Quantification of Protein "Biomarkers" in Wheat-Based Food Systems: Dealing with Process-Related Issues.

Selected food proteins may represent suitable markers for assessing either the presence/absence of specific food ingredients or the type and intensity of food processes. A fundamental step in the quantification of any protein marker is choosing a proper protocol for solubilizing the protein of interest. This step is particularly critical in the case of solid foods and when the protein analyte is prone to undergo intermolecular disulfide exchange reactions with itself or with other protein components in the system as a consequence of process-induced unfolding. In this frame, gluten-based systems represent matrices where a protein network is present and the biomarker proteins may be either linked to other components of the network or trapped into the network itself. The protein biomarkers considered here were wheat gluten toxic sequences for coeliac (QQPFP, R5), wheat germ agglutinin (WGA), and chicken egg ovalbumin (OVA). These proteins were considered here in the frame of three different cases dealing with processes different in nature and severity. Results from individual cases are commented as for: (1) the molecular basis of the observed behavior of the protein; (2) the design of procedure aimed at improving the recovery of the protein biomarker in a form suitable for reliable identification and quantification; (3) a critical analysis of the difficulties associated with the plain transfer of an analytical protocol from one product/process to another. Proper respect for the indications provided by the studies exemplified in this study may prevent coarse errors in assays and vane attempts at estimating the efficacy of a given treatment under a given set of conditions. The cases presented here also indicate that recovery of a protein analyte often does not depend in a linear fashion on the intensity of the applied treatment, so that caution must be exerted when attributing predictive value to the results of a particular study.

Towards a metabolomic approach to investigate iron-sulfur cluster biogenesis.

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.

Enzymatic and Chemical In Vitro Reconstitution of Iron-Sulfur Cluster Proteins.

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.

Monitoring the carryover of egg proteins in pasta making to support allergen risk management.

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).

Cefoxitin treatment of MRSA leads to a shift in the IL-12/IL-23 production pattern in dendritic cells by a mechanism involving changes in the MAPK signaling.

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of ß-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 ß-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.

Effect of Sprouting on Proteins and Starch in Quinoa (Chenopodium quinoa Willd.).

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.

Probiotic Administration Increases Amino Acid Absorption from Plant Protein: a Placebo-Controlled, Randomized, Double-Blind, Multicenter, Crossover Study.

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.

Bio-Functional and Structural Properties of Pasta Enriched with a Debranning Fraction from Purple Wheat.

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.

Inhibition of Pancreatic α-amylase by Resveratrol Derivatives: Biological Activity and Molecular Modelling Evidence for Cooperativity between Viniferin Enantiomers.

To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids-in particular, viniferin isomers- were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.

Effects of starch addition on the activity and specificity of food-grade lipases.

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.

Greetings from foodland: Teaching biochemistry to BS students in food-related courses in Italy.

Biochemistry has always been a mandatory topic within BS courses aimed at food science students at the University of Milan, namely: Food Science & Technology and Catering Sciences. Addressing biochemistry topics in this peculiar setting requires: (i) specific focus on topics that are seldom considered in courses offered in bio-medical curricula; (ii) close integration with other area disciplines, such as food biotechnology; (iii) ad hoc design of laboratory classes; and (iv) an array of elective courses covering specific aspects of biochemistry. In this context, for example, protein chemistry is presented by using food proteins of known structure and discussed in terms of structural features in the raw materials and of structural and chemical modifications occurring upon processing. Along the same lines, metabolic pathways and their regulation are presented starting from widespread metabolism-related issues and to issues related to food safety (including food allergies and intolerances). A similar "hands on" approach is used for laboratory classes, that cover about one third of total credits and are aimed at providing fundamental-type information by analyzing practical situations in the food chain. In spite of their inherent complexity and volume, biochemistry courses score very well with the students in mandatory anonymous surveys. Our approach to biochemistry courses seems to help the students in "visualizing" the practical implications of concepts acquired in other courses within their curricula. The students' appreciation is confirmed by the sizeable attendance to elective and specialized biochemical-themed courses. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):394-403, 2019.

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells.

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-ß), 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-1ß 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.

Topological features of the intermolecular contacts in gluten-forming proteins: Exploring a novel methodological approach based on gold nanoparticles.

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.

The Bio-Functional Properties of Pigmented Cereals may Involve Synergies among Different Bioactive Species.

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.

Enriching gluten-free rice pasta with soybean and sweet potato flours.

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.

Structural consequences of the interaction of puroindolines with gluten proteins.

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.

Understanding the influence of buckwheat bran on wheat dough baking performance: Mechanistic insights from molecular and material science approaches.

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.

Physicochemical properties of sorghum flour are selectively modified by combined germination-fermentation.

The combined effects of grain germination and of subsequent fermentation on the physicochemical properties of sorghum flour were investigated by studying the structural changes occurring in the starch and protein fractions and by assessing their effects on physical properties of the resulting materials most relevant to end use. The sequential treatments were more effective than either individual treatment in the modification of starch-related properties, whereas modification of the protein components only occurs in the fermentation step, almost regardless of a previous germination step. The resulting profile of physicochemical traits offers several hints as for the suitability of flour from treated sorghum as an ingredient for various types of gluten-free food products, and provides a basis for expanding the use of processed sorghum in applications other than traditional African foods.

Macromolecular Traits in the African Rice Oryza glaberrima and in Glaberrima/Sativa Crosses, and Their Relevance to Processing.

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. PRACTICAL APPLICATION: 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.

Stabilization of beta-lactoglobulin by polyols and sugars against temperature-induced denaturation involves diverse and specific structural regions of the protein.

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.