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.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.     

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.     

Potential probiotic characteristics of Lactobacillus and Enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization

Traditional fermented buffalo milk in Indonesia (dadih) has been believed to have a beneficial impact on human health, which could be related to the properties of the lactic acid bacteria (LAB) involved in its fermentation process. In previous studies, it was discovered that strains of dadih lactic isolates possessed some beneficial properties in vitro. In the present study, the adhesion capacity of specific LAB isolates from dadih to intestinal mucus was analyzed. Further, the ability to inhibit model human pathogens and displace them from mucus was assessed. The adhesion of tested LAB strains was strain-dependent and varied from 1.4 to 9.8%. The most adhesive Lactobacillus plantarum strain was IS-10506, with 9.8% adhesion. The competition assay between dadih LAB isolates and pathogens showed that a 2-h preincubation with L. plantarum at 37 degrees C significantly reduced pathogen adhesion to mucus. All tested LAB strains displaced and inhibited pathogen adhesion, but the results were strain-specific and dependent on time and pathogen strains. In general, L. plantarum IS-10506 showed the best ability against pathogen adhesion.     

Gastrointestinal transit survival of an Enterococcus faecium probiotic strain administered with or without vancomycin

The primary aim of this study was to evaluate if an ingested probiotic, containing viable Enterococcus faecium could survive gastrointestinal transit and if so, correlate the amount of the recovered probiotic strain with the host's own enterococci. The second aim was to investigate if simultaneous vancomycin intake influenced the survival and persistence of the probiotic strain and the stability of endogenous enterococci strains. Twenty healthy volunteers were given the probiotic product once daily for 10 days. Half of the subjects were simultaneously given vancomycin. Isolates of E. faecium strains were genotypically or phenotypically analysed with pulsed-field gel electrophoresis (PFGE) and the PhenePlate system, respectively. In eight of the ten volunteers given only the probiotic, the ingested E. faecium could be detected on day 10, while in none on day 31. From subjects given both probiotic and vancomycin no ingested E. faecium could be detected on day 10 or day 31. The estimated amount of ingested E. faecium recovered from faeces on day 10 ranged from 1.2 x 10(3) to 4.2 x 10(6) colony forming units per gram faeces, which in several cases were a substantial part of the total amount of E. faecium. The E. faecium isolated before probiotic plus vancomycin administration showed no close relationship to the ones isolated 3 weeks after ceased intake in any subjects. In conclusion, the ingested E. faecium strain can survive gastrointestinal transit. After intake, the E. faecium probiotic strain might become a large part of the total E. faecium population. The occurrence of the probiotic strain in the human gut seems to be transient after intake stop. Re-colonization of E. faecium after simultaneous probiotic plus vancomycin intake occurs mainly with strains without close genetic relationship to the strains harboured before treatment or to the ingested E. faecium strain.     

Characterisation of anti-Listeria monocytogenes bacteriocins from Enterococcus faecium strains isolated from dairy products

Bacteriocin-producing lactic acid bacteria were isolated from 34 samples of dairy products. Nine bacteriocin producers were phenotypically and genotypically identified as Enterococcus faecium . By means of PCR-techniques, enterocin A was characterised in all of the nine bacteriocin-producing Enterococcus isolates. Enterocin-producing lactic acid bacteria were the most abundant in dairy products collected from different areas in Iran. Maximum bacteriocin production by Enterococcus faecium strains was detected in the stationary phase of growth. Bacteriocins produced by all isolates were found to have anti-listerial activity in sterile milk. The purified bacteriocins were identified as <6.5 kDa peptide by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The molecular weights of bacteriocins were found to be the same in all strains. This bacteriocin might be useful as a natural preservative.     

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.     

自然发酵乳中粪肠球菌和屎肠球菌的4种鉴定方法的比较

采用传统生理生化鉴定方法,16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),对分离于自然发酵乳中的9株粪肠球菌和6株屎肠球菌进行鉴定,并对4种鉴定方法进行比较和评价。结果表明,16S-23S rRNA间区序列多态性分析技术和DGGE技术不但可以快速、精确地区分粪肠球菌和屎肠球菌,而且能够将粪肠球菌和屎肠球菌种内的不同基因亚型区分开,而传统生理生化鉴定方法和16S rRNA基因序列分析技术较以上两种方法区分效果略差。     

屎肠球菌扩大发酵的培养基优化

通过正交试验对扩大发酵中的碳源、氮源、番茄汁以及无机盐进行优化,得到最佳培养基为:蔗糖0.5%,豆粕4%,麸皮2.5%,番茄汁15mL/L,K2HPO4 0.2%,NaAC 0.3%,MgSO.47H2O0.08%,MnSO.44H2O 0.03%,结果表明,发酵液的菌体量和抑菌效果明显优于原培养基。     

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.     

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.     

产酪胺粪肠球菌和屎肠球菌PCR检测方法的建立

目的：建立快速简便地检测产酪胺粪肠球菌(Enterococcus faecalis)和屎肠球菌(Enterococcus faecium)的PCR方法。方法：将粪肠球菌和屎肠球菌的酪氨酸脱羧酶基因与GenBank数据库中已公布的细菌的酪氨酸脱羧酶基因进行比对,根据它们的非保守序列,分别设计粪肠球菌和屎肠球菌的特异性引物,建立检测产酪胺粪肠球菌和屎肠球菌的PCR方法。结果：根据非保守序列,分别设计粪肠球菌和屎肠球菌的特异性引物,用27株细菌对这两对引物分别进行反复验证,结果显示,所设计的两对引物都只对其目的菌株产生特异性扩增,对其他菌株没有扩增,方法的检测限可达到1.0×102CFU/mL。结论：本方法具有良好的特异性、稳定性和灵敏性,可用作食品中产酪胺粪肠球菌和屎肠球菌的检测。     

Potentially probiotic acetic acid bacteria isolation and identification from traditional dairies microbiota

Among probiotics, acetic acid bacteria (AAB) can be used as preservative agents because of their high fermentation and acidification activities. This study aimed to isolate, identify and biologically characterise Acetobacter strains from traditional Iranian dairy products. Acetobacter strains were identified by catalase assay, Gram staining, and combined repetitive sequence‐based PCR [(GTG)₅‐PCR fingerprints] and 16S rDNA gene sequencing. We identified eight strains belonging to four species, including Acetobacter aceti, Acetobacter indonesiensis, Acetobacter cibinongensis and Acetobacter syzygii. The molecular techniques could be used as an effective and rapid alternative tool to identify and characterise dairy‐associated AAB. Primary probiotic assessments, including low pH and high bile salt tolerance tests, antagonistic activity test against pathogens, and antibiotic susceptibility confirmed the probiotic properties of these AAB, particularly A. cibinongensis 34L strain, which was isolated from curd. Therefore, this strain can be introduced as novel candidate probiotics that could be used in the food industry.     

Preliminary characterization of bacteriocins from Lactococcus lactis,Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima)

The aim of this work was the characterization of new strains of lactic acid bacteria (LAB) from farmed fish and with potential application as biopreservatives against both Listeria monocytogenes and Staphylococcus aureus. Twenty-five strains of LAB isolated from the muscle of farmed turbot were investigated. Genetic identification of the bacteriocin-producing LAB strains was performed by means of a PCR method using novel BAL1/BAL2 16S ribosomal-RNA-targeted primers. Maximum bacteriocin production by Lactococcus lactis ssp. lactis USC-39, Enterococcus faecium USC-46 and Enterococcus mundtii USC-51 was detected in the stationary phase of growth. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition. In contrast, the antimicrobial activity of all three LAB strains was inactivated by the addition of proteinase K, thus confirming the proteinaceous nature of the inhibition. The activity against L. monocytogenes was maintained in the 3.5–5.5 or 3.5–6.5 pH range, depending on the LAB strain. Likewise, inhibition of S. aureus strains was observed in the 3.5–4.5 and in the 3.5–5.5 pH ranges, depending on the LAB strain and on the S. aureus strain tested. Bacteriocin activity was stable in all three strains after heating the cell-free extract for 60 min at 100 °C, or even for 15 min at 121 °C, in all the three LAB strains. The acidic and heat-resistant bacteriocins produced by the three LAB strains isolated from turbot, able to inhibit the growth of both L. monocytogenes and S. aureus may find application as biopreservatives in fermented and/or heated food products.     

Protective effect of Enterococcus faecium J96, a potential probiotic strain, on chicks infected with Salmonella Pullorum

Enterococcus faecium J96 was isolated from a healthy free-range chicken and it inhibited Salmonella Pullorum, in vitro, due to its lactic acid and bacteriocin production. In vivo assays were carried out with 30-h-old broiler chicks. The lactic acid bacteria (approximately 1 x 10(9) cells per chick) were orally administered as preventive and as therapeutic treatments. In the first case they were given to the chicks twice a day for 3 consecutive days. In the second case the lactic bacteria were administered in the same way after a 24-h challenge by Salmonella Pullorum (in both instances the salmonella dose was 1 x 10(5) cells per chick). Cecal contents, liver, and spleens were analyzed and liver and spleen fragments were also fixed in formaldehyde (pH 7.00) in order to determine salmonella translocation. The chickens that were preventively treated with E. faecium J96 survived the Salmonella Pullorum challenge. Those that were infected on the first day and then inoculated with lactic bacteria died 4 days later. Salmonellae were isolated from their livers and spleens. From these results we may conclude that E. faecium J96 can protect newly hatched chicks from Salmonella Pullorum infection but cannot act as a good therapeutic agent.     

屎肠球菌R2发酵豆腐黄浆水的代谢作用

为研究屎肠球菌R2对豆腐黄浆水的代谢作用,采用高效液相色谱法测定发酵过程中豆腐黄浆水中的低聚糖、有机酸及大豆异黄酮的组分和含量,对α-半乳糖苷酶和β-葡萄糖苷酶进行初步定位及活性测定,并测定发酵黄浆水对DPPH自由基的清除能力.结果表明,发酵过程中,豆腐黄浆水中的水苏糖、棉籽糖、葡萄糖、蔗糖和果糖均被屎肠球菌R2利用,...     

屎肠球菌冷冻干燥保护剂筛选及稳定性比较研究

屎肠球菌在微生态产品中的应用效果受产品活菌量、贮存稳定性影响明显,为寻找适于屎肠球菌规模化生产的冻干保护剂,本研究通过正交试验设计,及屎肠球菌冷冻干燥保护剂4℃、37℃贮存稳定性比较,优化适合屎肠球菌的冻干保护剂配方为:脱脂乳5g/100mL、蔗糖1g/100mL、麦芽糊精5g/100mL。采用此保护剂配方制备的屎肠球菌冻干样品,在铝箔袋密封包装、温度37℃、相对湿度75%条件下存放4周,菌存活率为97.90%。