Microbiological Analysis of Manufacturing Processes and Microbial Hazard Assessment of Quality and Safety of Commercial Salted Shrimp (Saeu-jeot)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Material and methods

Please indicate the scientific name of the species used in the experiments.

Fig. 1 – authors should replace the figure1 b with one with better quality.

Line 98 – “all samples” - how do authors know that the commercial samples were prepared using that proportion of shrimp and salt?

Line 99 – please revise the size of the shrimps.

Line 101 – “analysis was performed within 15 days…” it is necessary to include information about the expiring date (or best before), data of manufacture, and date of analysis. Otherwise, it is not possible to understand whether the analyzes were carried out with the product close to its expiration date, or shortly after it was prepared.

Lines 128-130 – the surface inspection was done in one of the facilities? The results in table 1 seem to represent only on facility, in only just one moment. Also, the results are expressed per gram. It should be corrected since the surfaces were done in a selected area.

Lines 143-147 – saeu-jeot was contaminated with S. aureus, and then the sample was mixed and stored at 20 ºC, for how long? During that time, the sample was mixed?  Which is the proportion of shrimps that are immersed (lower layer) and those that are in the upper layer.

Line 148 - Please discuss the application of UV-irradiation in the context of an industry, considering that the tests were done in a layer of 0.5 cm thickness.

Line 149 – please explain here why authors choose irradiation treatments with a wavelength of 253.7 nm.

 

Results and discussion

Line 167 - Please revise the expression ”Microbial contamination” throughout the text. Contamination refers to the non-intended or accidental introduction of microorganisms.

Line 190 - It would be important to explain the possible origin of that contamination, and the consequences of the presence of the bacteria.

Fig.2 – please indicate the number of samples for each group (certified and normal).

Fig. 3 – it is not clear what is represented in axis XX and YY.

Line 240 – total bacteria in the raw material was 4.2 log CFU/g. Do authors have the results of total bacteria in the final product?

Section 3.4. “effect of salt and UV…”. The results of the effect of salt were not presented. Please modify accordingly.

Line 270 - “effect of saltwater immersion process” – the shrimps were mixed with salt, or with a seawater solution?

Lines 270-274 - The saeu-jeot was prepared and then inoculated with S. aureus, and mixed, or it was inoculated with S. aureus before the salting and aging process? It is not clear how the assay was performed. Was this an immediate effect of saltwater immersion, or the counts were done after a predefined period?

Table 2 – please correct the units of time. Also check section 2.3, since it is said that a treatment of 180 seconds was done, and it was not included in the table. The results presented in the table correspond to the upper layer? Please specify in the table. Are there any replicates of this assay?

Were the results obtained here compared to results obtained in previous studies with similar species, or with other treatment methods?

Conclusions

Lines 309-310 – this conclusion is not in accordance with the discussion (see lines 281-282).

Lines 311-312 – how can it inhibit the growth? the test had to be carried out over time

Lines 313-314 – “can suppress cross contamination…” the treatment reduced less than 1 log CFU/g. the conclusions seem too ambitious.

Author Response

Material and methods
Please indicate the scientific name of the species used in the experiments.
Response: The species used in saeu-jeot is Acetes japonicus. I have included a brief mention of this on line 45 of the revised manuscript.

Fig. 1 – authors should replace the figure1 b with one with better quality.
Response: Figure 1 has been replaced with an appropriately high-quality image. (Figure 1(b))

Line 98 – “all samples” - how do authors know that the commercial samples were prepared using that proportion of shrimp and salt?
Response: According to South Korea's food labeling standards, in order to use the term 'saeu-jeot,' the shrimp content and ingredients used must be indicated on the packaging. It is a legal obligation to specify the proportion of shrimp paste in all samples tested in the experiment.

Line 99 – please revise the size of the shrimps.
Response: I have made modifications to the units used to describe the size of the shrimp. I have also included the term 'about' prior to the value to emphasize that these are approximate values.

Line 101 – “analysis was performed within 15 days…” it is necessary to include information about the expiring date (or best before), data of manufacture, and date of analysis. Otherwise, it is not possible to understand whether the analyzes were carried out with the product close to its expiration date, or shortly after it was prepared.
Response: Typically, the shelf life of saeu-jeot in South Korea is set at 1 to 2 years. It is presumed that quality changes will be minimized when the product is stored at low temperatures. Mention of this expiration date is indicated on line 104.

Lines 128-130 – the surface inspection was done in one of the facilities? The results in table 1 seem to represent only on facility, in only just one moment. Also, the results are expressed per gram. It should be corrected since the surfaces were done in a selected area.
Response: The microbial contamination of products was tested in terms of log CFU/g, while the results of the manufacturing process were analyzed based on surface contamination and expressed as log CFU/cm². This has been modified accordingly in the manuscript.

Lines 143-147 – saeu-jeot was contaminated with S. aureus, and then the sample was mixed and stored at 20 ºC, for how long? During that time, the sample was mixed?  Which is the proportion of shrimps that are immersed (lower layer) and those that are in the upper layer.
Response: After inoculation shrimp paste was inoculated, it was homogenized and then divided into upper and lower parts at a 5:5 ratio. Please refer to line 160 for more detailed information regarding the experimental protocol.

Line 148 - Please discuss the application of UV-irradiation in the context of an industry, considering that the tests were done in a layer of 0.5 cm thickness.
Response: I have added content outlining the experimental plan based on its industrial applications. Please refer to line 160 for more detailed information regarding the experimental protocol.

Line 149 – please explain here why authors choose irradiation treatments with a wavelength of 253.7 nm.
Response: This has been detailed in the Introduction section of the manuscript. “The UV-C treatment at 253.7 nm wavelength causes DNA modification and damage to microorganisms; the survival rate decreases rapidly with increasing irradiation dose” (Line 84).

Results and discussion
Line 167 - Please revise the expression ”Microbial contamination” throughout the text. Contamination refers to the non-intended or accidental introduction of microorganisms.
Response: This is a commonly used term in the field of microbiology, particularly since the study was intended to evaluate microbial contamination. If there are any other suitable terms, we are willing to change this appropriately.

Line 190 - It would be important to explain the possible origin of that contamination, and the consequences of the presence of the bacteria.
Response: In the Discussion section of the manuscript, I have described it as originating from the raw materials. Please see line 323 of the manuscript.

Fig.2 – please indicate the number of samples for each group (certified and normal).
Response: I have applied appropriate numerical labels to Figure 2.

Fig. 3 – it is not clear what is represented in axis XX and YY.
Response: I have added explanations for the x and y axes in the caption of Figure 3. Y-axis: Probability Density; X-axis: Contamination Level.

Line 240 – total bacteria in the raw material was 4.2 log CFU/g. Do authors have the results of total bacteria in the final product?
Response: Total bacteria was confirmed to be 3.3 log CFU/g. However, the results regarding general bacteria from products manufactured by the company were excluded to avoid duplicate application as they were included in the commercially available products.

Section 3.4. “effect of salt and UV…”. The results of the effect of salt were not presented. Please modify accordingly.
Response: UV-C and salting do not imply complete sterilization. However, it has been confirmed that these processes have an inhibitory effect on the growth of pathogenic microorganisms. This content has been revised accordingly. Please refer to lines 328-335.

Line 270 - “effect of saltwater immersion process” – the shrimps were mixed with salt, or with a seawater solution?
Response: After capturing the shrimp, there is a separate step in the manufacturing process where they are mixed with salt. This process is commonly referred to as salting.

Lines 270-274 - The saeu-jeot was prepared and then inoculated with S. aureus, and mixed, or it was inoculated with S. aureus before the salting and aging process? It is not clear how the assay was performed. Was this an immediate effect of saltwater immersion, or the counts were done after a predefined period?
Response: The description of the experimental method for shrimp paste salting has been revised (Section 2.3, line 154).

Table 2 – please correct the units of time. Also check section 2.3, since it is said that a treatment of 180 seconds was done, and it was not included in the table. The results presented in the table correspond to the upper layer? Please specify in the table. Are there any replicates of this assay?
Response: In the initial experimental plan, we also intended to confirm this at the 180-second stage, but it was determined that there would be no significant difference, so this content has been removed from the revised manuscript.

Were the results obtained here compared to results obtained in previous studies with similar species, or with other treatment methods?
Response: While this was not conducted on the same species, other research has confirmed the effects of UV on dried pollack and mackerel fillets. This information has been briefly discussed in the Discussion section of the manuscript.

Conclusions
Lines 309-310 – this conclusion is not in accordance with the discussion (see lines 281-282).
Response: UV-C and salting do not imply complete sterilization. However, they have been found to inhibit the growth of pathogenic microorganisms. This information has been amended, see lines 328-335

Lines 311-312 – how can it inhibit the growth? the test had to be carried out over time
Response: I am aware of the points you mentioned. 

The main objective of this study was to assess microbial contamination in manufacturing facilities implementing the HACCP system to determine the effectiveness of its application. 

I will incorporate this suggestion into our next study.

Lines 313-314 – “can suppress cross contamination…” the treatment reduced less than 1 log CFU/g. the conclusions seem too ambitious.
Response: I have made revisions such as changing the expression from 'reduction' to 'inhibition' to better reflect the overall content of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors verified the quality and safety of typical Korean salted shrimp by microbiological assessment. The paper needs a lot of improvement regarding the number of samples to be examined. The number of samples analyzed is too small to have an impact on the microbiological evaluation of this product. The authors should increase the pool of samples to be examined.

Materials and methods

line 101: It could be better if authors consider changes over time not only at 15 days after.

The figure are of scarse quality.

Line 159: Have the AA verified the assumption of normality of distribution? If yes, what type of test was used?

In figure 2, what the round circles correspond to and what the black bar?

 

 

Author Response

The Authors verified the quality and safety of typical Korean salted shrimp by microbiological assessment. The paper needs a lot of improvement regarding the number of samples to be examined. The number of samples analyzed is too small to have an impact on the microbiological evaluation of this product. The authors should increase the pool of samples to be examined.
Response: Thank you for raising this important point. 

The main objective of this study was to verify the effectiveness of implementing the HACCP system by examining microbial tests in both traditionally manufactured saeu-jeot and in facilities applying the HACCP system. 

We collected a total of 49 representative saeu-jeot products from Korea, including those imported domestically, for analysis. The production of saeu-jeot varies depending on the catch of shrimp, and it is influenced by seasonal characteristics and marine environments. Due to these factors, there are not many saeu-jeot manufacturers, and furthermore, the number of facilities in Korea with HACCP certification is limited, which limited the number of samples that could be tested in this study 

We made efforts to obtain accurate result data considering these circumstances. Please take these points into consideration.

Materials and methods
line 101: It could be better if authors consider changes over time not only at 15 days after.
Response: Saeu-jeot is typically consumed within 15 days under refrigerated conditions in Korea, which may vary depending on the packaging capacity. 

Due to this reason, we have deemed it appropriate to conduct the experiment under similar conditions. 

Based on your feedback, we will expand the application of these considerations to our future research endeavors.

The figure are of scarse quality.
Response: This figure has been replaced with a high-resolution version. (Figure 1(b))

Line 159: Have the AA verified the assumption of normality of distribution? If yes, what type of test was used?
Response: The data used in the paper were analyzed using ANOVA, as described in line 179.

In figure 2, what the round circles correspond to and what the black bar?
Response: The round circles denote outliers, while the black bars indicate the median. Another perspective is presented with detailed values in Figure 2.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

This manuscript presents the evaluation of microbiological properties of commercially available Korean traditional salted shrimp (saeu-jeot) and the assessment of the effects of salt water immersion and UV irradiation on reduction of Staphylococcus aureus in saeu-jeot. The microbiological testing for total viable bacteria, coliforms, Escherichia coli, S. aureus and Vibrio parahaemolyticus was performed in 56 commercially available saeu-jeot samples from the Korean, Chinese, and Vietnamese markets. The manuscript is well written and organised, comprising the abstract, introduction, material and methods, results and discussion, conclusions, 24 references, four figures and two tables.

However, some issues must be addressed before considering for publication, as it is difficult to understand the presented results, due to missing information and some incoherencies in the sections of “Material and methods” and “Results and Discussion”.

Further information should be included concerning: the studied samples (Certificated vs. Normal, raw material, packaging, surfaces, etc), conducting hazards analysis, microbial methods and statistical analyses. The detection/quantification limits of microbial methods should also be addressed. It is difficult to understand how some results for Staphylococcus aureus are presented as “.00 Log CFU/g”, without stating the quantification limit. For example, when inoculating 0.1 mL of serial dilutions, prepared from the homogenate of 10g of sample in 90 mL of diluent, how were you able to quantify 0.2 Log CFU/g Staphylococcus aureus (as stated in line 179)?

Concerning statistical analysis, please explain how data was treated/transformed (eg. log₁₀ transformation) and analysed. It is unclear how data analysis (averages/medians, standard deviations, ranges, significance of differences) was performed for parameters with values below the quantification level. Why do you refer data “expressed as mean and standard deviation”, but present results with minimum, maximum, median values and position of the lower and upper quartiles (box plots) in figure 2?

Moreover, some data is presented in an unclear way. For example, in table 1, why are results of surface contamination expressed as CFU/g and not CFU/cm2?

These issues make difficult to assess the sections of “Material and methods” and “Results and discussion”. So please revise accordingly.

Please see below for specific comments:

 

1. Introduction

 

Lines 58-59: “The Ministry of Food and Drug Safety of Korea regulates the microorganism standard of salted fish, including saeu-jeot”. Please include a reference for this statement.

 

Line 62: “However, of ~500 companies”. Please replace “~” by “approximately”.

 

Lines 90-93: “Therefore, the aim of this study was ... and analyze their microbiological safety. Furthermore, ... analyzed the microbiological contamination levels”. Please clarify and improve the description of the aim of the study, as the microbiological parameters studied also involved total viable bacteria, coliforms and Escherichia coli, which are not hazards. Besides, the application of saltwater immersion and UV treatments was also studied.

 

2. Materials and Methods

 

Lines 105-106: “… were analyzed according to the microbial quantitative test method presented in the Food Code (Jetokal)”. Please include a reference for this statement and state the detection limit of each microbial quantitative test.

 

Lines 108-109: Please explain the reason for the collection/inoculation only of the upper layer. Also replace “~0.1 mL” by “approximately 0.1 mL”.

 

Lines 127-133: Please include relevant information concerning the samples of raw and subsidiary materials (shrimp, salt and empty bottles - packaging materials) and surfaces studied, such as the number of samples by type, manufactured with or without HACCP certification, and the performed microbial tests.

 

Line 141: Please describe the strains of S. aureus tested (eg. reference strains and/or wild strains isolated from…).

 

Line 142: Please describe the meaning of the abbreviation CFU.

 

Line 155: Please better explain how experiments and/or testing were performed in triplicates.

 

3. Results and Discussion

  Lines 171-175: For total viable bacteria the results for “samples manufactured with and without HACCP certification was 3.8 ± 0.1 and 3.9 ± 0.1 log CFU/g, respectively (p > 0.05). ...This difference could be because...”. Please rephrase, as if p > 0.05, no differences were verified for the parameter of total viable bacteria.   Lines 190-191: Figure 2 caption, please include the number of samples for both groups (“Certificated” and “Normal”).   Lines 218-233: Please consider changing these two paragraphs to the section of “Material and methods” as the results only begin with the presentation of the three CCP of figure 4.   Lines 237-238: Figure 4 caption, please explain the meaning of abbreviations (eg, CCP-1P, CCP-2B, CCP-3P and SUS, Fe).   Lines 239-244: Please revise the units of results for surfaces (UFC/cm2?) and subsequent discussion.   Line 272-273: “The count of S. aureus in the upper layer was 6.1 ± 0.0 log CFU/g and in the lower layer was 4.7 ± 0.1 log CFU/g (Table 3).” Please present table 3.   4. Conclusion   Please revise, as some statements are not supported by the results (eg. “… it can be concluded that the salting and UV-C treatment processes can effectively inhibit the growth of microorganisms without heat treatment in saeu-jeot”).     Best Regards,  

 

Reviewer

Author Response

This manuscript presents the evaluation of microbiological properties of commercially available Korean traditional salted shrimp (saeu-jeot) and the assessment of the effects of salt water immersion and UV irradiation on reduction of Staphylococcus aureus in saeu-jeot. The microbiological testing for total viable bacteria, coliforms, Escherichia coli, S. aureus and Vibrio parahaemolyticus was performed in 56 commercially available saeu-jeot samples from the Korean, Chinese, and Vietnamese markets. The manuscript is well written and organised, comprising the abstract, introduction, material and methods, results and discussion, conclusions, 24 references, four figures and two tables.
However, some issues must be addressed before considering for publication, as it is difficult to understand the presented results, due to missing information and some incoherencies in the sections of “Material and methods” and “Results and Discussion”.
Further information should be included concerning: the studied samples (Certificated vs. Normal, raw material, packaging, surfaces, etc), conducting hazards analysis, microbial methods and statistical analyses. The detection/quantification limits of microbial methods should also be addressed. It is difficult to understand how some results for Staphylococcus aureus are presented as “.00 Log CFU/g”, without stating the quantification limit. For example, when inoculating 0.1 mL of serial dilutions, prepared from the homogenate of 10g of sample in 90 mL of diluent, how were you able to quantify 0.2 Log CFU/g Staphylococcus aureus (as stated in line 179)? 
Response: The detection limit is 1.0 log CFU/g or less, which has been detailed in the manuscript. 
Additionally, for the data mentioned on line 179, excluding the values not detected ranging from 0.2±0.2 to 2.5±0.2 log CFU/g, the minimum average value was calculated. Furthermore, the overall mean is 0.6 ± 0.6 log CFU/g.

Concerning statistical analysis, please explain how data was treated/transformed (eg. log10 transformation) and analysed. It is unclear how data analysis (averages/medians, standard deviations, ranges, significance of differences) was performed for parameters with values below the quantification level. Why do you refer data “expressed as mean and standard deviation”, but present results with minimum, maximum, median values and position of the lower and upper quartiles (box plots) in figure 2?
Response: To enhance readability for subscribers, the data for the 56 samples has been represented using box plots, as it would be unwieldy to present the data for this many samples in a tabular form. Box plots are commonly used visualization tools that simultaneously display data distribution and outliers, facilitating easy comparison between different data groups. This was done to compare facilities applying the HACCP system with those that do not, as mentioned in the text. 

Additionally, box plots are also utilized for microbial analysis. 

Reference: MAZURKIEWICZ, Karolina; JEŻ-WALKOWIAK, Joanna; MICHAŁKIEWICZ, Michał. Physicochemical and microbiological quality of rainwater harvested in underground retention tanks. Science of the Total Environment, 2022, 814: 152701.

Moreover, some data is presented in an unclear way. For example, in table 1, why are results of surface contamination expressed as CFU/g and not CFU/cm2?
Response: The microbial contamination of products was tested in terms of log CFU/g, while the results of the manufacturing process were analyzed based on surface contamination and expressed in terms of log CFU/cm2. This has been appropriately corrected in the manuscript.

These issues make difficult to assess the sections of “Material and methods” and “Results and discussion”. So please revise accordingly.

Please see below for specific comments:

1. Introduction

Lines 58-59: “The Ministry of Food and Drug Safety of Korea regulates the microorganism standard of salted fish, including saeu-jeot”. Please include a reference for this statement.
Response: I have added a citation regarding Korean food-related regulations (Reference 5).

Line 62: “However, of ~500 companies”. Please replace “~” by “approximately”.
Response: This suggestion has been incorporated into the revised manuscript and the overall analysis has been verified.

Lines 90-93: “Therefore, the aim of this study was ... and analyze their microbiological safety. Furthermore, ... analyzed the microbiological contamination levels”. Please clarify and improve the description of the aim of the study, as the microbiological parameters studied also involved total viable bacteria, coliforms and Escherichia coli, which are not hazards. Besides, the application of saltwater immersion and UV treatments was also studied.
Response: I have described the main objectives aimed to be confirmed through each microbial analysis used in the study and revised the manuscript to focus on the experimental results regarding the effects of salting and UV irradiation utilized by HACCP-certified manufacturing facilities.

2. Materials and Methods

Lines 105-106: “… were analyzed according to the microbial quantitative test method presented in the Food Code (Jetokal)”. Please include a reference for this statement and state the detection limit of each microbial quantitative test.
Response: I have added the references to the revised manuscript you mentioned and indicated the microbial detection limits.

Lines 108-109: Please explain the reason for the collection/inoculation only of the upper layer. Also replace “~0.1 mL” by “approximately 0.1 mL”.
Response: During the preliminary experiment, after homogenization, some red spots from the shrimp shells were inadvertently mixed into the agar, which resulted in errors in counting. 

To minimize experimental errors, only the supernatant was separated and used for the experiment. As per your suggestion, this has been changed to ‘approximately’.

Lines 127-133: Please include relevant information concerning the samples of raw and subsidiary materials (shrimp, salt and empty bottles - packaging materials) and surfaces studied, such as the number of samples by type, manufactured with or without HACCP certification, and the performed microbial tests.
Response: As stated in section 2.2 of the manuscript, we investigated the saeu-jeot manufacturing processes of HACCP-certified facilities located in Hongseong, Buan, and Ganggyeong.

Line 141: Please describe the strains of S. aureus tested (eg. reference strains and/or wild strains isolated from…).
Response: The S. aureus strain used in the experiment was a standard and commercial strain obtained from the ATCC. Due to strain variations in S. aureus, 5 strains were each cultured and then used as a mixture in the experiment.

Line 142: Please describe the meaning of the abbreviation CFU.
Response: I have provided additional details regarding the microbial experiments. The abbreviation has been appropriately defined in the revised manuscript (Lines 116).

Line 155: Please better explain how experiments and/or testing were performed in triplicates.
Response: I have provided additional details regarding the microbial experiments performed in the analysis. (Lines 133-136).

3. Results and Discussion
Lines 171-175: For total viable bacteria the results for “samples manufactured with and without HACCP certification was 3.8 ± 0.1 and 3.9 ± 0.1 log CFU/g, respectively (p > 0.05). ...This difference could be because...”. Please rephrase, as if p > 0.05, no differences were verified for the parameter of total viable bacteria.  
Response: I have added a sentence indicating that no significant difference was observed.

 Lines 190-191: Figure 2 caption, please include the number of samples for both groups (“Certificated” and “Normal”).   
Response: I have labeled Figure 2 with numbers appropriate to the figure.

Lines 218-233: Please consider changing these two paragraphs to the section of “Material and methods” as the results only begin with the presentation of the three CCP of figure 4.   Lines 237-238: Figure 4 caption, please explain the meaning of abbreviations (eg, CCP-1P, CCP-2B, CCP-3P and SUS, Fe).  
Response: I have depicted the CCP processes commonly utilized by HACCP-certified manufacturing facilities. The caption for Figure 4 now reflects the content mentioned in the manuscript.

 Lines 239-244: Please revise the units of results for surfaces (CFU/cm2?) and subsequent discussion.  
Response: I have revised the unit accordingly.

Line 272-273: “The count of S. aureus in the upper layer was 6.1 ± 0.0 log CFU/g and in the lower layer was 4.7 ± 0.1 log CFU/g (Table 3).” Please present table 3.  
Response: Since there were only three data points for this matter, I have expressed it in a sentence. Additionally, Table 3 has been removed from the revised manuscript.

 4. Conclusion   Please revise, as some statements are not supported by the results (eg. “… it can be concluded that the salting and UV-C treatment processes can effectively inhibit the growth of microorganisms without heat treatment in saeu-jeot”). 
Response: I have revised the content based on the research results, changing the term 'reduction' to 'inhibition' and making other appropriate adjustments to the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors analyzed the risks associated with the production and consumption of traditional fermented saeu-jeot shrimp.

The manuscript is interesting, but I think it is of low scientific importance. The researchers determine the presence of microorganisms in commercially available products, which is important, especially since they observed an improvement in the quality of saeu-jeot, and they are investigated a method that, according to the manuscript, is already used in the industry, although rarely.

If the manuscript is accepted for further processing, it must be corrected.

Introduction:

In the methodology, the authors specify that the products came not only from Korea but also from China and Vietnam, maybe it would be worth supplementing the paragraph (l: 58-63) with some information about the quality of the products and Haccap systems also from these countries.

Line 63 is missing a citation

Material and methods:

Figure 1 does not contribute anything to the manuscript

Line 106- Jetokal- reference should be provided, it is impossible to find it using search websites

The authors determined the presence of V. parahaemolyticus, which is typical for this geographical location, so despite the lack of confirmation, why was it limited to determining the effectiveness of UV-C only for S. aueus if only ATTC strains were worked on?

Results

Lines 178-179-what does it mean that detection limit is 1 log CFU/g or less

In Figure two and text I think using word normal- referring not certificated plant is unfortunate, please change

Line 205- other ingredients, please specify, from introduction or figure 4 we do not expect addition of any other ingredients

Table 1. evidence of bacteria were investigated so it should be less than…. (limit of method) not “ND” which is used for detection in 25g.

Line 269- general bacteria- please change for total counts/ total viable or just lower number of bacteria

Conclusions

Authors used strong defined effect of UV-C as effective- which for me doesn't correspond with results, I would rewrite the sentence to indicate that is more likely to inhibit growth than actual reducing or slightly reducing

References

All references are old, only 16.6% of references is published after 2014

Author Response

The authors analyzed the risks associated with the production and consumption of traditional fermented saeu-jeot shrimp.

The manuscript is interesting, but I think it is of low scientific importance. The researchers determine the presence of microorganisms in commercially available products, which is important, especially since they observed an improvement in the quality of saeu-jeot, and they are investigated a method that, according to the manuscript, is already used in the industry, although rarely.

If the manuscript is accepted for further processing, it must be corrected.

Introduction:

In the methodology, the authors specify that the products came not only from Korea but also from China and Vietnam, maybe it would be worth supplementing the paragraph (l: 58-63) with some information about the quality of the products and Haccap systems also from these countries.
Response: I have explored the CODEX specifications and Chinese standards, and included a brief description of them in the Introduction section of the revised manuscript (Line 58–64)

Line 63 is missing a citation
Response: I have added a citation regarding Korean food-related regulations (Reference 5).

Material and methods:

Figure 1 does not contribute anything to the manuscript
Response: Due to the fact that saeu-jeot is a traditional food consumed in some Asian countries and not widely recognized globally, Figure 1 was deemed necessary to enhance readers' understanding of the nature of saeu-jeot.

Line 106- Jetokal- reference should be provided, it is impossible to find it using search websites
Response: I have added a citation regarding Korean food-related regulations in the revised manuscript (Reference 5).

The authors determined the presence of V. parahaemolyticus, which is typical for this geographical location, so despite the lack of confirmation, why was it limited to determining the effectiveness of UV-C only for S. aueus if only ATTC strains were worked on?
Response: Typical colonies of S. aureus were detected in commercial products, while V. parahaemolyticus was not detected. This is described in lines 182-185. For this reason, we conducted UV-C experiments on S. aureus.

Results
Lines 178-179-what does it mean that detection limit is 1 log CFU/g or less
Response: The detection limit is 1.0 log CFU/g. It was expressed after a 10-fold dilution and converted to a logarithmic scale. However, since the results were expressed in terms of the mean after conducting repeated experiments, the values are less than 1.0 log CFU/g.

In Figure two and text I think using word normal- referring not certificated plant is unfortunate, please change
Response: I agree with your opinion. Thank you for pointing out this error. I have removed the term ‘normal’ and replaced it with ‘not standardized’ (see Figure 2).

Line 205- other ingredients, please specify, from introduction or figure 4 we do not expect addition of any other ingredients
Response: In saeu-jeot, only shrimp and salt are used in the preparation of saeu-jeot without the addition of any other ingredients. However, according to the cited paper, contamination due to prolonged storage during the salting process for salted foods was mentioned, so I have revised the content to reflect this.

Table 1. evidence of bacteria were investigated so it should be less than…. (limit of method) not “ND” which is used for detection in 25g.
Response: As Table 1 represents surface analysis, it is appropriate to express it in cm2.

Line 269- general bacteria- please change for total counts/ total viable or just lower number of bacteria
Response: I have revised this to 'total viable bacteria’.

Conclusions

Authors used strong defined effect of UV-C as effective- which for me doesn't correspond with results, I would rewrite the sentence to indicate that is more likely to inhibit growth than actual reducing or slightly reducing
Response: UV-C and salting do not necessarily imply complete sterilization. However, it has been confirmed that these processes have inhibitory effects on the growth of pathogenic microorganisms. I have amended the manuscript accordingly. Please refer to lines 332-342.

References
All references are old, only 16.6% of references is published after 2014
Response: I have expanded the citation of papers published after 2014 from 16.6% to 50.0%. I hope that this change to the referencing within the manuscript is sufficient.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

The manuscript is improved, compared to the first version. However, many of the raised issues remain unanswered, in particular those concerned with methods and results, and still need to be properly addressed. So, please see below the specific comments:

 

Line 90: The expression “or less” is unclear “(detection limit 1.0 log colony-forming units [CFU]/g or less)”. As previously raised, the detection/quantification limits of each microbial methods need to be included. So, please also revise accordingly the section of results/discussion.

 

Line 91-92: Please clarify the sample preparation for all the microbial methods, as the sentence “To quantify the total viable bacterial population, a 10-g sample was homogenized in 90 mL of 0.1% buffered peptone water for 60 s” only refers total viable bacteria. Were samples prepared the same way for the other microbial methods? Please clarify and include all the information.

 

Line 92: “The homogenate was serially diluted”. Please specify which kind of dilution, decimal?

 

Lines 95-96: “S. aureus was cultured at 37 °C for 48 h after the test solution was spread on Baird–Parker agar medium (Difco BD Biosciences, Franklin Lakes, NJ, USA).” Which “test solution” (initial suspension) was used? Was it a primary decimal suspension of 10-g sample homogenized in 90 mL of 0.1% buffered peptone water? And what was the amount of “test solution” (initial decimal suspension) spread on Baird–Parker, also 0.1 mL?

 

Lines 145-146: The concerns related to statistical analysis were not adequately clarified/supported. How was data analysis performed for parameters with values below the quantification level? The expression of results remains incongruent and unclear. It is stated “The data are expressed as mean ± standard deviation”. So, why is data not presented as a bar chart, showing sample means with standard deviation, instead of boxplot with whiskers (showing sample mendians, quartiles and range), as shown in figure 2? Besides, the caption of figure 2 lacks specifying the meaning of the graphic presentation (Lines 180/181). Please complete.

 

Lines 168-169 “The amount of S. aureus in the samples detected (detection limit 1.0 log CFU/g or less) varied from 0.2 ± 0.2 to 2.5 ± 0.2 log CFU/g with an average of 0.6 ± 0.6 log CFU/g.” For example, in the case S. aureus quantification, if an initial suspension was prepared with 10-g sample plus 90 mL of diluent, 0.1 mL of the initial decimal suspension was spread on Baird–Parker plates and one colony was counted and confirmed, then the amount is 1x10x10 = 100 = 2 log CFU/g. In that case, it was not possible to have neither a detection limit of 1.0 log colony-forming units [CFU]/g nor of 0.2 log CFU/g... Please check/revise the presentation of results, in particular Figure 2b, supported by the description of the method, to include in the section “Material/Methods”.

 

These issues need to be adequately, explicitly and effectively addressed, in order to accept the manuscript for publication.

 

  Best Regards,   Reviewer

 

 

Author Response

Thanks for your comment.

Line 90: The expression “or less” is unclear “(detection limit 1.0 log colony-forming units [CFU]/g or less)”. As previously raised, the detection/quantification limits of each microbial methods need to be included. So, please also revise accordingly the section of results/discussion.

Response: We revised as your comments in Line 199.  

 

Line 91-92: Please clarify the sample preparation for all the microbial methods, as the sentence “To quantify the total viable bacterial population, a 10-g sample was homogenized in 90 mL of 0.1% buffered peptone water for 60 s” only refers total viable bacteria. Were samples prepared the same way for the other microbial methods? Please clarify and include all the information.

Response: All microbiological experiments were conducted using consistent method. We revised as your comments in Line 117.  

 

Line 92: “The homogenate was serially diluted”. Please specify which kind of dilution, decimal?

 

Response: A decimal dilution method was conducted in the microbiological experiment. We revised as your comments in Line 118.  

 

Lines 95-96: “S. aureus was cultured at 37 °C for 48 h after the test solution was spread on Baird–Parker agar medium (Difco BD Biosciences, Franklin Lakes, NJ, USA).” Which “test solution” (initial suspension) was used? Was it a primary decimal suspension of 10-g sample homogenized in 90 mL of 0.1% buffered peptone water? And what was the amount of “test solution” (initial decimal suspension) spread on Baird–Parker, also 0.1 mL?

 

Response: All microbiological experiments were conducted using consistent method. We revised as your comments in Line 117.  

 

Lines 145-146: The concerns related to statistical analysis were not adequately clarified/supported. How was data analysis performed for parameters with values below the quantification level? The expression of results remains incongruent and unclear. It is stated “The data are expressed as mean ± standard deviation”. So, why is data not presented as a bar chart, showing sample means with standard deviation, instead of boxplot with whiskers (showing sample mendians, quartiles and range), as shown in figure 2? Besides, the caption of figure 2 lacks specifying the meaning of the graphic presentation (Lines 180/181). Please complete.

Response: The 'Results and Discussion' section discusses the rationale behind utilizing the box plot. We revised as your comments in Line 209 to 212.

 Also, the graphs illustrating the mean and standard deviation of microbial contamination results were adjusted.

 

 

Lines 168-169 “The amount of S. aureus in the samples detected (detection limit 1.0 log CFU/g or less) varied from 0.2 ± 0.2 to 2.5 ± 0.2 log CFU/g with an average of 0.6 ± 0.6 log CFU/g.” For example, in the case S. aureus quantification, if an initial suspension was prepared with 10-g sample plus 90 mL of diluent, 0.1 mL of the initial decimal suspension was spread on Baird–Parker plates and one colony was counted and confirmed, then the amount is 1x10x10 = 100 = 2 log CFU/g. In that case, it was not possible to have neither a detection limit of 1.0 log colony-forming units [CFU]/g nor of 0.2 log CFU/g... Please check/revise the presentation of results, in particular Figure 2b, supported by the description of the method, to include in the section “Material/Methods”.

Response: All microbiological experiments were conducted using consistent method. We revised as your comments in Line 117.  

 

We have also made some corrections to errors.

 

Best Regards

Reviewer 4 Report

Comments and Suggestions for Authors

The authors answered all my concerns, and manuscript is visibly improved. 

Author Response

Thanks for your comments.

 

Best Regards.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

 

Dear Authors,

 

The manuscript is improved; however, many of the raised issues remain unanswered. Besides, the revision of the manuscript should be comprehensive and not only directed to a specific sentence/paragraph, as there is the risk of adjacent sentences/paragraphs remaining out of the context. So, please make a critical revision of the overall document, as it presents several incongruences.

 

1) Concerning the detection limit the expression “or less” in “detection limit 1.0 log colony-forming units [CFU]/g or less” the questions wasn´t neither clarified nor answered. As previously raised, the detection/quantification limits of each microbial methods need to be included. Moreover, For example, in the case S. aureus quantification, if an initial suspension was prepared with 10-g sample plus 90 mL of diluent, 0.1 mL of the initial decimal suspension was spread on Baird–Parker plates and one colony was counted and confirmed, then the amount is 1x10x10 = 100 = 2 log CFU/g. In that case, it was not possible to have neither a detection limit of 1.0 log colony-forming units [CFU]/g, i.e 10 cfu/g nor of 0.2 log CFU/g... So, please also revise accordingly the manuscript.

 

2) Concerning data analysis performed for parameters with values below the quantification level questions wasn´t neither clarified nor answered. Besides, you state that “(All average data of experimental results from all samples, includes not detections)”. If you have a value below 1.0 log colony-forming units [CFU]/g, i.e <10 cfu/g, it means your sample can present between 0 and 9, so you could not make an average out of that range? So, if you had to make assumptions please explain and revise the manuscript accordingly.

 

3) There is text that after previous corrections needs critical revision; if “The average bacterial count contamination in samples manufactured with and without HACCP certification was 3.8 ± 0.1 and 3.9 ± 0.1 log CFU/g, respectively. There was no significant difference (p > 0.05).” ; If there is no difference the justification of finding differences is no more adequate (“This difference could be because, in recent times, most saeu-jeot is produced under established sanitary management systems. ”). Please revise the manuscript accordingly.

 

These issues need to be adequately, explicitly and effectively addressed, in order to accept the manuscript for publication.

 

 

Best Regards,

 

Reviewer

 

 

Author Response

We deeply appreciate your comments.

We were able to make revisions to the manuscript based on your comment.

Furthermore, we made some minor adjustments to enhance the completeness of the manuscript.

 

1) Concerning the detection limit the expression “or less” in “detection limit 1.0 log colony-forming units [CFU]/g or less” the questions wasn´t neither clarified nor answered. As previously raised, the detection/quantification limits of each microbial methods need to be included. Moreover, For example, in the case S. aureus quantification, if an initial suspension was prepared with 10-g sample plus 90 mL of diluent, 0.1 mL of the initial decimal suspension was spread on Baird–Parker plates and one colony was counted and confirmed, then the amount is 1x10x10 = 100 = 2 log CFU/g. In that case, it was not possible to have neither a detection limit of 1.0 log colony-forming units [CFU]/g, i.e 10 cfu/g nor of 0.2 log CFU/g... So, please also revise accordingly the manuscript.

Response: Thank you for raising this important point.

Based on the information you provided, we conducted the discussion. Ultimately, it was confirmed to be a 1 mL suspension. Thank you for pointing out the significant oversight, allowing us to make the necessary corrections.

We revised as your comments in Line 114. 

2) Concerning data analysis performed for parameters with values below the quantification level questions wasn´t neither clarified nor answered. Besides, you state that “(All average data of experimental results from all samples, includes not detections)”. If you have a value below 1.0 log colony-forming units [CFU]/g, i.e <10 cfu/g, it means your sample can present between 0 and 9, so you could not make an average out of that range? So, if you had to make assumptions please explain and revise the manuscript accordingly.

 

Response: As you mentioned, it could also be confirmed as 0 ~ 0.9 log CFU/g. However, excluding data below the detection limit from the entire sample may yield misleading information. Therefore, we assumed data below the detection limit as zero to conduct quantification of the entire sample.

I have noted this information in the manuscript to ensure clarity for the readers.

Please check Line 192 ~ 197

 

3) There is text that after previous corrections needs critical revision; if “The average bacterial count contamination in samples manufactured with and without HACCP certification was 3.8 ± 0.1 and 3.9 ± 0.1 log CFU/g, respectively. There was no significant difference (p > 0.05).” ; If there is no difference the justification of finding differences is no more adequate (“This difference could be because, in recent times, most saeu-jeot is produced under established sanitary management systems. ”). Please revise the manuscript accordingly.

Response: The applied or not applied HACCP systems results in differences in maximum total bacteria counts. and also, According to previous study(reference 15), it was confirmed that the maximum total bacteria counts have decreased.

We revised as your comments in Line 183 ~ 189.