Health Beneficial Bioactivities of Faba Bean Gastrointestinal (In Vitro) Digestate in Comparison to Soybean and Pea.

Faba beans are a promising emerging plant-based protein source to be used as a quality alternative to peas and soy. In this study, the potential health beneficial activities of three Canadian faba bean varieties (Fabelle, Malik and Snowbird) were investigated after in vitro gastrointestinal digestion and compared to two commonly used legumes (peas and soy). The results revealed that the faba beans had a higher antioxidant activity than peas when assessed with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, except for the Fabelle variety. In the oxygen radical absorbance capacity (ORAC) and the iron chelating assays, the faba beans had a lower antioxidant activity than soy. Interestingly, Fabelle and Snowbird showed a higher antioxidant effect than the peas and soy at the cellular level. The antihypertensive properties of Fabelle and Malik varieties were significantly higher than peas but lower than soy. The in vitro antidiabetic activity was higher for soy, but no differences were found at the cellular level. The faba bean peptides were further fractionated and sequenced by mass spectrometry. Eleven peptides with in silico predicted bioactivities were successfully identified in the faba bean digestate and support validating the health-promoting properties of peptides. The results demonstrate the bioactive potential of faba beans as a health-promoting food ingredient against non-communicable diseases.

Antioxidant and Angiotensin-Converting Enzyme Inhibitory Activity of Faba Bean-Derived Peptides After In Vitro Gastrointestinal Digestion: Insight into Their Mechanism of Action.

Faba bean flour, after in vitro gastrointestinal digestion, showed important antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities. In the present study, 11 faba bean- derived peptides were synthesized to confirm their bioactivities and provide a deeper understanding of their mechanisms of action. The results revealed that 7 peptides were potent antioxidants, namely, NYDEGSEPR, TETWNPNHPEL, TETWNPNHPE, VIPTEPPH, VIPTEPPHA, VVIPTEPPHA, and VVIPTEPPH. Among them, TETWNPNHPEL had the highest activity in the ABTS (EC50 = 0.5 ± 0.2 mM) and DPPH (EC50 = 2.1 ± 0.1 mM) assays (p < 0.05), whereas TETWNPNHPE had the highest activity (p < 0.05) in the ORAC assay (2.84 ± 0.08 mM Trolox equivalent/mM). Synergistic and/or additive effects were found when selected peptides (TETWNPNHPEL, NYDEGSEPR, and VVIPTEPPHA) were combined. Four peptides were potent ACE inhibitors, where VVIPTEPPH (IC50 = 43 ± 1 µM) and VVIPTEPPHA (IC50 = 50 ± 5 µM) had the highest activity (p < 0.05), followed by VIPTEPPH (IC50 = 90 ± 10 µM) and then VIPTEPPHA (IC50 = 123 ± 5 µM) (p < 0.05). These peptides were noncompetitive inhibitors, as supported by kinetic studies and a molecular docking investigation. This study demonstrated that peptides derived from faba beans have multifunctional bioactivities, making them a promising food-functional and nutraceutical ingredient.

Improved in vitro gastrointestinal digestion protocol mimicking brush border digestion for the determination of the Digestible Indispensable Amino Acid Score (DIAAS) of different food matrices.

The Digestible Indispensable Amino Acid Score (DIAAS) is the new gold standard method for the assessment of protein nutritional quality. The DIAAS is evaluated with in vivo models, that are complex, constraining and costly. There is still no established method to assess it in vitro. In this study, we proposed to add a jejunal-ileal digestion phase to the standardized in vitro gastrointestinal digestion protocol developed by the International Network of Excellence on the Fate of Food in the Gastrointestinal Tract (INFOGEST protocol) to mimic brush border digestion and to enable DIAAS assessment in vitro in a more physiologically relevant manner. This jejunal-ileal digestion phase was performed with a porcine intestinal aminopeptidase as an alternative to brush border membrane extract, which is more difficult to obtain in a standardized way. This modified INFOGEST protocol was applied to various food matrices (faba bean, pea and soy flours, whey protein isolate and caseins) and the results were compared to published in vivo data to assess the model's physiological relevance. The addition of the jejunal-ileal digestion phase lead to a significant (p < 0.05) increase of 31 and 29 % in free and total amino acid digestibility, respectively, and of 83 % on average for the in vitro DIAAS values for all food matrices. Although the in vitro DIAAS remained underestimated compared to the in vivo ones, a strong correlation between them was observed (r = 0.879, p = 0.009), stating the relevance of this last digestion phase. This improved digestion protocol is proposed as a suitable alternative to evaluate the DIAAS in vitro when in vivo assays are not applicable.

Bioaccessibility and Antioxidant Activity of Faba Bean Peptides in Comparison to those of Pea and Soy after In Vitro Gastrointestinal Digestion and Transepithelial Transport across Caco-2 and HT29-MTX-E12 Cells.

In this study, the transepithelial transport of bioactive peptides derived from faba bean flour gastrointestinal digestates was investigated, in vitro, using a Caco-2 and HT29-MTX-E12 coculture monolayer, in comparison to those of pea and soy. The profile of transported peptides was determined by mass spectrometry, and the residual antioxidant activity was assessed. The ORAC value significantly (p < 0.05) decreased after transepithelial transport (24-36% reduction) for all legumes, while the antioxidant activity in ABTS assay significantly (p < 0.05) increased, as shown by the EC50 decrease of 26-44%. Five of the nine faba bean peptides that crossed the intestinal cell monolayer exhibited antioxidant activity. Two of these peptides, TETWNPNHPEL and TETWNPNHPE, were further hydrolyzed by the cells' brush border peptidases to smaller fragments TETWNPNHP and TWNPNHPE. These metabolized peptides were synthesized, and both maintained high antioxidant activity in both ABTS (EC50 of 1.2 ± 0.2 and 0.4 ± 0.1 mM, respectively) and ORAC (2.5 ± 0.1 and 3.4 ± 0.2 mM of Trolox equivalent/mM, respectively) assays. These results demonstrated for the first time the bioaccessibility of faba bean peptides produced after in vitro gastrointestinal digestion and how their bioactive properties can be modulated during transepithelial transport.

A graphene-based label-free voltammetric immunosensor for sensitive detection of the egg allergen ovalbumin.

A graphene-based label-free voltammetric immunosensor for the sensitive detection of the egg white allergen ovalbumin has been developed. Graphene-modified screen printed carbon electrodes have been covalently functionalized using electrochemical reduction of in situ generated aryl diazonium salt forming a carboxyphenyl film on the graphene surface. The blocking property of the carboxyphenyl film grafted on to the graphene electrodes using different cyclic voltammetry cycles has been characterized using differential pulse voltammetry in [Fe(CN)6](3-/4-) solution. Then, the terminal carboxylic groups on the graphene surface were activated using EDC/NHS and used to immobilize the ovalbumin antibody and construct the immunosensor. The fabrication steps of the immunosensor have also been characterized using differential pulse voltammetry. The decrease in the [Fe(CN)6](3-/4-) reduction peak current after the immunochemical reaction with ovalbumin has been used for the ovalbumin detection. The developed immunosensor has been used for ovalbumin detection in the concentration range of 1 pg mL(-1) to 0.5 µg mL(-1) with a detection limit of 0.83 pg mL(-1) in PBS buffer. The food matrix effect studied with ovalbumin spiked cake extract showed a good percentage of recovery, indicating the possible applicability of the developed immunosensor in real food samples.

Investigation of the nutritional quality of raw and processed Canadian faba bean (Vicia faba L.) flours in comparison to pea and soy using a human in vitro gastrointestinal digestion model.

Faba bean is an ancient legume that is regaining interest due to its environmental and nutritional benefits. Very little is known on the protein quality of the new faba bean varieties. In this study, the digestibility and the Digestible Indispensable Amino Acid Score (DIAAS) of the protein quality of three Canadian faba bean varieties (Fabelle, Malik and Snowbird) were compared to pea and soy using the harmonized in vitro digestion procedure developed by the International Network of Excellence on the Fate of Food in the Gastrointestinal Tract (INFOGEST). The impact of boiling on the nutritional quality of faba bean flours was also ascertained. Protein content in faba bean (28.7-32.5%) was lower than defatted soy (56.6%) but higher than pea (24.2%). Total phenolics and phytate content were higher (p < 0.05) in faba bean (2.1-2.4 mg/g and 11.5-16.4 mg/g respectively) and soy (2.4 mg/g and 19.8 mg/g respectively) comparatively to pea (1.3 mg/g and 8.9 mg/g). Trypsin inhibitor activity was significantly higher (p < 0.05) in soy (15.4 mg/g) comparatively to pea (0.7 mg/g) and faba bean (0.8-1.1 mg/g). The digestibility of free amino acids of raw faba bean flours ranged from 31 to 39% while the digestibility of total amino acids ranged from 38 to 39%. The in vitro Digestible Indispensable Amino Acid Score (IV-DIAAS) of raw faba bean flours ranged from 13 to 16 (when calculated based on free amino acid digestibility) to 32-38 (when calculated based on total amino acid digestibility) and was in a similar range to pea (13-31) and soy (11-40). Boiling modified the protein electrophoretic profile and decreased trypsin inhibitor activity (30-86% reduction), while total phenolics and phytate content were unaffected. The IV-DIAAS significantly decreased in all boiled legumes, possibly due to an increased protein aggregation leading into a lower protein digestibility (18-32% reduction). After boiling, the nutritional quality of faba bean was significantly lower (p < 0.05) than soy, but higher than pea. Our results demonstrate that faba bean has a comparable protein quality than other legumes and could be used in similar food applications.

Assessment of Protein Nutritional Quality of Novel Hairless Canary Seed in Comparison to Wheat and Oat Using In Vitro Static Digestion Models.

Hairless canary seed (Phalaris canariensis L.) is a novel true cereal that is now approved for human consumption in Canada and the United States. This true cereal grain has higher protein content (22%) than oat (13%) and wheat (16%) and represents a valuable source of plant proteins. Assessment of canary seed protein quality is therefore essential to evaluate its digestibility and ability to provide sufficient amounts of essential amino acids for human requirements. In this study, the protein nutritional quality of four hairless canary seed varieties (two brown and two yellow) were evaluated in comparison to oat and wheat. The assessment of anti-nutrients contents (phytate, trypsin inhibitor activity, and polyphenols) showed that brown canary seed varieties had the highest content in phytate and oat the highest in polyphenols. Trypsin inhibitor level was comparable among studied cereals, but slightly higher in the brown canary seed Calvi variety. In regard to protein quality, canary seed had a well-balanced amino acid profile and was particularly high in tryptophan, an essential amino acid normally lacking in cereals. The in vitro protein digestibility of canary seeds as determined by both the pH-drop and INFOGEST (international network of excellence on the fate of food in the gastrointestinal tract) protocols appears slightly lower than wheat and higher than oat. The yellow canary seed varieties showed better overall digestibility than the brown ones. For all studied cereal flours, the limiting amino acid was lysine. The calculated in vitro PDCAAS (protein digestibility corrected amino acid score) and DIAAS (digestible indispensable amino acid score) were higher for the yellow C05041 cultivar than the brown Bastia, similar to those of wheat, but lower than those of oat proteins. This study demonstrates the feasibility and utility of in vitro human digestion models for the assessment of protein quality for comparison purpose.

Faba Bean: An Untapped Source of Quality Plant Proteins and Bioactives.

Faba beans are emerging as sustainable quality plant protein sources, with the potential to help meet the growing global demand for more nutritious and healthy foods. The faba bean, in addition to its high protein content and well-balanced amino acid profile, contains bioactive constituents with health-enhancing properties, including bioactive peptides, phenolic compounds, GABA, and L-DOPA. Faba bean peptides released after gastrointestinal digestion have shown antioxidant, antidiabetic, antihypertensive, cholesterol-lowering, and anti-inflammatory effects, indicating a strong potential for this legume crop to be used as a functional food to help face the increasing incidences of non-communicable diseases. This paper provides a comprehensive review of the current body of knowledge on the nutritional and biofunctional qualities of faba beans, with a particular focus on protein-derived bioactive peptides and how they are affected by food processing. It further covers the adverse health effects of faba beans associated with the presence of anti-nutrients and potential allergens, and it outlines research gaps and needs.

Combining phenolic grafting and laccase-catalyzed cross-linking: Effects on structures, technofunctional properties and human immunoglobulin E binding capacity of egg white proteins.

The efficiency of laccase-catalyzed protein cross-linking can be impacted by substrate protein structure and competing reactions. In this study, chemical grafting of ferulic acid (FA) on protein surface was applied to modulate the cross-linking of two inflexible globular proteins, lysozyme (LZM) and ovalbumin (OVA). The extent of FA-grafting was positively correlated with protein cross-linking extent, and determined the molecular weight profile and structures of the cross-linked product. While laccase-catalyzed reactions (with or without free FA mediator) did not lead to evident cross-linking of the native proteins, oligomeric (up to 16.4%), polymeric (up to 30.6%) FA-LZMs and oligomeric FA-OVA (5.1-31.1%) were obtained upon the enzymatic treatments. The cross-linking on the grafted FA sites occurred mainly through the formation of 8-5'-noncyclic-dehydro-diferulic linkages. The effects of investigated cross-linking approach on the emulsifying, foaming properties and the immunoglobulin E (IgE) binding capacity of LZM and OVA were also evaluated in relation to the structural properties of cross-linked proteins.

Health Promoting Bioactive Properties of Novel Hairless Canary Seed Flour after In Vitro Gastrointestinal Digestion.

The bioactive properties and health-promoting effects of two novel yellow (C09052, C05041) and two brown (Calvi, Bastia) hairless canary seed (Phalaris canariensis L.) cultivars were investigated in comparison to two common cereal grains (wheat and oat). The cereal flours were digested using the standardized INFOGEST in vitro human gastrointestinal digestion model. The three-kilo dalton molecular weight cutoff (3 kDa MWCO) permeate of the generated digestates was assessed in vitro for their antioxidant, chelating, antihypertensive and antidiabetic activities. The results showed no significant differences in studied bioactivities between yellow and brown canary seed cultivars, except for antioxidant activity by the DPPH and chelating Fe2+ assays, where brown cultivars had higher activities. Canary seeds had superior or equivalent antioxidant activity than those from oat and wheat. The anti-hypertensive activity (Angiotensin-converting enzyme (ACE) inhibition) in yellow canary seed cultivars was significantly higher than that of oat and wheat, particularly for C09052 and Calvi varieties. Peptides exhibiting the highest antihypertensive activity from the permeate of the C09052 canary seed variety were further fractionated and identified by mass spectrometry. Forty-six peptides were identified belonging to 18 proteins from the Pooideae subfamily. Fourteen of the parent proteins were homologous to barley proteins. Peptides were analyzed in silico to determine potential bioactivity based on their amino acid composition. All 46 peptides had potential anti-hypertensive and anti-diabetic activities and 20 had potential antioxidant activity, thereby validating the in vitro assay data. Canary seed peptides also exhibited potential antiamnestic, antithrombotic, immunostimulating, opioid and neuro-activity, demonstrating important potential for health promoting effects, particularly against cardiovascular disease.

Immunoglobulin E-Binding Pattern of Canadian Peanut Allergic Children and Cross-Reactivity with Almond, Hazelnut and Pistachio.

Peanut allergic individuals can be both co-sensitized and co-allergic to peanut and tree nuts. At the moment, standard diagnostic approaches do not always allow differentiation between clinically relevant sensitization and nonsignificant cross-reactions, and the responsibility of each allergen remains unclear. The objective of this study was therefore to determine a peanut sensitization profile in a cohort of Canadian peanut allergic children and assess the immunoglobulin E (IgE) molecular cross-reactivity between peanut, almond, hazelnut and pistachio. The specific IgE (sIgE) levels of each patient serum were determined by ImmunoCAP, indirect ELISA and immunoblot to examine their sIgE-binding levels and profiles to peanut proteins. Reciprocal inhibition ELISA and immunoblotting were used to study sIgE cross-reactions between peanut and the selected tree nuts using an adjusted and representative serum pool of the nine allergic patients. The results showed that the prepared peanut and tree nut protein extracts allowed for the detection of the majority of peanut and selected tree nut known allergens. The reciprocal inhibition ELISA experiments showed limited sIgE cross-reactivities between peanut and the studied tree nuts, with peanut being most likely the sensitizing allergen and tree nuts the cross-reactive ones. In the case of hazelnut and pistachio, a coexisting primary sensitization to hazelnut and pistachio was also demonstrated in the serum pool. Reciprocal inhibition immunoblotting further revealed that storage proteins (2S albumin, 7S vicilin and 11S legumin) could possibly account for the observed IgE-cross-reactions between peanut and the studied tree nuts in this cohort of allergic individuals. It also demonstrated the importance of conformational epitopes in the exhibited cross-reactions.

Scale up fractionation of components from novel glabrous brown and yellow canary seeds (Phalaris canariensis L.) and techno-functional properties of the resulting protein isolates.

Glabrous canary seed (Phalaris canariensis L.) is a novel true cereal grain produced primarily in Western Canada which has been approved for human consumption by the U.S. Food and Drug Administration and Health Canada in 2016. Due to its high protein content (22%), this new edible grain is emerging as an alternative source of plant proteins. In the present work, protein extractability from four novel glabrous (2 yellow and 2 brown) canary seeds varieties was improved based on the selection of optimal pH of protein solubilisation and precipitation. Solubilisation at pH 12 followed by acid precipitation at pH 5 were retained as optimal conditions. Scale up of the protein optimized wet fractionation process resulted in highly purified canary seed protein isolates (purity of 91 to 93%) with protein recovery yield of 65 to 69%. In parallel, for the others canary seed components, a good recovery yields were obtained for the oil fraction (6.1-6.7 g/100 g flour), starch fraction (48.1-54 g/100 g flour), and crude fiber fraction (15.1-19.7 g/100 g flour). The study of the functional properties of the obtained canary protein isolates revealed, higher solubility at acidic than alkaline region; enhanced fat and water holding capacities and notably higher foaming and emulsifying capacities than control soy protein isolate. With growing global demand for protein, glabrous canary seed has high potential in the food industry, particularly as a good source of functional gluten free cereal proteins.

Assessment of the effect of lactic acid fermentation on the gastroduodenal digestibility and immunoglobulin E binding capacity of soy proteins via an in vitro dynamic gastrointestinal digestion model.

Fermentation by lactic acid bacteria is helpful in reducing soy protein immunoreactivity. However, how lactic acid fermentation influences the gastroduodenal digestibility and immunoglobulin E (IgE) binding capacity of soy proteins remains unclear. In this study, the protein digestion of a fermented soybean protein isolate (FSPI) was investigated and compared with that of a soybean protein isolate (SPI). The effect on their respective IgE binding capacities at the gastric and duodenal phases was also explored by using a novel in vitro dynamic gastrointestinal digestion model (Bionic Rat Model II+). Medium pH was measured, microstructural analysis was performed, peptide distribution and free amino acid content were determined, and SDS-PAGE analysis was performed to assess the differences between SPI and FSPI. The results showed that FSPI had lower pH (3.76), larger protein aggregates (>60 µm), and higher low-molecular-weight peptides than SPI. During the first 30 min of gastric and duodenal digestion, the extent of hydrolysis of FSPI was higher than that of SPI, and the gastric transition time of the former was longer than that of the latter. Conversely, differences tended to be narrower in the next 30-180 min of gastric and duodenal digestion. As a result, the IgE binding capacity of FSPI was significantly lower than that of SPI at 30 min of gastric and duodenal digestion. Therefore, fermentation by lactic acid bacteria affected the digestibility rate of soy proteins, especially at the initial phases of gastric and duodenal digestion, thereby reducing the exposure of intact epitopes in the duodenum. This study helped to elucidate how lactic acid fermentation affected the digestive behavior of soy proteins and its implication in IgE immunoreactivity reduction.

Properties of selected hemicellulases of a multi-enzymatic system from Penicillium funiculosum.

A multi-enzymatic system from Penicillium funiculosum displayed alpha-L-arabinofuranosidase, endo-1,4-beta-D-xylanase, beta-D-xylosidase and endo-1,3-1,4-beta-D-glucanase activities at high levels over a wide acidic pH range of 2.0 to 5.5. Moreover, the pH stability was particularly extended over the wide range of pH of 2.0 to 8.0 with endo-1,3-1,4-beta-D-glucanase and endo-1,4-beta-D-xylanase; however, alpha-L-arabinofuranosidase and beta-D-xylosidase exhibited higher stability in the pH range of 2.0 to 5.5. The results indicate that the optimal temperature of alpha-L-arabinofuranosidase (65 degrees C) and beta-D-xylosidase (70 degrees C) as well as their thermal stability were higher than those of endo-1,3-1,4-beta-D-glucanase (60 degrees C) and endo-1,4-beta-D-xylanase (50 degrees C). Although V(maxapp) of beta-D-xylosidase and endo-1,4-beta-D-xylanase was higher than that of alpha-L-arabinofuranosidase and endo-1,3-1,4-beta-D-glucanase, respectively, their catalytic efficiency was lower. High levels of ferulolyl esterase, alpha-D-galactosidase, beta-D-mannosidase and endo-1,4-beta-D-mannanase activities were also detected in the multi-enzymatic system. The overall features of the multi-enzymatic system from P. funiculosum reveal its potential for degrading and modifying plant cell walls from a variety of food and feedstuffs.

Detection and identification of allergens from Canadian mustard varieties of Sinapis alba and Brassica juncea.

Currently, information on the allergens profiles of different mustard varieties is rather scarce. Therefore, the objective of this study was to assess protein profiles and immunoglobulin E (IgE)-binding patterns of selected Canadian mustard varieties. Optimization of a non-denaturing protein extraction from the seeds of selected mustard varieties was first undertaken, and the various extracts were quantitatively and qualitatively analyzed by means of protein recovery determination and protein profiling. The IgE-binding patterns of selected mustard seeds extracts were assessed by immunoblotting using sera from mustard sensitized and allergic individuals. In addition to the known mustard allergens-Sin a 2 (11S globulins), Sin a 1, and Bra j 1 (2S albumins)-the presence of other new IgE-binding protein bands was revealed from both Sinapis alba and Brassica juncea varieties. Mass spectrometry (MS) analysis of the in-gel digested IgE-reactive bands identified the unknown ones as being oleosin, ß-glucosidase, enolase, and glutathione-S transferase proteins. A bioinformatic comparison of the amino acid sequence of the new IgE-binding mustard proteins with those of know allergens revealed a number of strong homologies that are highly relevant for potential allergic cross-reactivity. Moreover, it was found that Sin a 1, Bra j 1, and cruciferin polypeptides exhibited a stronger IgE reactivity under non-reducing conditions in comparison to reducing conditions, demonstrating the recognition of conformational epitopes. These results further support the utilization of non-denaturing extraction and analysis conditions, as denaturing conditions may lead to failure in the detection of important immunoreactive epitopes.

Hairless Canaryseed: A Novel Cereal with Health Promoting Potential.

Glabrous canaryseeds were recently approved for human consumption as a novel cereal grain in Canada and the United States. Previously, canaryseeds were exclusively used as birdseed due to the presence of carcinogenic silica fibers; therefore the nutritional value of the seeds has been seriously overlooked. Two cultivars of glabrous canaryseeds (yellow and brown) were created from the hairy varieties. They are high in protein compared to other cereal grains, and contain high amounts of tryptophan, an amino acid normally lacking in cereals, and are gluten-free. Bioactive peptides of canaryseeds produced by in vitro gastrointestinal digestion have shown antioxidant, antidiabetic, and antihypertensive activity. The seeds contain other constituents with health promoting effects, including unsaturated fatty acids, minerals, and phytochemicals. Anti-nutritional components in the seeds are comparable to other cereal grains. Because of their beneficial health effects, canaryseeds should be regarded as a healthy food and have immense potential as a functional food and ingredient. Further research is required to determine additional bioactive peptide activity and capacity, as well as differences between the yellow and brown cultivars.

Ionic strength and pH-induced changes in the immunoreactivity of purified soybean glycinin and its relation to protein molecular structure.

This study examined the immunogenic response of glycinin under varying conditions of pH and ionic strength using enzyme-linked immunosorbent assay. Differential scanning calorimetric (DSC) analysis and Fourier transform infrared spectroscopy (FTIR) were used to investigate the conformational changes induced as a result of these conditions, and the correlation with the changes observed in glycinin immunoreactivity were determined. A highly purified glycinin obtained by isoelectric precipitation followed by native preparative continuous flow electrophoresis was used for these studies. Purity was confirmed by two-dimensional-polyacrylamide gel electrophoresis and mass spectroscopy. DSC and FTIR results suggest that glycinin immunoreactivity is affected by changes in the tertiary and secondary packing of the protein, when flexibility, stability, and accessibility of certain substructures are modified. Aggregation and/or increased compactness of glycinin subcomponents could have potentially prevented epitopes from reacting with the IgG antibodies.

Quantitative and qualitative optimization of allergen extraction from peanut and selected tree nuts. Part 1. Screening of optimal extraction conditions using a D-optimal experimental design.

A D-optimal design was constructed to optimize allergen extraction efficiency simultaneously from roasted, non-roasted, defatted, and non-defatted almond, hazelnut, peanut, and pistachio flours using three non-denaturing aqueous (phosphate, borate, and carbonate) buffers at various conditions of ionic strength, buffer-to-protein ratio, extraction temperature, and extraction duration. Statistical analysis showed that roasting and non-defatting significantly lowered protein recovery for all nuts. Increasing the temperature and the buffer-to-protein ratio during extraction significantly increased protein recovery, whereas increasing the extraction time had no significant impact. The impact of the three buffers on protein recovery varied significantly among the nuts. Depending on the extraction conditions, protein recovery varied from 19% to 95% for peanut, 31% to 73% for almond, 17% to 64% for pistachio, and 27% to 88% for hazelnut. A modulation by the buffer type and ionic strength of protein and immunoglobuline E binding profiles of extracts was evidenced, where high protein recovery levels did not always correlate with high immunoreactivity.

Quantitative and qualitative optimization of allergen extraction from peanut and selected tree nuts. Part 2. Optimization of buffer and ionic strength using a full factorial experimental design.

A full factorial design was used to assess the single and interactive effects of three non-denaturing aqueous (phosphate, borate, and carbonate) buffers at various ionic strengths (I) on allergen extractability from and immunoglobulin E (IgE) immunoreactivity of peanut, almond, hazelnut, and pistachio. The results indicated that the type and ionic strength of the buffer had different effects on protein recovery from the nuts under study. Substantial differences in protein profiles, abundance, and IgE-binding intensity with different combinations of pH and ionic strength were found. A significant interaction between pH and ionic strength was observed for pistachio and almond. The optimal buffer system conditions, which maximized the IgE-binding efficiency of allergens and provided satisfactory to superior protein recovery yield and profiles, were carbonate buffer at an ionic strength of I=0.075 for peanut, carbonate buffer at I=0.15 for almond, phosphate buffer at I=0.5 for hazelnut, and borate at I=0.15 for pistachio. The buffer type and its ionic strength could be manipulated to achieve the selective solubility of desired allergens.

Allergenicity of potato proteins and of their conjugates with galactose, galactooligosaccharides, and galactan in native, heated, and digested forms.

The effect of glycation of potato proteins on their immunoreactivity was studied by using a pool of human sera with specific IgE to potato proteins. Patatin conjugates were more immunoreactive than protease inhibitors ones. To better understand this behavior, the changes in patatin structure upon glycation and heat treatment were investigated. Patatin demonstrated an increase in total immunoreactivity when glycated with galactose and galactooligosaccharides. However, galactan conjugation to patatin resulted in a decrease in immunoreactivity by restricting IgE's access to the epitopes. Although the heat treatment resulted in a decrease in patatin's immunoreactivity through aggregation, it was less effective when patatin conjugates were used due to the decrease in aggregation and the secondary structural changes. Upon digestion, native patatin exhibited the largest decrease in immunoreactivity resulting from the disruption of both conformational and sequential epitopes. Patatin conjugates were less digested and had higher IgE-immunoreactivity as compared to the digested patatin.