Candida utilis Biosurfactant from Licuri Oil: Influence of Culture Medium and Emulsion Stability in Food Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript explores licuri oil (Syagrus coronata) as a carbon source for producing Candida utilis biosurfactant, tests medium factors using a 2^4 factorial design, characterizes CMC and surface-tension reduction, assesses stability across different pH levels, temperatures, and salinity, and applies the biosurfactant (alone or combined with lecithin/egg yolk) in salad-dressing (“mayonnaise-type”) emulsions stored for up to 240 days. Furthermore, the research, described in an interesting way, effectively integrates biotechnology and food technology.

1. The abstract mentions “high purity.” Still, it provides no compositional data (such as FTIR/NMR/TLC, sugar/lipid content, HPLC/GC–MS) to support the claim or to identify the biosurfactant class.
2. Visual stability over 240 days is promising. Still, quantitative measurements (droplet-size distribution, zeta potential, viscosity/rheology, pH, etc.) are necessary to support “physical integrity,” compare to controls, and uncover mechanisms (co-emulsifier vs. primary emulsifier).
3. Consider adding a reference emulsifier like Tween 80 for ST/CMC benchmarking, along with a no-BS control in the kinetic ST curve, to provide context for performance. This will help readers compare your BS to established standards.
4. Table 1 – How were the concentrations determined? Why, in some cases, is it summed to 3.5, while in others it's 3, etc.?
5. Figures 2 and 3 are unnecessary because the same results are shown in Table 2.
6. Table 4 requires a statistical analysis of the results, specifically ANOVA and post-hoc tests.

Minor suggestions:

1. Line 58 – The Latin name should be written in italics.
2. Line 75 – „nuts” instead of „almonds”
3. All the abbreviations found in Table 2 should be defined in the Table footer, i.e., X1-X4, -1, 0, 1 etc.

Author Response

Reviewer 1:

Response: We sincerely appreciate your valuable comments and suggestions, which have greatly helped us enhance the clarity and overall quality of the manuscript. In response to your feedback, we have carefully revised the abstract to improve its readability. The specific revisions are as follows.

1.The abstract mentions “high purity.” Still, it provides no compositional data (such as FTIR/NMR/TLC, sugar/lipid content, HPLC/GC–MS) to support the claim or to identify the biosurfactant class.

Response: Thank you very much for your suggestion. We agree that the term "high purity" was inaccurate given that compositional analyses (FTIR/NMR/HPLC) were not within the scope of this work. Our study focuses on the bioprocess (production kinetics), physical properties (surface tension/CMC), and the novel application in food emulsions. Therefore, we have rephrased the statement in the Abstract (line 28) to emphasize the surface activity and efficiency of the isolated product, rather than its chemical purity.

2.Visual stability over 240 days is promising. Still, quantitative measurements (droplet-size distribution, zeta potential, viscosity/rheology, pH, etc.) are necessary to support “physical integrity,” compare to controls, and uncover mechanisms (co-emulsifier vs. primary emulsifier).

Response: We sincerely thank the reviewer for this recommendation. We agree that quantitative data is essential to validate visual stability. To address this, we requested an extension from the Editor and performed additional analyses on the stable formulations. Specifically, we have now included:

Physicochemical measurements: pH and apparent viscosity of the emulsions were evaluated to confirm physical integrity (lines 153-159)

Comparative Control: We included a control formulation using a commercial surfactant (Tween 80) to benchmark the performance of the biosurfactant (lines 145-146)

Microbiological analysis: To ensure that the stability was not compromised by microbial growth during storage. These new data have been Tadded to the Methods and Results sections, demonstrating that the biosurfactant formulations maintained rheological properties comparable to the control and remained microbiologically safe (lines 162-171).

3.Consider adding a reference emulsifier like Tween 80 for ST/CMC benchmarking, along with a no-BS control in the kinetic ST curve, to provide context for performance. This will help readers compare your BS to established standards.

Response: We appreciate the reviewer's suggestion to benchmark our product against established standards.

Reference Emulsifier (Tween 80): As detailed in our response to Comment 2, we have introduced Tween 80 as a positive control in the emulsion stability assays (viscosity and pH) to validate the functional performance of our biosurfactant in the food matrix (lines 145-146, lines 153-159)

Kinetic Control: Regarding the kinetic profile, we consider the measurement at Time 0 h as the adequate baseline control. The initial surface tension (~44 mN/m) reflects the medium composition (mineral salts + Licuri oil) before significant microbial activity. The subsequent drop to 31.55 mN/m confirms the biological production of surface-active compounds.

4.Table 1 – How were the concentrations determined? Why, in some cases, is it summed to 3.5, while in others it's 3, etc.?

We apologize for the oversight regarding the sum of percentages in Table 1. Indeed, these were typographical errors, and we have corrected the table to accurately reflect the experimental design, ensuring all values are consistent. Regarding the chosen concentrations, we clarify that Brazilian legislation (ANVISA) does not specify a mandatory quantitative limit for this class of emulsifiers in sauces. Therefore, we adopted the methodology proposed by Ribes et al. (2021) as a guideline, setting the maximum concentration at 3.0% (w/w) to ensure functional stability without compromising the formulation's sensory profile.

5.Figures 2 and 3 are unnecessary because the same results are shown in Table 2.

Response: We agree with the reviewer regarding the redundancy. To improve the manuscript's conciseness and avoid data duplication, we have removed Figures 2 and 3. The experimental results for all runs are fully detailed in Table 2.

6.Table 4 requires a statistical analysis of the results, specifically ANOVA and post-hoc tests.

Response: We appreciate the reviewer's guidance to improve the data interpretation. We have performed an Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test (p < 0.05) for the stability results presented in Table 4. Superscript lowercase letters have been added to the mean values to indicate statistically significant differences within each parameter group (Temperature, NaCl, and pH). A footnote explaining the statistical notation was also included.

Minor suggestions:

1.Line 58 – The Latin name should be written in italics.

Response: Corrected.

2. Line 75 – „nuts” instead of „almonds”

Response: Thank you for your feedback. Term replaced

3. All the abbreviations found in Table 2 should be defined in the Table footer, i.e., X1-X4, -1, 0, 1 etc.

Response: Thank you for your feedback. We have added a legend at the end of Table 2 with the terms that refer to the independent variables. (lines 204-206).

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript requires major revisions before it can be considered for publication. Key issues pertain to data consistency, methodological depth, structural characterization, and the interpretation of results, which currently limit the impact and reproducibility of the study.

1. The minimum surface tension is reported as 31.55 mN·m⁻¹ in the kinetics experiment (Fig. 1) and abstract. However, the CMC determination (Fig. 5) shows a minimum surface tension of 25.17 mN·m⁻¹ for the isolated biosurfactant. This significant discrepancy is not explained.
2. The study relies solely on functional properties (surface tension, E24) without any chemical structural analysis (e.g., FTIR, HPLC-MS, NMR). The type of biosurfactant (e.g., glycolipid, lipopeptide, protein-polysaccharide complex) produced by C. utilis on this substrate remains unknown. This is a major omission, as structure dictates function, stability, and potential regulatory approval for food use.
3. The stability tests do not cover the full range of conditions relevant to food processing. The temperature test stops at 60°C, omitting pasteurization or sterilization temperatures (e.g., 80-121°C). Furthermore, only acidic to neutral pH (3-7) is tested, neglecting alkaline conditions common in some food systems. This limits the claim of "robustness."
4. The stability of the salad dressings is assessed solely by visual observation ("remained stable," "phase separation"). This is highly subjective. Quantitative measures such as droplet size distribution, zeta potential, rheological measurements, or centrifugation tests are necessary to provide objective, reproducible data on emulsion stability.
5. The experimental design for the salad dressings lacks a positive control containing a standard commercial emulsifier (e.g., Tween 80, Mono- and diglycerides). Without this comparison, it is impossible to objectively evaluate the performance of the BS relative to current industry standards, thus weakening the application claim.
6. The extraction and isolation protocol is described, but no data on the yield (g BS / g substrate) or purity of the final isolated product is provided. This information is crucial for assessing the economic feasibility and for correlating concentration with functional properties in the CMC experiment.

Author Response

Reviewer 2:
The manuscript requires major revisions before it can be considered for publication. Key issues pertain to data consistency, methodological depth, structural characterization, and the interpretation of results, which currently limit the impact and reproducibility of the study.

Response: We sincerely thank the reviewer for the thorough evaluation and the constructive criticism regarding the depth and consistency of our study. We have carefully considered each comment and provided detailed, point-by-point responses below. To address the concerns about methodological depth and data consistency, we have:

Performed new experimental analyses: Including apparent viscosity, pH stability, and microbiological safety of the emulsions.

Added Controls: We included a comparative analysis with a commercial surfactant (Tween 80) to benchmark performance.

Refined Data Analysis: We applied rigorous statistical methods (ANOVA and Tukey’s test) to validate the results.

Corrected Inconsistencies: Typographical errors in formulation tables were corrected, and the discussion on structural characterization was adjusted to align with the analytical scope. A summary of the major revisions is provided below. Should there be any remaining issues, we would be grateful for the opportunity to make further improvements.

1.The minimum surface tension is reported as 31.55 mN·m⁻¹ in the kinetics experiment (Fig. 1) and abstract. However, the CMC determination (Fig. 5) shows a minimum surface tension of 25.17 mN·m⁻¹ for the isolated biosurfactant. This significant discrepancy is not explained.

Response: We thank the reviewer for highlighting this difference. We clarify that this variation is attributed to the matrix effect. The value of 31.55 mN·m⁻¹ in the kinetics experiment refers to the cell-free culture broth, which contains residual nutrients, salts, and metabolites that can interfere with interfacial packing. In contrast, the value of 25,17 mN·m⁻¹ was obtained using the isolated biosurfactant redissolved in pure distilled water. The extraction and partial purification process effectively removed these interfering compounds and concentrated the surface-active moieties, allowing for better interfacial saturation and, consequently, a lower minimum surface tension. We have added a sentence to the Discussion to clarify this purification effect (lines 331-335),

2.The study relies solely on functional properties (surface tension, E24) without any chemical structural analysis (e.g., FTIR, HPLC-MS, NMR). The type of biosurfactant (e.g., glycolipid, lipopeptide, protein-polysaccharide complex) produced by C. utilis on this substrate remains unknown. This is a major omission, as structure dictates function, stability, and potential regulatory approval for food use.

Response: We appreciate your feedback. Indeed, the use of Candida utilis in this substrate is unprecedented. However, the focus of this publication was to produce the biosurfactant and apply and evaluate it as a potential additive/emulsifier in the stability of mayonnaise-type sauces. Further studies on the characterization of the biosurfactant are already underway, but it was not possible to add them to this article.

3.The stability tests do not cover the full range of conditions relevant to food processing. The temperature test stops at 60°C, omitting pasteurization or sterilization temperatures (e.g., 80-121°C). Furthermore, only acidic to neutral pH (3-7) is tested, neglecting alkaline conditions common in some food systems. This limits the claim of "robustness."

Response: We understand and value your point of view on this issue. However, the data presented, which includes temperature, salt concentration, and pH parameters, refers to the emulsification index with two types of oils. Since the proposal involved the production of emulsions stored at refrigeration temperatures, using already pasteurized ingredients such as egg yolk, and other foods that do not require pasteurization, it was decided to test them at lower temperatures. Regarding pH, it is also known that most raw and processed foods have a very to slightly acidic pH, with only a few examples, such as milk, in the neutral range, and raw egg white and yolk in the alkaline range. Furthermore, conventional mayonnaises and those obtained in these experiments presented pH values between 2.37 and 3.78, encompassing the range of acidic foods.

4. The stability of the salad dressings is assessed solely by visual observation ("remained stable," "phase separation"). This is highly subjective. Quantitative measures such as droplet size distribution, zeta potential, rheological measurements, or centrifugation tests are necessary to provide objective, reproducible data on emulsion stability.

Response: We appreciate your feedback. Some quantitative parameters were measured with the six stable emulsions and are presented in this revised version, such as pH and viscosity measurements. However, due to the short time available for the production of the biocoating, which prevented the performance of additional analyses, such as zeta potential, and the need to send samples for scanning electron microscopy (SEM) analysis, it was not possible to include them in this article. Even so, we emphasize that these analyses will be considered in future experiments.

5. The experimental design for the salad dressings lacks a positive control containing a standard commercial emulsifier (e.g., Tween 80, Mono- and diglycerides). Without this comparison, it is impossible to objectively evaluate the performance of the BS relative to current industry standards, thus weakening the application claim.

Response: Thank you for your feedback. Following the review, we have re-prepared all twelve emulsion formulations containing Tween 80 as a positive control, as requested. The updated data has now been incorporated into this new revised version.

6.The extraction and isolation protocol is described, but no data on the yield (g BS / g substrate) or purity of the final isolated product is provided. This information is crucial for assessing the economic feasibility and for correlating concentration with functional properties in the CMC experiment.

Response: We appreciate the reviewer's query regarding yield. We clarify that the gravimetric yield was indeed monitored as a dependent variable during the experimental design. However, the results obtained in the flask-scale assays were consistently low and exhibited low variance across the different conditions, making them unsuitable for statistical modeling compared to the surface tension and emulsification index, which were highly responsive. Therefore, we prioritized surface activity metrics as the primary indicators of production efficiency.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been significantly improved. The authors addressed all the comments and suggestions.

I have only one suggestion - In Table 6, results below the detection limit are denoted as “0”. I recommend using “ND” or “<10” instead, as “0” may be interpreted as an actual zero count.

Reviewer 2 Report

Comments and Suggestions for Authors

I think this manuscript can be accepted