Selection of Microbial Targets for Treatments to Preserve Fresh Carrot Juice

Round 1

Reviewer 1 Report

The manuscript provides information about the impact of storage on the physicochemical and microbial quality of carrot juices produced from local market carrots and company-provided carrots and also investigated their correlation with microbial indicators as a source of the juice spoilage. However, the paper does not read very well, and it needs correction through the manuscript to improve the writing. A better presentation of results will strengthen the manuscript. I believe, in general, the microbial part of the study needs more clarification on the methodology and the result& discussion sections.

There are a few things through the manuscript that need to be corrected:

Introduction: It needs more literature review.

Section 2.1: Did the carrots provided by the company are juiced similar to the lab-made juices? Or the company provided the carrot juices, namely “C1,C2,C3”?

Line 99: please have “e” and “c” as subscripts!

Table 1: are the values an average of lab-made or company batches? If not, please indicate which batches are you referring? L1,L2,…C1,C2,..?

Table 1: Was there any interaction between the type of carrot juice (lab or company) and storage days?

Figure 2: the quality of the figure is not good! Please provide a better quality!

Figure 2: In total, the F1 and F2 explain less than 60% variability? How do you explain this?

Table 2 is a little confusing! What does the “units of peak surface” mean? Also, please add “of carrot juices” in the table's caption after batches.

Table 2: I suggest having the cell named “batch” on top of the “L1,L2,L3,L4” AND in place of “Batch” write down the “volatile compounds”. This helps a better reading of the table.

Table 2: in this table, the compound changes are indicated as “▲ ▼ ≈” Could you explain why some of the compounds do not show any changes indicator? Also, please explain what the p-value is explaining.

Table 3: what are the codes before the name of bacterial isolates? Are there any differences for sources with similar titles? For example, there are two rows for juice, day of preparation or for juice, stored for 3 days! 

Table 3: what do the different colors mean?

Line 352: what are those 7 isolates?

Line 379: anoxic conditions or anoxia

Lines 386-391: Please explain what the codes like 31C6,..39D1 stand for.

Author Response

Comments and Suggestions for Authors

The manuscript provides information about the impact of storage on the physicochemical and microbial quality of carrot juices produced from local market carrots and company-provided carrots and also investigated their correlation with microbial indicators as a source of the juice spoilage. However, the paper does not read very well, and it needs correction through the manuscript to improve the writing. A better presentation of results will strengthen the manuscript. I believe, in general, the microbial part of the study needs more clarification on the methodology and the result& discussion sections.

We thank you for your careful reading and suggestions of improvement of our manuscript. Please see below detailed answers.

 There are a few things through the manuscript that need to be corrected:

Introduction: It needs more literature review.

All the data used from previous studies to describe the context and scientific questions were referred to a literature reference. We have more references in results and discussion.

Section 2.1: Did the carrots provided by the company are juiced similar to the lab-made juices? Or the company provided the carrot juices, namely “C1,C2,C3”?

The carrots bought to the company were already washed and shredded. Moreover, their origin from the Island was not certified. For that reason, we preferred consider the product as different, though carrots were juiced similarly than those from local market. A sentence was added in the materials and methods to make it clearer.

Line 99: please have “e” and “c” as subscripts!

Done

Table 1: are the values an average of lab-made or company batches? If not, please indicate which batches are you referring? L1,L2,…C1,C2,..?

The legend was modified.

Table 1: Was there any interaction between the type of carrot juice (lab or company) and storage days?

The batches were independent and independently stored, at 4°C. As indicated in materials and methods, one sample prepared at day 0 was discarded at each indicated time.

Figure 2: the quality of the figure is not good! Please provide a better quality!

The figure was enlarged as were the fonts and symbols.

Figure 2: In total, the F1 and F2 explain less than 60% variability? How do you explain this?

To reach 90% of representation of the variance of data we should have 9 axes, which cannot be represented. The third axis represented 8.2% of the variability. A sum of 60% for this kind of analysis is a good score.

Table 2 is a little confusing! What does the “units of peak surface” mean? Also, please add “of carrot juices” in the table's caption after batches.

The legend was modified and the content range was described with µg/100g butanol equivalent.

Table caption was modified.

Table 2: I suggest having the cell named “batch” on top of the “L1,L2,L3,L4” AND in place of “Batch” write down the “volatile compounds”. This helps a better reading of the table.

Thank you, the change was applied.

Table 2: in this table, the compound changes are indicated as “▲ ▼ ≈” Could you explain why some of the compounds do not show any changes indicator? Also, please explain what the p-value is explaining.

p-value indicates the significance of the difference of means, and the sign of change was indicated only if the variation was considered as significant. Table footer was modified to explain that.

Table 3: what are the codes before the name of bacterial isolates? Are there any differences for sources with similar titles? For example, there are two rows for juice, day of preparation or for juice, stored for 3 days! 

Thank you for your remark. We realized the table was not easy to read and modified it. The column titles were modified, as well as their order, and the table caption.

Table 3: what do the different colors mean?

This was added in the legend.

Line 352: what are those 7 isolates?

The number was corrected to 6 and reference to Table 3 added.

Line 379: anoxic conditions or anoxia

Modified.

Lines 386-391: Please explain what the codes like 31C6,..39D1 stand for.

These are our internal references, as indicated in table 4.

Reviewer 2 Report

Please see all my comments in the annotated manuscript attached. Globally Ithe study is well done and the results clearly presented. However, I do not understand why the spoiled juices with bad odor were discarded (lines 87 and 207), since the aim was to isolate microorganisms potentially involved in juice spoilage?

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

Please see all my comments in the annotated manuscript attached. Globally the study is well done and the results clearly presented. However, I do not understand why the spoiled juices with bad odor were discarded (lines 87 and 207), since the aim was to isolate microorganisms potentially involved in juice spoilage?

Your remark is fully relevant, but the spoiled juices were heterogeneous, with solidified parts, and it was not possible to work with them.

1. 49: log CFU/ml in clear when first used

Done.

1. 56 pathogens, which (with a comma before which)

Done.

1. 63 of raw material, which

Done.

1. 66 spores, which

Done.

1. 86-87 : why did you discard spoiled juices, since the aim was to isolate microorganisms potentially involved in juice spoilage ?

The spoiled juices were heterogeneous, with solidified parts, and it was unfortunately not possible to work with them.

1. 104 and 105: min

The change was applied.

1. 113: in water ?

It was methanol: this has been added.

1. 125: 70 eV

Done.

1. 139: PsyB to be suppressed (not re-used)

Right, removed.

1. 151: Please give the region of 16S rRNA gene targeted, the criteria of choice of primers and a reference

There was an error in primer sequences and names. This was corrected and the reference was added (Weisburg et al., 1991). Those primers lead to the amplification of a 1.4 kb region.

1. 164-169: Only primer concentration and PCR cycle changed, adjust section

This was modified.

1. 174 TAE in clear when first cited; Na2-EDTA 2 as indice

This was modified.

1. 193: Ryan, Einot, Gabriel, Welsh Studentized Range Q (REGWQ) Please briefly explain the choice of this tool for mean comparison

A sentence and a reference were added.

1. 207: Could you describe the type of "bad odor" perceived? Was this off-flavor accompagnied by pH changes or other changes?

Thank you. The qualifying term “rotting” was added, as well as texture change.

Was the off-flavor the same for company juice and the lab-made juices discarded?

Not obviously.

Table 1: Please give in the legend the number and type (analytical, biological...) of replicates

The legend was modified accordingly.

1. 237: Is color change considered as a defect? Is there a threshold of acceptable deltaE change?

As indicated in the introduction, a color change of carrot juice is among spoilage indicators. A color difference of 5 can be assigned to different colors (Mokrzycki, W. S., et M. Tatol. « Colour difference DELTA(E) - a survey ». Machine Graphics & Vision International Journal 20, no 4 (1 avril 2011): 383‑411).

1. 252: Please give the ranges observed in previous studies

This was added in text.

Figure 1: Would it be possible to find representations to facilitate the comparison between the four microbial groups targeted? For example in the current Figure 1, it is difficult to see which group is dominant over the time-course of storage.

Another graphical representation with bars was proposed, and the legend was modified accordingly.

1. 269: higher

Done.

1. 276: It could have been interesting to perform a multivariate analysis of all these data (pH, microbial counts, color change,...) to illustrate these observations, as done for volatile compounds. A PCA for example helps visualising correlations between variables.

We added a figure with PCA analysis: the figure shows clearly the observations reported.

L; 286: n=16

Done.

L; 290: and 12 compounds of other chemical families:

Done.

1. 298: I agree that F1 separated batch L4 from the other 3 batches, but it aslo separated replicates of batch L4, regardless of the storage time.

We agree with that remark, but it applies mainly for 7 days and, in a lesser extent, 10 days of storage.

Table 2: Please add the calculated RI and an identifier (e.g. the CAS number) for each compound. You may also add the odor/flavor found in flavornet or other databases

Retention index and CAS numbers were added in the table, as well as odor data if available. Sesquiterpenes A and B were identified as cis-γ-bisabolene and trans-γ-bisabolene.

1. 319: It is preferable to use However at the beginning of a sentence

Done.

1. 321: How do you explain that the concentration of terpenes increase over the storage time ? Could you give in Table 2, not only the statistical significance, but also the fold-changes observed? Which were the compounds that showed the greatest fold-changes?

We do not have hypotheses to explain the increase in some terpenes in some batches but this effect was already observed. Some sentences have been added (L. 359-362). We added in table 2 the percent of change. This is somewhat informative but some compounds were merely detected at the first day of storage and then, a huge increase of % was observed.

1. 322: was

Done.

1. 329: do you mean that these indicators are not enough concentrated to be used as potential early markers of microbial spoilage?

Exactly. The sentences were modified to make them clearer.

Table 3: It is not clear for me if these values are bacterial concentrations or increase of bacterial concentration. Do you have any idea of the initial concentrations added as inoculant?

The column title was modified. The inoculation of carrot juice (10 mL) was performed with 0.2 mL of microbial 48h-culture, which means an inoculation level of ca. 7 log(CFU/mL).

Column Color modification: here and everywhere in Table 3: it is not clear for me if this mention means that odor modification was observed for all the isolates cited in this line (here six isolates)?

A column was added and the table and column titles were modified to make it clearer.

1. 340: here and everywhere in Table 3: it is not clear for me if this mention means that odor modification was observed for all the isolates cited in this line (here six isolates)?

This is the case. A sentence was added (L. 367-368) and the table was modified.

1. 347: do you mean L. miyukkimchii ?

In Patterson et al (2012), they observed the species Leuconostoc kimchi.

1. 348: Each of the 30 isolates

The reported observation was for all tested isolates, not only those identified. The sentence was modified (actual L. 377).

1. 353: Could you describe this "terrible" odor? sulfurous? putrefaction odor? ...

We replaced “terrible” by “rotten”.

1. 356: delete "of"

Done.

1. 357: Why didn't you extend the storage time

The observed modifications were enough to conclude to spoilage ability.

1. 360: in two batches in which Leuconostoc sp. isolates were identified?

In juices inoculated with either Leuconostoc or Weissella isolated.

1. 361: has not been showed

Modified.

1. 366: 6?
2. 367: 10?

Yes, we modified that.

1. 368: of the 19

Done.

1. 369: to grow

Done.

1. 385: has been

Done.

1. 387: for

Done.

Table 4: why didn't you not the texture of this culture? I guess that a sticky juice is quite easy to detect?

We agree with you, but we prefer to indicate that it was not determined since this was not clearly detected.

1. 416: in

Done.

References:

9, 10, 11, 13, 14: the capital lettre used results in spelling errors for species names

14: PH

Thank you. This was caused by the use of automatic style. We checked all references and modified accordingly.

Reviewer 3 Report

The topic of the manuscript is very interesting and a lot of experimental work was done. Nevertheless, some things have to be clarified in order to improve the manuscript quality. Introduction Ln 59 Salmonella Typhimurium -> Salmonella Typhimurium (Italic) Materials and Methods Ln 91 Please, explain why you have used 0.05 M NaOH for measuring titratable acidity? Results and Discussion Ln 285 Please, mention it in the text which exactly were samples L1-L4. These which haven't changed their odor during storage or some other samples? Ln 318 According to me the number of compounds were 8, 7, 16 and 8, respectively. Table 3 Please, explain which batches you have used for isolation - lab or commercial, with or without odor changed Table 4 is written more understandable than Table 3

Author Response

Comments and Suggestions for Authors

The topic of the manuscript is very interesting and a lot of experimental work was done. Nevertheless, some things have to be clarified in order to improve the manuscript quality. Introduction

Ln 59 Salmonella Typhimurium -> Salmonella Typhimurium (Italic)

Typhimurium is the serotype and as such should not be italicized.

Materials and Methods Ln 91 Please, explain why you have used 0.05 M NaOH for measuring titratable acidity?

The concentration of sodium hydroxide was reduced so that titration was more accurate.

Results and Discussion Ln 285 Please, mention it in the text which exactly were samples L1-L4. These which haven't changed their odor during storage or some other samples?

Those batches were chosen because they exhibited different behavior during storage. A sentence was added (actual L. 311-313).

Ln 318 According to me the number of compounds were 8, 7, 16 and 8, respectively.

We checked that and modified.

Table 3 Please, explain which batches you have used for isolation - lab or commercial, with or without odor changed Table 4 is written more understandable than Table 3

We modified Table 3 to make it clearer. We preferred not to add isolation batches as it makes the tables unreadable and does not bring usable information.

Round 2

Reviewer 1 Report

The manuscript has been further improved and authors provided more information. Here are two comments that need to be addressed please:

Regarding Figures 2 and 3, the F1 and F2 components explain a smaller proportion of the total variation in the data set? Can authors explain this. 

Line 323: Please rephrase "Axes F1 and F2 represented 56.6% of data".

Author Response

The reviewer is right as in the two figures (numbered 2 and 3) the projection in the plan represented 50-60% of data variation. The sree plots did not show a clear elbow but rather a gradual decrease in the slope with the increase in the number of dimensions (see attached file).

A sentence was modified (L. 292-293) by replacing the verb "showed" by "indicated".

The sentence L. 323 has been modified as: Projection of variables in the plan former by the axes F1 and F2 represented only 56.6% of data variability.

Author Response File: Author Response.docx