Changes in the Concentration and Profile of Free Fatty Acids during the Ripening of a Spanish Blue-Veined Cheese Made from Raw and Pasteurized Cow and Goat Milk

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is clearly written and structured, with adequate method descriptions.

However, there are inappropriate references, the experimental plan description is lacking, and the article needs to be revised for more clarity.

All comments are listed below point by point.

 

Abstract

The abstract is repetitive, and the results obtained are not clearly explained, so it should be rewritten to report how maturation, season and pasteurization affect the FFA profile and concentration.

In my opinion, the importance of lipolysis on cheese aroma and flavour should be emphasized and its impact on the final quality, especially in blue cheese, should be highlighted.

 

Introduction

In the introduction, there are inappropriate bibliographic citations (e.g. [11] and [15]).

Avoid generic references and check that the citations refer to the free fatty acids (FFA content) and not to the fatty acid composition (FA content) in cheese. Please, include recent and specific references and check the reference list. Reference [14] is missing in the introduction.

L 49-51 Please check this sentence, it is incomplete.

 

Materials and Methods

L 61-65 Please, explain why only the liquid suspension of P. roqueforti spores was added to raw milk and not a starter culture as in pasteurized milk. It should be justified because cheeses obtained with different production processes are compared.

Please specify more details about the experimental plan. Explain figure1 in the text.

If I understand correctly figure 1, cheese from pasteurized milk was produced in different seasons of the year while cheese from raw milk was not. It is not clear whether raw milk is sampled in different seasons or only in one season of the year. Raw and pasteurized milk from the same season should be compared.

Rephrase Figure 1 and the relative caption.

L 95 Check the composition of the standard solution. The name of the C2:0 is missing.

Table 1 is incomplete. Check the acronyms used for fatty acids (replace C4 with C4:0, ....) in tables 1 and 2.

The list of fatty acids reported in the text should be the same as that reported in table 1 and table 2.

 

Results and discussion

L 117 Please, add “and Discussion”.

L 126-127 Reference [19] is inappropriate not considering the free fatty acids but the total fatty acid composition. Avoid generic references and check that the citations refer to free fatty acids.

L 138-140 It is not clear whether you calculated the n3/n6 ratio of free fatty acids. Citation [19] refers to n3/n6 of total FA and not of FFA.

L 152-153 Specify if the reported data are referred to cheese at the end of ripening (120 d).

Table 2, Figures 1 and 2 Adding the letters would help to see significant differences.

L 175-177 It is not clear what the ROGOSA and KAA counts represent and that these results are reported in previous work.

L 179, L 188, L 193, L 207 Replace FAA with FFA. Check the acronym FFA in all the text.

L 184 Reference [29] is inappropriate not considering SCFAs decomposition to aromatic compounds.

L 194 They are free fatty acids.

L 210-212 Please, revise these sentences. The reported results should correspond to figure 5. Are the values observed in summer compared with the values of the other seasons?

L 212-213 Is there an explanation for the causes of this different FFA profile in summer? And on the higher total concentration of FFA in summer?

Figure 7 Only one point is reported for each ripening time. Does it represent an average sample? Several points should be represented for each ripening time, considering all the repetitions.

 

Conclusions

In my opinion, the conclusion should report the impact of these findings on cheese aroma and flavour.

The particular interest in the lipolysis of blue cheese should be highlighted and the importance of these results for its sensory characteristics should be emphasized.

The obtained results could suggest how to improve the quality of Valdeón cheese.

Author Response

Manuscript ID dairy-2115910

 

Firstly, we should like to thank the reviewer for their advice and suggestions, as well as for the corrections of the manuscript, which, without any doubt, will aid in improving its quality.

 

Reviewer 1

Principio del formulario

Comments and Suggestions for Authors

Comments and Suggestions for Authors

The manuscript is clearly written and structured, with adequate method descriptions.

However, there are inappropriate references, the experimental plan description is lacking, and the article needs to be revised for more clarity.

All comments are listed below point by point.

 

Abstract

Reviewer (Re): The abstract is repetitive, and the results obtained are not clearly explained, so it should be rewritten to report how maturation, season and pasteurization affect the FFA profile and concentration.

In my opinion, the importance of lipolysis on cheese aroma and flavour should be emphasized and its impact on the final quality, especially in blue cheese, should be highlighted.

Authors (Au): The abstract has been rewritten to avoid being repetitive, also indicating the impact on the flavour (lines 21-22 of the new version).

Introduction

Re: In the introduction, there are inappropriate bibliographic citations (e.g. [11] and [15]).

Au: The authors agree with the recommendations and the references have been revised, changing some and renumbering others.

Re: Avoid generic references and check that the citations refer to the free fatty acids (FFA content) and not to the fatty acid composition (FA content) in cheese. Please, include recent and specific references and check the reference list. Reference [14] is missing in the introduction.

L 49-51 Please check this sentence, it is incomplete.

Au: The authors agree. The text has been revised to avoid referring to the fatty acid composition of cheese. The reference list has been amended and the incomplete sentence has been rewritten.

Materials and Methods

Re: L 61-65 Please, explain why only the liquid suspension of P. roqueforti spores was added to raw milk and not a starter culture as in pasteurized milk. It should be justified because cheeses obtained with different production processes are compared.

Au: The cheese makers who make Valdeón cheese do not add any lactic starter to the raw milk. The cheeses used in the study come from the production carried out at the cheese factory, so the conditions were not modified from the usual ones.

Re: Please specify more details about the experimental plan. Explain figure1 in the text.

Au: Figure 1 was explained in text (section 2.1)

Re: If I understand correctly figure 1, cheese from pasteurized milk was produced in different seasons of the year while cheese from raw milk was not. It is not clear whether raw milk is sampled in different seasons or only in one season of the year. Raw and pasteurized milk from the same season should be compared.

Au: The reviewer is right. Several batches of Valdeón cheese were indeed made with pasteurized milk at different times of the year, as this is the largest production of this type of cheese. However, cheeses made with raw milk represent a smaller percentage of production and are only made at certain times of the year, mainly in spring. Therefore, the influence of seasonality has not been applied to cheeses made from raw milk. This information is included in the new version of the article (lines 69-71 of the new version).

Re: Rephrase Figure 1 and the relative caption.

Au: Figure 1 has been rephrased and the relative caption.

Re: L 95 Check the composition of the standard solution. The name of the C2:0 is missing.

Au: The composition of the standard solution has been checked. Thank you.

Re: Table 1 is incomplete. Check the acronyms used for fatty acids (replace C4 with C4:0, ....) in tables 1 and 2.

Au: Tables 1 and 2 (now Table 3) have been completed and corrected.

Re: The list of fatty acids reported in the text should be the same as that reported in table 1 and table 2.

Au: The list of FFA in the tables and text has been revised and corrected. Thank you.

 

Results and discussion

Re: L 117 Please, add “and Discussion”.

Au: Thank you.

 

Re: L 126-127 Reference [19] is inappropriate not considering the free fatty acids but the total fatty acid composition. Avoid generic references and check that the citations refer to free fatty acids.

Au: The authors agree. The text has been revised to avoid considering references to total fatty acid composition and keep only those referring to FFA.

 

Re: L 138-140 It is not clear whether you calculated the n3/n6 ratio of free fatty acids. Citation [19] refers to n3/n6 of total FA and not of FFA.

Au: n6/n3 ratio of the LFAs was calculated and has been included in table 2. Citation 19 has been deleted.

 

Re: L 152-153 Specify if the reported data are referred to cheese at the end of ripening (120 d).

Au: In the new version, the sampling point corresponding to specific FFA values given in the text has been added. In addition, all values have been expressed as a percentage in dry matter.

 

Re: Table 2, Figures 1 and 2 Adding the letters would help to see significant differences.

Au: Differences have been indicated in the last columns of the tables to avoid even larger and more complex tables. Where necessary, these differences are also indicated in the values in the tables and in the text, with reference to the variable concerned.

In addition, two tables have been incorporated in the new version. Differences of the values between pasteurized and raw milk cheeses have been added in each ripening time.

Figure 2 does not include differences because there were no significant differences with respect to season.

In figure 3 the significant differences with respect to the heat treatment have been included.

Re: L 175-177 It is not clear what the ROGOSA and KAA counts represent and that these results are reported in previous work.

Au: Information in this respect has been included (lines 251-253 of the new version).

Re: L 179, L 188, L 193, L 207 Replace FAA with FFA. Check the acronym FFA in all the text.

Au: Thank you.

 

Re: L 184 Reference [29] is inappropriate not considering SCFAs decomposition to aromatic compounds.

Au: reference 29 has been checked and removed. Thank you

 

Re: L 194 They are free fatty acids.

Au: All right. Thank you.

 

Re: L 210-212 Please, revise these sentences. The reported results should correspond to figure 5. Are the values observed in summer compared with the values of the other seasons?

Au: Figures 4, 5 and 6 have been revised and are referenced in the text. The following paragraph was included: “Cheeses made in summer showed the highest concentration of total FFA after 30 days of ripening, reaching values of 10,149.8 mg 100 g-1 of dry matter at the end of ripening. On the other hand, the lowest concentration was found in winter cheeses (5,879.1 mg 100 g-1 of dry matter, at 120 days of ripening)”. (Lines 229-233 of the new version).

 

Re: L 212-213 Is there an explanation for the causes of this different FFA profile in summer? And on the higher total concentration of FFA in summer?

Au: It is indeed difficult to find an explanation for the high FFA concentration in summer, as the microbial counts at that time of the year are not particularly high compared to the rest of the year, with the exception of those found in MSA, which are surprisingly 2 log units higher in summer than in the rest of the year, reaching counts of around 7 log cfu g^-1. In any case, the high temperatures in the cheese factory in summer and the time that the cheeses spend draining, as they are not pressed, could justify a higher enzyme activity.

 

Re: Figure 7 Only one point is reported for each ripening time. Does it represent an average sample? Several points should be represented for each ripening time, considering all the repetitions.

Au: In accordance with your and other reviewers' suggestions, section 3.2 on Principal component analysis has been deleted. 

A further section on sensory analysis has been included.

 

Conclusions

Re: In my opinion, the conclusion should report the impact of these findings on cheese aroma and flavour. The particular interest in the lipolysis of blue cheese should be highlighted and the importance of these results for its sensory characteristics should be emphasized. The obtained results could suggest how to improve the quality of Valdeón cheese.

Au: Information on sensory analysis for aroma and taste has been incorporated in section 3.3 about sensory analysis, and in conclusion.

 

Submission Date

07 December 2022

Date of this review

16 Dec 2022 17:16:20

Final del formulario

© 1996-2023 MDPI (Basel, Switzerland) unless otherwise state

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript reports a study aimed at characterizing the lipolysis in Valdeòn cheese, a blue veined cheese manufactured from cow’s milk added with 10% ewe’s milk. The cheesemaking process implied use of either raw or pasteurized milk, and was repeated in the four seasons of the year. A total of 12 cheese batches were produced and analysed at 6 different ripening times up to 120 days. The content of free fatty acids was determined on the cheeses by GC/MS and data were statistically evaluated.

Although the topic may be of interest to dairy industry, particularly at local level, the manuscript does not provide significant new knowledge to the scientific community. The content of FFA changed depending on the ripening time and was much higher in raw milk cheeses than in pasteurized milk cheeses, this is not surprising. However, due to the low number of samples that are considered for each tested variable, the observed variability is extremely high and comparing the mean values is not satisfactory. The data of FFA are expressed on cheese (w/w) basis, without considering that the fat content of the cheese also changes during ripening, as a consequence of moisture loss and possibly of milk pasteurization. At this regard, relevant information lacks on the analytical data collection. The sampling procedure is not described. The composition of cheese is very different between the inner and outer part of the wheel, especially in the first months of ripening. The number of replicates is not indicated, therefore the “weight” of the analytical error is not deductible. Differences between single FFA that could be traced back to the tested variables are very few, while this would have been an interesting output of the study.   A discussion on possible microbiological, chemical or biochemical aspects supporting the evidences arising from the results almost lacks. The reference to existing literature is not always justified or accompanied by convincing comments. An example is the sentence at Line 182-184. The cited paper [29] refers to Feta cheese, which is not a blue veined cheese, and formation of the mentioned compounds was directly observed, differently from what occurred here.

Impressively, the authors produced 9 figures and 3 tables from the determination of one parameter on 12 samples. Actually, the relevant information on single FFA is given in Table 1 but, unfortunately, the total FFA amounts are only mentioned within the text. No statistical evaluation is shown in Figures 2 and 3.  Therefore, comments at Lines 152-156 looks unjustified. The total FFA contents mentioned there can not be find in Figure 2. General recommendation: avoid using two decimal digits in large numbers (e.g., 6,657.26).

Another major concern is about PCA analysis. In all of the plots (Figures 7-9), samples positioned along PC1, which already explained 97-99% of variance depending on the plot. This indicates that PCA does not give additional information with this set of data. I recommend to delete this part.

 

Additional remarks

-       Abstract. Lines 16 and Line 20 report the same information.

-       Paragraph 3 should be titled “Results and Discussion”.

-       Line 138-140. The n3/n6 ratio is not defined and is not shown in Table 2. Therefore, related comments are not pertinent unless the ratio is shown.

-       Line 178. Short-, medium-, and long-chain fatty acids shall be defined.

Author Response

Manuscript ID dairy-2115910

 

Firstly, we should like to thank the reviewer for their advice and suggestions, as well as for the corrections of the manuscript, which, without any doubt, will aid in improving its quality.

 

Reviewer 2

 

Comments and Suggestions for Authors

Re: The manuscript reports a study aimed at characterizing the lipolysis in Valdeòn cheese, a blue veined cheese manufactured from cow’s milk added with 10% ewe’s milk. The cheese-making process implied use of either raw or pasteurized milk, and was repeated in the four seasons of the year. A total of 12 cheese batches were produced and analysed at 6 different ripening times up to 120 days. The content of free fatty acids was determined on the cheeses by GC/MS and data were statistically evaluated.

Although the topic may be of interest to dairy industry, particularly at local level, the manuscript does not provide significant new knowledge to the scientific community. The content of FFA changed depending on the ripening time and was much higher in raw milk cheeses than in pasteurized milk cheeses, this is not surprising. However, due to the low number of samples that are considered for each tested variable, the observed variability is extremely high and comparing the mean values is not satisfactory. The data of FFA are expressed on cheese (w/w) basis, without considering that the fat content of the cheese also changes during ripening, as a consequence of moisture loss and possibly of milk pasteurization. At this regard, relevant information lacks on the analytical data collection. The sampling procedure is not described. The composition of cheese is very different between the inner and outer part of the wheel, especially in the first months of ripening. The number of replicates is not indicated, therefore the “weight” of the analytical error is not deductible. Differences between single FFA that could be traced back to the tested variables are very few, while this would have been an interesting output of the study.   A discussion on possible microbiological, chemical or biochemical aspects supporting the evidences arising from the results almost lacks. The reference to existing literature is not always justified or accompanied by convincing comments. An example is the sentence at Line 182-184. The cited paper [29] refers to Feta cheese, which is not a blue veined cheese, and formation of the mentioned compounds was directly observed, differently from what occurred here.

Au: The Protected Geographical Identification of Queso de Valdeón includes both cheeses made with raw milk and those made with pasteurized milk, so the aim of the study was to find out to what extent both types of cheese differ in lipolysis intensity, through the study of the FFA, during ripening, considering the differences depending on the type of milk during ripening and seasonality in the cheeses that were made with pasteurized milk.

We agree that it is not very appropriate to express FFA on cheese, although in the literature some authors refer to it as such. Consequently, all data have been recalculated for expression on 100 g of total solids.

Regarding the sampling procedure, the cheeses were crushed and homogenised before the analyses were carried out (all of them in duplicate and some of them in triplicate). This has been indicated in lines 76-77 of the new version.

The cheeses were made by the 'Picos de Europa' cheese factory, which is part of the 'Queso de Valdeón' PGI Regulatory Council. Eight batches were made from pasteurised milk (two per season) and a whole cheese was collected at each ripening point (i.e. six cheeses): a total of 48 samples of cheeses made from pasteurised milk. A further 24 cheeses were made from raw milk. The authors consider that working with 72 cheeses is not a low number of samples. The sampling procedure has been indicated in the lines 73-77 of the new version.

The evolution of the FFA content during cheese ripening has been interrelated with the evolution of physicochemical and microbiological parameters. The higher values obtained during the ripening of cheeses made with pasteurized milk for chemical parameters such as dry matter and salt/moisture ratio and the lower values for aw could condition the lipolytic activity of P. roqueforti. A new table of values concerning chemical and physico-chemical parameters (Table 2 in the new version) has been included for pasteurized and raw milk cheeses during ripening. The differences between the cheeses have also been indicated and the text relates the influence they may have on the course of lipolysis in both types of cheeses during ripening.

References not related to blue cheeses have been deleted.

Re: Impressively, the authors produced 9 figures and 3 tables from the determination of one parameter on 12 samples. Actually, the relevant information on single FFA is given in Table 1 but, unfortunately, the total FFA amounts are only mentioned within the text. No statistical evaluation is shown in Figures 2 and 3.  Therefore, comments at Lines 152-156 looks unjustified. The total FFA contents mentioned there cannot be find in Figure 2. General recommendation: avoid using two decimal digits in large numbers (e.g., 6,657.26).

Au: For the study, 72 samples of cheese were used.

Total amounts of FFA have been included in Table 2.

Figure 2 does not include differences because there were no significant differences with respect to season.

In figure 3 the significant differences with respect to the heat treatment have been included.

The use of two decimal places in large numbers has been avoided

 

Re: Another major concern is about PCA analysis. In all of the plots (Figures 7-9), samples positioned along PC1, which already explained 97-99% of variance depending on the plot. This indicates that PCA does not give additional information with this set of data. I recommend to delete this part.

Au: Authors are in accordance with delete part concern PCA analysis and in the new version it has been deleted. Thank you.

A new section referred to Sensory analysis (aroma and flavour) has been included in the new version of article.

Re: Additional remarks

-       Abstract. Lines 16 and Line 20 report the same information.

Au: The summary has been rewritten.

-       Paragraph 3 should be titled “Results and Discussion”.

Au: Paragraph 3 has been corrected.

-       Line 138-140. The n3/n6 ratio is not defined and is not shown in Table 2. Therefore, related comments are not pertinent unless the ratio is shown.

Au: The n6/n3 ratio is now shown in the new Table 4.

-       Line 178. Short-, medium-, and long-chain fatty acids shall be defined.

Au: Short, medium and long chain fatty acids have been defined (lines 260-261 of the new version).

Short-chain fatty acids = C4:0-C6:0

Medium-chain FA = C8:0-C12:0

Long-chain FA = ≥ C14:0. C14:0+C16:0+C18:0+MUFA (C14:1+C16:1+C18:1)+PUFA (C18:2+C18:3).

 

Reviewer 3 Report

1. Title. It should be re-consider In lines 44-45 the authors say: "Valdeón cheese is a traditional blue-veined cheese made in the province of León 44 (Spain) from raw or pasteurized cow's milk or a mixture of cow's, sheep's and/or goat's milk ..." So, in the title and text, the mixture of cow's and goat's milk (90:10, respectively) should be specified. And, Why 90:10 ratio, by regulation of PGI? or What?

2. Line 16. The authors say "However, pasteurization of the milk did not influence the FFA profile ..." However, in the next sentences mentioned the effect of pasteurization on the level of FFA. There is a discrepancy here.

3. Figure 1. Why did you produce 4 batches of raw milk cheese? Other are 2 batches of each season. And, Why not a season effect in raw milk cheese's FFA?

4. Line 110. Please write the statistical method in correct order. What was your treatment effect, random effect, etc.

5. Table 2. None of data for each season; however, you give the statistical evaluation for season (S) at the right side of table. Please give the data for each season and discuss in the text.

6. Table 2. Please give the total FFAs for P and R cheeses (as meant and SD values) at the bottom line of the table. You're already discuss in the text. 

7. Figure 2. Please explain the high SD values of the total FFAs. Example, SD for total FFAs in summer (at 90 d) ranging from ca. 3000 to 12000 mg/100 g. What means of the huge wide range?

8. Please combine the data of Figs 2 and 3  in Table 2, FOR: easier interpretation and comparison, and also save page.

9. Is it necessary to give the Figs 4-6. 

10. Tavle 3. Delete please and give a PCA bi-plot to explain your treatment effects. Figs 7-8 not acceptable, what means of 99.1% PC1 on Fig 7. Is it meaningful? Instead of three figs on PCA, you can give one bi-plot PCA and discuss the effect of season, pasteurization and ripening time on the same plot. 

11. Lines 287-289. "Regardless of the effect studied oleic, palmitic and, to a lesser 287 extent, butyric and myristic acids were the FFAs that showed the best correlation with 288 the variables (ripening time, season and raw or pasteurized milk)" What is correlation coefficient for each factor? Please discuss only your data, even if statistical tests.

Author Response

Manuscript ID dairy-2115910

 

Firstly, we should like to thank the reviewer for their advice and suggestions, as well as for the corrections of the manuscript, which, without any doubt, will aid in improving its quality.

Reviewer 3

Au: Firstly, we should like to thank the reviewer for their advice and suggestions, as well as for the corrections of the manuscript, which, without any doubt, will aid in improving its quality.

 

Comments and Suggestions for Authors

Re: 1. Title. It should be re-consider In lines 44-45 the authors say: "Valdeón cheese is a traditional blue-veined cheese made in the province of León 44 (Spain) from raw or pasteurized cow's milk or a mixture of cow's, sheep's and/or goat's milk ..." So, in the title and text, the mixture of cow's and goat's milk (90:10, respectively) should be specified. And, Why 90:10 ratio, by regulation of PGI? or What?

Au: The indication regarding the type of milk used has been included in the title but the proportion used is not indicated as this may vary, there is no specific proportion required by the PGI Queso de Valdeón.

The Regulatory Board of PGI 'Valdeón cheese' provides for the use of cow's and/or goat's and/or sheep's milk for the production of this variety. The proportion of mixed cow's and goat's milk indicated is that normally used by the industry to make the cheeses. The cheeses were all made by the PGI 'queso de Valdeón' cheese factory.

Re: 2. Line 16. The authors say "However, pasteurization of the milk did not influence the FFA profile ..." However, in the next sentences mentioned the effect of pasteurization on the level of FFA. There is a discrepancy here.

Au: The authors want to indicate that pasteurization influenced the FFA concentration but not the FFA profile, i.e. the relative proportion of each free fatty acid.

Re: 3. Figure 1. Why did you produce 4 batches of raw milk cheese? Other are 2 batches of each season. And, Why not a season effect in raw milk cheese's FFA?

Au: 48 cheeses were produced with pasteurized milk, two batches per station (8 batches) and one cheese per sampling point (6). The largest production of cheese by the industry is with pasteurized milk. On the other hand, the production of cheese with raw milk is occasional, mainly in spring-summer. Therefore, 4 batches of these cheeses with raw milk x 6 sampling points (24 cheeses) were collected without taking into account the effect of the season.

Re: 4. Line 110. Please write the statistical method in correct order. What was your treatment effect, random effect, etc.

Au: The variables ripening time, season and treatment were included in lines 129-131 of the new version.

Re: 5. Table 2. None of data for each season; however, you give the statistical evaluation for season (S) at the right side of table. Please give the data for each season and discuss in the text.

Au: Table 2, now Table 3 shows the mean FFA values for cheeses made from pasteurised milk in the different seasons and throughout the year. Statistical differences by seasonal effect and between raw and pasteurised milk cheeses throughout the ripening are included in column S. Differences in values between pasteurised and raw milk cheeses at each sampling point were also included after each value when there were significant differences.

Re: 6. Table 2. Please give the total FFAs for P and R cheeses (as meant and SD values) at the bottom line of the table. You're already discuss in the text. 

Au: The total FFA of P and R cheeses (as mean and SD values) have been included in the new table 4.

Re: 7. Figure 2. Please explain the high SD values of the total FFAs. Example, SD for total FFAs in summer (at 90 d) ranging from ca. 3000 to 12000 mg/100 g. What means of the huge wide range?

Au: The variability between cheeses in summer was reduced by expressing the values on dry matter rather than on cheese.

The deviations that can still be observed could be related to the temperature in the manufacturing room which can be high at some point and affect the enzyme activity. It should be noted that the cheeses remain in the room draining as they are not pressed.

Re: 8. Please combine the data of Figs 2 and 3 in Table 2, FOR: easier interpretation and comparison, and also save page.

Au: In our opinion, including more data in table 2 would result in a very data-heavy and extensive table; we believe that the figures and data explained in the new version of the article in more detail can now facilitate a better understanding. Total FFA for pasteurized and raw milk cheese are shown in table 4.

Re: 9. Is it necessary to give the Figs 4-6. 

Au: Figures 4-6 show the ratios of short, medium and long chain FFA to total FFA to indicate seasonal and treatment-dependent differences in FFA throughout maturation for the influence of the studied variables on the FFA profile.

Re: 10. Table 3. Delete please and give a PCA bi-plot to explain your treatment effects. Figs 7-8 not acceptable, what means of 99.1% PC1 on Fig 7. Is it meaningful? Instead of three figs on PCA, you can give one bi-plot PCA and discuss the effect of season, pasteurization and ripening time on the same plot. 

Au: We agree with the reviewer. Figs 7-8 are not acceptable 99.1% PC1 in Fig 7. Therefore, in line with the opinion of the reviewer and other reviewers, section 3.2 on principal component analysis (PCA) has been deleted.

Another new section 3.2 over sensory analysis has been included.

Re: 11. Lines 287-289. "Regardless of the effect studied oleic, palmitic and, to a lesser 287 extent, butyric and myristic acids were the FFAs that showed the best correlation with 288 the variables (ripening time, season and raw or pasteurized milk)" What is correlation coefficient for each factor? Please discuss only your data, even if statistical tests.

Au: This information had been derived from the results of the PCAs, so this information has also been removed from the new version of the article.

 

Round 2

Reviewer 2 Report

The reviewer acknowledge that the manuscript has been improved. However, some criticisms remain in the revised version.

- From 9 figures and 3 tables in the first submission, Authors have changed to 7 figures and 5 tables. The reviewer stresses once more that some of these are redundant. Figures 2 and 3 show the same data reported in Table 4, including the statistically significant differences. Therefore, the data can be fully discussed on the basis of Table 4. Same comment applies to Figure 7 that shows the data of Table 5. The reviewer recommends to delete Figures 2, 3, and 7. These can be given as Supplementary material.

- Table 2. Data of protein and fat content are given on dry matter basis, salt on moisture. This approach is questionable. Cheese composition is usually given as cheese main fractions (moisture, protein, fat, salt). Taking into consideration its relevance in the present manuscript, it may be useful giving fat content also on dry matter basis. Beside these basic comments, it is unexpected that the fat content on DM basis significantly increases during ripening in both pasteurized and raw milk cheeses, while the protein content remains unchanged (not significant differences). What happened there? There are also doubts on single cheese composition: e.g. cheese from raw milk at 2 days of ripening: 36.86 (protein) + 48.84 (fat)= 86.7 g/100g DM. Assuming that salt is 3-4%, about 10% of DM is missed. The standard deviations of the data do not explain their inconsistency. The authors should carefully check data of Table 2 and adopt more useful expression.

- Lines 139, 141, 144. Table 1 shall be changed into Table 2. Please, check through the whole manuscript.

- Conclusions. Lines 355-356. It is stated that raw milk cheeses “were better assessed in the sensory analysis than cheeses made with pasteurized milk”. However, based on scores shown in Table 5, this is not true because no significant differences were obtained. The same incorrect thought is in the Abstract (Lines 20-21). Please, change both statements. Furthermore, in the discussion of sensory analysis, scores are discussed in terms of “higher” or “lower” without a reference to the best aroma and taste for the studied type of cheese. How do readers with no familiarity with Valdeon cheese know whether high spiciness, persistency, lactic aroma, etc. are positive traits? Please, comment on that.

Author Response

Manuscript ID dairy-2115910

Firstly, we should like to thank the reviewer for their advice and suggestions, as well as for the corrections of the manuscript, which, without any doubt, will aid in improving its quality.

Author’s replay to the Reviewer 2:

Re: From 9 figures and 3 tables in the first submission, Authors have changed to 7 figures and 5 tables. The reviewer stresses once more that some of these are redundant. Figures 2 and 3 show the same data reported in Table 4, including the statistically significant differences. Therefore, the data can be fully discussed on the basis of Table 4. Same comment applies to Figure 7 that shows the data of Table 5. The reviewer recommends to delete Figures 2, 3, and 7. These can be given as Supplementary material.

Au: The reviewer is correct and, as suggested, the authors have deleted Figures 2, 3 and 7 to avoid redundancy. This information is provided as supplementary material. Some of the information relating to figure 3 has been included in lines 213 to 226 and that relating to figure 2 in lines 247 to 252.

Re: - Table 2. Data of protein and fat content are given on dry matter basis, salt on moisture. This approach is questionable. Cheese composition is usually given as cheese main fractions (moisture, protein, fat, salt). Taking into consideration its relevance in the present manuscript, it may be useful giving fat content also on dry matter basis. Beside these basic comments, it is unexpected that the fat content on DM basis significantly increases during ripening in both pasteurized and raw milk cheeses, while the protein content remains unchanged (not significant differences). What happened there? There are also doubts on single cheese composition: e.g. cheese from raw milk at 2 days of ripening: 36.86 (protein) + 48.84 (fat)= 86.7 g/100g DM. Assuming that salt is 3-4%, about 10% of DM is missed. The standard deviations of the data do not explain their inconsistency. The authors should carefully check data of Table 2 and adopt more useful expression.

Au: The composition of the cheese has been expressed in dry matter because the intention is to show its evolution during ripening, thus avoiding, to a certain extent, the fluctuations due to the increase in dry matter during ripening. It is often expressed in this way when referring to evolution.

To explain why the fat content on dry matter increases significantly during ripening in both pasteurized and raw milk cheeses, while the protein content remains unchanged (non-significant differences), we have to take into account that 2 batches were made per season, thus obtaining 8 cheeses at each sampling point.

The variability observed for the % fat on dry matter, for example after 2 days in pasteurized milk cheeses, is indicated by values ranging from 56% to 52%, depending on the batches and replicates, with the % fat generally higher in the winter batches. There were also variations in dry matter. The lowest values of fat on dry matter were obtained at 2 and 15 days of ripening and set the starting point for increasing the fat on dry matter. It should be borne in mind that these cheeses are not pressed but drained under their own weight, which in our opinion could lead to greater moisture losses during sampling, particularly in cheeses at the beginning of ripening. This would result in slightly higher dry matter values than expected and would influence the unexpected increase in fat content over dry matter during ripening.

If the composition of each batch at a given time, e.g. at two days of ripening, is summed individually, it is close to 100, bearing in mind that it is also necessary to add the % lactose and D- and L-lactic acid, which are in fact higher at the beginning of ripening.

The determination of lactose and D- and L-lactic acid have been included in lines 80-82 of the Material and methods section and in lines 150-159 of the new version. Table 2 of the new version now also shows the lactose and D- and L-lactic acid contents expressed as a percentage of dry matter.

Re: - Lines 139, 141, 144. Table 1 shall be changed into Table 2. Please, check through the whole manuscript.

Au: Thank you. This has been corrected on lines 142, 144 and 147 of the new version.

Re: - Conclusions. Lines 355-356. It is stated that raw milk cheeses “were better assessed in the sensory analysis than cheeses made with pasteurized milk”. However, based on scores shown in Table 5, this is not true because no significant differences were obtained. The same incorrect thought is in the Abstract (Lines 20-21). Please, change both statements. Furthermore, in the discussion of sensory analysis, scores are discussed in terms of “higher” or “lower” without a reference to the best aroma and taste for the studied type of cheese. How do readers with no familiarity with Valdeón cheese know whether high spiciness, persistency, lactic aroma, etc. are positive traits? Please, comment on that.

Au: Although it was stated that the differences observed were not significant, it is true that such a statement may lead to misinterpretation. Therefore, as suggested by the reviewer, this information has been modified to indicate that there were no significant differences in the sensory analysis parameters between raw and pasteurized milk cheeses (lines 352-353 in the new version). The same information has been modified in the Abstract (lines 21-22 in the new version).

The scale of values for each sensory attribute was set at 1, e.g. non-lactic, non-spicy or non-sweet, to give an example, while 7 would be extremely lactic, extremely spicy or extremely sweet. The following rating scale for sensory attributes (1- no aroma or taste perception, 4 - moderate perception and 7 – very strong perception) was indicated on lines 128-129 of the new version and in the foot of Table 5. On the other hand, the 20 tasters were trained in the optimal characteristics of Valdeón cheese (lines 123-124).