Heterotrophic Plate Count for Bottled Water Safety Management
Round 1
Reviewer 1 Report
Line 84. The author listed six (6) collection stages for the processed samples to equal 20, however, there are only five (5) listed in the table and they equal to 20. The raw samples aren't listed. And when they are listed the total number will have to be amended in line 84.
Line 100. Is is suppose to be "analyzed for..."
Lines 107, 109. Extra spaces after Bactident
Lines 147, 148. Reported 450 and 266 CFU/mL so how are these numbers lower than 100 CFU/mL?
Line 158. It might be clearer to the reader if the author begins the sentence with "On the TSA agar, the bacterial morphotypes..." since earlier in the paragraph there was a distinction made for the GSP agar.
And since bottled water is use, should processed water but used instead of process water?
Author Response
We would like to take this opportunity to express sincere thanks to the Reviewers who identified areas of the manuscript in need of correction and modification. Our point-by point answers are presented bellow:
Line 84. The author listed six (6) collection stages for the processed samples to equal 20, however, there are only five (5) listed in the table and they equal to 20. The raw samples aren't listed. And when they are listed the total number will have to be amended in line 84.
Corrections have been done. L: 86; Table 1
Line 100. Is is suppose to be "analyzed for..."
It has been corrected. L: 115-116
Lines 107, 109. Extra spaces after Bactident
It has been corrected.
Lines 147, 148. Reported 450 and 266 CFU/mL so how are these numbers lower than 100 CFU/mL?
Thank you very much for your suggestion. This text has been modified. L: 151-156
Line 158. It might be clearer to the reader if the author begins the sentence with "On the TSA agar, the bacterial morphotypes..." since earlier in the paragraph there was a distinction made for the GSP agar.
Thank you very much for your suggestion. This text has been modified. L: 175-177
And since bottled water is use, should processed water but used instead of process water?
The term „process water” has been changed into „processed water” in the manuscript
Reviewer 2 Report
The manuscript is very interesting, references are correctly selected and used correctly, however, it needs to be rewritten and completed before being allowed to be published.
2. Materials and Methods
2.1. Water analysis
At what intervals did the samples be taken from the production plant installation? Were there several days between sampling or was it the same day?
The number of samples given in the manuscript text does not match with the information given in Table 1. I am asking for information on how many water samples from the installation were collected: how many still mineral water samples, how many lightly carbonated water samples and how many carbonated water samples, separately. At what stage of the installation is the water saturated with carbon dioxide?
Why was the water sample filtered through a 0.45 μm-pore-size filter before the microbiological test?
2.3. Assessment of bacterial adhesion
Why the samples were incubated with agitation on a laboratory shaker since research has shown that the largest population of tested microorganisms in the installation is located in places where water stands? How do the results of these experiments translate into reality?
3. Results and discussion
3.1. Bacteriological analysis of process water and bottled water
How to explain the fact that CO2 treatment does not reduce the HPC 37oC population, but only the HPC 22oC (see figure 3)? In my opinion, the results should also be commented on with regard to at what stage of the production process carbonation of water occurs? Please comment on the results obtained.
3.2. Adhesion abilities
No discussion of the results obtained. The obtained results should be commented on with reference to what I wrote above (to point 2.3.).
Biofilm formation depends on the size of the bacterial population. How to relate the obtained results of the described experiments to the confirmed population of bacteria in industrial conditions? In my opinion, this should be discussed.
Autorzy piszą, że „According to the 202 literature, biofilm formation may be stimulated in water environments poor in carbon sources [24]”. How does this relate to the fact that water with varying carbon dioxide levels has been studied? How much does carbon dioxide saturation affect biofilm formation? In my opinion, this should be discussed.
Author Response
We would like to take this opportunity to express sincere thanks to the Reviewers, who identified areas of the manuscript in need of correction and modification. Our point-by point answers are presented bellow:
The manuscript is very interesting, references are correctly selected and used correctly, however, it needs to be rewritten and completed before being allowed to be published.
- Materials and Methods
2.1. Water analysis
At what intervals did the samples be taken from the production plant installation? Were there several days between sampling or was it the same day?
The number of samples given in the manuscript text does not match with the information given in Table 1. I am asking for information on how many water samples from the installation were collected: how many still mineral water samples, how many lightly carbonated water samples and how many carbonated water samples, separately. At what stage of the installation is the water saturated with carbon dioxide?
Thank you very much for these questions. The text of manuscript has been changed and modified.
In the installation only one type of water was processed. Carbonization took place just before the bottling. L: 86-90, Table 1
Why was the water sample filtered through a 0.45 μm-pore-size filter before the microbiological test?
This sentence has been corrected. L: 103
2.3. Assessment of bacterial adhesion
Why the samples were incubated with agitation on a laboratory shaker since research has shown that the largest population of tested microorganisms in the installation is located in places where water stands? How do the results of these experiments translate into reality?
Thank you very much for this suggestions. In this study we have made following assumption: if bacteria have the ability to biofilm formation with regular water flow (9rotary shaker), then in industrial conditions, when water stagnates, the formation of biofilms will be even more likely. We have discussed this problem L: 237-240.
- Results and discussion
3.1. Bacteriological analysis of process water and bottled water
How to explain the fact that CO2 treatment does not reduce the HPC 37oC population, but only the HPC 22oC (see figure 3)? In my opinion, the results should also be commented on with regard to at what stage of the production process carbonation of water occurs? Please comment on the results obtained.
Thank you very much for your suggestion. It helped us to identify this weak point of discussion. We have corrected and completed this text. L: 154-167
3.2. Adhesion abilities
No discussion of the results obtained. The obtained results should be commented on with reference to what I wrote above (to point 2.3.).
Biofilm formation depends on the size of the bacterial population. How to relate the obtained results of the described experiments to the confirmed population of bacteria in industrial conditions? In my opinion, this should be discussed.
Authors write: „According to the literature, biofilm formation may be stimulated in water environments poor in carbon sources [24]”. How does this relate to the fact that water with varying carbon dioxide levels has been studied? How much does carbon dioxide saturation affect biofilm formation? In my opinion, this should be discussed.
Discussion has been corrected and extended. L: 224-247
Reviewer 3 Report
I think the research topic is interesting;
Title: biofilm should be mentioned in the title;
Abstract:
Line 13-15:I tink the phrase is not conceptually correct: not critical points that contaminate the final product, but the critical points where contamination occurs or increases;
Material and Methods
It's known that the ability of bacteria to form biofilms depends on the genus, species and microbial strain, but also on the type of material that makes up surface of the equipments;
The authors should have carried out samples on the filters, on the equipment and any sediments;
The authors should have tested the formation of biofilms by isolated strains, not only on plastic, but also on the materials that make up the filters, tanks, etc.
In addition, they would have to test ATCC strains to have a comparison with isolated strains;
I think the research design is not appropriate.
Rsults and Discussion
Line 133-135: the filters, tanks, etc. were probably not clean;
The authors should have had information on the cleaning and sanitization procedures of the plant.
I think that the manuscript may be more appropriate and useful for a national journal.
Author Response
We would like to take this opportunity to express sincere thanks to the Reviewers, who identified areas of the manuscript in need of correction and modification. Our point-by point answers are presented bellow:
Abstract:
Line 13-15:I tink the phrase is not conceptually correct: not critical points that contaminate the final product, but the critical points where contamination occurs or increases;
Thank you for your suggestion. It has been corrected. L: 14-15
Material and Methods
It's known that the ability of bacteria to form biofilms depends on the genus, species and microbial strain, but also on the type of material that makes up surface of the equipments;
The authors should have tested the formation of biofilms by isolated strains, not only on plastic, but also on the materials that make up the filters, tanks, etc.
Thank you for your suggestion. These issues have been discussed. L: 230-236
The authors should have carried out samples on the filters, on the equipment and any sediments;
Authors had limited possibilities of taking water samples from the entire plant installation. Samples were taken from places provided by the manufacturer.
In addition, they would have to test ATCC strains to have a comparison with isolated strains; I think the research design is not appropriate.
According to our experience wild isolates are more adhessive than refference strains cultivated for a long time in laboratory conditions. Therefore we had limited our experiments to wild bacterial strains
Rsults and Discussion
Line 133-135: the filters, tanks, etc. were probably not clean;
The authors should have had information on the cleaning and sanitization procedures of the plant.
I think that the manuscript may be more appropriate and useful for a national journal.
The information about sanitization procedures has been included. L: 83-84
According to our knowledge, there are no articles describing both the process and quality of the final products. In our opinion, the article contains valuable process, technological and scientific data.
Round 2
Reviewer 3 Report
I'm not totally agree with the authors, but they have sufficiently integrated the manuscript
