The Riboflavin Metabolism in Four Saccharomyces cerevisiae Wine Strains: Assessment in Oenological Condition and Potential Implications with the Light-Struck Taste.

Riboflavin (RF), or vitamin B2, is an essential compound for yeast growth and a precursor of the flavin coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in redox and non-redox processes. RF is a photosensitive compound involved in the light-struck taste (LST), a fault causing the formation of off-flavors that can develop when the wine is exposed to light in the presence of methionine (Met), as well. As both RF and Met can be associated with detrimental changes in wines, a better comprehension of its yeast-mediated production is relevant to predict the maintenance of the desired character of the wine. This study aims at assessing the production of flavin derivatives (FDs) and Met by S. cerevisiae oenological starters under laboratory conditions. The results showed the presence of extra- and intracellular FDs, and Met is a strain-dependent characteristic being also affected by the initial content of RF in the medium. This finding was confirmed when the winemaking was carried out in a relevant environment. Our results evidenced the important impact of the yeast strain on the content of RF and its derivatives.

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods.

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier "indolome" to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us "indolome") in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100-1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition.

Assessment of Tryptophan, Tryptophan Ethylester, and Melatonin Derivatives in Red Wine by SPE-HPLC-FL and SPE-HPLC-MS Methods.

Melatonin (MEL) is an indoleamine produced mainly by the pineal gland in vertebrates. It plays a significant role in the regulation of circadian rhythms, mitigation of sleeping disorders, and jet lag. This compound is synthetized from tryptophan (TRP) and it has been found in seeds, fruits, and fermented beverages, including wine. Wine is also a source of other tryptophan derivatives, the tryptophan ethylester (TEE) and MEL isomers (MISs), for which the biological properties need to be elucidated. An analytical method for the simultaneous quantification of TRP, TEE, and MEL was developed by a Solid Phase Extraction (SPE) of a preconcentration of wine followed by high performance liquid chromatography (HPLC) analysis either with fluorescence or mass spectrometer detectors. The analytical method showed a relative standard deviation (RSD) lower than 8%, except for TRP (RSD 10.5% in wine). The recovery was higher than 76%. The versatility of SPE preconcentrations allowed for the adequate preconcentration of wine sample as well as detection of low concentrations, an important aspect especially for MEL (detection limit 0.0023 µg/L). The proposed method proved to be suitable for assessing the investigated compounds in some red wine samples, where 74.4⁻256.2 µg/L and 0.038⁻0.063 µg/L of TEE and MEL were detected, respectively. Five MISs were also found in wine samples in concentrations up to 1.97 µg/L.

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.

Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure.

Brettanomyces bruxellensis displays a high degree of genotypic and phenotypic polymorphism and is the main yeast species involved in wine spoilage. The innate resistance of 108 B. bruxellensis strains to the antimicrobial agent SO2 used in winemaking was investigated. Nineteen strains (17.6%) were sensitive to SO2 , failing to grow at the lowest concentration tested (0.1 mg L(-1) molecular SO2). Twenty-nine strains (26.8%) grew at 0.1 mg L(-1), 42 strains (38.9%) grew at 0.2 mg L(-1) , and 16 strains (14.8%) were able to grow as high as 0.4 mg L(-1) mol. SO2. Two strains able to grow in the presence of 0.6 mg L(-1) mol. SO2 were further studied by GCMS-TOF analysis to define the metabolic response to SO2 treatment. Two hundred and fifty-three intracellular metabolites were detected. The main effect observed was a decrease in cytoplasmic levels of polyols and an increase in levels of some amino acids, alanine, glutamic acid, glycine, proline, 5-oxoproline, serine and valine, which were significantly accumulated in the presence of SO2. No alteration in the pentose phosphate pathway was observed, suggesting NADPH usage could be diverted to other pathways. Finally, a change in metabolites involved in the glycerophospholipid pathway (glycerol-3-phosphate and myo-inositol) was also found.

Assessment of transduction of Escherichia coli Stx2-encoding phage in dairy process conditions.

In the environment, bacteriophages are regarded as natural vector for the transmission of Shiga-toxin genes among Shiga-toxin Escherichia coli strains. The possibility of transduction has been noticed in intestinal tract of various animals but experimental observations on this phenomenon in food processes are lacking. To investigate the transduction in milk at different temperature profiles and cell concentrations, an experimental plan including two different Stx(2)-phages (ϕGV2412 and ϕL34), induced respectively from E. coli O157:H7 181181/2 and E. coli O157:H7 EC34, and two recipient E. coli strains (CNCTC 6896, WG5) was performed. The donor strains were generated by lysogenization of CNCTC 6896 with ϕGV2412 and ϕL34 respectively. Spectinomycin resistance gene (aadA) was inserted into stx(2) operon in order to select transduced cells. Transductants were never observed at 4°C up to 24 h, whereas after a treatment at 37°C for 2 h and at 25°C for 22 h they were detected in 67% of the trials with a ratio of transduction varying from 1.13 10(-6) to 7.87 10(-8). A treatment at 48°C for 2 h followed by a second step at 25°C for 22 h showed an occurrence of transduction events in only 19% of cases with a ratio of transduction varying from 2.22 10(-7) to 2.67 10(-8). The generation of transductants and the spontaneous induction of phages in milk were not affected by initial or final concentration of the donor or recipient strains. The results show that transduction phenomenon occurs when the cells are metabolically active and it does not take place at low temperatures. Therefore, the maintenance of the chilling chain proved to be a main factor to prevent the spread of Stx-genes in dairy processes.