Effects of Phenyllactic Acid on Fermentation Parameters, Nitrogen Fractions and Bacterial Community of High-Moisture Stylo Silage
Round 1
Reviewer 1 Report
The study entitled: Effects of phenyllactic acid on fermentation parameters, nitro- gen fractions and bacterial community of high-moisture stylo silage, is a well designed and written study that can be manipulated further after responding to the following comments:
Please, through the introduction, please add some information on Stylo, such as scientific name, common chemical analysis, particularly protein content, its importance as a roughage in animal feeding worldwide.
Line 69: add information on the stylo used in the study, fore example the season of collection, the age of plant, the part used for fermentation, ….
Line 86: , where pH was measured by a glass …start with new paragraph (Start as following: The values of pH were……)
For bacterial community analysis: add reference for the procedure.
Statistical analysis: add more details on the model used for statistical analysis of data ( according two tables two way anova was used considering treatment, time, and interaction). Please, clarify in statistical analysis section.
Table 2: the interaction between time and treatment is not significant for pH. Please, revise and clearly show on which base the letters of significance were added, according to the D, T, or their interaction. Moreover, keep the letters of significance for the significant differences between means only. For example, do not use A, A, A if there is not significant difference between means. There are missed data for coliforem, revise. What ,-, means for the DM loss data????
Revise all tables according to the comments provided for table 2.
Conclusion: suggest a level and duration for fermintation of Stylo using PLA according to the results of your study.
Minor revision of English editing is required.
Author Response
First of all, thanks for your revised suggestions.
Please, through the introduction, please add some information on Stylo, such as scientific name, common chemical analysis, particularly protein content, its importance as a roughage in animal feeding worldwide.
R: We have added the related introduction of stylo as suggested.
Line 69: add information on the stylo used in the study, for example the season of collection, the age of plant, the part used for fermentation, ….
R: We have added the related introduction of stylo as suggested.
Line 86: , where pH was measured by a glass …start with new paragraph (Start as following: The values of pH were……)
R: We have revised the text as suggested.
For bacterial community analysis: add reference for the procedure.
R: We have added the references as suggested.
Statistical analysis: add more details on the model used for statistical analysis of data ( according two tables two way anova was used considering treatment, time, and interaction). Please, clarify in statistical analysis section.
R: We have added the model as suggested.
Table 2: the interaction between time and treatment is not significant for pH. Please, revise and clearly show on which base the letters of significance were added, according to the D, T, or their interaction. Moreover, keep the letters of significance for the significant differences between means only. For example, do not use A, A, A if there is not significant difference between means. There are missed data for coliforem, revise. What ,-, means for the DM loss data????
Revise all tables according to the comments provided for table 2.
R: We have revised the statistic analysis and cleared the mistakes as suggested.
Conclusion: suggest a level and duration for fermintation of Stylo using PLA according to the results of your study.
R: We have rewritten the conclusion to make it more clear as suggested.
Reviewer 2 Report
General comments
The manuscript submitted examined the effect of phenyllactic acid as a silage additive. The subject seems interesting and falls within the aims and scopes of the journal. However, there are concerns in relation to Materials and Methods, which I am not how to revamp the manuscript.
Firstly, it is not easy to accept the true, authentic silage quality prepared by the laboratory- scale described in this manuscript. Three hundred grams of fresh and chopped forage is only a handful, and it is again challenging to extrapolate the results into a larger scale of silage production. In the literature, over one- or two-liter size glass jars or much bigger size (i.e., 20 liters plastic containers) containers should be used in order to examine silage additives. Smaller-scale silage preparation is an easier way to start an experiment, but as a reviewer, it is not easy to accept the methodology of silage preparation.
Secondly, phenyllactic acid is employed as an “antimicrobial” agent, and I do not understand why an antimicrobial agent was applied for silage preparation. In the early era of silage making, an agent such as formic acid was applied to drop pH as quickly as possible and promote a dominant lactic acid bacteria fermentation environment. However, due to its complexity in silage making, formic acid has been replaced with a more human-friendly microorganism (lactic acid bacteria). Therefore, before its application, the safety issue should be discussed heavily.
Author Response
First of all, thanks for your revised suggestions.
Firstly, it is not easy to accept the true, authentic silage quality prepared by the laboratory- scale described in this manuscript. Three hundred grams of fresh and chopped forage is only a handful, and it is again challenging to extrapolate the results into a larger scale of silage production. In the literature, over one- or two-liter size glass jars or much bigger size (i.e., 20 liters plastic containers) containers should be used in order to examine silage additives. Smaller-scale silage preparation is an easier way to start an experiment, but as a reviewer, it is not easy to accept the methodology of silage preparation.
R: We admit that 300 gram bag silage is small in material size, and such bag silages might be not wholly consistent with the result of silo silage. As lab-level bag silage is an easy way to investigate the variation of silage fermentation and the function mechanism of silage additives, especially in a warm environment condition, there are still a lot of research studies using these similar equipment. We believe the results would get a good representativeness of the ensiling treatments.
Secondly, phenyllactic acid is employed as an “antimicrobial” agent, and I do not understand why an antimicrobial agent was applied for silage preparation. In the early era of silage making, an agent such as formic acid was applied to drop pH as quickly as possible and promote a dominant lactic acid bacteria fermentation environment. However, due to its complexity in silage making, formic acid has been replaced with a more human-friendly microorganism (lactic acid bacteria). Therefore, before its application, the safety issue should be discussed heavily.
R: As known, PLA can be produced with phenylalanine (a common amino acid in animals and plants) as raw material via LAB fermentation, such as Lactobacillus casei, Lactobacillus plantarum and Leuconostoc mesenteroides. Thus, it is believed that some LAB species may owe the ability to generate PLA naturally. The application of PLA or PLA-producing LAB in silage production might help to inhibit the proliferation of undesirable microorganisms in silage thus resulting in improved silage quality. Therefore, PLA is a promising antimicrobial agent for fermented foods and feeds in the perspective of safety and effectiveness, and the related functional LAB might have a well application in silage fermentation.
Reviewer 3 Report
General points about the manuscript: The manuscript brings interesting information regarding the utilization of phenyllactic acid on stylo silages. Authors evaluated 3 levels of phenyllactic acid, including control, on fermentation quality, nitrogen fractions and bacterial community of stylo silages. Additionally, authors collected samples after 3, 7, 14 and 30 days of fermentation period. The manuscript is very well written, with sufficient information in the different sections. However, I kindly pointed in this letter some issues that must be reviewed and fixed by the authors, or at least they should explain them better.
Specific considerations:
Keywords: For indexing reasons, use as keywords different words than those already mentioned in the title.
Insert references in the text according to Journal’s guideline.
L30: “during ensiling” or “during storage”, but not “during ensiling storage”.
Searching in literature, this reference sounds also very appropriate in this manuscript (https://doi.org/10.3389/fanim.2022.1080535). Please include if you think it’s convenient.
L39: …much effort has been made…
L50: Should “mesenteroides” be with lower case letter?
L62: …the present study aimed to investigate…
L63: style?
L65: Why only up to 30 days? Some of the parameters regarding silage fermentation quality and possibly bacterial communities still haven’t reached plateau.
L65: I’m not sure if “…were unsealed for samples collection…” is indeed very clear for the reader. Please consider revising.
L68: Please describe a bit more the raw material, for instance the growth stage, when and where it was harvested, fertilization, and so on.
L77: How come that bags were unsealed? Preservation of silage should be done under anaerobic conditions.
Section 2.2: Why some abbreviations are in bold? Is it a guideline from another journal? Please check and remove bold if that’s the case.
L93: Check use of parenthesis. At least one is missing.
Reference for NDF, ADF and ADL is missing. Please add.
L115: The statistical design allows both linear and quadratic evaluation for the PLA effect. Why didn’t you consider linear and quadratic effect of PLA?
L123: style?
L124: style?
L125: The number does not match with the number in the Table, but anyhow, do not repeat in the text those numbers already mentioned in the Table. Please check throughout the manuscript.
The first paragraph of Results section should be completely revised. The results presented in the Table must not be repeated in the text. The description of results should be done in a way to present the results, however not repeating the numbers in both Table and text. Please fix accordingly.
Table 2: Where is propionic acid?
L158: How can you say “linearly dose effect” if you evaluated treatment effect, instead of linear and quadratic effects? Please see my suggestion in the statistical evaluation section. Please consider including linear and quadratic effects, which might be even more important than the interaction DxT.
Title of Figure 1 is in different font sizes. The same in other Figures. Please fix.
L192: “after fermentation” or “after ensiling”, but not “after ensiling fermentation”.
L219: Scientific names should be in italic. Please check throughout.
L222: Revise “ensiling fermentation”. Check throughout the manuscript.
L239: Why are these abbreviations described if they are not in the Figure? Instead, add a description regarding the colour and size of the dots.
Figure 5: Are these correlations described in Material and Methods? Please include this description.
L268: “linearly”? Statistically that is not how you evaluate. Please consider evaluating the linear and quadratic effects of PLA on stylo silages.
L333: This reference is not in the reference list. Please add and check the conformity, if all references in text are in the reference list and vice versa.
L365: ‘Moreover’ is misspelled.
L392: “…bacteria in silage in that they…” might require revision.
L394: Add space after period.
L408: “…Pantoea abundance is negatively related to silage ammonia-N concentrations…” Are you considering including a correlation between the communities and the fermentation quality parameters in your study?
L414: “gallic or vallic acids”
L415: “It might be due to the action of these silage additives as methy- donors promote…”
Conclusion: Instead of summarizing the results, just add a conclusion regarding the finds of this study especially considering the treatments utilized.
Include references according to journal’s guidelines.
Kind regards.
English can be improved. Some comments were given regarding that.
Author Response
First of all, thanks for your revised suggestions.
Keywords: For indexing reasons, use as keywords different words than those already mentioned in the title.
R: we have substituted the keywords as suggested.
Insert references in the text according to Journal’s guideline.
R: We have revised it across the manuscript.
L30: “during ensiling” or “during storage”, but not “during ensiling storage”.
R: We have revised it across the manuscript.
Searching in literature, this reference sounds also very appropriate in this manuscript (https://doi.org/10.3389/fanim.2022.1080535). Please include if you think it’s convenient.
R: We have cited it in the manuscript.
L39: …much effort has been made…
R: We have revised it.
L50: Should “mesenteroides” be with lower case letter?
R: We have revised it.
L62: …the present study aimed to investigate…
R: We have revised it.
L63: style?
R: We have revised it.
L65: Why only up to 30 days? Some of the parameters regarding silage fermentation quality and possibly bacterial communities still haven’t reached plateau.
R: The selection of ensiling duration is based on the results of our previous studies, such as the dynamic changes of pH value, microbial population, organic acid concentration.
L65: I’m not sure if “…were unsealed for samples collection…” is indeed very clear for the reader. Please consider revising.
R: We have revised it.
L68: Please describe a bit more the raw material, for instance the growth stage, when and where it was harvested, fertilization, and so on.
L77: How come that bags were unsealed? Preservation of silage should be done under anaerobic conditions.
R: In total, 36 silage bags (3 treatments × 4 time-points × 3 replicates) were individually prepared using polythene plastic bags, thus we unsealed 3 bag silages of each treatment group on one sampling day.
Section 2.2: Why some abbreviations are in bold? Is it a guideline from another journal? Please check and remove bold if that’s the case.
R: We have revised it.
L93: Check use of parenthesis. At least one is missing.
R: We have revised it.
Reference for NDF, ADF and ADL is missing. Please add.
R: We have added it.
L115: The statistical design allows both linear and quadratic evaluation for the PLA effect. Why didn’t you consider linear and quadratic effect of PLA?
R: We have supplemented the analysis as suggested.
L123: style?
L124: style?
R: We have revised it.
L125: The number does not match with the number in the Table, but anyhow, do not repeat in the text those numbers already mentioned in the Table. Please check throughout the manuscript.
The first paragraph of Results section should be completely revised. The results presented in the Table must not be repeated in the text. The description of results should be done in a way to present the results, however not repeating the numbers in both Table and text. Please fix accordingly.
R: We have reedited the manuscript.
Table 2: Where is propionic acid?
R: Propionic acid was undetected in the study and we have added the annotations.
L158: How can you say “linearly dose effect” if you evaluated treatment effect, instead of linear and quadratic effects? Please see my suggestion in the statistical evaluation section. Please consider including linear and quadratic effects, which might be even more important than the interaction DxT.
R: We have supplemented the analysis as suggested.
Title of Figure 1 is in different font sizes. The same in other Figures. Please fix.
R: We have revised it.
L192: “after fermentation” or “after ensiling”, but not “after ensiling fermentation”.
R: We have revised it.
L219: Scientific names should be in italic. Please check throughout.
R: We have revised it.
L222: Revise “ensiling fermentation”. Check throughout the manuscript.
R: We have revised it.
L239: Why are these abbreviations described if they are not in the Figure? Instead, add a description regarding the colour and size of the dots.
R: We have revised it.
Figure 5: Are these correlations described in Material and Methods? Please include this description.
R: We have added the information.
L268: “linearly”? Statistically that is not how you evaluate. Please consider evaluating the linear and quadratic effects of PLA on stylo silages.
R: We have supplemented the analysis as suggested.
L333: This reference is not in the reference list. Please add and check the conformity, if all references in text are in the reference list and vice versa.
R: We have revised it.
L365: ‘Moreover’ is misspelled.
R: We have revised it.
L392: “…bacteria in silage in that they…” might require revision.
R: We have revised it.
L394: Add space after period.
R: We have revised it.
L408: “…Pantoea abundance is negatively related to silage ammonia-N concentrations…” Are you considering including a correlation between the communities and the fermentation quality parameters in your study?
R: We have added correlation heatmap.
L414: “gallic or vallic acids”
R: We have revised it.
L415: “It might be due to the action of these silage additives as methy- donors promote…”
R: We have revised it.
Conclusion: Instead of summarizing the results, just add a conclusion regarding the finds of this study especially considering the treatments utilized.
R: We have revised it.
Include references according to journal’s guidelines.
R: We have revised it.
Reviewer 4 Report
Material and methods
L26 – The authors indicate that the samples were taken randomly on different days. They should explain this situation better, since the samples must be taken randomly, but from each treatment, this is not what is described.
In point 2.2 they must separate the methodology of “Determination of microbial population” from the rest of the methodology. They make reference to the “fermentation parameters”, however what they actually do are the chemical analyzes of the silages (DM, ADF, NDF, ADL).
In the statistical analysis, they use the “General linear model”, to carry out this statistical test it is necessary to test the normality and homogeneity. This statistical test is not the most suitable for the experimental design carried out.
Results
In point 3.1 of the L123 results, they immediately indicate the chemical composition of the samples, in the material and methods they refer to as “fermentation parameters” they must always use the same terminology, being the correct one in this case chemical composition.
Table 2
I didn't understand what they mean by DM loss; The pH only drops at 30 days, but only with the addition of PLA.
Discussion
In each of the points the chemical composition is designated differently, in the discussion it is called “General characteristics”
As this plant is a legume, the protein content (13.35%) is low.
There is no reference to low MS content in the discussion.
They state that a pH between 4.3 – 5 is acceptable for leguminous silages. Isn't the value of 5 a bit high for silages, especially with such a low DM content?
Author Response
First of all, thanks for your revised suggestions.
L26 – The authors indicate that the samples were taken randomly on different days. They should explain this situation better, since the samples must be taken randomly, but from each treatment, this is not what is described.
R: We have rewritten it to make it more clear.
In point 2.2 they must separate the methodology of “Determination of microbial population” from the rest of the methodology. They make reference to the “fermentation parameters”, however what they actually do are the chemical analyzes of the silages (DM, ADF, NDF, ADL).
R: We have revised it.
In the statistical analysis, they use the “General linear model”, to carry out this statistical test it is necessary to test the normality and homogeneity. This statistical test is not the most suitable for the experimental design carried out.
R: We have replaced GLM model with MIXED model, as well the table data.
Results
In point 3.1 of the L123 results, they immediately indicate the chemical composition of the samples, in the material and methods they refer to as “fermentation parameters” they must always use the same terminology, being the correct one in this case chemical composition.
R: We have revised it.
Table 2
I didn't understand what they mean by DM loss; The pH only drops at 30 days, but only with the addition of PLA.
R: With initial weight and final weight recorded, dry matter content of raw material and silage was determined to calculated the difference of total dry matter weight before and after ensiling, i.e., dry matter loss.
Discussion
In each of the points the chemical composition is designated differently, in the discussion it is called “General characteristics”
R: We have revised it.
As this plant is a legume, the protein content (13.35%) is low.
R: Referring to reported results and our previous results, the crude protein content of fresh stylo is 11%-16% on a dry matter basis.
There is no reference to low MS content in the discussion.
R: We have added it.
They state that a pH between 4.3 – 5 is acceptable for leguminous silages. Isn't the value of 5 a bit high for silages, especially with such a low DM content?
R: Such a statement is based on a meta analysis of a lot of studies conducted by Kung et al. (2018). Generally, the pH value of natural leguminous silages like alfalfa silage or stylo silage falls in this range.
Round 2
Reviewer 1 Report
The article is modulated as recommended. It can be published in its present form.
The editing and quality of English is fair enough.
Reviewer 2 Report
No further comments to be made.
Reviewer 4 Report
The authors modified the work statistics and presented the results in more detail.
The discussion was practically the same as the previous version, they could have improved taking into account the new results they obtained.
