Novel bifidobacteria strains isolated from nonconventional sources. Technological, antimicrobial and biological characterization for their use as probiotics.

AIM: To characterize four novel autochthonous bifidobacteria isolated from monkey faeces and a Bifidobacterium lactis strain isolated from chicken faeces by evaluating their technological and biological/functional potential to be used as probiotics. Different stressors, including food process parameters and storage, can affect their viability and functionality. METHODS AND RESULTS: The resistance to frozen storage, tolerance to lyophilization and viability during storage, thermal, acidic and simulated gastric resistance, surface hydrophobicity and antimicrobial activity against pathogens were studied. Bifidobacterium lactis Bb12 and INL1 were used as reference strains. The results obtained demonstrated that the new isolates presented strain-dependent behaviour. Good results were obtained for thermal resistance, frozen storage at -80°C and lyophilized powders maintained at 5°C. Cell viability during refrigerated storage was higher when the strains were resuspended in milk at pH 5·0 than at 4·5. The surface hydrophobicity ranged between 7 and 98% depending on the strain. The simulated gastric resistance was improved for the strains incorporated in cheese. Regarding antimicrobial activity, bifidobacteria isolated from monkey presented higher inhibitory capacity than the reference strains. CONCLUSION: This research provides a deeper insight into new strains of bifidobacteria isolated from primates and chicken that have not been previously characterized for their potential use in dairy products and confirm the most robust stress tolerance of B. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility of expanding the available bifidobacteria with the potential to be added to a probiotic food necessarily implies characterizing them from different points of view, especially when considering unknown species. For monkey isolates (which showed higher antimicrobial activity against pathogens), more in-depth knowledge is needed before applying strategies to improve their performance. On the contrary, the chicken isolate B. lactis P32/1 showed similar behaviour to the references B. lactis strains; therefore, it could be considered as a potential probiotic candidate.

Transcriptomic approach and membrane fatty acid analysis to study the response mechanisms of Escherichia coli to thyme essential oil, carvacrol, 2-(E)-hexanal and citral exposure.

AIMS: The application of essential oils (EOs) and their components as food preservatives is promising but requires a deeper understanding of their mechanisms of action. This study aims to evaluate the effects of thyme EO, carvacrol, citral and 2-(E)-hexenal, on whole-genome gene expression (the transcriptome), as well as the fatty acid (FA) composition of the cell membranes of Escherichia coli K12. METHODS AND RESULTS: Therefore, we studied the response against 1 h of exposure to sublethal concentrations of natural antimicrobials, of exponentially growing E. coli K12, using DNA microarray technology and a gas chromatographic method. The results show that treatment with a sublethal concentration of the antimicrobials strongly affects global gene expression in E. coli for all antimicrobials used. Major changes in the expression of genes involved in metabolic pathways as well as in FA biosynthesis and protection against oxidative stress were evidenced. Moreover, the sublethal treatments resulted in increased levels of unsaturated and cyclic FAs as well as an increase in the chain length compared to the controls. CONCLUSIONS: The down-regulation of genes involved in aerobic metabolism indicates a shift from respiration to fermentative growth. Moreover, the results obtained suggest that the cytoplasmic membrane of E. coli is the major cellular target of EOs and their components. In addition, the key role of membrane unsaturated FAs in the response mechanisms of E. coli to natural antimicrobials has been confirmed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The transcriptomic data obtained signify a further step to understand the mechanisms of action of natural antimicrobials also when sublethal concentrations and short-term exposure. In addition, this research goes in deep correlating the transcriptomic modification with the changes in E. coli FA composition of cell membrane identified as the main target of the natural antimicrobials.

Incidence of West Nile virus infection in the Dallas-Fort Worth metropolitan area during the 2012 epidemic.

The 2012 West Nile virus (WNV) epidemic was the largest since 2003 and the North Texas region was the most heavily impacted. We conducted a serosurvey of blood donors from four counties in the Dallas-Fort Worth area to characterize the epidemic. Blood donor specimens collected in November 2012 were tested for WNV-specific antibodies. Donors positive for WNV-specific IgG, IgM, and neutralizing antibodies were considered to have been infected in 2012. This number was adjusted using a multi-step process that accounted for timing of IgM seroreversion determined from previous longitudinal studies of WNV-infected donors. Of 4971 donations screened, 139 (2·8%) were confirmed WNV IgG positive, and 69 (1·4%) had IgM indicating infection in 2012. After adjusting for timing of sampling and potential seroreversion, we estimated that 1·8% [95% confidence interval (CI) 1·5-2·2] of the adult population in the Dallas-Fort Worth area were infected during 2012. The resulting overall estimate for the ratio of infections to reported WNV neuroinvasive disease (WNND) cases was 238:1 (95% CI 192-290), with significantly increased risk of WNND in older age groups. These findings were very similar to previous estimates of infections per WNND case, indicating no change in virulence as WNV evolved into an endemic infection in the United States.

Survival of the functional yeast Kluyveromyces marxianus B0399 in fermented milk with added sorbic acid.

In this study, the survival of the functional yeast Kluyveromyces marxianus B0399 in an industrially produced fermented milk was evaluated. In particular, the yeast viability was assessed throughout the entire shelf-life of the product (30 d) to ensure the presence of the effective yeast dose (20 million viable cells for each serving of 125 g) while avoiding, by sorbic acid addition, yeast growth, which could affect product quality and stability. To find the best combination of yeast and sorbic acid concentration, 13 different combinations were tested, and then 2 of them were chosen for industrial production. In production at lower concentrations (30 million viable cells, 150 mg/kg of sorbic acid) the effective dose was maintained only at 4 and 6°C, whereas at higher dosages (70 million viable cells, 250 mg/kg of sorbic acid) the effect of temperature was less evident. In all the trials, the concentration of sorbic acid was not affected by microbial metabolism and remained stable throughout the entire shelf-life.

Effect of a sublethal high-pressure homogenization treatment on the fatty acid membrane composition of probiotic lactobacilli.

UNLABELLED: High-pressure homogenization (HPH) has been proposed to be applied directly to lactic acid bacterial cells at sublethal levels to enhance some functional properties. As the principal target of HPH are the cell surface envelope structures, the aim of this work was to study the effect of a HPH treatment, applied at 50 MPa, on cell membrane stress responses of already-known functional strains, isolated from Argentinean products. Specifically, the membrane fatty acid composition of cells before and after the sublethal treatment was investigated, and the results showed that plasma membranes, their level of unsaturation and their composition are involved in response mechanisms adopted by microbial cells when subjected to a sublethal HPH stress. In fact, the data obtained demonstrated that the treatment was able to modify the fatty acid profile of the different strains, although a uniform response was not observed. Further studies are necessary both to elucidate the role of each fatty acid in the cell response mechanisms and to clarify the changes in membrane compositions induced by HPH treatment also in relation to the applicative potential of this technique. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to understand the response mechanisms activated in cells exposed to pressure stress. It has been demonstrated that high-pressure homogenization (HPH) treatments, conducted at sublethal levels, could increase some important functional and technological characteristics of nonintestinal probiotic strains. The findings of this paper can contribute to elucidate the mechanisms through which these treatments can modify these strain probiotic properties that are related to outermost cell structures, also principal target of HPH.

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Development of multiplex PCR-ligase detection reaction assay for detection of West Nile virus.

We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

Probiotic Crescenza cheese containing Lactobacillus casei and Lactobacillus acidophilus manufactured with high-pressure homogenized milk.

High-pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment of food preservation and diversification. Its effectiveness on the deactivation of pathogenic and spoilage microorganisms in model systems and real food is well documented. To evaluate the potential of milk treated by HPH for the production of Crescenza cheese with commercial probiotic lactobacilli added, 4 types of cheeses were made: HPH (from HPH-treated milk), P (from pasteurized milk), HPH-P (HPH-treated milk plus probiotics), and P-P (pasteurized milk plus probiotics) cheeses. A strain of Streptococcus thermophilus was used as starter culture for cheese production. Compositional, microbiological, physicochemical, and organoleptic analyses were carried out at 1, 5, 8, and 12 d of refrigerated storage (4 degrees C). According to results obtained, no significant differences among the 4 cheese types were observed for gross composition (protein, fat, moisture) and pH. Differently, the HPH treatment of milk increased the cheese yield about 1% and positively affected the viability during the refrigerated storage of the probiotic bacteria. In fact, after 12 d of storage, the Lactobacillus paracasei A13 cell loads were 8 log cfu/ g, whereas Lactobacillus acidophilus H5 exhibited, in P-P cheese, a cell load decrease of about 1 log cfu/g with respect to the HPH-P cheese. The hyperbaric treatment had a significant positive effect on free fatty acids release and cheese proteolysis. Also, probiotic cultures affected proteolytic and lipolytic cheese patterns. No significant differences were found for the sensory descriptors salty and creamy among HPH and P cheeses as well as for acid, piquant, sweet, milky, salty, creamy, and overall acceptance among HPH, HPH-P, and P-P Crescenza cheeses.

Effect of high-pressure homogenization, nonfat milk solids, and milkfat on the technological performance of a functional strain for the production of probiotic fermented milks.

The aim of this research was the evaluation of the effects of milkfat content, nonfat milk solids content, and high-pressure homogenization on 1) fermentation rates of the probiotic strain Lactobacillus paracasei BFE 5264 inoculated in milk; 2) viability loss of this strain during refrigerated storage; and 3) texture parameters, volatile compounds, and sensorial properties of the coagula obtained. The data achieved suggested a very strong effect of the independent variables on the measured attributes of fermented milks. In fact, the coagulation times were significantly affected by pressure and added milkfat, and the rheological parameters of the fermented milk increased with the pressure applied to the milk for added nonfat milk solids concentrations lower than 3%. Moreover, the polynomial models and the relative response surfaces obtained permitted us to identify the levels of the 3 independent variables that minimized the viability loss of the probiotic strain used during refrigerated storage.

The emergence of West Nile virus in North America: ecology, epidemiology, and surveillance.

In late summer 1999, the first domestically acquired human cases of WN encephalitis were documented in the USA. Aggressive vector-control and public education efforts by state and local public health officials limited the extent of human involvement. The discovery of virus-infected, overwintering mosquitoes during the winter of 1999-2000, predicted renewed virus activity for the following spring, and prompted early season vector-control activities and disease surveillance efforts in NYC and the surrounding areas. These surveillance efforts were focused on identifying WN virus infections in birds and mosquitoes as predictors of the potential risk of transmission to humans. By the end of the 2000 mosquito-borne disease transmission season, WN virus activity had been documented as far north as the states of Vermont and New Hampshire, and as far south as the state of North Carolina. The ongoing impacts that WN virus will have on wildlife, domestic animal and human populations of the western hemisphere are not yet known. Plans are in place for public health officials and scientists to monitor the further expansion of WN virus with the establishment or enhancement of vector-borne disease surveillance and control programs throughout the eastern seaboard. The valuable lessons learned from the detection and response to the introduction of WN virus into NYC should prove useful if and when subsequent intrusions of new disease agents occur.

Use of Yarrowia lipolytica strains for the treatment of olive mill wastewater.

The principal aim of this research was to evaluate the ability of different Yarrowia lipolytica strains, having different origin, to grow in olive mill wastewater (OMW) and reduce its COD level. All the strains were able to grow in undiluted OMW; the comparison between the data obtained in a semi-synthetic medium and in OMW suggests that lipases with different specificity can be produced in relation to the medium composition. Under the adopted conditions, the reduction of the OMW COD values varied from 1.47% and 41.22% of the initial value. Some strains determined a significant reduction of polyphenol content, while other ones caused its apparent increase. Moreover, some Y. lipolytica strains, isolated from chilled foods, produced the highest citric acid concentrations. These results evidenced that some Y. lipolytica strains are good candidates for the reduction of the pollution potential of OMW and for the production of enzymes and metabolites such as lipase and citric acid.

First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley province, Kenya.

West Nile virus is a mosquito borne flavivirus endemic over a large geographic area including Africa, Asia, and the Middle East. Although the virus generally causes a mild, self-limiting febrile illness in humans, it has sporadically caused central nervous system infections during epidemics. An isolate of West Nile virus was obtained from a pool of four male Culex univittatus complex mosquitoes while we were conducting an investigation of Rift Valley fever along the Kenya-Uganda border in February-March 1998. This represents the first field isolation of West Nile virus from male mosquitoes and strongly suggests that vertical transmission of the virus occurs in the primary maintenance mosquito vector in Kenya. A phylogenetic analysis of the complete amino acid sequence of the viral envelope glycoprotein demonstrated a sister relationship with a Culex pipiens mosquito isolate from Romania made in 1996. This unexpected finding probably reflects the role of migratory birds in disseminating West Nile virus between Africa and Europe.

Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes.

Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic were conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, including 5 of 13 ducks (38%), 1 of 1 goose, 19 of 52 chickens (37%), 1 of 1 peahen, and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 26) from the urban Bucharest site was not significantly different (P = 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46). Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted for 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obtained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97-50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained from a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenetic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest the existence of two large lineages of viruses. Bootstrap analysis of the nucleotide alignment indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Algeria, and west, central, and east Africa, all of the European isolates, those from France and Romania, an Israeli isolate, and an isolate of KUN virus from Australia. The nucleotide sequence of RO97-50 was identical to the sequence of a WN virus isolate obtained from Cx. neavei mosquitoes from Senegal and Cx. univittatus mosquitoes from Kenya. The phylogenetic analyses were compatible with the introduction of virus into Romania by birds migrating from sub-Saharan Africa, to northern Africa, and into southern Europe.

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458.

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.

Direct sequencing of large flavivirus PCR products for analysis of genome variation and molecular epidemiological investigations.

The polymerase chain reaction (PCR) was used to amplify viral cDNAs from selected regions of dengue genomic RNA by using appropriate 'consensus' primers. DNA amplicons containing the structural genes from all 4 dengue serotypes were prepared and directly sequenced using dengue-virus-specific primers. This method can characterize reliably flavivirus field isolates at the molecular level without extensive virus propagation and molecular cloning, and will be a valuable tool for molecular epidemiological studies.

Survey of the physiological properties of the most frequent yeasts associated with commercial chilled foods.

A comparative analysis of the initial and final population of yeasts, lactic acid bacteria and psychrotrophic bacteria, in a number of chilled foods, varying in ingredients, physico-chemical characters and origin, gave evidence that yeasts could play a significant role in the spoilage. The yeast populations appeared to be unexpectedly uniform and comprised principally strains of Yarrowia lipolytica, Debaryomyces hansenii and Pichia membranaefaciens. A survey of 62 isolates, comprising physiological characteristics such as growth temperatures, proteolytic and lipolytic activities, hydrophobicity, aw and preservative tolerance, in addition to organic acid production, indicated that these dominant species have very few common characters and that they are endowed with a spoilage potential probably linked to different physiological properties. The isolates of Y. lipolytica exhibited the strongest proteolytic and lipolytic activities and a pronounced hydrophobicity while D. hansenii isolates were characterized by a high growth rate at low temperature and at intermediate aw. P. membranaefaciens isolates showed a remarkable tolerance to acetic acid as a sole selective factor. A hypothesis of separate growth loci, in multicomponent or polyphasic food systems, was formulated.

Growth modelling of Listeria monocytogenes and Yersinia enterocolitica in food model systems and dairy products.

The growth potential of Listeria monocytogenes, measured with a gaschromatographic method, was evaluated in model systems simulating dairy products in which NaCl concentration, pH and lipid concentration ranged from values corresponding to matured hard cheeses to some light cheeses and other dairy products. The growth data from the model systems, calculated on the basis of the polynomial models obtained, were compared with values observed for dairy products. Growth parameters of Yersinia enterocolitica in traditional and light cheeses were also available. The comparison between predicted and observed data suggested that individual or interactive effects of chemicophysical variables such as pH, salt and lipid concentration are not sufficient to predict the fate of L. monocytogenes in dairy products. Microstructural factors as well as the pH effect on water binding capacity of proteins could also be implicated.

Suitability of log-linear models to evaluate the microbiological quality of baby clams (Chamelea gallina L.) harvested in the Adriatic Sea.

The presence of fecal coliforms or Escherichia coli in baby clams (Chamelea gallina L.) is considered an indicator related to their safety because they can be correlated with the presence of pathogenic bacteria. For this reason the Italian regulation has defined limits for these microorganisms. The presence of these microbial indicators is dependent on various environmental variables. In this work all the variables considered are categorical and, consequently, the traditional approach of predictive microbiology was not applicable. The data were summarized by means of a cross-tabulation and analyzed using the log-linear model technique. This statistical technique is widely used in social and economic studies but only partially developed in food microbiology. The suitability of the log-linear model to analyse microbiological data in relation to environmental variables was evaluated. In particular, the microbiological quality of baby clams harvested in five different areas of the Adriatic Sea coast in Emilia Romagna (Italy) was considered. The influence of the season and geographical origin on microbiological standards was assessed. A logit model was developed to predict the frequencies, depending on geographical origin and season, of samples with concentrations of the indicator organisms below or above the legal standards provided by Italian regulation.

Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase.

It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk. Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment. The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment. Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species. Although L. monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C. The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity. This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L. plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

Occurrence and characterization of yeasts isolated from milks and dairy products of Apulia region.

One hundred and five strains of yeasts isolated from milk of different animal origin and from typical Apulian cheeses were studied to identify and characterize yeast strains for further selection as starter cultures for cheese production. The most prevalent isolates belonged to the species Trichosporon cutaneum (15.24%), Candida catenulata (10.48%) and Yarrowia lipolytica (8.57%). In order to evaluate the potential use as starter cultures, the occurrence of some selected properties, such as fermentation of glucose and lactose, assimilation of lactic acid, citric acid and lactose, growth at 4 degrees C and production of lipolytic and proteolytic enzymes in the strains belonging to the most frequent species was tested. C. catenulata and C. zeylanoides were positive for assimilation of lactic and citric acids and showed psychrotrophic aptitude. T. cutaneum was positive for all properties tested except for glucose and lactose fermentations. Y. lipolytica was endowed with remarkable lipolytic activity also at 4 degrees C and was positive for assimilation of lactic and citric acids, growth at 4 degrees C and proteolytic activity.