Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

Thermally-Induced Lactosylation of Whey Proteins: Identification and Synthesis of Lactosylated ß-lactoglobulin Epitope.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, ß-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a ß-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

The Sortase-Dependent Fimbriome of the Genus Bifidobacterium: Extracellular Structures with Potential To Modulate Microbe-Host Dialogue.

Bifidobacteria are important gut commensals of mammals, including humans, of any age. However, the molecular mechanisms by which these microorganisms establish themselves in the mammalian gut and persist in this environment are largely unknown. Here, we analyzed the genetic diversity of the predicted arsenal of sortase-dependent pili of known and sequenced members of the Bifidobacterium genus and constructed a bifidobacterial sortase-dependent fimbriome database. Our analyses revealed considerable genetic variability of the sortase-dependent fimbriome among bifidobacterial (sub)species, which appears to have been due to horizontal gene transfer events and for which we were able to perform evolutionary mapping. Functional assessment by transcriptome analysis and binding assays involving different substrates demonstrates how bifidobacterial pili are pivotal in promoting various abilities for adhesion to glycans and extracellular matrix proteins, thereby supporting the ecological success of bifidobacteria in the mammalian gut.IMPORTANCE Adhesion of bifidobacterial cells to the mucosa of the large intestine is considered a hallmark for the persistence and colonization of these bacteria in the human gut. In this context, we analyzed the genetic diversity of the predicted arsenal of sortase-dependent pili of known and sequenced members of the Bifidobacterium genus, and constructed a bifidobacterial sortase-dependent fimbriome database. Our analyses revealed considerable genetic variability of the sortase-dependent fimbriome among bifidobacterial (sub)species, which appears to have been due to horizontal gene transfer events. In addition, functional assessment by transcriptome analysis and binding assays involving different substrates demonstrates how bifidobacterial pili are crucial in promoting various abilities for adhesion to glycans and extracellular matrix proteins, thereby supporting the ecological success of bifidobacteria in the mammalian gut. This study represents a complete genomic study regarding the presence of fimbriae in the genus Bifidobacterium.

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.

The degradation of curcuminoids in a human faecal fermentation model.

In the present study, the colonic metabolism of three curcuminoids (80.1% curcumin, 15.6%, demethoxycurcumin (DMC) and 2.6% bis-demethoxycurcumin (Bis-DMC)) was evaluated using an in vitro model containing human faecal starters. The breakdown products formed were identified and characterized using different analytical platforms. Following in vitro incubation, the relative amounts of degraded curcuminoids and the produced metabolites were analyzed using a UHPLC coupled with a linear ion trap mass spectrometer, with the addition of hybrid ion trap-Orbitrap Mass Spectrometer when required. Up to ∼24% of curcumin, ∼61% of demethoxycurcumin (DMC) and ∼87% of bis-demethoxycurcumin (Bis-DMC) were degraded by the human faecal microbiota after 24 h of fermentation in vitro. Three main metabolites, namely tetrahydrocurcumin (THC), dihydroferulic acid (DFA) and a metabolite with an accurate mass of 181.08734, which was tentatively identified as 1-(4-Hydroxy-3-methoxyphenyl)-2-propanol were detected in the fermentation cultures containing the curcuminoids. The data presented here provide insights into curcuminoid colonic metabolism, showing that bacterial breakdown products should be considered in further studies on both bioavailability bioactivity of curcumin.

Qualitative and quantitative determination of peptides related to celiac disease in mixtures derived from different methods of simulated gastrointestinal digestion of wheat products.

During wheat digestion, gluten-derived proteolytic resistant peptides are generated, some of them involved in celiac disease. In vitro digestion models able to mimic the peptides generated in the human gastrointestinal tract are extremely useful to assess the pathogenicity of wheat-derived products. In this paper, samples belonging to three different durum wheat varieties were taken at six different steps of the pasta production chain and two different digestion models present in the literature were assessed on the different samples: a more complex one using artificial fluids simulating the exact composition of digestive juices, and a simplified method based on a peptic-tryptic/chymotryptic treatment of wheat ethanolic extract. An extensive characterization of the peptides generated using two in vitro digestion models was performed through LC-MS/MS techniques and the two methods were compared in order to evaluate qualitative and quantitative differences and their possible implications for varietal screening. Strong differences in the type of peptides produced with the two methods were detected, indicating that the simplified method can still be used for a varietal screening but is not representative of the peptides really generated after physiological human digestion. Results indicate a clear necessity of physiologically accurate models for simulating human gastrointestinal digestion of wheat products.

Comparison of protein quality and digestibility between plant-based and meat-based burgers.

Nowadays, consumers are increasingly inclined toward plant-based meat analogues for sake of food security, safety, and sustainability. This growing interest, not only from consumers but also from food companies, brought the offer on the market to be wide and vast. From our previous study it emerged that the market supply, especially the Italian one, is diversified both in terms of protein sources and nutrient content. Although these products are increasingly consumed, for most of the meat analogues today on the market, little is still known about their actual protein quality and digestibility. To fill this gap, in this study different commercial plant-based burgers (2 soy-based and 2 pea-based) were selected and compared to two beef burgers, as controls, in terms of protein quality and digestibility. The findings of this study demonstrated the essential amino acidic profile lacks lysine for almost all burgers (including the meat-based ones) compared to the amino acid scoring pattern set by FAO/WHO (for older children and adults), even if the sum of essential amino acids was within the range of sufficiency. All samples showed good initial protein integrity with low hydrolysis (above 6%) and percentage of D-enantiomers (above 15%). The study of the digestibility, performed by the validated INFOGEST in vitro model, showed better protein solubilisation in the case of meat burgers (63 ± 3% and 61 ± 8%), but a good digestibility also in the case of plant-based ones (from 55% to 40%). The degree of hydrolysis of the solubilised proteins was very high in all samples (from 65% to 40%) indicating a very good protein accessibility to digestive enzymes. The analysis of the peptide fraction of digestates indicated a high prevalence of collagen proteins in beef burgers and of reserve proteins in plant-based burgers. This study showed that the differences between these products are mostly dependent on the quality of the raw materials used, rather than on the vegetal or animal protein source. Therefore, to have a product with a good protein quality and digestibility, independently from the protein origin, the consumer needs to make an accurate choice, carefully reading the ingredient list.

Molecular characterization and allergenicity assessment of different samples of Mung Bean.

Legumes represent a promising nutritional alternative source of proteins to meat and dairy products. Additionally, Novel Foods (Regulation EU 2015/2283) can help meet the rising protein demand. However, despite their benefits, emerging allergenicity risks must be considered. The aim of this work is the molecular characterization of the Novel Food Mung bean protein isolate for allergenicity prediction with High Resolution Mass Spectrometry analysis. The assessment of the allergenicity was evaluated in silico by comparing protein sequences of the Novel Food with other known legume allergens, using bioinformatic databases. The results highlighted similarity higher than 60 % of the protein structure of Mung bean with two known allergens of soybean and pea. Furthermore, enzymatic hydrolysis effects on allergenic potential was evaluated by immunoblotting analysis using sera of patients allergic to legumes. The protein hydrolysates obtained showed a high nutritional quality and a reduced allergenic potential, making them suitable for hypoallergenic food formulations.

Effectiveness of enzymatic hydrolysis for reducing the allergenic potential of legume by-products.

The interest in agri-food residues and their valorization has grown considerably, and many of them are today considered to be valuable, under-exploited sources of different compounds and notably proteins. Despite the beneficial properties of legumes by-products, there are also some emerging risks to consider, including their potential allergenicity. In this work the immunoreactivity of chickpea, pea, and white bean by-products was assessed, and whether the production of enzymatic hydrolysates can be an effective strategy to reduce this allergenic potential. The results presented clearly indicate that the efficiency of this strategy is strongly related to the enzyme used and the food matrix. All legume by-products showed immunoreactivity towards serum of legume-allergic patients. Hydrolysates from alcalase did not show residual immunoreactivity for chickpea and green pea, whereas hydrolysates from papain still presented some immunoreactivity. However, for white beans, the presence of antinutritional factors prevented a complete hydrolysis, yielding a residual immunoreactivity even after enzymatic hydrolysis with alcalase.

Molecular composition of lipid and protein fraction of almond, beef and lesser mealworm after in vitro simulated gastrointestinal digestion and correlation with the hormone-stimulating properties of the digesta.

The current production of meat presents many disadvantages for the environment and much research focuses on alternative protein sources. Insects are novel protein sources highly valued for their nutritional and sustainable potential. However, many aspects concerning biological and nutritional properties of the insects after digestion, in comparison with other protein sources, are still overlooked. In this work, a comparative study on three different protein sources, namely almond, lean beef and insect Alphitobius diaperinus (lesser mealworm), was performed after in vitro simulated gastrointestinal digestion. An in-depth characterization of the chemical composition of the solubilized protein and lipid fractions of the digesta was performed by applying different analytical techniques, including chromatographic methods coupled to mass spectrometry and 1H NMR spectroscopy. Beef and insect were proven to be very similar in amino acid composition and protein solubilization after digestion, when considering the proper corrections for the chitin content. Lipid fraction from insects was solubilized during digestion as the one of almonds, but with a fastest kinetics. Thus, lesser mealworms are a good source of both lipids and highly nutritional proteins. Then, the amino acid composition of raw and digested protein fraction from the three sources was related to the PYY, ghrelin, GLP-1 and CCK release and rats' food intake. The composition of amino acids in insect digesta was found to be related to specific effects on enterohormone release, and the modulation of food intake in rats.

Peptide fingerprinting of Hermetia illucens and Alphitobius diaperinus: Identification of insect species-specific marker peptides for authentication in food and feed.

Insects have been proposed as new source of proteins to meet the growing demand connected to the increasing world population. In the EU the inclusion of insect proteins in feed and food is strictly regulated. Hence, analytical methods able to discriminate and identify different insect species in food and feed are a necessity. In this work, a peptidomic approach was applied to determine peptide biomarkers for two edible insect species: lesser mealworm and black soldier fly. Three species specific peptide biomarkers were identified using LC-MS/MS. The two insects were mixed with fish standard feed at different concentrations, to evaluate the feasibility of their use as markers in complex matrices. The detection of marker peptides was confirmed down to 1% insect amount. The data here reported constitutes the first proof of concept for the potential application of the peptide marker approach for the identification and quantification of insect ingredients.

Assessment of Enzymatic Improvers in Flours Using LC-MS/MS Detection of Marker Tryptic Peptides.

Enzymatic improvers are enzymes obtained from microbial or fungal cultures, added as technical adjuvants to flour, with the aim of improving the dough characteristics in bakery products. They are used in a low ppm range and, being technical adjuvants, can go undeclared on the label. Many types of enzymatic improvers are present on the market, such as amylases, lipases, proteases, xylanases, glucose oxidases, and others, each with a different function. Analytical methods capable of detecting these enzymes are needed, particularly for bakery companies, in order to monitor the quality of raw materials and to detect any undeclared presence. In the present work, specific peptide markers, obtained by enzymatic digestion, have been used to detect the presence of enzymatic improvers by LC-MS/MS techniques. Promising results were obtained for some enzymes acting on the carbohydrate fraction (glucoamylase, glucose oxidase, xylanase) in which amounts as low as 20 ppm could be identified in blind flour samples. For lipases and proteases the method proved to be very effective in terms of specific identification, even if less sensitive.

Shotgun proteomics, in-silico evaluation and immunoblotting assays for allergenicity assessment of lesser mealworm, black soldier fly and their protein hydrolysates.

Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG- and IgE-immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. The relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. IgG-immunoblotting analysis with anti-Tropomyosin I antibodies and IgE-immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. The immunoassays were carried out also on protein hydrolysates extracted by treating insects with Protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While IgG-immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, IgE-immunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate.

A Complete Mass Spectrometry (MS)-Based Peptidomic Description of Gluten Peptides Generated During In Vitro Gastrointestinal Digestion of Durum Wheat: Implication for Celiac Disease.

Resistance of gluten to gastrointestinal digestion is involved in immune-mediated adverse reactions to wheat, since several peptides produced by the incomplete digestion are able to trigger, in predisposed individuals, the immune response responsible, for instance, of celiac disease (CD) and other adverse reactions. Even if several peptides have been identified, an exhaustive description of the peptidome generated by wheat digestion is lacking. To this end, in the present work, durum wheat proteins were fractionated, digested, and then subjected to various proteomic techniques, including single stage and multiple stage mass spectrometry (MS) (SDS-PAGE, UPLC/ESI-MS, UPLC/ESI-MS/MS, and LTQ-Orbitrap). Based on SDS-PAGE, although proteins were severely degraded after in vitro gastrointestinal digestion, some differences were observed among protein profile of the different digests. Through untargeted UPLC techniques, 227 peptide sequences were identified, with only few sequences shared by the different digests. In particular, 9 gluten peptides involved in CD were identified. Based on target proteomic, the quantification of these peptides revealed significant (p ≤ 0.05) differences among the different extracts. Taken together, all the proteomic tools confirmed that gluten digestion is closely related to the matrix regardless of wheat genotype.

Food wastes from agrifood industry as possible sources of proteins: A detailed molecular view on the composition of the nitrogen fraction, amino acid profile and racemisation degree of 39 food waste streams.

The nitrogen fraction of 39 food waste streams was characterized by Kjeldahl analysis, amino acid analysis, protein analysis and racemization degree, for assessing their potential for further valorization. For every waste streams the specific nitrogen-to-protein conversion factor was calculated, allowing to assess the accurate protein content. The results indicated which streams are most rich in relevant proteins (all wastes of dairy origin, beer yeast, malted barley germs, brewing cake, rapeseed press cake, sea buckthorn spent pulp, leek leaves, parsley waste, pumpkin kernel cake, and mushroom waste), which ones have valuable proteins, but in too little amount, and also which ones are rich in proteins, but of low nutritional value. Specific data also indicated, for every waste stream, its possible use for supplementing specific amino acids. To date, this represents the most complete characterization with homogeneous methodologies of the nitrogen fraction in food waste streams ever reported in the literature and outlines in unprecedented molecular details the potentialities and the limitations of many waste streams to be used as source of proteins.

Comparison of gluten peptides and potential prebiotic carbohydrates in old and modern Triticum turgidum ssp. genotypes.

Old wheat genotypes are perceived by consumers as healthier than modern ones. The release of gluten peptides with in vitro digestion and the content of potentially prebiotic carbohydrates (i.e. resistant fraction of starch and cell-wall associated dietary fiber) were evaluated in tetraploid wheats, namely 9 old and 3 modern Triticum turgidum ssp. genotypes. Simulated digestion of wholemeal flours yielded 152 major peptides, 59 of which were attributed a sequence. Principal component analysis revealed that peptide profiles were variable in old genotypes, unlike in modern ones. Digestion of old genotypes generally yielded peptides in greater concentration. In particular, 5 peptides of γ-gliadin, known to trigger the adaptive immune reaction, and two peptides of α-gliadin, known to be toxic to celiac patients, were particularly abundant in some old varieties. Resistant starch (RS) was negligible in modern genotypes (<0.6%), but it was remarkably abundant in some old varieties, reaching the highest value in Dauno III (8.5%, P < 0.05). Dauno III also presented the highest amount of soluble fiber (4.2%, P < 0.05). Pasta was made with an old and a modern genotype (Dauno III and PR22D89, respectively) with opposite RS content. Pasta making and cooking affected starch digestibility, overtaking differences between genotypes and yielding the same amount of RS for both the varieties (approx. 1.7%). The data herein presented suggest that the wholemeal flours of old tetraploid wheat genotypes could not boast particular claims associated to a lower exposure to gluten peptides and, if cooked, to a prebiotic potential.

The Aryl Hydrocarbon Receptor (AhR) Mediates the Counter-Regulatory Effects of Pelargonidins in Models of Inflammation and Metabolic Dysfunctions.

Pelargonidins are anthocyanidins thought to be beneficial for the human health, although controversies exist over the doses needed and the unclear mechanism of action, along with poor systemic bioavailability. One putative target of pelargonidins is the aryl hydrocarbon receptor (AhR). A synthetic pelargonidin (Mt-P) was synthesized by the methylation of the pelargonidin (the natural compound indicated as P). Mt-P transactivated the AhR with an EC50 of 1.97 µM and was ~2-fold more potent than the natural compound. In vitro Mt-P attenuated pro-inflammatory activities of Raw264.7 macrophage cells in an AhR-dependent manner. In vivo, administration of the Mt-P in Balb/c mice resulted in a dose-dependent attenuation of signs and symptoms of colitis induced by TNBS. A dose of 5 mg/kg Mt-P, but not the natural compound P, reversed intestinal inflammation and increased expression of Tnf-α, Ifn-Æ´, and Il-6, while promoted the expansion of regulatory T cells and M2 macrophages. In C57BL/6J mice fed a high fat diet (HFD), Mt-P attenuated body weight gain, intestinal and liver inflammation, and ameliorated insulin sensitivity, while worsened liver steatosis by up-regulating the liver expression of Cd36 and Apo100b. These effects were abrogated by AhR gene ablation. Mt-P is a synthetic pelargonidin endowed with robust AhR agonist activity that exerts beneficial effects in murine models of inflammation and metabolic dysfunction.

Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis.

PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.

PNA conjugated to high-molecular weight poly(ethylene glycol): synthesis and properties.

The conjugation of a bioactive, fluorescent PNA sequence to high-molecular weight poly(ethylene glycol) (PEG) is described and the properties of the PEG-PNA conjugate are evaluated.

Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.