Banca de QUALIFICAÇÃO: VINÍCIUS JOSÉ MOREIRA NOGUEIRA

Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.
DISCENTE: VINÍCIUS JOSÉ MOREIRA NOGUEIRA
DATA: 04/12/2025
HORA: 09:00
LOCAL: DMV08
TÍTULO:

Microbiome of Minas Artisanal Cheese Experimentally Contaminated with Brucella abortus during ripeness

PALAVRAS-CHAVES:

16S rRNA Sequencing, Microbial Diversity, Cheese Maturation, Food Safety, Brucellosis Control

PÁGINAS: 60
GRANDE ÁREA: Ciências Agrárias
ÁREA: Medicina Veterinária
SUBÁREA: Medicina Veterinária Preventiva
RESUMO:

The in-depth knowledge of food microbiomes is important, as fermented products are

intrinsically dependent on microbial activity for their production and unique

characteristics. Cheese stands out as a fundamental example of a food whose identity

is profoundly shaped by its microbiota, being more pronounced in artisanal cheeses.

Despite the benefits of the microorganisms present in these products, it is necessary to

monitor and prevent the maintenance of those considered pathogenic, such as Brucella

spp., which is present in Brazilian cattle herds and is transmitted through raw milk and

its derivatives. The objective of this study was to characterize the microbiota of Minas

Artisanal Cheese, produced under laboratory conditions and experimentally

contaminated with Brucella abortus, during the maturation process, in addition to

investigating the temporal interaction and the potential inter-relationship between the

artisanal cheese microbiota and the inoculated pathogen. Forty-two samples of Minas

Artisanal type cheese were produced with milk and whey starter ("pingo") from a farm

in the process of certification as brucellosis-free. These were experimentally

contaminated with 103 CFU/mL and 106 CFU/mL of Brucella abortus 2308, and the

cheeses underwent seven maturation times (1, 8, 15, 22, 28, 49, and 60 days). DNA

extraction was performed using the QIAamp DNA Microbiome Kit. High-throughput

paired-end sequencing of the V3/V4 regions of the 16S rRNA gene was performed by

Neoprospecta Microbiome Technologies, using the MiSeq Sequencing System.

Bioinformatic analysis included: quality control of reads (FASTQC and QIIME-2);

denoising with DADA2; removal of reads with a Phred score below 30; and classification

of ASVs using the SILVA 138 database. QIIME-2 data were exported to the R software,

where taxonomic classification and alpha and beta diversity analyses (without

rarefaction) were performed. For alpha diversity, the Shannon and Faith_PD indices

were used (Kruskal-Wallis Test). For beta diversity, Bray-Curtis dissimilarity and

Weighted UniFrac distance were used (PERMANOVA Test). The adopted statistical

significance level was p < 0.05. A total of 6,804,916 high-quality sequences were

obtained, resulting in 6,888 ASVs and 301 bacterial genera. 103 CFU/g Group: on day

1, the predominant genera (>1%) were Lactococcus, Leuconostoc, Staphylococcus,

Rothia, Lactiplantibacillus, Weissella, Streptococcus, and Enterococcus, with the top

three representing over 78%. From day 8 of maturation, a shift occurred, and

Lactiplantibacillus, Lactococcus, Lactobacillus, and Leuconostoc became the most

prevalent (>1%) until day 60. 106 CFU/g Group: this group followed a similar pattern but

with the presence of Brucella among the most prevalent. On day 1, Lactococcus,

Brucella, and Leuconostoc represented over 78% of the genera. On days 48 and 60 of

maturation, the concentration of Brucella decreased, accompanied by an increase in

Lactiplantibacillus and Lactococcus. Although the alpha diversity was numerically

different at the first maturation time point, it showed no statistical difference in any

analysis. However, in the beta diversity, a statistical difference was observed in the

Weighted UniFrac when comparing maturation days within each inoculum,

demonstrating that the initial concentration of Brucella profoundly impacted the

phylogenetic structure of the microbial community and drove a gradual substitution of

lineages throughout maturation. It was possible to confirm the presence of Brucella in

both groups. Maturation acts as a protective factor, as there was a decrease in the

Brucella DNA count overtime. Furthermore, its concentration apparently interferes with

the microbial ecology of other bacterial genera in Minas Artisanal type cheeses.

MEMBROS DA BANCA:
Externo ao Programa - ROBERTA HILSDORF PICCOLI - DCA/ESAL (Membro)
Presidente - ELAINE MARIA SELES DORNELES (Membro)
Externo à Instituição - DIRCÉIA APARECIDA DA COSTA CUSTÓDIO - UFLA (Suplente)
Interno - CARINE RODRIGUES PEREIRA (Membro)
Externo à Instituição - ANDREY PEREIRA LAGE - UFMG (Membro)
Interno - ALESSANDRO DE SA GUIMARAES - EMBRAPA (Suplente)