Luana Martins Perin

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese.

To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity.

Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk.

The present study aimed to investigate the virulence, antibiotic resistance and biogenic amine production in bacteriocinogenic lactococci and enterococci isolated from goat milk in order to evaluate their safety. Twenty-nine bacteriocinogenic lactic acid bacteria (LAB: 11 Lactococcus spp., and 18 Enterococcus spp.) isolated from raw goat milk were selected and subjected to PCR to identify gelE, cylA, hyl, asa1, esp, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc genes. The expression of virulence factors (gelatinase, hemolysis, lipase, DNAse, tyramine, histamine, putrescine) in different incubation temperatures was assessed by phenotypic methods, as well as the resistance to vancomycin, gentamicin, chloramphenicol, ampicillin and rifampicin (using Etest®). The tested isolates presented distinct combinations of virulence related genes, but not necessarily the expression of such factors. The relevance of identifying virulence-related genes in bacteriocinogenic LAB was highlighted, demanding for care in their usage as starter cultures or biopreservatives due to the possibility of horizontal gene transfer to other bacteria in food systems.

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese.

Lactic acid bacteria (LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n=2) and Lactococcus lactis ssp. lactis (n=18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.

Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis.

BackgroundThe raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains.ResultsThe obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum.ConclusionsRaw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action.

Comparison of phenotypic and molecular tests to identify lactic acid bacteria

Twenty-nine lactic acid bacteria (LAB) isolates were submitted for identification using Biolog, API50CHL, 16S rDNA sequencing, and species-specific PCR reactions. The identification results were compared, and it was concluded that a polyphasic approach is necessary for proper LAB identification, being the molecular analyzes the most reliable.

Technological properties and biogenic amines production by bacteriocinogenic lactococci and enterococci strains isolated from raw goat's milk

Technological properties and biogenic amine production were analyzed in 56 bacteriocinogenic lactococci and enterococci strains isolated from raw goats milk. Fifteen lactococci strains were able to reduce milk pH to 5.3 or lower after 6 h, while enterococci strains were initially slow in producing acids. Lactococcus lactis subsp. lactis GLc06 and three strains of Enterococcus faecalis (GEn20, GEn22, and GEn23) presented high proteolytic activity. L. lactis subsp. lactis GLc06 and E. faecalis GEn22 also showed a high percentage of autolysis after only 4 h, reaching 71.11 and 97.67%, respectively, after 24 h. No strain was able to secrete exopolysaccharides, and L. lactis subsp. lactis GLc22 and 25 of the Enterococcus strains were able to produce diacetyl. L. lactis subsp. lactis GLc05 and 23 of the Enterococcus strains presented a high tolerance to NaCl at 10% (wt/vol). Regarding biogenic amine production, 12 strains (5 lactococci and 7 enterococci) were capable of forming tyramine and 4 strains (1 lactococcus and 3 enterococci) were capable of forming 2-phenylethylamine, but in very low amounts. GLc06 presented great acidifying, proteolytic, and autolytic activities, and GLc05 was capable of growing at high NaCl concentrations (10%, wt/vol), possessing medium autolytic and proteolytic activities. Some enterococci strains produced diacetyl and high autolytic and extracellular proteolytic activities and also presented resistance to high NaCl concentrations. The interesting technological properties presented by some bacteriocinogenic strains can justify their use by the dairy industry, with the aim of ensuring both safety due to bacteriocin production and technological transformations in fermented products.

Glucuronidase activity of Escherichia coli isolated from chicken carcasses

To identify Escherichia coli through the production of β-D-glucuronidase (GUD), 622 suspect cultures were isolated from chicken carcasses and plated in Petrifilm™ EC. Of these cultures, only 44 (7.1%) failed to produce GUD. This result indicates the usefulness of GUD production for estimating E. coli populations in chicken.To identify Escherichia coli through the production of β-D-glucuronidase (GUD), 622 suspect cultures were isolated from chicken carcasses and plated in PetrifilmTM EC. Of these cultures, only 44 (7.1%) failed to produce GUD. This result indicates the usefulness of GUD production for estimating E. coli populations in chicken.

Cow’s Milk Protein Allergy and Lactose Intolerance

Abstract Allergies to cow milk and lactose intolerance are problems that are increasingly being diagnosed by patients. This chapter addresses the main characteristics of both conditions, presenting their concepts, physiopathology, symptoms, diagnosis, and possible treatments for patients, aiming their recovering. Finally, the impacts and consequences of the exclusion diet are presented.

In vitro evaluation of the safety and probiotic and technological potential of Pediococcus pentosaceus isolated from sheep milk

Six isolates (Ac1Pd, Ac3Pd, Ac4Pd, Ac5Pd, Ac7Pd, and Ac22Pd) of Pediococcus pentosaceus from sheep milk were tested for safety and for probiotic and technological potential. The results showed that none of the isolates were able to produce biogenic amines or virulence factors. The isolates tested showed low hydrophobicities, high auto-aggregation capacities and co-aggregation with L. monocytogenes ATCC 7644, L. sakei ATCC 15521 and E. faecalis ATCC 19444, but none produced β-galactosidase and bacteriocins. The isolates did not show growth at pH values 3 and 12, while in a pH range from 4 to 10 the growth was variable. In the absence of bile, all the isolates showed growth, with suppression at bile concentrations of 0.1%, 0.3%, 0.6% and 1.0%. In the disc-diffusion test, the isolates tested were resistant to oxacillin, sulfatrimethoprim and vancomycin but were sensitive to chloramphenicol and tetracycline. The isolates showed variable responses to penicillin G and were resistant to most of the drugs tested, except for amoxicillin trihydrate and ibuprofen. All cultures showed a high milk-acidification capacity after 24 hours and none produced exopolysaccharides. The isolates of P. pentosaceus were able to produce diacetyl; however, no culture showed extracellular proteolytic activity and the autolysis varied from 21.3% to 30.5% after 24 h. The isolates grew at NaCl concentrations of 4.0 and 6.0%, but the growth was lower at 10.0%. Finally, all the isolates were found to be safe but had limited application as probiotics and in some technological uses.

The Relevance of Biogenic Amines in Dairy Products

Abstract Biogenic amines (BA) are nitrogenous compounds involved in natural biological processes, and their main exogenous sources are fermented foods and beverages. In foods BA are formed by microorganisms, such as yeast, Gram-negative and positive bacteria capable to decarboxylate amino acids. In dairy products the main BA formers are lactic acid bacteria. The intake of foods with high BA contents constitutes a health hazard and BA formation and accumulation should be avoided mainly by good hygienic practices as well as usage of high quality of raw material. In the case of fermented products, it is important to choose as starters or coadjutants strains that are not BA-forming. This chapter will present current information concerning the occurrence of BA in dairy products, mainly in cheeses, and the health risks and strategies to avoid BA accumulation in foods.