All fermented foods are subject to the risk of biogenic amine contamination. Histamine and tyramine are among the most toxic amines for consumers' health, exerting undesirable effects on the central nervous and vascular systems, but putrescine and cadaverine can also compromise the organoleptic properties of contaminated foods. These compounds are produced by fermenting microbial flora that decarboxylate amino acids to amines. Little is known of the factors which induce biosynthesis of decarboxylating enzymes and/or which modulate their catalytic activity: the accumulation of amines is generally considered to be a mechanism that contrasts an acidic environment and/or that produces metabolic energy through coupling amino acid decarboxylation with electrogenic amino acid/amine antiporters. Two Lactobacillus strains, Lactobacillus sp. 30a (ATCC 33222), and a Lactobacillus sp. strain (w53) isolated from amine-contaminated wine, carrying genetic determinants for histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), were studied and the influence of some environmental and nutritional parameters on amine production and protein biosynthesis was analyzed through a proteomic approach; this is the first report of a proteomic analysis of amine-producing bacteria. HDC and ODC biosynthesis were shown to be closely dependent on the presence of high concentrations of free amino acids in the growth medium and to be modulated by the growth phase. The stationary phase and high amounts of free amino acids also strongly induced the biosynthesis of an oligopeptide transport protein belonging to the proteolytic system of Lactic Acid Bacteria. At least two isoforms of glyceraldehyde-3-phosphate dehydrogenase, with different M(r), pI and expression profiles, were identified from Lactobacillus sp. w53: the biosynthesis of one isoform, in particular, is apparently repressed by high concentrations of free amino acids. Other proteins were identified from the Lactobacillus proteome, affording a global knowledge of protein biosynthesis modulation during biogenic amine production.

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of beta-phenylethylamine. Kinetics of tyramine and beta-phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine-enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to beta-phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2-DE and MALDI-TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and beta-phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down-regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy-supplying routes. The most interesting finding is a membrane-bound TDC highly over-expressed during amine production. This is the first evidence of a true membrane-bound TDC, longly suspected in bacteria on the basis of the gene sequence.

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Lactic acid bacteria (LAB) are very ancient organisms that can't obtain metabolic energy by respiration without external heme supplementation. Since the gain in ATP from lactic fermentation is inadequate to support efficient growth, they developed alternative strategies for energy production. Three main energy generating routes are present in LAB: amino acid decarboxylation, malate decarboxylation and arginine deimination (ADI pathway). These routes, apart from supplying energy, also play a role in pH control. Lactic fermentation, which leads to lactic acid accumulation, causes a pH decrease that amino acid decarboxylations, originating basic amines, and the ADI pathway, giving rise to ammonia, may partially contrast. In the present mini-review, the reciprocal relationships among these metabolic pathways are considered, on the basis of proteomic results obtained from four different LAB strains, all of which possess the ADI pathway, but express different amino acid decarboxylases. The strains have been isolated and selected from different habitats and the role of some inducing molecules as well as of the growth phases is discussed. The overall results have revealed that LAB are complex biosystems able to set up a sophisticated metabolic regulation through a complex network of proteins that also include stress responses, as well as protease activation or inhibition.

Selenium (Se), Se-cysteines and selenoproteins have received growing interest in the nutritional field as redox-balance modulating agents. The aim of this study was to establish the Se-concentrating and Se-metabolizing capabilities of the probiotic Lactobacillus reuteri Lb2 BM, for nutraceutical applications. A comparative proteomic approach was employed to study the bacteria grown in a control condition (MRS modified medium) and in a stimulated condition (4.38 mg/L of sodium selenite). The total protein extract was separated into two pI ranges: 4-7 and 6-11; the 25 identified proteins were divided into five functional classes: (i) Se metabolism; (ii) energy metabolism; (iii) stress/adhesion; (iv) cell shape and transport; (v) proteins involved in other functions. All the experimental results indicate that L. reuteri Lb2 BM is able to metabolize Se(IV), incorporating it into selenoproteins, through the action of a selenocysteine lyase, thus enhancing organic Se bioavailability. This involves endo-ergonic reactions balanced by an increase of substrate-level phosphorylation, chiefly through lactic fermentation. Nevertheless, when L. reuteri was grown on Se a certain degree of stress was observed, and this has to be taken into account for future applicative purposes. The proteomic approach has proven to be a powerful tool for the metabolic characterization of potential Se-concentrating probiotics.

Insects have been proposed as a high quality, efficient and sustainable alternative protein source for humans and animals, and a vast selection of edible products is currently available in many European countries. However, respiratory allergy among professional and domestic breeders and food allergy among consumers are known. 1,2We here report the case of 2 patients (Pt#1 and Pt#2, both male, 24 and 27 years old, respectively) employed in the production of insect flour made of yellow mealworm larvae (Tenebrio molitor larvae, TML) and black soldier fly larvae (Hermetia illucens larvae, HIL).The patients did not show the previous history of food or respiratory allergies. In the same factory, a total of ten workers handled TML, eight out of ten without developing any symptom. The first symptoms of the 2 patients (rhinoconjunctivitis, itching and contact erythema) appeared after some weeks of repetitive exposure to TML. They did not report any symptoms when exposed to HIL.The symptoms were particularly severe during the sifting of TML from their faeces (TMF), and when the patients entered the rearing room after the sifting operation, they disappeared after 24 hours.The sensitization therefore presumably occurred through a percutaneous route and/or by inhalation. After developing symptoms, Pt#1 continued to work using adequate protection devices, while Pt#2 left the job. The 2 patients were used to eat such edible insects as the greater wax moth, crickets and HI. They experienced an oral allergy syndrome (OAS) characterized by oral pruritus and tightness in the throat, as soon as they start eating for the first time a TML hamburger. Symptoms recovered spontaneously in about 40 minutes. Following this episode, they continued to tolerate other edible insects, but never tried to taste TML again and refused to undergo provocation test.In order to characterize the patients' allergic profile, we performed: skin prick tests (SPTs) with a standard battery of inhalant and food allergens, specific IgE for TM, Der p10, Dermatophagoides pteronyssinus, Dermatophagoides farinae and Prawn (ImmunoCap Thermo Fisher Scientific Inc) and basophil activation test (BAT) with TML and TMF protein extracts. Immunoblot analyses of both protein extracts and LC-MS/MS analysis followed by protein database screening were performed in order to identify the allergens involved in the TM sensitization (see Appendix S1).The SPTs with inhalant and food allergens were negative in both patients, except for Grass in Pt#1 and Alternaria in Pt#2. Specific TM IgEs were present in both patients (21.00 and 10.63 kU A /L, respectively), while no specific IgEs were found for house dust mites, Prawn and Der p10. BAT were positive in both subjects for TMF and TML (Figure 1).Immunoblotting showed a 15 kDa reactive band in both the TML and TMF extracts, when incubated with both patients' sera ( Figure 2, panel A). Three additional aspecific bands were also detected in the healthy control patients' serum pool, but only in the TML extract ( Figure 2). The cockroach all...

Background: Shrimp sensitization is common in the general population, but the presence of symptoms is only moderately related to sensitization. A point still at issue is which in vivo and/or in vitro tests (food challenge, component-resolved diagnosis, house dust mite [HDM] sensitization) can help in distinguishing shrimp-allergic subjects from subjects that are sensitized but tolerant. Methods: The aim of this study was to evaluate the role of IgE to the different shrimp and mite allergens in distinguishing shrimp challenge-positive from challenge-negative patients. Subjects with suspected hypersensitivity reactions to shrimp, positive skin prick tests (SPTs), and/or anti-shrimp IgE were submitted to open and double-blind placebo-controlled food challenges (DBPCFC). Specific IgE to shrimp, mites, and the recombinants rPen a 1, rDer p 1, 2, and 10 were tested using ImmunoCAP-FEIA. IgE immunoblotting was performed to identify the patients' allergenic profiles. Results: In total, 13 out of 51 (25.5%) patients with reported reactions to shrimp were truly shrimpallergic (7 DBPCFC positive and 6 with documented severe reactions). These patients had significantly higher skin test wheal diametersthan nonallergic patients,as well as higher levels of IgE to rPen a 1 and rDer p 10. HDM-induced asthma and thesimultaneous presence of anti-nDer p 1, 2, and 10 IgE levels increased the risk of true shrimp allergy. Conclusion: Food challenge tests are mandatory for the diagnosis of shrimp allergy. Tropomyosin is associated with clinical reactivity. HDM-induced asthma and anti-mite IgE are risk factors for shrimp allergy.