Bacterial Biodiversity of Extra Virgin Olive Oils and Their Potential Biotechnological Exploitation

Round 1

Reviewer 1 Report

The manuscript microorganisms-669105 is focused on bacterial diversity detected in monovarietal olive oils. The authors used cultural-dependent and molecular methods to identify the bacterial isolates, and performed biochemical, enzymatic, biofilm and antibiotic susceptibility assay to characterize them. The results indicate that the olive oil may be a source of bacteria with potentially useful biotechnological applications. The experimental design was clearly illustrated in the manuscript, and the experiments were well conducted, leading to interesting findings.

I recommend this manuscript to be published, with some minor revisions.

The English used in the manuscript is good, but a careful re-read of the text is required to correct typos and formatting errors.

In the manuscript text the authors use terms such as “a rich microflora” (Introduction, line 30) or “bacterial microbiota” (Results and Discussion, line 185; line 189) to describe bacteria that occur in the olive oil samples. The presence of an “olive oil microbiota” is not generally accepted. I suggest replacing the above-indicated terms with “bacterial community”. In the paragraph “Isolation of bacteria from oil samples” (Materials and methods, ln. 67-68) the authors describe the process used to obtain the olive oils. However, there is no indication about the procedure to avoid/minimize environmental and/or human contamination. This should be reported in the revised manuscript. In the paragraph “DNA isolation, cluster analyses, and molecular characterization of bacteria”, details about the amplicon sequencing methods, and reference about the universal primers W001 and W002 should be provided. Please indicate the amplicon length (1500 bp) refers to E. coli 16S rRNA gene, and also specify the reason why 800/900 bp were aligned in spite of the available 1500 bp. Consistently with the experiments that were described, the paragraph title “3.3. Potential ability to survive in the gastrointestinal tract” (page 9, line 319) should be replaced by “Acid and Bile Salt Resistance of bacterial isolates”. Bacterial names and gene names should be in italics. The references have to be reported according to MPDI guidelines for the authors. Typing errors: Page 1, line 16 (for the first) Page 1, line 43 (a highly specific microflora) Page 2, line 54 (bacteria is are related) Page 2, line 75 (termophilic thermophilic bacteria; ,) Page 2, line 82 (Brain heart Heart Infusion Medium) Page 3, line 99 (Ribosomal Database project Project) Page 3, line 103 (as the substrate) Page 3, line 108-109 (to the hydrolysis) Page 3, line 128 (a 100 mL) Page 3, line 129 (were was added) Page 3, line 138 (accorded according) Page 4, line 144 (was were added) Page 4, line 160 (was were assessed) Page 4, line 174 (of the following) Page 4, line 177 (by the use) Page 4, line 188 (to Eelucidate) Page 4, line 189 (to the the bacterial) Page 5, line 194 (pression pressure) Page 7, line 250 (being is) Page 7, line 253 (plants,) Page 7, line 262 (who were was unable) Page 8, line 292 (in terms of lipolytic) Page 8, line 298 (in this case, bacteria) Page 8, line 299 (in particular,) Page 9, line 302 (gastrointestinal) Page 9, line 312 (as a signal) Page 10, line 394 (coagulase-negative) Page 12 lines 410-411 (due to the due to) Page 14 line 423 (In this work,) Page 14 line 425 (on the olive) Page 14 line 429 (growth-promoting)

Author Response

Reviewer 1

The manuscript microorganisms-669105 is focused on bacterial diversity detected in monovarietal olive oils. The authors used cultural-dependent and molecular methods to identify the bacterial isolates, and performed biochemical, enzymatic, biofilm and antibiotic susceptibility assay to characterize them. The results indicate that the olive oil may be a source of bacteria with potentially useful biotechnological applications. The experimental design was clearly illustrated in the manuscript, and the experiments were well conducted, leading to interesting findings.

I recommend this manuscript to be published, with some minor revisions.

The English used in the manuscript is good, but a careful re-read of the text is required to correct typos and formatting errors.

In the manuscript text the authors use terms such as “a rich microflora” (Introduction, line 30) or “bacterial microbiota” (Results and Discussion, line 185; line 189) to describe bacteria that occur in the olive oil samples. The presence of an “olive oil microbiota” is not generally accepted.  

I suggest replacing the above-indicated terms with “bacterial community”.

R. We really thank the reviewer for the suggestion, but both “rich microflora” and “olive oil microbiota” have been previously used by other authors and in our previous work (Food Microbiology 70, 2018, 65e75).

 

In the paragraph “Isolation of bacteria from oil samples” (Materials and methods, ln. 67-68) the authors describe the process used to obtain the olive oils. However, there is no indication about the procedure to avoid/minimize environmental and/or human contamination. This should be reported in the revised manuscript.

R. We really thank the reviewer. We add “under aseptic condition” in the new version of the manuscript

 

In the paragraph “DNA isolation, cluster analyses, and molecular characterization of bacteria”, details about the amplicon sequencing methods, and reference about the universal primers W001 and W002 should be provided.

R. We add the reference

 

Please indicate the amplicon length (1500 bp) refers to E. coli 16S rRNA gene, and also specify the reason why 800/900 bp were aligned in spite of the available 1500 bp.

R. We used 800 900 bp of 16s rDNA only because, after trimming e visually control of all sequences, is the portion of the sequence that perfectly overlaped after alignment. In the new version of the manuscript we used all 1500 pb of 16s rDNA as suggested by the reviewer. The new analysis, as expected, confirmed previous results.

 

Consistently with the experiments that were described, the paragraph title “3.3. Potential ability to survive in the gastrointestinal tract” (page 9, line 319) should be replaced by “Acid and Bile Salt Resistance of bacterial isolates”.

R. we replaced the title as suggested by the reviewer.

 

Bacterial names and gene names should be in italics.

R. We changed the names in italics. It was a problem of copy and paste.

 

 The references have to be reported according to MPDI guidelines for the authors.

R. We changed accordingly. It was also a problem of copy and paste.

 

Typing errors: Page 1, line 16 (for the first) Page 1, line 43 (ahighly specific microflora) Page 2, line 54 (bacteria is are related) Page 2, line 75 (termophilicthermophilic bacteria; ,) Page 2, line 82 (Brain heart Heart Infusion Medium) Page 3, line 99 (Ribosomal Database project Project) Page 3, line 103 (as the substrate) Page 3, line 108-109 (to the hydrolysis) Page 3, line 128 (a 100 mL) Page 3, line 129 (were was added) Page 3, line 138 (accorded according) Page 4, line 144 (was were added) Page 4, line 160 (was were assessed) Page 4, line 174 (of the following) Page 4, line 177 (by the use) Page 4, line 188 (to Eelucidate) Page 4, line 189 (to the the bacterial) Page 5, line 194 (pression pressure) Page 7, line 250 (being is) Page 7, line 253 (plants,) Page 7, line 262 (who were was unable) Page 8, line 292 (in terms of lipolytic) Page 8, line 298 (in this case, bacteria) Page 8, line 299 (in particular,) Page 9, line 302 (gastrointestinal) Page 9, line 312 (as a signal) Page 10, line 394 (coagulase-negative) Page 12 lines 410-411 (due to the due to) Page 14 line 423 (In this work,) Page 14 line 425 (on the olive) Page 14 line 429 (growth-promoting) 

R. thanks to the reviewer. We accepted many of the suggestions.

Reviewer 2 Report

The authors investigate the bacterial diversity associated to Italian Olive varieties. They identify and characterize bacterial isolates from the virgin olive oils. Furthermore they studied the enzymatic activity, the biofilm formation, the antimicrobial effect and the screening for tolerance to acidic pH and bile of the characterized isolates. However, the work is descriptive without major emphasize on the utility of these isolates. The manuscript is well done written and presented.

General comments:
1. The authors must better present experimental data that emphasize the potential of their isolates.
2. Study the novelty of the research: Compare the bacteial diversity described in this work to other described bacterial diversities of olives in other parts of the world.
3. Statistical analysis are missing in this work specially in activity description. About the experiments, some crucial information (on methods, statistics, data) are not provided.

The aim of the work is not well defended in the manuscript.

Specific comments:
Table2 : In enzymatic activity test, it is mentioned as ranked data N: No activity; W: Weak activity, M: Moderate activity; S: Strong activity. However, it should be presented as measurements and please check your results in this table.

(The same comment for Table 3 and 4).

Author Response

Reviewer 2

 

The authors investigate the bacterial diversity associated to Italian Olive varieties. They identify and characterize bacterial isolates from the virgin olive oils. Furthermore they studied the enzymatic activity, the biofilm formation, the antimicrobial effect and the screening for tolerance to acidic pH and bile of the characterized isolates. However, the work is descriptive without major emphasize on the utility of these isolates. The manuscript is well done written and presented.

General comments:
1. The authors must better present experimental data that emphasize the potential of their isolates.

R. We did some changes in the new version of the manuscript.

Study the novelty of the research: Compare the bacteial diversity described in this work to other described bacterial diversities of olives in other parts of the world.

R. Unfortunately, there are no many works on the bacterial microflora of olive oils, the only present in literature are cited in the manuscript. At the same time, to describe bacterial diversities of olives and not olive oil is, to our opinion, far from the aim of this work.

Statistical analysis are missing in this work specially in activity description. About the experiments, some crucial information (on methods, statistics, data) are not provided.

R. To our opinion, all the experiments are well described in the manuscript, and, when not described, the references related to the methods used are always indicated. For the statistical analyses, as our aim was not directed towards the evaluation of any statistical hypothesis about the significance of differences among strains, or their activities, we consider useless and not necessary any test of statistical hypothesis through statistical inference methods. Data reported in tables were intended to describe the phenotypic characteristics of the isolates, as shown below.

The aim of the work is not well defended in the manuscript.

R. We modified the aim in the new version of the manuscript.

Specific comments:
Table2 : In enzymatic activity test, it is mentioned as ranked data N: No activity; W: Weak activity, M: Moderate activity; S: Strong activity. However, it should be presented as measurements and please check your results in this table.

R. We used the ranking data according to the following published works on the same topic:

International Journal of Food Microbiology 75 (2002) 111 –118

Food Microbiology 23 (2006) 60–67

Food Microbiology 27 (2010) 1035e1042

Food Microbiology 27 (2010) 487e492

Food Microbiology 36 (2013) 70e78

Food Microbiology 70, 2018, 65e75

Food Microbiology 84 (2019) 103250

We checked the results of all the presented tables.

(The same comment for Table 3 and 4).

R. Please see the answer above.

Reviewer 3 Report

The topic of the present study is the characterization of the bacterial biodiversity in 15 extra olive oils samples obtained by equal number of Italian olive varieties. The 16S rDNA sequencing analysis for 51 bacterial isolates revealed that they mainly belonged to Bacillus spp. (17 isolates), Brevibacillus spp. (22 isolates), Micrococcus spp. (3 isolates), Staphylococcus spp. (4 isolates), Pantoea spp. (3 isolates), Lysinibacillus spp. (one isolate), Kocuria spp. (one isolate), and Lactobacillus spp (one isolate). Forty of the strains were identified at species level while the rest ones, at genus level only. The enzymatic activity of the isolates was evaluated with regard to β-glucosidase, β-glucanase, lipase, decarboxylase and catalase activity. Finally the biofilm formation, acidic and bile tolerance, as well as antimicrobial agent’s susceptibility was elucidated for all the above isolates in order to evaluate their probiotic potential as well as their safety issues.

 

Strength of the article:

The topic of the study has a merit as the olive oil microbiology has not yet been extensively studied in the literature.

 

Limitations of the article:

The study is rather simplistic and its design is relative superefficient. Taking into account the availability of experimental tools nowadays, the experimental methodology applied for the characterization of the isolates and the results obtained should be characterized as preliminary and more detailed experiments should be performed for the characterization of olive oil microbiota (e.g. by metagenomics). Correlation of the microbial enzymatic activity with the quality of the olive oil (e.g. by GC analysis), or in vitro evaluation of this enzymatic activity could improve the impact of the paper. Finally, in my opinion, the claims in Discussion section are not supported sufficiently by the results obtained in the study.

 

In the case of resubmission of this work, the author should take into account the following:

In the Abstract authors mention “All bacterial isolates were genotyped using RAPD and REP-PCR methods ….” but in the Materials and Methods section there is no mention to the REP-PCR analysis. The introduction section should be expanded, it is rather short. Please indicate from which plates each of the 51 isolates were isolated. The number of initial isolates is not reported in the manuscript (the authors mention only the number of the 51 isolated strains, obtained as representative of each cluster). In the Results section, there is no mention to the results of the RAPD fingerprint analysis and to the number of clusters obtained. The sum of the strains referred in lanes 200-206 as well as in Table 1, is 52 strains instead of 51 which the authors refer as the total number of the isolates analyzed. The format of Table 1 and Table 5 is rather confusing, it needs improvement. There is no mention in the manuscript of the possible correlation between olive variety and bacterial biodiversity. In lanes 290-296 authors analyze the importance of lipolytic activity without any other mention throughout the manuscript about the importance of the rest enzymatic activities examined in this study. In lanes 332 authors mention “… probiotic potential” but the experiments performed are very few to support such a claim. In the Discussion section (lane 424) authors say: “ ….. is rich of different bacterial species” This characterization is rather overvalued taking into account the number of different bacterial species isolated. The Latin names of the microbes are not in italics.

Author Response

Reviewer 3

 

The topic of the present study is the characterization of the bacterial biodiversity in 15 extra olive oils samples obtained by equal number of Italian olive varieties. The 16S rDNA sequencing analysis for 51 bacterial isolates revealed that they mainly belonged to Bacillus spp. (17 isolates), Brevibacillus spp. (22 isolates), Micrococcus spp. (3 isolates), Staphylococcus spp. (4 isolates), Pantoea spp. (3 isolates), Lysinibacillus spp. (one isolate), Kocuria spp. (one isolate), and Lactobacillus spp (one isolate). Forty of the strains were identified at species level while the rest ones, at genus level only. The enzymatic activity of the isolates was evaluated with regard to β-glucosidase, β-glucanase, lipase, decarboxylase and catalase activity. Finally the biofilm formation, acidic and bile tolerance, as well as antimicrobial agent’s susceptibility was elucidated for all the above isolates in order to evaluate their probiotic potential as well as their safety issues.

 

Strength of the article: 

The topic of the study has a merit as the olive oil microbiology has not yet been extensively studied in the literature.

 

Limitations of the article:

The study is rather simplistic and its design is relative superefficient.

R. I am very sorry with the reviewer but we don’t agree with this description of our work. Even considering that at the moment this is one of the first work on this topic. We understand that this work could be preliminary but for sure not “simplistic”.

Taking into account the availability of experimental tools nowadays, the experimental methodology applied for the characterization of the isolates and the results obtained should be characterized as preliminary and more detailed experiments should be performed for the characterization of olive oil microbiota (e.g. by metagenomics).

R. Thanks for the suggestion, but we not agree with the reviewer that judged our work preliminary only because a metagenomic approach has not been used (other available experimental tools have not been cited). Techniques have to be applied in the right way depending on the matrix that has to be analyzed. Olive oil is characterized by low microbial biomass (this is the reason why we used the enrichment method), so the utilization of metagenomics in samples characterized by low microbial biomasses only brings to cross-contaminations and confounding results https://doi.org/10.1016/j.tim.2018.11.003., but, and this is the primary reason, it was not the experimental design of this work. Anyway, our idea of work it was to use the olive oil as reservoir of microorganisms with biotechnological exploitations. So, the culture-dependent method, and the plethora of assays done gave us a clear picture of the bacterial biodiversity present in olive oils and particularly, as already underlined, the possibility to use the isolated bacteria for biotechnological scopes.

Correlation of the microbial enzymatic activity with the quality of the olive oil (e.g. by GC analysis), or in vitro evaluation of this enzymatic activity could improve the impact of the paper.

R. We thanks the reviewer but the aim of this work was primary the utilization of the olive oil as reservoir of biodiversity for biotechnological scopes. For this reason, we consider this correlation as out of the scope of this work. Maybe this correlation could be the topic of the next research in this field.

Finally, in my opinion, the claims in Discussion section are not supported sufficiently by the results obtained in the study.

R. In the results and discussion we just described the different species found and, in order to reinforce the discussion itself, we made some hypotheses about their biotechnological exploitations.

In the case of resubmission of this work, the author should take into account the following:

In the Abstract authors mention “All bacterial isolates were genotyped using RAPD and REP-PCR methods ….” but in the Materials and Methods section there is no mention to the REP-PCR analysis.

R. Thanks to the reviewer. We add the missing part in the M&M.

 

The introduction section should be expanded, it is rather short.

R. Thanks to the reviewer for the suggestion, but to have concise introduction is our style.

 

Please indicate from which plates each of the 51 isolates were isolated.

R. If the reviewer is referring to the media used, they are indicated in the paragraph 2.1.

 

The number of initial isolates is not reported in the manuscript (the authors mention only the number of the 51 isolated strains, obtained as representative of each cluster).

R. We reported the initial number of the isolates in the new version of the manuscript (see beginning of paragraph 3.1)

 

In the Results section, there is no mention to the results of the RAPD fingerprint analysis and to the number of clusters obtained.

R. See paragraph 3.1 new version of the manuscript.

 

The sum of the strains referred in lanes 200-206 as well as in Table 1, is 52 strains instead of 51 which the authors refer as the total number of the isolates analyzed.

R. We changed the number of isolates

 

 The format of Table 1 and Table 5 is rather confusing, it needs improvement.

R. The reviewer is totally right but it was not our fault. Once I pasted the tables in the word format of the journal, the tables changed. I resolved the problem in the new version of the manuscript.

 

There is no mention in the manuscript of the possible correlation between olive variety and bacterial biodiversity.

R. I really thank the reviewer for the suggestion, but for this kind of correlation we should have the at least three years of harvest, and in this work, we analyzed only one season. Anyway, there is a sentence that postulate possible correlations in the conclusions in lines 446-447, that, although not really exhaustive, but, at least says something about possible correlations.

 

In lanes 290-296 authors analyze the importance of lipolytic activity without any other mention throughout the manuscript about the importance of the rest enzymatic activities examined in this study.

R. Considering the number of the isolates characterized by lipolytic activity, and for its important in different biotechnology fields, we concentrate our attention only on that enzymatic activity. Anyway, we add some info about the other enzymatic activities in the new version of the manuscript.

 

In lanes 332 authors mention “… probiotic potential” but the experiments performed are very few to support such a claim.

R. In fact it is potential, and for us it is not a claim. There are hundreds of papers that, performing the same type of experiments, use the same way to say.

 

In the Discussion section (lane 424) authors say: “ ….. is rich of different bacterial species” This characterization is rather overvalued taking into account the number of different bacterial species isolated.

R. We changed the sentence and cancelled “rich”.

 

The Latin names of the microbes are not in italics. 

R. done.

 

Round 2

Reviewer 3 Report

My main objection with this manuscript is that the experimental methodology applied and the results obtained for the characterization of the isolates as well as for the evaluation of their biotechnological and probiotic potential is rather preliminary, for publication in a journal of the level of Microorganisms with an impact factor around 4. On the other hand I have to mention that the topic is interesting and the manuscript is well written. Finally, my minor corrections and suggestions were done by the authors.

As my initial suggestion was the rejection of the article, mainly due to the above mentioned reason, I would not like to review it again.