The Influence of Binding of Selected Mycotoxin Deactivators and Aflatoxin M1 on the Content of Selected Micronutrients in Milk
, Ivan Jurak 2, Dario Lasic 1, Ksenija Markov 3, Zeljko Jakopovic 3 and Jadranka Frece 3Round 1
Reviewer 1 Report
The article contributes with concise conclusions regarding the use of ATM1, that is an important alternative for treating contaminated milk. A novel and very interesting contribution is establishing the length of binding at which the nutritional composition of milk is not affected.
Author Response
Dear reviewer,
On behalf of all the co-authors, I would like to sincerely thank you for the effort and time you spent reviewing our work. The review we received was very thorough, extremely dedicated, professionally written, and completely focused on improving the article itself.
We tried to extract every single remark from your review and respond to them. As a rule, wherever we could, and it concerns almost all suggestions, we accepted and changed what was suggested. We hope that you will be satisfied with the changes made and the effort we have invested in the work to ultimately receive the green light from all reviewers involved.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
GENERAL OVERVIEW: the authors presented a manuscript regarding the influence of mycotoxin deactivator’s binding with aflatoxin M1 in milk micronutrient composition. The aflatoxin contamination in milk is a global human health concern, mainly for children. The article is easy to follow, but flaws must be corrected. The significant corrections are described below:
SPECIFIC COMMENTS:
Abstract: if possible, I suggest including some numerical values and p-values. These abstract results are too descriptive, and the numbers may clarify the understanding.
Introduction: The authors must reduce this section without loss of quality.
L.134: Please state the hypothesis of this study before or after the objectives.
L.134-141: Remove this part. This is a repetition of abstract, as well as Material and Methods.
Material and Methods: This section could be better if the authors provided subsections.
L.143: Please include the selection criteria for choosing this Croatian producer.
L.179: This nonparametric method is not more robust than longitudinal methods. Also, the current techniques allow you to correct the homoscedasticity of the variance. Please consider changing the model or justifying with references why this limited class of nonparametric methods is used.
Table 1: I’m afraid this number of replicates is too small. Please provide references to corroborate this number.
- Results: The tables and figures do not need to be introduced in the text. Go directly to the point.
- Figures 1 and 2: If you use the ggplot2 on R software, you may perform better graphics than those. Please improve the graphical quality aiming for the final publication.
- The conclusions are the repetition of Results and Discussion. This section must be reduced without loss of quality.
- I would be pleased if the authors would make the algorithms (analysis script) and the database available to the scientific community, if possible. In this case, I don’t think a prior authorisation to the authors is necessary.
- The references are different from the Processes’ template. Please adjust it to the Instruction for Authors.
Author Response
Dear reviewer,
On behalf of all the co-authors, I would like to sincerely thank you for the effort and time you spent reviewing our work. The review we received was very thorough, extremely dedicated, professionally written, and completely focused on improving the article itself.
We tried to extract every single remark from your review and respond to them. As a rule, wherever we could, and it concerns almost all suggestions, we accepted and changed what was suggested. We hope that you will be satisfied with the changes made and the effort we have invested in the work to ultimately receive the green light from all reviewers involved.
Author Response File: Author Response.pdf
Reviewer 3 Report
The following comments were made in order to improve the manuscript.
a) Is necessary to improve English style throughout the whole manuscript.
b) About Introduction: special attention is required, contains many grammatical errors and information is repeated in different sections. The first section on minerals is extensive and unnecessary, it can be summarized.
c) It is necessary to Improve the approach of the general objective.
d) About the materials and methods section:
• Statistical Analysis:
• The experimental design used and number of repetitions per treatment it is necessary to be described.
• Also, it is necessary to express the linear model fitted.
• The random effects included in the model, as well as the covariance structure used, are required to be described.
• it must specifically include the experimental design and the statistical analysis used, as well as the number of samples measured during the 21 days.
b) About Results and discussion section:
• Improving the description of the tables is required. include Type of data presented, scale, number of observations, etc.
• Comparison of the results obtained with those reported by other authors is required.
Comments for author File: Comments.pdf
Author Response
Dear reviewer,
On behalf of all the co-authors, I would like to sincerely thank you for the effort and time you spent reviewing our work. The review we received was very thorough, extremely dedicated, professionally written, and completely focused on improving the article itself.
We tried to extract every single remark from your review and respond to them. As a rule, wherever we could, and it concerns almost all suggestions, we accepted and changed what was suggested. We hope that you will be satisfied with the changes made and the effort we have invested in the work to ultimately receive the green light from all reviewers involved.
Author Response File: Author Response.pdf
Reviewer 4 Report
The study aimed to examine the impact of selected mycotoxin deactivators (beta-glucan from yeast and oats, and live and dead lactic acid bacteria) on the nutritional composition of milk after binding to intentionally added aflatoxin M1 to milk. This should be the only aim as the statistics is always part that decides whether the impact is significant or not. In general, the submission is too wordy and could be shortened substantially. For example, first two pages in the introduction are very general knowledge and could be summarized in a few sentences. Everybody knows that milk is nutritious, no need to dilute the importance of the study by this information. The part on the aflatoxin is appropriate. Now where the problems start is the materials and methods. First the authors stated that the concentration of aflatoxin was 0.1 ug/kg (line 134) then later it was stated to be 0.5 ug/kg. The even bigger problem is the description of the sample treatment. First of all, the numbers in Table 2 appear fairly low in comparison to the numbers in literature – why not analysing the milk prior to addition? While the authors may state that “0” was used, it is never 0 time as the deactivators needed to be mixed thus the binding have already done their job (Table 4), thus the time has already passed. For the true assessment, the control sample is a must here. Another, and probably the main issue, is how the samples were analyzed. Were the deactivators removed? If they did how? If not, how come that the mineral levels were reduced? So at the moment, here’s a brief description of the method as outlined in the paper – deactivators were added to milk, and samples were taken at assigned time intervals. Such samples were digested and assessed by ICP – where did the minerals go then if the complex between deactivators and aflatoxin was not removed from the milk? Also how did you measure bound aflatoxin? I think you are missing a big chunk in your materials and methods and could easily remove Table 1 as these are standard parameters. Not sure why you decided to use nonparametric data analysis since this can be analyzed as a repeated measurement in time using a general linear model. The paper requires a good overhaul so it can be understandable. It also appears to be heavily focused on statistics instead on science.
Author Response
Dear reviewer,
On behalf of all the co-authors, I would like to sincerely thank you for the effort and time you spent reviewing our work. The review we received was very thorough, extremely dedicated, professionally written, and completely focused on improving the article itself.
We tried to extract every single remark from your review and respond to them. As a rule, wherever we could, and it concerns almost all suggestions, we accepted and changed what was suggested. We hope that you will be satisfied with the changes made and the effort we have invested in the work to ultimately receive the green light from all reviewers involved.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Thank you for your attention regarding my suggestions. The authors followed all my considerations. Thus, I recommend the acceptance of this manuscript.
Author Response
Dear reviewer
Thank you for the time and effort you invested in reviewing the changes made to the paper - We thank you for accepting all our changes and the explanations we provided and for not noticing in the second round of reviews that you had additional requests for the manuscript.
With respect
Authors of the paper
Reviewer 3 Report
The authors responded appropriately to the comments made to the manuscript.
Author Response
Dear reviewer
Thank you for the time and effort you invested in reviewing the changes made to the paper - We thank you for accepting all our changes and the explanations we provided and for not noticing in the second round of reviews that you had additional requests for the manuscript.
With respect
Authors of the paper
Reviewer 4 Report
I believe the author's didn't understand the request regarding the controls. For the proper assessment, they MUST include additional column in table 2 for example which shows the concentration of minerals BEFORE addition of deactivators. Then they need to include this level into statistical analysis. At present the true comparison is not possible.
Author Response
Dear reviewer
Thank you for the second review of our article.
After the changes in the work in which we answered all the requests of all 4 reviewers, in the second round we received two of your remarks, which we tried to answer.
The article was re-sent to a professional translator who corrected the omissions you pointed out, and everything was entered into the work and the changes entered in blue are visible.
The second part of the remarks was related to the way of processing and displaying the results, in which you suggested adding an additional column to table two and using difference statistics (t-test) to compare the initial values ​​with those after the analytical procedure. In accordance with the instructions of the reviewers, in the first round of the review, a very complex statistical processing of the results was carried out in such a way that the change in the composition of micronutrients was monitored at certain time points in order to determine the most adequate time and the most adequate method, and the results were interpreted in this sense. The initial values ​​for all samples were the same because they were initial samples from the same initial package, and the measured values ​​were entered in the text of the paper in lines 166 - onwards.
We could not apply the approach that you suggest afterward because the complete processing of the results presented in the paper would have taken a different form, which deviates from our conception of the presentation of the results, which, I repeat, was requested by the other two reviewers. Since we could not do what you asked us to do, we put the same in the limitations of the paper and separated it at the end of the paper, as an incentive for future research.
We hope that the explanations provided to you will be acceptable so that the work can proceed further in the process. In any case, we respect your approach and it is possible, but our processing and interpretation of the results were carried out in a more complex way, although the conclusions drawn would probably be the same.
Changes made in the text:
Lines 166-167:
All micronutrients were analyzed and quantified in milk in the same manner and under the same conditions before the addition of mycotoxin deactivators. Before adding aflatoxin AF M1 to milk for human consumption with 2.8% milk fat, the amounts of micronutrients were determined and the following values were obtained: Na 302.97 mg/l; Mg 75.16 mg/l; K 1188.86 mg/l; and Ca 864.7 mg/l, which is within acceptable and expected values for milk on the Croatian market. The stated values are mean values based on 5 measurements of micronutrients’ composition using the same initial sample. All the analyses in the further experiments were carried out in relation to the same initial milk sample, which is the reason for the basic values of micronutrients being considered as the same in all the conducted experiments. The design of the experiment and the analyses and the subsequent statistical processing of the data were carried out so as to monitor and compare the changes between the micronutrients' composition at 4 time points, and not in relation to the initial sample.
New chapter upon reviewers' suggestions and requests:
Limitations of the study
The limitation of the study can be the fact that all micronutrients were analyzed and quantified in milk in the same manner and under the same conditions before adding the mycotoxin deactivators. The following amounts of micronutrients and values ​​were obtained before adding aflatoxin AF M1 to milk for human consumption with 2.8% milk fat: At 302.97 mg/l, Mg 75.16 mg/l, K 1188.86 mg/l, and Ca 864.7 mg/l, which is within the acceptable and expected values ​​for milk on the Croatian market. The stated values ​​are mean values ​​based on 5 measurements of micronutrients’ composition using the same initial sample. All the analyses in the further experiments were carried out in relation to the same initial milk sample, which is the reason for the basic values ​​of micronutrients being considered the same in all the conducted experiments. The design of the experiment and the analyses and the subsequent statistical processing of the data was carried out so as to monitor and compare the changes between the micronutrients' composition at 4 time points, and not in relation to the initial sample.
Round 3
Reviewer 4 Report
The request is basically related to the fundamental statistical approach - inclusion of the controls. If this is ignored, regardless what others reviewers requested, this is NOT a limitation, this is a fundamental flaw. It appears that the data is available then it has to be included, otherwise this can't be published. The inclusion will also provide the paper with additional dimension and it is actually disappointing that the authors keep avoiding to address the fundamental request.
Author Response
Dear reviewer,
Respected reviewer,
We have taken time since receiving your last comment on our work (28.10. 2022.) and we have once again passed through all the changes that we successively made through three rounds of the review process.
We want to point out once again that we received comments and advice from 4 different reviewers and for three reviewers we fully answered all the requirements and they approved the work. We have also responded positively to all your remarks and fulfilled almost everything you asked for except one. Regarding your only remaining request, which you even said in the last review that "we don't want to act on it even though there are data", I clearly emphasize that it is not about any stubbornness or insistence that you may not be right from your point of view. Simply, we do not agree with that one remark of yours and from our point of view, but for us, it seems that it is not a matter of difference of opinion, because of which you should accuse us of not wanting to react to something. We, as the authors of the work, conducted the work, objectives, sampling, and analyses in accordance with the research purpose and the answers we were looking for. Maybe we could have done it differently at the start, but at the moment we have research and analysis, and interpretations that we fully stand behind.
It is your right as a reviewer to object to something, our right as authors is to say that we do not agree in that one single segment and explain our answer, and then let the editor judge the fate of the work. I am also the editor-in-chief of a scientific journal, and every day I am in a position to analyze the author's answers to the reviewers' requests and explanations that sometimes do not agree with the expectations of one of the reviewers. And that's normal, it's not a reason for malice or cynicism. It's just a process. It would be very strange indeed if we all thought the same way about everything. I hope that our answer, which we are attaching, will be acceptable to you and that we can get out of this situation in a win-win way. Attached is the answer to your request, and I sincerely thank you for all the other constructive remarks.
Reviewer: „The request is basically related to the fundamental statistical approach - inclusion of the controls. If this is ignored, regardless of what others reviewers requested, this is NOT a limitation, this is a fundamental flaw. It appears that the data is available then it has to be included, otherwise this can't be published. The inclusion will also provide the paper with additional dimension and it is actually disappointing that the authors keep avoiding addressing the fundamental request.“
Answer: Thank you for your comment. However, we kindly disagree with your assessment of the fundamentals. All experimental groups were drawn from the same milk sample. Before drawing experimental groups from that sample, the amounts of micronutrients were measured (5 times) and means were calculated and presented in the manuscript. From that sample, experimental groups were drawn and divided into smaller samples (observations). After adding mycotoxin deactivators samples were measured at 0, 2, 4, and 24 hours.
We disagree with your assessment that this was a fundamental flaw. We performed posttest-only control group experiment. The posttest-only control group design is a basic experimental design individual samples get assigned to one of the experimental groups, and then the outcome of interest is measured only after the intervention takes place in order to determine its effect. This is one of the most basic types of experimental design (1). Under the assumption that groups were similar at baseline (all groups were drawn from the exact same larger sample), this is a valid experiment. This type of experiment was chosen for its well-known benefit - decreasing unneeded testing. Its limitations, namely, potential baseline differences between groups are addressed by our above-mentioned assumption, though we absolutely acknowledge better control of baseline differences when the classic pretest-posttest experiment is performed. Also, we would like to point out that even that method does not completely remove potential pretesting bias. In our experiment, we were primarily concerned with the effect of mycotoxin deactivators on milk content across four-time points with the clear goal of finding the mycotoxin deactivator that has the least amount of influence on micronutrient content up to 24 hours.
We address this limitation in our manuscript and in the future, we will probably allocate more resources to pre-tests even when we believe that they are not entirely necessary if only to avoid future misunderstandings such as this.
(1) Mucherah, Winnie. (2018). The SAGE Encyclopedia of Educational Research, Measurement, and Evaluation Cross-Cultural Research.
Author Response File: Author Response.pdf
