Study on the Antibacterial Rule in Fermented Feed with Different Amounts of CaCO3 by Quantitative Real-Time Polymerase Chain Reaction
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study is relevant and interesting. The author needs to rephrase some of the sentences for a clearer understanding. The conclusion needs to be stronger.
See comments in attached file.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe Ms Ferm -2585795 by Li et al. entitled “ study on the antibacterial rule in fermented feed with different amounts of CaCO3 by quantitative real-time PCR ” examined new microbial quantitative real-time PCR analysis method to investigate the antibacterial rule in fermented feed with different amounts of CaCO3.. The current results indicated that The CaCO3 addition promoted the fermenting strain growth, especially lactic acid bacteria, and a large amount of organic acid production, which made the fermented feed have a certain antibacterial effect. Thus, the current MS meets the journal readership and has scientific and practical merits, and provides guidance for the antibacterial rule of high pH fermented feed with different amounts of CaCO3. However, I have some recommendations that need to further address all comments to improve the outcomes of this project.
Title, plz adjust as:
1. Title: The title fits well with the contents of the Ms.
2. Abstract: Please add the tested dose of CaCO3 in the text and in the closing statement of the abstract.
3. Keywords, Don’t repeat words from the title, use new words to improve the MS readership.
4. Introduction, it seems that it was well written, please add the novelty and add value of your work and update references for 2023 and with:
-Attia Y. A., M.A. Al-Harthi and H. A. M. Abo El-Maaty (2020). Calcium and cholecalciferol levels on late phase-laying hens’ diets: effects on productive and egg quality traits, blood biochemistry and immune responses. Front. Vet. Sci. 7:389. doi: 10.3389/fvets.2020.00389
5. Materials and method section:
- The quality of Figure 1. needs to be improved and the source of figure should be added
6. The high level of CaCO3 (12%, w/w) is higher than the level used in dietary formulation of laying hens diets 8-10%
7. Plz provide details of the statistical analyses such as experimental design, model, unit, test for mean differences, normality of the data and adequate sample size determination.
8. I see under this experimental set-up that CaCO3 used 0, 2, 4, 8 and 12, BLR may be tested to identify the optimum CaCo3 level.
9. In the conclusion section, this statements should be adjusted based on CaCo3 level
10. The overall recommendation is major revision, however, the Ms is well written and presented and discussed but further improvements are essential before final acceptance. Best wishes
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this study, the authors proposed the use of the qPCR method for the determination of pathogens in fermented foods for animal feeding. The results showed that the addition of calcium carbonate strongly influences the growth of inoculated pathogens, with a clear increase in the early stages of fermentation and a decrease at the end of the process. This is also applicable to Saccharomyces. However, for lactobacilli, the increase in viability by CaCO3 addition appears to be unaffected over time, assuming their ability to survive and counteract the growth of selected pathogens. The work makes use of quantification, by qPCR, of genomic DNA rather than cDNA, previously obtained from RNA retrotranscription. The limitation associated with the quantification of genomic DNA is that it does not allow one to distinguish viable from non-viable cells, so there will be an overestimation of copy number in amplification. In my opinion, for the quantification of food and feed pathogens, it is recommended to make a true estimate of viable cells in the food sample, since the reduction in pH is not always an indication of good fermentation or reduction of pathogenic microorganisms.
The following are some suggestions and corrections to be applied.
1 1. Introduction: I suggest that the authors argue more about the use of probiotics in animal feeding and related claims to which they should respond.
2. Line 109: According to the new nomenclature and genus-level reclassification proposed by Zheng et al., 2020, for lactobacillus species, plantarum species should be replaced with Lactiplantibacillus plantaraum and rhamnosus species with Lacticaseibacillus rhamnosus, respectively. Please recheck the entire body of the manuscript, and replace the species names with the new nomenclature.
3. Lines 115-126: I suggest that the authors unify section 2.3 and 2.4, as both report information on the type of medium, time and incubation temperature used for the selected species.
4. Line 143: Please specify the amplification protocol used for the quantitative-PCR, including the holding and the melting temperatures. If is the same protocol already reported, please specify it.
5. Line 166: Please specify the cell density of the initial inoculum of the pathogenic strains.
6. Line 167: Please increase the resolution of Figure 1, it appears grainy.
7. Line 170: Specify the instrument (brand and company) used for pH detection.
8. Lines 173, 177: Do you mean the sampling times during the fermentation process? Please replace day 0.5 as 12 hours from the start of fermentation. In addition, For a clearer understanding of the materials and methods, I suggest unifying sections 2.8 and 2.11 as they all relate to chemical determination with different amounts of CaCO3, specifying sampling times, respectively. And unify sections 2.9, 2.10 and 2.12 as microbial detection with qPCR with information regarding DNA extraction and the qPCR protocol used.
9. Line 191: Is the determination related to fermented feeds? Please specify and, if so, also include this paragraph in 2.13.
10. Lines 200-202: What kind of statistical analysis was conducted and with what kind of software? Please specify.
11. Lines220-222. In order to support your data, I suggest that you also report, in Table 2, the efficiency coefficient of each reaction, that you obtain from the slope values, and the cycle threshold values (Ct).
12. Lines 239 and 241: According to the copies obtained, are they 2.63x10^7 and 2.14x10^7? Please correct them.
13. Suggestion for discussion: Please discuss the potential of using quantitative analysis for the determination of pathogens in food and any limitations of it, supported by studies.
14. Suggestions for conclusions: As future purposes, specify or argue the possibility of applying some methods for the detection of viable bacteria in animal feed.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors has done good improvements in the revised copy and addressed the comments adequately
