Impact of Probiotic and Bioprotective Cultures on the Quality and Shelf Life of Butter and Buttermilk

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work is interesting, provides valuable and innovative contributions to the field, and is written in a fluid and clear manner. However, the data presentation is very one-dimensional, lacking dynamism and integration. The work lacks greater statistical support and precision in data presentation. The discussion is well-referenced but sometimes general and not focused on the objectives of the work, the treatments, and the results obtained. I recommend a major revision before reconsideration for publication. Below are detailed comments on each line and paragraph to be revised (attached).

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The quality of the English is quite good; the writing is fluid and coherent. There are a few minor errors in the use of articles or British variants of words

Author Response

REVIEWER1

 

Journal: Dairy (ISSN 2624-862X) Manuscript ID: dairy-3165989 Title: Butter and buttermilk produced with cream fermented by aromatic or probiotic starters, with or without bioprotective cultures.

General comments: The work is interesting, provides valuable and innovative contributions to the field, and is written in a fluid and clear manner.

However, the data presentation is very one dimensional, lacking dynamism and integration. The work lacks greater statistical support and precision in data presentation.

R: Significant correlations between measured parameters of butter and buttermilk samples were included in the discussion of results. PCA did not add useful information.

The discussion is well-referenced but sometimes general and not focused on the objectives of the work, the treatments, and the results obtained.

R: We tried to focus the discussion regarding the objectives of the work.

I recommend a major revision before reconsideration for publication.

Below are detailed comments on each line and paragraph to be revised.

Line 1-2: Try reworking the title to better reflect the results, here are some suggestions: "Impact of Probiotic and Bioprotective Cultures on the Quality and Shelf Life of Butter and Buttermilk"; or "Evaluating the Influence of Fermentation Cultures on Butter and Buttermilk: Physicochemical, Rheological, and Sensory Properties"; or "Probiotic Versus Aromatic Cultures in Butter Production: Effects on Texture, Colour, and Shelf Life"; or "The Role of Probiotics and Bioprotective Cultures in Enhancing Butter and Buttermilk Quality and Longevity"; or "Comparative Analysis of Butter and Buttermilk Produced with Different Fermentation Cultures: A Study of Physicochemical and Sensory Attributes"; or "Rheological and Microbiological Characteristics of Butter and Buttermilk with Probiotic and Bioprotective Cultures.

R: We corrected the title as indicated.

Line 83-87: The term "Creamy goodness" seems a bit confusing to me, as well as the last sentence of the paragraph. Could you rewrite it in a way that more broadly addresses the product's quality parameters that could be improved?

R: Corrected.

Line 107: It would be very enriching for the manuscript and very useful for overall understanding if a visual diagram or flowchart were presented that encompasses all the materials and methods described, focusing mainly on point 2.1 but also including, in a very simplified manner, the subsequent analyses performed. The figure should clearly indicate the treatments used for later analyses and their respective codes as referenced throughout the text.

R: Corrected. A flow diagram of the experiment steps was added.

Line 107-139: Why were no processing replicates performed in the study? How can processing variability be identified and assessed? At the end of the process, only one product from each treatment (butter block and buttermilk bottle) was analyzed for each of the subsequent tests, without technical replicates?

R: The tests were performed with the same original cream (60 L) that was divided in four portions of 15 L which were fermented with the different cultures. From each batch of cream were produced ca. 6 kg of butter, which were packed in 0.5 L polypropylene boxes. The resulting buttermilk samples were packaged in 500 mL PET bottles. At each sampling time one box of butter and one bottle were used and replicates of analysis were performed with the same butter block or with the same buttermilk bottle.

Ideally the overall procedure should have been repeated with other batches of cream to consider the variations resulting from processing variability. This is a limitation of the study. However, the experimental design intended to evaluate the differences resulting from the treatments applied to the cream. Repeating the processing steps with different batches of cream would have the advantage of evaluating the differences resulting from process variations but would be difficult to clarify the causes for the differences (i.e. resulting from the fermentations conditions or from the different cream batches).

Line 154: Include the model of the Gerber butyrometer used.

R: Corrected. Inserted: (SuperVario-N Funke Gerber™ centrifuge, Berlin, Germany).

Line 168: Were three measurements taken from the same block of butter? Please clarify.

R: Yes. Corrected.

Line 193: As suggested by Stone and Sidel (2004) chapter 7, since the test was conducted with only 25 people, I recommend naming this item as 'Laboratory Acceptance Sensory Test".

R: Corrected. In fact, this is a more appropriate definition.

Line 200: Was the test submitted to an internal ethics committee? Is that what you meant in this line? If so, please be more specific and, if possible, cite the identifier of the document submitted to the committee and the date.

R: No. Sensory tests were not submitted to an internal ethics committee. However, the informed and consented form approved by the ethics committee was used. We clarified this point. Please refer to the Institutional Review Board Statement: The execution of sensory analysis is not a procedure submitted to the appreciation of the ethics commission (EC) of the Polytechnic of Coimbra. Most of the panelists are members of the staff of the institution and frequently perform such types of tests. However, when consumer sensory tests are carried out, the panelists are informed about the objectives of the work and sign the informed consent form provided by the EC (CIEIPC_CILE_02). Hence, this research did not necessitate formal ethical approval as per the situation at that time. In the course of the implementation of this study, no experiments violating human or animal laws were performed. This research follows Law No. 58/2019, of 8 August, the GDPR the Declaration of Helsinki and the Oviedo Convention.

Table1: Normally, when there is no significant difference, there is no need to place superscript letters in the columns where there is no difference between the samples. Please include a final row in the table with the p-value for each analysis, if possible.

R: Corrected.

Line 217: Instead of naming the color variables as 'parameter L' or 'a' or 'b', always use 'Lightness (L*); green-red hue (a*); blue-yellow hue (b*)'. This should apply to Table 2 as well as throughout the entire manuscript.

R: Corrected.

Table 2: See comments on Table 1.

R: Corrected.

Table 3: I believe this table is unnecessary; the text is already quite clear and sufficient regarding the results presented here.

R: The table presents the results of the color differences observed between samples. We believe that this information is important since it indicates if a common observer can or cannot detect color differences.

Line 227-235: How does this discussion serve the purpose of the manuscript? Could you delve deeper into the perceived differences regarding the possible causes related to the treatments studied in this work? In other words, why should the PBC treatment theoretically show a more yellowish color? Can we assert that this is not just a fluctuation in the process itself?"

R: Although significant differences between the color parameters (L*, a* and b*) of butter samples were observed, the values of the calculated color differences (ΔEab*) indicate that a common observer cannot detect the differences in color of butter samples. Hence, it can be considered that the cultures used had a negligible effect on the color of butter samples. (please see lines 246-249).

Line 236-251: I don’t understand the objective of evaluating the texture parameters at 10 and 20 degrees, aside from the obvious fact that at lower temperatures the samples will be harder. It might be more interesting to compare how the influence affects each pair of samples (with p value). Additionally, the discussion based on another study where not only the analysis temperature was different but, more importantly, the raw material used for the butter was different doesn’t seem appropriate, as the triacylglycerol profiles are different and, therefore, the complexity of the melting profile curve will be entirely different. Check it on Devi & Shatkar, (2016).

R: Texture measurements were made at temperatures that intend to replicate usual conditions of butter consumption. 10 ºC would correspond to butter after removal from the refrigerator, while 20 ºC would correspond to butter kept out of the refrigerator. So, the idea of doing the tests at 10 and 20 ºC was to mimic the conditions at which butter is consumed.

Please see an example in which texture measurements were made at 5 ºC and 23 ºC. Texture of Butter from Cows with Different Milk Fatty Acid Compositions G. Bobe, E. G. Hammond, A. E. Freeman, G. L. Lindberg, and D. C. Beitz. J. Dairy Sci. 86:3122–3127. Besides, in the discussion data from other authors was also obtained at different temperatures. Please see lines 720-726.

Line 256-276: See comments for line 236-251. Once again, I do not understand the actual contribution of comparing the rheological properties of the products at 10 and 20 degrees to the title and objective of the work. How could the variables studied in each treatment (probiotic culture, bioprotective, etc.) alter these profiles? The data analysis and discussion should be oriented in this direction.

R: Since the temperature of butter has a strong influence on its textural parameters, we decided to evaluate them at the temperatures at which butter is usually consumed. This would allow for comparison with data of other authors.

Line 337-349: Present the data more precisely and with statistical support. Report the absolute values, standard deviations, and, most importantly, the statistical differences at a given level of significance. In scientific papers, subjective terms like 'similar' or 'almost identical' are typically not used, especially given the small number of testers employed.

Figure 5. You can use a radar chart like this, but with statistical support such as error bars or at least asterisks indicating where there are statistically significant differences

R: Corrected. New graphs were produced. In fact, the previous graphs had an error of identification of samples.

Line382-384: This is the part of the discussion that is most related to your objectives; develop it further

R: The discussion was reformulated.

Line 296-399: If the analysis conducted by other authors is not replicable in your work, it should not be included in your discussion. Ideally, you should explain the different values you obtained based on your results and concrete factors such as varying raw materials, process variability, or ideally, the differences in your treatments.

R: Agreed. We reformulated the discussion.

Line 400-425: What is the relationship between color and the variables studied in each treatment in this work?

R: As previously indicated, the treatments had a negligible impact on butter color. (please see lines 246-249).

Line 440-452: What is the relationship between your sensory results and the variables studied in each treatment in this work?

R: Significant correlations between parameters were inserted in results.

Line 425: The work and discussion would benefit greatly from a multivariate correlation analysis (Pearson) and a PCA to elucidate and correlate all the variables of interest.

Line 454-459: Rewrite the conclusion after the statistical reevaluation and more in-depth discussion. Clearly specify how each section of analysis and results contributes to addressing the objective and to making sense of the title of the work.

R: Corrected

REVIEWER2

The authors planned and performed the experiment very well, and the paper is reasonably written. However, additional corrections are still needed. The authors should correct and address the following issues:

2.2. Physico chemical Analysis

Measurement of Butter Color:
In the equation ΔEab* = √[(L* - L0)² + (a - a0)² + (b - b0)²], the terms L0, a0, and b0 should be written with an exponent of 0, and the meaning of these parameters should be explained.

R: Corrected. Please see note below equation. Please see lines 181,182.

Line 168: Three measurements were taken for each sample. It should be added that the results were expressed as the mean value.

R: Corrected as proposed.

3. Results

Line 236: The authors should state which sample is shown in Figure 1 and explain why that sample was chosen.

R: Corrected. Sample used as example was identified. The pattern of the texture plots is similar in all samples.

Throughout the entire paragraph discussing texture results, the terms “the higher positive and negative peaks” are used. This is not a scientific explanation. The terminology should be precise and reflect the characteristics of the analyzed sample (e.g., spreadability, hardness, cohesiveness). Additionally, the effects of these characteristics on the product should be explained.

R: Corrected.

We tried to clarify all parameters evaluated using appropriate definitions. However, the information available in the literature uses different terms to express the same measure. We used the definitions provided by Lis et al. 2021 (https://doi.org/10.1080/10942912.2021.1999262)

Line 255: In Table 4, the terms "positive peak" and "negative peak" should be replaced with appropriate names.

R: Corrected.

Line 287: The metabolites of microorganisms should be referenced.

R: Sentence was reformulated.

Figure 4: The quality of Figure 4 in my copy of the manuscript is blurry and needs improvement.

R: The figure was enlarged. However, if the situation is not solved, we will reformulate it.

Discussion

The authors explain the differences among the analyzed samples as consequences of various parameters (fatty acids, fat globules, packaging) which were not analyzed in their work. This discussion should be more closely connected with their previous results or framed as plans for future experiments.

R: Discussion was reformulated.

Do you plan to analyze the microbiological quality of butter samples in terms of pathogenic microorganisms?

R: No. Butter samples were produced with pasteurized cream and the presence of pathogenic microorganisms is not expectable in the butter samples produced in such conditions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

The authors planned and performed the experiment very well, and the paper is reasonably written. However, additional corrections are still needed. The authors should correct and address the following issues:

2.2. Physico chemical Analysis

• Measurement of Butter Color:
  In the equation ΔEab* = √[(L* - L0)² + (a - a0)² + (b - b0)²], the terms L0, a0, and b0 should be written with an exponent of 0, and the meaning of these parameters should be explained.
• Line 168: Three measurements were taken for each sample. It should be added that the results were expressed as the mean value.

3. Results

• Line 236: The authors should state which sample is shown in Figure 1 and explain why that sample was chosen.
• Throughout the entire paragraph discussing texture results, the terms “the higher positive and negative peaks” are used. This is not a scientific explanation. The terminology should be precise and reflect the characteristics of the analyzed sample (e.g., spreadability, hardness, cohesiveness). Additionally, the effects of these characteristics on the product should be explained.
• Line 255: In Table 4, the terms "positive peak" and "negative peak" should be replaced with appropriate names.
• Line 287: The metabolites of microorganisms should be referenced.
• Figure 4: The quality of Figure 4 in my copy of the manuscript is blurry and needs improvement.

Discussion

• The authors explain the differences among the analyzed samples as consequences of various parameters (fatty acids, fat globules, packaging) which were not analyzed in their work. This discussion should be more closely connected with their previous results or framed as plans for future experiments.
• Do you plan to analyze the microbiological quality of butter samples in terms of pathogenic microorganisms?

Author Response

REVIEWER1

 

Journal: Dairy (ISSN 2624-862X) Manuscript ID: dairy-3165989 Title: Butter and buttermilk produced with cream fermented by aromatic or probiotic starters, with or without bioprotective cultures.

General comments: The work is interesting, provides valuable and innovative contributions to the field, and is written in a fluid and clear manner.

However, the data presentation is very one dimensional, lacking dynamism and integration. The work lacks greater statistical support and precision in data presentation.

R: Significant correlations between measured parameters of butter and buttermilk samples were included in the discussion of results. PCA did not add useful information.

The discussion is well-referenced but sometimes general and not focused on the objectives of the work, the treatments, and the results obtained.

R: We tried to focus the discussion regarding the objectives of the work.

I recommend a major revision before reconsideration for publication.

Below are detailed comments on each line and paragraph to be revised.

Line 1-2: Try reworking the title to better reflect the results, here are some suggestions: "Impact of Probiotic and Bioprotective Cultures on the Quality and Shelf Life of Butter and Buttermilk"; or "Evaluating the Influence of Fermentation Cultures on Butter and Buttermilk: Physicochemical, Rheological, and Sensory Properties"; or "Probiotic Versus Aromatic Cultures in Butter Production: Effects on Texture, Colour, and Shelf Life"; or "The Role of Probiotics and Bioprotective Cultures in Enhancing Butter and Buttermilk Quality and Longevity"; or "Comparative Analysis of Butter and Buttermilk Produced with Different Fermentation Cultures: A Study of Physicochemical and Sensory Attributes"; or "Rheological and Microbiological Characteristics of Butter and Buttermilk with Probiotic and Bioprotective Cultures.

R: We corrected the title as indicated.

Line 83-87: The term "Creamy goodness" seems a bit confusing to me, as well as the last sentence of the paragraph. Could you rewrite it in a way that more broadly addresses the product's quality parameters that could be improved?

R: Corrected.

Line 107: It would be very enriching for the manuscript and very useful for overall understanding if a visual diagram or flowchart were presented that encompasses all the materials and methods described, focusing mainly on point 2.1 but also including, in a very simplified manner, the subsequent analyses performed. The figure should clearly indicate the treatments used for later analyses and their respective codes as referenced throughout the text.

R: Corrected. A flow diagram of the experiment steps was added.

Line 107-139: Why were no processing replicates performed in the study? How can processing variability be identified and assessed? At the end of the process, only one product from each treatment (butter block and buttermilk bottle) was analyzed for each of the subsequent tests, without technical replicates?

R: The tests were performed with the same original cream (60 L) that was divided in four portions of 15 L which were fermented with the different cultures. From each batch of cream were produced ca. 6 kg of butter, which were packed in 0.5 L polypropylene boxes. The resulting buttermilk samples were packaged in 500 mL PET bottles. At each sampling time one box of butter and one bottle were used and replicates of analysis were performed with the same butter block or with the same buttermilk bottle.

Ideally the overall procedure should have been repeated with other batches of cream to consider the variations resulting from processing variability. This is a limitation of the study. However, the experimental design intended to evaluate the differences resulting from the treatments applied to the cream. Repeating the processing steps with different batches of cream would have the advantage of evaluating the differences resulting from process variations but would be difficult to clarify the causes for the differences (i.e. resulting from the fermentations conditions or from the different cream batches).

Line 154: Include the model of the Gerber butyrometer used.

R: Corrected. Inserted: (SuperVario-N Funke Gerber™ centrifuge, Berlin, Germany).

Line 168: Were three measurements taken from the same block of butter? Please clarify.

R: Yes. Corrected.

Line 193: As suggested by Stone and Sidel (2004) chapter 7, since the test was conducted with only 25 people, I recommend naming this item as 'Laboratory Acceptance Sensory Test".

R: Corrected. In fact, this is a more appropriate definition.

Line 200: Was the test submitted to an internal ethics committee? Is that what you meant in this line? If so, please be more specific and, if possible, cite the identifier of the document submitted to the committee and the date.

R: No. Sensory tests were not submitted to an internal ethics committee. However, the informed and consented form approved by the ethics committee was used. We clarified this point. Please refer to the Institutional Review Board Statement: The execution of sensory analysis is not a procedure submitted to the appreciation of the ethics commission (EC) of the Polytechnic of Coimbra. Most of the panelists are members of the staff of the institution and frequently perform such types of tests. However, when consumer sensory tests are carried out, the panelists are informed about the objectives of the work and sign the informed consent form provided by the EC (CIEIPC_CILE_02). Hence, this research did not necessitate formal ethical approval as per the situation at that time. In the course of the implementation of this study, no experiments violating human or animal laws were performed. This research follows Law No. 58/2019, of 8 August, the GDPR the Declaration of Helsinki and the Oviedo Convention.

Table1: Normally, when there is no significant difference, there is no need to place superscript letters in the columns where there is no difference between the samples. Please include a final row in the table with the p-value for each analysis, if possible.

R: Corrected.

Line 217: Instead of naming the color variables as 'parameter L' or 'a' or 'b', always use 'Lightness (L*); green-red hue (a*); blue-yellow hue (b*)'. This should apply to Table 2 as well as throughout the entire manuscript.

R: Corrected.

Table 2: See comments on Table 1.

R: Corrected.

Table 3: I believe this table is unnecessary; the text is already quite clear and sufficient regarding the results presented here.

R: The table presents the results of the color differences observed between samples. We believe that this information is important since it indicates if a common observer can or cannot detect color differences.

Line 227-235: How does this discussion serve the purpose of the manuscript? Could you delve deeper into the perceived differences regarding the possible causes related to the treatments studied in this work? In other words, why should the PBC treatment theoretically show a more yellowish color? Can we assert that this is not just a fluctuation in the process itself?"

R: Although significant differences between the color parameters (L*, a* and b*) of butter samples were observed, the values of the calculated color differences (ΔEab*) indicate that a common observer cannot detect the differences in color of butter samples. Hence, it can be considered that the cultures used had a negligible effect on the color of butter samples. (please see lines 246-249).

Line 236-251: I don’t understand the objective of evaluating the texture parameters at 10 and 20 degrees, aside from the obvious fact that at lower temperatures the samples will be harder. It might be more interesting to compare how the influence affects each pair of samples (with p value). Additionally, the discussion based on another study where not only the analysis temperature was different but, more importantly, the raw material used for the butter was different doesn’t seem appropriate, as the triacylglycerol profiles are different and, therefore, the complexity of the melting profile curve will be entirely different. Check it on Devi & Shatkar, (2016).

R: Texture measurements were made at temperatures that intend to replicate usual conditions of butter consumption. 10 ºC would correspond to butter after removal from the refrigerator, while 20 ºC would correspond to butter kept out of the refrigerator. So, the idea of doing the tests at 10 and 20 ºC was to mimic the conditions at which butter is consumed.

Please see an example in which texture measurements were made at 5 ºC and 23 ºC. Texture of Butter from Cows with Different Milk Fatty Acid Compositions G. Bobe, E. G. Hammond, A. E. Freeman, G. L. Lindberg, and D. C. Beitz. J. Dairy Sci. 86:3122–3127. Besides, in the discussion data from other authors was also obtained at different temperatures. Please see lines 720-726.

Line 256-276: See comments for line 236-251. Once again, I do not understand the actual contribution of comparing the rheological properties of the products at 10 and 20 degrees to the title and objective of the work. How could the variables studied in each treatment (probiotic culture, bioprotective, etc.) alter these profiles? The data analysis and discussion should be oriented in this direction.

R: Since the temperature of butter has a strong influence on its textural parameters, we decided to evaluate them at the temperatures at which butter is usually consumed. This would allow for comparison with data of other authors.

Line 337-349: Present the data more precisely and with statistical support. Report the absolute values, standard deviations, and, most importantly, the statistical differences at a given level of significance. In scientific papers, subjective terms like 'similar' or 'almost identical' are typically not used, especially given the small number of testers employed.

Figure 5. You can use a radar chart like this, but with statistical support such as error bars or at least asterisks indicating where there are statistically significant differences

R: Corrected. New graphs were produced. In fact, the previous graphs had an error of identification of samples.

Line382-384: This is the part of the discussion that is most related to your objectives; develop it further

R: The discussion was reformulated.

Line 296-399: If the analysis conducted by other authors is not replicable in your work, it should not be included in your discussion. Ideally, you should explain the different values you obtained based on your results and concrete factors such as varying raw materials, process variability, or ideally, the differences in your treatments.

R: Agreed. We reformulated the discussion.

Line 400-425: What is the relationship between color and the variables studied in each treatment in this work?

R: As previously indicated, the treatments had a negligible impact on butter color. (please see lines 246-249).

Line 440-452: What is the relationship between your sensory results and the variables studied in each treatment in this work?

R: Significant correlations between parameters were inserted in results.

Line 425: The work and discussion would benefit greatly from a multivariate correlation analysis (Pearson) and a PCA to elucidate and correlate all the variables of interest.

Line 454-459: Rewrite the conclusion after the statistical reevaluation and more in-depth discussion. Clearly specify how each section of analysis and results contributes to addressing the objective and to making sense of the title of the work.

R: Corrected

REVIEWER2

The authors planned and performed the experiment very well, and the paper is reasonably written. However, additional corrections are still needed. The authors should correct and address the following issues:

2.2. Physico chemical Analysis

Measurement of Butter Color:
In the equation ΔEab* = √[(L* - L0)² + (a - a0)² + (b - b0)²], the terms L0, a0, and b0 should be written with an exponent of 0, and the meaning of these parameters should be explained.

R: Corrected. Please see note below equation. Please see lines 181,182.

Line 168: Three measurements were taken for each sample. It should be added that the results were expressed as the mean value.

R: Corrected as proposed.

3. Results

Line 236: The authors should state which sample is shown in Figure 1 and explain why that sample was chosen.

R: Corrected. Sample used as example was identified. The pattern of the texture plots is similar in all samples.

Throughout the entire paragraph discussing texture results, the terms “the higher positive and negative peaks” are used. This is not a scientific explanation. The terminology should be precise and reflect the characteristics of the analyzed sample (e.g., spreadability, hardness, cohesiveness). Additionally, the effects of these characteristics on the product should be explained.

R: Corrected.

We tried to clarify all parameters evaluated using appropriate definitions. However, the information available in the literature uses different terms to express the same measure. We used the definitions provided by Lis et al. 2021 (https://doi.org/10.1080/10942912.2021.1999262)

Line 255: In Table 4, the terms "positive peak" and "negative peak" should be replaced with appropriate names.

R: Corrected.

Line 287: The metabolites of microorganisms should be referenced.

R: Sentence was reformulated.

Figure 4: The quality of Figure 4 in my copy of the manuscript is blurry and needs improvement.

R: The figure was enlarged. However, if the situation is not solved, we will reformulate it.

Discussion

The authors explain the differences among the analyzed samples as consequences of various parameters (fatty acids, fat globules, packaging) which were not analyzed in their work. This discussion should be more closely connected with their previous results or framed as plans for future experiments.

R: Discussion was reformulated.

Do you plan to analyze the microbiological quality of butter samples in terms of pathogenic microorganisms?

R: No. Butter samples were produced with pasteurized cream and the presence of pathogenic microorganisms is not expectable in the butter samples produced in such conditions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

General comments: The quality of the manuscript has significantly improved in terms of clarity in some key areas; however, methodological flaws and issues with the experimental design prevent me from feeling comfortable recommending the acceptance of the article. Some of the concerns either confirmed previous apprehensions regarding important aspects of the manuscript, were not addressed satisfactorily, or were only partially resolved.

Line 107: The required and presented diagram does not clearly and thoroughly organize the treatments used in the experiment, their respective codes, the methodologies applied, the sample population, and other relevant details.

Line 107-139: Biological and technological processes have intrinsic cascading variations that can significantly obscure instrumental results of medium or high precision by the end of the experimental process. In a study where the central pillar of the hypothesis is in the area of food technology, as in this case, it is essential to isolate variables through the replication of the technological process itself.

Line 168: Just as food technological processes are highly variable, their products, and even different spatial regions within those products, also exhibit high variability. In international journals of medium and high impact, you will not find texture analyzer experiments where analyses are conducted in triplicate only. Typically, 5 to 10 (or even more) repetitions are used due to the high variability of the measurements. The standard deviation of the dataset was not shown. Finally, there is a critical flaw in taking measurements from the same point or product, as the first compression or cutting of the product alters its structure, preventing a true repetition of the analysis conditions.

Line 227: The entire discussion still does not address the differences in treatments applied to each sample (starter cultures). Consequently, the article does not present new insights or relevant discussions for the field and does not meet its objectives.

Line 236: I understand the motivation behind the analysis conditions. However, my concern lies with the objective of the analysis under these conditions. What do these results reveal that is significant and insightful? As it stands, the results become accurate and well-articulated assertions but are ultimately quite obvious and have little impact on the field. Additionally, they fail to address the relationship between the chosen variables for treatments/samples.

Line 337: Although the data are presented in a more scientific manner with some statistical grounding, they remain unclear and difficult to interpret as a whole. The presentation lacks the means, standard deviations, and sample sizes, which hinders effective comparison.

Line 425: The presentation of the correlation analyses is superficial and isolated in separate blocks of results. Some correlations are nonsensical, such as the one described in lines 308-309. How could there be strong negative and positive correlations, yet the PCA did not add useful information?

Line 492: The data, results, and discussions presented do not convince me of the claims made or the final conclusions drawn.

Author Response

General comments: The quality of the manuscript has significantly improved in terms of clarity in some key areas; however, methodological flaws and issues with the experimental design prevent me from feeling comfortable recommending the acceptance of the article. Some of the concerns either confirmed previous apprehensions regarding important aspects of the manuscript, were not addressed satisfactorily, or were only partially resolved.

R: Taking in attention the reviewer's comments, according to his opinion, all the experiment would have to be repeated. This is unfeasible, currently. However, it has to be taken in attention that each batch of butter samples weighed ca. 7 kg and, at each time of sampling, 0,5 kg of butter were used, being each analysis repeated at least 3 times.  So, we can consider that the sampling plan was acceptable for the purpose of the manuscript.

We must stress that the main objective of the work was to evaluate the possibility of fermenting cream with probiotic and bioprotective cultures and comparing that samples with conventional starter currently used in butter production (aromatic), with, or without bioprotective culture.

So, taking in attention the results of the microbiological analysis, one can consider that both probiotic and bioprotective cultures could be used in butter production, allowing for the obtention of a probiotic butter and the correspondent buttermilk. It has to be noted that level of lactobacilli and lactococci exceeded log 7 UFC/g (in butter) or mL (in buttermilk), indicating the probiotic potential of both products. Besides, it was observed that the probiotic culture used together with the bioprotective culture was able to reduce the counts of yeasts and molds in buttermilk, by one log cycle, at the end of storage.

So, we consider that the main objective of the work was attained. Naturally that we consider that the repetition of all the experiments with other batches of cream would strengthen the evaluation of results. However, it can also be argued that different batches of cream would introduce non controlled variations resulting from variations in cream from different batches.

Line 107: The required and presented diagram does not clearly and thoroughly organize the treatments used in the experiment, their respective codes, the methodologies applied, the sample population, and other relevant details.

R: Diagram was reformulated. We also consider that the text of the M&M section satisfactorily explains all the experimental procedure and, with the aid of diagram, one can clearly understand the layout of the experiment.

Line 107-139: Biological and technological processes have intrinsic cascading variations that can significantly obscure instrumental results of medium or high precision by the end of the experimental process. In a study where the central pillar of the hypothesis is in the area of food technology, as in this case, it is essential to isolate variables through the replication of the technological process itself.

R: We agree. But as said above, the sampling plan is strong enough to evaluate the feasibility of fermenting cream with probiotic and bioprotective cultures, and to evaluate the survival of such cultures over the storage period. Besides the repeatability of the measurements made (i.e. the observed standard deviations) indicate that data can be interpreted with some security.

Line 168: Just as food technological processes are highly variable, their products, and even different spatial regions within those products, also exhibit high variability. In international journals of medium and high impact, you will not find texture analyzer experiments where analyses are conducted in triplicate only. Typically, 5 to 10 (or even more) repetitions are used due to the high variability of the measurements. The standard deviation of the dataset was not shown. Finally, there is a critical flaw in taking measurements from the same point or product, as the first compression or cutting of the product alters its structure, preventing a true repetition of the analysis conditions.

R: Right. Five or more repetitions would be preferred. But 3 repetitions are also commonly reported in the literature in experiments involving food samples.

All tables clearly indicate the standard deviation of the samples and the results of the ANOVA, thus allowing to identify significative differences between samples, so we cannot agree with the reviewer’s comment regarding this point.

Besides, taking in attention the observed standard deviations, it is clear that the variability observed in the experiments is not as high as to indicate the need of a repetition of the experiment.

Line 227: The entire discussion still does not address the differences in treatments applied to each sample (starter cultures). Consequently, the article does not present new insights or relevant discussions for the field and does not meet its objectives.

R: The principal component analysis results were included on the manuscript (Figs. 5 and 6). As we previously indicated, this tool although providing useful information could not discriminate all the butter samples. Only sample P appears as a separate group of results. The results obtained for buttermilk samples could discriminate all samples.

Line 236: I understand the motivation behind the analysis conditions. However, my concern lies with the objective of the analysis under these conditions. What do these results reveal that is significant and insightful? As it stands, the results become accurate and well-articulated assertions but are ultimately quite obvious and have little impact on the field. Additionally, they fail to address the relationship between the chosen variables for treatments/samples.

R: Works reporting about the use of probiotic or bioprotective bacteria in butter are scarce in the literature. So, research on this subject is worthwhile and can have practical application in the industry. Hence, we do not agree with the sentence “they fail to address the relationship between the chosen variables for treatments/samples”.

Notice that no differences were observed regarding butter samples composition, that adequate amounts of probiotic bacteria were found in both products and that products were well accepted by consumers. Modified samples P, PBC and ABC obtained higher sensory scores than the conventional product (A), both regarding butter and buttermilk. We believe that this information is meaningful for the industry.

Line 337: Although the data are presented in a more scientific manner with some statistical grounding, they remain unclear and difficult to interpret as a whole. The presentation lacks the means, standard deviations, and sample sizes, which hinders effective comparison.

R: We do not understand why the reviewer refers that the presentation “lacks the means, standard deviations and sample sizes”.

All tables present means, standard deviations, and results of the Anova tests. In the material and methods section, it is clearly indicated that physicochemical analyses were performed at least in triplicate.

Besides, regarding to the evaluation of color parameters the ΔEab values were calculated based on the measured parameters L*, a* and b*, and this is the best way to evaluate colour differences in the samples. Please see:

A Simple Review of CIE ΔE* (Colour Difference) Equations. Available online: https://techkonusa.com/a-simple-review-of-cie-%CE%B4e-colour-difference-equations/ 

With regard to texture parameters, two methods were used: texture analysis and evaluation of rheological parameters, and both methods indicated comparable patterns of samples. So, at least, 6 measures confirmed the textural behaviour of butter samples.

Line 425: The presentation of the correlation analyses is superficial and isolated in separate blocks of results. Some correlations are nonsensical, such as the one described in lines 308-309. How could there be strong negative and positive correlations, yet the PCA did not add useful information?

R: The PCA analysis results were included in the manuscript.

Line 492: The data, results, and discussions presented do not convince me of the claims made or the final conclusions drawn.

R: The conclusion is short and intends to highlight the main findings that can be of interest for the industry:

“Overall, it can be concluded that the fermentation of cream with probiotic and bioprotective cultures represents an interesting opportunity to enhance the functional properties of butter and allows for the obtention of the correspondent probiotic buttermilk. Both butter and buttermilk products exceeded the probiotic cell counts over the storage period (90 days for butter and 28 days for buttermilk). However, further work is needed to improve the sensory properties of buttermilk samples”.

Microbial counts of butter are clearly superior than the ones reported in other works in which probiotics were used to ferment cream used for butter production:

(e.g. Bellinazo, P.; Vitola, H.; Cruxen, C.; Braun, C.; Hackbart, H.; Silva, W.; Fiorentini, Â. Probiotic butter: viability of lactobacillus casei strains and bixin antioxidant effect (bixa orellana l.). J. Food Process. Preservation 2019, 43(9); Gaba, K.; Anand, S.; Syamala, A. Development of value-added butter by incorporating whey protein hydrolysate- encapsulated probiotics. Microorganisms 2023, 11, 1139).

What statement does not convince the reviewer?

That the use of probiotic and bioprotective cultures represent and interesting opportunity to enhance the functional properties of butter (namely the presence of adequate numbers of probiotic bacteria in the product)?

or

That both in butter and buttermilk products exceeded the probiotic’s cell counts required for probiotic products?

We rephrased the sentences in order to clarify the conclusions.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

none