Ultrasound-Assisted Extraction of Polysaccharides from Pleurotus ostreatus By-Products: Box–Behnken Optimization and Low-Fat Cookies Formulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well-organized and the writing is smooth, but there are still some issues that the author should address and revise.
1. This paper studies the effect of ultrasonic treatment on polysaccharide extraction. Should the power, temperature and time of ultrasonic extraction be taken into account? How does the author consider this and it needs to be explained in the text.
2. The total dietary fiber (TDF) recovery rate is used as the response value instead of the polysaccharide yield. How is this considered? Does it affect the polysaccharide and what is the relationship between the two?
3. Is Figure 1 meaningful? Its practical value is not clear.
4. There is still a significant difference between the central group experiment (15 groups) and the other two groups. What causes this?
5. Is the model reliable? The predicted TDF of the optimal condition is 39.60%, but the actual measurement is only 30.82%, with a relative error of 22.2%.
6. The protein content of the extract is 14.0g/100g, which is significantly higher than that of the raw material. The author attributes this to the ultrasonic solubilization and ethanol co-precipitation. Should a protein removal control experiment be conducted to verify this mechanism?
7. The oil-holding capacity of the extract is 1.39g/g and the emulsifying activity is only 5%, but the emulsifying stability is as high as 80%. This "low activity, high stability" phenomenon lacks reasonable data support.
8. The color difference ΔE is all greater than 3, but there is no difference in sensory evaluation. What causes this?
9. The reference format is not uniform. Some references only have the doi without page numbers, and the abbreviations of journal names are inconsistent.

Author Response

The manuscript is well-organized and the writing is smooth, but there are still some issues that the author should address and revise.

A: Thank you for this overall positive evaluation of the manuscript. We appreciate the reviewer’s acknowledgment of the structure and clarity of the writing. We have carefully addressed all the specific comments raised and revised the manuscript accordingly to improve methodological clarity, consistency, and the interpretation of the results.

Q1: This paper studies the effect of ultrasonic treatment on polysaccharide extraction. Should the power, temperature and time of ultrasonic extraction be taken into account? How does the author consider this and it needs to be explained in the text.

A1: Thank you for this important comment. We agree that ultrasonic extraction parameters such as power, temperature, and time are critical factors. In our study, the ultrasonic device operated at a nominal power of 2000 W and was used at 100% amplitude in all experiments to ensure consistent operating conditions.

To standardize the extraction process, the treatment was controlled based on the total energy input (expressed as J/mL) rather than sonication time. The total energy applied to each sample was expressed as Wh, and calculated as Wh = (J/mL × volume)/3600s, allowing us to apply a defined and comparable energy dose to each sample. This approach was selected because the effective sonication time required to reach a given energy input may vary depending on sample volume, viscosity, and temperature, which influence the actual power transmitted to the medium. Therefore, by fixing the total energy input instead of time, we ensured that all samples received an equivalent treatment, minimizing variability associated with process dynamics.

In addition, for the Box–Behnken experimental design, all experiments were conducted at laboratory scale using a working volume of 500 mL. Subsequently, the optimized conditions were scaled up to a semi-pilot scale (20 L) to obtain sufficient material for further analyses. In this context, the use of specific energy input allowed the extraction conditions defined at laboratory scale to be consistently transferred to the larger scale.

The extraction temperature was continuously monitored and maintained between 70 and 80 °C throughout the process. This explanation has now been clarified in the revised manuscript.

Q2: The total dietary fiber (TDF) recovery rate is used as the response value instead of the polysaccharide yield. How is this considered? Does it affect the polysaccharide and what is the relationship between the two?

A2: Thank you for this insightful comment. In this study, both polysaccharide yield and total dietary fiber (TDF) recovery were initially evaluated as potential response variables. However, the statistical fitting of the model showed a much better adjustment for TDF recovery (higher R² values and more adequate model significance), whereas the polysaccharide yield exhibited a weaker fit and lower predictive capacity under the same experimental conditions. For this reason, TDF recovery was selected as the main response variable for process optimization, as it provided a more robust and reliable representation of the extraction efficiency within the studied design. This choice is also consistent with the objective of the study, which focused on the development of a fiber-enriched cookie.

Regarding the relationship between both parameters, the water-soluble polysaccharides (WSP) obtained from the extraction are largely associated with the soluble fraction of dietary fiber, particularly hemicellulosic compounds. This is supported by the composition of the raw SMS, which showed a hemicellulose content of 15.41%. Therefore, WSP recovery can be considered closely related to the soluble dietary fiber fraction. An additional clarification has now been included in the manuscript (lines 370–374).

Q3: Is Figure 1 meaningful? Its practical value is not clear.

A3: Thank you for this comment. Figure 1 shows photographs of the cookies prepared with different levels of dietary fiber extract incorporation. We consider that the figure provides visual evidence of the technological feasibility of incorporating the Pleurotus ostreatus SMS extract into a cookie formulation, yielding cohesive and well-formed products. However, to improve the flow of the main manuscript and in response to the reviewer’s suggestion, this figure has now been moved to the Supplementary Material.

Q4: There is still a significant difference between the central group experiment (15 groups) and the other two groups. What causes this?

A4: Thank you for this comment. We would like to clarify that the experimental design consists of a Box–Behnken design with 15 runs, including 12 factorial points and 3 replicates at the central point. Therefore, there are no other two groups. However, we regret that we may not fully understand the specific concern raised in this comment. We would be grateful if the reviewer could kindly reformulate or clarify the question so that we can address it more accurately.

Q5: Is the model reliable? The predicted TDF of the optimal condition is 39.60%, but the actual measurement is only 30.82%, with a relative error of 22.2%.

A5: Thank you for this comment. We agree that the difference between the predicted TDF value (39.60%) and the experimental validation result (30.82%) may raise concerns regarding model reliability. However, this deviation can be explained by the fact that the predicted value corresponds to the laboratory-scale optimization conditions, whereas the extract characterization was performed at semi-pilot scale. As discussed in the revised manuscript, the Box–Behnken experimental design was conducted at laboratory scale (500 mL), while the optimized conditions were subsequently scaled up to semi-pilot scale (20 L) for further analyses. Therefore, the observed discrepancy is likely attributable to scale-up effects, which may influence mass and energy transfer during extraction. To clarify this aspect, the scale-up procedure has now been explicitly stated in the Materials and Methods section.

Q6: The protein content of the extract is 14.0g/100g, which is significantly higher than that of the raw material. The author attributes this to the ultrasonic solubilization and ethanol co-precipitation. Should a protein removal control experiment be conducted to verify this mechanism?

A6: Thank you for this insightful comment. We agree that performing a protein removal control experiment could have provided additional confirmation of the proposed mechanism. However, in this study the removal of proteins was intentionally not carried out, since the presence of protein–polysaccharide complexes was considered potentially beneficial from both a technological and nutritional perspective, and extract purification was therefore not deemed relevant to the objectives of the work. Nevertheless, we acknowledge that such a control could help to further clarify the extraction mechanism, and this will be considered in future studies.

Q7: The oil-holding capacity of the extract is 1.39g/g and the emulsifying activity is only 5%, but the emulsifying stability is as high as 80%. This "low activity, high stability" phenomenon lacks reasonable data support.

A7: Thank you for this insightful comment. We agree that the apparent discrepancy between low emulsifying activity (EC = 5%) and high emulsifying stability (ES = 80%) requires clearer justification. Accordingly, we have revised the discussion to provide a more detailed physicochemical explanation. In the revised manuscript, we clarify that EC reflects the initial ability to form an emulsion, while ES describes the capacity to maintain it over time. We further explain that the low EC may be related to the limited interfacial adsorption capacity of the extract, consistent with its relatively low WHC (0.24 g/g) and OHC (1.39 g/g). In contrast, the high ES is attributed to the stabilizing effect of polysaccharide–protein interactions and the increase in viscosity of the continuous phase, which reduces droplet coalescence and enhances emulsion stability.

Q8: The color difference ΔE is all greater than 3, but there is no difference in sensory evaluation. What causes this?

A8: Thank you for this comment. We would like to clarify that the sensory evaluation assessed color liking rather than the ability to discriminate color differences. Therefore, although instrumental measurements indicated ΔE values greater than 3, this does not imply that panelists did not perceive differences, but rather that these differences did not negatively influence their acceptance. We acknowledge that this point was not clearly expressed in the original manuscript and have revised the sentence accordingly to improve clarity.

Q9: The reference format is not uniform. Some references only have the doi without page numbers, and the abbreviations of journal names are inconsistent.

A9: Thank you for this observation. The reference list has been carefully revised to ensure a uniform format throughout the manuscript. Journal name abbreviations have been standardized, and missing bibliographic information (including page numbers where applicable) has been completed.

Reviewer 2 Report

Comments and Suggestions for Authors

The present study investigates the valorization of spent mushroom substrate (SMS) through ultrasound-assisted extraction (UAE) of dietary fiber and its subsequent application as a fat-mimetic ingredient in low-fat cookies. The topic is relevant and fits well within the scope of Foods, particularly in the context of circular economy and functional food development. However, several aspects of the manuscript require clarification, correction, or improvement in terms of experimental reporting, data presentation, and interpretation of results. The following specific comments are provided to help the authors enhance the quality and clarity of the work.

 

 

Line 49: Have there been any previous reports on the non-conventional use of SMS?

 

Sections 2.2–2.4 should be merged into a single section, as they all refer to the same extraction experiments

 

Please specify the extraction time used in the UAE experiments and describe how this time was established

 

Section 2.12: The statistical method used to analyze the sensory analysis results should be clearly stated.

 

Lines 313–317: To avoid confusion, please express the energy unit as watts (W), which links energy to time

 

Lines 331–334: A better way to present and discuss the effect of the independent variables on TDF would be to use response surface plots.

 

I suggest that Tables 4 and 5 be presented as supplementary material.

 

Lines 378–381: This sentence is unclear. The estimated effect indicates that increasing yield results from combining large particle size with a high feed/solvent ratio. This corresponds to high sample concentration and large particle size — conditions that typically imply low mass transfer. Please clarify.

 

Figure 2: Axis title should read "Solute-to-Solvent..." instead of "Soluto-to-Solvent...".

 

Line 408: The experimental design and Figure 2b present the S/S ratio in units of percentage (%). Please report the optimal conditions using the same units for consistency.

 

Tables 6 and 7 should be revised for better presentation.

 

Table 6: The ash units shown are incorrect.

 

Table 9: The units for each measured parameter should be indicated in the table

 

Lines 558–560: The authors should also consider that the changes in parameters a and b could be due to the initial color of the extract. Furthermore, the correlation between color changes and Maillard reactions should be supported by data on the amino acid composition of the proteins present in the extract.

 

Lines 589–590: Please specify which volatile compounds may be present in the extracts and describe how changes in aromatic notes could affect these compounds. Include detailed information on this topic in the manuscript.

 

Table 9: Column title should read "Samples" instead of "Muestras".

 

Line 66: The reported pGI of 69.65 ± 0.54 falls on the borderline of the high GI category. Consequently, the conclusions drawn from these results are not fully conclusive.

 

 

 

Author Response

Revisor 2:

The present study investigates the valorization of spent mushroom substrate (SMS) through ultrasound-assisted extraction (UAE) of dietary fiber and its subsequent application as a fat-mimetic ingredient in low-fat cookies. The topic is relevant and fits well within the scope of Foods, particularly in the context of circular economy and functional food development. However, several aspects of the manuscript require clarification, correction, or improvement in terms of experimental reporting, data presentation, and interpretation of results. The following specific comments are provided to help the authors enhance the quality and clarity of the work.

A: Thank you for your positive assessment of the relevance and scope of our study, as well as for your constructive and detailed comments. We appreciate the recognition of the manuscript’s alignment with the topics of circular economy and functional food development. We have carefully addressed all the specific comments provided and revised the manuscript accordingly to improve clarity, methodological description, data presentation, and interpretation of the results.

 Q1: Line 49: Have there been any previous reports on the non-conventional use of SMS?

Thank you for your comment. We have revised the manuscript to acknowledge previous reports on the non-conventional use of SMS. Specifically, the following sentence has been added to the text (Lines 49-51):

"However, in recent years, non-conventional uses of SMS have been explored, including its application in bioremediation, animal feed, biogas production, or enzyme extraction."

We hope this addition adequately addresses your concern.

Q2: Sections 2.2–2.4 should be merged into a single section, as they all refer to the same extraction experiments

A2: Thank you for this suggestion. We agree that Sections 2.2–2.4 refer to the same extraction experiments, and they have now been merged into a single section to improve clarity and organization of the manuscript.

Q3: Please specify the extraction time used in the UAE experiments and describe how this time was established

A3: Thank you for this important comment. In our study, extraction time was not defined as an independent controlled variable in the UAE experiments. Instead, the process was designed and controlled based on specific energy input (J/mL), as this parameter integrates the combined effect of power, time, and sample properties on the extraction process. For this reason, the effective sonication time was not fixed beforehand but was automatically adjusted by the equipment until the target specific energy input was reached for each experimental condition. This means that differences in sample volume and matrix characteristics could lead to variations in processing time, while still ensuring that all samples received the same energy dose.

To clarify this aspect, we have now included an explicit explanation in the Materials and Methods section describing how the UAE process was controlled based on specific energy input and how extraction time was consequently dependent on the energy requirement rather than predefined as a fixed factor.

Q4: Section 2.12: The statistical method used to analyze the sensory analysis results should be clearly stated.

A4: Thank you for this comment. The statistical analysis section has been revised to clearly state that sensory analysis data were included in the statistical treatment. Specifically, it has now been indicated that hedonic sensory data, together with all other determinations, were analyzed using one-way ANOVA and Tukey’s post hoc test.

Q5: Lines 313–317: To avoid confusion, please express the energy unit as watts (W), which links energy to time

A5: Thank you for this comment. We understand the concern regarding the use of J/mL instead of W. However, in this section we are referring to previously published studies cited in the text, which express ultrasonic treatment intensity in terms of watts (W). For this reason, the unit was maintained to ensure consistency with the cited literature. The relevant references have been reintroduced after this statement to clearly indicate that the discussion is based on previously reported methodologies.

Q6: Lines 331–334: A better way to present and discuss the effect of the independent variables on TDF would be to use response surface plots.

A6: Thank you for this suggestion. We agree that response surface plots provide a more comprehensive visualization of the effect of the independent variables on TDF. In the revised manuscript, we have clarified the interpretation of TDF results and better linked this discussion to the corresponding response surface plots presented later in the section, which illustrate the observed trends more clearly.

Q7: I suggest that Tables 4 and 5 be presented as supplementary material.

A7: Thank you for this suggestion. We understand the reviewer’s point, however we would prefer to retain Tables 4 and 5 in the main manuscript, as they contain the ANOVA results and the statistical parameters for model validation, which are essential to support the reliability and robustness of the developed model. We believe that including these tables in the main text improves transparency and allows readers to properly evaluate the adequacy of the statistical analysis.

Q8: Lines 378–381: This sentence is unclear. The estimated effect indicates that increasing yield results from combining large particle size with a high feed/solvent ratio. This corresponds to high sample concentration and large particle size — conditions that typically imply low mass transfer. Please clarify.

A8: Thank you for this comment. We believe there may have been a misunderstanding regarding the expression of the solute-to-solvent ratio. In our study, this parameter was calculated as mL of solvent per g of solute, rather than the inverse, although it was expressed in percentage, which may have led to confusion. We sincerely apologize for this lack of clarity. The terminology and units will be revised throughout the manuscript to ensure consistency and correct interpretation, and an explicit clarification will be added in the Materials and Methods section. This correction resolves the apparent inconsistency regarding mass transfer conditions highlighted by the reviewer.

Q9: Figure 2: Axis title should read "Solute-to-Solvent..." instead of "Soluto-to-Solvent...".

A9: Thank you for pointing out this typographical error. The axis title has been corrected from “Soluto-to-Solvent” to “Solute-to-Solvent” in Figure 2.

Q10: Line 408: The experimental design and Figure 2b present the S/S ratio in units of percentage (%). Please report the optimal conditions using the same units for consistency.

A10: Thank you for this observation. We agree that consistency in reporting units is important. The optimal conditions have now been revised to express the S/S ratio in percentage (%) to match the experimental design and Figure 2b.

Q11: Tables 6 and 7 should be revised for better presentation.

A11: Thank you for this suggestion. Tables 6 and 7 have been revised to improve their presentation. Specifically, formatting has been adjusted to comply with the journal guidelines (including line spacing), and in Table 7 the SEM has been aligned on the same line as the mean values to enhance clarity and readability.

 

Q10: Table 6: The ash units shown are incorrect.

A10: Thank you for this comment. We have carefully checked Table 6 and confirm that ash content is correctly expressed as g/100 g on a dry weight basis, which is consistent with the rest of the compositional data reported in the manuscript.

Q13: Table 9: The units for each measured parameter should be indicated in the table

A13: Thank you for this comment. The units for each measured parameter have now been added to Table 9 to improve clarity and ensure consistency in data presentation.

Q14: Lines 558–560: The authors should also consider that the changes in parameters a and b could be due to the initial color of the extract. Furthermore, the correlation between color changes and Maillard reactions should be supported by data on the amino acid composition of the proteins present in the extract.

Thank you for this insightful comment. We agree that the changes in color parameters may also be influenced by the intrinsic color of the SMS extract, and that the potential contribution of Maillard reactions should be interpreted with caution in the absence of amino acid composition data. Accordingly, we have revised the discussion to better reflect these considerations, explicitly stating that color changes are likely associated with both the inherent color of the extract and formulation effects, rather than attributing them solely to Maillard reactions. We have also clarified that the role of Maillard reactions is only suggested and cannot be confirmed within the scope of the present study due to the lack of specific amino acid data.

Q15: Lines 589–590: Please specify which volatile compounds may be present in the extracts and describe how changes in aromatic notes could affect these compounds. Include detailed information on this topic in the manuscript.

 A15: We thank the reviewer for this insightful suggestion. We have expanded the discussion in the manuscript (Lines [637-647]) to provide a detailed chemical basis for the aromatic contribution of the SMS extract.

Specifically, we have clarified that:

1. Chemical Composition: The extract is rich in C8 volatile compounds, such as 1-octen-3-ol, 3-octanol, and 3-octanone, which are the primary contributors to the characteristic "mushroom" and "earthy" aroma of Pleurotus species.
2. Thermal Transformation: We described how high baking temperatures (180–200 °C) lead to the partial loss of these fresh-mushroom volatiles while promoting the formation of pyrazines and Strecker aldehydes through Maillard reactions.
3. Sensory Impact: These changes shift the aromatic profile from raw, earthy notes to toasted, nutty, and savory notes.

The manuscript has been updated to reflect these details, providing a more robust explanation of how the SMS extract influences the sensory properties of the final product.

Q16: Table 9: Column title should read "Samples" instead of "Muestras".

A16: Thank you for this correction. The column title has been changed from “Muestras” to “Samples” in Table 9.

Q17: Line 66: The reported pGI of 69.65 ± 0.54 falls on the borderline of the high GI category. Consequently, the conclusions drawn from these results are not fully conclusive.

A17: Thank you for this insightful comment. We agree that the reported pGI value of 69.65 ± 0.54 is at the borderline between medium and high GI, and therefore the initial interpretation could be considered too conclusive. The text has been revised accordingly to reflect a more cautious interpretation of the results. We now clarify that G1 lies at the upper limit of the medium-GI range and may also be considered borderline high GI. Additionally, we emphasize that, despite this, the enriched cookie showed a clear reduction in pGI compared to the control (85.88), highlighting the beneficial effect of the dietary fiber extract. Finally, we have included a statement acknowledging this limitation and suggesting that further formulation optimization could help achieve a more substantial reduction in pGI while maintaining sensory acceptability.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript discusses the Ultrasonic-assisted extraction of polysaccharides from Ploreutus Ostreatus. The authors optimized the extraction yield using response surface design. The topic of the manuscript should be of interest to the journal’s readership. Still, the manuscript must be improved mainly in the Methodology section by an inclusion of additional information. Some reported discussions also require additional clarification. The authors should also pay a specific attention to the reference style and include references based on journal guidelines (either author date or numerical format).

Comments:

1. In line 52, please check if the reference discusses prolonged extraction times and provide another reference instead.

 

2. In lines 108-109, explicitly mention the tested energy inputs, solute-to-solvent ratios, and particle size.

 

3. The degree sign “ ° ” in °C should be corrected throughout the manuscript.

 

4. In line 162, provide an adequate reference for color analysis.

 

5. References should be either in author date format or numeric. Please correct based on journal guidelines and present consistently.

 

6. In section 2.10, add additional information if participants provided informed consent.

 

7. In line 256, provide the complete term for GOPOD.

 

8. Provide a reference for equation 2.

 

9. Relevant to equations 5 and 6, add definitions of I and pGI.

 

10. In line 348, provide a reference for Durbin-Watson statistics.

 

11. Referring to line 395, add a brief explanation on the reason for the observed behaviour. Why TDF increased with increased particle size?

 

12. In line 593, “crispiness” should be corrected to “crispness”.

 

13. In lines 662 and 676, Table 9 should be Table 10.

 

 

Author Response

Revisor 3:

The manuscript discusses the Ultrasonic-assisted extraction of polysaccharides from Ploreutus Ostreatus. The authors optimized the extraction yield using response surface design. The topic of the manuscript should be of interest to the journal’s readership. Still, the manuscript must be improved mainly in the Methodology section by an inclusion of additional information. Some reported discussions also require additional clarification. The authors should also pay a specific attention to the reference style and include references based on journal guidelines (either author date or numerical format).

A: Thank you for this overall assessment and for recognizing the relevance of the topic for the journal’s readership. We appreciate the reviewer’s constructive feedback. We have carefully revised the manuscript with particular attention to the Methodology section by adding the missing information and improving the level of detail where required. We have also clarified and strengthened the discussion where necessary. In addition, the reference list and in-text citations have been thoroughly revised to ensure full compliance with the journal’s formatting guidelines and consistency throughout the manuscript.

Q1: In line 52, please check if the reference discusses prolonged extraction times and provide another reference instead.

 A1: Thank you for this comment. We added two additional references that specifically support the statement regarding prolonged extraction times in ultrasound-assisted extraction processes. This provides stronger and more appropriate bibliographic support for the claim.

Q2: In lines 108-109, explicitly mention the tested energy inputs, solute-to-solvent ratios, and particle size.

A2: Thank you for this suggestion. The tested energy inputs, solute-to-solvent ratios, and particle size have now been explicitly specified in lines 124-126  of the revised manuscript.

Q3: The degree sign “ ° ” in °C should be corrected throughout the manuscript.

 A3: Thank you for this observation. The degree symbol formatting in °C has been corrected throughout the manuscript to ensure consistency.

Q4: In line 162, provide an adequate reference for color analysis.

A4: Thank you for this comment. An appropriate reference has been added in line 182 to support the color analysis methodology based on the CIELAB system. Specifically, the study by Lucas-González et al. (2017) has been included, as it describes the use of CIELAB coordinates and the calculation of derived color parameters in food coproducts, which is consistent with the approach used in this work.

• Lucas-González, R., Viuda-Martos, M., Pérez-Álvarez, J. Á., & Fernández-López, J. Evaluation of Particle Size Influence on Proximate Composition, Physicochemical, Techno-Functional and Physio-Functional Properties of Flours Obtained from Persimmon (Diospyros kaki Trumb.) Coproducts. Plant Foods Hum. Nutr. 2017, 72(1), 67–73. https://doi.org/10.1007/s11130-016-0592-z

Q5: References should be either in author date format or numeric. Please correct based on journal guidelines and present consistently.

A5: Thank you for this comment. The reference style has been carefully revised throughout the manuscript to ensure full consistency with the journal guidelines. All in-text citations and the reference list have been checked and unified to follow the required numeric format. In addition, formatting inconsistencies have been corrected to ensure a homogeneous presentation of all references.

Q6: In section 2.10, add additional information if participants provided informed consent.

 A6: Thank you for this comment. We confirm that all participants provided informed consent before their involvement in the study. This information has now been added in Section 2.10 to clarify the ethical considerations of the sensory evaluation.

Q7: In line 256, provide the complete term for GOPOD.

 A7: Thank you for this comment. The complete term for GOPOD has now been provided in lines 288-289, where it is defined as glucose oxidase–peroxidase.

Q8: Provide a reference for equation 2.

A8: Thank you for this comment. Equation 2 is based on the protocol provided in the total starch assay kit (Megazyme, Bray, Ireland), which follows AOAC Official Method 996.11. This has now been clarified in the manuscript to indicate the source of the equation.

Q9: Relevant to equations 5 and 6, add definitions of I and pGI.

A9: Thank you for this comment. We would like to clarify that the symbol “I” was a typographical error and has been corrected to “HI” in equation 5. In addition, the definitions of HI, pGI, and AUC have now been included in relation to Equations 5 and 6 to improve clarity and completeness.

 

 

Q10: In line 348, provide a reference for Durbin-Watson statistics.

A10: Thank you for this comment. The following relevant references for the Durbin–Watson statistic have now been added in Section 2.10 (Statistical Analysis) of the Materials and Methods.

• Durbin, J., & Watson, G. S. (1950). Biometrika Trust Testing for Serial Correlation in Least Squares Regression: I (Vol. 37, Issue 3).
• Durbin, J., & Watson, G. S. (1951). Biometrika Trust Testing for Serial Correlation in Least Squares Regression. II (Vol. 38, Issue 1).

 

Q11: Referring to line 395, add a brief explanation on the reason for the observed behaviour. Why TDF increased with increased particle size?

A11: Thank you for this comment. We have now added a brief mechanistic explanation in the revised manuscript clarifying the observed trend. Specifically, the increase in TDF with particle size is explained by a possible limitation in cavitation intensity and fiber solubilization due to increased viscosity caused by excessive grinding. This clarification is consistent with and supports the interpretation previously discussed in the ANOVA results section.

Q12: In line 593, “crispiness” should be corrected to “crispness”.

Thank you for this correction. The term “crispiness” has been replaced with “crispness” in line 651.

Q13: In lines 662 and 676, Table 9 should be Table 10.

Thank you for this observation. The references have been corrected from Table 9 to Table 10.

Reviewer 4 Report

Comments and Suggestions for Authors

The aim of the study is to assess the possibility of using the fiber extracted from spent mushroom substrate as a fat substitute in the production of cookies. Additionally, the fiber ultrasound-assisted extraction process was optimized using Box–Behnken experimental design.

Line 31, 39 – “[1], [2]“ this way of citing literature is incorrect, it should be “[1,2]”.

Line 34 – “During the commercialization of fruiting bodies”. Shouldn't there be “production”?

It is worth adding in the introduction what benefits come from using methods such as: Box-Behnken Design.

Line 98 – “17% Ash”, it should be „17.00% ash”.

Line 145 – “stove”, the better word is “dryer”.

Line 147 – it should be “muffle furnace”.

Line 146-149 – “Total ash was determined using a 10-PR/400 146
muffle (Hobersal, Barcelona, Spain) at 550 ◦C after 5 h crude protein was determined using 147
the Kjeldahl method using a Kjeltec System 2200 nitrogen distiller (FOSS IBERIA, Barce- 148
lona, Spain) and a digestion block”. Please edit the text. The sentence should be divided into two. The manufacturer's name of digestion block is missing.

Line 150 – „31]The”, the space and dot are missing.

Line 187 – “Coffmann and Garcia (2007)”, the year of publication is not needed.

Line 188-189 – “a stirring beaker”?

Line 198 – “(G3) In”, dot is missing.

Why samples of dough were stored for 10 days?

Line 212-226 – I suggest sticking to word “cookie”.

Line 217 – “sensory analysis “ will be better.

Line 239, 258 – it should be “CaCl₂”.

Line 228-261 – The degree designation is inconsistent in text: 100ºC, 200 °C. In this section once there is a space between the value and the unit, in another place there is no.

Line 252 – „Dilution”, should be „dilution”.

Line 274, 276 – “each studied bread formulation”, “using white bread as reference food” It was calculated for bread or for cookie? Please check the description and equation 5 if they are correct.

Equation 5 – „I” or „HI”?

Line 347 – “Durbin-Watson statistic” was not mentioned in section Statistical Analysis.

Line 384 – standard error is not given in the table as an abbreviation.

Line 416 – “substrate” Isn't this already a product?

Line 446 – it should be Regulation (EC) No 1924/2006.

Table 6, 9 – The table gives the results of selected mineral components, but the methodology does not state that such an analysis was performed.

Table 7 – there is no aw method description in the methodology.

Table 9 – there is no information about the energy C, ΔE in the methodology.

Figure 4 – “Total starch hydrolysis rate of bread during gastrointestinal digestion” or cookie?

Line 660-662, 676 – it should be table 10.

Line 676-679, table 9 – abbreviations should be marked below the table. What is “CT” and “Muestras”?

References - some of the literature items do not comply with the journal's requirements.

Author Response

Revisor 4:

The aim of the study is to assess the possibility of using the fiber extracted from spent mushroom substrate as a fat substitute in the production of cookies. Additionally, the fiber ultrasound-assisted extraction process was optimized using Box–Behnken experimental design.

A: Thank you for summarizing the scope and aim of the study. We appreciate the reviewer’s concise description of the work and agree with the stated objectives.

Q1: Line 31, 39 – “[1], [2]“ this way of citing literature is incorrect, it should be “[1,2]”.

A1: Thank you for this correction. The in-text citations have been revised throughout the manuscript, and the format “[1], [2]” has been corrected to “[1,2]” to ensure consistency with the journal guidelines.

Q2: Line 34 – “During the commercialization of fruiting bodies”. Shouldn't there be “production”?

A2: Thank you for this suggestion. We agree that “production” is more appropriate in this context. The sentence has been revised accordingly, replacing “commercialization” with “production” to improve accuracy.

Q3: It is worth adding in the introduction what benefits come from using methods such as: Box-Behnken Design.

A3: Thank you for your valuable suggestion. A sentence has been added in the Introduction to highlight the advantages of using the Box–Behnken Design in the optimization process (Lines 87-90).

Q4: Line 98 – “17% Ash”, it should be „17.00% ash”.

A4: Thank you for this correction. The value has been revised to “17.00% ash” to ensure consistent formatting and precision in reporting.

Q5: Line 145 – “stove”, the better word is “dryer”.

A5: Thank you for this comment. The term “stove” has been replaced with “dryer” in line 159 to improve accuracy and clarity of the description.

Q6: Line 147 – it should be “muffle furnace”.

A6: Thank you for this correction. The term has been revised to “muffle furnace” in line 160 for accuracy and consistency.

Q7: Line 146-149 – “Total ash was determined using a 10-PR/400 146
muffle (Hobersal, Barcelona, Spain) at 550 ◦C after 5 h crude protein was determined using 147 the Kjeldahl method using a Kjeltec System 2200 nitrogen distiller (FOSS IBERIA, Barce- 148 lona, Spain) and a digestion block”. Please edit the text. The sentence should be divided into two. The manufacturer's name of digestion block is missing.

A7: Thank you for this comment. We have revised the sentence to improve clarity by splitting it into two separate sentences. The manufacturer's information for the digestion block is FOSS IBERIA, Barcelona, Spain.

Q8: Line 150 – „31]The”, the space and dot are missing.

A8: Thank you for pointing out this typographical error. The missing space and punctuation have been corrected in line 150.

Q9: Line 187 – “Coffmann and Garcia (2007)”, the year of publication is not needed.

A9: Thank you for this observation. The year of publication has been removed from “Coffmann and Garcia (2007)” in line 207 of the revised manuscript.

Q10: Line 188-189 – “a stirring beaker”?

A10: Thank you for this comment. The term “stirring beaker” has been corrected to “blender” to more accurately describe the equipment used.

Q11: Line 198 – “(G3) In”, dot is missing.

A11: Thank you for pointing out this typographical error. The missing punctuation has been corrected in line 217 of the revised manuscript.

Q12: Why samples of dough were stored for 10 days?

A12: Thank you for this comment. It was not feasible to prepare, bake, and perform all analytical determinations on the same day due to the large number of formulations and analyses included in the study. For this reason, freezing was incorporated as an additional processing step to allow all samples to be handled and analyzed under comparable conditions.

The dough samples were produced in independent batches on different days and stored at −18 °C for no longer than 10 days before baking. Importantly, within each batch containing all the studied formulations, samples were stored for the same number of days to ensure consistent treatment across formulations. Therefore, any potential influence of frozen storage would affect all samples equally and would not bias comparisons among treatments.

To clarify this aspect, a sentence has been added in the Materials and Methods section indicating that all samples were subjected to the same freezing conditions prior to baking.

Q13: Line 212-226 – I suggest sticking to word “cookie”.

A13: Thank you for this suggestion. We have revised the manuscript accordingly and replaced the term “biscuit” with “cookie” throughout the manuscript.

Q14: Line 217 – “sensory analysis “ will be better.

A14: Thank you for this suggestion. The term has been revised to “sensory analysis” and this change has been applied consistently throughout the entire manuscript to ensure uniform terminology.

Q15: Line 239, 258 – it should be “CaCl₂”.

A15: Thank you for this correction. The notation has been revised to “CaCl₂ in the revised manuscript to ensure proper chemical formatting.

Q16: Line 228-261 – The degree designation is inconsistent in text: 100ºC, 200 °C. In this section once there is a space between the value and the unit, in another place there is no.

A16: Thank you for this observation. The degree symbol formatting has been standardized throughout the manuscript, ensuring consistent spacing between values and units.

Q17: Line 252 – „Dilution”, should be „dilution”.

A17: Thank you for this correction. The term “Dilution” has been changed to “dilution” in line 285 to ensure correct formatting.

Q18: Line 274, 276 – “each studied bread formulation”, “using white bread as reference food” It was calculated for bread or for cookie? Please check the description and equation 5 if they are correct.

A18: Thank you for this comment. We have reviewed the text and confirmed that “each studied bread formulation” refers to cookies. Regarding “using white bread as reference food” and Equation 5, both remain correct as originally presented, as white bread is used as the reference food for the calculation. We provide articles below, where this same approach is used for comparison purposes:

• Naknaen, P., Itthisoponkul, T., Sondee, A., & Angsombat, N. (2016). Utilization of watermelon rind waste as a potential source of dietary fiber to improve health promoting properties and reduce glycemic index for cookie making. Food Science and Biotechnology, 25(2), 415–424. https://doi.org/10.1007/s10068-016-0057-z
• Lucas-González, R., Ángel Pérez-Álvarez, J., Moscaritolo, S., Fernández-López, J., Sacchetti, G., & Viuda-Martos, M. (2021). Evaluation of polyphenol bioaccessibility and kinetic of starch digestion of spaghetti with persimmon (Dyospyros kaki) flours coproducts during in vitro gastrointestinal digestion. Food Chemistry, 338, 128142–128152. https://doi.org/10.1016/j.foodchem.2020.128142

Q19: Equation 5 – „I” or „HI”?

A19: Thank you for this comment. We confirm that the correct notation in Equation 5 is “HI” and not “I”.

Q20: Line 347 – “Durbin-Watson statistic” was not mentioned in section Statistical Analysis.

A20: Thank you for this observation. The Durbin–Watson statistic has now been included in Section 2.10 (Statistical Analysis) to ensure consistency between the Methods and Discussion sections and to properly describe the statistical approach used in the model evaluation.

Q21: Line 384 – standard error is not given in the table as an abbreviation.

A21: Thank you for this observation. The abbreviation has been removed in the revised manuscript to avoid confusion.

Q22: Line 416 – “substrate” Isn't this already a product?

A22: Thank you for this comment. In this context, “substrate” refers specifically to spent mushroom substrate (SMS), which is the material remaining after mushroom cultivation. The term has been used in a shortened form for readability, but we acknowledge that this may not be sufficiently clear. We will revise the text to explicitly indicate “spent mushroom substrate (SMS)” to avoid ambiguity.

 

Q23: Line 446 – it should be Regulation (EC) No 1924/2006.

A23: Thank you for this correction. The reference has been updated to “Regulation (EC) No 1924/2006” in line 486 of the revised manuscript.

Q24: Table 6, 9 – The table gives the results of selected mineral components, but the methodology does not state that such an analysis was performed.

Q24: Thank you for this comment. We confirm that the mineral composition analysis was performed but was not clearly described in the original Methods section. This has now been addressed by adding the corresponding methodology in the “Proximate composition and mineral profile” section, ensuring consistency with the results presented in Tables 6 and 9.

Q25: Table 7 – there is no aw method description in the methodology.

A25: Thank you for this comment. The methodology for water activity (a_w) determination has now been included in Section 2.4 (pH, water activity and instrumental color) of the Materials and Methods to ensure consistency with the results presented in Table 7.

Q26: Table 9 – there is no information about the energy C, ΔE in the methodology.

A26: Thank you for this comment. The methodology for the calculation of C and color difference (ΔE) has now been included in Sections 2.4 and 2.7 of the Materials and Methods, respectively, to ensure consistency with the results presented in Table 9. In addition, the calculation of energy (kcal/100 g) has been described in Section 2.7.

Q27: Figure 4 – “Total starch hydrolysis rate of bread during gastrointestinal digestion” or cookie?

A27: Thank you for this comment. The figure caption has been corrected to refer to cookies instead of bread, as the results correspond to cookie samples.

Q28: Line 660-662, 676 – it should be table 10.

A28: Thank you for this observation. The references have been corrected from Table 9 to Table 10 to ensure consistency with the revised table numbering throughout the manuscript.

 

 

Q29: Line 676-679, table 9 – abbreviations should be marked below the table. What is “CT” and “Muestras”?

A29: Thank you for this comment. The abbreviations have now been added below the table as requested. In addition, “Muestras” has been corrected to “Samples” to ensure consistency in terminology. The abbreviation “CT” has also been changed to control.

Q30: References - some of the literature items do not comply with the journal's requirements.

A30: Thank you for this comment. We have carefully reviewed the reference list and revised it to ensure compliance with the journal’s formatting and quality requirements. In particular, references that did not meet the journal standards have been removed, including a conference proceedings paper and a low-impact journal article with inconsistent bibliographic format. The remaining references have also been checked to ensure consistency in journal name abbreviations, author formatting, and complete citation information, including DOIs and page numbers where applicable.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All comments and suggestion were addressed correctly. I recoment that for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

My comments to the authors are addressed and I have no additional comment.