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Bromelain is a mixture of cysteine endopeptidase usually extracted from pineapple juice and is used for the treatment of various human diseases and in industrial applications. Bromelain demand is quickly increasing, and its recovery from pineapple residues appears to be a sustainable waste management strategy. Pineapple core is among the most significant waste materials in the production of canned pineapple and is richer in bromelain than other pineapple residues. In this project, we compared the enzymatic properties and composition of bromelain extracts from either pineapple core or pulp to address the recovery of bioactive bromelain from pineapple core, thus contributing to the valorization of this waste material. Although significant differences were detected in the protein content of the two preparations, no differences could be detected for their proteolytic activity and for the effect of pH on their enzymatic activity. Mass spectrometry (MS) approaches identified the same peptidases in the fruit and in the core. This confirmed the possibility of using pineapple core to obtain relevant amounts of bioactive bromelain by applying a relatively simple procedure, thus paving the way to implementing a circular economy in this specific industrial sector.

Iron-sulfur clusters are prosthetic groups that are assembled on their acceptor proteins through a complex machine centered on a desulfurase enzyme and a transient scaffold protein. Studies to establish the mechanism of cluster formation have so far used either in vitro or in vivo methods, which have often resulted in contrasting or non-comparable results. We suggest, here, an alternative approach to study the enzymatic reaction, that is based on the combination of genetically engineered bacterial strains depleted of specific components, and the detection of the enzymatic kinetics in cellular extracts through metabolomics. Our data prove that this ex vivo approach closely reproduces the in vitro results while retaining the full complexity of the system. We demonstrate that co-presence of bacterial frataxin and iron is necessary to observe an inhibitory effect of the enzymatic activity of bacterial frataxin. Our approach provides a new powerful tool for the study of iron-sulfur cluster biogenesis.

Bromelain is a mixture of proteolytic enzymes that is extracted primarily from pineapples. It is present in almost all the aerial parts of the plant—the peels, leaves, stems and fruit—although only the fruit and stems contain significant amounts. The market-demand for bromelain is quickly increasing, especially in the health sector as it can be used as a drug and/or nutraceutical. Although the complete molecular mechanism has not been fully identified, bromelain possesses several properties, including anti-inflammatory, anti-oedema, antithrombotic and fibrinolytic, mucolytic, anticancer and cicatrizing effects that have been evaluated in several clinical trials. However, one of the main limitations for the clinical use of this supplement is the varying composition of extracts, which leads to heterogeneity in results and, therefore, difficulty in making evidence-based prescriptions. In fact, different geographic locations, land and modes of cultivation, as well as extraction methods, can all give extracts with different activities, depending on the plant part used. Additionally, although novel extraction techniques have been developed to improve bromelain purification and extraction and give higher yields without loss in enzymatic activity, these methods are still expensive and challenging. This review will describe the state of the art in the main conventional and unconventional extraction and purification methods of bromelain and discuss the advantages and limitations of these strategies. Pharmacokinetic and pharmacodynamic profiles and the clinical applications that have arisen from randomized controlled clinical trials are also discussed. Finally, future perspectives for bromelain extracts will be presented.

Iron-sulfur (Fe-S) clusters are key cofactors for proteins involved in essential cellular processes such as DNA replication and repair, ribosome biogenesis, tRNA thio-modification, and co-enzyme synthesis. Fe-S clusters can assemble spontaneously from inorganic compounds, but their biogenesis requires dedicated machineries to circumvent the toxic nature of iron and sulfur. To address how these machines work, different laboratories have applied various biochemical and biophysical approaches, both in vivo and in vitro. Fe-S cluster enzymatic and chemical formation in vitro is the most efficient way to follow Fe-S cluster biogenesis in a controlled environment and investigate each component of the machinery at the molecular level. In this review, we detail and discuss an efficient protocol for an in vitro Fe-S cluster enzymatic and chemical formation, which we successfully developed to study Fe-S cluster formation. We underline the applications of this approach to the study of an essential biological system.

Iron-sulfur (Fe-S) clusters are key cofactors for proteins involved in essential cellular processes such as DNA replication and repair, ribosome biogenesis, tRNA thio-modification, and co-enzyme synthesis. Fe-S clusters can assemble spontaneously from inorganic compounds, but their biogenesis requires dedicated machineries to circumvent the toxic nature of iron and sulfur. To address how these machines work, different laboratories have applied various biochemical and biophysical approaches, both in vivo and in vitro. Fe-S cluster enzymatic and chemical formation in vitro is the most efficient way to follow Fe-S cluster biogenesis in a controlled environment and investigate each component of the machinery at the molecular level. In this review, we detail and discuss an efficient protocol for an in vitro Fe-S cluster enzymatic and chemical formation, which we successfully developed to study Fe-S cluster formation. We underline the applications of this approach to the study of an essential biological system.

Egg proteins are among the major food allergens. Very often, the same pasta-making plants are used for industrial production of egg-based pasta (EBP) and semolina-only pasta (SP), so that residual egg proteins may be present in SP. This calls for defining the amount of semolina pasta that should be discarded when switching production lines. In this study, the egg proteins content was measured in pasta samples taken at various times after switching production lines from EBP to SP Both long and short pasta shapes were sampled before and after a drying step. Protocols meant to circumvent the difficulties associated with detecting egg proteins in a complex matrix after processing were set up for using commercial ELISA kits to monitor the disappearance of egg proteins from the products. The use of both denaturants and disulphide reductants to solubilise egg proteins was found to be mandatory, as verified by ovalbumin detection by ELISA and by using mass spectrometry to assess residual egg white lysozyme. Appropriate sample preparation protocols were used to monitor the progressive disappearance of egg proteins in the products when shifting production lines in an industrial pasta plant, providing a basis for credible, reliable, and consistent self-control procedures. For lines with a production capacity of 2200-2400 kg h-1, the amount of material to be discarded to ensure that products meet the strictest analytical requirements has been found to be around 2000-3000 kg (for long pasta) and 3000-4000 kg (for short pasta).

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of β-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that β-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus.

The development of innovative rice products is a way to exploiting and adding value to low-grade African rice varieties. To this purpose, rice-based pasta was enriched with flours from soybean and orange-fleshed sweet potato, that are common ingredients in the African tradition. Four different formulations based on pre-gelatinized rice flour and liquid egg albumen, and containing soybean and/or sweet potato (up to 20%) were prepared and characterized via a multidisciplinary approach. Soybean and sweet potato enrichment leads to a decrease in the pasta consistency and in significant changes in the color of the resulting samples, likely due to Maillard-type reactions. E-sensing approaches indicated that the sensory profile of the various pasta products strongly depends on the type of enrichment. Data collected after cooking suggest that both soybean and sweet potato have a role in defining the firmness and water absorption, as well as the optimum cooking time. Structural characterization of proteins in the uncooked products indicates the presence of protein aggregates stabilized by hydrophobic interactions and disulfide bonds in all samples, although structural properties of the aggregates related to specific compositional traits.

The effects of fermentation of teff flour by a mixture of lactic acid bacteria and yeasts present in a gluten-free sourdough have been considered. Fermentation had a major impact on the physicochemical properties of teff starch and on its pasting behavior, and a somewhat more limited impact on teff proteins, leaving essentially intact protein components of possible relevance for formation of a protein network. Either fermented or non-fermented teff were added to a 25% level to a commercial corn-based gluten-free bread mix, containing chemical leavening agents. The bread enriched with fermented teff had improved physical properties and a lower staling rate with respect to a non-enriched control or to a bread enriched with non-fermented teff flour.

Gluten replacement in gluten-free (GF) products presents major challenges for the food industry in terms of sensorial, technological and nutritional characteristics. The absence of gluten reportedly affects starch digestibility, thus increasing the postprandial glycaemic response. However, the role of ingredients and processing conditions has been addressed only seldom. We investigated the in vitro starch digestibility of 9 commercial GF products (5 Italian pasta and 4 Oriental noodles) differing in formulation and processing conditions. Content of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were assessed and combined with information on starch pasting properties and on the overall protein organization. Oriental noodles presented higher relative levels of RS and RDS than Western-style pasta, that often had SDS levels compatible with low rates of starch digestion. As regard formulation, presence of multiple ingredients seems to likely increase the RDS level, as did the different protein organization in the various samples.

This study aims at understanding the relation among sprouting time (from 12 up to 72 h), changes in protein and starch components, and flour functionality in quinoa. Changes related to the activity of sprouting-related proteases were observed after 48 h of sprouting in all protein fractions. Progressive proteolysis resulted in relevant modification in the organization of quinoa storage proteins, with a concomitant increase in the availability of physiologically relevant metals such as copper and zinc. Changes in the protein profile upon sprouting resulted in improved foam stability, but in impaired foaming capacity. The increased levels of amylolytic enzymes upon sprouting also made starch less prompt to gelatinize upon heating. Consequently, starch re-association in a more ordered structure upon cooling was less effective, resulting in low setback viscosity. The nature and the intensity of these modifications suggest various possibilities as for using flour from sprouted quinoa as an ingredient in the formulation of baked products.

The fate of dietary protein in the gut is determined by microbial and host digestion and utilization. Fermentation of proteins generates bioactive molecules that have wide-ranging health effects on the host. The type of protein can affect amino acid absorption, with animal proteins generally being more efficiently absorbed compared with plant proteins. In contrast to animal proteins, most plant proteins, such as pea protein, are incomplete proteins. Pea protein is low in methionine and contains lower amounts of branched-chain amino acids (BCAAs), which play a crucial role in muscle health. We hypothesized that probiotic supplementation results in favorable changes in the gut microbiota, aiding the absorption of amino acids from plant proteins by the host. Fifteen physically active men (24.2 ± 5.0 years; 85.3 ± 12.9 kg; 178.0 ± 7.6 cm; 16.7 ± 5.8% body fat) co-ingested 20 g of pea protein with either AminoAlta™, a multi-strain probiotic (5 billion CFU L. paracasei LP-DG® (CNCM I-1572) plus 5 billion CFU L. paracasei LPC-S01 (DSM 26760), SOFAR S.p.A., Italy) or a placebo for 2 weeks in a randomized, double-blind, crossover design, separated by a 4-week washout period. Blood samples were taken at baseline and at 30-, 60-, 120-, and 180-min post-ingestion and analyzed for amino acid content. Probiotic administration significantly increased methionine, histidine, valine, leucine, isoleucine, tyrosine, total BCAA, and total EAA maximum concentrations (Cmax) and AUC without significantly changing the time to reach maximum concentrations. Probiotic supplementation can be an important nutritional strategy to improve post-prandial changes in blood amino acids and to overcome compositional shortcomings of plant proteins. ClinicalTrials.gov Identifier: ISRCTN38903788

A colored and fiber-rich fraction from the debranning of purple wheat was incorporated at 25% into semolina- and flour-based pasta produced on a pilot-plant scale, with the aim of increasing anthocyanin and total phenolic content with respect to pasta obtained from whole pigmented grains. The debranning fraction impaired the formation of disulfide-stabilized protein networks in semolina-based systems. Recovery of phenolics was impaired by the pasta making process, and cooking decreased the phenolic content in both enriched samples. Cooking-related losses in anthocyanins and total phenolics were similar, but anthocyanins in the cooked semolina-based pasta were around 20% of what was expected from the formulation. HPLC (High Performance Liquid Chromatography) profiling of phenolics was carried out on extracts from either type of enriched pasta both before and after cooking and indicate possible preferential retention of specific compounds in each type of enriched pasta. Extracts from cooked samples of either enriched pasta were tested as inhibitors of enzymes involved in glucose metabolism and uptake, as well as for their capacity of suppressing the response to inflammatory stimuli. Results of both biological tests indicate that the phenolics in extracts from both cooked pasta samples had inhibitory capacities higher than extracts of the original debranning fraction at identical concentrations of total bioactives.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

The development of innovative legume-enriched rice products is a promising way to exploit rice varieties with a low sensory grade. In this work, a multidisciplinary approach was applied to the characterization of extruded breakfast cereals prepared from African-grown Oryza glaberrima (cv. Viwonor) or Oryza sativa (cv. Jasmine 85) enriched with 30% cowpea flour, obtained from sprouted or nonsprouted cowpea. Regardless of the rice species, addition of sprouted cowpea flour conferred a peculiar volatiles profile, rich in sour, bitter, and astringent taste. Protein structural indices provided molecular insights about the macroscopic differences among samples. Extruded products from O. glaberrima were characterized by lower expansion rates with respect to those obtained from O. sativa, regardless of the type of cowpea flour. Sprouting time had a positive influence on the hardness of extruded glaberrima-based products, facilitating formation of a more compact matrix, but it did not influence sativa-based produc...

Lipases are surface-active enzymes, acting on their substrates at the polar/nonpolar interface in emulsions. This study was aimed to test whether their activity, specificity, and the rates of formation/degradation of the various hydrolysis intermediates (i.e., mono- and diglycerides of interest as surface-active agents) could be modulated by adhesion of the triglyceride substrates as a thin layer on the surface of solids. These hypotheses were tested by using an array of food-grade lipases used in bakery, testing various types of starch as the "solid" phase. Starch-dependent increase in the hydrolysis rate was tested by pH-stat techniques on pure triglycerides and on food-grade oils in diluted emulsions. Starch-related improvements in the rate of fatty acids release were most evident at temperatures above 40 °C, and when using maize starch instead of wheat starch. Starch-dependent changes in the nature of the hydrolysis products were tested by chromatographic profiling of ethyl ether extracts from aqueous slurries containing up to 33% fat and 33% starch. Accumulation of mono- and diglycerides as hydrolysis intermediates was found to be modulated by the type of oil being used, by the reaction conditions, as well as by the enzyme nature and amount.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production. IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Bioactives specific to pigmented grains are colored compounds belonging to the anthocyanin family. These compounds have been shown to have antioxidant and immunoregulatory properties in humans. In addition, they are of interest in sustainability terms, as they make grains resistant to pests and pathogens, thus minimizing phytochemical use. The nature and amount of pigments present in various classes of pigmented cereals are discussed, along with the possibility of retaining them as useful bioactives when cereals are transformed in various types of foods for direct human consumption.

This study was aimed at characterizing the anthocyanins and phenolics profile in different varieties of pigmented corn and wheat and in some of their milling fractions. Acid/ethanol extracts were used to assess total anthocyanins, overall antioxidant activity, the overall polyphenol profile, and for evaluating the inhibition of pancreatic α-amylase and of intestinal α-glucosidase. Both enzymes were inhibited in a dose-dependent manner by all extracts, but individual extracts had specific effects on each enzyme. Anti-inflammatory response was evaluated by using acid-free extracts and Caco-2 cells transiently transfected with a luciferase reporter gene responding to cytokine stimulation. The immune response of interleukin-stimulated cells decreased significantly in a dose-dependent manner in the presence of 20-50 μM/l anthocyanins from all grains extracts, again with a different efficiency. The inhibitory ability and the anti-inflammatory capability of these extracts are in most cases higher than in similar extracts from other sources, suggesting that activities in each extract may imply specific synergies between anthocyanins and other phenolics.

This work introduces a novel methodological approach to study both the geometry of complex protein networks and the nature of the interacting proteins. This approach is based on the high reactivity of Au + ions on the surface of gold nanoparticles (AuNPs) towards thiols, that allows fast formation of a covalent bond between accessible protein thiols and AuNPs. In the case of the durum wheat semolina used in the exploratory studies reported here, the nature of proteins covalently bound to AuNPs is expected to be affected by both the compactness of the protein network and by the AuNPs size. Simple centrifugation procedures allowed recovery of the protein-loaded AuNPs that remained soluble, and the protein(s) covalently bound on the surface of soluble AuNP were identified by MS analysis of their proteolytic fragments. Gluten-forming proteins were found to be bound to soluble AuNPs only when detergents or chaotropes were added to the semolina/AuNPs suspension at room temperature. AuNPs-bound proteins also included gluten-forming proteins with no reported free thiols, suggesting that they are piggybacked on other thiol-containing gluten-forming proteins via disulfide bonds already present in the otherwise untreated semolina. The potential of this approach is discussed in terms of the possibility of developing a methodology suitable for further clarification of the geometrical features of protein networks, of the nature of the involved proteins, and of the type of interaction they establish, as well as any modifications of these features upon processing.

An attempt was made to evaluate gluten structural changes in refined and whole wheat pasta from hard white winter wheat to elucidate the impact of whole wheat components on the formation and structure of the gluten network in pasta. Attenuated total reflectance–FTIR spectroscopy was used to track gluten secondary structure through most of the major steps in pasta processing: raw material, mixing, drying, and cooking. Protein solubility, accessible thiols, and SDS-PAGE data were also collected to provide additional information on the nature of protein interactions and network composition. Few secondary structural differences were observed between refined and whole wheat flours from hard white wheat. However, mixing induced a significant shift to β-sheet structures in refined dough that was not equally matched by whole wheat dough. Drying under both high temperature, short time (HT) and low temperature, long time (LT) conditions resulted in a reversion to structural distributions similar to those for flour ...

Enrichment with other components may improve the textural and nutritional characteristics of rice-based pasta, often used in gluten-free diets. In this work, formulations based on enrichment with sorghum flour were tested. Sorghum flour was used in the absence of treatments, after fermentation by lactic acid bacteria, or was prepared from sprouted grains. Both fermentation and sprouting affected sorghum proteins, but with different mechanisms. Different proteins were affected by fermentation and sprouting, that also had a different impact on the thiol/disulfide balance. Sprouting, but not fermentation, resulted in significant breakdown of sorghum starch. As a result of these modifications, it was not possible to prepare acceptable rice-based pasta upon enrichment with sprouted sorghum flour. Conversely, fermented sorghum flour gave pasta with improved cooking properties with respect to both rice-only pasta and to pasta enriched with untreated sorghum flour.

The effect of puroindolines (PINs) on structural characteristics of wheat proteins was investigated in Triticum turgidum ssp. durum (cv. Svevo) and Triticum aestivum (cv. Alpowa) and in their respective derivatives in which PIN genes were expressed (Soft Svevo) or the distal end of the short arm of chromosome 5D was deleted and PINs were not expressed (Hard Alpowa). The presence of PINs decreased the amount of cold-SDS extractable proteins and the accessibility of protein thiols to specific reagents, but resulted in facilitated solvation of gluten proteins, as detected by tryptophan fluorescence measurements carried out on minimally mixed flour/water mixtures. We propose that PINs and gluten proteins are interacting in the grain or flour prior to mixing. Hydrophobic interactions between PINs and some of the gluten proteins modify the pattern of interactions among gluten proteins, thus providing an additional mechanistic rationale for the effects of PINs on kernel hardness.

Thiol–disulfide exchange reactions, are major contributors to the formation of a covalently-linked protein network in many foods, where disulfides represent the most “natural” type of interprotein covalent bond. Thiol–disulfide exchange reactions occur also as a function of the accessibility of the involved thiols, which in turn depends on structural features of the involved proteins. Thiols in soluble and insoluble food proteins were covalently labeled by 5-iodoacetamide-fluorescein in the absence or in the presence of 4 M urea, a procedure that allowed to evaluate thiols accessibility before and after protein unfolding and dissociation of non-covalently linked protein complexes. Proteins labeled under either condition, along with unlabeled proteins, were then solubilized by treatment with disulfide reductants (and urea, when not added before) and separated either by SDS-PAGE or by two-dimensional electrophoresis. The 5-iodoacetamidofluorescein labeling procedures were also applied to soft wheat flours, and to semolina from durum wheat. Results highlight the different accessibilities of thiols in specific protein components in these materials, suggesting a possible role of minor protein components as for promoting rearrangement in the thiol pattern in wheat proteins upon processing and pointing out the relevance of structural issues in addition to compositional ones.

Molecular properties of proteins and starch were investigated in 2 accessions of Oryza glaberrima and Oryza sativa, and in one NERICA cross between the 2 species, to assess traits that could be relevant to transformation into specific foods. Protein nature and organization in O. glaberrima were different from those in O. sativa and in NERICA. Despite the similar cysteine content in all samples, thiol accessibility in O. glaberrima proteins was higher than in NERICA or in O. sativa. Inter-protein disulphide bonds were important for the formation of protein aggregates in O. glaberrima, whereas non-covalent protein-protein interactions were relevant in NERICA and O. sativa. DSC and NMR studies indicated only minor differences in the structure of starch in these species, as also made evident by their microstructural features. Nevertheless, starch gelatinization in O. glaberrima was very different from what was observed in O. sativa and NERICA. The content of soluble species in gelatinized starch from the various species in the presence/absence of treatments with specific enzymes indicated that release of small starch breakdown products was lowest in O. glaberrima, in particular from the amylopectin component. These findings may explain the low glycemic index of O. glaberrima, and provide a rationale for extending the use of O. glaberrima in the production of specific rice-based products, thus improving the economic value and the market appeal of African crops.The structural features of proteins and starch in O. glaberrima are very different from those in O. sativa and in the NERICA cross. These results appear useful as for extending the use of O. glaberrima cultivars in the design and production of specific rice-based products (for example, pasta), that might, in turn, improve the economic value and the market appeal of locally sourced raw materials, by introducing added-value products on the African market.

A molecular and material science approach is used to describe the influence of coarse and fine buckwheat bran on wheat dough properties and bread textural quality. Focus is given on (i) gluten solvation and structural arrangements in presence of bran as studied by front-face fluorescence; (ii) thermo-mechanical behavior of dough during heating studied by dynamic mechanical thermal analysis and (iii) texture of bread crumb analyzed in terms of a cellular solid. The thermo-mechanical behavior of dough was found to be largely related to starch phase transitions during heating. The use of thermodynamic approaches to biopolymer melting revealed that key transitions such as the onset of starch gelatinization were function of the interplay of water and bran volume fractions in the dough. Front-face fluorescence studies in wheat dough revealed that gluten solvation and structural arrangements were delayed by increasing bran addition level and reduction in particle size, as indicated by the drastic decrease in the protein surface hydrophobicity index. Variations in gluten structure could be strongly related to dough baking performance, i.e. specific volume. With regards to texture, the approach revealed that crumb texture was controlled by variations in density, moisture and bran volume fractions. Overall, this study elucidates a number of physical mechanisms describing the influence of buckwheat bran addition to dough and bread quality. These mechanisms strongly pointed at the influence of bran on water partitioning among the main polymeric components. In the future, these mechanisms should be investigated with bran material of varying source, composition and structure. © 2017 Elsevier Ltd

Most of the unique properties of waxy wheat have been associated with the lack of amylose, that in turn may affect the mutual interactions between starch and proteins. To address this particular aspect, we carried out molecular, rheological, and calorimetric studies on flours from two waxy wheat lines that were compared with a non-waxy one. Dough thermal properties and water binding capacity were investigated by Differential Scanning Calorimetry (DSC) and by thermogravimetric analysis, respectively. Protein solvation, aggregation, and thiol accessibility were also investigated, together with dough mixing properties and stickiness. Proteins in waxy wheat samples needed more water to complete solvation, likely because of the water-retaining capacity of waxy wheat starch. In waxy wheat dough, water was tightly bound to starch, and DSC studies indicated an increase in gelatinization temperature. Moreover, the low water mobility in waxy wheat resulted in low and retarded gluten hydration and in high stickiness. In samples with the highest stickiness, protein aggregates were stabilized mainly by hydrophobic interactions. Differences between waxy wheat lines may be attributed to a different structural organization of components within each class of biopolymers.

Pulse flour may be used to improve nutritional traits of gluten and gluten-free formulations in traditional food such as bread or pasta. However, owing to some intrinsic nutritional, textural, and sensory properties, the use of pulses as ingredients for production of enriched food remains limited. In this study, we investigated the modification in macromolecules and micronutrients in industrial-scale flour from partially sprouted chickpeas to define its possible use as an ingredient in cereal-based foods. Controlled sprouting resulted in significant decrease of antinutritional compounds (e.g., phytic acid and serine protease inhibitors) and in an increase of free minerals and vitamins. Sprouting also affected the overall structural organization of proteins (such as aggregate formation) and their thiol/disulfide balance, and it promoted release of peptides. All of these had a positive effect on dough mixing properties, in particular for dough development. Formulations with enrichment in sprouted chickpea f...

Temperature sensitivity of bovine milk beta-lactoglobulin (BLG) was assessed in the presence/absence of non-reducing sugars (sucrose and trehalose) and polyols (glycerol and sorbitol). None of them affected the structural features of the protein at room temperature, where the only observed effect was an increased affinity towards hydrophobic probes in the presence of all co-solutes but glycerol. Although most of the observed effects in temperature-ramp experiments are due to entropic effects (fitting within the “preferential exclusion” theory of protein stabilization), this study indicates that each co-solute exhibit different efficacy at stabilizing specific regions of BLG, suggesting that each of them acts in a specific way on the solvent/protein system. The relevance of these observations with respect to systems of practical relevance is discussed, given the widespread use of heat-polymerizing proteins – such as BLG – in many food formulations that very often include significant amounts of sugars and/or polyols. © 2017 Elsevier Ltd

Snacks were produced by extruding blends of partially-defatted soybean flour with flours from milled or parboiled African-grown rice. The interplay between composition and processing in producing snacks with a satisfactory sensory profile was addressed by e-sensing, and by molecular and rheological approaches. Soybean proteins play a main role in defining the properties of the protein network in the products. At the same content in soybean flour, use of parboiled rice flour increases the snack's hardness. Electronic nose and electronic tongue discriminated samples containing a higher amount of soybean flour from those with a lower soybean flour content.

The ability of high hydrostatic pressure processing to promote changes in both the structural properties of fiber and the interaction of fiber with water were addressed. Both coarse and fine bran from milling of common wheat were considered. Treatment-induced morphological changes were most pronounced in fine bran, whereas treatment of coarse bran resulted in the largest change in water-holding capacity. The significance of the process-induced changes is discussed in terms of their practical relevance in the production of fiber-enriched foods.

Active cellulose-based packaging materials were prepared by binding lysozyme to paper modified with anionic polyelectrolytes. Polyelectrolytes improve the paper binding capacity towards the positively charged lysozyme and play a protective role towards lysozyme activity during the paper-making process. The charge density of paper was increased by incorporating carboxymethylcellulose (CMC) or polygalacturonic acid (PGA). The presence of either CMC or PGA greatly increased the amount of bound lysozyme, and the stability of its binding towards buffers or towards non-ionic chaotropes which disrupt the three dimensional structure in macromolecules (urea, 8 M). Binding of lysozyme to modified papers was sensitive to anionic detergents (sodium dodecyl sulphate, 1%) and to non-chaotrope salts (NaCl, 0.5M). These data provide information about the nature of the interactions between the protein and the various types of paper, and provide insights on how to preserve the activity of lysozyme-loaded paper. The polymers used for lysozyme immobilization were found to affect only marginally the structural and functional properties of the antimicrobial protein, and to facilitate the recovery of structural features of the protein after heat treatment. Presence of a negative polyelectrolyte (and of CMC in particular) improves the thermal stability of the protein, making it resistant to thermal inactivation, even under the conditions used for drying processes without compromising the paper's mechanical properties. The activity measurements showed that paper-bound lysozyme retains its lytic (and therefore, antimicrobial) activity against the cell walls of Micrococcus lysodeikticus, the target microrganism used as standard for lysozyme bioassays. Copyright © 2009 John Wiley & Sons, Ltd.

The digestion of the seed storage proteins is a finely regulated process operated by several proteases whose action is influenced by the exposure of specific regions, which became progressively available upon their action. We focused our study on the initial stages of germination, where more subtle modifications to the storage proteins are expected. Small-size peptides containing cysteine residues and other possible metalbinding regions are de facto produced but are not released from the “parental” protein since they remain bound trough disulphide bridges. The meaning of these findings is discussed.

Adsorption on the surface of sub-micrometric oil droplets resulted in significant changes in the tertiary structure of bovine beta-lactoglobulin (BLG), a whey protein broadly used as a food ingredient and a major food allergen. The adsorbed protein had increased sensitivity to trypsin, and increased immunoreactivity towards specific monoclonal antibodies. In spite of the extensive tryptic breakdown of emulsion-bound BLG, some sequence stretches in BLG became trypsin-insensitive upon absorption of the protein on the fat droplets. As a consequence - at contrast with free BLG - proteolysis of emulsion-bound BLG did not decrease the immunoreactivity of the protein, and some of the large peptides generated by trypsinolysis of emulsion-bound BLG were still recognizable by specific monoclonal antibodies. Structural changes occurring in emulsion-bound BLG and their consequences are discussed in comparison with those occurring when the tertiary structure of BLG is modified by lipophilic salts, by urea, or upon interaction with solid hydrophobic surfaces. Such a comparison highlights the relevance of situation-specific structural modifications, that in turn may affect physiologically relevant features of the protein.

The scaffold protein IscU is involved in the assembly/transfer of FeS clusters. IscU exists in both open and closed conformation. The clusterless open conformation of IscU adheres to the hydrophobic surface of polystyrene nanobeads, as observed for other proteins. Increased accessibility of the ligand cysteines in bound IscU facilitates assembly of a 2Fe2S cluster, and the cluster-bearing structured form of IscU does not interact with the nanobeads, thus ensuring turnover. The dependence of accelerated cluster assembly on the nanobeads concentration pointed to steric and crowding effects as for promoting cluster formation, and confirms the requirement for structural flexibility of IscU .

The effects of adding egg albumen or whey proteins to pasta made from parboiled rice flour (PR) were investigated. Pasta quality was evaluated in terms of color, furosine content, and cooking properties (water absorption, cooking loss, and consistency at the optimal cooking time). The surface heterogeneity of the cooked and uncooked materials was studied, and some starch properties (pasting properties and starch susceptibility to α-amylase hydrolysis) were assessed, along with the features of the protein network as determined by conditional solubility studies and with ultrastructural features of the cooked products. Egg albumen improved pasta appearance and gave a product with low cooking loss, firmer, and nutritionally more valuable than the other ones. In albumen-enriched pasta, small starch granules appear homogeneously surrounded by a protein network. In the uncooked product, the protein network is stabilized mostly by hydrophobic interactions, but additional disulfide interprotein bonds form upon cooking. Thus, addition of 15 % liquid albumen to PR results in significant improvement of the textural and structural features of rice-based gluten-free pasta.

Front-face fluorescence was used to assess changes in the structural features of proteins in wheat flour dough upon addition of different exogenous lipids. Structural changes resulting from the solvation of proteins and from the mechanical deformation of proteins in dough or dough containing lipids were measured. The effects of lipid type and addition on changes in overall protein surface hydrophobicity were estimated by titrating soft and hard wheat flours, mixed with water and varying the lipid levels, with increasing concentrations of the fluorescence hydrophobic probes 1,8 anilino-napthalene-sulphonate (ANS) and thioflavin T. The lipid type and level modified the exposure of the probe to the solvent. The effects of lipids were more apparent with soft wheat flour having low-affinity hydrophobic sites on the protein surface. The dough was then characterized upon consistent mixing and physical modification in the farinograph at constant water and ANS/thioflavin T concentration, while varying the type and amount of lipids. Lipid-dependent shifts toward longer wavelengths in the probe fluorescent emission with low-protein flour suggest differences in protein coating effects related to lipid structure and protein quality.

Gluten-free pasta represents a challenge for food technologists and nutritionists since gluten-free materials used in conventional formulations have poor functional and nutritional properties. A novel extrusion-cooking process was set up to improve the textural characteristics of rice-based pasta, and to enrich it with amaranth. Mineral and fiber content, and protein digestibility were improved by amaranth enrichment. Extrusion-cooking of a 75/25 mixture of rice flour and amaranth prior to pasta-making gave the best results as for the textural characteristics of the final product. The firmness of cooked pasta increased due to the extrusion-cooking process, that also decreased protein solubility in the amaranth-enriched pasta. The content in accessible thiols also decreased in amaranth-enriched pastas, indicating that amaranth proteins may be involved in forming disulphide bonds during the pasta-making process. Our results suggest that starch in rice flour interacts best with amaranth proteins when starch gelatinization occurs simultaneously to protein denaturation in the extrusion-cooking process.

Scope: To improve our understanding of the interaction of food allergens with cells of the immune system, the endocytosis by human monocytes of bovine β-lactoglobulin (BLG) and ovomucoid (OM) – two major food allergens – and human serum albumin (HSA) was studied. Methods and results: BLG was covalently conjugated to dextran-coated magnetic nanoparticles (MNPs) without affecting its structure and immunoreactivity. BLG-conjugated MNPs were taken up by human monocytes much more efficiently than non-conjugated MNPs, allowing easy magnetic separation of cells that had adsorbed the allergen. BLG, OM, and HSA were conjugated to MNPs also labeled with a fluorescent probe. The uptake of these materials by human monocytes was monitored through flow cytometry, and compared with fluorescent MNPs and the free fluorescently labeled proteins, confirming higher uptake of the BLG-conjugated MNPs versus non-conjugated MNPs. OM but not HSA conjugation to particles enhanced uptake of the MNPs. Confocal microscopy provided direct evidence of the actual internalization of BLG–MNP conjugates into the cytoplasm. Conclusions: These results contribute to the current understanding of the interaction between food allergens and antigen-presenting cells, and demonstrate that the BLG is readily endocytosed by monocytes both as the single protein and as a conjugate.