments and Suggestions for Authors

None