Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present study, "Microbiological analysis of traditional sausage during production and storage " by Ajazi et al studied the  traditional fermented sausage in terms of technology and hygiene, throughout the production and storage phases. Overall, the the manuscript is poorly written and have several concerns that need to be addressed before further processing of this manuscript.

1. The present manuscript is a preliminary reports and may not be suitable for full-length research paper. Please check for any other suitable article type.
2. Why only three butcher shops were considers for the study? Does it represent the traditional sausage production across the region? 
3. The Abstract aims to analyze the  traditional fermented sausage in terms of technology along with hygiene, however,  didn't conclude anything about the technological aspects of the sausage-making process.
4. Please write briefly the historical aspects of sausage production.
5. Write the current national and international status of  sausage consumption and associated issues
6. Please write more on the research gap. For example, how limited information about microbial quality of traditional sausage can impacts its food safety, quality, or culinary heritage?
7. Figure 1 is not required. 
8. Please write the "Production Practices" in methodology.
9. How did you perform the Principal component analysis?
10. Please write the Results and Discussion separately? Moreover, discussion needs to improve significantly.
11. What is the need for Figure 7 and 8. How its results are different from Figure 5.

Author Response

Dear Reviewer,
Thank you for your comments!

Comments 1: The present manuscript is a preliminary reports and may not be suitable for full-length research paper. Please check for any other suitable article type.

Answers 1: The manuscript has undergone substantial revisions and improvements compared to the previous version. We respectfully request that it be considered for publication as an original research article. 

Comments 2: Why only three butcher shops were considers for the study? Does it represent the traditional sausage production across the region?

Answers 2: Thank you for the question. 

The butchers were selected based on traditional producing methods without the use of starter cultures, which aligned with our research objectives.
The aim of the study was to focus on sausage produced on a small-scale, with traditional methods that inherited pass down through generations and that have been declared not using any additives in the product. We acknowledge that further studies including a broader geographic scope and more producers would be beneficial for a more comprehensive regional representation. 

Comments 3: The Abstract aims to analyze the traditional fermented sausage in terms of technology along with hygiene, however,  didn't conclude anything about the technological aspects of the sausage-making process.

Answers 3: Thank you for pointing this out. This sentence is added in the abstract (in line 39): “No differences regarding the production practices and storage of traditional sausage were observed, based on data from butchers who participated in this study.”

Comments 4: Please write briefly the historical aspects of sausage production. 

Answers 4: This paragraph is added in lines 44-52: "Historical records from ancient Mesopotamia, Greece, and Rome document sausage-making as a method for meat preservation [a]. Traditionally, sausages were produced by combining ground meat with salt, fat, and spices, then encasing the mixture in natural casings and fermenting or drying it to extend its shelf life[b]. Over the centuries, various cultures have developed distinctive variations influenced by local ingredients, climate, and culinary customs. This artisanal knowledge has been transmitted across generations and continues to underpin many traditional sausage production methods, including those still practiced in Kosovo today. 

Comment 5: Write the current national and international status of sausage consumption and associated issues.

Answers 5: This paragraph is added in lines 518-526: Based on our knowledge of Kosovar cuisine, sausage consumption is high and occupies a very important place on consumers' tables. However, accurate data regarding the statistics of consumption of this product in Kosovo is lacking. Regarding the consumption of meat products on a global level today, concerns have increased for health reasons [c], however, it is important to emphasize that products such as sausage or other meat-based products are a good source of protein for our diet. Another issue related to the consumption of meat products is the impact of their production on the environment [d]. contributing to greenhouse gas emissions and land use. Despite all this, beef production increased in 2024, reflecting the increase in slaughter rates and higher carcass weights [e].

Comments 6: Please write more on the research gap. For example, how limited information about microbial quality of traditional sausage can impacts its food safety, quality, or culinary heritage?

Answers 6: This paragraph is added in lines 148-154: Despite the long-standing tradition of sausage-making in Kosovo there is a notable lack of scientific literature and systematic studies on the microbial quality of traditionally produced sausages. The relevant research regarding the quality and safety of traditional sausage could be used as a referent study in the case of standardization and certification of this traditional product in the future. This study takes a first step in that direction by analyzing the microbiological changes during the production and storage of traditionally fermented sausage in Kosovo. 

Comments 7: Figure 1 is not required. 

Answers 7: Figure 1 is removed from the article.

Comments 8: Please write the "Production Practices" in methodology

Answers 8: The subsection "Production Practices" has been moved to the "Materials and Methods" (in line 197). 

Comments 9: How did you perform the Principal component analysis? 

Answers 9: As recommended by other reviews, we have revised the PCA plot, and the updated model is now presented in figure 5 (line 193) . Principal Component Analysis (PCA) biplot (correlation circle) was conducted using RStudio, employing the FactoMineR and factoextra packages. Prior to analysis, the dataset was standardized to ensure comparability among variables with differing units of measurement. 

Comments 10: Please write the Results and Discussion separately? Moreover, discussion needs to improve significantly.

Answers 10: Thank you for pointing this out. We agree with your recommendation. Now the results and discussion are presented separately.

Comments 11: What is the need for Figure 7 and 8. How its results are different from Figure 5.

Answers 11: Figure 5 presents the PCA results separated by meat processing facilities, allowing for a comparative analysis of patterns and variable behavior between individual facilities. In contrast, Figures 7 and 8 combine data from all facilities into a single analysis, aiming to assess the overall relationships and trends of all parameters in relation to time. This broader approach provides a more general overview of how the variables evolve collectively across all samples, regardless of facility-specific differences. 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors submitted a manuscript on microbial groups during fermentation and storage of traditional sausage in the Republic of Kosovo using culture-dependent methods. The approach is promising but the following aspects need to be strengthened:

 

>Title:

 

Adding where traditional sausages are produced from.

 

>Abstract

 

Emphasize the biotechnological potential or participation of microbial groups in fermentation.

 

>Introduction

 

Please describe the fermentation phases of traditional sausages and the changes in the microbiota. What are the consequences of these changes on the organoleptic characteristics.

 

Include examples of which metabolites or microbial products are associated with the flavors and odors of traditional sausages.

 

Mention in more detail the role of yeasts.

 

Describe general characteristics of LAB as well as bacteriocin production.

 

Include examples of bacteriocins that cause inhibition of microorganisms.

 

Include information on the dynamics of the microbiota on traditional sausage fermentation using metagenomic approach, what is known to date?

 

Include the hypotheses of your research and how you intend to experimentally evaluate them.

 

 

>Materials and methods

 

Mention more details of the sampling, what were the criteria for the selection of butchers and when the sampling was carried out. Also, what the sausages are made of.

 

Include the composition of the solutions and culture media used.

 

Mention the N or replicates corresponding to each trial or experiment.

 

L-125, there is an error in Eneterobacteria, check that the scientific names are spelled correctly.

 

Mention if the pH was measured in a suspension.

 

 

It is recommended to use a canonical correspondence analysis to analyze the relationship between physicochemical and biological variables, since they are two groups of variables with contrasting compartments.

 

Mention the statistical programs that were used.

 

 

>Results and discussion:

 

Mention if the ingredients used in the preparation are disinfected to know their contribution to the microbiota of sausages.

 

Indicate the range of environmental temperatures.

 

The discussion needs to be based on explaining the findings and not just comparing with other studies, e.g., what causes the fluctuations in the microbial groups and what are the consequences on the sausages.

 

It is also required to identify what variables are different in previous studies that led to different results in your research.

 

Describe the abbreviations in the figures

 

Make sure that in all figures it is clear whether or not statistical differences exist.

 

It is curious that the total variance of your results is distributed in 2 components of the PCA. Please include a table of the principal components generated during the analysis, including % their eigenvalues, percentage of variance, cumulative percentage of variance and loadings.

Include a Pearson or Spearman correlogram between variables to identify potential associations during fermentation.

 

Discuss the limitations of your study in relying on culture-dependent methods.

 

 

>Conclusion

 

Include perspectives of the study and future directions

Comments on the Quality of English Language

There are many typos and many scientific names of microorganisms are not spelled correctly.

Author Response

Comments 1: Adding where traditional sausages are produced from.

Answers 1: The title has changed: "Microbiological analysis of traditional sausage during production and storage in Prishtina, Republic of Kosovo"

Comments 2: Emphasize the biotechnological potential or participation of microbial groups in fermentation.

Answers 2: This sentence is added in lines 17-20:In sausage fermentation usually participate natural microbiota such as lactic acid bacteria (LAB) and Micrococcaceae that are not only critical for ensuring product safety and flavor development but also represent significant biotechnological potential. 

Comments 3: Please describe the fermentation phases of traditional sausages and the changes in the microbiota. What are the consequences of these changes on the organoleptic characteristics?

Answers 3: The text is added in lines 63-70: "In the fermentation phase of traditional sausage, we usually have a dominance of BAL and coagulase-negative staphylococci which can affect the reduction of the pH of the product, leading to the reduction of pathogenic and spoilage microorganisms. In an experimental study have shown that BAL are active throughout all phases of production and maturation/storage of this product with a dominance of the genus Lactobacillus (76.96%) and Staphylococcus (6.44%) on day 0, while on day 12 of maturation there was a decrease in Lactobacillus by 63.03% and an increase in Staphylococcus by 22.29%."

Comments 4:Include examples of which metabolites or microbial products are associated with the flavors and odors of traditional sausages.

Answers 4: The paragraphs are added in lines 71-76: "Due to the releasing free amino acids, proteolytic and lipolytic activity of the microbial community in traditional sausage, we consequently have the production of volatile organic components leading to their increase in the maturation phase [g]. All these changes in the microbial community and production components such as aldehydes, ketones, alcohols and sulfur-containing components contribute to the development of organoleptic characteristics and safety of the final product"

Comments 5: Mention in more detail the role of yeasts.

Answers 5: The text is added in lines 78-83: Yeasts also can play an important role in sausage fermentation and organoleptic characteristics development. Particularly species such as Debaryomyces hansenii and Yarrowia lipolytica contribute to the development of aroma and flavor through the production of esters, alcohols, and sulfur-containing compounds. They exhibit lipolytic activity, breaking down fats into free fatty acids that serve as precursors to volatile compounds. Furthermore, they contribute to surface deacidification, pigment stability, and prevention of lipid oxidation, which enhance the appearance and shelf life of the product.

Comments 6: Describe general characteristics of LAB as well as bacteriocin production.

Answers 6: Thank you for the comment, the text is added in lines 85-91

"They are Gram-positive, non-spore-forming, generally non-motile, catalase-negative microorganisms that grow under anaerobic or microaerophilic conditions. They are known for their fermentative metabolism, primarily converting sugars into lactic acid, which contributes to the acidification and preservation of fermented foods and are known for their probiotic properties. LABs are naturally present in raw meat and are dominant during sausage fermentation due to their acid tolerance and fast growth in low-oxygen environments...."

Comments 7:Include examples of bacteriocins that cause inhibition of microorganisms.

Answers 7: The paragraphs are included in lines 102-109: Examples of these bacteriocins are nisin leucocin and pediocin [23]. Nisin produced by Lactococcus lactis, is effective against Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Clostridium botulinum [m]. Leucocin A, produced by Leuconostoc carnosum DH25, also inhibits Listeria monocitogenes rather than factor like pH and water activity (aw) [n]. Pediocin, from Pediococcus spp., has potent antilisterial activity and is often used in meat preservation [o]. Bacteriocin production enhances the microbial safety of fermented sausages and supports natural preservation without chemical additives.

Comments 8:Include information on the dynamics of the microbiota on traditional sausage fermentation using metagenomic approach, what is known to date?

Answers 8: The text is adedd in lines 110-118: Using high-throughput sequencing, researchers have identified complex consortia of bacteria in traditional urutan sausage, analysis showed that Bacillota was the most abundant phylum and at the genus level were dominated by Latilactobacillus, Macrococcoides, Lactococcus, and Weissella. The genera that were positively correlated to the pH, water activity (aW), and acidity of fermented urutan were Staphylococcus, Lactococcus, Mammalicoccus, Macrococcoides, and Citrobacter [p]. While traditional culture-based methods provide quantitative insight into dominant microbial groups, metagenomic studies have offered a more comprehensive view of microbial succession and diversity during sausage fermentation.

Comments 9:Include the hypotheses of your research and how you intend to experimentally evaluate them.

Answers 9: The text is adedd in lines 155-167: Using high-throughput sequencing, researchers have identified complex consortia of bacteria in traditional urutan sausage, analysis showed that Bacillota was the most abundant phylum and at the genus level were dominated by Latilactobacillus, Macrococcoides, Lactococcus, and Weissella....

Comments 10: Mention more details of the sampling, what were the criteria for the selection of butchers and when the sampling was carried out. Also, what the sausages are made of.

Answers 10: Thank you for the comment, the text is added in lines 180-187: These butchers were selected based on the following criteria: (i) they had a long-standing tradition of producing fermented sausages using artisanal methods, (ii) they produced sausages without the addition of starter cultures, and (iii) they used consistent, repeatable processes in sausage preparation. All shops operated under similar hygienic standards and had local customer bases. The traditional sausages were composed of locally sourced beef meat, including rib and neck cuts, mixed with beef fat. No commercial starter cultures or curing agents were added, in line with traditional preparation methods.

Comments 11:Include the composition of the solutions and culture media used.

Answers 11: The text is adedd in lines 225-254: The microbiological media and solutions used were as follows: 

BPW (tipical formula was: Pancreatic digest of casein 1.0g/l; sodium chloride 4.3g/l; sodium phosphate monobasic anhydrous 5.77 g/l; potassium phosphate dibasic 3.56g/l and final pH 7.0±0.2 at 25°C) , samples were plated for bacterial enumeration according to the pour plate method. Briefly, 1 ml aliquots of the diluted samples were inoculated directly into the molten media...

Comments 12:Mention the N or replicates corresponding to each trial or experiment.

Answers 12: Thank you for the comment. The trial for the experiment is mentioned in line 191: 
“The traditional sausage was analyzed in three different stages of production and storage, on day zero 0 (before drying process), day 7 (after drying/fermentation) and day 14 (after two weeks of storage) at a temperature of +4 C, two samples per each stage with 2 repetitions (n=2).” 

Comments 13: L-125, there is an error in Eneterobacteria, check that the scientific names are spelled correctly.

Answers 13: Agree. According to your comment the text is revised  

Comments 14: Mention if the pH was measured in a suspension.

Answers 14: Thank you for pointing this out. The text is added in line 260-263
"For pH determination, 10 grams of sausage sample were homogenized with 90 mL of distilled water in a sterile blender to create a 1:10 (w/v) suspension. The pH of the resulting homogenate was measured using a calibrated pH meter. Measurements were performed in duplicate for each sample."

Comments 15: It is recommended to use a canonical correspondence analysis to analyze the relationship between physicochemical and biological variables, since they are two groups of variables with contrasting compartments.

Answers 15: Thank you for your suggestion regarding the use of Canonical Correspondence Analysis (CCA). We have carefully considered this recommendation; however, we chose to apply PCA biplot for both methodological and practical reasons. Our dataset includes a limited number of physicochemical and microbiological variables, with relatively stable distributions and no significant zero inflation—conditions under which the use of CCA is not particularly advantageous. In this context, PCA biplot provides a more flexible and interpretable approach for identifying patterns and similarities among the samples. 

Comments 16: Mention the statistical programs that were used.

Answers 16: Thank you for the comment. The text is adedd in lines 272-276

Comments 17: Mention if the ingredients used in the preparation are disinfected to know their contribution to the microbiota of sausages.

Answers 17: The paragraph is adedd in lines 210-212: The ingredients used in the sausage preparation, such as beef meat, onions, and bread, were not subjected to any disinfection or pasteurization procedures before processing

Comments 18: Indicate the range of environmental temperatures.

Answers 18: The environmental temperatures involved in the sausage production and storage ranged from 0°C during refrigeration to 34°C during fermentation. (in the line 217-219)

Comments 19: The discussion needs to be based on explaining the findings and not just comparing with other studies, e.g., what causes the fluctuations in the microbial groups and what are the consequences on the sausages.

Answers 19: These paragraphs are added in line 466: This could be indicated by the presence of normal flora in raw meat and other ingredients that have been used for sausage production and must be unique for this product. The increase in TNC and LAB by day 7 is likely due to favorable fermentation conditions (27–34°C), presence of fermentable substrates (e.g., carbohydrates from minced bread), and the lack of starter culture, allowing spontaneous fermentation. LAB proliferation leads to acidification, as observed in the pH drop from 6.64± to 5.07± by day 14. 

Comments 20: It is also required to identify what variables are different in previous studies that led to different results in your research.

Answers 20: Thank you for the comment. The text is added in lines 539-548: "The differences between our findings and those of previous studies can be attributed to several key variables. The absence of added starter cultures in our sausages allowed the natural microbiota from raw ingredients (meat and seasonings) to dominate fermentation, which contrasts with studies that used selected LAB or CNS strains to standardize microbial dynamics [r]. The fermentation in this study was performed under traditional room-temperature conditions (27–34°C) without humidity or airflow control which may have influenced microbial succession differently than in chamber-controlled processes [s]. Finally, the composition of the sausage—particularly the inclusion of onions, bread and local vegetable seasonings—provides fermentable substrates and endogenous enzymes do not present in standardized formulations."

Comments 21: Describe the abbreviations in the figures

Answers 21: Thank you for the comment. The abbreviations are included in the figures as required.

Comments 22: Make sure that in all figures it is clear whether or not statistical differences exist.

Answers 22: It was not possible to statistically determine significant differences in all figures, as in Figure 4 the number of measurements was small, which limited the ability to detect statistically significant differences with high accuracy. 

Comments 23: It is curious that the total variance of your results is distributed in 2 components of the PCA. Please include a table of the principal components generated during the analysis, including % their eigenvalues, percentage of variance, cumulative percentage of variance, and loadings.

Answers 23: Agree. The new PCA biplot has been constructed (Figure 5 line 392), and the relevant data—including eigenvalues, percentage of variance, cumulative percentage of variance, and loadings—are provided in the supporting material. 

Comments 24: Include a Pearson or Spearman correlogram between variables to identify potential associations during fermentation.

Answers 24: The Pearson correlogram is presented in figure 6 (line 424) 

Comments 25: Discuss the limitations of your study in relying on culture-dependent methods.

Answers 25: Thank you for suggestions. The text is added in line 526: "Despite providing information, for the first time, regarding the number of microbial groups and production methods of sausage produced in Prishtina, our study has a limitation regarding the methods used for identification of microorganisms. The culture-dependent method can provide information regarding the number of some microorganisms but can exclude the growth of some others, which may not have been cultivated but are present in the sausage. Furthermore, this method cannot provide us with detailed data regarding the diversity of the microbial community in traditional sausage..."

Comments 26: Include perspectives of the study and future directions

Answers 26: Thank you for suggestions. The paragraphs are added in lines 562-580:"It is needed to increase the quality of sausage and other meat products produced by traditional methods, through improving their hygienic parameters, labeling and packaging, which can affect the increase in export and product value. Additional investigation may focus on analyzing this product for the presence of pathogens such as Salmonella Thyphimurium, Listeria monocytogenes and Staphylococcus aureus that poses a risk to consumer health and to characterize the full microbial community using culture-independent methods, such as 16S rRNA sequencing or metagenomics, to better understand the roles of uncultured and functionally important organisms..."

Reviewer 3 Report

Comments and Suggestions for Authors

The presented paper (manuscript ID: microbiolres-3755449) reports on the microbiological analysis of traditional sausage during production and storage. Below are some comments and suggestions for authors.

1. In subsection 2.2 it should be clarified whether all studied groups of microorganisms were cultivated under aerobic conditions? It is also recommended to identify all abbreviations used, such as TNC (total number of colonies), LAB (lactic acid bacteria), MC (Micrococcaceae) and EC (Enterobacteria and Coliforms).
2. In subsection 2.3, add a reference to a standard or literature method for determining the pH of sausage. In particular, it is unclear whether the pH measurement procedure included prior homogenization of the sample in a liquid electrolyte.
3. Apparently, the authors used the common logarithm (lg) to represent the results of microbiota counting in Petri dishes. In addition, the SI system uses the symbol ''g'' (not ''gr'') to denote a gram. Therefore, throughout the manuscript, including Figures 3 and 4, the unit of measurement ''lg cfu/g'' should be used.
4. Check the text and all values in subsection 3.2. Here are some examples: (i) according to the sentence ''After 14 days of sausage storage +4 C, the number of mesophilic bacteria and LAB had a slight decrease to 6.68 log cfu/gr, respectively'' in line 169, two values should be given; (ii) apparently in line 170 should be ''While EC''; (iii) according to line 163, the average TNC value at stage D0 is 7.08 lg cfu/g, whereas further in line 176 the value is reported as 7.06 lg cfu/g; (iv) according to line 171, the average EC value at stage D14 is 1.6 lg cfu/g, whereas further in line 195 the value is reported as 1.66 lg cfu/g.
5. The interpretation of Figure 6 in lines 234-238 is not entirely accurate. First, notice that all vectors start from the origin. Secondly, from the presented graph we can speak about a moderate correlation between the parameters TNC and MC, EC and LAB, as well as a strong correlation between the parameters EC and Aw, Aw and LAB. At the same time, the pH indicator does not correlate with any of the studied parameters.
6. In Figure 6, the authors presented the first PCA plot for the parameters, showing the distribution and correlation between the studied parameters in vector space. However, the manuscript would be greatly improved if the authors added a second PCA plot for the samples, showing the distribution of sausage samples in vector space depending on the production and storage stage.
7. Figures 7 and 8 appear identical. Apparently Figure 7 should be replaced by a corresponding graph for pH. In addition, these figures should include boxplots, so the sentences ''The PCA results for aw are reported in Figure 7'' (line 242) and ''The PCA results for pH are reported in Figure 8'' (line 255) should be reworded.

Author Response

Comments 1: In subsection 2.2 it should be clarified whether all studied groups of microorganisms were cultivated under aerobic conditions? It is also recommended to identify all abbreviations used, such as TNC (total number of colonies), LAB (lactic acid bacteria), MC (Micrococcaceae) and EC (Enterobacteria and Coliforms).

Answers 1: Thank you for the suggestion. The text is included in lines 233-234: Yes, all groups of microorganisms were incubated in aerobic conditions. It is corrected in subsection 2.2. Were clarified all abbreviations used in paper.

Comments 2: In subsection 2.3, add a reference to a standard or literature method for determining the pH of sausage. In particular, it is unclear whether the pH measurement procedure included prior homogenization of the sample in a liquid electrolyte.

Answers 2: Thank you for the comment. The text is added in lines: 260-263: "For pH determination, 10 grams of sausage sample were homogenized with 90 mL of distilled water in a sterile blender to create a 1:10 (w/v) suspension. The pH of the resulting homogenate was measured using a calibrated pH meter. Measurements were performed in duplicate for each sample."

Comments 3: Apparently, the authors used the common logarithm (lg) to represent the results of microbiota counting in Petri dishes. In addition, the SI system uses the symbol ''g'' (not ''gr'') to denote a gram. Therefore, throughout the manuscript, including Figures 3 and 4, the unit of measurement ''lg cfu/g'' should be used.

Answers 3: Thank you. The unit is revised as required. 

Comments 4:Check the text and all values in subsection 3.2. Here are some examples: (i) according to the sentence ''After 14 days of sausage storage +4 C, the number of mesophilic bacteria and LAB had a slight decrease to 6.68 log cfu/gr, respectively'' in line 169, two values should be given; (ii) apparently in line 170 should be ''While EC''; (iii) according to line 163, the average TNC value at stage D0 is 7.08 lg cfu/g, whereas further in line 176 the value is reported as 7.06 lg cfu/g; (iv) according to line 171, the average EC value at stage D14 is 1.6 lg cfu/g, whereas further in line 195 the value is reported as 1.66 lg cfu/g.

Answers 4: Thank you for the suggestions. It is corrected in the text and figures. Now is lg cfu/g throughout the manuscript. 

Comments 5: The interpretation of Figure 6 in lines 234-238 is not entirely accurate. First, notice that all vectors start from the origin. Secondly, from the presented graph we can speak about a moderate correlation between the parameters TNC and MC, EC and LAB, as well as a strong correlation between the parameters EC and Aw, Aw and LAB. At the same time, the pH indicator does not correlate with any of the studied parameters.

Answers 5: The new PCA biplot has been constructed, and the relevant data—including eigenvalues, percentage of variance, cumulative percentage of variance, and loadings—are provided in the supporting material. 

Comments 6:In Figure 6, the authors presented the first PCA plot for the parameters, showing the distribution and correlation between the studied parameters in vector space. However, the manuscript would be greatly improved if the authors added a second PCA plot for the samples, showing the distribution of sausage samples in vector space depending on the production and storage stage.

Answers 6: The new PCA biplot has been constructed, and the relevant data—including eigenvalues, percentage of variance, cumulative percentage of variance, and loadings—are provided in the supporting material. 

Comments 7: Figures 7 and 8 appear identical. Apparently Figure 7 should be replaced by a corresponding graph for pH. In addition, these figures should include boxplots, so the sentences ''The PCA results for aw are reported in Figure 7'' (line 242) and ''The PCA results for pH are reported in Figure 8'' (line 255) should be reworded.

Answers 7: Thank you, it was a technical issue...now the pH boxplot is revised. 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for making the required corrections. Please remove the unnecessary bold formatting in the genus and or species name. 

Author Response

Comments 1: Please remove the unnecessary bold formatting in the genus and or species name. 

Answers 1: Dear reviewer,  Thank you for your comments. The bold formatting is now removed from the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This version of the manuscript is much more structured. The introduction clearly explains the state of the art, the methods are more thoroughly explained, and the results are described in greater detail. However, the figure captions need more description, and the statistical tests used should be specified. When comparing more than two treatments, it is important to use letters to indicate which treatments differ. The discussion and conclusion are adequate.

Author Response

Comments 1: This version of the manuscript is much more structured. The introduction clearly explains the state of the art, the methods are more thoroughly explained, and the results are described in greater detail. However, the figure captions need more description, and the statistical tests used should be specified. When comparing more than two treatments, it is important to use letters to indicate which treatments differ. The discussion and conclusion are adequate.

Answer 1: Thank you for your comments.
All figure captions have been revised to include more detailed descriptions, and the statistical analyses are now explained with greater clarity.

No treatments or interventions were applied during the sausage production process; instead, the same product was analyzed at three distinct phases of production and storage. These phases are consistently referred to using the abbreviations D0, D7, and D14, corresponding to day 0, day 7, and day 14, respectively.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, the manuscript has been significantly improved, but there are some inconsistencies in the newly added subsection 3.4 (Pearson Correlation).

1) Line 338 of the revised manuscript reports that ꞌꞌtake the striking 0.92 correlation between pH and TNCꞌꞌ, but Figure 6 reports a value of 0.14 at the intersection of pH and TNC.

2) Further on in line 341 it is reported that ꞌꞌthe influence of pH on LAB is also noteworthy, with a correlation of 0.80ꞌꞌ, but in Figure 6 the intersection of pH and LAB values shows a value of 0.92.

3) Finally, line 350 report that ꞌꞌ0.94 correlation between TNC and ECꞌꞌ, but Figure 6 reports a value of -0.10 at the intersection of TNC and EC.

Can the authors comment on the observed contradictions and, if necessary, make appropriate changes? After this, the manuscript can be accepted for publication.

Author Response

Dear reviewer, Thank you for your valuable and insightful comments.

Comments 1: 1) Line 338 of the revised manuscript reports that ꞌꞌtake the striking 0.92 correlation between pH and TNCꞌꞌ, but Figure 6 reports a value of 0.14 at the intersection of pH and TNC.

Answers 1: Thank you for your insightful comments. All three points raised are valid, and we acknowledge that certain technical errors occurred during the preparation of the manuscript. Accordingly, the paragraph in question has been removed from the revised version.

Comments 2: Further on in line 341 it is reported that ꞌꞌthe influence of pH on LAB is also noteworthy, with a correlation of 0.80ꞌꞌ, but in Figure 6 the intersection of pH and LAB values shows a value of 0.92.

Answers 2: The correlation numbers are replaced as suggested.

Comments 3: Finally, line 350 report that ꞌꞌ0.94 correlation between TNC and ECꞌꞌ, but Figure 6 reports a value of -0.10 at the intersection of TNC and EC.

Answers 3: Our intention was to present the correlation between TNC and MC (r=0.94). Additionally, we have included a further comparison between LAB and EC (r = 0.99) in the corresponding line of the text.