Search

Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α -lactalbumin ( α -LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Y n , n = 3–8) was found in addition to di-tyrosine (Y 2 ). Two stages of cross-linking of α -LA were identified: a) 1–2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3–4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α -LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates. [ABSTRACT FROM AUTHOR]

Background: Many agricultural and industrial food by-products are rich in cellulose and xylan. Their enzymatic degradation into monosaccharides is seen as a basis for the production of biofuels and bio-based chemicals. Lytic polysaccharide monooxygenases (LPMOs) constitute a group of recently discovered enzymes, classified as the auxiliary activity subgroups AA9, AA10, AA11 and AA13 in the CAZy database. LPMOs cleave cellulose, chitin, starch and ß-(1 ? 4)-linked substituted and non-substituted glucosyl units of hemicellulose under formation of oxidized gluco-oligosaccharides. Results: Here, we demonstrate a new LPMO, obtained from Myceliophthora thermophila C1 (MtLPMO9A). This enzyme cleaves ß-(1 ? 4)-xylosyl bonds in xylan under formation of oxidized xylo-oligosaccharides, while it simultaneously cleaves ß-(1 ? 4)-glucosyl bonds in cellulose under formation of oxidized gluco-oligosaccharides. In particular, MtLPMO9A benefits from the strong interaction between low substituted linear xylan and cellulose. MtLPMO9A shows a strong synergistic effect with endoglucanase I (EGI) with a 16-fold higher release of detected oligosaccharides, compared to the oligosaccharides release of MtLPMO9A and EGI alone. Conclusion: Now, for the first time, we demonstrate the activity of a lytic polysaccharide monooxygenase (MtLPMO9A) that shows oxidative cleavage of xylan in addition to cellulose. The ability of MtLPMO9A to cleave these rigid regions provides a new paradigm in the understanding of the degradation of xylan-coated cellulose. In addition, MtLPMO9A acts in strong synergism with endoglucanase I. The mode of action of MtLPMO9A is considered to be important for loosening the rigid xylan-cellulose polysaccharide matrix in plant biomass, enabling increased accessibility to the matrix for hydrolytic enzymes. This discovery provides new insights into how to boost plant biomass degradation by enzyme cocktails for biorefinery applications. [ABSTRACT FROM AUTHOR]

Enzymatic protein hydrolysis is typically described by the degree of hydrolysis and by the enzyme specificity. While the specificity describes which cleavage sites can potentially be cleaved, it does not describe which are preferred. To identify the relative rate at which each individual cleavage site is hydrolysed, enzyme selectivity has recently been introduced. To test the effect of pH on selectivity, whey protein isolate (WPI) was hydrolysed with Bacillus licheniformis protease (BLP) at pH 7.0, 8.0 or 9.0. At all pH values, large differences in the enzyme selectivity (from <0.004% to 27%) towards the different cleavage sites of β-lactoglobulin were observed. The changes in selectivity as a function of pH were significant (up to 80% increase or decrease). This significant variation in selectivity shows that this parameter may be useful in understanding peptide release kinetics in enzymatic hydrolysis under different conditions. [ABSTRACT FROM AUTHOR]

Enzyme selectivity is introduced as a quantitative parameter to describe the rate at which individual cleavage sites in a protein substrate are hydrolyzed relative to other cleavage sites. Whey protein isolate was hydrolyzed by Bacillus licheniformis protease, which is highly specific for Glu and Asp residues. The molar concentration of all peptides (58) from β-lactoglobulin formed during hydrolysis was determined from the UV signal. The quality of identification and quantification of the peptides were described by newly defined parameters: the peptide sequence coverage (on average 94 %) and the molar sequence coverage (on average 75 %). The selectivity was calculated from the rate of hydrolysis of each cleavage site, and showed differences of up to a factor of 5,000. The ability to quantitatively discriminate the enzyme preference towards individual cleavage sites is considered essential to the understanding of enzymatic protein hydrolysis. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Peptides and amino acids generated during cocoa bean fermentation are known to be the most important precursors for the development of cocoa aroma. Although cocoa fermentation and aroma development have been extensively studied, the cocoa oligopeptide fraction is under-investigated. In particular, the identification of specific peptide sequences and the quantification of cocoa peptides are scarce in the literature. The aim of this study was to investigate the presence of oligopeptides in fermented cocoa beans. Peptides were analysed by reversed phase LC/ESI-MS and LC/ESI-MS/MS, and the molecular masses of 44 different peptides were obtained by analysing the mass spectra associated with the most intense chromatographic peaks. Peptides were identified based on the exact molecular masses, mass fragmentation patterns and by comparison with vicilin and 21 kDa cocoa seed protein sequences. Semi-quantitative data on peptide presence in fermented cocoa samples of different geographic origin, different fermentation levels and on roasted products were also provided. [ABSTRACT FROM AUTHOR]

Background: From patients' reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow's milk (CM) allergic patients. Objective and Methods: To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, alactalbumin (ALA), b-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. Results: The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly αS1-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. Conclusions: 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected. [ABSTRACT FROM AUTHOR]

Abstract: For the first time, the identification of N-γ-glutamyl-, N-pyroglutamyl- and N-lactoyl-amino acids in Parmigiano-Reggiano cheese is reported. Their structures were assigned by electrospray mass spectrometric analysis and demonstrated for several compounds by the synthesis of authentic specimens and sample spiking. The comparison with the synthesised compounds also allowed the determination of their absolute stereochemistry (l,l), which indicated a common enzymatic origin. All these molecules were shown to accumulate in Parmigiano-Reggiano during ripening, probably because of a resistance to proteolysis due to their unusual structures. Thus, these compounds can be used as markers of ripening time and may be involved in the development of the pleasant flavour of aged cheeses. Their presence in other cheeses was also demonstrated. [Copyright &y& Elsevier]

Abstract: The possibility of using short peptides in cheeses, derived from the proteolysis of milk caseins, as molecular markers for the origin of the milk (cows'' or sheep) in fresh sheep milk cheeses has been explored. By using a liquid chromatography/mass spectrometry (LC/MS) technique, two peptides generated during the proteolytic process (fragments 1-14 and 1-23 from αS1 casein), which are slightly different in sheep and cows, were detected separately. The method easily allowed for the detection of casein-derived peptides from cows'' caseins even when cows'' milk was present at very low concentrations, thus revealing the presence of undeclared cows'' milk. This method is a possible simpler alternative to the official electrophoretic methods. [Copyright &y& Elsevier]

Abstract: A new submonomeric approach for the Fmoc solid phase synthesis of chiral PNAs is reported here. The design and synthesis of a new d-lysine-based Fmoc submonomer obtained by replacing the nucleobase residue with an Alloc group, compatible with Fmoc chemistry, is described starting from d-lysine and Fmoc-aminoacetaldehyde. The desired submonomer was obtained in high yield with no racemization. The conditions for synthesizing a chiral PNA dimer on solid support by a submonomeric approach have been optimized, allowing to obtain the desired PNA with a very high optical purity. [Copyright &y& Elsevier]

Abstract: The 3,3′-diethylthiacarbocyanine (DiSC2(5)) dye is able to aggregate on full matched PNA–DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-β-cyclodextrin (Succ-β-CyD) to the solutions containing PNA–DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC2(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples. [Copyright &y& Elsevier]

Abstract: The synthesis of new chiral PNA analogues based on lysine is reported. In particular, l- and/or d-lysine-based PNA submonomers bearing two lysine side chains exactly spaced as in the dipeptide Lys-Lys were synthesized and incorporated in the middle of decameric PNA strands, obtaining four diastereomeric (LD, DL, LL and DD) lysine-based chiral PNAs. The hybridization with their complementary antiparallel DNA strand was studied by melting temperature determination and compared with the analogue achiral PNA and chiral PNAs bearing one residue with either of the two lysine enantiomers. The binding abilities were shown to be strongly dependent on the configuration of the stereogenic centres. [Copyright &y& Elsevier]

The study of unsubstituted and disubstituted β-cyclodextrins (β-CDs) by ESI-mass spectrometry is reported, applying a cone-induced fragmentation in the presence of a twofold excess of sodium chloride, in order to gain information about the fragmentation of the different regioisomers. On the basis of the fragmentation pattern observed for the unsusbstituted β-CD, a statistical model shows that the fragments generated by every regioisomer of a disubstituted CD (AB, AC, and AD) are expected to differ in their relative intensity and, therefore, they can be used for correctly identifying the three different regioisomers. The model was tested on the three regioisomeric (AB, AC, and AD) diamino-β-CDs and ditosyl-β-CD and on the AC and AD regioisomers of dimesitylenesulphonyl-β-CD, allowing in every case through statistical analysis of the fragmentation pattern the correct assignment of every regioisomer on the basis of an ESI mass spectrum (single quadrupole analyzer, high cone voltage) of the pure compounds. The absolute intensities of the fragmentation peaks were voltage-dependent but their ratios was voltage-independent, indicating that no mass bias in peak ratios is introduced by the analyzer. Given the fast time of analysis and its general applicability, independently from the substituents, we propose our method as an easy way to identify the regioisomers of disubstituted β-CDs. [Copyright &y& Elsevier]

Within a project aimed at studying the peptide fraction in dry-cured Parma hams, a peptide was purified by means of reversed phase-high performance liquid chromatography (RP-HPLC) and identified by its molecular mass and amino acid sequence analysis. The peptide showed a very high degree of homology with the N-terminal part of different mammalian pyruvate kinases reported in databases and was accordingly identified as the N-terminal part of swine pyruvate kinase, whose sequence had never been reported before. The peptide was determined quantitatively by comparison with a suitable internal standard (Phe-Phe) in commercial ham samples with different age degrees. The peptide was found to be ubiquitous in Parma ham and its amount to increase during ageing even if a large variability was found within each assayed maturing time. The correlation found with the ham proteolysis degree (P<0.01) suggests that this peptide is related to the endopeptidasic activity. The peptide did not show a significant relationship with bitterness perception in assayed dry-cured ham, in agreement with the hypothesis that this taste is more related to free amino acids and low molecular weight peptides. [Copyright &y& Elsevier]

Abstract Insects are being explored as novel protein sources in order to overcome the future food demands connected to world growing population. Insects for food/feed uses are currently slowly killed through freezing by most insect rearing companies, and typically, enzymatic browning takes place in the insect proteins fractions. However, very little is known about the influence of these enzymatic reactions on the protein physical, chemical, nutritional and technological properties. In this work a metabolomics and proteomic study was conducted on Black Soldier Fly (Hermetia illucens) prepupae, killed by two different methods: freezing (commonly used), and blanching (with the aim to inhibit the enzymatic activities). Proton nuclear magnetic resonance (1H NMR) metabolomics demonstrated that slow killing method by freezing, compared with blanching, elicits the activation of several enzymatic pathways, among them melanisation with tyrosine consumption, energetic metabolism and lipolysis. These metabolic changes have an impact also on protein nutritional quality, with a loss of cysteine and lysine, likely involved in the process of melanisation and enzymatic browning. A strong effect was also observed on protein extractability: proteins from prepupae killed by blanching were found to be more extractable in milder conditions by chemical methods, and more prone to enzymatic digestion (97% of proteins released in solution upon proteolysis) than proteins from prepupae killed by freezing. All these data indicate that killing by blanching inhibits the browning reaction and other enzymatic changes occurring during slow killing by freezing, increasing the extractability of proteins in aqueous solutions, avoiding essential amino acid loss, and improving enzymatic digestibility. Graphical abstract Unlabelled Image Highlights • Blanching and freezing were explored as killing methods for black soldier fly prepupae. • Blanching reduced the browning of proteins caused by phenol oxidase. • 1H NMR metabolomics proved that blanching inactivates several enzymatic pathways which are active in frozen preupae. • Proteomic analysis demonstrated modification on amino acids profile and protein quality upon freezing. • Blanching improved protein extractability and susceptibility to enzymatic digestion. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editors discusses a series of papers honoring Professor Rosangela Marchelli with works ranging from organic synthesis to biomedical applications of peptide nucleic acids (PNA).

Germination is already a well-accepted process by consumers with many products made from sprouted seeds or containing limited amounts of flour form sprouted grains. The present work aimed assessing the usefulness of germination in reducing gluten peptides associated with celiac disease, at the same time evaluating some technological features of the obtained germinated wheat. In the first part of the work, celiac disease (CD)-triggering peptides were tracked as a function of germination kinetics (from day 1 to day 6). Using simulated gastrointestinal digestion and liquid chromatography coupled to mass spectrometry, ten celiac disease triggering peptides were identified: seven peptides presumably involved in the adaptive immune response (TI) and three peptides mainly involved in the innate immune response (TT). All the identified peptides belonged to gliadins. TI track pattern showed three phases: the first two days displayed a significant degradation, a stability phase was observed from day 3 to day 5, and finally a drastic reduction occurred on the 6th day. For TT peptides, important degradation was exclusively observed at the 6th day. In the second part, some techno-functional features of germinated whole wheat flour were assessed to estimate its potential as an alternative to conventional flour. Functionality comparison of the non-germinated versus germinated flours revealed that germination significantly influenced solvents retention capacities as well as swelling and solubility. Thus, with a reduced amount of celiac disease triggering peptides, but also with different technological behavior compared to traditional wheat flour. [ABSTRACT FROM AUTHOR]

The extent of protein enzymatic hydrolysis is considered to be mostly determined by protease specificity and the number of cleavage sites (CS) on the substrate. However, this theoretical maximum is typically not reached. The limited hydrolysis of certain CS may be due to the differences in enzyme preference resulting from neighbouring amino acids (AA) of the CS (secondary specificity). This study aims to find the link between enzyme secondary specificity and the relative hydrolysis rate constants (selectivity) of individual CS in a protein, to better predict the maximum experimental degree of hydrolysis. Bovine tryptic hydrolysis of α-lactalbumin and β-casein showed that ≥50% of the CS were inefficiently hydrolysed. The selectivity depended on the number of charged AA at P2 and P2′ positions of a CS. Trypsin efficiently cleaves CS with neutral AA at these two positions. The selectivity towards 12 out of 18 (67%) CS in β-lactoglobulin was correctly predicted. The predicted maximum degree of hydrolysis is within ∼13% error of the experimental value, which is ∼5 times better than the prediction based only on enzyme specificity. This work helps to estimate the extent of hydrolysis and the peptide formation of bovine tryptic hydrolysis of other substrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A significant quantity of bone-rich poultry by-products must be disposed of by poultry processors. These products still contain a significant amount of nutritionally valuable animal proteins. In the present work, a hydrolysis protocol was optimized to recover the protein fraction of bone-rich poultry by-products while simultaneously minimizing the amount of water required for hydrolysis (thus reducing drying costs) and recycling the hydrolytic broth up to 3 times, to reduce the cost of the proteolytic enzyme. The final hydrolysis conditions involved the use of (protease from B. licheniformis , ≥2.4 U/g; 0.5 V/w of raw material) and a hydrolysis time of 2 h at 65°C. The protein hydrolysate obtained has a high protein content (79–86%), a good amino acid profile (chemical amino acid score equal to 0.7–0.8) and good gastric digestibility (about 30% of peptide bonds are already hydrolyzed before digestion). This supports its use as an ingredient in food, pet food or animal feed formulations. [ABSTRACT FROM AUTHOR]

It is expected that during the hydrolysis of proteins with specific enzymes only peptides are formed that result from hydrolysis of the specific cleavage sites (i.e. specific peptides). It is, however, quite common to find a-specific peptides (i.e. resulting from a-specific cleavage), which are often ignored, or explained by impurities in the enzyme preparation. In recent work in a whey protein isolate (WPI) hydrolysate obtained with the specific Bacillus licheniformis protease (BLP), 13 peptides of 77 identified were found to be the result of a-specific cleavage. These were formed after degradation of 6 specific peptides, after 5 different types of amino acids. The fact that other peptides were not hydrolyzed after these 5 amino acids suggests that the cleavages were not the result of a contamination with a different enzyme. In other systems, certain peptide sequences have been described to degrade chemically, under relatively mild conditions. This process is referred to as spontaneous cleavage. To test if the a-specific peptides observed in the WPI hydrolysis are the results of spontaneous cleavages, the parental peptides were synthesized. Surprisingly, 4 of the 5 synthesized peptides were indeed spontaneously cleaved under the mild conditions used in this study (i.e. 40 °C and pH 8) showing that peptides are less stable than typically considered. The rate of cleavage on the a-specific bonds was found to be enhanced in the presence of BLP. This suggests that the formation of a-specific peptides is not due to side activity but rather an enhancement of intrinsic instability of the peptides. [ABSTRACT FROM AUTHOR]

In recent years, due to the significant increase in hypertension, peptides which are able to reduce blood pressure have gained special interest by scientific research and food industry. Several bioactive peptides with ascertained ACE-inhibitory activity have been found in Parmigiano Reggiano (PR) cheese and/or mixtures deriving from its digestion in vitro, and this may be predictive of its potential antihypertensive effect in vivo. This study investigated the long-term effect of feeding (PR) cheese on blood pressure (BP) of spontaneously hypertensive rats (SHRs). A total of 30 male SHRs, 13 weeks old, were subdivided into 6 groups balanced for body weight and BP, to receive daily dietary supplementation with: 0.1–0.2–0.4–0.6 g PR/rat, captopril, and water. Systolic and diastolic BP were recorded every two weeks, for 10 weeks. Blood samples were collected at the end of the trial. Dietary integration with PR led to a transitory reduction in rats' pressure in the first 35 days of treatment and pressure decreased in a dose-dependent manner. In the second part of the study, the beneficial effect of PR antihypertensive peptides may have been masked and reduced by the increase in BP of rats linked to the rise in age of animals. No PR derived peptides were detected in rats' serum. Highlights: Parmigiano Reggiano (PR) cheese led to a transitory reduction in rats' pressure in the first 35 days of treatment. This effect was PR dose dependent. The highest amounts of PR tested did not increase both systolic and diastolic blood pressures of hypertensive rats. [ABSTRACT FROM AUTHOR]

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH 2 (belonging to β-lactoglobulin, position 48–60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved. [Display omitted] • Racemization reduced the proteolytic process impairing the nutritional value • D-amino acid appears to affect more pepsin activity than trypsin/chymotrypsin • The slowing down of cleavage is not only observed at the preferred sites • Protein cleavability changed depending on the enzyme and/or amino acid involved [ABSTRACT FROM AUTHOR]

Sulfite salts are widely used as antibrowning agents in food processing. Nevertheless, the exact mechanism by which sulfite prevents enzymatic browning has remained unknown. Here, we show that sodium hydrogen sulfite (Na HSO3) irreversibly blocks the active site of tyrosinase from the edible mushroom Agaricus bisporus, and that the competitive inhibitors tropolone and kojic acid protect the enzyme from Na HSO3 inactivation. LC- MS analysis of pepsin digests of Na HSO3-treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO3 upon MS2 fragmentation. These peptides were found to be homologous peptides containing two of the three histidine residues that form the copper-B-binding site of mushroom tyrosinase isoform PPO3 and mushroom tyrosinase isoform PPO4, which were both present in the tyrosinase preparation used. Peptides showing this neutral loss behavior were not found in the untreated control. Comparison of the effects of Na HSO3 on apo-tyrosinase and holo-tyrosinase indicated that inactivation is facilitated by the active site copper ions. These data provide compelling evidence that inactivation of mushroom tyrosinase by Na HSO3 occurs through covalent modification of a single amino-acid residue, probably via addition of HSO3− to one of the copper-coordinating histidines in the copper-B site of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms, respectively “light” and “heavy”, by using light-derivatized standards for the quantification of the heavy-derivatized analytes, and vice versa. To this purpose, 2,4-dinitro-3,5,6-trideuterophenylhydrazine (d 3 -DNPH) has been synthesized and used as “heavy” reagent in combination with commercial “light” DNPH. Using the AIDA method, any unknown concentration of the analyte in the matrix can be calculated without the need of a calibration curve. An external calibration method has been also investigated for comparative purpose. The stability of DNPH and d 3 -DNPH derivatives was verified by excluding any exchange of hydrazones with each other. In the range of concentrations of biological interest, e.g., 2–40nM for MDA and 0.5–10nM for 4-HNE, the derivatization reactions of MDA and 4-HNE with DNPH and d 3 -DNPH showed overlapping kinetics and comparable yields. The MS response of both DNPH and d 3 -DNPH hydrazones was similar. The precision of AIDA, calculated as %RSD, was within 3.2–8% for MDA and 4.5–11% for 4-HNE. Accuracy was tested by analyzing a spiked EBC pool sample and acceptable results (accuracy within 98–108% for MDA and 93–114% for 4-HNE) were obtained by AIDA after subtraction of the blank, which was not negligible. The results of quantitative analysis of MDA and 4-HNE in EBC samples obtained by AIDA assay with four analyses per sample were in good agreement with those obtained by external calibration method on the same samples. [Copyright &y& Elsevier]

Peptides obtained from whey proteins have been associated with different biological properties, but most peptides are usually degraded during the digestive process. In this work, hydrolysates were prepared from a whey protein concentrate obtained by ultrafiltration of Colombian traditional cheese whey and subsequent enzymatic hydrolysis using alcalase, chymotrypsin and flavourzyme. The antioxidant and angiotensin-converting enzyme (ACE) inhibitory properties were evaluated, and peptide profile was determined by ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Changes in the above biological properties and peptide profile after simulated gastrointestinal digestion were then investigated. Before enzymatic hydrolysis, the alcalase hydrolysate showed higher 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity, whereas an inhibition greater than 85% of ACE activity was observed for the alcalase and chymotrypsin hydrolysates. In both tests, the flavourzyme hydrolysates showed lower activities. Different changes in the above biological activities were observed after gastrointestinal digestion of the alcalase and chymotrypsin hydrolysates: ABTS radical scavenging ability increased, whereas ACE inhibitory capacity decreased significantly. The alcalase hydrolysate obtained from ultrafiltrated whey protein concentrate showed high antioxidant and ACE inhibition capacities and higher survivability of peptides after in vitro gastrointestinal digestion. • Bioactivity of Columbian cheese whey hydrolysates was tested. • Whey hydrolysates were subjected to INFOGEST in vitro digestion. • In vitro digestion affected bioactivity and peptide profile. [ABSTRACT FROM AUTHOR]

• IgE of patients allergic to shrimp or mites cross-react with TPM of H. illucens. • The main structure in solution of both TPM variants is a α-helices tetramer. • In vitro gastrointestinal digestion reduces the number of potential epitopes. Insect consumption could address the increasing protein demand in compliance with environmental sustainability. Hermetia illucens (black soldier fly, BSF) is a promising insect for human diet and it is essential to assess the related allergenic risk, meant as primary sensitization or cross-reactivity with known allergens. In this work, we investigate the allergenicity of two tropomyosin variants identified in the BSF genome and produced as recombinant proteins. Immunoblot experiments showed that both proteins were recognized by sera of patients allergic to shrimp or mites highlighting the cross-reactivity risk. CD spectroscopy, cross-linking assays and size-exclusion chromatography showed a structure composed of alpha-helices oligomers for both variants. These proteins were quite stable to pH but sensitive to increasing temperatures. In vitro simulated digestion associated to mass-spectrometry allowed the identification of peptides resistant to gastrointestinal conditions which were compared with epitopes of Arthropoda and Mollusca allergens to predict the persistence of allergenicity upon digestion. [ABSTRACT FROM AUTHOR]

High-quality proteins from farmed fish, including sea bream, represent a cheaper and healthier alternative to proteins from other animals, especially for people affected by chronic diseases. Aquaculture has also the possibility to move towards more sustainable growth and production. Therefore, the present study was designed to evaluate the impact of a starvation period before slaughtering in comparison to full feeding on quality attributes of sea bream fillet focused on their health promoting values. SDS-PAGE, Western blotting and high-resolution MS/MS analyses of the protein profiles showed that in the fillets from starved fish, only the amount of myosin light chains (MLCs) was markedly reduced at slaughter, suggesting their higher susceptibility to proteolysis. In the full feeding samples, MLCs degradation was evident only during storage at 4 °C. Conversely, skeletal alpha-actin fragmentation was similar regardless of the pre-slaughter feeding system. In vitro gastric digestibility of the myosin complex from starved fish fillets was much higher at pH 4, a condition that may occur in drug-treated patients affected by the gastroesophageal reflux disease (GERD). Using UHPLC–MS/MS, ACE-inhibitor gastric-resistant bioactive peptides potentially coming from MLCs (VF, MF) were found in all the digested samples, with one of these peptides (MF) being present in larger amount in the starved samples. Pre-slaughter starvation, other than decreases the environmental impact of fish aquaculture, is an effective tool in obtaining good-quality sea bream fillets that provide potential health benefits for hypertensive and GERD consumers. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

[Display omitted] • The essential amino acids profile is comparable for both analogue and beef burgers. • Protein integrity of plant- and animal-based products was not affected by treatments. • INFOGEST in vitro model showed good protein solubilisation for both protein sources. • High prevalence of collagen-deriving peptides was observed in beef digestates. • The quality of raw material affects digestibility regardless of the protein source. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • 39 food waste streams of vegetal and dairy origin have been investigated. • The nitrogen fraction was deeply characterized at the proteins and amino acid level. • Protein amount and quality was assessed in all sources. • New sources of high value proteins were identified in still unexploited agro wastes. Abstract 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. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Gluten peptides, fiber, and starch were analyzed in old and modern wheat genotypes. • Peptides associated to celiac disease were released abundantly in some old genotypes. • Resistant starch is abundant in the wholemeal of some old wheat genotypes. • Pasta cooking overtook differences in resistant starch content between genotypes. • Old wheats cannot claim prebiotic potential or low gluten compared with the new ones. [ABSTRACT FROM AUTHOR]

Abstract Whey is a valuable by-product of the dairy industry, due to the high nutritional, biochemical and functional properties of the contained proteins. As for other dairy products, to guarantee the microbiological safety of whey, thermal treatments are generally used. However, these processes can have a significant impact on the quality of the product. Thus, in recent years, interest in the use of non-thermal treatments in the dairy industry is significantly grown. In the present work, the use of UV irradiation as stabilizing treatment has been investigated on whey obtained from the Parmigiano-Reggiano cheese production. Two treatments were tested: UV treatment "1" which is characterized by three passages and UV treatment "2" by six ones. On skimmed whey samples, microbiological tests showed UV treatment 2 and the thermal treatment have the same effectiveness on Enterobacteriaceae, but UV treatment 2 is more effective than thermal treatment on total viable counts. Molecular characterization of the protein and total amino acid content was used as markers for the quality of the product. The results highlight that UV treatment can be equivalent to thermal pasteurization in terms of microbiological stabilization, and better preserving the whey quality. Highlights • UV irradiation and pasteurization allow comparable microbiological stabilization. • The impact of UV irradiation on whey protein precipitation is neglectable. • UV irradiation do not alter the nutritional value of the whey. [ABSTRACT FROM AUTHOR]

Abstract Food frauds are a critical issue in the field of food safety and quality. Given the high added value, and the complexity of the matrix, processed meat products are among those most susceptible of adulteration. Despite all the efforts made by the official control authorities and by the food industry to counteract these frauds, the undeclared replacement of meat species with cheaper ones is still widespread. The meat species allowed for food consumption are many, and their specific and accurate detection in highly processed food products requires very sensitive and selective analytical methods. In this work, a LC-MS method was developed to identify and quantify eight different meat species (duck, rabbit, chicken, turkey, buffalo, equine, deer and sheep) in a complex food matrix, such as Bolognese sauce. After protein extraction and trypsin digestion, a species-specific peptide marker for each species was chosen for qualification and quantification. The method was validated on real Bolognese sauce samples prepared in an industrial environment, showing a good sensitivity (LOD 0.2–0.8% on whole finished product) and the possibility, using specifically defined calibration lines, to quantify the amount of meat present coming from different species. Highlights • Species-specific marker peptides were identified for eight meat species. • A LC-MS based method was developed for the detection of the marker peptides. • Bolognese sauces were prepared with increasing amount of each species. • Using proper calibration curves the method can quantify the meat species present. • A validation was performed analyzing blind Bolognese sauce samples. [ABSTRACT FROM AUTHOR]

Abstract Black soldier fly (BSF, Hermetia illucens) represents a valuable source of biomolecules and it also constitutes an economic way to valorise residual biomasses. BSF prepupae contain high amounts of lipids (37% DM basis). The present investigation aimed at studying the composition of BSF lipids and the effect of killing/storage on their quality. The main fatty acid was lauric acid, sterols were represented primarily by beta-sitosterol and campesterol. Global fatty acid and sterol profiles, determined by GC–MS, were only slightly affected by the killing procedure, while lipid classes distribution, determined by 1H NMR, strongly changed. Prepupae killed by freezing showed a drastic reduction of acylglycerols during storage and a relevant release of free fatty acids, likely due to activation of lipases. On the contrary, prepupae killed by blanching have a stable lipid fraction constituted mainly by triacylglycerols. Therefore, killing procedure strongly influences BSF oil composition and the potential applications. Graphical abstract Unlabelled Image Highlights • A detailed molecular characterization of black soldier fly prepupae lipids is provided. • Killing method of prepupae determinates the lipid composition. • Prepupae killed by freezing have an unstable lipid fraction with high acidity. • Prepupae killed by blanching have a stable lipid fraction suitable for food uses. [ABSTRACT FROM AUTHOR]

Using Western blotting and mass spectrometry we investigated the changes in trout muscle proteins as affected by pre-slaughter nutrient supply practices, including starvation and feed restriction, in comparison to full feeding. A number of fillet quality attributes were also analyzed. During cold storage, fillet firmness was not influenced by the feeding practices, while the initial pH was higher in the treated trout. The early appearance of an alpha-actin 26 kDa-fragment and the detection of enigma protein were both signs of impaired muscle integrity, observed only in the restricted conditions, where water holding capacity was initially lower. The detection of reticulon involved in authophagy and the early finding of apolipoprotein A-I-1 fragments in these restricted samples was consistent with their early degradation in comparison to full ration controls. Since specific protein changes reflected differences in feed management, the use of selected muscle proteins as sensitive indicators of fillet quality is proposed. [ABSTRACT FROM AUTHOR]

Abstract Proteolysis is the most important event occurring during maturation of dry-cured hams: it strongly influences the flavour and the texture of the aged ham by the accumulation of peptides and free amino acids released by protein hydrolysis. Apart from compounds of proteolytic origin, it has been demonstrated that also non-proteolytic amino acyl derivatives (γ-glutamyl amino acids, pyroglutamyl-amino acids and lactoyl-amino acids) may accumulate during ripening of cheese, and they can be also found in fermented soy sauce, where they contribute to the umami taste of the products. Using a semi-quantitative analysis, in this paper we report the occurrence of significant amounts of γ-glutamyl amino acids and, for the first time, pyroglutamyl-amino acids and lactoyl-amino acids, in aged ham. The amino acid counterparts were mainly found to be hydrophobic amino acids. The amount of these compounds was found to increase with time, because they are not degraded by proteolytic activity. They were also found to be stable to simulated gastrointestinal digestion. Angiotensin Converting Enzyme inhibitory activity was also tested, but they were not found to be characterized by significant ACE-inhibitory activity. Graphical abstract Unlabelled Image Highlights • Non proteolytic aminoacyl derivatives (NPADs) were quantified in dry cured ham • NPADs stability to gastrointestinal digestion was assessed • The enzymatic origin of NPADs in water soluble extracts of ham was demonstrated • ACE inhibitory activity of NPADs was studied [ABSTRACT FROM AUTHOR]

The aim of this research was to valorize onion skins, an under-utilized agricultural by-product, into pectic oligosaccharides (POS), compounds with potential health benefits. To achieve high hydrolysis performance with the multi-activity enzyme Viscozyme L, an innovative approach was investigated based on a cross-flow continuous membrane enzyme bioreactor (EMR). The influence of the various process conditions (residence time, enzyme concentration, substrate concentration) was investigated on productivity and yield. The composition of the POS mixtures in terms of mono- and oligosaccharides was assessed at the molecular level. At optimized conditions, a stable POS production with 22.0 g/L/h volumetric productivity and 4.5 g/g POS/monosaccharides was achieved. Compared to previous results obtained in batch for the enzyme Viscozyme L, EMR provided a 3–5× higher volumetric productivity for the smallest POS. Moreover, it gave competitive results even when compared to batch production with a pure endo-galacturonase enzyme, demonstrating its feasibility for efficient POS production. [ABSTRACT FROM AUTHOR]

Abstract The present work had a dual objective: to assess if the evolution of durum wheat from landraces to modern genotypes as function of breeding programs impacted proteins and starch fractions, as well as starch properties. Flours and starches isolated from Tunisian durum wheat landraces, old and modern lines were thoroughly characterized for their compositional, morphological, and gel properties. Statistical results showed significant (p ≤ 0.05) differences among the studied set. Protein and starch fractions assessment revealed that modern genotypes had the highest total starch and albumin contents, old genotypes had the highest amylose and glutenin contents, and landraces had the highest protein and gliadin contents. Starch properties screening allowed several findings: no significant (p > 0.05) differences were found among starch granules morphology, significant (p ≤ 0.05) differences were recorded in terms of technological properties, and old genotypes had the highest starch gel hardness. Overall, these results indicated that the influence of genotype on flour and starch properties was more relevant than breeding history. Highlights • Landraces, modern and old genotypes properties were compared. • High genetic diversity was revealed among the studied genotypes. • Low variability in starch granules morphology and technological properties. • Genotype was more relevant than breeding history. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simplified whey protein-catechins system was prepared in the same ratio as in milk chocolate. • Interaction between whey proteins and catechins determined by (chiro-)optical spectroscopy. • Protein hydrolysis/solubilisation of whey proteins during digestion unaffected by catechins. • Milk chocolate proteins completely digested. • Interactions of catechins with milk proteins does not impair in vitro protein digestibility. Flavonoids are largely present in plant food such as cocoa and derived products. These compounds can interact with proteins inherently contained in the food matrix and/or the proteolytic enzymes involved in gastrointestinal digestion. The flavonoid/protein interaction might hamper protein bioaccessibility and digestibility, affecting the nutritional quality. However, information on the digestion fate of proteins in food matrices containing both proteins and flavonoids is limited. The aim of this work was to evaluate the interaction between proteins and flavonoids and verify the potential effects of this interaction on protein digestibility. Taking milk chocolate as model, first a simple whey proteins/catechins mixed system was evaluated, and then the effects on digestibility were also verified in a real sample of commercial milk chocolate. The effects of the catechins/whey proteins interaction in the model system were evaluated by optical and chiro-optical spectroscopy, outlining a slight protein structure modification upon interaction with catechins. The digestibility of the protein fraction both in the model system, with and without catechins, and also in milk chocolate, was then determined by the application of INFOGEST in vitro digestion method: the bioaccessibility was evaluated in terms of protein hydrolysis and protein solubilisation, and major peptides generated by the digestion were also determined by LC/HR-MS. Despite the slight interaction with proteins, flavonoids were found to not hinder nor modify protein solubilization, protein hydrolysis and peptide profile by digestive enzymes. Also protein digestibility in milk chocolate, evaluated by SDS-PAGE, was found to be complete. The present data clearly indicate that the interaction of the proteins with the flavonoids present in the cocoa matrix does not to affect protein bioaccessibility during digestion. [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPβ transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD. [ABSTRACT FROM AUTHOR]

Abstract: The protein content and allergen composition was studied of cashews from 8 different origins (Benin, Brazil, Ghana, India, Ivory Coast, Mozambique, Tanzania, Vietnam), subjected to different in‐shell heat treatments (steamed, fried, drum‐roasted). On 2D electrophoresis, 9 isoforms of Ana o 1, 29 isoforms of Ana o 2 (11 of the acidic subunit, 18 of the basic subunit), and 8 isoforms of the large subunit of Ana o 3 were tentatively identified. Based on 1D and 2D electrophoresis, no difference in allergen content (Ana o 1, 2, 3) was detected between the cashews of different origins (P > 0.5), some small but significant differences were detected in allergen solubility between differently heated cashews. No major differences in N‐ and C‐terminal microheterogeneity of Ana o 3 were detected between cashews of different origins. Between the different heat treatments, no difference was detected in glycation, pepsin digestibility, or IgE binding of the cashew proteins. [ABSTRACT FROM AUTHOR]

Black soldier fly (BSF, Hermetia illucens ) constitutes an economic way to convert residual biomasses into a valuable source of biomolecules, such as proteins, lipids and chitin. The present investigation was undertaken to evaluate the feasibility of applying different extraction protocols, either chemical extractions or enzymatic assisted extraction, to recover pure fat, protein and chitin fractions. First, exact proximate composition, total amino acids, fatty acids profile, and N -acetylglucosamine content of the prepupae samples were determined. BSF prepupae biomass contained, expressed on dry weight, 32% proteins, 37% lipids, 19% minerals, 9% chitin. The lipid fraction was easily recovered by organic solvents, while the most challenging issue was the separation of protein from chitin. The best separation was obtained by alkali extraction of proteins (96% of protein recovered) albeit with loss in their integrity as indicated by the measurement of the degree of hydrolysis with the o-phthaldialdehyde method. To avoid protein damage in alkali media, a stepwise protein extraction adopting milder conditions was also explored based on Osborne fractionation method, allowing the recovery of > 85% of BSF high purity and high quality proteins, and the obtainment of chitin-enriched fraction as well. The possibility of using an enzymatic assisted extraction of proteins was also explored, obtaining a maximum nitrogen solubilisation in the best case (with Bacillus licheniformis protease) of about 60%. In this latter case, the chitin fraction obtained also had a significant residual protein content. [ABSTRACT FROM AUTHOR]

A huge amount of by-products, wastes and chemicals are generated during leather processing, originating environmental problems due to the great amount of pollutants released, in particular for the unhairing step. Due to this reason, several strategies are studied today to reduce its impact. In this work two different methods, developed at a pilot scale level in semi-industrial reactors, were compared: an unhairing method based on proteolytic enzymes (Alcalase from Bacillus licheniformis ) and an oxidative one based on chemical agents (sodium hydroxide and hydrogen peroxide). The efficiency of the unhairing was evaluated in both cases. The impact of both treatments on the nitrogen fraction in wastewaters was determined in order to assess the suitability of these by-products for a possible recovery in food/feed industry: the protein amount, the total and free amino acids, peptides and the percentage of amino acid racemization were determined on broths, washing residues and skins. The results indicated that both methods have the potential to be more sustainable alternatives to the current ones. The oxidative method seemed to be the most efficient and less expensive unhairing methodology, and does not require a further step after the end of the treatment as in the case of enzymatic unhairing process. On the other side, the wastewaters of enzymatic treatments, quite rich in good quality proteins, could be recovered for feed/food formulations, whereas the wastewaters coming from the oxidative treatments, due to the oxidative damage, only for lesser value applications. [ABSTRACT FROM AUTHOR]

In this research, for the first time, the valorization of soursop seeds, a poorly studied biomass, was investigated to simultaneously produce oil and proteins within a biorefinery approach. A new mild protein extraction method (Pressurized Water Extraction, PWE) was developed and optimized via response surface methodology and compared with conventional alkaline extraction. Furthermore, the impact of the de-oiling technique, i.e. , n -hexane or cold pressing, was investigated. The extracted proteins were evaluated in terms of physicochemical composition, amino acid content, and techno-functional properties. The type of de-oiling technique was found to have a minor impact. Instead, considerable differences were observed between the two protein extraction methodologies. At optimized conditions (pH 8.1 and 40 °C), PWE resulted in a more protein-rich extract (48 vs. 30 wt% for alkaline water), better preservation of the amino acid content, and higher solubility and in vitro digestibility. A mixed behavior was found for the techno-functional properties. Emulsification and oil holding capacity were similar for both techniques, while improved foaming was observed after alkaline water extraction. This research showed that the non-exploited soursop seeds could be a valuable source of oil and protein. Moreover, the novel PWE enables protein extraction with reduced impact on the protein structure. [Display omitted] • Soursop seeds allows the simultaneous valorization of proteins and oils in a biorefinery approach. • The type of de-oiling technology had a minor influence on the recovered oil and proteins. • Pressurized water extraction (PWE) is a viable alternative technology for the extraction of proteins from soursop seeds. • PWE proteins had better preservation of nutritional composition and in vitro digestibility as alkaline extracted proteins. • PWE proteins had similar emulsifying and oil-holding, but reduced foaming capacity as alkaline extracted proteins. [ABSTRACT FROM AUTHOR]

Parmigiano Reggiano (PR) is a raw-milk, hard cooked, long-ripened cheese of high quality and nutritional value. Long ripening times allow for extensive proteolysis of milk proteins to yield a number of peptides, some of which have potential healthy bioactive properties. This study aimed to: i) determine the peptide profile of PR cheese subjected to simulated gastrointestinal transit; ii) evaluate in vitro whether the peptides could support growth of beneficial microbial groups of the gut microbiota. PR samples were subjected to in vitro digestion, simulating oral, gastric, and duodenal transit. Liquid chromatography coupled with tandem mass spectrometry revealed that digestion caused the disappearance of the serum proteins and most of the original peptides, while 71 new peptides were found, all ranging from 2 to 24 residues. The digests were given as sole nitrogen source to pure cultures of Bifidobacterium (27 strains) and Lactobacillus (30 strains), and to bioreactor batch cultures of human gut microbiota. Most of bifidobacteria and lactobacilli grew more abundantly on PR digests than on the control peptone, and exhibited strain- or species-specific peptide preferences, as evidenced by principal component analysis. Bifidobacteria generally consumed a greater amount of peptides than lactobacilli, in terms of both the mean peptide consumption and the number of peptides consumed. For bifidobacteria, peptide preferences were very diverse, but a core of 10 peptides with 4 or 5 residues were consumed by all the strains. Lactobacilli behaved more homogenously and consumed nearly only the same 6 peptides, mostly dipeptides. The peptide preferences of the different groups of bifidobacteria and lactobacilli could not be ascribed to features such as the length of the peptide or the abundance of residues with peculiar properties (hydrophobicity, polarity, charge) and likely depend on specific proteases and/or peptide transporters preferentially recognizing specific sequence motifs. The cultures of human colonic microbiota confirmed that PR digest promoted the growth of commensal bifidobacteria. This study demonstrated that peptides derived from simulated gastrointestinal digestion of PR supported the growth of most lactobacilli and bifidobacteria. [ABSTRACT FROM AUTHOR]

Objective: An efficient breast-feeding monitoring system should be in place in every country to assist policy makers and health professionals plan activities to reach optimal breast-feeding rates. Design/Setting/Subjects From March to June 2015, breast-feeding rates at 3 and 5 months of age were monitored in Emilia-Romagna, an Italian region, using four questions added to a newly developed paediatric immunization database with single records for each individual. Data were collected at primary-care centres. Breast-feeding definitions and 24 h recall as recommended by the WHO were used. Direct age standardization was applied to breast-feeding rates. Record linkage with the medical birth database was attempted to identify maternal, pregnancy and delivery factors associated with full breast-feeding rates at 3 and 5 months of age.Results: Data on breast-feeding were collected for 14044 infants. The mean regional full breast-feeding rate at 3 months was 52 %; differences between local health authorities ranged from 42 to 62 %. At 5 months of age, the mean regional full breast-feeding rate dropped to 33 % (range between local health authorities: 26 to 46 %). Record linkage with the birth certificate database was successful for 93 % of records. Total observations more than doubled with respect to the previous regional survey.Conclusions: The new monitoring system implemented in 2015 in Emilia-Romagna region, totally integrated with the immunization database, has proved to be feasible, sustainable and more efficient than the previous one. This system can be a model for other regions and countries where the vast majority of mothers obtain vaccinations from public health facilities and that already have an immunization database in place. [ABSTRACT FROM AUTHOR]

Food frauds have become a very important issue in the field of food quality and safety. The risk of food adulteration is higher in highly processed food and mainly affects high added value foodstuff. The methods currently available to face this issue, PCR and ELISA, are very sensitive and specific, but they have some limitations. In the present work, tandem mass spectrometry is presented as an emerging approach to detect beef and pork meat in very complex and highly processed food matrices, such as Bolognese sauce, both in qualitative than in quantitative way. The detection is achieved using two different marker peptides, specific for beef and pork meat, both deriving from α2-collagen chain. Then, a calibration curve is set up using real sauces made by different percentages of pork and beef meat in a working range from 0 to 100%. The method here developed allows to quantify beef and pork meat in a complex product such as Bolognese sauce. [ABSTRACT FROM AUTHOR]

Coeliac disease is an autoimmune enteropathy that develops in genetically predisposed subjects after the ingestion of gluten or related proteins. Coeliac disease has an increasing incidence in the last years in western countries and it has been suggested that wheat breeding might have contributed to select more toxic forms of gluten. In this work, we analysed gluten peptides generated by in vitro digestion of different old and modern Triticum varieties, using LC-MS. We concluded that old varieties analysed produced a higher quantity of peptides containing immunogenic and toxic sequences than modern ones. Thus old wheat lines are not to be considered “safer” for subjects that are genetically predisposed to celiac disease. [ABSTRACT FROM AUTHOR]