Description of the bacteriocins produced by two strains of Enterococcus faecium isolated from Italian goat milk

In this study two strains of Enterococcus faecium, M241 and M249, isolated from goat milk, were studied for their capability to produce antibacterial compounds. It was determined that the bacteriocins produced by both strains were active towards Listeria monocytogenes and Clostridium butyricum, and they did not have any activity with respect to other species of lactic acid bacteria. Enterocins A and B were targeted by polymerase chain reaction (PCR) and sequenced, after cloning, in both strains. The bacteriocins contained in the cell free supernatants were stable when subjected to treatments at high and low temperatures or with lipase, catalase and alpha-amylase. Whereas, the activity was lost when proteinases were used. Lastly, a co-culture experiment with L. monocytogenes in skimmed milk was performed. In the presence of the E. faecium strains, the pathogen showed a delay in the growth of about 6h and it reached a maximum counts of about 10(6) colony forming unit, two orders of magnitude low with respect to the control. This result suggests the possibility to use the strains studied as starter cultures to enhance food safety of dairy products.     

Characterisation of histamine-producing bacteria from farmed blackspot seabream (Pagellus bogaraveo) and turbot (Psetta maxima)

Turbot (Psetta maxima) and blackspot seabream (Pagellus bogaraveo) represent two of the most important emerging farmed fish species in European countries. However, no information of the presence and development of histamine-producing bacteria on them has been reported so far. Accordingly, the aim of this study was to isolate and identify the main histamine-producing bacteria in farmed turbot and blackspot seabream. For this study, 24 isolates (12 from turbot and 12 from blackspot seabream) were preliminarily selected on Niven medium. Two of these isolates were confirmed as prolific histamine producers by HPLC. Thus, Pseudomonas fragi (isolated from turbot) and Pseudomonas syringae (isolated from blackspot seabream) were able to produce 272 ± 69 ppm and 173 ± 45 ppm of histamine in vitro, respectively, after incubation at 30 °C/24 h. While turbot fillets proved to be quite resistant to histamine formation at temperatures below 10 °C, blackspot seabream fillets inoculated with P. syringae and the prolific histamine former Morganella morganii accumulated 696 ± 84 and 760 ± 59 ppm histamine, respectively, under such conditions. Genetic identification based on 16S rRNA sequencing was performed in parallel with the investigation of characteristic mass spectral profiles of the isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS analyses provided species-specific fingerprints, which allow rapid identification and classification of the isolates. Six genus-specific mass peaks in the range of 2218-4434 m/z were shared by both strains. Bacterial identification was achieved by the identification of six species-specific mass peaks in the ranges of 2534-7183 m/z and 2536-9113 m/z for P. fragi and P. syringae, respectively.     

Identification and characterization of two bacteriocin-producing strains of Lactococcus lactis isolated from vegetables.

Isolated from mixed salad and fermented carrots, 123 strains of lactic acid bacteria were screened for bacteriocin production. Two strains, D53 and 23, identified as Lactococcus lactis by DNA-DNA hybridizations, produced heat stable bacteriocins which were resistant to trypsin and pepsin, but were inactivated by alpha-chymotrypsin and proteinase K. The bacteriocins were active from pH 2 to 9 and inhibited species of Listeria, Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Bacillus and Staphylococcus. Strain D53 produced bacteriocin at pH values of 4.5-8.0 and from 10 to 37 degrees C.     

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

电子鼻结合气质联用法分析大菱鲆冷藏过程中挥发性成分变化

通过测定大菱鲆在4℃冷藏过程中的感官品质、菌落总数和TVB-N值等指标的变化,确定贮藏20d时已达腐败阶段。利用电子鼻检测新鲜及腐败鱼肉的气味差异,并对所获数据进行主成分分析,同时结合顶空固相微萃取(HS-SPME)和气质联用技术(GC-MS)对新鲜及腐败鱼肉的挥发性成分进行分析鉴定。结果表明:电子鼻对新鲜及腐败鱼肉整体风味的区分快速、灵敏,其分析结果与常规新鲜度评价指标的变化致;采用GC-MS法在新鲜及腐败鱼肉中分别检测出27及28种挥发性物质,主要为醛类、酮类、醇类、酯类、烃类和胺类化合物等,其中醛类、酮类和醇类物质在新鲜鱼肉中的含量较高,而胺类物质在腐败鱼肉中含量较高。     

Phenotypic and genetic diversity of Lactococcus lactis and Enterococcus spp. strains isolated from Northern Spain starter-free farmhouse cheeses

To evaluate a previous phenotypic classification of lactococci, 39 presumed lactococcal strains were classified by molecular techniques. The strains were also subjected to several typing techniques to estimate the phenotypic and genetic diversity present in original populations from starter-free farmhouse cheeses. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with either restriction enzyme MboII or HhaI divided these isolates into four distinctive groups. Sequencing of representative amplicons identified 29 isolates as belonging to Lactococcus lactis subsp. lactis (24) and Lactococcus lactis subsp. cremoris (5). The remaining 10 isolates were shown to be Enterococcus durans (8) and Enterococcus faecalis (2), which were misclassified by the traditional tests. Thus, partial ARDRA was successfully used to classify wild Lactococcus-like strains into Lactococcus and Enterococcus species. The technique also allowed differentiation of L. lactis strains at subspecies level. The 29 strains of L. lactis showed five different fermentation profiles, four distinct Random Amplification of Polymorphic DNA (RAPD) profiles, and 14 unrelated profiles by both Restriction Fragment Length Polymorphism analyzed by Pulsed Field Gel Electrophoresis (RFLP-PFGE) and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Using the same techniques, the 10 enterococcal strains showed four fermentation profiles, four RADP, and six by RFLP-PFGE and SDS-PAGE, respectively. Several typing techniques, especially RFLP-PFGE and SDS-PAGE, revealed wide phenotypic and genetic variability in both the lactococcal and enterococcal isolates. Two simple, rapid and cheap techniques (partial ARDRA and SDS-PAGE) are proposed as reliable tools for the classification and typing of new lactococcal-like isolates.     

Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Spanish aquacultured turbot (Psetta maxima)

The human population is exposed to dioxins (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) mainly through diet; bioaccumulation and biomagnification in aquatic environment results in fishery products and by-products being an important vector to humans. The determination of PCDD/Fs and dl-PCBs in fillets of young turbots (Psetta maxima) (0-2 years) from aquaculture plant (Galicia, Spain) (N = 21) and in feeding stuffs were carried out, and dietary accumulation values and lipid-normalized biomagnification factors (BMF) relating concentration in fish and in feed were calculated. Levels found in feeding stuffs (0.5 pg TEQ-PCDD/F/g and 1.6 pg TEQ-dl-PCB/g), and turbots (0.13-0.27 pg TEQ-PCDD/F/g fresh weight and 0.35-1.2 pg TEQ-dl-PCB/g fresh weight) were below maximum permitted levels set by EC. Levels of toxic compounds in feeding stuff are reflected in fish fillets; predominant isomers are 2,3,7,8-TCDF, OCDD, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF and 1,2,3,4,6,7,8-HpCDD, and PCBs 118, 105, 156 and 167. Relevant compounds accounting for total toxicity are the same congeners in feeding stuff and turbots: 2,3,4,7,8- PeCDF; 2,3,7,8-TCDF; 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD, and PCB 126. Higher accumulation efficiency values were obtained for dl-PCBs (30-46%); tetra- and penta-chloro substituted PCDD/Fs showed the highest values (27-34%) of the PCDD/F group. Biomagnification was shown for these compounds (BMF around 1.5).     

Distribution of lipids and trace minerals in different muscle sites of farmed and wild turbot (Psetta maxima)

Lipid and trace mineral composition were studied in different sites of the edible flesh of farmed and wild turbot (Psetta maxima). Lipid matter (total content, sterols, tocopherols) showed to accumulate in the edge zone (EZ), except for phospholipid (PL), which provided a distribution that was found to be independent from the kind of turbot and the zone considered. Fatty acid composition of total lipids showed a non‐homogeneous distribution, as the EZ exhibited a different fatty acid group composition (higher monounsaturated and lower polyunsaturated and ω3/ω6 ratio values) than the other zones considered; fewer differences were observed by considering the PL fatty acid composition. Most minerals (Ca, Cu, Fe, Mn and Se) studied showed to be homogeneously distributed along the muscle sites of the wild fish, while more differences were obtained when considering the farmed one. For both kinds of turbots, the most important difference was obtained in the case of Zn, as a largely higher content in the EZ than in the other zones was detected. A close relationship between Zn and total lipid contents (r² = 0.90 and 0.76 for farmed and wild turbots, respectively) was observed.     

Characterization of semipurified enterocins produced by Enterococcus faecium strains isolated from raw camel milk

Food safety has become an issue of great interest worldwide. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to control in the dairy industry. The use of lactic acid bacteria (LAB) and their antimicrobial substances against Listeria is promising in food applications. Here, we report the isolation from raw camel milk of LAB displaying antilisterial activity. Two isolates were selected for their secretion of bacteriocin(s) and identified by 16S rRNA sequencing as Enterococcus faecium S6 and R9. The produced bacteriocins were partially purified by ammonium sulfate precipitation and then biochemically characterized. Antimicrobial activity was estimated to be 6,400 and 400 AU (arbitrary units)/mL for E. faecium S6 and R9, respectively. The proteinaceous nature of the bacteriocins was confirmed via enzymatic reactions. Moreover, lipolytic and glycolytic enzymes completely inactivated the antimicrobial effect of the bacteriocins. These bacteriocins were heat-resistant and stable over a wide range of pH (2.0 to 10.0). To confirm its inactivation by lipolytic and glycolytic enzymes, the bacteriocin of E. faecium S6 was further purified by gel filtration, which suggested the existence of carbohydrate and lipid moieties. In addition, enterocin-coding genes were identified by PCR, showing DNA fragments corresponding in size to enterocins A, B, and P for E. faecium S6 and to enterocins B and P for E. faecium R9. In conclusion, these results indicate that partially purified bacteriocins from E. faecium S6 and R9 may be beneficial in controlling Listeria in the dairy industry.     

Screening for natural defence mechanisms of Lactococcus lactis strains isolated from traditional starter cultures

Three different bacterial defence mechanisms were identified in the seventeen Lactococcus lactis isolates from starter cultures in three Slovenian dairy plants. Isolates MB18, KR7, PT4, PT13 and PT19 inhibited phage adsorption by means of exopolysaccharides production. The most extensive polysaccharides production was detected in PT19 isolate, which was susceptible only to phage ΦPT19. Eight isolates exhibited nuclease activity, and seven of them were susceptible up to four phages out of thirteen from our collection. Eight isolates possessed the abiB gene, fourteen isolates abiH, two isolates abiJ and one isolate abiQ. Isolates PT27 and PT28 possessed AbiB, AbiH and AbiJ mechanisms as well as inhibition of phage adsorption. Isolate MB18, which was susceptible to one phage only, possessed the abiQ gene, nuclease activity and ability to prevent adsorption of most phages. Isolates PT67 and PT70, possessing only AbiH mechanism, were susceptible to only two phages.     

Cross-inhibition among wild strains of Lactococcus lactis isolated from the same ecological niche.

The cross-inhibition between 23 Lactococcus lactis subsp. lactis strains and 9 L. lactis subsp. cremoris strains with different randomly amplified polymorphic DNA patterns, all isolated from the same ecological niche--cheese made in the spring at a single factory from raw milk without added lactic starter cultures-was investigated. Cross-inhibition, as determined by the agar well diffusion assay, was recorded in 130 cases (12.7%) out of 1.024 total cases, with 109 cases due to supernatants of L. lactis subsp. lactis strains and 21 cases due to supernatants of L. lactis subsp. cremoris strains. L. lactis strains isolated in April, May, and June showed differences in their inhibitory activities, with cross-inhibition against each other in 34.7, 14.1, and 6.1% of the cases, respectively. Polymerase chain reaction techniques using specific primers for nisin, lacticin 481, and lactococcin A only revealed the presence of the structural gene of lacticin 481 in two L. lactis subsp. lactis strains.     

屎肠球菌源谷氨酸脱羧酶的制备及其酶学性质研究

为了开发谷氨酸脱羧酶(glutamate decarboxylase,GAD),以Enterococcus faecium为gadB基因供体、纤维素结合域(cellulose-binding domain,CBD)为亲和标签,利用内含肽DnaB自剪切作用分离GAD,对融合酶CBD-DnaB-GAD的构建、表达、GAD纯...     

Genetic diversity of Enterococcus faecium isolated from Bryndza cheese

One hundred and seventy-six Enterococcus faecium isolates from Slovak dairy product Bryndza were tested for the presence of plasmid DNA. Eighty-two isolates were positive and their plasmid DNA was isolated and digested by EcoRI and HindIII restriction endonucleases. The patterns obtained were compared with those obtained after pulsed-field gel electrophoresis of macrorestriction fragments (PFGE), (GTG)(5)-PCR and ERIC-PCR. All these molecular approaches were applied for the study of genetic variability and determination of strain relatednesses among plasmid-positive isolates of E. faecium. In general, all methods revealed a considerable genetic diversity of E. faecium isolates. Plasmid profiling and ERIC-PCR have offered a higher resolution than PFGE and (GTG)(5)-PCR.     

Purification and characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis H-559 isolated from kimchi

Lactic acid bacteria were isolated from kimchi and screened for bacteriocin production. Strain H-559, identified as Lactococcus lactis subsp. lactis, exhibited the strongest antibacterial activity among them and was active against pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus as well as many lactic acid bacteria. The antimicrobial substance produced by L. lactis subsp. lactis H-559 was inactivated by alpha-chymotrypsin, and protease type IX and XIV and was confirmed to be a bacteriocin. The bacteriocin activity was stable from pH 2.0-11.0 and up to 10 min heating at 100 degrees C. The bacteriocin was sequentially purified by ammonium sulfate precipitation, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC). Its molecular weight was determined to be 3343.7 Da by MALDI-mass spectrometry. Isoleucine was detected as the first N-terminal amino acid residue but the remaining amino acid sequence could not be determined by the Edman degradation method. It was different from other bacteriocins in terms of pH stability, molecular weight, amino acid composition, and the partial amino acid sequences of peptides obtained by acid hydrolysis.     

Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains

Lactococcus lactis ssp. lactis is one of the most important starter bacteria used in dairy technology and it is of great economic importance because of its use in the production of dairy products, including cheese, butter, cream, and fermented milks. Numerous studies have evaluated the biochemical and probiotic properties of lactococci; however, limited studies on the probiotic characteristics of lactococci were conducted using strains originating from raw milk and dairy products. Characterizing the probiotic properties of strains isolated from raw milk and fermented milk products is important in terms of selecting starter culture strains for the production of functional dairy products. In this study, biochemical properties (including antibiotic sensitivity, lipolytic activity, amino acid decarboxylation, antioxidant activity) and probiotic properties (including antimicrobial activity, growth in the presence of bile salts, bile salts deconjugation, and hydrophobicity) of 14 Lactococcus lactis strains isolated from raw milk and kefir grains were investigated. Strains originating from kefir grains had better characteristics in terms of antimicrobial activity and bile salt deconjugation, whereas strains from raw milk had better hydrophobicity and antioxidant activity characteristics. None of the strains were able to grow in the presence of bile salt and did not show amino acid decarboxylation or lipolytic activities. Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties. However, these strains did not possess all of the properties required to meet the definition of a probiotic.     

豆酱中产细菌素屎肠球菌的筛选及特性分析

从农家传统发酵豆酱中分离出164株疑似乳酸菌,其中有84株球菌。试验筛选到1株产细菌素的屎肠球菌R1并对其理化性质进行研究。该细菌素对金黄色葡萄球菌、单核细胞增生李斯特菌、大肠杆菌等具有较好的抑制作用,对酸和热稳定。经鉴定该细菌素种类为肠球菌素P。菌株R1在p H3.0模拟胃液中3 h存活率达86.35%,在模拟肠液中6 h存活率达87.72%,对人工消化液耐受性良好。药敏试验结果表明,菌株R1对青霉素、氨苄西林、环丙沙星、氯霉素和庆大霉素敏感,对诺氟沙星和红霉素不敏感,未检测出毒力因子,具有潜在安全性。     

Phenotypic,genetic and technological characterization of Lactococcus garvieae strains isolated from a raw milk cheese

A series of Lactococcus garvieae strains isolated as the majority population of a Spanish traditional, starter-free cheese made from raw milk were phenotypically and genotypically characterised to address their biochemical potential, safety requirements, and technological properties. As expected, all L. garvieae cheese strains fermented lactose but grew slowly in UHT-treated milk. Enzymatic activities of L. garvieae were similar to those of Lactococcus lactis, although higher esterase and lipase activities were recorded for L. garvieae strains. Profiles of the volatile compounds produced from milk by L. garvieae and L. lactis strains were also comparable. L. garvieae strains did not produce haemolysin, gelatinase and the biogenic amines tyramine and histamine. Five L. garvieae stains showed tetracycline resistance encoded by a tet(M) gene. The use of L. garvieae strains as starter or adjunct cultures might be recommended for certain cheese types, provided that the safety of the strains has been demonstrated.     

Growth characteristics of Candida kefyr and two strains of Lactococcus lactis subsp. lactis isolated from Zimbabwean naturally fermented milk.

Lactic acid bacteria (LAB) and yeasts constitute part of the microflora in Zimbabwean traditional fermented cows' milk, amasi. The present study was carried out to investigate the growth characteristics of Candida kefyr 23, Lactococcus lactis subsp. lactis biovar. diacetylactis C1 and L. lactis subsp. lactis Lc261, previously isolated from amasi, in ultrahigh temperature (UHT)-treated cows' milk. The strains were inoculated into the UHT milk as both single and yeast     

Quality retention during the chilled distribution of farmed turbot (Psetta maxima): effect of a primary slurry ice treatment

The quality and shelf‐life of chilled farmed turbot (Psetta maxima) was evaluated by sensory, microbiological and biochemical procedures after being subjected to a two‐step refrigeration storage in slurry ice and flake ice, respectively. Turbot specimens were stored for 10 or 17 days in slurry ice, and then were transferred to flake ice for 1–3 days to simulate the sale conditions in the market. The results were compared with control batches stored only in flake ice and processed in parallel. Storage of turbot in the two‐step strategy resulted in a better maintenance of sensory quality, especially with regard to its mucus production and gill odour development, better control of microbial activity, especially of aerobes, and the slowing down of some biochemical degradation mechanisms such as the nucleotide degradation pathway and trimethylamine production. As a consequence, the shelf‐life was extended significantly. From these results it can be concluded that the refrigerated transport of farmed turbot in slurry ice enhances its shelf‐life, before its transfer into flake ice in the retail market.