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).

Ohmic heating processing of milk for probiotic fermented milk production: Survival kinetics of Listeria monocytogenes as contaminant post-fermentation, bioactive compounds retention and sensory acceptance.

The survival kinetics of Listeria monocytogenes (9 log CFU/mL) as a post-fermentation contaminant in probiotic fermented milk (Lactobacillus acidophilus La-5, 8-9 log CFU/mL) processed with milk subjected to ohmic heating (0, 4, 6, and 8 V/cm; CONV, OH4, OH6, OH8, 90-95 °C/5 min) was investigated using Weibull predictive model. Additionally, the presence of bioactive compounds (antioxidant activity, inhibition of the enzymes α-glucosidase, α-amylase, and angiotensin-converting) and sensory analysis (consumer test) of probiotic fermented milks were evaluated. Overall, OH provided a decrease in the viability of Listeria monocytogenes, suitable Lactobacillus acidophilus counts, and satisfactory results in the gastrointestinal tract survival. The Weibull model presented an excellent fit to the data of all conditions. Furthermore, lower δ values (217-298 against 665 h, CONV), and increased R2 values (0.99 against 0.98, CONV) were obtained for the OH-treated samples, emphasizing the best performance of OH data. In addition, OH improved the generation of bioactive compounds as well as the sensory acceptance. Indeed, considering functional and safety purposes, OH presented as an interesting technology to be used in milk for manufacturing probiotic fermented milk.

Can sucrose-substitutes increase the antagonistic activity against foodborne pathogens, and improve the technological and functional properties of sheep milk kefir?

The effect of different types of sugar (sucrose, demerara, brown, fructose, coconut sugar, and honey) on sheep milk kefir was evaluated. Microbial counts (Lactobacillus, Lactococcus, Leuconostoc, yeast), antagonistic activity against foodborne pathogens, microstructure (scanning electron microscopy), and antiproliferative activity of cancer cells were evaluated. Furthermore, the antioxidant activity (DPPH), inhibitory activity of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase, lactose content, lactic and acetic acids and ethanol, fatty acid profile and volatile organic compounds were determined. The addition of sugars increased the Lactobacillus population (up to 2.24 log CFU/mL), metabolites concentration, antagonistic activity against pathogens, antioxidant activity (11.1 to 24.1%), ACE inhibitory activity (27.5 to 37.6%), α-amylase inhibition (18 to 37.4%), and anti-proliferative activity. Furthermore, it improved the fatty acid profile and volatile compounds. The results suggest that the replacement of sucrose with different types of sugar constitute an interesting option to be used in sheep milk kefir formulations.

Biopreservation and probiotic potential of a large set of lactic acid bacteria isolated from Brazilian artisanal cheeses: From screening to in product approach.

The increasing interest in products with functional properties has encouraged the search for new lactic acid bacteria (LAB) present in natural sources, as traditional fermented foods. In this study, a large set of wild LAB isolates (n = 220) from Brazilian artisanal cheeses (BAC) were assessed for their probiotic and biopreservation potential. First, the rational selection was performed. From the tested isolates, 92 (41.8 %) were resistant to low pH (2 and 3). These isolates were submitted to bile salt (0.4 % Oxgall powder) resistance, and 22 were selected and submitted to adhesion assays. The autoaggregation values ranged from 68.5-99% and were considered moderate to high (20-70 %). Hydrophobicity values varied significantly between LAB (5.0-64.3%), and seven isolates presented values higher than 40 %. All selected LAB (n=22) were capable of adhering to Caco-2 (> 70 %) cells, and none isolate displayed any tested gene for biogenic amine production. Most isolates (18/22) showed less than 1 log CFU reduction after passage through the simulated gastrointestinal tract (GIT) conditions. A total of twenty isolates satisfied key in vitro criteria to be considered as probiotics. A hierarchical cluster analysis (HCA) was performed, and two clusters were observed, showing high variability between Lactobacillus plantarum isolates regarding adhesion properties and survival to GIT stress and one influence of the source of isolation on these properties. After screening, the antimicrobial activity of Lb. plantarum (1QB77) was tested in microcheeses in which survival of two relevant pathogenic bacteria (Staphylococcus aureus and Listeria monocytogenes) was monitored along ripening and after a simulated GIT passage, concomitantly. L. plantarum (1QB77) has shown the ability to reach high counts (∼ 9 log CFU/g) at the end of ripening. This isolate was also able to reduce counts of S. aureus and L. monocytogenes in microcheeses in approx. 2.3 and 2.5 log CFU/g, respectively, until the 21st day of ripening; and about 3.2 and 3.5 log CFU/g after simulated GIT passage. Overall, the assessment of the probiotic properties of a large set of LAB was fundamental for gaining insights on the technological, functional, and potential regional traits of wild LAB isolates that can be used to develop starter cultures for tailored applications.

Brazilian artisanal cheeses are rich and diverse sources of nonstarter lactic acid bacteria regarding technological, biopreservative, and safety properties-Insights through multivariate analysis.

In this study a total of 220 isolates of lactic acid bacteria (LAB) recovered from 10 types of Brazilian artisanal cheeses marketed in 4 main regions of Brazil were evaluated regarding their safety and ability to produce diacetyl (a precursor of aromatic compounds), exopolysaccharides (EPS; from different sugar sources), and antagonistic activity against Listeria monocytogenes and Staphylococcus aureus. The results indicated that 131 isolates (59.6%) were classified as strong (40.5%) and moderate (19.1%) diacetyl producers; 28 isolates (12.7%) stood out due to their remarkable production of EPS from different sugars, including sucrose (3.2%), fructose (2.3%), lactose (2.3%), and glucose (6%). Furthermore, 94.1% and 95.9% of isolates presented antagonistic activity against S. aureus and L. monocytogenes, respectively, even though only 27 isolates (12.3%) exhibited positive results in the bacteriocin production test. None of the isolates tested presented hemolytic activity, and 117 were classified as safe, due to their intrinsic resistance to a maximum of 4 different antibiotics. The data obtained for assessment of antibiogram profile and technological potential (moderate and high production of diacetyl, EPS, and bacteriocins) were submitted to a multiple correspondence analysis to correlate them with the cheese of isolation. Regarding the antimicrobial profile of LAB strains, it was possible to verify an association between isolates from Minas artisanal cheeses from Araxá and resistance to tetracycline; Minas artisanal cheeses from Serro and resistance to erythromycin; Coalho and Minas artisanal cheese from Cerrado and resistance to penicillin; and isolates from Serrano and Colonial cheeses with clindamycin and ceftazidime resistance. Although the susceptibility of strains to these antibiotics was considered high (71.8-80.5%), these data may be related to the horizontal transfer of genes in the production chain of these cheeses. Results of multiple correspondence analysis also showed that isolates with antagonistic activity were mostly isolated from Manteiga, Colonial, and Coalho cheeses. The isolates with high or moderate EPS-producer ability from sucrose, glucose, and fructose were mainly associated with Minas artisanal cheeses from Cerrado. In contrast, isolates with high or moderate EPS-producer ability from lactose were isolated from Serrano, Minas artisanal cheeses from Canastra, and Campo das Vertentes microregions. Finally, isolates from Minas artisanal cheeses (from Araxá microregion), Coalho, and Caipira cheeses were associated with moderate/high diacetyl production. To the best of the authors' knowledge, this study provides, for the first time, data indicating that the dominant technological, biopreservative, and safety properties of LAB isolates can be correlated with the type of Brazilian artisanal cheeses, which denotes its singularity. This knowledge is of utmost relevance for the development of starter or adjunct cultures with tailored properties.

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.