A Snapshot, Using a Multi-Omic Approach, of the Metabolic Cross-Talk and the Dynamics of the Resident Microbiota in Ripening Cheese Inoculated with Listeria innocua.

Raw milk cheeses harbor complex microbial communities. Some of these microorganisms are technologically essential, but undesirable microorganisms can also be present. While most of the microbial dynamics and cross-talking studies involving interaction between food-derived bacteria have been carried out on agar plates in laboratory-controlled conditions, the present study evaluated the modulation of the resident microbiota and the changes of metabolite production directly in ripening raw milk cheese inoculated with Listeria innocua strains. Using a proxy of the pathogenic Listeria monocytogenes, we aimed to establish the key microbiota players and chemical signals that characterize Latteria raw milk cheese over 60 days of ripening time. The microbiota of both the control and Listeria-inoculated cheeses was analyzed using 16S rRNA targeted amplicon sequencing, while direct analysis in real time mass spectrometry (DART-HRMS) was applied to investigate the differences in the metabolic profiles of the cheeses. The diversity analysis showed the same microbial diversity trend in both the control cheese and the inoculated cheese, while the taxonomic analysis highlighted the most representative genera of bacteria in both the control and inoculated cheese: Lactobacillus and Streptococcus. On the other hand, the metabolic fingerprints revealed that the complex interactions between resident microbiota and L. innocua were governed by continuously changing chemical signals. Changes in the amounts of small organic acids, hydroxyl fatty acids, and antimicrobial compounds, including pyroglutamic acid, hydroxy-isocaproic acid, malic acid, phenyllactic acid, and lactic acid, were observed over time in the L. innocua-inoculated cheese. In cheese that was inoculated with L. innocua, Streptococcus was significantly correlated with the volatile compounds carboxylbenzaldheyde and cyclohexanecarboxylic acid, while Lactobacillus was positively correlated with some volatile and flavor compounds (cyclohexanecarboxylic acid, pyroxidal acid, aminobenzoic acid, and vanillic acid). Therefore, we determined the metabolic markers that characterize a raw milk cheese inoculated with L. innocua, the changes in these markers with the ripening time, and the positive correlation of flavor and volatile compounds with the resident microbiota. This multi-omics approach could suggest innovative food safety strategies based on the enhanced management of undesirable microorganisms by means of strain selection in raw matrices and the addition of specific antimicrobial metabolites to prevent the growth of undesirable microorganisms.

Authentication of Edible Insects' Powders by the Combination of DART-HRMS Signatures: The First Application of Ambient Mass Spectrometry to Screening of Novel Food.

This feasibility study reports the use of direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) in profiling the powders from edible insects, as well as the potential for the identification of different insect species by classification modeling. The basis of this study is the revolution that has occurred in the field of analytical chemistry, with the improved capability of ambient mass spectrometry to authenticate food matrices. In this study, we applied DART-HRMS, coupled with mid-level data fusion and a learning method, to discriminate between Acheta domesticus (house cricket), Tenebrio molitor (yellow mealworm), Locusta migratoria (migratory locust), and Bombyx mori (silk moth). A distinct metabolic fingerprint was observed for each edible insect species, while the Bombyx mori fingerprint was characterized by highly abundant linolenic acid and quinic acid; palmitic and oleic acids are the statistically predominant fatty acids in black soldier fly (Hermetia illucens). Our chemometrics also revealed that the amino acid proline is a discriminant molecule in Tenebrio molitor, whereas palmitic and linoleic acids are the most informative molecular features of the house cricket (Acheta domesticus). Good separation between the four different insect species was achieved, and cross-validation gave 100% correct identification for all training samples. The performance of the random forest classifier was examined on a test set and produced excellent results, in terms of overall accuracy, sensitivity, and specificity. These results demonstrate the reliability of the DART-HRMS as a screening method in a future quality control scenario to detect complete substitution of insect powders.

Serum Metabolomic Profiles of Paratuberculosis Infected and Infectious Dairy Cattle by Ambient Mass Spectrometry.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis [Johne's disease (JD)], a chronic disease that causes substantial economic losses in the dairy cattle industry. The long incubation period means clinical signs are visible in animals only after years, and some cases remain undetected because of the subclinical manifestation of the disease. Considering the complexity of JD pathogenesis, animals can be classified as infected, infectious, or affected. The major limitation of currently available diagnostic tests is their failure in detecting infected non-infectious animals. The present study aimed to identify metabolic markers associated with infected and infectious stages of JD. Direct analysis in real time coupled with high resolution mass spectrometry (DART-HRMS) was, hence, applied in a prospective study where cohorts of heifers and cows were followed up annually for 2-4 years. The animals' infectious status was assigned based on a positive result of both serum ELISA and fecal PCR, or culture. The same animals were retrospectively assigned to the status of infected at the previous sampling for which all JD tests were negative. Stored sera from 10 infected animals and 17 infectious animals were compared with sera from 20 negative animals from the same herds. Two extraction protocols and two (-/+) ionization modes were tested. The three most informative datasets out of the four were merged by a mid-level data fusion approach and submitted to partial least squares discriminant analysis (PLS-DA). Compared to the MAP negative subjects, metabolomic analysis revealed the m/z signals of isobutyrate, dimethylethanolamine, palmitic acid, and rhamnitol were more intense in infected animals. Both infected and infectious animals showed higher relative intensities of tryptamine and creatine/creatinine as well as lower relative abundances of urea, glutamic acid and/or pyroglutamic acid. These metabolic differences could indicate altered fat metabolism and reduced energy intake in both infected and infectious cattle. In conclusion, DART-HRMS coupled to a mid-level data fusion approach allowed the molecular features that identified preclinical stages of JD to be teased out.

Determination of Deoxynivalenol Biomarkers in Italian Urine Samples.

Deoxynivalenol (DON) is a mycotoxin mainly produced by Fusariumgraminearum that can contaminate cereals and cereal-based foodstuff. Urinary DON levels can be used as biomarker for exposure assessment purposes. This study assessed urinary DON concentrations in Italian volunteers recruited by age group, namely children, adolescents, adults, and the elderly. In addition, vulnerable groups, namely vegetarians and pregnant women, were included in the study. To determine the urinary DON, its glucuronide and de-epoxydated (DOM-1) forms, an indirect analytical approach was used, measuring free DON and total DON (as sum of free and glucuronides forms), before and after enzymatic treatment, respectively. Morning urine samples were collected on two consecutive days, from six different population groups, namely children, adolescent, adults, elderly, vegetarians and pregnant women. Total DON was measured in the 76% of the collected samples with the maximum incidences in children and adolescent age group. Urine samples from children and adolescent also showed the highest total DON levels, up to 17.0 ng/mgcreat. Pregnant women had the lowest positive samples per category (40% for day 1 and 43% for day 2, respectively), low mean levels of total DON (down to 2.84 ng/mgcreat) and median equal to 0 ng/mgcreat. Estimation of DON dietary intake reveals that 7.5% of the total population exceeds the TDI of 1 µg/kg bw/day set for DON, with children showing 40% of individuals surpassing this value (male, day 2).

Direct analysis real-time-high-resolution mass spectrometry for Triticum species authentication.

Due to favourable climate condition, Italy is a prominent producer of different wheat varieties. Several wheat baked goods are produced, but the most typical Italian foods, like pasta, pizza and bread, are made of durum and common wheat flour. Because of the great importance of wheat in the Italian food market, authenticity represents an essential quality parameter not only for the producers and regulatory bodies but also for consumers. The aim of our study was to test the effectiveness of an unconventional non-targeted method for the discrimination of Triticum species using direct analysis real time-high-resolution mass spectrometry (DART-HRMS). For this purpose, 60 wheat samples including durum, common and hulled wheat varieties were collected over two consecutive harvest years. Chemometric evaluation revealed an optimal sample clustering according to the wheat species and the presence of 18 significant markers able to discriminate the groups. The discrimination power obtained is promising since the use of DART-HRMS can significantly reduce the analysis time compared to chromatographic techniques. A plausible future commercial and industrial scenario could see the application of this analytical approach especially to evaluate the risk of substitution of higher value wheat species with lower value flours.

Occurrence of deoxynivalenol in an elderly cohort in the UK: a biomonitoring approach.

Deoxynivalenol (DON) is a Fusarium toxin, to which humans are frequently exposed via diet. Although the elderly are speculated to be sensitive to the toxic effects of DON as a result of age-related conditions, disease and altered DON metabolism, there is lack of available data on DON biomarkers in this age group. This study characterised urinary DON concentrations and its metabolites in elderly aged ≥65years (n = 20) residing in Hull, UK. Morning urinary specimens were collected over two consecutive days together with food records to assess dietary intake over a 24h-period prior to each urinary collection. Free DON (un-metabolised), total DON (sum of free DON and DON-glucuronides or DON-GlcA) and de-epoxy deoxynivalenol (DOM-1) were analysed using a validated LC-MS/MS methodology. Total DON above the limit of quantification 0.25 ng/mL was detected in the urine from 90% of elderly men and women on both days. Mean total DON concentrations on day 1 were not different from those on day 2 (elderly men, day 1: 22.2 ± 26.3 ng/mg creatinine (creat), day 2: 28.0 ± 34.4 ng/mg creat, p = 0.95; elderly women, day 1: 22.4 ± 14.6 ng/mg creat, day 2: 29.1 ± 22.8 ng/mg creat, p = 0.58). Free DON and DON-GlcA were detected in 60-70% and 90% of total urine samples, respectively. DOM-1 was absent from all samples; the LoQ for DOM-1 was 0.50 ng/mL. Estimated dietary intake of DON suggested that 10% of the elderly exceeded the maximum provisional tolerable daily intake for DON. In this single-site, UK-based cohort, elderly were frequently exposed to DON, although mean total DON concentrations were reported at moderate levels. Future larger studies are required to investigate DON exposure in elderly from different regions of the UK, but also from different counties worldwide.

Assessment of Urinary Deoxynivalenol Biomarkers in UK Children and Adolescents.

Deoxynivalenol (DON), the mycotoxin produced mainly by Fusarium graminearum and found in contaminated cereal-based foodstuff, has been consistently detected in body fluids in adults. Available data in children and adolescents are scarce. This study assessed urinary DON concentrations in children aged 3-9 years (n = 40) and adolescents aged 10-17 years (n = 39) in the UK. Morning urine samples were collected over two consecutive days and analysed for free DON (un-metabolised form), DON-glucuronides (DON-GlcA), deepoxy deoxynivalenol (DOM-1), and total DON (sum of free DON, DON-GlcA, and DOM-1). Total DON was detected in the urine of >95% of children and adolescents on both days. Mean total DON concentrations (ng/mg creatinine) were 41.6 and 21.0 for children and adolescents, respectively. The greatest total DON levels were obtained in female children on both days (214 and 219 ng/mg creatinine on days 1 and 2, respectively). Free DON and DON-GlcA were detected in most urine specimens, whereas DOM-1 was not present in any sample. Estimation of dietary DON exposure suggested that 33-63% of children and 5-46% of adolescents exceeded current guidance regarding the maximum provisional tolerable daily intake (PMTDI) for DON. Although moderate mean urinary DON concentrations were shown, the high detection frequency of urinary DON, the maximum biomarker concentrations, and estimated dietary DON exposure are concerning.