Biogenic Amine Content in Retailed Cheese Varieties Produced with Commercial Bacterial or Mold Cultures

Biogenic amines (BAs) are considered a potential microbiological toxicological hazard in aged cheese. Risk mitigation strategies include good hygiene practice measures, thermal treatment of milk and the use of competitive dairy cultures. The aim of this study was to evaluate the amount of BAs—tryptamine, β-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine—in the core and rind of cheeses ripened by bacteria (n = 61) and by mold cultures (n = 8). The microbial communities were counted, and the dominant lactic acid bacteria (LAB) were identified, corresponding to the BA concentrations. The total BA content was highest in the core of semi-hard cheeses (353.98 mg/kg), followed by mold cheeses (248.99 mg/kg) and lowest in hard cheeses (157.38 mg/kg). The highest number of BAs was present in the rind of cheeses with mold (240.52 mg/kg), followed by semi-hard (174.99 mg/kg) and hard cheeses (107.21 mg/kg). Tyramine was the most abundant BA, represented by 75.4% in mold cheeses, 41.3% in hard cheese and 35% of total BAs in semi-hard cheeses. Histamine was present above the defined European maximum level (ML) of 100 mg/kg in only two semi-hard and three hard cheeses. High amount of BAs (above 600 mg/kg) in cheeses, mainly tyramine, were associated with the presence of Enterococcus durans, while negligible BA concentrations were found in cheeses ripened with Lacticaseibacillus rhamnosus, Lactococcus lactis or Lacticaseibacillus paracasei cultures. This study has shown that retailed cheese varieties produced with commercial bacterial or mold cultures have acceptable levels of biogenic amines with respect to consumers.

Probiotic Potential Analysis and Safety Evaluation of Enterococcus durans A8-1 Isolated From a Healthy Chinese Infant

To evaluate the probiotic characteristics and safety of Enterococcus durans isolate A8-1 from a fecal sample of a healthy Chinese infant, we determined the tolerance to low pH, survival in bile salts and NaCl, adhesion ability, biofilm formation, antimicrobial activity, toxin gene distribution, hemolysis, gelatinase activity, antibiotic resistance, and virulence to Galleria mellonella and interpreted the characters by genome resequencing. Phenotypically, E. durans A8-1 survived at pH 5.0 in 7.0% NaCl and 3% bile salt under aerobic and anaerobic condition. The bacterium had higher adhesion ability toward mucin, collagen, and Bovine Serum Albumin (BSA) in vitro and showed high hydrophobicity (79.2% in chloroform, 49.2% in xylene), auto-aggregation activity (51.7%), and could co-aggregate (66.2%) with Salmonella typhimurium. It had adhesion capability to intestinal epithelial Caco-2 cells (38.74%) with moderate biofilm production and antimicrobial activity against several Gram-positive pathogenic bacteria. A8-1 can antagonize the adhesion of S. typhimurium ATCC14028 on Caco-2 cells to protect the integrity of the cell membrane by detection of lactate dehydrogenase (LDH) and AKP activities. A8-1 also helps the cell relieve the inflammation induced by lipopolysaccharide by reducing the expression of cytokine IL-8 (P = 0.002) and TNF-α (P > 0.05), and increasing the IL-10 (P < 0.001). For the safety evaluation, A8-1 showed no hemolytic activity, no gelatinase activity, and had only asa1 positive in the seven detected virulence genes in polymerase chain reaction (PCR), whereas it was not predicted in the genome sequence. It was susceptible to benzylpenicillin, ampicillin, ciprofloxacin, levofloxacin, moxifloxacin, tigecycline, nitrofurantoin, linezolid, vancomycin, erythromycin, and quinupristin/dalofopine except clindamycin, which was verified by the predicted lasA, lmrB, lmrC, and lmrD genes contributing to the clindamycin resistance. The virulence test of G. mellonella showed that it had toxicity lower than 10% at 1 × 107 CFU. According to the results of these evaluated attributes, E. durans strain A8-1 could be a promising probiotic candidate for applications.

Slovak Local Ewe’s Milk Lump Cheese, a Source of Beneficial Enterococcus durans Strain

Slovak ewe’s milk lump cheese is produced from unpasteurized ewe’s milk without any added culture. Because of the traditional processing and shaping by hand into a lump, this cheese was given the traditional specialty guaranteed (TSG) label. Up till now, there have existed only limited detailed studies of individual microbiota and their benefits in ewe’s milk lump cheese. Therefore, this study has been focused on the beneficial properties and safety of Enterococcus durans strains with the aim to contribute to basic dairy microbiology but also for further application potential and strategy. The total enterococcal count in cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on a MALDI-TOF mass spectrometry evaluation, the strains were allotted to the species E. durans (score, 1.781–2.245). The strains were gelatinase and hemolysis-negative (γ-hemolysis) and were mostly susceptible to commercial antibiotics. Among the strains, E. durans ED26E/7 produced the highest value of lactase enzyme β-galactosidase (10 nmoL). ED26E/7 was absent of virulence factor genes such as Hyl (hyaluronidase), IS 16 element and gelatinase (GelE). To test safety, ED26E/7 did not cause mortality in Balb/c mice. Its partially purified bacteriocin substance showed the highest inhibition activity/bioactivity against Gram-positive indicator bacteria: the principal indicator Enterococcus avium EA5 (102,400 AU/mL), Staphylococcus aureus SA5 and listeriae (25,600 AU/mL). Moreover, 16 staphylococci (out of 22) were inhibited (100 AU/mL), and the growth of 36 (out of 51) enterococcal indicators was as well. After further technological tests, E. durans ED26E/7, with its bacteriocin substance, can be supposed as a promising additive to dairy products.

New Water-Soluble Condensed Heterocyclic Compounds with Antimicrobial Activity Based on Annulation Reactions of 8-Quinolinesulfenyl Halides with Natural Products and Alkenes

The annulation reactions of 8-quinolinesulfenyl halides with natural products and alkenes affording new water-soluble [1,4]thiazino[2,3,4-ij]quinolin-4-ium derivatives in high or quantitative yields are developed in this study. The reactions with styrene derivatives and terminal alkenes including allyl arenes proceed in a regioselective manner but with the opposite regiochemistry. The reactions with terminal alkenes including allyl arenes occur in an anti-Markovnikov fashion (regarding addition of the 8-quinolinesulfenyl electrophile to the double bond) to give 2-organyl-2Н,3Н-[1,4]thiazino[2,3,4-ij]quinolin-4-ium halides, while the reactions with styrene derivatives proceed in a Markovnikov fashion, leading to 3-substituted condensed heterocyclic compounds. In general, styrene derivatives demonstrate higher reactivity in the annulation reactions compared to the terminal alkenes. Antimicrobial activity of novel water-soluble compounds against Enterococcus durans, Bacillus subtilis and Escherichia coli are evaluated. The compounds with high antimicrobial activity are found. The annulation products of the reactions of 8-quinolinesulfenyl halides with 1H-indene, eugenol, methyl eugenol and 1-heptene, are superior in their activity compared to the antibiotic gentamicin.

A Novel Family of [1,4]Thiazino[2,3,4-ij]quinolin-4-ium Derivatives: Regioselective Synthesis Based on Unsaturated Heteroatom and Heterocyclic Compounds and Antibacterial Activity

A novel family of [1,4]thiazino[2,3,4-ij]quinolin-4-ium derivatives was synthesized by annulation reactions of 8-quinolinesulfenyl chloride with unsaturated heteroatom and heterocyclic compounds. It was found that the reactions with 4-pentenoic and 5-hexenoic acids, allyl chloride and bromide, allyl cyanate and vinyl heterocyclic compounds (N-vinyl pyrrolidin-2-one and 1-vinylimidazole) proceeded in a regioselective mode but with the opposite regiochemistry. The reactions with vinyl heterocyclic compounds included electrophilic addition of the sulfur atom of 8-quinolinesulfenyl chloride to the β-carbon atom of the vinyl group. In the case of other substrates, the annulation proceeded with the attachment of the sulfur atom to the α-carbon atom of the vinyl group. The antibacterial activity of novel water-soluble compounds against Enterococcus durans, Bacillus subtilis and Escherichia coli was evaluated. Compounds with high antibacterial activity were found.

Autochthonous Enterococcus durans PFMI565 and Lactococcus lactis subsp. lactis BGBU1–4 in Bio-Control of Listeria monocytogenes in Ultrafiltered Cheese

Nowadays, consumers are interested in cheese produced without chemical additives or high-temperature treatments, among which, protective lactic acid bacteria (LAB) cultures could play a major role. In this study, the aims were to isolate, identify and characterize antilisterial LAB from traditionally produced cheese, and utilize suitable LAB in cheese production. Among 200 isolated LAB colonies, isolate PFMI565, with the strongest antilisterial activity, was identified as Enterococcus durans. E. durans PFMI565 was sensitive to clinically important antibiotics (erytromicin, tetracycline, kanamycin, penicillin, vancomycin) and had low acidifying activity in milk. E. durans PFMI565 and the previously isolated bacteriocin producer, Lactococcus lactis subsp. lactis BGBU1–4, were tested for their capability to control Listeria monocytogenes in experimentally contaminated ultrafiltered (UF) cheeses during 35 days of storage at 4 °C. The greatest reductions of L. monocytogenes numbers were achieved in UF cheese made with L. lactis subsp. lactis BGBU1–4 or with the combination of L. lactis subsp. lactis BGBU1–4 and E. durans PFMI565. This study underlines the potential application of E. durans PFMI565 and L. lactis subsp. lactis BGBU1–4 in bio-control of L. monocytogenes in UF cheese.

Effect of diet supplementation with Enterococcus durans ED26E/7 and its durancin EntED26e/7 on growth performance, caecal enzymatic activity, jejunal morphology and meat properties of broiler rabbits

The present study investigates the effects of Enterococcus durans ED26E/7 beneficial strain and its enterocin - durancin EntED26E/7 on selected parameters in rabbits: growth performance, caecal enzymatic activity, jejunal morphometry and meat physico-chemical characteristics. Seventy-two rabbits (aged five weeks, M91 meatline, both sexes) were divided into experimental groups E1 (E. durans ED26E/7 strain; dose 500 μL/animal/day, concentration 109 CFU/mL) and E2 (durancin EntED26E/7; dose 50 µL/animal/day, with activity 12 800 AU/mL) and control group (C). The additives were administered in drinking water for a period of 21 days. All animals remained in good health during the experiment. The highest body weight gain (increase by 1.5% compared to C) was noted in E1 group during ED26E/7 strain application (P<0.001). Both bioactive compounds positively influenced („reduced“) the feed conversion ratio (P<0.001). The ED26E/7 strain and its durancin EntED26E/7 application stimulated the activity of most enzymes tested in the caecum; only the amylolytic and inulolytic activity in E2 group decreased during durancin EntED26E/7 addition. Both additives, but mainl the ED26E/7 strain, showed a tendency to improve the jejunal morhological parameters till the end of the experiment (day 42). The meat physico-chemical parameters were no negatively influenced by the application of E. durans ED26E/7 strain and its durancin EntED26E/7. The diet supplementation with bacteriocinogenic and probiotic E. durans ED26E/7 strain and its durancin EntED26E/7 may improve the growth performance, caecal enzymatic activity and jejunal morphometry of rabbits, without any negative effect on rabbit meat quality.

Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania (Romania)

The composition and production technology of the cheese are extremely diverse. There are a wide variety of microbial species on their surface, with a much smaller number inside of the product. The microbiota of the cheese may be composed of beneficial microorganisms, spoilage and foodborne pathogens. Identification and characterization of the microorganisms present in these products are important nutrition, food safety and technological aspects. During our work we evaluated the prevalence of allochthonous bacteria and microscopic fungi in traditionally processed cheeses from northeastern region of Transylvania, with classical microbiological culture methods. Based on the results the microbiota of the analysed cheeses was highly diversified. The identified bacteria with the highest prevalence from different selective media, were as follows: Escherichia coli, Enterococcus durans, Enterococcus faecalis, Shigella flexnerii, Proteus vulgaris, Stenotrophomonas maltophilia, Staphylococcus equorum subsp. equorum, Staphylococcus equorum subsp. linens, Halomonas alkaliphila, Kocuria rhizophila, Hafnia paralvei, Bacillus licheniformis and Klebsiella michiganensis.