Influence of cheese making process on STEC bacteriophage release.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens implicated in diseases including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). The main virulence factor are Shiga toxins; their production and secretion are by-products of the expression of late genes of prophages upon sub-lethal environmental stimuli exposure. Hence, the lysogenic prophage after a stress switch to lytic cycle spreading the Stx phages. In the present study, 35 STEC were screened for the presence and the ability to release Shiga toxin-encoding bacteriophages. Three bacterial strains showed signals of prophage presence both in plate and in PCR. Subsequently, these bacterial strains were subjected to stressors that simulate cheese manufacturing conditions: NaCl (1, 1.5 and 2% w/v), lactic acid (0.5, 1.5 and 3% v/v), anaerobic growth, pasteurization (72°C for 15 s), UV irradiation. The ability to release prophage was evaluated by Real Time qPCR. Induction of the prophages showed that the addition of NaCl at 1.5 and 2% significantly increased viral release compared to control. Conversely, the addition of lactic acid had a significant repressive effect. The other applied stressors had no significant effect in phage release according to the experimental conditions adopted.

Characterization of Two Zymomonas mobilis Wild Strains and Analysis of Populations Dynamics during Their Leavening of Bread-like Doughs.

Two Zymomonas mobilis wild strains (UMB478 and 479) isolated from water kefir were characterized for their biomass production levels and leavening performance when used as the inoculum of a real bread-like dough formulation. The obtained baked product would be consumable by people with adverse responses to Saccharomyces cerevisiae. In liquid cultures, the two strains reached similar biomass concentration (0.7 g CDW/L). UMB479 showed an interesting resistance to NaCl (MBC 30 g/L), that may be useful in the bakery sector. When inoculated in doughs, UMB479 produced the maximum dough volume (650 mL) after 5 h, glucose was almost consumed and 1 g/100 g of ethanol produced, +200% respective to UMB478. Using S. cerevisiae for comparison purposes, the dough doubled its volume fast, in only 2 h, but reached a final level of 575 mL, lower than that achieved by Z. mobilis. The analysis of bacterial and fungal population dynamics during dough leavening was performed through the Automated Ribosomal Intergenic Spacer Analysis (ARISA); doughs leavened by UMB479 showed an interesting decrease in fungal richness after leavening. S. cerevisiae, instead, created a more complex fungal community, similar before and after leavening. Results will pave the way for the use of Z. mobilis UMB479 in commercial yeast-free leavened products.

Development of quantitative real-time PCR and digital droplet-PCR assays for rapid and early detection of the spoilage yeasts Saccharomycopsis fibuligera and Wickerhamomyces anomalus in bread.

In the present study, for the first time, high sensitive quantitative polymerase chain reaction (qPCR) and digital droplet polymerase chain reaction (ddPCR) assays were developed to detect and quantify total eumycetes with potential application in several food matrices and to specifically determine the level of contamination by Saccharomycopsis fibuligera and Wickerhamomyces anomalus cells directly in bread. Among the candidate target genes used to develop the assays, car1 gene was chosen to detect the two spoilage yeasts S. fibuligera and W. anomalus. The specificity of the PCR assays was tested using purified genomic DNA from 36 bacterial and fungal strains. The sensitivity of the assays was defined by using tenfold serial dilutions of genomic DNA starting from 106 cfu/mL to 1 cfu/mL of S. fibuligera and W. anomalus. Validation of the assays was achieved by enumeration of S. fibuligera and W. anomalus DNA copies from samples of artificially contaminated bread homogenates detecting up to 10 cfu/mL (0.06 ± 0.01 copies/µL) of W. anomalus by using ddPCR. In conclusion, the developed qPCR and ddPCR assays demonstrate strong performance in the early detection of S. fibuligera and W. anomalus in bread, representing promising tools for applying high-throughput approaches to regularly monitor bread quality.

Critical Aspects in the Preparation of Extemporaneous Flecainide Acetate Oral Solution for Paediatrics.

The availability of liquid oral preparations compounded by pharmacists is essential to meet paediatric needs which remain unanswered by the pharmaceutical industry. Unfortunately, compendial monographs are often not available and, in many cases, pre-formulation studies (e.g., compatibility with other excipients and solubility evaluations) are not performed in-depth, leading, in some rare cases, to the inadvertent administration of a toxic dose. In this study, the preparation of an oral liquid formulation for paediatric use, containing flecainide acetate at different strengths, was considered, taking into account the possible effects of conventionally used excipients. First, the optimal vehicle was selected based on a solubility study, evidencing some unexpected formations of precipitates. As a matter of fact, the buffers commonly used for oral solutions significantly reduced flecainide solubility, and the concomitant presence of citrate buffer and methylparaben even caused the formation of non-resuspendable crystals. Then, chemical, physical, and microbiological stability were assessed. Solutions at strengths of 10 and 20 mg/mL flecainide acetate were stable up to 8 weeks when compounded by using a 40% sucrose solution as a vehicle. Microbiological data showed that the use of methylparaben was not necessary over this time period.

Application of Bacteriophages on Shiga Toxin-Producing Escherichia coli (STEC) Biofilm.

Shiga toxin-producing Escherichia coli are pathogenic bacteria able to form biofilms both on abiotic surfaces and on food, thus increasing risks for food consumers. Moreover, biofilms are difficult to remove and more resistant to antimicrobial agents compared to planktonic cells. Bacteriophages, natural predators of bacteria, can be used as an alternative to prevent biofilm formation or to remove pre-formed biofilm. In this work, four STEC able to produce biofilm were selected among 31 different strains and tested against single bacteriophages and two-phage cocktails. Results showed that our phages were able to reduce biofilm formation by 43.46% both when used as single phage preparation and as a cocktail formulation. Since one of the two cocktails had a slightly better performance, it was used to remove pre-existing biofilms. In this case, the phages were unable to destroy the biofilms and reduce the number of bacterial cells. Our data confirm that preventing biofilm formation in a food plant is better than trying to remove a preformed biofilm and the continuous presence of bacteriophages in the process environment could reduce the number of bacteria able to form biofilms and therefore improve the food safety.

Sisymbrium Officinale (the Singers' Plant) as an Ingredient: Analysis of Somatosensory Active Volatile Isothiocyanates in Model Food and Drinks.

Sisymbrium officinale (L.) Scop. (hedge mustard) is a wild common plant of the Brassicaceae family. It is known as "the singers' plant" for its traditional use in treating aphonia and vocal disability. The plant is rich in glucosinolates and isothiocyanates; the latter has been demonstrated to be a strong agonist in vitro of the Transient Receptor Potential Ankirine 1 (TRPA1) channel, which is involved in the somatosensory perception of pungency as well as in the nociception pathway of inflammatory pain. Volatile ITCs are released by the enzymatic or chemical hydrolysis of GLSs (glucosinolates) during sample crushing and/or by the mastication of fresh plant tissues when the plant is used as an ingredient. Some functional food and drink model preparations have been realised: honey enriched with seeds and flowers, infusions, cold drink (voice drink), artisanal beer, and a fermented tea (kombucha). Using SPME-GCMS chromatography, we analysed samples of the plant and of the food preparations adopting conditions that simulate the release of isothiocyanates (ITCs) during oral assumption. Two active compounds, iso-propylisothiocyanate and 2-butylisothiocyanate, have been assayed. The concentration of ITCs varies according to temperature, pH, grinding conditions, and different plant organs used. Kombucha-type fermentation seems to eliminate the ITCs, whereas they are retained in beer. The ITCs' concentration is higher when entire seeds and flowers are used.

Evaluation of a Potential Bacteriophage Cocktail for the Control of Shiga-Toxin Producing Escherichia coli in Food.

Shiga-toxin producing Escherichia coli (STEC) are important foodborne pathogens involved in gastrointestinal diseases. Furthermore, the recurrent use of antibiotics to treat different bacterial infections in animals has increased the spread of antibiotic-resistant bacteria, including E. coli, in foods of animal origin. The use of bacteriophages for the control of these microorganisms is therefore regarded as a valid alternative, especially considering the numerous advantages (high specificity, self-replicating, self-limiting, harmless to humans, animals, and plants). This study aimed to isolate bacteriophages active on STEC strains and to set up a suspension of viral particles that can be potentially used to control STEC food contamination. Thirty-one STEC of different serogroups (O26; O157; O111; O113; O145; O23, O76, O86, O91, O103, O104, O121, O128, and O139) were investigated for their antibiotic resistance profile and sensitivity to phage attack. Ten percent of strains exhibited a high multi-resistance profile, whereas ampicillin was the most effective antibiotic by inhibiting 65% of tested bacteria. On the other side, a total of 20 phages were isolated from feces, sewage, and bedding material of cattle. The viral particles proved not to carry genes codifying Shiga-toxins and intimin. No STEC was resistant to all phages, although some strains revealed weak sensitivity by forming turbid plaques. Three different bacteriophages (forming the "cocktail") were selected considering their different RAPD (Random Amplification of Polymorphic DNA) profiles and the absence of virulence-encoding genes and antibiotic-resistance genes. The lytic ability against STEC strains was investigated at different multiplicity of infection (MOI = 0.1, 1, and 10). Significant differences (p < 0.05) among mean values of optical density were observed by comparing results of experiments at different MOI and controls. An effective reduction of bacterial population was obtained in 81% of cases, with top performance when the highest MOI was applied. The efficacy of the phage cocktail was tested on fresh cucumbers. Results showed a reduction in pathogenic E. coli by 1.97-2.01 log CFU/g at 25°C and by 1.16-2.01 log CFU/g at 4°C during 24 h, suggesting that the formulated cocktail could have the potential to be used in bio controlling STEC different serogroups.

Yeast-Free Doughs by Zymomonas mobilis: Evaluation of Technological and Fermentation Performances by Using a Metabolomic Approach.

This research focuses on the leavening performances and development of volatile compounds of three strains of Zymomonas mobilis in the production of yeast-free doughs. Z. mobilis DSM 3580, 424, and 473 were used in doughs supplemented with glucose and with or without NaCl. Z. mobilis produced about 10 mg ethanol/g dough, with maximum dough volumes (640-680 mL) being reached after 2 h leavening. NaCl addition postponed this parameter up to 6 h. Among organic acids, hexanoic acid resulted the highest produced compound; DSM 424 and 473 formed more propanoic, butanoic and pentanoic acid, being both negatively affected by NaCl. Esters were mainly discriminated on NaCl addition, with octanoic acid (DSM 3580), butanoic acid (DSM 424), and propanoic acid (DSM 473) ethyl esters as main components. DSM 3580 specifically produced 2-heptanal, DSM 424 2-hexadecenal, (E) and DSM 473 octanal, while DSM 424 and DSM 473 produced 2-butanone-4-hydroxy better than DSM 3580. Z. mobilis unique signatures were the production of nonanoic and undecanoic acids, 2-hexadecenal, (E), L(+)-tartaric acid diethyl ester and 3-decen-5-one, 4-methyl, (E). This outcome can pave the way for using Z. mobilis in baking goods, providing innovation possibilities in the area of yeast-free leavened products.

Valorization of cheese whey using microbial fermentations.

Cheese whey (CW), the liquid resulting from the precipitation and removal of milk casein during cheese-making, and the second cheese whey (SCW) derived from the production of cottage and ricotta cheeses are the main byproducts of dairy industry. The major constituent of CW and SCW is lactose, contributing to the high BOD and COD content. Because of this, CW and SCW are high-polluting agents and their disposal is still a problem for the dairy sector. CW and SCW, however, also consist of lipids, proteins, and minerals, making them useful for production of various compounds. In this paper, microbial processes useful to promote the bioremediation of CW and SCW are discussed, and an overview on the main whey-derived products is provided. Special focus was paid to the production of health-promoting whey drinks, vinegar, and biopolymers, which may be exploited as value-added products in different segments of food and pharmaceutical industries.

Ripe and Raw Pu-Erh Tea: LC-MS Profiling, Antioxidant Capacity and Enzyme Inhibition Activities of Aqueous and Hydro-Alcoholic Extracts.

Herein, we reported a detailed profiling of soluble components of two fermented varieties of Chinese green tea, namely raw and ripe pu-erh. The identification and quantification of the main components was carried out by means of mass spectrometry and UV spectroscopy, after chromatographic separation. The antioxidant capacity towards different radical species, the anti-microbial and the enzyme inhibition activities of the extracts were then correlated to their main constituents. Despite a superimposable qualitative composition, a similar caffeine content, and similar enzyme inhibition and antimicrobial activities, raw pu-erh tea extract had a better antioxidant capacity owing to its higher polyphenol content. However, the activity of raw pu-erh tea seems not to justify its higher production costs and ripe variety appears to be a valid and low-cost alternative for the preparation of products with antioxidant or antimicrobial properties.

Assessment of Microbial Populations in the Manufacture of Vacuum-Packaged Ready-to-Eat Roast Beef and in a Related Production Plant.

Some microbiological criteria were monitored for 6 months in vacuum-packaged roast beef (15 production batches), raw beef (10 batches), and other meat products (12 batches) produced in an Italian small to medium-size enterprise. Fifty-five environmental swab samples also were analyzed. The main bacterial groups were identified by cultural methods according to International Organization for Standardization standards. Listeria monocytogenes was enumerated with the most-probable-number protocol, and species identification was confirmed with a specific PCR assay. Immediately after vacuum packaging, all ready-to-eat (RTE) products had low mean aerobic colony counts (<102 to 2.4 × 102 CFU g-1), anaerobic colony counts (1.6 to 6.5 × 101 CFU g-1), Enterobacteriaceae counts (1.1 to 1.4 × 101 CFU g-1), and Escherichia coli counts (generally below the detection limit). Nevertheless, the prevalence of L. monocytogenes in these samples was 3.7%. In roast beef samples, the aerobic and anaerobic colony counts reached unacceptable levels (>106 CFU g-1) after 14 days of refrigerated storage. Because the prevalence of L. monocytogenes increased to 13.3% during storage, a substantial reduction in the shelf life of these products is recommended. Surfaces without direct contact with food (floors and drains) had the highest mean counts for aerobic colonies (8.0 × 103 to 9.5 × 105 CFU/cm2), anaerobic colonies (2.9 × 103 to 3.2 × 104 CFU/cm2), Enterobacteriaceae (1.5 × 101 to 8.4 × 101 CFU/cm2), and E. coli (6.0 to 7.7 CFU/cm2). The levels of L. monocytogenes on direct food contact surfaces were below the detection limit, but more than 25% of floor samples were contaminated. These results reveal the persistence of L. monocytogenes in food processing environments, although at very low levels, posing a high risk of postcooking recontamination for RTE products. To improve hygienic conditions and reduce cross-contamination, an increase in operator awareness and a reassessment of surface sanitization protocols are needed.

Data on thermal and hydrolytic stability of both domiphen bromide and para-bromodomiphen bromide.

In this data article, HPLC analyses were applied to investigate the hydrolytic and thermal stability of domiphen bromide a FDA approved OTC ingredients endowed with antimicrobial activity. The data obtained by stressing domiphen bromide in acid, base and thermal conditions enlarge the research article published by Fumagalli et al. [2]. The chromatograms herein presented reveal that domiphen bromide is stable under acidic and thermal stress while the treatment with base yield to a by-product. The para-bromo derivative, p-bromodomiphen bromide shows the same behavior under the up mentioned stressed conditions.

Stressed degradation studies of domiphen bromide by LC-ESI-MS/MS identify a novel promising antimicrobial agent.

Nowadays, parabens have been replaced by domiphen bromide, which is widely used in pharmaceutical and cosmetic products. The main aim of this study was to investigate stressed degradation products of domiphen bromide by mean of a rapid, specific and reliable LC-ESI MS/MS since phenyl bromination may occur due to the oxidation of bromide counter ion under oxidative conditions. LC-ESI-MS/MS have characterized a new compound, p-bromodomiphen, as the only degradation product and structure elucidation was also confirmed by the synthesis of the standard. Notably, the resulting p-bromodomiphen bromide is more stable then domiphen bromide in oxidizing conditions since no di-bromoderivatives were detected by MS studies; both domiphen and its p-bromo derivative were tested for antibacterial activity and were more effective on Gram positive (Staphylococcus aureus ATCC25923 and Bacillus cereus DSM31) compared to Gram negative bacteria (Escherichia coli ATCC25922 and Pseudomonas aeruginosa DSM22644). In conclusion, stressed degradation studies by LC-ESI-MS/MS have characterized a new compound that comprises an alternative to domiphen bromide since its antimicrobial activity is comparable to, if not better than, the parental compound.

Can Zymomonas mobilis Substitute Saccharomyces cerevisiae in Cereal Dough Leavening?

Baker’s yeast intolerance is rising among Western populations, where Saccharomyces cerevisiae is spread in fermented food and food components. Zymomonas mobilis is a bacterium commonly used in tropical areas to produce alcoholic beverages, and it has only rarely been considered for dough leavening probably because it only ferments glucose, fructose and sucrose, which are scarcely present in flour. However, through alcoholic fermentation, similarly to S. cerevisiae, it provides an equimolar mixture of ethanol and CO2 that can rise a dough. Here, we propose Z. mobilis as a new leavening agent, as an alternative to S. cerevisiae, overcoming its technological limit with different strategies: (1) adding glucose to the dough formulation; and (2) exploiting the maltose hydrolytic activity of Lactobacillus sanfranciscensis associated with Z. mobilis. CO2 production, dough volume increase, pH value, microbial counts, sugars consumption and ethanol production were monitored. Results suggest that glucose addition to the dough lets Z. mobilis efficiently leaven a dough, while glucose released by L. sanfranciscensis is not so well fermented by Z. mobilis, probably due to the strong acidification. Nevertheless, the use of Z. mobilis as a leavening agent could contribute to increasing the variety of baked goods alternative to those leavened by S. cerevisiae.

One step access to oxindole-based ß-lactams through Ugi four-center three-component reaction.

A multicomponent Ugi reaction involving isatin, isocyanide and ß-amino acid components has been developed. The reactions proceeded smoothly to give ß-lactam-containing 3,3-disubstituted oxindoles in only one step and generally high yields. When chiral, non racemic, ß-amino acids were used, products were obtained as enantiomerically pure ß-lactams diastereoisomers, whose relative stereochemistry was determined by X-ray analysis. For one compound, a weak antibacterial activity has been preliminarily highlighted.

Use of Native Yeast Strains for In-Bottle Fermentation to Face the Uniformity in Sparkling Wine Production.

The in-bottle fermentation of sparkling wines is currently triggered by few commercialized Saccharomyces cerevisiae strains. This lack of diversity in tirage yeast cultures leads to a prevalent uniformity in sensory profiles of the end products. The aim of this study has been to exploit the natural multiplicity of yeast populations in order to introduce variability in sparkling wines throughout the re-fermentation step. A collection of 133 S. cerevisiae strains were screened on the basis of technological criteria (fermenting power and vigor, SO2 tolerance, alcohol tolerance, flocculence) and qualitative features (acetic acid, glycerol and H2S productions). These activities allowed the selection of yeasts capable of dominating the in-bottle fermentation in actual cellar conditions: in particular, the performances of FX and FY strains (isolated in Franciacorta area), and OX and OY strains (isolated in Oltrepò Pavese area), were compared to those of habitually used starter cultures (IOC18-2007, EC1118, Lalvin DV10), by involving nine wineries belonging to the two Consortia of Appellation of Origin. The microbiological analyses of samples have revealed that the indigenous strains showed an increased latency period and a higher cultivability along the aging time than the commercial starter cultures do. Results of chemical analyses and sensory evaluation of the samples after 18 months sur lies have shown that significant differences (p < 0.05) were present among the strains for alcoholic strength, carbon dioxide overpressure and pleasantness, whereas they were not observed for residual sugars content, titratable acidity or volatile acidity. Indigenous S. cerevisiae exhibited comparable values respect to the commercial starter cultures. The ANOVA has also proven that the base wine formulation is a key factor, by significantly affecting (p < 0.01) some oenological parameters of wine, like alcoholic strength, volatile acidity, carbon dioxide overpressure, titratable acidity and dry extract. The use of native yeast strains for the re-fermentation step can be considered a convenient way for introducing differentiation to the final product without modifying the traditional technology. In a perspective of "precision enology," where the wine is designed on specific vine cultivars and microorganisms, this work underlines that exploring yeast biodiversity is a strategic activity to improve the production.

Genotypic characterization and biofilm formation of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) are recognized as one of the most dangerous foodborne pathogens. The production of Shiga toxins together with intimin protein is among the main virulence factors. However, the ability to form biofilm can protect bacteria against environmental factors (i.e. desiccation, exposure to UV rays, predation, etc.) and sanitization procedures (cleaning, rinsing, chlorination), increasing their survival on food products and in manufacturing plants. Forty-five isolates collected from food and fecal samples were genotyped by pulsed field gel electrophoresis analysis with XbaI restriction enzyme and investigated by searching for toxins (stx1, stx2) and intimin (eae) genes and serogroup (O157, O26, O145, O111, O103 and O104). Afterward, the ability to develop biofilm in microtiter assay and the production of adhesive curli fimbriae and cellulose on agar plates were tested. Our study demonstrated that biofilm formation has a great variability among STEC strains and cannot be related to a specific pulsotype nor even to serogroup or presence of virulence genes.

Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese.

Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a "third-generation PCR") provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4 × 106 to 4 × 101 CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

Strain Diversity of Pseudomonas fluorescens Group with Potential Blue Pigment Phenotype Isolated from Dairy Products.

The blue discoloration in Mozzarella cheese comes from bacterial spoilage due to contamination with Pseudomonas. Fourteen Pseudomonas fluorescens strains from international collections and 55 new isolates of dominant bacterial populations from spoiled fresh cheese samples were examined to assess genotypic and phenotypic strain diversity. Isolates were identified by 16S rRNA gene sequencing and tested for the production of the blue pigment at various temperatures on Mascarpone agar and in Mozzarella preserving fluid (the salty water in which the cheese is conserved, which becomes enriched by cheese minerals and peptides during storage). Pulsed-field gel electrophoresis analysis after treatment with the endonuclease SpeI separated the isolates into 42 genotypes at a similarity level of 80%. Based on the pulsotype clustering, 12 representative strains producing the blue discoloration were chosen for the multilocus sequence typing targeting the gyrB, glnS, ileS, nuoD, recA, rpoB, and rpoD genes. Four new sequence typing profiles were discovered, and the concatenated sequences of the investigated loci grouped the tested strains into the so-called ''blue branch'' of the P. fluorescens phylogenetic tree, confirming the linkage between pigment production and a specific genomic cluster. Growth temperature affected pigment production; the blue discoloration appeared at 4 and 14°C but not at 30°C. Similarly, the carbon source influenced the phenomenon; the blue phenotype was generated in the presence of glucose but not in the presence of galactose, sodium succinate, sodium citrate, or sodium lactate.