Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors describe an interesting comparison of the effectiveness of vegetable-derived rennets for cheese making. The manuscript fits the scope of the journal and requires some revision before its acceptance for publication.
Introduction
Line 88, when they are used for
Line 100, latex, and papaya extracts were further investigated as …
Materials and Methods
Line 116, pasteurized milk is not a chemical, but a raw material; suggestion to change 2.1 to Chemicals and raw materials
Line 130 and 202, please indicate the centrifugation speed in g (rotor-independent)
Lines 140 and 141, triplicates and two independent experiments. – What was done exactly; each biological replicate (triplicate) was analysed twice?
Line 155, it should be explained that the slab gel was stained with Coomassie Blue.
Line 162, of fat
Line 207, so the samples were dissolved in 55 % B (starting conditions HPLC)?
Line 215, using an
Line 229, MUFA, PUFA, SFA should once be spelled out
Results
Line 250, lowest pH in Table 1 is 5.0
Line 251, last part of the sentence should be rephrased for clarity
Line 293, The latter
Lines 298-299, For this reason, the MCA was measured in the vegetable extracts of interest.
Line 333, in duplicate
Line 342, but the 72 g are indistinguishable with the respective STDEVs from the mean values of artichoke, cardoon, papaya and fig milky sap
Lines 375-376, this is contradicting Figure 4 (peak heights in the chromatogram).
Line 423, I am not sure about the meaning of the upper graph in Figure 6A; if these are ratios between 1 and 2, then they do not fit with what the lower graph, e.g. for cardoon there is about 20 % OFA and 80% DFA, so the ratio should 4 and not ~1.8? H/H (also in line 427) means height/height?
Lines 431-432, how do these value fit with Figure 6A lower graph?
Discussion
Line 449, were prepared
Line 475, 4 – 10 % higher according to Table 2
Author Response
Reviewer 1
Comments and Suggestions for Authors:
The authors describe an interesting comparison of the effectiveness of vegetable-derived rennets for cheese making. The manuscript fits the scope of the journal and requires some revision before its acceptance for publication.
Introduction
Line 88, when they are used for
Done.
Line 100, latex, and papaya extracts were further investigated as …
Done.
Materials and Methods
Line 116, pasteurized milk is not a chemical, but a raw material; suggestion to change 2.1 to Chemicals and raw materials
Done.
Line 130 and 202, please indicate the centrifugation speed in g (rotor-independent)
Done.
Lines 140 and 141, triplicates and two independent experiments. – What was done exactly; each biological replicate (triplicate) was analysed twice?
Done. We apologize for the error. We performed two measurements, starting from two independent preparations.
Line 155, it should be explained that the slab gel was stained with Coomassie Blue.
Done. We stained the gel by using Coomassie brilliant blue, and we added this explanation in the text (line 155).
Line 162, of fat
Done.
Line 207, so the samples were dissolved in 55 % B (starting conditions HPLC)?
No, the samples were dissolved in the most suitable solvent, which was n-hexane (LC-MS grade), to provide the complete dissolution of all the constituents.
Line 215, using an
Done.
Line 229, MUFA, PUFA, SFA should once be spelled out
Done.
Results
Line 250, lowest pH in Table 1 is 5.0
Done. We apologize because in the text we reported a wrong value: the correct value is 5.0 as reported in Table 1.
Line 251, last part of the sentence should be rephrased for clarity
Done. We have modified this sentence with the aim of clarifying that the pH values are within the range observed in curd.
Line 293, The latter
Done.
Lines 298-299, For this reason, the MCA was measured in the vegetable extracts of interest.
Done, accepted.
Line 333, in duplicate
Done.
Line 342, but the 72 g are indistinguishable with the respective STDEVs (deviazione standard) from the mean values of artichoke, cardoon, papaya and fig milky sap
Done. We rewritten it, indicating that the values are comparable with calf rennet (line 346).
Lines 375-376, this is contradicting Figure 4 (peak heights in the chromatogram).
It is not contradictory. In fact, the most intense peak was attributed to oleic acid (18:1), which is a MUFA and not a PUFA. As it can be observed in the chromatogram, at 3.5 min there is a co-elution of a-linolenic acid (18:3) and myristic acid (14:0). Thus, the peak intensity reflects both compounds. The occurrence of linoleic and a-linolenic acids has been further explained in Figure 5.
Line 423, I am not sure about the meaning of the upper graph in Figure 6A; if these are ratios between 1 and 2, then they do not fit with what the lower graph, e.g. for cardoon there is about 20 % OFA and 80% DFA, so the ratio should 4 and not ~1.8? H/H (also in line 427) means height/height?
The H/H ratio is the ratio of hypocholesterolemic and hypercholesterolemic fatty acids and was calculated according to the fatty acid composition using the following formula:
H/H = (C18:1n-9 + C18:2n-6 + C18:3n-3)/(C12:0 + C14:0 + C16:0)
The reference was reported in Materials and Methods section. The figure caption has been changed to avoid a misunderstanding.
Lines 431-432, how do these value fit with Figure 6A lower graph?
The OFA and DFA quality indices are calculated based on the following formulas:
DFA = UFA + C18:0
OFA = C12:0 + C14:0 + C16:0
Thus, they are not directly related to the content of linoleic and a-linolenic acids. The statement at lines 431-432 fit with Figure 5A.
Discussion
Line 449, were prepared
Done. Accepted.
Line 475, 4 – 10 % higher according to Table 2
We reported the percentage ratio of values measured for vegetables-like rennet respect to the value measured with calf rennet, and the range is between 10 to 30% higher. We modified this sentence to better explain the concept.
Reviewer 2 Report
Comments and Suggestions for AuthorsManuscript title: Exploring new vegetable-derived rennets for cheese-making.
Manuscript #: applsci-2888971
The manuscript is about the vegetable rennet obtained from various sources. The topic is good, and some research done is better, however the writing can be improved and clarified. Many of the things are not properly mentioned. In some places, the English needs to be improved. The following points will help improve the article.
General:
Please remove “s” from the rennets in the title -Exploring new vegetable-derived rennets for cheese-making
The paper is on vegetable-derived rennet but pineapple, fig, and some are fruits- please consider changing the title and text in the abstract and the whole manuscript.
Cheese paste is an unusual choice of a word, it should be cheese curd – please use this in the whole manuscript.
Many scientists now use milk-clotting enzymes instead of rennet when the extraction is not from a standard source.
Line 26: please give the scientific name of cardoon.
Line 43: the cheese number is low. It has more than 1800 different varieties. Please check.
Line 51: caseins- be specific κ-casein, as this is a rennet paper.
Line 60: it should be fig and then the scientific name.
Line 75: please check the casein spelling.
Lines 87-88: please rewrite the sentence- formation is wrong.
Line 107: please standardize the paper for British or US English- flavor vs flavour- please check the whole manuscript.
Line 115: and have can be replaced with having.
Lines 119-131: what are references for extractions for each process so the readers can benefit?
Line 174: 50 or 500 mL milk – comparison with Table 1 data
Line 175: why citric acid why not lactic acid? Citric acid may have a different approach in cheese making.
Line 234: The statistical analysis is very weak. How many replications of each treatment were done? How the treatments were compared.
Table 1: How many replications were done and, in each treatment, how many measurements were done? What is SU please explain at the bottom of the table.
Table 1: how color and odor were measured please explain in materials and method. If visual and smell, please say so.
The MCA varies too much so how do you compare them in cheese making? Did you use or adjust the amount to one single strength – it doesn’t seem the way it's written.
When you know the MCA, then you should standardize the additional rate for better comparison. Here the addition rate of each rennet is different, and you are comparing them (Figure 2)
Line 316-326: many of the things are repeats of materials and methods. Please consider deleting repeated things.
Table 2: why not use statistics when you have the data?
Line 357: humidity is different in the cheese industry – please consider using moisture as in the table.
Figure 4: Please explain (theor. m/z) at the bottom.
Figure 5: Some readers may not understand the photographs of mushrooms and others please write the name directly for clarity along with photographs of vegetables and fruit.
Line 421: latex fig- earlier only used fig- be consistent and consider rewriting this.
Line 430: the claim of omega3- and 6- seems exaggerated so please consider it rewriting. It is not so easy to develop omega fatty acids in milk using only rennet or rennet addition. Needs a strong scientific basis.
Line 503-513: question claim on omega fatty acids- consider modifying significantly.
Comments on the Quality of English LanguageNeeds improvement in certain areas.
Author Response
Reviewer 2
Comments and Suggestions for Authors:
The manuscript is about the vegetable rennet obtained from various sources. The topic is good, and some research done is better, however the writing can be improved and clarified. Many of the things are not properly mentioned. In some places, the English needs to be improved. The following points will help improve the article.
General:
Please remove “s” from the rennets in the title -Exploring new vegetable-derived rennets for cheese-making
We apologize. We corrected in the text “rennets” with “rennet”.
The paper is on vegetable-derived rennet but pineapple, fig, and some are fruits- please consider changing the title and text in the abstract and the whole manuscript.
We thank the reviewer for the suggestion. We modified the title in “….. new fruit- and vegetable-derived rennet ……”,and correct it in the whole text.
Cheese paste is an unusual choice of a word, it should be cheese curd – please use this in the whole manuscript.
Done, we modified as request from the reviewer.
Many scientists now use milk-clotting enzymes instead of rennet when the extraction is not from a standard source.
We prefer to use the term vegetable or fruit rennet to indicate that the activity is not related exclusively to the curd formation, but also refers to the ripening step, because this extract, as we have demonstrated, contains other non-proteolytic enzymes important for the cheese flavor formation.
Line 26: please give the scientific name of cardoon.
Done.
Line 43: the cheese number is low. It has more than 1800 different varieties. Please check.
Done. We checked it, and modified in “there are about 2000 different types of artisanal and industrial cheeses in the world, depending also from the milk origin”.
Line 51: caseins- be specific κ-casein, as this is a rennet paper.
Done.
Line 60: it should be fig and then the scientific name.
Done.
Line 75: please check the casein spelling.
Done.
Lines 87-88: please rewrite the sentence- formation is wrong.
Done. We rewrite the sentence to better explain the concept (lines 89-91).
Line 107: please standardize the paper for British or US English- flavor vs flavour- please check the whole manuscript.
We apologize for the double indication of flavor in the text. We reported in the whole text “flavor”.
Line 115: and have can be replaced with having.
Done.
Lines 119-131: what are references for extractions for each process so the readers can benefit?
Done. We followed a procedure described by Liburdi et al. (reference n. 4) with some modifications that are reported in the text (lines 131-135).
Line 174: 50 or 500 mL milk – comparison with Table 1 data
As reported in Materials, the milk clotting activity was measured starting from 10 mL of milk; mini-curd making was preliminarily tested by using 50 mL of milk and then the final analysis was done by using 500 mL of milk. In table 1 are summarized these results. In the capture of Table 1 we have specified it.
Line 175: why citric acid why not lactic acid? Citric acid may have a different approach in cheese making.
The use of citric acid is accepted in laboratory studies that concern cheeses; we have previous experiences in the use of citric acid in lab cheese making.
Line 234: The statistical analysis is very weak. How many replications of each treatment were done? How the treatments were compared.
As reported in the section 2.9 Statistical analysis (line 240), the measurements were in triplicate.
Table 1: How many replications were done and, in each treatment, how many measurements were done? What is SU please explain at the bottom of the table.
Done.
Table 1: how color and odor were measured please explain in materials and method. If visual and smell, please say so.
Done. At line 135-136 in M&M, we added the evaluation of these two parameters.
The MCA varies too much so how do you compare them in cheese making? Did you use or adjust the amount to one single strength – it doesn’t seem the way it's written.
No, we have not changed the amount of the different vegetable or fruit rennet, because we have standardized the extraction step by using the same amount of dried material in the same amount of water, and for the same time of extraction. This was done because varying the amount of extract, also changes the other components present in the extract and, more importantly, the amount of proteases that could give a different pattern of k-casein digestion (see figure 1), negatively altering the flavor of the final product.
When you know the MCA, then you should standardize the additional rate for better comparison. Here the addition rate of each rennet is different, and you are comparing them (Figure 2)
As shown in figure 1, the level of proteolytic activity of the extracts is not comparable with the milk clotting activity; in fact the proteolytic activity for artichoke and cardoon is double compared to calf rennet but their MCA value is 10-fold lower to that of calf rennet, because the proteases present in the extract and in calf rennet have different specificities. Therefore increasing the amount of extracts to increase the MCA rate would cause a strong hydrolysis of milk proteins, resulting in the production of small peptides, which they would give the cheese a bitter taste. As mentioned in the previous point, this is the reason why we have standardized the extraction method.
Line 316-326: many of the things are repeats of materials and methods. Please consider deleting repeated things.
Done. We delete the text from line 329 to 333.
Table 2: why not use statistics when you have the data?
We agreed, in fact the data reported in Table 2 are expressed as %. We specified it at line 361.
Line 357: humidity is different in the cheese industry – please consider using moisture as in the table.
We agree with the reviewer. We modified it.
Figure 4: Please explain (theor. m/z) at the bottom.
The explanation has been added in the Figure caption.
Figure 5: Some readers may not understand the photographs of mushrooms and others please write the name directly for clarity along with photographs of vegetables and fruit.
An explanation has been added in the Figure caption, as suggested.
Line 421: latex fig- earlier only used fig- be consistent and consider rewriting this.
Done. Accepted.
Line 430: the claim of omega3- and 6- seems exaggerated so please consider it rewriting. It is not so easy to develop omega fatty acids in milk using only rennet or rennet addition. Needs a strong scientific basis.
In the text, the authors’ aim was not to demonstrate the development of fatty acids in milk but to highlight that the use of vegetable rennet could also enhance the lipid profile in the final products. The n6/n3 ratio mentioned at line 430 and the following lines are derived from the amounts calculated in each sample based on the calibration curve. It is reasonable to assume that the FFA content in the plant matrix used in the cheese-making process is responsible for some variation in their profile in the final products.
Line 503-513: question claim on omega fatty acids- consider modifying significantly.
Done. We modify this part (line 516-527).
Comments on the Quality of English Language: Needs improvement in certain areas.
Done. We asked our English editing assistants, Bernard Loeffer and Francesca Varrone, to revise the manuscript. For this reason, we inserted both in the “Acknowledges” section. Dr Loeffer and Dr Varrone agreed to be in the Acknowledges section.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsStatistical analysis: Taking one treatment and analyzing it three times is not a replication. Its repetition. For replication, the authors should have taken 3 different lots of milk and analyzed each 3 times-This is more of a replication.
The rest of the decision lies with the editor.
Author Response
"Statistical analysis: Taking one treatment and analyzing it three times is not a replication. Its repetition. For replication, the authors should have taken 3 different lots of milk and analyzed each 3 times-This is more of a replication.”
Done.
We rewritten the paragraph 2.9 (page 5, lines 242-244) to better explain how we conducted the analysis.
In the paragraph 2.7, (page 4, line 197) is reported that cheeses analysis was in triplicate. To better explain this point we specify it by adding “three independent replicates”.
In other two points we refer to the statistical analysis:
-In paragraph 2.3 (page 3, lines 144-146) is clearly reported that were made two independent preparations of extract, and assays were carried out in duplicate: “Assays were carried out in duplicate, and the results were expressed as the means of two independent preparations.”
-In the figure 3 legend is clearly reported that the mini-curd preparation was made in duplicate (page 8, line 339).
