Lactic Bacteria in Artisanal Cheese: Characterization through Metagenomics

Round 1

Reviewer 1 Report

The manuscript sent by Erhardt M.M, et al describes the presence of Lactic acid bacteria in artisanal cheese from southern region of brazil by using metagenomis. In my opinion, the ms is well conducted, nevertheless this reviewer has some doubts and questions that require answers from the authors.

Please describe the Sentinel pipeline used to process the sequence data. 

Which quality controls were performed on the raw sequence? 

Which software and database were used for OTU taxonomic assignment? 

Rarefaction curves were checked?

Please indicate in materials and methods which correlation test was applied. 

Please check the naming of the microorganisms and the errors in them throughout the text.

Minor questions:

Which types of cheese were sampled (soft, hard, etc)? 

Please define artisanal cheese? Do producers use raw milk and spontaneously fermentation or they use commercial starter cultures from international or local producers? 

Does the cheese production have a cultural influence from Dutch or Italian producers, regarding the use of L. lactis vs S. thermophilus?

Author Response

Manuscript ID: 2109525

To: Fermentation

Manuscript Tittle: Lactic bacteria in artisanal cheese: characterization through metagenomics

Subject: Answers to reviewers’ comments

Dear Editors and Reviewers,

We greatly appreciate the contributions made to the review in our manuscript. They made our work even clearer and more complete to Fermentation readers. We believe that the article will significantly contribute with relevant information and data for the scientific community, especially focused on bacterial characterization, mainly lactic acid bacteria, in artisanal cheeses.

The suggested corrections and complementary information are presented in the same order as reviewers suggested. All changes made are highlighted in yellow in the manuscript submitted for re-analysis.

Best Regards,

Prof. Dr. Magnolia Erhardt – corresponding author

1. Please describe the Sentinel pipeline used to process the sequence data.

Thank you for the comment. By default, the bioinformatics pipeline performs the analysis against its own database. The sequence bank for the 16S rRNA genes has complete gene sequences (mostly), which contain sequences retrieved from genomes, unambiguous and filtered for chimera sequences. More detailed description of the methodology used in the treatment of NGS data was added in item 2.3. Metagenomic analysis, in line 148-164.

2. Which quality controls were performed on the raw sequence?

Thank you for the comment. Fastq files are evaluated for Phred quality (QP) using the FastQC v.0.11.8 program (ANDREWS, 2010). Next, the fastq files are subjected to primer and sequence trimming with low quality (Phred < 20). The proprietary software used for this purpose was built in Python v.3.6, which is inspired by the features of the BioPython project (COCK et al. 2009). As the data is paired-end, before the trimming step, two pairs of files (R1 and R2) are merged into a single file using pandaseq v.2.11 (MASELLA et al., 2012). Clusters with abundance less than 5 are removed from the analysis, as such structures are usually related to chimera sequences (SMYTH et al., 2010).

REFERENCES

ANDREWS, S. FastQC: a quality control tool for high throughput sequence data. 2010. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc.

COCK, P. J. A et al. Biopython: freely available Python tools for computational molecular

biology and bioinformatics. Bioinformatics, v. 25, n. 11, p. 1422–1423, 2009.

MASELLA, A. P. et al. PANDAseq: paired-end assembler for illumina sequences. BMC

Bioinformatics, v. 13, n. 31, 2012.

QUAST C. Et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl. Acids Res., v. 41, n. D1, D590-D596, 2013.

SMYTH, R. P. et al. Reducing chimera formation during PCR amplification to ensure accurate genotyping. Gene, v. 469, p. 45–51, 2010.

3. Which software and database were used for OTU taxonomic assignment?
   Thank you for the comment. Taxonomic identifications are performed with blastn v.2.6.0+ (ALTSCHUL et al., 1990), using a proprietary database from the company Neoprospecta as reference. As for the definition of a species, among the 20 hits returned for each cluster, an instruction in Python evaluates whether one of the three requirements would be met by the hits:

1) higher bit-score; 2) lower e-value; and 3) taxonomies with greater representation. At

species are defined using 99% identity. Hits that met one of the previous items were chosen as the representative species. The analyses of DMD Bacteria were performed against reference databases for 16S rRNA genes.

The sequence banks that the laboratory uses for 16S rRNA genes have complete gene sequences (mostly), which contain sequences retrieved from genomes, unambiguous and filtered for chimera sequences.

REFERENCES

ALTSCHUL et al. Basic local alignment search tool. J. Mol. Biol., v. 215, n. 3, p. 403- 410, 1990.

4. Rarefaction curves were checked?

Thank you for the suggestion. The rarefaction curve was checked and it revealed that the total diversity assessed by the 16S rRNA analysis was high in the artisanal cheese samples. The rarefraction curve approached a plateau at the end of the curve, reaching a clear saturation, indicating that there was a good representation of the microbial community throughout the samples, as most of the abundant species are represented with some rare species. This result shows that the collected samples were sufficient to represent the bacterial diversity of the local artisanal cheese. The rarefaction curve image has been added as supplemental material and information added in the abstract (line 23-24), methodology (line 161-164), results (line 269-275) and conclusion (line 480-483). This analysis brought greater reliability to the results. We thank you for your contribution.

5. Please indicate in materials and methods which correlation test was applied.

Thank you for the suggestion. In item 2.4 - Statistical analysis (line 194-195) the following sentence was added: Spearman's non-parametric correlation test was used with a 95% confidence interval (version 8.2, GraphPad Software, Inc., La Jolla, CA).

6. Please check the naming of the microorganisms and the errors in them throughout the text.

Thank you for the comment. The nomenclatures were revised.  

7. Which types of cheese were sampled (soft, hard, etc)?
   Thank you for the comment. Para deixar mais claro o perfil das amostras de queijo coletadas adicionamos no item 2.2 Sample collection (line 119-120) as informações do tipo de queijo - macio e a classificação quanto a umidade - alta umidade. Com essas informações caracterizamos melhor o tipo de queijo analisado.

8. Please define artisanal cheese? Do producers use raw milk and spontaneously fermentation or do they use commercial starter cultures from international or local producers?

Thank you for the comment. To clarify the concept of artisanal cheese here in Brazil, we added to the text (introduction - line 55-59) the description of this type of cheese based on the most current Brazilian legislation Decree No. 11,099, of June 21, 2022. This decree describes the concept of artisan cheese. Producers who sell artisanal cheese in southern Brazil use raw milk to produce this dairy product. In addition, they use commercially acquired starter cultures to help, along with the indigenous population present in raw milk, the fermentation process. This information was also added to the text in item 2.2 Sample collection (line 119-120).

9. Does the cheese production have a cultural influence from Dutch or Italian producers, regarding the use of L. lactis vs. S. thermophilus?

In Rio Grande do Sul State, the immigration cycle took place from 1875 to 1914, where 84,000 Italians settled, mainly from Lombardy, Veneto and Tyrol. Today, Brazil has 31 million descendants (Scarparo, 2011).

Specifically, in the southern region of Brazil, the influence is Italian, although the Germans arrived in the region in 1824 (coming from different regions of Germany). The group was made up of farmers, artisans and soldiers and, until the arrival of the Italians, they did not produce cheese (Rosa, 2005; Pauls et al., 2012; Costa, 2017).

In the present work, of the 13 municipalities where artisanal cheeses were collected, three of them (Lajeado, Arroio do Meio, Roca Sales) have influence from German colonization, and the others have mostly Italian.

The Dutch, in Brazil, immigrated, in the first phase between 1630 and 1654, they occupied part of the current Northeast region of Brazil, more specifically Pernambuco, however it is not known how many Dutch people lived in Brazil or how many remained after the resumption of the territory by the Portuguese. In the second phase of immigration, between 1946 and 1976, 6,098 Dutch immigrated to Brazil, less expressive than other groups of immigrants, but formed cooperatives and agricultural companies in all regions where they settled, collaborating with the development of milk production (1951) and the Brazilian economy (Gallas & Gallas, 2012).

In the present work, the cheeses were all produced with raw milk, therefore, they have the natural microbiota of the pastures consumed by dairy animals, and that is, the highest percentage found was of Lactococcus lactis, consistent with the climate of the region of the rural properties and also commercial starter cultures are added. The cheeses presented a native biodiversity, which has great potential to be used in the improvement of elaborated products.

Brazil still does not have the technology to isolate, prepare and market the lactic cultures naturally present in artisanal cheeses produced from fresh milk, because Brazilian regions have specific climates and microbiomes. For this reason, most cheeses made with pasteurized milk use freeze-dried culture from foreign companies, such as Chr. Hansen. To obtain the desired acidity, essential in the manufacture of artisanal cheeses, especially in the maturation stage, cultures of the CHOOZIT® MA type containing mesophilic and thermophilic microorganisms (Lactococcus lactis subsp. lactis and Streptococcus thermophilus) homofermentative that produce a mild flavor and adequate amount of diacetyl, are used in the preparation of semi-cooked cheeses.

To make the cultural influence on the production of the cheeses analyzed in this study clearer to the reader, we added some information in item 2.1 Characterization of the region in line 114-117.

REFERENCES

COSTA F. R. Colônias Italianas Dona Isabel e Conde d’Eu. 3ª ed. Porto Alegre: EST Edições. 2017. 480p. 1ª ed. Porto Alegre: EST Edições. 2005. 328p.;

GALLAS A.O.G., GALLAS F.D. Holandeses no Brasil, 100 anos de imigração positiva. São Paulo: Editora Gallas & Disperati, 2012. 273p.;

PAULS A., DEFRAIN A., VITECK A.S., VITECK H. Imigração Alemã no Paraná: 180 anos, 1829-2009. Marechal Cândido Rondon: Editora Germânica, 2012. 472 p.;

ROSA G.J. Imigrantes alemães. 1824-1853.

Reviewer 2 Report

The article is well-written and focused, and conforms to the aim and scope of the journal, so I suggest a minor revision of this manuscript. The article is generally well written, the English language is appropriate and understandable, and only minor spell check is required.

Line 369:  Please delete 'fresh'

The Conclusions section can be improved and include more relevant results. 

The number of bibliographic sources is adequate, more than 50% of the total bibliographic sources are from the last 5 years.

Congratulations, an interesting research article. Good results, with pertinent explications.

Author Response

Manuscript ID: 2109525

To: Fermentation

Manuscript Tittle: Lactic bacteria in artisanal cheese: characterization through metagenomics

Subject: Answers to reviewers’ comments

Dear Editors and Reviewers,

We greatly appreciate the contributions made to the review in our manuscript. They made our work even clearer and more complete to Fermentation readers. We believe that the article will significantly contribute with relevant information and data for the scientific community, especially focused on bacterial characterization, mainly lactic acid bacteria, in artisanal cheeses.

The suggested corrections and complementary information are presented in the same order as reviewers suggested. All changes made are highlighted in yellow in the manuscript submitted for re-analysis.

Best Regards,

Prof. Dr. Magnolia Erhardt – corresponding author

We thank you for your availability to revise the text of this article and we are grateful for the positive comments expressed. The team was very dedicated to carry out an excellent analysis of the results and present them in the best way and quality.

1. Line 369: Please delete 'fresh

Thank you for the suggestion. The word “fresh” has been deleted from the text.

2. The Conclusions section can be improved and include more relevant results.

Thank you for the comment and suggestion. The conclusion was simple and with little information about the results, we obtained in our study. Based on your observation, we were able to improve and make our main/best results clearer and more prominent. We believe that with the modifications made we have corrected the deficiency pointed out by the reviewer and enriched the conclusion of our work. The item has been entirely restructured.