Effects of zein extractions on the structural properties of SPI-zein composite gels: Implications for gluten-free plant-based products.

The growing global interest in physical and environmental health has led to the development of plant-based products. Although soy protein and wheat gluten are commonly utilized, concerns regarding gluten-related health issues have driven exploration into alternative proteins. Zein has emerged as a promising option. This research investigated the impact of extraction methods on zein characteristics and the structures of SPI-zein composite gels. Different extraction methods yielded zein with protein contents ranging from 48.12 % to 64.34 %. Ethanol-extracted Z1 and Z3, obtained at different pH conditions, exhibited zeta potential of -3.25 and 5.43 mV, respectively. They displayed similar characteristics to commercial zein and interacted comparably in composite gels. Conversely, alkaline-extracted Z2 had a zeta potential of -2.37 mV and formed distinct gels when combined with SPI. These results indicated that extraction methods influence zein behaviour in composite gels, offering possibilities for tailored formulations and expanding zein's applications, particularly in gluten-free plant-based products.

Insight into the effect of gluten-starch ratio on the properties of Chinese steamed bread (Mantou).

Chinese steamed bread (CSB) is one of the traditional staple foods of Chinese people, and its quality is mainly affected by wheat gluten and starch. Herein, four different ratios of wheat gluten and starch were selected to investigate its effects on the properties of CSB. It was observed that the surface of CSB gradually became darker, yellower, and shrank with increasing gluten-starch ratio. The hardness and chewiness of CSB decreased with the increasing of gluten-starch ratio, as well as the network structure of CSB was dense and porous. The increase of gluten content could effectively control the migration of water in the CSB. Moreover, with increasing gluten-starch ratio, the crystallinity of starch was reduced from 9.95% to 2.03%. As a result, the ratio of gluten-starch mainly affected the development of gluten network structure and starch gelatinization through the competition of water between gluten and starch in the system, which in turn affected the quality of CSB. Thus, it will provide the basis for the adaptability of wheat flour from different origins as the raw material of CSB processing, and also provide guidance for consumers to select flour with different gluten protein content to prepare steamed bread according to their preferences.

Mass spectrometry of in-gel digests reveals differences in amino acid sequences of high-molecular-weight glutenin subunits in spelt and emmer compared to common wheat.

High-molecular-weight glutenin subunits (HMW-GS) play an important role for the baking quality of wheat. The ancient wheats emmer and spelt differ in their HMW-GS pattern compared to modern common wheat and this might be one reason for their comparatively poor baking quality. The aim of this study was to elucidate similarities and differences in the amino acid sequences of two 1Bx HMW-GS of common wheat, spelt and emmer. First, the sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) system was optimized to separate common wheat, spelt and emmer Bx6 and Bx7 from other HMW-GS (e.g., 1Ax and 1By) in high concentrations. The in-gel digests of the Bx6 and Bx7 bands were analyzed by untargeted LC-MS/MS experiments revealing different UniProtKB accessions in spelt and emmer compared to common wheat. The HMW-GS Bx6 and Bx7, respectively, of emmer and spelt showed differences in the amino acid sequences compared to those of common wheat. The identities of the peptide variations were confirmed by targeted LC-MS/MS. These peptides can be used to differentiate between Bx6 and Bx7 of spelt and emmer and Bx6 and Bx7 of common wheat. The findings should help to increase the reliability and curation status of wheat protein databases and to understand the effects of protein structure on the functional properties. Graphical abstract.

Effects of Ultrasonic-Assisted Extraction on the Physicochemical Properties of Different Walnut Proteins.

The effects of ultrasonic-assisted extraction (UAE, 200 W, 20 min) on the yield and physicochemical properties of different walnut proteins (WNPs, including albumin, globulin, and glutelin) were investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that UAE could result in protein molecular fragmentation of albumin, but did not affect the major bands of globulin and glutelin. The CD spectra demonstrated that different WNPs obtained by UAE had different changes in their secondary structure. Under UAE, there was an increase in surface hydrophobicity (H0) of albumin and gluten and no change in the fluorescence intensity, while decreases were observed in the H0 and fluorescence intensity of globulin; and the contents of total and surface free sulfhydryl in albumin dramatically decreased. UAE reduced the size of the particles and the dimension of the microstructures in albumin and gluten, indicating that ultrasound could unfold protein aggregates. In addition, UAE increased the solubility, emulsifying activity (EA), foaming capacity (FC), and foam stability (FS) of the obtained proteins. The above results indicate that ultrasound extraction is a promising approach to improve the extraction yield and properties of walnut proteins.

Assessing the Utility of Multiplexed Liquid Chromatography-Mass Spectrometry for Gluten Detection in Australian Breakfast Food Products.

Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten that is associated with gastrointestinal issues, including diarrhea, abdominal pain, and malabsorption. Gluten is a general name for a class of cereal storage proteins of wheat, barley, and rye that are notably resistant to gastrointestinal digestion. After ingestion, immunogenic peptides are subsequently recognized by T cells in the gastrointestinal tract. The only treatment for CD is a life-long gluten-free diet. As such, it is critical to detect gluten in diverse food types, including those where one would not expect to find gluten. The utility of liquid chromatography-mass spectrometry (LC-MS) using cereal-specific peptide markers to detect gluten in heavily processed food types was assessed. A range of breakfast products, including breakfast cereals, breakfast bars, milk-based breakfast drinks, powdered drinks, and a savory spread, were tested. No gluten was detected by LC-MS in the food products labeled gluten-free, yet enzyme-linked immunosorbent assay (ELISA) measurement revealed inconsistencies in barley-containing products. In products containing wheat, rye, barley, and oats as labeled ingredients, gluten proteins were readily detected using discovery proteomics. Panels comprising ten cereal-specific peptide markers were analyzed by targeted proteomics, providing evidence that LC-MS could detect and differentiate gluten in complex matrices, including baked goods and milk-based products.

Catcher of the Rye: Detection of Rye, a Gluten-Containing Grain, by LC-MS/MS.

Rye, wheat, and barley contain gluten, proteins that trigger immune-mediated inflammation of the small intestine in people with celiac disease (CD). The only treatment for CD is a lifelong gluten-free diet. To be classified as gluten-free by the World Health Organization the gluten content must be below 20 mg/kg, but Australia has a more rigorous standard of no detectable gluten and not made from wheat, barley, rye, or oats. The purpose of this study was to devise an LC-MS/MS method to detect rye in food. An MS-based assay could overcome some of the limitations of immunoassays, wherein antibodies often show cross-reactivity and lack specificity due to the diversity of gluten proteins in commercial food and the homology between rye and wheat gluten isoforms. Comprehensive proteomic analysis of 20 rye cultivars originating from 12 countries enabled the identification of a panel of candidate rye-specific peptide markers. The peptide markers were assessed in 16 cereal and pseudocereal grains, and in 10 breakfast cereals and 7 snack foods. One of two spelt flours assessed was contaminated with rye at a level of 2%, and trace levels of rye were found in a breakfast cereal that should be gluten-free based on its labeled ingredients.

Phase separation dynamics of gluten protein mixtures.

We investigate by time-resolved synchrotron ultra-small X-ray scattering the dynamics of liquid-liquid phase-separation (LLPS) of gluten protein suspensions following a temperature quench. Samples at a fixed concentration (237 mg ml-1) but with different protein compositions are investigated. In our experimental conditions, we show that fluid viscoelastic samples depleted in polymeric glutenin phase-separate following a spinodal decomposition process. We quantitatively probe the late stage coarsening that results from a competition between thermodynamics that speeds up the coarsening rate as the quench depth increases and transport that slows down the rate. For even deeper quenches, the even higher viscoelasticity of the continuous phase leads to a "quasi" arrested phase separation. Anomalous phase-separation dynamics is by contrast measured for a gel sample rich in glutenin, due to elastic constraints. This work illustrates the role of viscoelasticity in the dynamics of LLPS in protein dispersions.

Preparation and characterization of redox-sensitive glutenin nanoparticles.

In recent thirty years, protein-based nanoparticles have attracted considerable attention, and they are being widely used in the food, pharmaceutical, and biomedical fields. Wheat glutenin, an important natural vegetable protein, has been demonstrated to be nutritive and biocompatible. This study aimed to develop a new type of redox-sensitive protein nanoparticles. The glutenin nanoparticles (GNPs) were synthesized with glutenin concentrations (0.082%, 0.5%, and 0.83%) through the adoption of an antisolvent titration technique and the use of hydrogen peroxide (H2O2) oxidative cross-linking for different periods. At a glutenin concentration of 0.83% and oxidation time of 20 h, the obtained GNPs were spherical in shape and approximately 100-300 nm in size, as measured by transmission electron microscopy and dynamic light scattering. The formation of disulfide was confirmed by Raman spectroscopy. The turbidity values of the GNP suspensions were decreased by half after the addition of ß-mercaptoethanol. Nile blue A, a model hydrophilic substance, was entrapped in the GNPs with 77.67% loading efficiency. The newly developed GNPs can be used as redox-responsive carriers for delivering hydrophilic active substances.

Rheological and microstructural characteristics of low molecular weight glutenin subunits of commercial wheats.

A study was conducted on the effects occurred in rheological properties of base flour dough by the addition of gluten, glutenin and purified low molecular weight glutenin subunits (LMW-GS) using a 4 g sample Microdoughlab (MDL). Incorporation of these elements brought about a significant increase in the dough strength in the order of LMW-GS < gluten < glutenin. LMW-GS from variety C 306 brought a decrease in the dough development time (DDT; 2.03 min), dough stability (DS; 3 min) and peak energy (EP; 2.90 Wh/kg) values. On the contrary, the effects of LMW-GS extracted from variety PBW 550 were more strong as indicated by an increase in DDT (2.75 min), DS (3.30 min) and EP (4.20 Wh/kg). The alterations in the microstructure of dough by the inclusion of gluten, glutenin and LMW-GS, which lacks resemblance among different samples, were contemplated subjecting it to Scanning Electron Microscopy (SEM).

A novel angiotensin-I converting enzyme inhibitory peptide derived from the glutelin of vinegar soaked black soybean and its antihypertensive effect in spontaneously hypertensive rats.

Vinegar soaked black soybean is a traditional Chinese food widely used for the treatment of hypertension. While its pharmacodynamic substance was not fully unveiled. It contained abundant glutelin, thus the purpose of this study was to obtain potent antihypertensive peptides from vinegar soaked black soybean. Black soybean was soaked with vinegar and then glutelin was first catalyzed by alcalase. Ultrafiltration, ion exchange chromatography and reversed-phase high performance liquid chromatography were sequentially applied to separate and purify the angiotensin-I converting enzyme (ACE) inhibitory peptides from glutelin hydrolysates. As a result, the fraction L1-4 with the highest ACE inhibitory activity (83.41%) at the final concentration of 0.01 mg/ml was obtained and five peptides were then identified. These peptides were further optimized by virtual screening combining with in silico proteolysis. Finally, a novel tetrapeptide Phe-Gly-Ser-Phe (FGSF) was obtained. FGSF exhibited high in vitro ACE inhibitory activity (IC50 = 117.11 µM) and in vivo hypotensive effect which maximally reduced systolic blood pressure of 21.95 mmHg at 20 mg/kg body weight in spontaneously hypertensive rats. Our study demonstrated that FGSF derived from vinegar soaked black soybean might be used as a promising ingredient for pharmaceuticals against hypertension and its related diseases.

Efficient Extraction and Digestion of Gluten Proteins.

Coeliac disease (CD) is a T-cell mediated autoimmune disorder triggered by ingestion of cereal gluten found in wheat (gliadins and glutenins), barley (hordeins), and rye (secalins). As the only treatment for CD is a lifelong gluten-free diet, the measurement of gluten in raw ingredients and processed food products is critical to protecting people with CD or gluten intolerance. The most commonly employed method is the enzyme-linked immunosorbent assay (ELISA), but more recently mass spectrometry has been employed wherein the extracted gluten proteins are digested to peptides that are then directly measured. To achieve the goal of accurate gluten quantitation, gluten must be efficiently extracted from the ingredient or food matrix and then digested to yield the peptides that are monitored by LC-MS. In this chapter, a rapid, simple, and reproducible protocol for extraction and digestion of gluten proteins is described.

Report- Immunological study of different fraction of wheat proteins.

Wheat allergy specifically refers to the adverse reaction involving IgE antibody to one or more protein fraction of wheat such as albumin, globulin, gliadin and glutenin (gluten). The majority of IgE-mediated reactions to wheat involve albumin and globulin fraction while gluten (gliadin & glutenin) also cause allergy (Celiac disease). Allergic reactions to wheat may be caused by ingestion of wheat containing foods or inhalation of flour (Bakers asthma). The present study was an effort to explore the antibody response of different proteins present in wheat. ELISA results revealed that the antibody response for albumin varied from 0.92-1.78, whereas, for globulin ranged from 1.39-1.60. Antibody response against glutenin and gliadin ranged from 0.57-1.05 and 0.98-1.95 respectively, among the different varieties of wheat. All the tested wheat varieties showed the significant difference antibody response against the different fractions of protein.

Comparison of different extraction methodologies for the analysis of volatile compounds in gluten-free flours and corn starch by GC/QTOF.

Gluten-free bakery products usually exhibit weak aroma. Their main constituents are flours and starches, which contain aroma precursors but can also contribute additional volatiles in low concentrations. Static headspace (SHS), solid-phase microextraction (SPME) and solvent-assisted flavour evaporation (SAFE) coupled to GC/QTOF were compared for their efficacy in the analysis of volatiles in corn starch. SPME-GC/QTOF was selected as the most suitable methodology based on the number of detected compounds, LODs, repeatability as well as simplicity. It was successfully applied for the quantification of volatiles in corn starch and qualitative comparison of different gluten-free flours. Hexanal, 3-methyl-1-butanol, 1-pentanol, 1-octen-3-ol, acetic acid, furfural, benzaldehyde, (E)-2-nonenal, phenylethyl alcohol and short-medium chain acids were found in all the flours and corn starch. Quinoa flour and corn starch showed the highest contents of pyrazines, terpenes and esters, while teff, buckwheat and rice flours presented the highest contents of 3/2-methyl-1-butanol, acetoin and organic acids.

Effect of premilling treatments on wheat gluten extraction and noodle quality.

Wheat cultivars were treated by four different treatments and their flours were obtained after milling. The quality of the obtained wheat flours was determined using various quality parameters and it was observed that premilling treatments significantly affect the gluten content, wet and dry gluten yield, gluten index, and water absorption capacity of gluten, respectively. Acid-treated wheat flours of all the four wheat varieties (C-306, Raj-3765, PBW-343, and KW-11) showed lower gluten content and quality, whereas conditioning and yeast treatment improve gluten quality as well as its content. Flours obtained after premilling treatments were also utilized for noodle preparation and their sensory and quality parameters were also determined. Also, it was observed that yeast treatment and conditioning improved the noodle quality, whereas acid treatment deteriorates the noodle quality in comparison to tempering. The cultivar PBW-343 produced the best quality noodle among four wheat cultivars. In different milling treatments acid-treated flours of all cultivars showed lowest sensory and quality characteristics, while yeast treatment showed highest.

Hydrolysis and transepithelial transport of two corn gluten derived bioactive peptides in human Caco-2 cell monolayers.

The objective of this paper was to investigate the transepithelial transport of two novel corn gluten-derived antioxidant peptides, YFCLT and GLLLPH, using Caco-2 cell monolayers. Results showed that both of YFCLT and GLLLPH could transport in intact form across Caco-2 cell monolayers with apparent permeability coefficient (Papp) values of (1.10±0.16)×10-7cm/s and (1.98±0.23)×10-7cm/s, respectively. However, it was found that the two peptides were susceptible and easily hydrolyzed by brush border membrane peptidases. In the presence of diprotin A, an inhibitor of dipeptidyl peptidase IV (DPPIV), the hydrolysis of YFCLT and GLLLPH decreased and their permeabilities increased significantly compared to control group (P<0.05). The results of transport routes revealed that Gly-Sar, a peptide transporter 1 (PepT1) substrate, had little effects on the transepithelial permeability (P>0.05), suggesting that the transport of YFCLT and GLLLPH across Caco-2 cell monolayers was not mediated by PepT1. However, it was found that cytochalasin d, a tight junctions (TJs) disruptor, increased the permeability significantly (P<0.05). While wortmannin, a transcytosis inhibitor, and sodium azide, an ATP synthesis inhibitor, both decreased the permeability significantly (P<0.05). It indicated that the TJs-mediated paracellular pathway and energy-dependent transcytosis were involved in the transport of YFCLT and GLLLPH across Caco-2 cell monolayers.

Molecular and Functional Properties of Protein Fractions and Isolate from Cashew Nut (Anacardium occidentale L.).

Some molecular and functional properties of albumin (83.6% protein), globulin (95.5% protein), glutelin (81.3% protein) as well as protein isolate (80.7% protein) from cashew nut were investigated. These proteins were subjected to molecular (circular dichroism, gel electrophoresis, scanning electron microscopy) and functional (solubility, emulsification, foaming, water/oil holding capacity) tests. Cashew nut proteins represent an abundant nutrient with well-balanced amino acid composition and could meet the requirements recommended by FAO/WHO. SDS-PAGE pattern indicated cashew nut proteins were mainly composed of a polypeptide with molecular weight (MW) of 53 kDa, which presented two bands with MW of 32 and 21 kDa under reducing conditions. The far-UV CD spectra indicated that cashew proteins were rich in ß-sheets. The surface hydrophobicity of the protein isolate was higher than that of the protein fractions. In pH 7.0, the solubility of protein fractions was above 70%, which was higher than protein isolate at any pH. Glutelin had the highest water/oil holding capacity and foaming properties. Protein isolate displayed better emulsifying properties than protein fractions. In summary, cashew nut kernel proteins have potential as valuable nutrition sources and could be used effectively in the food industry.

Contribution of specific amino acid and secondary structure to the antioxidant property of corn gluten proteins.

The composition, structure, and proper positioning of amino acid in a peptide are closely related to its antioxidant activity. In this study, we purified antioxidant peptides from corn protein hydrolysates (CPH) and identified novel antioxidant peptides from fraction CPH2-III as Ala-Gly-Ile/Leu-Pro-Met (AGI/LPM; 487.62Da) and His-Ala-Ile/Leu-Gly-Ala (HAI/LGA; 467.53Da). AGLPM and HALGA exhibited better oxygen radical absorbance capacities than AGIPM and HAIGA did (P<0.05), as assessed using HepG2 cells with the cellular antioxidant activity assay (CAA) and electron spin resonance (ESR) spectroscopy. Finally, the secondary structure was determined using circular dichroism (CD). ESR showed that the AGLPM and HALGA peptides had the strongest abilities to scavenge hydroxyl radicals, by 79.41±1.41% and 75.16±2.26%, respectively. Thus, corn gluten meal could be used as a potential source of antioxidant peptides for food applications. Additionally, the amino acid Leu compared with Ile may be a critical factor contributing to strong antioxidant activity than the Ile in the peptide sequence (not C-terminus or N-terminus) and CD showed that the lower α-helix and random coil are the main causes.

Improved Method for Reliable HMW-GS Identification by RP-HPLC and SDS-PAGE in Common Wheat Cultivars.

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differences in retention times (RTs) for subunits corresponding to HMW-GS alleles were determined using 16 standard wheat cultivars with known HMW-GS compositions. Subunits that were not identified unambiguously by RP-HPLC were distinguished by SDS-PAGE or inferred from association with linked subunits. The method was used to verify the allelic compositions of 32 Korean wheat cultivars previously determined using SDS-PAGE and to assess the compositions of six new Korean cultivars. Three cultivars contained subunits that were identified incorrectly in the earlier analysis. The improved RP-HPLC method combined with conventional SDS-PAGE provides for accurate, efficient and reliable identification of HMW-GS and will contribute to efforts to improve wheat end-use quality.

Comparison of Gluten Extraction Protocols Assessed by LC-MS/MS Analysis.

The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology (antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple "IPA/DTT" protocol and related "two-step" protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.

Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin.

The present study was carried out to characterize Angiotensin-converting enzyme (ACE) inhibitory peptides which are released from the trypsin hydrolysate of wheat gluten protein. The binding of two inhibitory peptide (P4 and P6) to human serum albumin (HSA) under physiological conditions has been investigated by multi-spectroscopic in combination with molecular modeling techniques. Time-resolved and quenching fluorescence spectroscopies results revealed that the quenching of HSA fluorescence by P4 and P6 in the binary and ternary systems caused HSA-peptides complexes formation. The results indicated that both peptides quenched the fluorescence intensity of HSA through a static mechanism. The binding affinities and number of binding sites were obtained for the HSA-peptides complexes. The circular dichroism (CD) data revealed that the presence of both peptides increased the α-helix content of HSA and induced the remarkable folding of the polypeptide of the protein. Therefore, the CD data determined that the protein structure has been stabilized in the percent of ACE inhibitory peptides in binary and ternary systems. The binding distances between HSA and both peptides were estimated by the Forster theory, and it was revealed that nonradiative energy transfer from HSA to peptides occurred with a high probability. ITC experiments reveal that, in the absence and presence of P6, the dominant forces are electrostatic in binary and ternary systems. Furthermore, molecular modeling studies confirmed the experimental results. Molecular modeling investigation suggested that P4 bound to the site IA and IIA of HSA in binary and ternary systems, respectively. This study on the interaction of peptides with HSA should prove helpful for realizing the distribution and transportation of food compliments and drugs in vivo, elucidating the action mechanism and dynamics of food compliments and drugs at the molecular level. It should moreover be of great use for understanding the pharmacokinetic and pharmacodynamic mechanism of the food compliments and drugs.