Microbial Diversity of Traditional Livno Cheese from Bosnia and Herzegovina

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have done a good piece of work. However, a major revision is needed before publication. The paper lacks a detailed description of the cheese production technology, considering that this is an important aspect that can influence the microflora of the product, the authors should include a description of the production technology in the text. 

Lines 101-102: What are the differences between the 3 productions? Which seasons were taken into account?

Line 173: Explain LAB

Line 209: Which LAB are mentioned in the table? Lactobacilli? explain

Line 273: Use either lactic acid bacteria or LAB, standardise in text and tables

Line 522: What does biofilm transmission mean? Can only be transmitted by biofilm formation? 

I advise the authors to revise and implement the conclusions, which seem obvious. 

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Reviewer,

please find our reply to your report bellow, and as a word document in attachment too.

Thank you.

Best,

Tarik Dizdarević.

Reviewer 1:

The authors have done a good piece of work. However, a major revision is needed before publication. The paper lacks a detailed description of the cheese production technology, considering that this is an important aspect that can influence the microflora of the product, the authors should include a description of the production technology in the text. 

Answer:

Dear reviewer,

we have done all necessary changes you proposed. As for cheese technology we didn't want to occupy to much space since one should be concise but we fully agree and understand that cheese production technology can influence the microflora of product. Therefore, we put into work description of cheese technology.

Lines 101-102: What are the differences between the 3 productions? Which seasons were taken into account?

Following cheese production processs and taking samples from three different producers and two seasons we tried to take into account differences due to -from day to day- variations in chemical and microbiological properties of cheese milk as well as cheese technology which normaly occour between producers and seasons. Two consecutive seasons were followed. The season of Livno cheese production is late spring-summer period.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate this work being done over two seasons. However this type of research is not new; there are now hundreds of similar papers that have been published over the last 10 years or so.

I have serious concerns  how  mesophilic cocci (lactococci, pediococci) and thermophilic cocci (streptococci) were enumerated. Firstly M17 is not a selective agar medium for lactic acid bacteria, many bacteria can grow on it. There are also  issues but less serious ones in using MRS even if it is acidified. My concern is that the authors show no awareness of these well known issues and have not controlled for them e.g. presumptive identification of colonies using basic microbiological tests.

It is disappointing that the authors have not adequately compared their results with the results of other similar studies.

Comments on the Quality of English Language

While the English could be improved it is satisfactory.

Author Response

Dear Reviewer,

please find our reply to your report bellow, and as a word document in attachment too.

Thank you.

Best,

Tarik Dizdarević.

Reviewer 2 – answers

Reviewer 2:

I appreciate this work being done over two seasons. However this type of research is not new; there are now hundreds of similar papers that have been published over the last 10 years or so.

I have serious concerns  how  mesophilic cocci (lactococci, pediococci) and thermophilic cocci (streptococci) were enumerated. Firstly M17 is not a selective agar medium for lactic acid bacteria, many bacteria can grow on it. There are also  issues but less serious ones in using MRS even if it is acidified. My concern is that the authors show no awareness of these well known issues and have not controlled for them e.g. presumptive identification of colonies using basic microbiological tests.

It is disappointing that the authors have not adequately compared their results with the results of other similar studies.

Answer:

Dear reviewer,

we accept all suggestions and we have made all required changes and additions. We did comparation with results of other authors.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The objective of this research was to examine the microbial biodiversity of the traditional Livno raw milk cheese made by various producers, during ripening and from two production seasons using culture-dependent and independent methods.

It is interesting to know the microbial composition of this traditional cheese, which does not use microbial starters, to identify the dominant microorganisms and develop an autochthonous starter. Highlighting the use of two different molecular methods (DGGE and ARISA) to determine the microbial compositions and compare it to culture methods is interesting.

Nevertheless, I would have appreciated it if the authors had included the determination of physicochemical or sensorial properties of the cheese of the different producers to link some typical characteristics of these cheeses with the abundance of different bacteria groups.

Below you will find some comments to improve the manuscript:

Abstract

L24. Clarify which culture-independent methods are used in the present research

L26. At the end, a summary of the advantages and disadvantages of the use of culture-dependent and culture-independent methods in the evaluation of Livno cheese microbiota… as the not detection of Enterococcus by culture-independent methods.

Introduction

L62-63. Correct microorganisms' names by adding cursive and upper letter

Material and methods

L112. How was it plated?

L119. Explain better the type of samples…Livno cheese curd, fresh salted cheese, and cheeses during ripening.

L121. Remove point.

L124. Add incubation temperature and time of aerobic bacteria enumeration.

L128. Correct the degree symbol, and review it in all the document

L129. Add point

L130. Correct Enterococci to Enterococcus

L131. Add the number of samples that were analysed at each sampling time. n?

L150. Replace spp. by subsp.

L155. Remove point

Results

L201. Replace by culture-dependent methods.

L207. What is the meaning of the asterisk?

Table 1. What is the distribution of microbial enumeration in each sampling time?

Correct Enterococci to Enterococcus

In data, replace comma by point

L235. Correct number of table

Table 2. Correct identification

L239. Review microorganism name

L245. Correct format

L250. Revise writing

L273. Correct PCR_DGGE

Figure 2. Revise the format of the index, include a space between genera and species, and correct the name of Enterococcus

L287. Revise the sentence because the 1st season presents a higher presence of Lc. garvieae than in the 2nd season.

L294. Specify if you are talking there about the last days of ripening because in the curd a higher presence of S. thermophilus we observe in producer B.

L296. Remove point

L299. Could you refer to L. plantarum in this sentence?

L304. Add cursive

L303-306. Detail that it occurred in the 1^st season

L308. Highlight the increase of S. thermophilus during ripening in the 2^nd season

L309-310. Could you refer to S. faecium in this sentence?

Discussion

L319. Does it mean at the end of 90 days of ripening?

L320. Explain if the value is the average of the 3 producers

L324. It is not the correct abbreviation

L331. Remove point

L342. It is necessary to describe the initials the first time that appear in the text

L373. Enterococci to Enterococcus

L387. Correct logarithmic units

L393. Add cursive

L395. Correct E. fecium to E. faecium

L423. Correct lover

L435. Replace spp. by subsp.

L445. Develop more the motive why was Enterococcus not detected.

L484. Add cursive

L494. Add a point

L504. I think that you refer there to cultivation-dependent methods.

L515. Remove point

L518. Remove one point

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

please find our reply to your report bellow, and as a word document in attachment too.

Thank you.

Best,

Tarik Dizdarević.

Reviewer 3:

The objective of this research was to examine the microbial biodiversity of the traditional Livno raw milk cheese made by various producers, during ripening and from two production seasons using culture-dependent and independent methods.

It is interesting to know the microbial composition of this traditional cheese, which does not use microbial starters, to identify the dominant microorganisms and develop an autochthonous starter. Highlighting the use of two different molecular methods (DGGE and ARISA) to determine the microbial compositions and compare it to culture methods is interesting.

Nevertheless, I would have appreciated it if the authors had included the determination of physicochemical or sensorial properties of the cheese of the different producers to link some typical characteristics of these cheeses with the abundance of different bacteria groups.

Answer:

We agreed with the last part but we didn't include it in this manuscript because of limited space and high quantity of results. Here we focused on studying of microbial biodiversity. A comprehensive research as we did requires more than one paper to elaborate all results and we feel that more results of different type to the ones we have presented would jeopardize the clarity of investigation results. We find reviewers thinking reasonable and logical and hope this explanation is justified, clear and understandable.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors revised the article and corrected all my comments. I believe that the article can be accepted for publication in its current form.

Comments on the Quality of English Language

Moderate editing of English language required

Reviewer 2 Report

Comments and Suggestions for Authors

I have serious concerns about the cultural studies. Other aspects are good. Unless they can conform that they have done the necessary work to confirm their isolates there is a real problem with this paper.

Work is not novel but this is an Editorial issue.

Author Response

Dear Reviewer 2.

Different nutrient media and incubation temperatures were used for the isolation of 1200 isolates of bacteria from traditional Livno cheese. 

During our experiment, we did the Catalase test and Gram staining. After number of 1200 isolates was reduced on 360. All 360 isolates were tested on:

• ability to produce CO2,
• the growth ability at different temperatures (10 ºC and 45 ºC)
• the growth ability at different salt concentrations (2%, 4.5% and 6.5% NaCl).

Also technological characterization of the isolated strains (results not shown) was examined based on the acidification rate of the isolates in milk. From a total of 360 isolates was seelected 200 microbial isolates and sequenced with 16 S rRNA.

From the total number of sequenced isolates, it was determined that the dominant microbial groups of Livno cheese were enterococci (36.50%), lactobacilli (31.00%), pediococci (11.50%), lactococci (8.50%), streptococci ( 7.00%) and leuconostoc (5.50%).

About vancomycin concentration use for Leuconostoc spp. we used MRS with addition of 20 µg/ml (20 mg/l) vancomycin.

Best,

Reviewer 3 Report

Comments and Suggestions for Authors

After introducing my suggestions in the manuscript and receiving your allegations, I have considered to accept your manuscript.

Only, I have identified some text editing mistakes:

L189: “itial denaturing program 95 °C for 5 min, 30 denaturation cycles at 95 °C for 30 sec, an-”. add the correct sec abbreviation

L438: “by PCR: E. faecium (71.2%), E. durans (20, 3%) and E. faecalis (8.5%) On the contrary, re-”. Change comma to a full stop in the value and add full stop at the end of the sentence.

Author Response

Dear Reviewer 3., text editing mistakes were corrected