Substitution of Asp29 with Asn29 in the metallochaperone UreE of Streptococcus thermophilus DSM 20617T increases the urease activity and anticipates urea hydrolysis during milk fermentation.

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

Shaping Future Foods through Fermentation of Side Streams: Microbial, Chemical, and Physical Characteristics of Fermented Blends from Sunflower Seed Press Cake and Cheese Whey.

The current food system suffers from the inefficient use of resources, including the generation of side streams of low economic value that still contain nutritional components. One potential approach to reach a more sustainable food system is to reintroduce such side streams into a circular value chain and valorise them in novel food products, preferably in an unrefined or minimally refined manner. Blending side streams from different industries might be a suitable way to improve the nutritional value of the final matrix. In this study, sunflower seed press cake and cheese whey were combined to obtain matrices containing valuable proteins, structuring polysaccharides, as well as lactose and minerals facilitating fermentation with three different co-cultures of lactic acid bacteria and yeasts. Fermentation for 48 h at 26 °C decreased the pH from ~6.3 to ~4.7 and enhanced the storage stability of the blends with no effect on their rheological properties and microstructure. This research demonstrates the potential of fermentation as a mean to stabilise side stream blends while only minimally affecting their physical appearance.

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.

Biotechnological Potential and Safety Evaluation of Dextran- and Riboflavin-Producing Weisella cibaria Strains for Gluten-Free Baking.

Gluten consumption causes several immunological and non-immunological intolerances in susceptible individuals. In this study, the dextran-producing Weissella cibaria BAL3C-5 and its derivative, the riboflavin-overproducing strain BAL3C-5 C120T, together with a commercial bakery yeast, were used to ferment gluten-free (GF)-doughs obtained from corn and rice flours at two different concentrations and supplemented with either quinoa, buckwheat, or chickpea to obtain laboratory-scale GF bread. The levels of dextran, riboflavin, and total flavins were determined in the fermented and breads. Both strains grew in fermented doughs and contributed dextran, especially to those made with corn plus quinoa (~1 g/100 g). The highest riboflavin (350-150 µg/100 g) and total flavin (2.3-1.75 mg/100 g) levels were observed with BAL3C-5 C120T, though some differences were detected between the various doughs or breads, suggesting an impact of the type of flour used. The safety assessment confirmed the lack of pathogenic factors in the bacterial strains, such as hemolysin and gelatinase activity, as well as the genetic determinants for biogenic amine production. Some intrinsic resistance to antibiotics, including vancomycin and kanamycin, was found. These results indicated the microbiological safety of both W. cibaria strains and indicated their potential application in baking to produce GF bread.

Valorisation of Bovine Sweet Whey and Sunflower Press Cake Blend through Controlled Fermentation as Platform for Innovative Food Materials.

The current environmental challenge is pushing food systems towards more sustainable models of production that require reorganizing of processes by re-using side products still containing nutrients. This work aimed at valorising a mix of bovine sweet whey and sunflower press cake, through targeted fermentation. After preliminary screening based on growth rate, final pH, lactose/galactose assimilation, phytase activity, six Lactic Acid Bacteria strains (Lacticaseibacillus casei, L. paracasei (2), Lactococcus lactis, Lentilactobacillus parakefiri and Leuconostoc pseudomesenteroides) and three yeasts (Kluyveromyces lactis, K. marxianus and Torulaspora delbrueckii) were co-cultivated in pairs in microcosms (1-part ground press cake: 4-parts whey). All tested microorganisms were able to grow and acidify the blend: the LAB counts increased during the incubation (26 °C for 48 h) of +2.80 log CFU/g, whereas yeasts counts were of +1.98 log CFU/g, with significant differences among the different associations (p < 0.01). Mould counts were always <3 log CFU/g. Interestingly, the bacterial contaminants count significantly varied in samples with different pairs of strains (p < 0.001). Acidification level, acetic acid and ethanol contents were the limiting factors affecting the growth of spoilage micro-organisms. Best performances were attained in microcosms inoculated with L. lactis or L. paracasei and K. lactis or K. marxianus.

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.

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.

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.

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens.

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action.