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.

Effect of extrusion temperature on the structure and emulsifying properties of soy protein isolate-oat ß-glucan conjugates formed during high moisture extrusion.

In this study, extrusion was used to induce Maillard reaction between soy protein isolate (SPI) and oat ß-glucan (OG) and effect of extrusion temperature (70, 90, 110 and 130 °C) on the structure and emulsifying properties of extruded SPI-OG was investigated. SDS-PAGE and fluorescence spectroscopy provided evidence for the formation of SPI-OG conjugates during extrusion. The results showed that 90 °C and 110 °C extruded SPI-OG had the highest level of degree of glycosylation (were 14.34% and 13.70%, respectively, p > 0.05). Structural analysis found that α-helix content of extruded SPI-OG decreased by 8.93-13.14% compared to mixture of SPI and OG. Meanwhile, extruded SPI-OG had lower protein solubility (29.83-34.38%) and surface hydrophobicity (1549-2027), larger average particle size (2363-4807 nm) and higher emulsion stability (74.33-90.15%). Therefore, these findings may provide a theoretical basis for the development of novel food emulsion stabilizers.

Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus.

TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored.

Soybean protein hydrolysate stimulated cholecystokinin secretion and inhibited feed intake through calcium-sensing receptors and intracellular calcium signalling in pigs.

Although soybean protein is the major component in livestock feeds, its effect on pigs' appetites is largely unknown. Recently, the importance of gut nutrient-sensing for appetite modulation by regulating anorectic gut hormone release has been recognised. This study investigates the roles of soybean proteins in appetite regulation, anorectic gut hormone secretion, and underlying mechanisms. The duodenal-cannulated piglets were used to evaluate the effects of soybean protein hydrolysate (SPH) on feed intake and anorectic hormone release, including cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) in the hepatic vein by infusing SPH. Identifying which nutrient-sensing receptor in pig duodenum response to SPH stimulation for gut hormone release was conducted. Using its antagonist, the role of the identified receptor in feed intake and anorectic hormone release was also investigated. Combination with an ex vivo perfusion system, the possible mechanism by which SPH exerts the effects in porcine duodenum was further illustrated. Results in vivo showed that intraduodenal infusion of SPH inhibited short-term feed intake in pigs and promoted CCK, PYY, and GIP secretion in the hepatic vein. SPH also increased duodenum calcium-sensing receptor (CaSR) expression. Pre-treated with CaSR antagonist NPS 2143, the feed intake of pigs tended to be attenuated by SPH (P = 0.09), and CCK release was also suppressed (P < 0.05), indicating that CaSR was involved in SPH-stimulated CCK release and inhibited feed intake in pigs. The ex vivo perfused duodenum tissues revealed that SPH-triggered CCK secretion was likeliest due to the activation of the intracellular Ca2+/TRPM5 pathway. Overall, this study's result illustrates that the diet soybean protein might decrease appetite in pigs by triggering duodenum CCK secretion by activating CaSR and the intracellular Ca2+/TRPM5 pathway.

Utilization of tofu processing wastewater as a source of the bioactive peptide lunasin.

The goal of our study was to design a simple and feasible method to obtain lunasin, a naturally-occurring bioactive peptide, from tofu whey wastewater. A combination of alcoholic precipitation of high-molecular weight proteins from the whey, isoelectric precipitation of lunasin enriched material, and purification via gel filtration chromatography was selected as the best approach using tofu whey prepared at the laboratory scale. This process was applied to tofu whey produced by a local tofu factory and 773 mg of 80% purity lunasin was obtained per kg of dry tofu whey. Significant reduction of nitric oxide, and pro-inflammatory cytokines TNF-α and IL-6 over lipopolysaccharide activated murine macrophages demonstrate its biological activity. Our three-step process is not only simpler and faster than the previously reported methods to obtain lunasin but provides a sustainable approach for the valorization of a waste product, promoting the better utilization of soybean nutrients and active compounds.

Separation of Storage Proteins (7S and 11S) from Soybean Seed, Meals and Protein Isolate Using an Optimized Method Via Comparison of Yield and Purity.

The primary purpose of this study was to extract ß-conglycinin (7S) and glycinin (11S) from soybean seed, soybean meals and soybean protein isolate and compare their yield and purity. The previous methods were modified for the extraction and isolation of 7S and 11S globulins. The adjustment mainly included sample to solution ratio of 1:10 (previously 1:15). Comparing the yield of 11S fraction in Tris-HCl and water as extractable solutions, it was almost doubled in soybean seed (16.97% to 32.41%) with purity from 96 to 98% respectively. In case of soybean meal, samples yield increased from 45.46 to 61.86% with purity from 94 to 98%. On contrary, 7S yield was significantly improved in soybean protein isolate sample from 30.33 to 53.81% along with no contamination in its purity while soybean seed and soybean meal samples had less increase in both yield and purity in Tris-HCl and water as extractable solutions. Results of this study will bring new insights into soybean 7S and 11S separation and purification techniques as well as pave the way for their application in food industry.

Soybean lectin induces autophagy through P2RX7 dependent activation of NF-κB-ROS pathway to kill intracellular mycobacteria.

BACKGROUND: Host-directed therapy is considered a novel anti-tuberculosis strategy in tackling the tuberculosis burden through autophagy induction by various inducers to curtail the growth of intracellular Mycobacterium tuberculosis. METHODS: In this study, we investigated the anti-tubercular role of soybean lectin, a lectin isolated from Glycine max (Soybean). Effect of SBL on intracellular mycobacterial viability through autophagy and the mechanism involved in differentiated THP-1 cells was studied using different experimental approaches. RESULTS: We initially performed a time kinetic experiment with the non-cytotoxic dose of SBL (20 µg/ml) and observed autophagy induction after 24 h of treatment. Abrogation of autophagy in the presence of 3-MA and an increase in LC3 puncta formation upon Baf-A1 addition elucidated the specific effect on autophagy and autophagic flux. SBL treatment also led to autophagy induction in mycobacteria infected macrophages that restricted the intracellular mycobacterial growth, thus emphasizing the host defensive role of SBL induced autophagy. Mechanistic studies revealed an increase in P2RX7 expression, NF-κB activation and reactive oxygen species generation upon SBL treatment. Inhibition of P2RX7 expression suppressed NF-κB dependent ROS level in SBL treated cells. Moreover, SBL induced autophagy was abrogated in the presence of either different inhibitors or P2RX7 siRNA, leading to the reduced killing of intracellular mycobacteria. CONCLUSION: Taken together, these results conclude that SBL induced autophagy exerts an anti-mycobacterial effect in P2RX7-NF-κB dependent manner through the generation of ROS. GENERAL SIGNIFICANCE: This study has provided a novel anti-mycobacterial role of SBL, which may play an important role in devising new therapeutic interventions.

Proteolytic activity of selected commercial Lactobacillus helveticus strains on soy protein isolates.

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.

Distribution and degradation of DNA from non-genetically and genetically modified soybean (Roundup Ready): Impact of soybean protein concentrate and soybean protein isolate preparation.

To improve genetically modified product labelling legislation and promote the development of genetically modified foods, the mass variations of genomic DNA and length distributions of DNA fragments in non-genetically and genetically modified soybean (Roundup Ready) and the variations in transgenic contents during soybean protein concentrate (SPC) and soybean protein isolate (SPI) preparation were monitored. The material masses throughout the process conformed to the law of mass conservation, and amounts of DNA recovered decreased with SPC and SPI preparation. The successive steps of ethanol extraction led to a decrease in the size distribution of the recovered DNA. For the LECTIN and CP4 EPSPS targets investigated, longer fragments (>800 bp) were more affected than smaller fragments (<200 bp), and both targets degraded slowly upon degradation into small fragments. DNA distribution and degradation thereby affected GMO quantification. After preparation procedures, the transgenic contents of SPC and SPI products were higher than that of raw soybean.

A biodegradable soy protein isolate-based waterborne polyurethane composite sponge for implantable tissue engineering.

A biodegradable soy protein isolate-based waterborne polyurethane composite sponge (SWPU) was prepared from soy protein isolate (SPI) and polyurethane prepolymer (PUP) by a process involving chemical reaction and freeze-drying. Effects of SPI content (0, 10%, 30%, 50%, 70%) on the micro-structure and physical properties of the composite sponges were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The results showed that the reaction between -NCO of PUP and -NH2 of SPI formed porous SPI-based WPU composite sponges. The results of the water absorption ratio measurement, solvent resistance measurement and compressive testing showed that water absorption, hydrophilicity, and tensile strength in the dry state of the composite sponges increased with the increase of SPI content. Especially, the tensile strength ranged from 0.3 MPa to 5.5 MPa with the increase in SPI content. The cytocompatibility and biodegradability of the composite sponges were evaluated by in vitro cell culture and in vivo implantation experiments. The results indicated that a certain SPI content in the sponges could promote the adhesion, growth, and proliferation of cells, enhance the cytocompatibility and accelerate the degradation speed of composite sponges. During the in vivo implanting period within 9 months, SWPU-50 sponge containing 50% of SPI brought out the lowest activated inflammatory reaction, most newly-regenerated blood capillaries, and best histocompatibility. All results indicated that SWPU-50 composite sponges had greatest potential for tissue engineering.

Enhanced separation and analysis of low abundant soy proteins by dual washing extraction process.

Soybean seeds provide a rich source of proteins, fats, carbohydrates, and micronutrients. Extraction and analysis of low abundant soybean seed proteins are challenging because of its complex seed composition. For characterizing various proteins, it is paramount to remove the other interfering components, primarily oils, and carbohydrates. In the present study, we used a sequential dual washing process initially with hexane to remove oil and non-polar interferences, followed by 80% ethanol washing to remove about 60% of the total soluble sugars. The extracted soluble sugars were quantified using a newly developed and validated high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This newly developed combined washings process significantly enhanced the separation of both low molecular weight and low abundant proteins using 1D (one dimensional)- and 2D (two dimensional) gel electrophoresis. The separated proteins were trypsinized and analyzed by using Bruker amazon speed ion trap mass spectrometer equipped with an ESI source. This combined washing process allowed the identification of 18 additional low abundant soy proteins as compared to the simple hexane washed samples. This purification process will allow researchers to identify and investigate the role of low molecular weight and low abundant proteins as it relates to plant functions, nutrition, and health.

Influence of soy and whey protein, gelatin and sodium caseinate on carotenoid bioaccessibility.

Proteins could alter carotenoid bioaccessibility through altering their fate during digestion, due to emulsifying properties of resulting peptides, or influencing access of digestion enzymes to lipid droplets. In this investigation, we studied whether whey protein isolate (WPI), soy protein isolate (SPI), sodium caseinate (SC) and gelatin (GEL), added at various concentrations (expressed as percentage of recommended dietary allowance (RDA): 0, 10, 25 and 50%) would influence the bioaccessibility of lycopene, ß-carotene or lutein, added as pure carotenoids solubilized in oil, during simulated gastro-intestinal (GI) digestion. Protein and lipid digestion as well as selected physico-chemical parameters including surface tension, ζ-potential and micelle size were evaluated. Adding proteins influenced positively the bioaccessibility of ß-carotene, by up to 189% (p < 0.001), but it resulted in generally decreased bioaccessibility of lutein, by up to 50% (p < 0.001), while for lycopene, the presence of proteins did not influence its bioaccessibility, except for a slight increase with WPI, by up to 135% (p < 0.001). However, the effect depended significantly on the type of protein (p < 0.001) and its concentration (p < 0.001). While ß-carotene bioaccessibility was greatly enhanced in the presence of SC, compared to WPI and GEL, the presence of SPI strongly decreased carotenoid bioaccessibility. Neglecting individual carotenoids, higher protein concentration correlated positively with carotenoid bioaccessibility (R = 0.57, p < 0.01), smaller micelle size (R = -0.83, p < 0.01), decreased repulsive forces (ζ-potential, R = -0.72, p < 0.01), and higher surface tension (R = 0.44, p < 0.01). In conclusion, proteins differentially affected carotenoid bioaccessibility during digestion depending on carotenoid and protein species, with both positive and negative interactions occurring.

Structure and functional properties of soy protein isolate-lentinan conjugates obtained in Maillard reaction by slit divergent ultrasonic assisted wet heating and the stability of oil-in-water emulsions.

Effects of a novel slit divergent ultrasound (SDU) treatment on soybean protein isolate (SPI)-lentinan conjugates via Maillard reaction was investigated. Besides, the stability of emulsions prepared by SPI and SPI-lentinan conjugates (ultrasound and un-ultrasound) as emulsifiers was compared. The results showed that ultrasonic treatment (40 min) markedly increased the degree of grafting (26.48%) by 1.91 times comparing with traditional heating method (2 h, 13.89%). In addition, structural analysis showed that the conjugates obtained by SDU treatment changed the secondary structure and had higher surface hydrophobicity and fluorescence intensity than those obtained by traditional heating method. Apart from this, SDU treatment could significantly improve the functional properties (solubility, foaming, emulsifying capacity, thermal stability, and viscosity) of conjugates. Furthermore, the emulsions prepared by the SPI-lentinan conjugates (ultrasound) as emulsifiers possessed the highest stability against environmental stresses. Taken together, SDU-assisted heating could be an excellent method to improve the functional properties of conjugates.

Physicochemical and structural properties of lunasin revealed by spectroscopic, chromatographic and molecular dynamics approaches.

Lunasin is a 43-amino acid peptide from seeds and grains with bioavailability in humans and potent chemotherapeutic action against several cancer cell lines. Here, we investigate new information about the physicochemical and structural properties of lunasin using circular dichroism (CD), fluorescence spectroscopy, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), size exclusion chromatography (SEC), molecular dynamics (MD), and bioinformatics. CD analysis and disorder prediction obtained by PONDR indicate that lunasin has a mostly unordered structure. Double wavelength [θ]222nm x [θ]200nm plot data suggests that lunasin is an intrinsically disordered peptide (IDP) in a pre-molten globule-like (PMG-like) state, while CD spectrum deconvolution and MD simulation indicate small ß-strand content. The presence of residual structure was supported by loss of CD signal at 222 nm after treatment with urea and by increasing fluorescence emission upon bis-ANS binding. Lunasin also demonstrated stability to heating up to the temperature of 100 °C, as verified by CD. MD and CD analyses in the presence of TFE and MoRFpred prediction indicated the helix propensity of lunasin. ESI-IMS-MS data revealed that lunasin shows a propensity to form disulfide bonds at the conditions used. MD data also indicated that disulfide bond formation affects the adopted structure, showing a possible role of aspartyl-end in structure stabilization and compaction. In conclusion, our data support a characterization of lunasin as a peptide with an intrinsic disorder in a PMG-like state and reveal new aspects about its structural stability and plasticity, as well as the effects of disulfide bond formation and electrostatic attractions.

Soy ß-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

We previously reported that soy ß-conglycinin (ßCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of ßCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared ßCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by ßCG peptide (ßCG peptide group). Consequently, rats in the ßCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that ßCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in ßCG peptide-fed OLETF rats. These findings suggest that peptidization of ßCG enhanced the anti-obese and hypolipidemic effects of ßCG.

Mass spectrometry combined with affinity probes for the identification of CP4 EPSPS in genetically modified soybeans.

Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low-abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5-enolpyruvylshikimate-3-phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography-multiple reaction monitoring tandem MS (nano LC-MS/MS-MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)-immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine-containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A-sepharose was used for the affinity capture of ß-conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable-isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time-consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.

Effects of native starch and modified starches on the textural, rheological and microstructural characteristics of soybean protein gel.

The effects of native starch (NS), acetylated starch (AS), and acetylated distarch phosphate (ADSP) on the gel properties of soybean protein thermal gel were investigated using texture analysis, low-field nuclear magnetic resonance (LF-NMR) spectroscopy, dynamic rheometry and scanning electron microscopy. The results of the textural profile analysis showed that 10% ADSP increased the hardness and chewiness of the mixed gel, while NS and AS led to decreases in the textural properties. The results of the LF-NMR analysis indicated that the AS improved the water-holding capacity of the mixed gel due to the transformation of weakly bound water to strongly bound water. During heating and cooling, the rheological profiles of the elastic (G') and viscous modulus (G″) of all the samples exhibited a two-stage pattern of decrease and then increase, and the final values of G' and G″ reached maxima when the ADSP content was 10%. The scanning electron microscopy images showed that the ADSP granules dispersed in the gel network. The integrity of the starch granules was crucial for regulating the properties of the soybean protein gel. These results provided information about the further design and preparation of soybean protein foods containing modified starch.

Improvement of the rheological and textural properties of calcium sulfate-induced soy protein isolate gels by the incorporation of different polysaccharides.

In this study, the effects of the addition of various polysaccharides (konjac gum, gellan gum, and curdlan gum) on the rheological and textural properties of calcium sulfate-induced soy protein isolate gels were investigated. The incorporation of konjac gum and curdlan gum at 0.3 and 0.5% (w/v) concentrations and gellan gum at 0.5% concentration significantly enhanced (P < 0.05) the hardness and water-holding capacity of the resultant gels. The increased elastic moduli during and after gelation, reinforced fracture stress, and lowered onset gelling temperature indicated that the addition of the abovementioned polysaccharides strengthened gel structures and accelerated gelation. Confocal laser scanning microscopy analysis revealed that the polysaccharides also improved gel microstructures, with the gels containing konjac gum displaying the highest homogeneity. The findings of this study may provide important information for the development of innovative soy protein isolate-based gel products with improved texture.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20 nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

Binding of aromatic compounds with soy protein isolate in an aqueous model: Effect of pH.

Interactions of the flavoring compounds hexyl acetate (HxAc), heptyl acetate (HpAc), linalyl formate (LiFo), linalyl acetate (LiAc), geraniol, linalool, limonene, and myrcene with soy protein isolate (SPI) were estimated in pH 3.0, 6.0, and 9.0 aqueous solutions using headspace solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS). The binding capacity of HxAc, HpAc, LiFo, LiAc, geraniol, and linalool increased in the pH of the medium from 3 to 9. For limonene and myrcene, an unexpected increase in headspace concentration or a "salting-out" effect was observed. Between pH 3 and 9, better accessibility to the primary hydrophobic sites as a result of a modification to the protein's flexibility was observed. PRACTICAL APPLICATIONS: SPME method is a technology of dynamic adsorption for flavors. The lowest level of lead be practicably detected in food as low as the practiced concentration of flavors (0.01-0.1 mM) in our study. At low concentrations of flavors, it is close to the actual flavor's concentration of food. In the previous studies, the technology, such as equilibrium dialysis, headspace-gas phase which need higher concentration of flavors (>0.2 mM). The interaction between flavors and protein has a different binding law at high and low concentrations. As we produced the acid fruit soy protein milk beverage, the off-flavors present in the beverage were due to the change in the interaction between denature SPI and flavors. The present work is aimed at paving the way for further research to elucidate flavor imbalances in acid fruit soy protein milk beverage.