Total and Free Fatty Acids Analysis in Milk and Dairy Fat
Round 1
Reviewer 1 Report
As in attached file
Comments for author File: 
Comments.pdf
Author Response
Point 1: “These columns consist of a 284 narrow bore tubing (0.1 to 0.3 mm internal diameter (i.d.) and 25 to 100 m length) of fused silica, the 285 inner wall of which is coated with the liquid phase [6]” IT IS SUPERFLUOUS…
Response 1: It has been deleted.
Point 2: Figure 2 → BETTER RESOLUTION.
Response 2: John Wiley and Sons has not provided the figure. We changed it improved by our means.
Point 3: Line 546: “MS” → IN THE FOLLOWING INFORMATION IT WILL BE USEFUL TO KNOW THE TYPE OF MS SPECTROMETER (SINGLE OR TRIPLE QUADRUPOLE, TOF, HIGH RESOLUTION )...
Response 3: Added in lines 547 and 548.
Point 4: Lines 549-566 → IT IS NOT USEFUL....THE REASERCHERS THAT WORK IN THIS FIELD KNOW VERY WELL THE MASS SPECTROMETRY FEATURES
Response 4: The paragraph has been shortened.
Point 5: References → THERE ARE SOME FORMATTING PROBLEM...YEAR IN BOLD BUT IN ANY CASE NO...OR OTHER LITTLE PROBLEMS
Response 5: All references have been reviewed and corrected.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The Review by Amores and Virto is interesting in its purposes and provide a useful survey of the chromatographic methods used for the general analysis of fatty acids in milk. In the opinion of this Reviewer, many section in the first part of Introduction are quite trivial and could be shortened or deleted.
Paragraph 4.1 is long, verbose and hard to follow. Probably, the information could be better summarized in a related Table. The section 4 renders the review unbalanced toward the chromatographic methods, which are undoubtedly the most important and widely used, but they require a series of complementary methods for a comprehensive analysis of milk fat and FA.
The manuscript should be throughout revised for unpleasant sentences, typos and formal mistakes, only some of which are listed below.
Minor and major specific issues
L 34. Minor amounts of glycolipids are also present in milk
L 34. Delete “l” after monoacylglycerols
L 36. TAG abbreviation introduced above should be used
L. 49 and elsewhere. Fatty acid abbreviation has been already introduced
L. 50 insert the year after Jensen
L 65. Establish
L. 95. Fractionation of lipid classes. The point “2” should be rephrased.
L 187. Please rephrase
L 215. Check the statement
L 271. Replace another with other
L 282. Probably form should be from
Figure 1. Please provide explanation why SCFA are significantly enriched in cheese if compared to milk
L 447 Maybe due to…
L 609. 13C NMR is a very effective method to detect and quantify milk fat SCFA. Butyric acid, for instance, could be monitored as a marker of milk fat using 13C NMR. The method also enables the determination of the regioisomeric distribution of FA which could be diagnostic for authenticity assessment. Please introduce this aspect.
It is important to underline the demand of adequate standard for the accurate (quantitative) analysis of FA in milk fat. The availability of a relevant reference standard at least for bovine milk fat should be indicated.
Some example of application of analysis of milk fat and FA in particular to real cases and for practical purposes should be at least briefly introduced.
MALDI mass spectrometry is a powerful (underestimated in my opinion) additional tools for the advanced analysis of both esterified and free FA, useful to many scopes.
Author Response
Point 1: The Review by Amores and Virto is interesting in its purposes and provide a useful survey of the chromatographic methods used for the general analysis of fatty acids in milk. In the opinion of this Reviewer, many section in the first part of Introduction are quite trivial and could be shortened or deleted.
Response 1: In the opinion of authors, the size of the introduction is correct. Nevertheless, some sentences have been deleted.
Point 2: Paragraph 4.1 is long, verbose and hard to follow. Probably, the information could be better summarized in a related Table. The section 4 renders the review unbalanced toward the chromatographic methods, which are undoubtedly the most important and widely used, but they require a series of complementary methods for a comprehensive analysis of milk fat and FA.
Response 2: Table 2 has been added and paragraph 4.1 shortened. A new point (point 6) summarizing complementary methods has been added.
Point 3: The manuscript should be throughout revised for unpleasant sentences, typos and formal mistakes, only some of which are listed below.
Minor and major specific issues
L 34. Minor amounts of glycolipids are also present in milk
L 34. Delete “l” after monoacylglycerols
L 36. TAG abbreviation introduced above should be used
L. 49 and elsewhere. Fatty acid abbreviation has been already introduced
L. 50 insert the year after Jensen
L 65. Establish
L. 95. Fractionation of lipid classes. The point “2” should be rephrased.
L 187. Please rephrase
L 215. Check the statement
L 271. Replace another with other
L 282. Probably form should be from
L 447 Maybe due to…
Response 3: The manuscript has been checked by a experienced English speaking colleague.
All mistakes found have been corrected.
Point 4: Figure 1. Please provide explanation why SCFA are significantly enriched in cheese if compared to milk.
Response 4: There was a mistake in the footnote. Both chromatograms corresponded to two different milk samples, not to a milk and a cheese elaborated with it.
Point 5: L 609. 13C NMR is a very effective method to detect and quantify milk fat SCFA. Butyric acid, for instance, could be monitored as a marker of milk fat using 13C NMR. The method also enables the determination of the regioisomeric distribution of FA which could be diagnostic for authenticity assessment. Please introduce this aspect.
Response 5: Applications of NMR in FA analysis have been reviewed in the new point 6.
Point 6: It is important to underline the demand of adequate standard for the accurate (quantitative) analysis of FA in milk fat. The availability of a relevant reference standard at least for bovine milk fat should be indicated.
Response 6: This issue has been addressed along the point 5.
Point 7: Some example of application of analysis of milk fat and FA in particular to real cases and for practical purposes should be at least briefly introduced.
Response 7: Some examples of application are mentioned in sentences of points 1 and 2. For example:
“…how milk fat changes according to different factors that influence its properties (animal species, breed, genotype, stage of lactation, feeding regime…)...” (L47-L48)
“…sensory and nutritional quality of milk and dairy fat are largely determined by its FA composition…” (L62-L63)
“…FFA have low flavor thresholds, especially short chain FFA (SCFFA) and provide the characteristic flavor and odor of many dairy products, particularly, the flavor of fermented dairy products, and especially of cheese [10,11]. However, elevated levels of SCFFA, especially C4:0, are also responsible for rancidity in milk and other dairy products. Rancid flavor generally becomes unacceptable to the consumer…” (L73-L77)
In the opinion of authors, a more extensive explanation of the results of these applications is beyond the scope of this review and it would extend it excessively.
Point 8: MALDI mass spectrometry is a powerful (underestimated in my opinion) additional tools for the advanced analysis of both esterified and free FA, useful to many scopes.
Response 8: Authors were not able to find any reference about the use of MALDI for these purposes, at least in milk and dairy fat analysis.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Even though the authors have been rather stingy to argument and motivate their changes, the review appears significantly improved and in my opinion it can be accepted for publication.
Fort what it is concerning the possibility of using the MALDI-MS techniques for the analysis of FA, there are more than a few examples of analysis of milk "esterified" FA (i. e. as acylglecerols). For example, see Picariello et al European Journal of Lipid Science and Technology 2007; Garcia JS et al., Food Chem 2012; Several papers by Calvano CD et al., Tsompa-Sosa DA et al European J Lipid Science and Technology 2018. MALDI has been used for the analysis of FFA as well (Ayorinde et al. 2000; Yu et al. Anal Biochem 2006), although as far I know it has not been applied to the analysis of milk lipids. But this could be a further perspective of the research, considered that the potentiality of the technique are largely underestimated, even for quantitative purposes (Ling et al J American Society Mass Spectrometry 2018).
For what it is concerning the use of MS in general there are several milestone papers that should be considered, such as Sokol E et al. European Journal of Lipid Science and Technology 2015,
Author Response
The subject of the review, as expressed in the title, is “Total and Free Fatty Acids analysis in milk and dairy fat”. Our purpose was to compile the principle methods used for the separation and quantification of total or free fatty acids present in milk and dairy products. Most of the FA present in milk fat are esterified in TAG or PL, and a variable amount is in the form of non-esterified FA (also called free fatty acids). This review deals with the methods used to extract FA from all lipid fractions (TFA) or from the FFA fraction, and to separate, identify and quantify all individual FA in both cases.
All references the reviewer mentions are about the analysis of lipid fractions, such as TAG or PL. These lipids fractions are made up of a multitude of individual molecular lipid species having different fatty acid moieties linked to a glycerol-backbone. In the referenced works, MS is used to identify lipid species based on their molecular composition (total carbon atoms and double bonds). They do not separate individual FA from these fractions neither identify and quantify them. Therefore, it is a different type of analysis. They provide different information about the composition of milk lipids. We agree that it is a very interesting information. Moreover, we want to thank the reviewer for all the references he/she has given us. Nevertheless, we believe that it is beyond the subject of our review.
It may be that in the text we have not made clear what we are talking about when we refer to "total fatty acids". To try to correct this, we have modified the sentence in the line 94, in an attempt to express it clearer than before.
