Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.     

Experimental challenge of bovine mammary glands with Enterococcus faecium during early and late lactation

Mammary glands of early and late lactation cows were challenged with Enterococcus faecium of bovine origin to determine in vivo pathogenicity and milk somatic cell count (SCC) responses. A total of 20 early lactation and 18 late lactation mammary glands were challenged. Two isolates highly adaptive and 2 isolates poorly adaptive for in vitro growth in mammary secretion were used as challenge strains of bacteria. Challenged quarters of early lactation cows were more susceptible to intramammary infection caused by E. faecium than those of late lactation cows. Intramammary challenge with isolates poorly adaptive for in vitro growth in mammary secretions resulted in 94.7% of quarters infected compared with 36.8% of the quarters infused with the isolates highly adaptive for in vitro growth in mammary secretions. Milk from quarters infused with the isolates poorly adaptive for in vitro growth had higher SCC and bacterial counts compared with quarters challenged with the isolates highly adaptive for in vitro growth. A stage of lactation effect within treatment groups was measured when milk SCC were compared between early and late lactation cows. Milk SCC in uninfused (negative control) quarters were lower in early lactation cows compared with late lactation cows. Conversely, in quarters infused with isolates poorly adaptive for in vitro growth, SCC were higher in early lactation cows compared with late lactation cows on d 2, 3, 4, 15, 16, and 17 postchallenge. In quarters infused with isolates highly adaptive for in vitro growth, SCC response did not differ between early and late lactation cows. In vitro growth of E. faecium in mammary secretion was inversely related to in vivo pathogenicity in the mammary glands of early and late lactation cows.     

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 °C and 37 °C, reaching a maximum production of 1.0 × 10⁹ AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.     

Effects of Enterococcus faecium (SF68) on immune function in mice

Probiotics exert health benefits on human and animals when administered in adequate amounts. The objective of this study was to assess the effects of Enterococcus faecium (SF68) on intestinal colonisation and immune function of BALB/c mice. Six-week-old female BALB/c mice were orally administered with E. faecium (SF68). Results showed that the total anaerobe and lactobacilli in the faeces increased (P < 0.05), while the number of faecal enterobacteria decreased (P < 0.05) in E. faecium-fed mice. Furthermore, supplementation of E. faecium (SF68) increased the percentage of double positive (DP) cells in peripheral blood, the concentration of plasma IgG, and the levels of interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-γ (IFN-γ) in splenocytes of the mice (P < 0.05). This study demonstrated that E. faecium SF68 affects the intestinal microbial flora and modulates the immune responses, which indicates a viable probiotic characteristic of E. faecium SF68 in modification of immune function.     

Effects of organic acids on thermal inactivation of acid and cold stressed Enterococcus faecium

In this study the adaptative response to heat (70 °C) of Enterococcus faecium using fresh and refrigerated (at 4 °C for up to 1 month) stationary phase cells grown in Brain Heart Infusion (BHI) buffered at pH 7.4 (non-acid-adapted cells) and acidified BHI at pH values of 6.4 and 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells) was evaluated. In all cases, the survival curves obtained were concave upward. A mathematical model based on the Weibull distribution accurately described the inactivation kinetic. The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, whereas the subsequent cold storage of cells reduced E. faecium thermal tolerance. Fresh acid-adapted cells showed t_2.5-values (time needed to obtain an inactivation level of 2.5 log₁₀ cycles) ranging from 2.57 to 9.51 min, while non-acid-adapted cells showed t_2.5-values of 1.92 min. The extent of increased heat tolerance varied with the acid examined, resulting in the following order: citric ≥ acetic > malic ≥ lactic > hydrochloric ≥ ascorbic. In contrast, cold storage progressively decreased E. faecium thermal resistance. The t_2.5 values found at the end of the period studied were about 2–3-fold lower than those corresponding to non-refrigerated cells, although this decrease was more marked (about 5-fold) when cells were grown in buffered BHI and BHI acidified at pH 5.4 with hydrochloric acid. These findings highlight the need for a better understanding of microbial response to various preservation stresses in order to increase the efficiency of thermal processes and to indicate the convenience of counterbalancing the benefits of the hurdle concept.     

Safety assessment and evaluation of probiotic potential of bacteriocinogenic Enterococcus faecium KH 24 strain under in vitro and in vivo conditions

In the present investigation, a previously isolated Enterococcus faecium KH 24 strain was evaluated for the presence of virulence determinants (agg, esp, efaAfm, gelE, cylA, cylB, clyM, cpd, cob, ccf, ace and hyl), sensitivity to various antibiotics and production of biogenic amines. No virulence determinants were detected, except efaAfm. KH 24 was found to be sensitive to most of the tested antibiotics and none of the biogenic amines were produced by it. Moreover, KH 24 showed good in vitro tolerance to biological barriers and furthermore, its survival in gut of mice was also evaluated. Mice group fed with E. faecium KH 24 strain showed better weight gain and nearly 1 log cfu/g decrease in Salmonella enteritidis counts in the intestines as compared to control (p < 0.05). Enhanced growth of lactobacilli (p < 0.05) and decrease in coliform counts (p < 0.05) were also observed in test group. E. faecium KH 24 is, therefore, found to be a safe strain and it may be used as protective culture or as a probiotic in food preparations.     

Volatile profiles of thua nao, a Thai fermented soy product

Thua nao, a Thai traditional fermented soybean has a unique odour due to the presence of several volatile components. In this investigation, the cooked non-fermented soybeans and soybean (thua nao) fermented by pure Bacillus subtilis TN51 (TNB51) and naturally fermented soybean (TNMX) were analysed for odour components by GC–MS, using the PDMS-SPME head space technique. The quantities of total volatiles in both TNB51 and TNMX were analogous, but the major volatile classes present in TNB51 and TNMX were pyrazines and ketones, respectively. The predominant volatiles in pure Bacillus-fermented thua nao included 2,5-dimethylpyrazine, 2-methylbutanoic acid, 2,3,5-trimethylpyrazine, 2-methylpropanoic acid and acetic acid, whereas the major volatiles in the naturally fermented soybean included 2,5-dimethylpyrazine, benzaldehyde, 5-methyl-3-hexanone, 2-butanone and 3-methyl-2-pentanone. In total, 85 volatile compounds were detected.     

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.     

Diversity of lactic acid bacteria from Hussuwa, a traditional African fermented sorghum food

The diversity of lactic acid bacteria associated with Hussuwa fermentation, a Sudanese fermented sorghum food, was studied using a polyphasic taxonomical approach. Predominant strains could be well characterised based on a combination of phenotypic tests and genotypic methods such as ARDRA, rep-PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Thus, the majority (128 of 220, 58.3%) of strains exhibited phenotypic properties typical of heterofermentative lactobacilli and of these, 100 strains were characterised more closely using the genotyping methods. The majority (97/100) strains could be characterised as Lactobacillus fermentum strains. Seventy-two of 220 strains (32.7%) showed phenotypic properties that are characteristic of pediococci. Of 41 selected strains investigated by genotyping techniques, 38 (92.7%) could be characterised as Pediococcus acidilactici strains, while three (7.3%) could be characterised as Pediococcus pentosaceus strains. The Hussuwa fermentation thus appears to be dominated by L. fermentum strains and P. acidilactici strains. For this reason, we selected representative and predominant strains as potential starter cultures for Hussuwa fermentation. These strains, L. fermentum strains BFE 2442 and BFE 2282 and P. acidilactici strain BFE 2300, were shown on the basis of RAPD-PCR fingerprinting to predominate in a model fermentation when used as starter cultures inoculated at 1 × 10⁶ CFU/g and to lower the pH of the fermentation to below pH 4.0 within 48 h. These cultures should be studied for further development as starter preparations in pilot scale studies in actual field fermentations.     

Impact of inulin and okara on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product and probiotic survival under in vitro simulated gastrointestinal conditions

The effect of inulin and/or okara flour on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product (FSP) and on probiotic survival under in vitro simulated gastrointestinal conditions were investigated throughout 28 days of storage at 4 °C. Employing a 2² design, four FSP trials were produced from soymilk fermented with ABT-4 culture (La-5, Bb-12, and Streptococcus thermophilus): FSP (control); FSP-I (with inulin, 3 g/100 mL of soymilk); FSP-O (with okara, 5 g/100 mL); FSP-IO (with inulin + okara, ratio 3:5 g/100 mL). Probiotic viabilities ranged from 8 to 9 log cfu/g during the 28 days of storage, and inulin and/or okara flour did not affect the viability of La-5 and Bb-12. Bb-12 resistance to the artificial gastrointestinal juices was higher than for La-5, since the Bb-12 and La-5 populations decreased approximately 0.6 log cfu/g and 3.8 log cfu/g, respectively, throughout storage period. Even though the protective effect of inulin and/or okara flour on probiotic microorganisms was not significant, when compared to a fresh culture, the FSP matrix improved Bb-12 survival on day 1 of storage and may be considered a good vehicle for Bb-12 and could play an important role in probiotic protection against gastrointestinal juices.     

Incidence of Staphylococcus aureus and associated risk factors in Nham,a Thai fermented pork product

Staphylococcus aureus is one of the most prevalent bacterial pathogens causing food-borne disease worldwide. Staphylococcal food poisoning is caused by ingestion of staphylococcal enterotoxins (SEs) pre-formed in the implicated food. In this study, the incidences of S. aureus and classical SEs (SEA-SEE) contamination in ‘Nham’, a traditional Thai fermented pork product, were determined. Among 155 Nham samples tested, as high as 39.35% of the samples were positive for S. aureus (2–3500 MPN/g), but none were positive for the SEs. The risk factors for S. aureus contamination were highly correlated with the manufacturer and the pH of the product. A predictive model determined the probability of the presence of S. aureus to be ≤0.24 at the pH ≤ 4.6. During the fermentation process, the number of S. aureus slightly increased in the first day and decreased afterward. S. aureus counts continued to decrease when Nham was stored refrigerated. The negative result for enterotoxins and low counts of S. aureus in Nham surveyed in this study, and reduction of the pathogen counts during fermentation and storage suggested that there is very low risk of staphylococcal food poisoning from consuming properly fermented Nham.     

High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts

Streptococcus thermophilus is a commonly used starter bacterium in dairy industry. It reduces the pH of milk rapidly and equilibrates the medium for the growth of Lactobacillus delbrueckii subsp. bulgaricus during yoghurt fermentation. Efforts to increase the diversity of artisanal yoghurt starters are not only important to bring new strains with novel and desirable characteristics, but also for the preservation of natural diversity which diminishes with the overuse and spread of industrial starters to natural resources. In the present study, 14 artisanal yoghurt samples were processed for the isolation of promising strains for yoghurt starter culture production and 66 strains were subsequently characterized. They were all identified as S. thermophilus using species-specific PCR and 16S rRNA gene sequencing. Genotypic diversity at the strain level was investigated by pulsed field gel electrophoresis (PFGE), and 22 homology groups were obtained. Further phenotypic characterization unearthed a significant phenotypic heterogeneity within homology groups, mostly with atypical novel character. Only 7 out of 66 strains showed S. thermophilus type-strain like phenotypic traits. Majority of the isolates were determined to be protease positive and fast milk acidifier to be used as yoghurt starter culture.     

Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods

Eleven Lactobacillus plantarum strains isolated from traditional Chinese fermented foods were investigated for their in vitro scavenging activity against hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, and their resistance to hydrogen peroxide. L. plantarum C88 at a dose of 10(10) CFU/ml showed the highest hydroxyl radical and DPPH scavenging activities, with inhibition rates of 44.31% and 53.05%, respectively. Resistance of intact cells to hydrogen peroxide was also found in all strains. L. plantarum C88 was the most resistant strain against hydrogen peroxide. When L. plantarum C88 was administered to senescent mice suffering oxidative stress induced by d-galactose, the serum superoxide dismutase activity, the glutathione peroxidase activity and the total antioxidant capacity in liver increased significantly, while the level of malondialdehyde in liver decreased significantly. L. plantarum C88 isolated from traditional Chinese fermented dairy tofu could be considered as a potential antioxidant to be applied in functional foods.     

Physicochemical characteristics and production of whole soymilk from Monascus fermented soybeans

Monascus fermented soybeans (MFS) were prepared by solid state fermentation. MFS showed higher solubility than the unfermented control within the acidic pH region but showed the opposite trend at pH 7 and 9. Although the emulsifying activity index of MFS was lower than that of the control, MFS had significantly higher emulsion stability at pH 7 and 9. Significant protein hydrolysis took place during fermentation, and the proportion of peak area less than 1.35 kDa was greatly increased in MFS. Whole soymilk prepared from MFS contained monacolin K (475 μg/g), and was enriched in isoflavone aglycones. Neither homogenisation nor pasteurisation caused significant changes in the isoflavone and monacolin K contents of the Monascus fermented soymilk. Total phenol content and ABTS radical scavenging activity were significantly higher in the Monascus fermented soymilk than in the control soymilk.     

Free-amino acid profiles of thua nao, a Thai fermented soybean

Thua nao, a rich source of free-amino acids, is a fermented soybean, usually used as seasoning or flavouring enhancer in northern Thailand. Free-amino acids (FAA) of unfermented/cooked soybeans, thua nao, fermented by pure Bacillus subtilis TN51 (TNB51), and a naturally fermented product (TNMX), were investigated by pre-column derivatisation with 9-fluorenylmethyl chloroformate, followed by reversed-phase HPLC. Total FAA and essential amino acids were found at significantly higher concentrations in TNB51 thua nao than in TNMX thua nao (naturally fermented). Both fermented thua nao had much higher concentrations of FAA than had their unfermented counterparts. With respect to taste-enhancing FAA, typical bitter attributes of thua nao came mainly from hydrophobic and basic FAA, whereas an umami attribute came predominantly from acidic FAA.     

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.     

Probiotic beverage from cashew apple juice fermented with Lactobacillus casei

This study optimized the conditions of Lactobacillus casei NRRL B-442 cultivation in cashew apple juice, as well as, determined the proper inoculum amount and fermentation time. Moreover, it was investigated the survivability ability of L. casei in cashew apple juice during refrigerated storage (4 °C) for 42 days. The optimum conditions for probiotic cashew apple juice production were initial pH 6.4, fermentation temperature of 30 °C, inoculation level of 7.48 Log CFU/mL (L. casei) and 16 h of fermentation process. It was observed that the L. casei grew during the refrigerated storage. Viable cell counts were higher than 8.00 Log CFU/mL throughout the storage period (42 days). The values of lightness, yellowness and total color change increased and the values of redness reduced along the fermentation and refrigerated storage periods. The fermented juice with L. casei is a good and healthy alternative functional food containing probiotics. Cashew apple juice showed to be as efficient as dairy products for L. casei growth.     

Sub-lethal stress effects on virulence gene expression in Enterococcus faecalis

Enterococci are ubiquitous lactic acid bacteria commonly associated with the human digestive tract as commensal organisms. Additionally, these organisms have a long history of use in foods improving flavor as well as providing protective mechanisms as either a probiotic or antimicrobial additive. However, Enterococcus faecalis accounts for up to 10% of all nosocomial infections of the bloodstream, wounds, urinary tract and heart. Knowledge about the regulation of virulence factors is limited and the involvement of environmental signals contributing to E. faecalis pathogenicity is poorly documented. In this study, two clinical E. faecalis isolates, TMW 2.63 and OG1RF, as well as one food isolate, TMW 2.629, were subjected to six sub-lethal food- and host-related stresses including 6.8% NaCl, 200 ppm nitrite, 51 °C, 80 MPa, pH 4.1 and 0.08% bile salts (cholic acid:chenodeoxycholic acid 1:1), respectively, reducing their growth rate to 10%. Relative gene expression of 15 stress and virulence-associated genes including dnaK, groEL, ctsR, clpPBCEX, gls24, efaAfs, ace, fsrB, gelE, sprE and cylB, was quantified by using real time PCR and Lightcycler^® technology (reference conditions: BHI broth, 37 °C, pH = 7.4). Apart from strain-dependent differences, sub-lethal environmental stress was capable of provoking significant alterations in the expression of virulence-associated genes in E. faecalis from clinical as well as food origins of isolation. These results help to avoid preconditioning enterococci in food production processes and to understand the complex mechanisms in E. faecalis' switch to pathogenicity.     

Technological and safety properties display biodiversity among enterococci isolated from two Egyptian cheeses, "Ras" and "Domiati"

The technological and safety properties of 35 indigenous strains of Enterococcus faecium isolated from two Egyptian cheeses were characterised in order to determine their ability for rational manufacture of diversified and typical dairy products in the Middle East. A great diversity was observed within the 35 strains on the basis of their technological properties. A statistical analysis made it possible to distribute the 35 strains of E. faecium into different groups. Three groups were identified in terms of their acidification activity, measured by the Cinac system: a group of strains that quickly acidified milk, a second that moderately acidified milk, and a last cluster that revealed weak acidification activity. On the basis of texturing properties that were evaluated using a texturometer and a viscometer, a cluster of strains produced viscous and firm gels, a second cluster included fairly viscous, firm and cohesive gels, and a last group generated slightly viscous, but firm and very cohesive gels. By considering the aroma profiles that were determined by gas chromatography coupled with mass spectrometry, four clusters were identified. One cluster displayed a high dimethyl disulfide level, a second group of strains was highly aromatic, a third cluster led to typical “lactic” products, and the last cluster made it possible to obtain low aromatic products. None of the 35 strains proved to be β-haemolytic on the basis of the characterisation of their safety properties. The resistance to 20 antibiotics was assessed by the disc diffusion method. The 35 isolates were sensitive to 12 antibiotics, and among them, one isolate was resistant to only two antibiotics (nalixidic acid and streptomycin). The resistance to eight antibiotics was strain-dependant. Finally, this study demonstrates that some indigenous strains of E. faecium displayed interesting technological properties for cheese manufacture, together with good safety characteristics. They could be useful for the manufacture of typical products in Egypt.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.