In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.     

Biochemical characteristics,virulence traits and antifungal resistance of two major yeast species isolated from kefir: Kluyveromyces marxianus and Saccharomyces unisporus

Kefir yeasts were investigated for their possible virulence using an in vitro system. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of 46 biochemical traits showed that all kefir isolates formed clusters distinct from Candida albicans. No isolate showed N‐acetylglucosaminidase activity, which is involved in resistance against host immune systems. All Kluyveromyces marxianus isolates formed pseudohyphae and grew at 42 °C, whereas Saccharomyces unisporus isolates did not. None of the kefir yeasts had proteolytic or haemolytic activities. All K. marxianus isolates were susceptible to fluconazole, whereas S. unisporus isolates were classified as resistant.     

Isolation of vancomycin-resistant Enterococcus faecium from Italian cheeses

Several industrial and artisan Italian cheeses were screened for the presence of vancomycin-resistant enterococci (VRE). One hundred and two strains, mostly belonging to the species Enterococcus faecium after species-specific PCR identification, were tested for antibiotic susceptibility by standard disc diffusion method and for resistance to vancomycin in liquid medium. About 25% of strains were resistant to both vancomycin and teicoplanin. PCR experiments showed that all resistant isolates harboured the vanA gene. Minimum inhibitory concentration (MIC) values of vancomycin for most of the vancomycin resistant E. faecium (VREF) ranged from 128 to 512 μg ml⁻¹(high level resistance). PCR fingerprinting of VREF indicated that multiple isolation of the same strains did not occur. The importance of finding VRE in cheeses is discussed.     

Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis

In this study 635 lactic acid bacteria of food origin were evaluated for their potential application as protective cultures in foods. A stepwise selection method was used to obtain the most appropriate strains for application as protective cultures in chicken meat. Specifically, all strains were examined for antimicrobial activity against various Gram positive and Gram negative pathogenic and spoilage bacteria. Strains exhibiting anti-bacterial activity were subsequently examined for survival in simulated food processing and gastrointestinal tract conditions, such as high temperatures, low pH, starvation and the presence of NaCl and bile salts. Selected strains where then examined for basic safety properties such as antibiotic resistance and haemolytic potential, while their antimicrobial activity was further investigated by PCR screening for possession of known bacteriocin genes. Two chosen strains were then applied on raw chicken meat to evaluate their protective ability against two common food pathogens, Listeria monocytogenes and Salmonella enteritidis, but also to identify potential spoilage effects by the application of the protective cultures on the food matrix. Antimicrobial activity in vitro was evident against Gram positive indicators, mainly Listeria and Brochothrix spp., while no antibacterial activity was obtained against any of the Gram negative bacteria tested. The antimicrobial activity was of a proteinaceous nature while strains with anti-listerial activity were found to possess one or more bacteriocin genes, mainly enterocins. Strains generally exhibited sensitivity to pH 2.0, but good survival at 45 °C, in the presence of bile salts and NaCl as well as during starvation, while variable survival rates were obtained at 55 °C. None of the strains was found to be haemolytic while variable antibiotic resistance profiles were obtained. Finally, when the selected strains Enterococcus faecium PCD71 and Lactobacillus fermentum ACA-DC179 were applied as protective cultures in chicken meat against L. monocytogenes and S. enteritidis respectively, a significantly reduced growth of these pathogenic bacteria was observed. In addition, these two strains did not appear to have any detrimental effect on biochemical parameters related to spoilage of the chicken meat.     

Survival of biogenic amine‐producing dairy LAB strains at pasteurisation conditions

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, synthesised by the microbial decarboxylation of precursor amino acids such as histidine, tyrosine or arginine. The consumption of food and beverages containing large amounts of BA can have toxicological effects. This paper reports the resistance of aminogenic lactic acid bacteria strains to pasteurisation (63 °C for 30 min). Skimmed milk inoculated with histamine‐ and tyramine‐producing strains of Enterococcus and Lactobacillus was subjected to pasteurisation. The viable number of Enterococcus durans IPLA 655 was reduced by 1 log, while that of Enterococcus faecalis BA64 was reduced by 3 logs. Lactobacillus brevis CECT 3810 did not survive pasteurisation, while the other Lactobacillus strains examined were more resistant; their viable numbers were reduced by about 2 logs. The two most resistant strains, E. durans IPLA 655 and Lactobacillus curvatus VI6, were subjected to higher temperatures. Enterococcus durans was completely inactivated at 78 °C, while some live cells of Lb. curvatus were observed. These results show that some native, metabolically active BA producers could be found in pasteurised milk owing to their resistance to the thermal treatment; their presence could have a negative impact on the final content of BAs in dairy products made from such milk.     

A survey of the occurrence and properties of methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus intermedius in water buffalo milk and dairy products in Turkey

Antimicrobial resistance, β‐lactamase activity and mecA gene of Staphylococcus aureus and Staphylococcus intermedius isolated from raw water buffalo milk and dairy products in Turkey were determined. All strains showed resistance to at least one antibiotic but none was resistant to vancomycin. Of the 97 S. aureus and 35 S. intermedius strains, 9 (9.2%) and 2 (5.7%) were resistant to oxacillin and harboured mecA gene. β‐lactamase activity of 13.4% and 5.7% of S. aureus and S. intermedius strains was positive, respectively. Overall, 2.5% and 0.55% of the samples were contaminated with methicillin‐resistant S. aureus and S. intermedius, respectively.     

Antibacterial potential of Enterococcus faecium strains isolated from ewes’ milk and cheese

The study was conducted to evaluate the antibacterial activity of three bacteriocin-producing Enterococcus faecium strains (TW15, TW20 and TW22) isolated from ewes’ milk and cheese sampled in the Patagonian region of Argentina. The strains were tested against spoilage and pathogens microorganisms showing antimicrobial activity towards 4 strains of Listeria monocytogenes, one strain of Listeria innocua and 2 strains of Staphylococcus aureus. E. faecium TW15, E. faecium TW20 and E. faecium TW22 were sensitive to vancomycin. Furthermore, investigation of virulence factors revealed the absence of the genes encoding them. The bacteriocin-like substances (BLISs) produced by the 3 strains were thermostable, pH resistant and can be expressed even in the presence of NaCl (3.0 g/100 g). Moreover, they prove to have a bactericidal mode of action. Results from physicochemical and biochemical characteristics of BLIS produced by these E. faecium strains make them potential candidates to aid in preservation of foods.     

Prevalence,characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey

The aim of the present work is to provide information about Enterococcus strains isolated from traditional Turkish cheese samples in Ankara (Turkey), focusing on their prevalence, phenotypic and genotypic characteristics, and antibiotic resistance. A total of 213 probable enterococcal isolates isolated from 215 samples were identified by phenotypic and genotypic methods. As a result of 16S rDNA sequence analysis, 88 of the 213 enterococci strains were identified as Enterococcus faecium and 125 as Enterococcus faecalis. The E. faecalis strains (58.7%) were identified as the dominant species isolated from cheese samples in Turkey. The 213 Enterococcus strains were tested for susceptibility to 12 different antimicrobial agents. The resistance phenotype were as follow: nalidixic acid (100%), kanamycin (98.6%), rifampicin (78.4%), ampicillin (48.8%), ciprofloxacin (45.5%), erythromycin (18.8%), tetracycline (11.7%), penicillin G (5.6%), chloramphenicol (4.2%), gentamycin (3.8%) and streptomycin (1.4%). None of the strains was resistant to vancomycin. E. faecium strains showed more resistant phenotypes than E. faecalis strains as shown by the antibiotic resistance levels. It was also observed that the resistance of E. faecium and E. faecalis strains against the antibiotics was statistically significant (p ? 0.05). In total, 100% of E. faecium and 88.8% of E. faecalis strains were resistant to multiple drugs.     

Bacterial and fungal microbiota of mould-ripened cheese produced in Konya

Bacterial and fungal diversities of 24 mould-ripened cheeses originating from Konya-Türkiye were examined by metagenomic analysis. Firmicutes phylum, Enterococcus, Clostridium sensu stricto and Lactobacillus (Levilactobacillus) genera were the dominant bacteria. Ascomycota phylum and Penicillium and Pichia genera and Penicillium roqueforti and Pichia membranifaciens species were dominant fungi. Enterococcus faecium (n = 30) and Enterococcus faecalis (n = 6) were identified, and all strains were susceptible to penicillin, ampicillin, vancomycin, teicoplanin, chloramphenicol and linezolid. The highest resistance (n = 14) was against rifampin. Tetracycline resistance was determined in two strains. Biofilm-forming ability was found in nine E. faecium and 1 E. faecalis. E. faecium strains revealed 40–88.9%, and E. faecalis showed 59.2–100% homology by pulsed field gel electrophoresis.     

Streptococcus thermophilus strains of plant origin as dairy starters: Isolation and characterisation

Streptococcus thermophilus strains have been isolated mainly from dairy environments. To prospect for new strains of S. thermophilus, isolation was made from different plant sources. In this study, 74 plant isolates were characterised as S. thermophilus by a polyphasic approach and by 16S rRNA sequencing. The isolates were further evaluated for their physiological and biochemical properties. Plant isolates exhibited good acid production and varying proteolytic activity. All the isolates showed acetaldehyde and capsular exopolysaccharide (EPS) production and few revealed diacetyl production. In contrast to industrial strains, six isolates were able to ferment galactose and 25 were found to have no urease activity. Both the plant isolates and reference dairy cultures were found to possess similar physiological and biochemical properties and can be considered for developing new starters.     

Phenolic composition and antioxidant activity of white mulberry (Morus alba L.) fruits

Eight major mulberry cultivars [Nakhonratchasima 60 (NS 60), Buriram 60 (BR 60), Chumphon (CP), Wavee (WV), Chaingmai (CM), Pikultong (PT), Kamphaengsaen (KS) and Kamnanchul (KJ)] cultivated in Thailand were assessed for their flavonoid and phenolic acid composition using HPLC and tested for antioxidant potential using 2,2‐diphenyl‐1‐picrylhydrazyl hydrate (DPPH) assay. The total phenolic content (TPC) ranged from 104.78 to 213.53 mg GAE/100 g DW, and total flavonoid content (TFC) ranged from 69.58 to 211.01 mg CE/100 g DW. The major flavonoid compounds in mulberry fruit cultivars were (+)‐catechin (309.26–750.01 mg/100 g DW), procyanidin B1 (62.59–224.41 mg/100 g DW), quercetin (5.36–58.42 mg/100 g DW), rutin (18.73–26.90 mg/100 g DW) and (?)‐epicatechin (8.47–29.21 mg/100 g DW). Gallic acid, cinnamic acid and p‐hydroxybenzoic acid were found to be the major phenolic acids in mulberry fruit cultivars. The gallic acid and cinnamic acid contents ranged from 7.33 to 23.90 mg/100 g DW and from 11.64 to 15.05 mg/100 g DW, respectively. p‐Hydroxybenzoic acid content ranged from 1.77 mg/100 g DW (PT) to 7.13 mg/100 g DW (KJ). DPPH‐scavenging ability was excellent for ethanolic extract of NS 60, and EC₅₀ value of NS 60 (241.83 μg mL^?1) was significantly lower than those of the others (P < 0.05). TPC and TFC of the mulberry fruit were positively correlated with the DPPH‐scavenging ability.     

Efficacy testing of teat dip solutions used as disinfectants for the dairy industry: Antimicrobial properties

The production of raw milk containing a limited number of bacterial contaminants, which retains its quality during storage, is the major task of the dairy industry. This can only be achieved with adequate regular cleaning and disinfection of the udder, milking equipment, the dairy parlour and animal housing areas. Studies were conducted to assess the efficacy of a range of dairy disinfectants on bacterial species isolated from cow udders as well as reference microbial strains (ATCC). Testing included the assessment of bacterial growth inhibition, biofilm inhibition or bacterial susceptibility to disinfection treatment. Findings show that Lir Analytical chemical disinfectants proved highly successful at inactivating a range of Gram‐positive (Bacillus, Enterococcus, Aeromonas) and Gram‐negative (Escherichia coli, Pseudomonas, Micrococcus) organisms, with up to 99.9% inactivation achieved. Additionally, test chemicals provided significant levels (P < 0.05) of biofilm inhibition for a number of test species. Furthermore, it was found that bacterial isolates from cow udders proved more sensitive to the test chemicals than their reference counterparts.     

Occurrence,heat and antibiotic resistance profile of Bacillus cereus isolated from raw cow and processed milk in Mezam Division,Cameroon

Bacillus cereus is the aetiologic agent of two distinct forms of food poisoning: the diarrhoeal and emetic syndromes. Little data exist on the prevalence of B. cereus in raw milk and milk products sold in Cameroonian towns. This study was aimed at investigating the occurrence, heat and antibiotic resistance of B. cereus isolated from raw milk and selected milk products in Mezam division, Cameroon. Bacillus cereus was isolated by inoculating samples onto mannitol‐egg yolk‐polymyxin B agar. Isolates were characterised morphologically and biochemically. The occurrence of B. cereus in raw milk (8.22%) was less than that in milk powder (13.33%). Bacillus cereus was not isolated from fermented milk. There was no significant difference (P < 0.05) between the B. cereus load in raw milk (2.6 × 10³ cfu/mL) and milk powder (3.0 × 10² cfu/mL). All the isolates showed haemolysin activity and were sensitive to tetracycline, gentamicin, chloramphenicol and nalidixic acid, but resistant to penicillin, ampicillin and trimethoprim/sulphamethoxazole. The detection of drug‐resistant, haemolysin‐positive isolates should serve as a warning for an impending health hazard following consumption of untreated milk. Heat resistance of isolates was assessed by determining the decimal reduction time; D‐value (time to inactivate 90% of the B. cereus spores); and the heat sensitivity, z (temperature increase leads to a tenfold reduction in the D‐value). The values for D₁₀₀ ranged from 0.5 to 3.5 min, and z‐values ranged from 10.0 to 32.6 °C. These results could be used in the dairy industry to evaluate the importance of heat treatment on B. cereus inactivation and calculation of process efficiency.     

Preparation of alcalase hydrolysed rice bran protein concentrate and its inhibitory effect on soybean lipoxygenase activity

Rice bran protein concentrate (RBPC) was prepared from defatted rice bran and hydrolysed by alcalase at different hydrolysis times. As the hydrolysis times increased, the degree of hydrolysis (DHs) increased. RBPC hydrolysate obtained at 50 min (RBPCH‐50) had the highest inhibitory efficiency on soybean lipoxygenase (LOX) activity (66%). The inhibition kinetics of the reaction analysed by Lineweaver–Burk plots indicates that RBPCH‐50 is a competitive inhibitor. RBPCH‐50 inhibited soybean LOX with an IC₅₀ of 11.73 μg μL^?1 RBPCH‐50, and the obtained K_I was 4.59 μg μL^?1 RBPCH‐50. LOX inhibitory activity of RBPCH‐50 was significantly higher than that of 50 and 100 ppm butylated hydroxyanisole (BHA) and 50, 100, and 200 ppm butylated hydroxytoluene (BHT) (P ≤ 0.05); however, LOX inhibitory activity of RBPCH‐50 was similar to that of 200 ppm BHA (P > 0.05). Therefore, RBPCH might potentially be used as a natural LOX inhibitor.     

Efficacy of Acorus calamus L. essential oil as a safe plant‐based antioxidant,Aflatoxin B1 suppressor and broad spectrum antimicrobial against food‐infesting fungi

The study explores the efficacy of Acorus calamus L. essential oil (EO) as a safe plant‐based broad spectrum antifungal, antiaflatoxin, antioxidant food additive. The oil completely inhibited the growth and toxin production of the toxigenic strain of Aspergillus flavus at 0.4 and 0.25 μL mL^?1, respectively. EO exhibited pronounced antifungal activity against sixteen food‐infesting fungal species at 0.5 μL mL^?1. The EO showed strong antioxidant efficacy (IC₅₀ 1.06 μL mL^?1) and nonphytotoxic nature on germination of chickpea seeds. The EO was found nonmammalian toxic showing high LD₅₀ (4877.4 μL kg^?1) for mice (oral, acute). The chemical profile of EO was determined through GC and GC–MS analysis. The findings strengthen the possibility of A. calamus EO as a plant‐based food additive in view of its favourable safety profile, antioxidant and antiaflatoxigenic efficacy and broad spectrum antimicrobial activity against food‐infesting fungi.     

Diversity of Antibiotic Resistance Genes in Enterococcus Strains Isolated from Ready‐to‐Eat Meat Products

The objective of the study was to answer the question of whether the ready‐to‐eat meat products can pose indirect hazard for consumer health serving as reservoir of Enterococcus strains harboring tetracyclines, aminoglycosides, and macrolides resistance genes. A total of 390 samples of ready‐to‐eat meat products were investigated. Enterococcus strains were found in 74.1% of the samples. A total of 302 strains were classified as: Enterococcus faecalis (48.7%), Enterococcus faecium (39.7%), Enterococcus casseliflavus (4.3%), Enterococcus durans (3.0%), Enterococcus hirae (2.6%), and other Enterococcus spp. (1.7%). A high percentage of isolates were resistant to streptomycin high level (45%) followed by erythromycin (42.7%), fosfomycin (27.2%), rifampicin (19.2%), tetracycline (36.4%), tigecycline (19.9%). The ant(6′)‐Ia gene was the most frequently found gene (79.6%). Among the other genes that encode aminoglycosides‐modifying enzymes, the highest portion of the strains had the aac(6′)‐Ie‐aph(2′′)‐Ia (18.5%) and aph(3′′)‐IIIa (16.6%), but resistance of isolates from food is also an effect of the presence of aph(2′′)‐Ib, aph(2′′)‐Ic, aph(2′′)‐Id genes. Resistance to tetracyclines was associated with the presence of tetM (43.7%), tetL (32.1%), tetK (14.6%), tetW (0.7%), and tetO (0.3%) genes. The ermB and ermA genes were found in 33.8% and 18.9% of isolates, respectively. Nearly half of the isolates contained a conjugative transposon of the Tn916/Tn1545 family. Enterococci are widely present in retail ready‐to‐eat meat products. Many isolated strains (including such species as E. casseliflavus, E. durans, E. hirae, and Enterococcus gallinarum) are antibiotic resistant and carry transferable resistance genes.     

Chemical composition,antioxidant activity and anti‐lipase activity of Origanum vulgare and Lippia turbinata essential oils

This study reported the chemical composition, phenolic content, antioxidant and anti‐lipase activity of oregano and Lippia essential oils. The major compounds found in oregano essential oil were γ‐terpinene (32.10%), α‐terpinene (15.10%), p‐cymene (8.00%) and thymol (8.00%). In Lippia essential oil, α‐limonene (76.80%) and 1,8‐cineole (4.95%) represented the major compounds. Oregano essential oil had higher phenolic content (12.47 mg gallic acid mL^?1) and DPPH scavenging activity (IC₅₀ 0.357 μg mL^?1) than Lippia essential oil (7.94 mg gallic acid mL^?1 and IC₅₀ 0.400 μg mL^?1, respectively). Both essential oils had similar antioxidant indexes (about 1.2) determined by Rancimat. Moreover, oregano essential oil had also higher anti‐lipase activity (IC₅₀ 5.09 and 7.26 μg mL^?1). Higher phenolic content in the essential oils was related with higher scavenging and anti‐lipase activities. Oregano and Lippia essential oils could be used as natural antioxidants on food products.     

Nutritional composition and sensory attributes of Alaskan flatfishes compared to plaice (Pleuronectes platessa)

Proximate composition, fatty acids profiles and other nutritional values were evaluated for fillets of Limanda aspera (yellowfin sole), Lepidopsetta bilineata (southern rock sole) and Lepidopsetta polyxystra (northern rock sole) and compared to North Sea plaice (Pleuronectes platessa). Additional information is given on the composition of fillets from arrowtooth flounder (Atheresthes stomias). Plaice (0.8% lipid) and Alaska soles (1.0–1.2% lipid) can be classified as lean species, resulting in low 0.3–0.5 g ∑EPA+DHA/100 g muscle, although the fatty acid profiles of the extracted lipids were characterised by high amount of n‐3 fatty acids (33.2–47.3%). Arrowtooth flounder belong to the medium‐fat species (4.3%). Taurine was the most prevalent free amino acid; mean values ranging between 221 mg100 g^?1 and 247 mg100 g^?1 wet weight. The selenium content varied between 130 and 310 μg kg^?1 ww. Sensory attributes of Southern and Northern rock sole were comparable to plaice.     

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.     

Determination of antibiotic susceptibility and decarboxylase activity of coagulase‐negative Staphylococcus and Macrococcus caseolyticus strains isolated from fermented Turkish sausage (sucuk)

The aim of this research was to detect antibiotic susceptibility patterns and amino acid decarboxylase activity of 61 presumptive coagulase‐negative Staphylococcus (CNS) strains from the Turkish dry fermented sausage (sucuk). The presumptive CNS strains were identified at species levels as 21 S. saprophyticus, 16 S. epidermidis, 10 Macrococcus caseolyticus, 4 S. xylosus, 3 S. sciuri, 2 S. hominis, 2 S. warneri, 1 S. cohnii, 1 S. pasteuri, and 1 S. vitulinus by 16S rDNA sequence analysis. S. saprophyticus and S. epidermidis species were found highly resistant to antibiotics than other species. In addition, most of the CNS and M. caseolyticus strains showed multiple antibiotic resistance profiles. None of the CNS and M. caseolyticus strains did not decarboxylate histidine, lysine, or ornithine, but only three CNS strains produced tyramine from tyrosine. A 100% correlation was found between the presence of tdc gene and tyramine production in tyraminogenic strains.

Practical applications

Coagulase‐negative Staphylococcus (CNS) strains play an important role in production of traditional fermented sausages such as sucuk. In this study, S. saprophyticus and S. epidermidis were found the most common isolated CNS species from sucuk. Antibiotic resistance was detected widespread in CNS and M. caseolyticus strains. Most of the CNS strains were found resistant to clinically relevant antibiotics. Antibiotic resistance strains may be dangerous for consumer health. These strains can be a potential reservoir to spread of antibiotic resistance gene between staphylococci and other species of bacteria. In addition, biogenic amine production was not found widespread in CNS strains.