Enterotoxigenic Staphylococcus aureus in Brazilian artisanal cheeses: Occurrence, counts, phenotypic and genotypic profiles.

The present study aimed to assess the occurrence and counts of Staphylococcus aureus in Brazilian artisanal cheeses (BAC) produced in five regions of Brazil: Coalho and Manteiga (Northeast region); Colonial and Serrano (South); Caipira (Central-West); Marajó (North); and Minas Artisanal cheeses, from Araxá, Campos das Vertentes, Cerrado, Serro and Canastra microregions (Southeast). The resistance to chlorine-based sanitizers, ability to attach to stainless steel surfaces, and antibiogram profile of a large set of S. aureus strains (n = 585) were assessed. Further, a total of 42 isolates were evaluated for the presence of enterotoxigenic genes (sea, seb, sec, sed, see, seg, sei, sej, and ser) and submitted to typing using pulsed-field gel electrophoresis (PFGE). BAC presented high counts of S. aureus (3.4-6.4 log CFU/g), varying from 25 to 62.5%. From the S. aureus strains (n = 585) assessed, 16% could resist 200 ppm of sodium hypochlorite, whereas 87.6% produced strong ability to attach to stainless steel surfaces, corroborating with S. aureus ability to persist and spread in the environment. Furthermore, the relatively high frequency (80.5%) of multidrug-resistant S. aureus and the presence of enterotoxin genes in 92.6% of the strains is of utmost attention. It reveals the lurking threat of SFP that can survive when conditions are favorable. The presence of enterotoxigenic and antimicrobial-resistant strains of S. aureus in cheese constitutes a potential risk to public health. This result calls for better control of cheese contamination sources, and taking hygienic measures is necessary for food safety. More attention should be paid to animal welfare and hygiene practices in some dairy farms during manufacturing to enhance the microbiological quality of traditional cheese products.

High throughput screening of technological and biopreservation traits of a large set of wild lactic acid bacteria from Brazilian artisanal cheeses.

This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).

Amplicon sequencing reveals the bacterial diversity in milk, dairy premises and Serra da Canastra artisanal cheeses produced by three different farms.

In this work, the amplicon sequencing of the 16 S rRNA gene was employed to investigate the bacterial diversity in ingredients, processing environment, and ripened cheeses collected from three farms producing Serra da Canastra artisanal cheese. The data obtained indicated a remarkable variability in the bacteria consortia of the milk, whey, and environmental samples collected in farms 1, 2, and 3, despite their location in the same city. On the other hand, the starter culture and final product (ripened cheese) presented more constant and similar microbiota no matter the farm. The findings suggest that Streptococcus and Lactococcus have competitive advantages throughout Serra da Canastra cheese-making/ripening, which is crucial for their high relative abundance in the final products. An exploratory assessment based on sequencing data available in the literature showed that the Serra da Canastra cheeses sequences clustered with specific cheese varieties that are also made from raw milk but ripened for very different periods. The findings of this study highlight that despite the variability of milk and whey microbiota among the three farms, the starter culture ("pingo") has strong relevance in shaping the microbiota of the final product.

Brazilian Artisanal Cheeses: An Overview of their Characteristics, Main Types and Regulatory Aspects.

A large variety of cheeses exist in Brazil, reflecting historical and cultural aspects. Brazilian artisanal cheeses present differences in the processing, ripening time (when applied), type of milk used, texture, size, shape, color, moisture content, flavor, the use or not of starter cultures, among others. This review describes the main artisanal cheeses produced in Brazil, focusing on general and particular characteristics associated with their making process and geographical identity. Overall, the high variability of the physicochemical data and deficiency of information on sensorial properties of Brazilian artisanal cheeses were noticed. On the other hand, culture-dependent methods were mostly used to expand the knowledge into the microbiology of these cheeses, whereas their microbial diversity has been recently discovered through the use of 16S rRNA gene sequencing-based methods. The certification of a geographical indication for Brazilian artisanal cheeses may encompass an essential milestone for adding value to these products. Regardless of their significance in the diet, culture, and economy of producing regions, taken together, the reviewed literature discloses the need of insightful studies to generate scientific data to support the expansion of the market, while ensuring the protection of historic aspects related to the production of Brazilian artisanal cheeses.

Brazilian cheeses: A survey covering physicochemical characteristics, mineral content, fatty acid profile and volatile compounds.

Chemical characteristics, mineral levels (Ca, Fe, K, Mg, Na, Zn, Cr, Cu, and Mn), fatty acid profile and volatile compounds of typically Brazilian cheeses (Minas Frescal, Minas Padrão, Prato and Coalho, n = 200, equally distributed) were investigated. The cheeses have proven to be a source of Ca, Cu and Zn (>15% RDI/30 g), with low atherogenic and thrombogenic indices, with ethanol as the main alcohol and butanoic acid as the main acid, and moderate to high sodium content. Minas Frescal cheese presented lower nutritional value (proteins, lipids, and minerals), while Prato cheese had a higher fatty acids concentration, including conjugated linoleic acid (0.013 g 100 g-1). Coalho cheese had a higher acetic acid level, while citric acid and lactic acid predominated in Minas Padrão cheese. The results provide essential information for Brazilian consumers, demonstrated that the manufacturing conditions are heterogeneous and suggested that a standardized manufacturing protocol for dairy processors is needed.

Selection of indigenous lactic acid bacteria presenting anti-listerial activity, and their role in reducing the maturation period and assuring the safety of traditional Brazilian cheeses.

Artisanal raw milk cheeses are highly appreciated dairy products in Brazil and ensuring their microbiological safety has been a great need. This study reports the isolation and characterization of lactic acid bacteria (LAB) strains with anti-listerial activity, and their effects on Listeria monocytogenes during refrigerated shelf-life of soft Minas cheese and ripening of semi-hard Minas cheese. LAB strains (n = 891) isolated from Minas artisanal cheeses (n = 244) were assessed for anti-listerial activity by deferred antagonism assay at 37 °C and 7 °C. The treatments comprised the production of soft or semi-hard Minas cheeses using raw or pasteurized milk, and including the addition of selected LAB only [Lactobacillus brevis 2-392, Lactobacillus plantarum 1-399 and 4 Enterococcus faecalis (1-37, 2-49, 2-388 and 1-400)], L. monocytogenes only, selected LAB co-inoculated with L. monocytogenes, or without any added cultures. At 37 °C, 48.1% of LAB isolates showed anti-listerial capacity and 77.5% maintained activity at 7 °C. Selected LAB strains presented a bacteriostatic effect on L. monocytogenes in soft cheese. L. monocytogenes was inactivated during the ripening of semi-hard cheeses by the mix of LAB added. Times to attain a 4 log-reduction of L. monocytogenes were 15 and 21 days for semi-hard cheeses produced with raw and pasteurized milk, respectively. LAB with anti-listerial activity isolated from artisanal Minas cheeses can comprise an additional barrier to L. monocytogenes growth during the refrigerated storage of soft cheese and help shorten the ripening period of semi-hard cheeses aged at ambient temperature.