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Microalgae are considered a valuable source of proteins that are used to enhance the nutritional value of foods. In this study, a standard vegetable cream recipe was reformulated through the addition of single-cell ingredients from Arthrospira platensis (spirulina), Chlorella vulgaris, Tetraselmis chui, or Nannochloropsis oceanica at two levels of addition (1.5% and 3.0%). The impact of microalgae species and an addition level on the amino acid profile and protein in vitro digestibility of the vegetable creams was investigated. The addition of microalgae to vegetable creams improved the protein content and the amino acid nutritional profile of vegetable creams, whereas no significant differences were observed in protein digestibility, regardless of the species and level of addition, indicating a similar degree of protein digestibility in microalgae species despite differences in their protein content and amino acid profile. This study indicates that the incorporation of microalgae is a feasible strategy to increase the protein content and nutritional quality of foods. [ABSTRACT FROM AUTHOR]

The beneficial effects of an insect-based diet on human health and, in particular, the regulatory ability of digested insects' proteins on the glycaemic response in humans are topics that need to be investigated deeper. In this work, we performed an in vitro study on the modulatory activity of gastrointestinal digested black soldier fly (BSF) prepupae on the enterohormone GLP-1 and its natural inhibitor, DPP-IV. We verified whether actions intended to valorise the starting insect biomass, i.e., insect-optimised growth substrates and prior fermentation, can positively impact human health. Our results highlight that the digested BSF proteins from all the prepupae samples had a high stimulatory and inhibitory ability on the GLP-1 secretion and the DPP-IV enzyme in the human GLUTag cell line. Gastrointestinal digestion significantly improved the DPP-IV inhibitory capacity of the whole insect protein. Moreover, it was seen that optimised diets or fermentation processes preceding the digestion, in any case, did not positively affect the efficacy of the answer. BSF was already considered one of the edible insects more suitable for human consumption for its optimal nutritional profile. The BSF bioactivity here shown, after simulated digestion, on glycaemic control systems makes this species even more promising. [ABSTRACT FROM AUTHOR]

Background: In Italy, there is scant evidence on the impact of Community Hospitals (CHs) on clinical outcomes. Aims: To assess the effectiveness of CHs versus long-term care hospital or inpatient rehabilitation facilities on mortality, re-admission, institutionalization, and activation of a home care programme in the Emilia-Romagna Region (ERR-Italy) after acute hospitalisation. Methods: We implemented a cohort study drawing upon the ERR Administrative Healthcare Database System and including hospital episodes of ERR residents subject ≥ 65 years, discharged from a public or private hospital with a medical diagnosis to a CH or to usual care between 2017 and 2019. To control for confounding, we applied a propensity score matching. Results: Patients transferred to CHs had a significantly lower risk of dying but an increased risk of being readmitted to community or acute hospital within 30/90 days from discharge. The hazard of institutionalisation within 30/90 days was significantly lower in the whole population of the CH exposed group but not among patients with cardiac or respiratory chronic diseases or diabetes. The activation of a home care program within 90 days was slightly higher for those who were transferred to a CH. Discussion: The findings of our study show mixed effects on outcomes of patients transferred to CHs compared to those who followed the post-acute usual care and should be taken with cautious as could be affected by the so-called 'confounding by indication'. Conclusions: The study contributes to the intermediate care available evidence from a region with a well-established care provision through CHs. [ABSTRACT FROM AUTHOR]

In a context where the commercial and nutritional interest in insect chitin is always increasing, an accurate and precise method to quantify this biopolymer, especially in food/feed, is required. In addition, quantification of insect crude protein through nitrogen determination is normally overestimated due to the presence of chitin. In this work, for the first time, an RP-UPLC-ESI/MS method for the simultaneous quantification in insects of chitin, as glucosamine (GlcN), and protein, as total amino acids, is presented. The method is based on acid hydrolysis and derivatization of amino acids and GlcN with the AccQ Tag reagent. Method was optimized and validated in terms of linearity, LOD and LOQ, intraday and inter-day repeatability, and accuracy. A hydrolysed commercial chitin was selected as reference standard for calibration. The instrumental LOD and LOQ correspond respectively to a concentration of 0.00068 mM and 0.00204 mM. The intraday precision satisfied the Horwitz ratio. Data from inter-day precision showed the necessity to perform the analysis within 1 week utilizing standard calibration solutions freshly prepared. A matrix effect was observed, which suggested the necessity to use an internal calibration curve or to work in a particular concentration range of GlcN. The chitin and protein content in black soldier fly (Hermetia illucens) and lesser mealworm (Alphitobius diaperinus) were found in agreement with results obtained by independent methods. The optimized method was also tested on two different commercial food supplements, suggesting its applicability on a wide range of matrices. This newly developed method proved to be simple, more accurate, and faster if compared to methods which separately analyse chitin and protein content. [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]

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]

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]

Cheese microbiota contribute significantly to the final characteristics of cheeses due to the growth and interaction between cheese microorganisms during processing and ripening. For raw milk cheeses, such as Parmigiano Reggiano (PR), the microbiota derive from the raw milk itself, the dairy environment, and the starter. The process of cheese making and time of ripening shape this complex ecosystem through the selection of different species and biotypes that will drive the quality of the final product by performing functions of their metabolism such as proteolysis. The diversity in the final peptide and amino acid composition of the cheese is thus mostly linked to the diversity of this microbiota. The purpose of this study was to get more insight into the factors affecting PR cheese diversity and, more specifically, to evaluate whether the composition of the bacterial community of cheeses along with the specific peptide composition are more affected by the ripening times or by the cheese making process. To this end, the microbiota and the peptide fractions of 69 cheese samples (from curd to cheese ripened 24 months) were analyzed during 6 complete PR production cycles, which were performed in six different dairies located in the PR production area. The relation among microbial dynamics, peptide evolution, and ripening times were investigated in this unique and tightly controlled production and sampling set up. The study of microbial and peptide moieties in products from different dairies – from curd to at least 12 months, the earliest time from which the cheese can be sold, and up to a maximum of 24 months of ripening – highlighted the presence of differences between samples coming from different dairies, probably due to small differences in the cheese making process. Besides these differences, however, ripening time had by far the greatest impact on microbial dynamics and, consequently, on peptide composition. [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]

Purpose: Poultry protein hydrolysates (PPHs) are obtained as by-product of poultry slaughter industry using bio catalytic hydrolysis. These protein hydrolysates have good antioxidant properties, which make them potential functional ingredient to prevent the oxidation of meat derived products. However, protein hydrolysates have a poor solubility in lipid media. Methods: To increase the solubilisation in the lipid phase, thus improving antioxidant properties on the fatty acids, several esterification reactions were carried out using different alcohols. The reactions were monitored by LC–MS and the shift in antioxidant activity was assessed with hydrophilic and lipophilic ABTS assay. Results: A good increase in the lipophilicity of the esterified PPHs is shown, proportional to the increase of the alcohol chain length. Accordingly, a significant increase of the lipophilic antioxidant activity, especially for longest chain alcohols, was also observed. Conclusions: These results indicated that the chemical esterification of PPHs with food-grade long chain alcohol can be a valuable approach to increase the lipophilic antioxidant properties of these products. [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]

Sugar beet pulp pectin is an attractive source for the production of pectic oligosaccharides, an emerging class of potential prebiotics. The main aim of the present work was to investigate a new process allowing to produce pectic oligosaccharides in a continuous way by means of a cross flow enzyme membrane reactor while using a low-cost crude enzyme mixture (viscozyme). Preliminary experiments in batch and semi-continuous setups allowed to identify suitable enzyme concentrations and assessing filtration suitability. Then, in continuous experiments in the enzyme membrane reactor, residence time and substrate loading were further optimized. The composition of the obtained oligosaccharide mixtures was assessed at the molecular level for the most promising conditions and was shown to be dominated by condition-specific arabinans, rhamnogalacturonans, and galacturonans. A continuous and stable production was performed for 28.5 h at the optimized conditions, obtaining an average pectic oligosaccharide yield of 82.9 ± 9.9% (w/w), a volumetric productivity of 17.5 ± 2.1 g/L/h, and a specific productivity of 8.0 ± 1.0 g/g E/h. This work demonstrated for the first time the continuous and stable production of oligosaccharide mixtures from sugar beet pulp using enzyme membrane reactor technology in a setup suitable for upscaling. [ABSTRACT FROM AUTHOR]

Abstract: The meat portion directly attached to bovine hides (fleshing meat) is a by-product of leather industry that is a potential new source of proteins. In literature different enzymatic and chemical methods have been proposed to hydrolyze and solubilize fleshing meat. Enzyme hydrolysis is preferable for the mildness and the lower environmental impact. Enzymatic hydrolysates have never been deeply characterized, nor the specific enzyme efficiency, deeply investigated. In this study, the activity of six proteolytic enzymes was tested in order to determine their efficiency in solubilizing fleshing meat and to characterize at the molecular level the composition of the nitrogen fraction of the obtained hydrolysates. Total nitrogen content and the degree of hydrolysis were determined by Kjeldhal and o-phthaldialdehyde (OPA) methods. Amino acids and peptides were analysed by high performance liquid chromatography (HPLC) and mass spectrometry techniques. The results showed that papain and Alcalase appear to be the most efficient enzymes, and the corresponding hydrolyzates were rich in peptides and amino acids characteristic of collagen, notably absent in the hydrolyzates obtained with other enzymes. Thus, the ability to efficiently hydrolyze collagen seems to be related to the efficiency in fleshing meat hydrolysis.Graphical Abstract: [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]

For centuries, ancient grains fed populations, but due to their low yield, they were abandoned and replaced by high-yielding species. However, currently, there is a renewed interest in ancient wheat and pseudocereal grains from consumers, farmers, and manufacturers. Ancient wheat such as einkorn, emmer, spelt, and Kamut®, are being reintegrated because of their low fertilizer input, high adaptability and important genetic diversity. New trends in pseudocereal products are also emerging, and they are mostly appreciated for their nutritional outcomes, particularly by the gluten-free market. Toward healthier lifestyle, ancient grains-based foodstuffs are a growing business and their industrialization is taking 2 pathways, either as a raw ingredient or a functional ingredient. This paper deals with these grain characteristics by focusing on the compositional profile and the technological potential. [ABSTRACT FROM AUTHOR]

Introduction Phlorotannins are complex mixtures of phloroglucinol oligomers connected via C-C (fucols) or C-O-C (phlorethols) linkages. Their uniformity in subunits and large molecular weight hamper their structural analysis. Despite its commercial relevance for alginate extraction, phlorotannins in Laminaria digitata have not been studied. Objective To obtain quantitative and structural information on phlorotannins in a methanolic extract from L. digitata. Methodology The combined use of 13C and 1H NMR spectroscopy allowed characterisation of linkage types and extract purity. The purity determined was used to calibrate the responses obtained with the colorimetric 2,4-dimethoxybenzaldehyde (DMBA) and Folin-Ciocalteu (FC) assays. Using NP-flash chromatography, phlorotannin fractions separated on oligomer size were obtained and enabled structural and molecular weight characterisation using ESI-MS and MALDI-TOF-MS. Results The fucol-to-phlorethol linkage ratio was 1:26 and the extract was 60.1% pure, determined by NMR spectroscopy. For DMBA, the response of the extract was 12 times lower than that of phloroglucinol, whereas there was no difference for FC. By accounting for differences in response, the colorimetric assays were applicable for quantification using phloroglucinol as a standard. The phlorotannin content was around 4.5% DM. Fucol- and phlorethol-linkage types were annotated based on characteristic MS n fragmentations. Structural isomers of phlorotannins up to a degree of polymerisation of 18 (DP18) were annotated and identification of several isomers hinted at branched phloroglucinol oligomers. With MALDI-TOF-MS phlorotannins up to DP27 were annotated. Conclusion By combining several analytical techniques, phlorotannins in L. digitata were quantified and characterised with respect to fucol-to-phlorethol linkage ratio, molecular weight (distribution), and occurrence of structural isomers. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Pectic oligosaccharides (POS) have been indicated as novel candidate prebiotics. Traditionally, POS are produced from pectin-rich by-products using a two-step process involving extraction of the pectin, followed by its hydrolysis into POS. A one-step approach, in which the POS is directly produced from the raw material, might provide a more efficient alternative. Thus, the main aim of this paper was to investigate a one-step enzymatic hydrolysis approach to directly produce POS from sugar beet pulp (SBP). The POS yield was investigated as a function of the process parameters, as well as raw material characteristics. A statistically-based response surface methodology, using a central composite design was applied, to investigate the individual as well as the combined influences of the diverse parameters. The model was confirmed by a validation experiment, carried out at 135 g/l substrate concentration, 0.75 FPU/g SBP enzyme concentration, 0.8 mm particle size and 3 h hydrolysis time. Under these conditions, a POS-rich hydrolysate was obtained, containing rhamnose, arabinose, galactose, xylose and galacturonic acid, at 0.9, 15.2, 5.1, 1.4, and 13.2 g/l, respectively, enzymes were added each at 20 FPU/g dry matter (DM). [ABSTRACT FROM AUTHOR]

Because of the continuous increase in the prevalence of gluten-related disorders, selection of wheat with a low content of immunogenic gluten epitopes could be an innovative alternative for prevention. In this review, the focus is on literature data concerning the deallergenization tools of wheat, which are mainly related to breeding approaches (classic and advanced) and processing operations (germination and fermentation). Until now, no safe wheat genotype has been identified, whereas decreasing wheat allergenicity is possible. On the other hand, the decrease of gluten or some of its epitopes can strongly affect technological properties. Thus, obtaining celiac-safe gluten without affecting the technological properties of wheat could be considered as a new challenge that scientists will be facing. Celiac-safe wheat-based product development could be a great revolution in the market of foods for special medical purposes. The present paper is aiming to: (a) review the strategies and the approaches used, or that can be used, for developing low allergenic wheat: their utilities and limits were also discussed and (b) screen the impact of gluten reduction or removal on the quality of wheat end-use products. [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]

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and forin vivoandin vitrostudies of important POS, so that they could be commercially exploited. [ABSTRACT FROM PUBLISHER]

Proteomics is the approach of choice to study the fate of specific proteins, and it is very useful in identifying quality molecular markers. A combination of immunochemical and mass spectrometry analysis was used to assess the occurrence of proteolytic changes of actin and myosin heavy chain (MHC) proteins in pig biceps femoris skeletal muscle during processing of three Italian PDO dry-cured hams. Early post-mortem muscle displayed low levels of actin and myosin fragments. In spite of a high proteolysis index and the presence of active cathepsin D until the final ripening phase, during dry-cured ham processing, very low actin proteolysis and no generation of fragments from α-skeletal muscle isoform were found, while the identified fragments derived mainly from the cardiac actin isoform. On the other hand, MHC showed a remarkable degradation of its catalytic head, generating a C-terminal 135-kDa fragment. Based on its ability to interact with actin in vitro, this MHC fragment might have a role in stabilisation of actin. In conclusion, these results suggest that maintenance of skeletal muscle α-actin could reflect limited dismantling of the sarcomeric structure and be a useful marker to monitor the events that result in the typical texture of dry-cured ham. [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]

Non-specific lipid transfer proteins (nsLTP) were shown to be among the most significant allergens, in particular in several fruits belonging to the Rosaceae family. The molecular features of LTPs, such as the presence of eight cysteine residues forming four disulfide bridges, confer a compact structure, decreasing the probability of degradation due to cooking or digestion, thereby increasing the chance of systemic absorption and severe allergic reactions. Few studies on LTP-induced allergies regarding almond (Prunus dulcisL) are available in the literature. In the present work, we describe for the first time the extraction and purification of an almond LTP, achieving its full characterisation by using liquid chromatography and exact mass spectrometry; the full sequence was identified by means of LC-ESI-Orbitrap™-MS applying a bottom-up approach. The characterised protein consists of 92 amino acids and has a calculated exact MW of 9579.0. The presence of four disulfide bridges was confirmed after reduction, as shown by a mass increment of 8 Da. Finally, its potential allergenicity was confirmed via anin silicoapproach. The results presented here demonstrate the enormous potential of advanced MS techniques for obtaining high-quality structural and functional data of allergenic proteins in a short time. [ABSTRACT FROM PUBLISHER]

Non-specific lipid-transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut-off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC-MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato. Copyright © 2014 John Wiley & Sons, Ltd. [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]

Antimicrobial peptides (AMPs) are natural compounds isolated from a wide variety of organisms that include microorganisms, insects, amphibians, plants, and humans. These biomolecules are considered as part of the innate immune system and are known as natural antibiotics, presenting a broad spectrum of activities against bacteria, fungi, and/or viruses. Technological innovations have enabled AMPs to be utilized for the development of novel biodetection devices. Advances in nanotechnology, such as the synthesis of nanocomposites, nanoparticles, and nanotubes have permitted the development of nanos-tructured platforms with biocompatibility and greater surface areas for the immobilization of biocomponents, arising as additional tools for obtaining more efficient biosensors. Diverse AMPs have been used as biological recognition elements for obtaining biosensors with more specificity and lower detection limits, whose analytical response can be evaluated through electrochemical impedance and fluorescence spectroscopies. AMP-based biosensors have shown potential for applications such as supplementary tools for conventional diagnosis methods of microorganisms. In this review, conventional methods for microorganism diagnosis as well new strategies using AMPs for the development of impedimetric and fluorescent biosensors are highlighted. AMP-based biosensors show promise as methods for diagnosing infections and bacterial contaminations as well as applications in quality control for clinical analyses and microbiological laboratories. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A new objective method has been developed for the identification of animal hair fibres, in particular wool, cashmere and yak. Enzymatic digestion of keratin extracted from these fibres and peptide analysis by ultra-performance liquid chromatography/electrospray–mass spectrometry (UPLC/ESI–MS) allows not only qualitative determination of the presence of fibres derived from these species but also a quantitative assessment of the relative percentages present in blends. Such an analysis will provide reliable objective data about the authenticity of commercial products. The effectiveness of the UPLC/ESI–MS method was assessed by analysing known samples of these fibres and confirmed using unknown wool/cashmere/yak blends, and the results were compared with those obtained by SEM method IWTO 58–00. [ABSTRACT FROM PUBLISHER]

Gamma-glutamyl-amino acids, lactoyl-amino acids and pyroglutamyl-amino acids, collectively named Non-Proteolytic Aminoacyl Derivatives (NPADs) are unusual aminoacyl derivatives of non-proteolytic origins found in consistent amount in several cheeses. Although their enzymatic origin arising from lactic acid bacteria has been demonstrated, the exact enzymes originating them, the ones eventually degrading them and also their resistance to digestive enzymes in the human gastrointestinal tract and in the blood serum after eventual absorption are still unknown. In this paper, pure enzymes and biological media were tested on NPAD and their aminoacidic precursors, for identifying the conditions favoring bioproduction and biodegradation of these compounds. Pure gamma-glutamyl-phenylalanine and its precursor (glutamic acid and phenylalanine), also in the isotopically labeled forms, were tested with Parmigiano-Reggiano extracts, blood serum and different pure enzymes, including typical digestion enzymes (pepsin, trypsin and chymotrypsin), gamma-glutamyl transpeptidase and carboxypeptidase. The data suggested that their production in cheese, and also their partial degradation, might be due to the action of peptidases and gamma-glutamyl transpeptidase. Anyway, under simulated gastrointestinal digestion and in blood serum these compounds turned out to be perfectly stable, suggesting a potential to be absorbed as such and possibly being transported to the body tissues. [ABSTRACT FROM AUTHOR]

In this work the antioxidant capacity of water soluble extracts of Parmigiano-Reggiano cheese (Water Soluble Extracts - WSEs) at different aging time was studied, by measuring their radical scavenging capacity with a standard ABTS assay. The WSEs were also fractionated by semi-preparative HPLC-UV and for each fraction the antioxidant capacity and the molecular composition was determined by LC/ESI-MS, in order to identify the most active antioxidant compounds. The antioxidant capacity was also determined after simulated in vitro gastrointestinal digestion of WSEs. The data indicated that antioxidant capacity in WSE from Parmigiano-Reggiano cheese, quite unaffected by ripening time and gastrointestinal digestion, is mostly due to free amino acids, mainly tyrosine, methionine and tryptophan, and only in minimal part to antioxidant peptides. [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]

The achievement of a sufficient acidity level by starters at early stages of manufacture is essential for a good quality Parmigiano Reggiano (PR) cheese. In this work, the chemical parameters and microbial characteristics of 18 PR cheeses during the moulding phase were analysed to determine how the microbial growth takes place conditioning acidification performances. Based on the mean values of lactic acid content, two groups of cheeses were observed. High numbers of culturable thermophilic bacteria and in particular Lactobacillus helveticus able to metabolise galactose but also grow faster in the cheese appeared to be essential conditions to achieve adequate lactic acid content. On the other hand, low acidification and residual galactose, as observed in the second group, may be associated with a lower amount of metabolically active L. helveticus and higher relevance of Streptococcus thermophilus contents in the cheese. While culturable thermophilic bacteria were fundamental for higher lactic acid production, non-culturable cells did not seem to contribute to the acidification. [ABSTRACT FROM AUTHOR]

In the present paper, a proteomic method for species determination in fibres has been developed. Keratin was extracted from yak, wool and cashmere fibres and digested by trypsin, providing peptide mixtures that were analyzed by liquid chromatography coupled with electrospray mass spectrometry (LC/ESI-MS) in order to identify peptidic species-specific markers able to differentiate the fibres. Several suitable peptide markers were identified and validated in different fibres of different origin and having undergone different technological treatments, showing 100% specificity and 100% selectivity. Most of the peptide markers were also identified by means of high-resolution mass spectrometry, confirming the origin from species-specific keratin sequences. Some peptides were also used for the quantification of the different species in mixed fibres by LC/ESI-MS. Validation experiments and blind tests confirmed their ability to act as very specific quantitative and qualitative markers. The method here developed is a valid complement to the standard benchmark methods for fibre identification and quantification and will be very useful for assessing the authenticity of textile products. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Qualitative and quantitative methods for the analysis of intact phytosteryl/phytostanyl esters in enriched dairy products with substantial amounts of protein and fat were established. Lipids were extracted after acid digestion in order to achieve a complete extraction of the esters. The extracted lipids were separated via liquid chromatography on a normal phase, and the fraction containing the phytosteryl/phytostanyl esters was subsequently transferred on-line and analyzed by means of capillary gas chromatography. The applicability of the method was demonstrated for the analysis of intact phytosteryl/phytostanyl fatty acid esters in enriched yogurt, cheese-based spread, and milk. The approach enables the determination of individual phytosteryl/phytostanyl esters as well as of their total contents and provides detailed information on the distributions of esterified phytosterols/phytostanols and fatty acids. The employed approach complements the existing GC-based methods for the analysis of phytosteryl/phytostanyl esters in low-fat and fat-based products and thus creates a base for authenticity assessments of these types of novel foods. [ABSTRACT FROM AUTHOR]

Bacterial growth media represent a high cost in industrial applications, and for this reason, it is economically important to find less expensive supplements to replace the traditional ones. In the present work, peptide hydrolysates obtained from poultry meat and bone residues (functional animal protein [FAP]) and from feathers (functional feather protein [FFP]) were studied to determine their ability for the production of microbial biomass with improved viability. The results obtained were compared with those obtained with other supplement nutritive compounds used in fermentation growth media. The molecular composition of the hydrolysates in terms of total and soluble nitrogen, molecular weight distribution, total and free amino acids, was determined. The growth and cellular state of Bifidobacterium and Lactobacillus strains were studied by turbidimetric measurements and direct count by fluorescence microscopy. Overall, this study suggested that by-products from poultry industry provide a good alternative to substitute expensive supplements for growth of Lactobacillus and Bifidobacterium with a high level of viability [ABSTRACT FROM AUTHOR]

RATIONALE Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceæ family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. METHODS Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH4HCO3 at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. RESULTS The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. CONCLUSIONS An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A method to detect the presence of common wheat in durum wheat flour samples was developed and tested. Flour samples, or ground wheat samples, were digested by pepsin and chymotrypsin, and the peptide mixture obtained was analyzed by LC/ESI-MS and LC/ESI-MS/MS, which led to the identification of two marker peptides. One peptide was coded only in the DD genome, and thus present only in common wheat; the second was present in all wheat samples (both common and durum), so it was used as marker of the total wheat content. The ratio of the chromatographic areas of these two peptides, as determined by LC/ESI-MS, was related to the proportion of common wheat in the sample using a calibration curve that was constructed with standards of known composition. The proportions of common wheat in samples obtained by mixing different common and durum wheat varieties were accurately determined by this method. Finally, the method was applied in a survey of several durum wheat flour brands present on the Italian market. The results of the survey revealed that contamination of durum wheat flour with common wheat is commonplace. [ABSTRACT FROM AUTHOR]

Fumonisins are a family of food-borne mycotoxins with a wide spectrum of toxicological activities, produced by Fusarium verticillioides. Twenty-eight fumonisin analogues have been characterised so far, which can be separated into four main groups, identified as fumonisin A, B, C and P, being fumonisin B the most widely occurring in maize and corn-based food. In this work, major and minor fumonisin analogues produced by F. verticillioides have been determined by the development of a suitable tandem mass spectrometry procedure for target compound identification and quantification. The method has been applied to the determination of the major fumonisins in culture media of F. verticillioides and in mouldy maize. In addition to the main fumonisins produced by F. verticillioides, also secondary compounds such as FB4, FB5, FAs and FCs have been detected in both fungal liquid cultures and contaminated maize samples. The use of this method for quantification of major and minor fumonisins may be useful for an exhaustive evaluation of their occurrence and toxicological relevance in food; moreover, it may be applied for a better definition of the fumonisin biosynthetic pathways in different growing media as well as in maize. Copyright © 2012 John Wiley & Sons, Ltd. [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]

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