Hemp (Cannabis sativa L.) protein: Impact of extraction method and cultivar on structure, function, and nutritional quality.

Hemp (Cannabis sativa L.) is increasingly gaining traction as a novel and sustainable source of plant protein. Accordingly, the aim of this study was to investigate the effectiveness of two protein extraction methods, alkaline extraction coupled with isoelectric precipitation (AE-IEP) and salt extraction coupled with ultrafiltration (SE-UF) in producing hemp protein isolates (pH-HPI and salt-HPI) with high purity and yield. Structural characterization as impacted by extraction method and cultivar was performed and related to functional performance and nutritional quality. Both extraction methods, with carefully selected parameters, resulted in HPI with high purity (86.6-88.1% protein) and protein extraction yields (81.6-87.3%). All HPI samples had poor solubility (∼9-20%) at neutral pH compared to commercial soy protein and pea protein isolates (cSPI, cPPI). A relatively high surface hydrophobicity and low surface charge contributed to such poor solubility of HPI. However, HPI demonstrated similar solubility at acidic pH (50-67%) and comparable gel strength (up to 24 N) to cSPI. Comparing experimental amino acid composition to the theoretical amino acid distribution in hemp protein provided insights to the functional performance of the protein isolates. While pH-HPI demonstrated better functionality than salt-HPI, minimal structural, functional, and nutritional differences were noted among the pH-HPI samples extracted from four different cultivars. Overall, results from this work could be used to guide future attempts to further develop successful protein extraction processes, and to provide valuable insights to propel breeding efforts that target enhanced hemp protein characteristics for food applications.

Salt Solubilization Coupled with Membrane Filtration-Impact on the Structure/Function of Chickpea Compared to Pea Protein.

The demand for pulse proteins as alternatives to soy protein has been steeply increasing over the past decade. However, the relatively inferior functionality compared to soy protein is hindering the expanded use of pulse proteins, namely pea and chickpea protein, in various applications. Harsh extraction and processing conditions adversely impact the functional performance of pea and chickpea protein. Therefore, a mild protein extraction method involving salt extraction coupled with ultrafiltration (SE-UF) was evaluated for the production of chickpea protein isolate (ChPI). The produced ChPI was compared to pea protein isolate (PPI) produced following the same extraction method in terms of functionality and feasibility of scaling. Scaled-up (SU) ChPI and PPI were produced under industrially relevant settings and evaluated in comparison to commercial pea, soy, and chickpea protein ingredients. Controlled scaled-up production of the isolates resulted in mild changes in protein structural characteristics and comparable or improved functional properties. Partial denaturation, modest polymerization, and increased surface hydrophobicity were observed in SU ChPI and PPI compared to the benchtop counterparts. The unique structural characteristics of SU ChPI, including its ratio of surface hydrophobicity and charge, contributed to superior solubility at both a neutral and acidic pH compared to both commercial soy protein and pea protein isolates (cSPI and cPPI) and significantly outperformed cPPI in terms of gel strength. These findings demonstrated both the promising scalability of SE-UF and the potential of ChPI as a functional plant protein ingredient.

Enhancement of pea protein solubility and thermal stability for acidic beverage applications via endogenous Maillard-induced glycation and chromatography purification.

A clean-label process to endogenously glycate and purify pea protein was investigated. The production of maltodextrin from pea starch with a specific dextrose equivalent (DE) was optimized. The produced maltodextrin (14.6 DE) was used to initiate a limited and controlled Maillard-induced glycation of pea protein. The partially glycated pea protein (PG-PP) was subjected to hydrophobic interaction chromatography to remove unreacted carbohydrate, followed by characterization of the purified product. The extent of Maillard-induced glycation was monitored by assessing changes in color, free amino groups, and protein/glycoprotein profiles. The purified PG-PP was evaluated for thermal denaturation, surface properties, protein secondary structure, protein solubility, thermal stability, and digestibility. Maillard-induced glycation was limited to initial stages and resulted in a moderate blockage of amine groups (∼30%). The purified PG-PP had a relatively low surface hydrophobicity, a markedly enhanced protein solubility (∼90%) at pH 3.4, and a nonimpacted protein in vitro digestibility (∼100%). This work provided the impetus needed for future scale-up and process optimization for the production of value-added pea protein ingredient intended for high protein beverage applications.

Transglutaminase-Induced Polymerization of Pea and Chickpea Protein to Enhance Functionality.

Pulse proteins, such as pea and chickpea proteins, have inferior functionality, specifically gelation, compared to soy protein, hindering their applications in different food products, such as meat analogs. To close the functionality gap, protein polymerization via targeted modification can be pursued. Accordingly, transglutaminase-induced polymerization was evaluated in pea protein isolate (PPI) and chickpea protein isolate (ChPI) to improve their functionality. The PPI and ChPI were produced following a scaled-up salt extraction coupled with ultrafiltration (SE-UF) process. Transglutaminase (TGase)-modified PPI and ChPI were evaluated in comparison to unmodified counterparts and to commercial protein ingredients. Protein denaturation and polymerization were observed in the TG PPI and TG ChPI. In addition, the TGase modification led to the formation of intermolecular ß-sheet and ß-turn structures that contributed to an increase in high-molecular-weight polymers, which, in turn, significantly improved the gel strength. The TG ChPI had a significantly higher gel strength but a lower emulsification capacity than the TG PPI. These results demonstrated the impact of the inherent differences in the protein fractions on the functional behavior among species. For the first time, the functional behavior of the PPI and ChPI, produced on a pilot scale under mild processing conditions, was comprehensively evaluated as impacted by the TGase-induced structural changes.

Structure-Function Guided Extraction and Scale-Up of Pea Protein Isolate Production.

The lack of adequate guidance and control of the extraction conditions as well as the gap between bench- and industrial-scale production, contributes to the poor functionality of commercial pea protein isolate (cPPI). Therefore, pea protein extraction conditions were evaluated and scaled up to maximize protein purity and yield, while maintaining structural integrity, following mild alkaline solubilization with isoelectric precipitation and salt solubilization coupled with membrane filtration. Both extraction methods resulted in high protein yield (>64%) and purity (>87%). Structure-function characterization illustrated the preserved structural integrity of PPI samples and their superior solubility, gelation, and emulsification properties compared to cPPI. Results confirmed, for the first time, that double solubilization at mild pH (7.5) can replace single solubilization at high alkalinity and achieve a similar yield while preserving structural integrity. Additionally, this study demonstrated, the scalability of the benchtop salt extraction coupled with ultrafiltration/diafiltration. Scaling up the production eliminated some structural and functional differences between the salt-extracted PPI and pH-extracted PPI. Scaling-up under mild and controlled conditions resulted in partial denaturation and a low degree of polymerization, coupled with the superior functionality of the produced isolates compared to cPPI. Results of this work can be used as a benchmark to guide the industrial production of functional pea protein ingredients.

Optimizing the extrusion conditions for the production of expanded intermediate wheatgrass (Thinopyrum intermedium) products.

In this study, the effects of extrusion conditions such as feed moisture content (20%, 24%, and 28%), screw speed (200, 300, and 400 rpm), and extrusion temperature (130, 150, and 170°C) on the physical and functional properties (moisture content, expansion ratio, bulk density, hardness, water absorption index [WAI], water solubility index [WSI]) of intermediate wheatgrass (IWG) were investigated for the first time. Response surface methodology was used to model and optimize the extrusion conditions to produce expanded IWG. The model coefficient of determination (R2 ) was high for all the responses (0.87-0.98). All the models were found to be significant (p < 0.05) and were validated with independent experiments. Generally, all the extrusion conditions were found to have significant effects on the IWG properties measured. Increasing the screw speed and decreasing the extrusion temperature resulted in IWG extrudates with a high expansion ratio. This also resulted in IWG extrudates with generally low hardness and bulk density. Screw speed was found to have the most significant effect on the WAI and WSI, with increasing screw speed resulting in a significant (p < 0.05) decrease in WAI and a significant (p < 0.05) increase in WSI. The optimum conditions for obtaining an IWG extrudate with a high expansion ratio and WAI were found to be 20% feed moisture, 200 -356 rpm screw speed, and 130-154°C extrusion temperature. PRACTICAL APPLICATION: Extrusion cooking was employed in the production of expanded IWG. This research could provide a foundation to produce expanded IWG, which can potentially be used as breakfast cereals and snacks. This is critical in the efforts to commercialize IWG for mainstream food applications.

Impact of plasma reactive species on the structure and functionality of pea protein isolate.

The impact of plasma-produced reactive oxygen and nitrogen species, in particular O3, NxOy, H2O2 and OH, on the structure and functionality of pea protein isolate (PPI) was evaluated. Reactive species were produced through a combination of controlled measurements and plasma treatments. Pronounced structural and functional effects were observed upon treatment with reactive species at pH 2. All reactive species induced protein denaturation and the formation of disulfide-linked soluble aggregates. A significant increase in surface hydrophobicity and ß-sheet content was only induced by treatment with O3 and OH. These specific changes resulted in significant enhancement in gelation and emulsification. While H2O2 enhanced PPI color by increasing whiteness, it had the least impact on protein structure and functionality. Results of this work can be used to optimize cold atmospheric plasma treatment of PPI to induce specific structural changes and a directed enhancement in functionality.

Efficient encapsulation of fish oil: Capitalizing on the unique inherent characteristics of whey cream and hydrolyzed whey protein.

The effects of protein concentration and of blending a phospholipid-rich whey coproduct, Procream (Salibra 700 Procream, Glanbia Nutritionals), with intact or hydrolyzed whey protein concentrate, on fish oil microencapsulation efficiency and oxidative stability were assessed. Trypsin and protease M, from Aspergillus oryzae, were used to produce 2 unique hydrolysates. All microcapsules had excellent encapsulation efficiencies (>92%) and good physical properties, regardless of protein content and Procream inclusion. Intact α-lactalbumin and ß-lactoglobulin and their peptides were involved in stabilizing oil droplets. Disulfide interchange resulted in formation of protein aggregates, which were more pronounced in samples containing Procream. Although all microcapsules had relatively good oxidative stability, most had better stability at 2 versus 0.5% protein. Protease M hydrolysate + Procream microcapsules had the highest stability, regardless of protein content. Results demonstrated that Procream, at a reduced protein inclusion level, can partially replace more expensive whey protein ingredients in microencapsulation, when blended with a select hydrolysate.

A proteomics approach to characterizing limited hydrolysis of whey protein concentrate.

Whey Protein Concentrate was hydrolyzed by trypsin and Protease M, a novel endo/exopeptidase mix from Aspergillus oryzae. Protein peptide profiling demonstrated that Protease M preferentially hydrolyzedα-lactalbumin (α-la), while trypsin targeted ß-lactoglobulin (ß-lg). Peptide fractions were analyzed by liquid chromatography coupled with tandem mass-spectrometry to characterize differences in enzyme specificity, peptide hydrophobicity, and bioactivity, using bioinformatics tools. While trypsin cleaved at the C-terminal end of lysine and arginine, Protease M contributed to pepsin-like endopeptidase activity coupled with carboxyl, amino, and leucine exopeptidase activity, resulting in relatively more hydrophilic peptides compared to those released by trypsin hydrolysis. While trypsin and Protease M had varying specificity, 9 bioactive peptides were common among the hydrolysates, which was attributed to the exopeptidase activity of Protease M. The proteomics coupled with bioinformatics approach provided fundamental knowledge needed to optimize whey protein hydrolysis in a direct and efficient manner for targeted applications.

Changes in protein structural characteristics upon processing of gluten-free millet pasta.

Proso millet exhibits favorable agronomic and nutritional properties but is currently under-utilized in the northern hemisphere. This study compared processing-induced changes in protein characteristics of commercial pasta to fresh gluten-free pasta from proso millet varieties differing in prolamin profile. Protein solubility, accessible thiols and secondary structures were measured in dough, sheeted and cooked pasta. Relationships between protein conformation and characteristics related to pasta quality were determined. Cooking significantly lowered protein solubility and induced exposure of thiol groups as well as a shift in secondary structure distribution, while sheeting only had a minor effect. Random structures positively and significantly (P < 0.05) correlated with solubility, cooking loss and protein digestibility. In contrast, ß-sheets, the main secondary structure in cooked pasta, negatively correlated with these properties. The utilization of proso millet in gluten-free pasta is promising, however, processing optimization to elicit targeted protein modifications to balance quality and nutritional attributes requires further investigation.

Physicochemical, melissopalynological and antioxidant properties of artisanal honeys from Lebanon.

Sixteen honeydew and 15 floral honeys from Lebanon were analyzed for pollen spectra and physicochemical parameters. A total of 37 families and 67 taxa were recorded with the honeybees producing honeydew honey exhibiting a more diverse foraging behavior than those making floral honeys. The honeydew and floral honeys exhibited differences in moisture content, pH, electrical conductivity, color, protein and Maillard reaction products. The honeydew honeys contained more total phenols, had higher antioxidant contents, and displayed higher antioxidant capacities than the floral samples in the Trolox equivalent antioxidant capacity, oxygen radical absorbance capacity, inhibition of superoxide dismutase activity and protection of red blood cells against hemolysis assays. The honey samples exhibited higher antioxidant capacities, in the aforementioned assays, than their corresponding methanol-extractable phenol fractions although the differences did not reach statistical significance in the floral samples. The relative antioxidant capacity indices which integrate measures of antioxidant capacity from the different assays of the honey samples and their corresponding extracts exhibited similar patterns (r = 0.9774, 0.9937) thereby indicating that the antioxidative behavior of the entire honeys is mirrored by their methanol-extractable phenolic fractions.

Limited hydrolysis combined with controlled Maillard-induced glycation does not reduce immunoreactivity of soy protein for all sera tested.

Combining proteolysis and Maillard-induced glycation was investigated to reduce the immunoreactivity of soy protein. Soy protein was hydrolyzed by Alcalase following response surface methodology utilizing three variables, temperature, time, and enzyme:substrate ratio, with the degree of hydrolysis (DH) and percent reduction in immunoreactivity as response variables. Western blots and ELISA were used to evaluate immunoreactivity using human sera. Data were fitted to appropriate models and prediction equations were generated to determine optimal hydrolysis conditions. The hydrolysate produced under optimized conditions was subjected to glycation with dextran. Hydrolysate produced under optimal conditions had 7.8% DH and a percent reduction in immunoreactivity ranging from 20% to 52%, depending on the sera used. Upon glycation, immunoreactivity was further reduced only when using serum that had the highest soy-specific IgE. This work revealed limitations and provided premises for future studies intended to prove the potency of the combined modification approach to produce a hypoallergenic protein ingredient.

Perennial Grain and Oilseed Crops.

Historically, agroecosystems have been designed to produce food. Modern societies now demand more from food systems-not only food, fuel, and fiber, but also a variety of ecosystem services. And although today's farming practices are producing unprecedented yields, they are also contributing to ecosystem problems such as soil erosion, greenhouse gas emissions, and water pollution. This review highlights the potential benefits of perennial grains and oilseeds and discusses recent progress in their development. Because of perennials' extended growing season and deep root systems, they may require less fertilizer, help prevent runoff, and be more drought tolerant than annuals. Their production is expected to reduce tillage, which could positively affect biodiversity. End-use possibilities involve food, feed, fuel, and nonfood bioproducts. Fostering multidisciplinary collaborations will be essential for the successful integration of perennials into commercial cropping and food-processing systems.

Structural characterization of proteins in wheat flour doughs enriched with intermediate wheatgrass (Thinopyrum intermedium) flour.

The high protein and fiber content of intermediate wheatgrass (IWG) - together with its interesting agronomic traits and environment-related benefits - make this perennial crop attractive also for human consumption. Structural characteristics of the proteins in IWG/hard wheat flour (HWF) doughs (at IWG:HWF ratios of 0:100, 50:50, 75:25 and 100:0) - including aggregate formation, thiols availability, and secondary structure changes during dough mixing - were investigated. Proteins in IWG-doughs had higher solubility and thiol content - as function of IWG content - suggesting that protein network was mostly based on non-covalent interactions. While 50% IWG-enrichment gave an increase in random structures, enrichment at ⩾75% resulted in a decrease in ß-sheets with an increase in random structures, indicating a decrease in structural order. The observed differences in protein molecular configuration and interactions in HWF compared to IWG doughs necessitate further investigation to establish their impact on the quality of IWG-enriched bread.

Malonylglucoside conjugates of isoflavones are much less bioavailable compared with unconjugated ß-glucosidic forms in rats.

Despite considerable interest in the physiologic effects of isoflavones, the in vivo bioavailability of the most common isoflavone forms, malonylglucoside conjugates, has not been determined. Differences in the bioavailability of malonylglucosides compared with the nonconjugated ß-glucoside forms may explain the inconsistent findings regarding the physiologic effects of isoflavones. Therefore, our objective was to determine the effect of malonyl- conjugation on isoflavone bioavailability in an animal model. Malonylgenistin and malonyldaidzin, and their corresponding nonconjugated glucosides, were extracted from soy grits and purified using liquid chromatography. Purity of the isolated forms was confirmed by nuclear magnetic resonance analysis. Male rats were gavaged with malonylgenistin, genistin, malonyldaidzin, or daidzin at a dose of 100 µmol/kg body weight. Blood and urine samples were collected at time intervals ranging from 0 to 48 h. Isoflavone metabolites in plasma and urine were determined using stable isotope dilution-liquid chromatography/mass spectrometry. Comparisons of pharmacokinetic variables were made between nonconjugated and conjugated glucosides and over time of plasma collection. The areas under the time-concentration curve of the metabolites in the plasma obtained after the administration of nonconjugated ß-glucosides were 1 to 6 times higher than those of their respective malonylglucosides (P ≤ 0.05). Additionally, maximum plasma concentration and urinary excretion of isoflavone metabolites were significantly higher (1-9 times; P ≤ 0.05) after the administration of nonconjugated ß-glucosides. To our knowledge, these results demonstrated, for the first time, that nonconjugated ß-glucosides are relatively more bioavailable than their respective malonylglucosides. These differences in the bioavailability of conjugated and nonconjugated ß-glucosides should be considered in future studies focused on the bioactivity of isoflavones.

Development of a simple, fast, and accurate method for the direct quantification of selective estrogen receptor modulators using stable isotope dilution mass spectrometry.

A rapid analytical procedure was developed to quantify major selective estrogen receptor modulators (SERMs) simultaneously using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein, were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The SIL analogues, which were mono- and dideutero substituted at the ortho positions, exhibited minimal deuterium isotope effects and were stable under the employed sample preparation protocol and MS analysis. An isotopic overlap correction was successfully employed to improve the accuracy and precision of the analytical method. The developed method, which was found to be sensitive, selective, precise and accurate, could be a valuable tool for research focused on determining the bioavailability of individual SERMs.

Structural characterisation of partially glycosylated whey protein as influenced by pH and heat using surface-enhanced Raman spectroscopy.

Maillard-induced glycosylation of whey protein improves solubility and thermal stability over a wide pH range. However, the relationship between structural changes and functional enhancement upon glycosylation is not well-characterized. Therefore, our objective was to characterise these structural changes and determine the protein conformation at various pH and thermal treatments, using surface-enhanced Raman-spectroscopy. The spectra of glycosylated protein revealed a new peak at 983 cm(-1) that can be used as a Raman marker for the early stage glycosylation. Upon glycosylation, structural variations were significant at the disulfide, hydrophobic, amide III, amide II, and amide I regions. Ionisation of carboxyl groups at all tested pH values, and increased ß-sheet configuration were also observed. The noted structural modifications imparted molecular rigidity and a consequent resistance to denaturation upon thermal treatment over a wide pH range. These findings can be used to explain various functional enhancements of whey protein upon glycosylation.

Angiotensin converting enzyme inhibitory activity of soy protein subjected to selective hydrolysis and thermal processing.

Soy protein isolate (SPI) and ß-conglycinin- and glycinin-rich fractions were hydrolyzed using papain and pepsin. Protein denaturation, profiling, and peptide identification were carried out following DSC, SDS-PAGE, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The in vitro antihypertensive activity of the hydrolysates was compared by determining the angiotensin converting enzyme (ACE) inhibitory activity. SDS-PAGE and LC-MS/MS analysis confirmed pepsin selectivity to glycinin and papain partial selectivity to ß-conglycinin when the protein is least denatured. Both the papain-hydrolyzed SPI and the papain-hydrolyzed ß-conglycinin-rich fraction had more than double the ACE inhibitory activity of that of pepsin-hydrolyzed SPI and pepsin-hydrolyzed glycinin-rich fraction. This observation indicated that ß-conglycinin is a better precursor for antihypertensive peptides than glycinin. Additionally, the inhibitory activity of the papain-hydrolyzed SPI was thermally stable. This work demonstrated, for the first time, that selective hydrolysis to release peptides with ACE inhibitory activity can be accomplished without inducing extensive hydrolysis and performing unnecessary fractionation.

The role of salivary proteins in the mechanism of astringency.

Understanding astringency has focused on the interaction of tannins with the salivary proline-rich proteins (PRPs), although it remains unclear if other astringents precipitate the PRPs or how this interaction relates to sensory perceptions of astringency. We used 2 approaches to compare how distinct classes of astringent compounds interacted with the salivary PRPs and mucins. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, we evaluated protein patterns and characterized the salivary proteins present in the supernatants and pellets of pooled saliva assayed with tannin, alum, and hydrochloric acid solutions. Tannins and alum precipitated many of the PRPs, but acid did not. Mucins were precipitated by both the acid and alum, but not by the tannins. From our research, it appears that the precipitation of salivary proteins may be involved in the mechanism of astringency, but the precipitation of PRPs is not requisite for the development of astringency. We also measured mucin and deoxyribonucleic acid content of expectorated solutions of astringents that panelists swished in their mouths to determine if astringency was associated with a loss of oral lubricating films.

Detection and structural characterization of thermally generated isoflavone malonylglucoside derivatives.

Malonylglucoside isomers were identified by high-performance liquid chromatography with ultraviolet/mass spectrometric detection and nuclear magnetic resonance (NMR). The formation and interconversions of the isomers were monitored in heated buffer and soymilk systems. Two positional or steroisomers of malonylgenistin and malonydaidzin, showing similar UV spectra and molecular weights yet different fragmentation patterns, were detected. NMR characterization of the malonylgenistin isomer revealed its structure to be 4''-O-malonylgenistin, suggesting a malonyl migration from the glucose-6 position to the glucose-4 position. Interconversions of malonylgenistin and its isomer were observed in both buffered and soymilk systems. While malonylgenistin partially converted to the isomer upon thermal treatment, conversion of the isomer to malonylgenistin was more spontaneous. The malonylgenistin isomer represented 6-9% of the total calculated genistein content in soymilk heated at 100 °C for various periods of time. Consequently, disregarding the content of malonylglucoside isomers in processed soy matrices can lead to isoflavone underestimation and misinterpretation of the biological contributions.