Soy Hydrolysate Research Articles

Изучение некоторых функциональных характеристик ферментативных гидролизатов пищевых белков

Food allergy is a matter of global concern, proteins being a popular allergen worldwide. Enzymatic protein hydrolysates serve as hypoallergenic components in functional foods. This article describes enzymatic hydrolysates of egg and soy proteins as potential antigens, sensitizers, and antioxidants. The research featured soy protein isolate and egg albumin. The protein hydrolysates were obtained in a two-step process with pepsin and trypsin to measure hydrolysis, total antioxidant activity, and residual antigenicity. The sensitizing ability of native proteins and their hydrolysates was studied using a delayed-type hypersensitivity (DTH) skin test of paw swelling in mice. The egg albumin and soy protein were hydrolyzed by sequentially adding the proteases, i.e., pepsin and trypsin. The degree of protein hydrolysis was calculated against total nitrogen and its contents in non-hydrolyzed raw materials and hydrolysate. It was 82.6% for the egg hydrolysate and 88.3% for the soy hydrolysate, the total antioxidant activity being 114.3 and 91.4 mg/100 mL, respectively. The residual antigenicity of the hydrolysates determined by ELISA in a sandwich version was 1.55×10–4 and 3.30×10–4 RU, respectively. The native egg and soy proteins demonstrated good allergenic properties, and the DTH response index increased fivefold. The hydrolysates reduced the DTH response indices by an average of 3.5 for egg hydrolysate and 2.6 times for soy hydrolysate. The resulting enzymatic hydrolysates of egg albumin and soy protein demonstrated a high degree of hydrolysis and antioxidant activity. They had low residual antigenicity (10–4) and low sensitizing ability. The resulting enzymatic hydrolysates could be used as hypoallergenic components and antioxidants in new functional foods.

NMR metabolomics of plant and yeast-based hydrolysates for cell culture media applications — A comprehensive assessment

Cultivated meat products, generated by growing isolated skeletal muscle and fat tissue, offer the promise of a more sustainable and ethical alternative to traditional meat production. However, with cell culture media used to grow the cells accounting for 55–95% of the overall production cost, achieving true sustainability requires significant media optimization. One means of dealing with these high costs is the use of low-cost complex additives such as hydrolysates to provide a wide range of nutrients, from small molecules (metabolites) to growth factors and peptides. Despite their potential, most hydrolysate products remain poorly characterized and many are thought to suffer from persistent issues of high batch-to-batch variability. Although there have been a number of isolated efforts to determine metabolic profiles for a handful of hydrolysate products, we present the first attempt at a more comprehensive metabolomic characterization of nine different products (four plant and five yeast-based) from two to four different lots each.NMR analysis identified 90 unique metabolites, with only 15 metabolites common to all hydrolysate products (including eight of the nine essential amino acids), and 16 metabolites found in only a single hydrolysate product. The different hydrolysate products were found to have substantial differences in metabolite concentrations (as a fraction of overall mass), ranging from a high of 43% in yeast extract to a low of 14% in soy hydrolysates. The proportion of various metabolites also varied between products, with carbohydrate concentrations particularly high in soy hydrolysates and nucleosides more prominent in two of the yeast products. Overall, yeast extract generally had higher metabolite concentrations than all the other products, whereas both yeast extract and cotton had the largest variety of metabolites. A direct calculation of batch-to-batch variability revealed although there are significant differences between lots, these are largely driven by a relatively small fraction of compounds. This report will hopefully serve as a useful starting point for a more nuanced consideration of hydrolysate products in cell culture media optimization, both in the context of cultivated meat and beyond.

Microfluidic assisted preparation of soybean hydrolyzed protein-curcumin nanocomplexes: Evaluation of structure, functional properties, and interaction mechanism

The instability and low bioavailability of curcumin are the main reasons that limit its application. In this study, soy protein hydrolysate was used as a carrier to prepare soy hydrolysate curcumin nanocomposites assisted by microfluidization to improve the practical application of curcumin. Preparation of nanocomplexes through microfluidization increased the stability, intestinal targeting and bioavailability of curcumin (74 %). The nanocomposite exhibits high encapsulation efficiency (98 %) and loading capacity (10 mg/g), as well as smaller average particle size (100 nm) and polydispersity index (0.25) and good oxidation resistance. In addition, Fourier transform infrared spectroscopy and fluorescence spectroscopy showed that curcumin and soy protein hydrolysate were combined mainly through hydrophobic interactions and hydrogen bonds. The introduction of curcumin changed the secondary structure of hydrolysate. This study provides a new method to increase the stability and bioavailability of curcumin, and provide a new strategy for the application of curcumin and soy protein in related foods and functional beverages.

Development, characterization and in vivo zinc absorption capacity of a novel soy meal hydrolysate-zinc complexes.

Zinc is an essential trace element for the human body. Recently, a novel Zn-binding peptide, Lys-Tyr-Lys-Arg-Gln-Arg-Trp (PP), was purified and identified from soy protein hydrolysates with high Zn-binding capacity (83.21 ± 2.65%) by our previous study. The preparation of soy meal hydrolysates (SMHs)-Zn complexes is convenient and low-cost, while PP (Lys-Tyr-Lys-Arg-Gln-Arg-Trp)-Zn complexes have a higher coordination rate but a relatively high cost. The aim of this study was to investigate the effect of soy meal hydrolysates (SMHs)-Zn complexes on zinc absorption in mice model, and synthetic soy peptide (PP)-Zn complexes with high Zn-binding capacity were used as control. Firstly, SMHs were prepared by enzymolysis, and the PP (Lys-Tyr-Lys-Arg-Gln-Arg-Trp) were synthesized based on previous studies. The binding mechanism of soy hydrolysates and zinc was analyzed by spectral analysis. Furthermore, the cytotoxicity of the SMHs-Zn complexes was also studied using the CCK-8 method. The effect of zinc absorption was evaluated based on Zn content, total protein and albumin content, relevant enzyme system, and the PeT1 and ZnT1 mRNA expression levels. The result showed that zinc was bound with carboxyl oxygen and amino nitrogen atoms on SMHs, with hydrophobic and electrostatic interactions as auxiliary stabilizing forces. SMHs-Zn were proved to have great solubility and a small particle size at different pH values, and it showed a beneficial effect on Caco-2 cells growth. Moreover, it was proved that SMHs-Zn and PP-Zn could increase the levels of zinc and the activity of Zn-related enzymes in mice. SMHs-Zn possessed higher PepT1 and ZnT1 mRNA expression levels than PP-Zn in the small intestine. SMHs-Zn with a lower Zn-binding capacity had similar effects on zinc absorption in mice as PP-Zn, suggesting that the bioavailability of peptide-zinc complexes in mice was not completely dependent on their Zn-binding capacity, but may also be related to the amino acid composition.

Efficacy of soy biopeptide on serum iron, serum ferritin and hemoglobin levels of adolescent girls in Pandeglang district in Indonesia

Soy hydrolysate contains biopeptides that can increase the absorption of iron. The study aimed to analyze the efficacy of a Soy-biopeptide in improving serum iron, serum ferritin and hemoglobin among female adolescents. For the product’s efficacy study, a one-group pre-test—post-test design was conducted. The products were given every day for 14 days. The serum iron, serum ferritin and hemoglobin were measured twice prior to administration and after day 14, for 52 respondents of female adolescent girls who met the inclusion criteria. The results showed that serum iron levels increased significantly from (66.87±32.44) mcg/dl to (103.77±55.87) mcg/dl (p = 0.000) in both groups. Meanwhile, serum ferritin levels increased significantly in the group with serum ferritin levels from (13.29±9.5) ng/ml to (17.48±12.18) ng/mL (p = 0.00), but there was no significant change in the group with serum ferritin levels from (28.06±23.7) ng/ml to (29.05±21.3) ng/ml (p = 0.64).The respondent's hemoglobin level before administration was (12.21±1.29) g/dl and did not change significantly after administration (12.05±1.31) g/dl (p = 0.016). In conclusion, soy-biopeptide has the health benefits of increasing serum iron and serum ferritin levels in respondents with low iron intake and can maintain normal hemoglobin levels.

Effects of soy protein hydrolysates on antioxidant activity and inhibition of muscle loss

Peptides show biological activity, and are more easily digested than complex proteins. In the present work, we evaluated the effects of soy hydrolysate on skeletal muscle. Soy protein isolate (SPI) was hydrolysed using 2% Alcalase (SPHA) and Flavourzyme (SPHF) at pH 8 for 3 h at 60°C, and at pH 7 for 3 h at 55°C, respectively. Antioxidant properties (total phenolic content and DPPH activity) and inhibition of muscle loss (myogenin, myosin heavy chain [MyHC], creatine kinase, and myostatin) by the SPI hydrolysates in C2C12 cells were compared with those of SPI. Alcalase produced more hydrolysed soy oligopeptides than Flavourzyme. Enzymatic hydrolysis increased the levels of essential amino acids, particularly in SPHF (2,466.85 mg/L) as compared to SPI (56.08 mg/L). The total phenolic contents of hydrolysates increased from 12.02 mg GAE/g in SPI to 22.87 and 18.67 mg GAE/g in SPHA and SPHF, respectively. The IC50 value of DPPH activity decreased four times after hydrolysis (SPI: 124.38, SPHA: 32.18, and SPHF: 30.21 mg/mL). SPHA and SPHF treatments increased the expression levels of both MyHC1 and MyHC3, as well as creatine kinase activity. A significant increase in MyHC3 expression was observed in SPHF at 10 µg/mL. Soy hydrolysates (SPHA: 93.5% and SPHF: 61%) induced a greater decrease in the expression of myostatin, a muscle reduction marker, than SPI (30.4%). In conclusion, soy hydrolysates may inhibit muscle loss, showing particularly better effects when Alcalase is used for hydrolysis.

High sensitivity and high-confidence compound identification with a flexible BoxCar acquisition method

Liquid chromatography-mass spectrometry (LC-MS) is in wide use for compound identification and quantification in complex matrices. While advances in mass spectrometry and the incorporation of new acquisition methods have resulted in greatly improved detection, there is an ongoing need to expand the limits of highly sensitive and confident identification of low abundance species in complex samples. The data acquisition method known as “BoxCar” was originally designed to achieve in-depth proteome profiling on an Orbitrap mass analyzer by decomposing ions into segments with narrow m/z windows. Using this method, selected segments are packaged in C-trap and all ions are then sent to Orbitrap for detection. In this study, we developed a flexible BoxCar acquisition method by placing more segments in the low m/z range for small molecule profiling. This new MS1 acquisition method was successfully integrated with iterative data dependent MS/MS acquisition by generating an inclusion list of ions detected in the flexible BoxCar to trigger the fragmentation of parent ions. The developed acquisition method was applied to the analysis of cell culture media, which plays a key role in antibody production. This challenging goal is of critical importance, as none of the currently available methods provide a comprehensive understanding of how individual components, metabolites, and impurities associated with the cell culture process might influence recombinant antibody production. Even when present at relatively low abundance, some components or impurities in the cell culture medium could have a profound impact on the quality and titer of the antibodies produced. The complex soy hydrolysate cell culture medium used in antibody generation has not been fully characterized. Using the developed flexible BoxCar acquisition method, we achieved 90 % higher sensitivity in experiments designed to detect spiked chemical substances at low abundance at the MS1 level compared to the full scan method. Iterative data-dependent acquisition (DDA) based on the targeted inclusion list generated much higher quality MS2 spectra and facilitated confident identification of low-abundance compounds. Our method achieved a 50 % increase in MS2 coverage of compounds present at low concentrations compared to conventional DDA methods. The results of our study demonstrate that this data acquisition workflow can be easily operated on Orbitrap mass spectrometers and used as a highly effective approach to improve sensitivity and high-confidence small molecule profiling in soy hydrolysate-based cell culture medium and thus provides significant support for therapeutic monoclonal antibody production.

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line.

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.

2,3-Butanediol production using soy-based nitrogen source and fermentation process evaluation by a novel isolate of Bacillus licheniformis BL1

2,3-Butanediol (2,3-BDO) has varied applications in chemical, pharmaceutical, & food industry. Microorganisms belonging to Klebsiella, Enterobacter & Serratia genera are well-known producers of 2,3-BDO. However, they have limited usage in industrial-scale owing to their pathogenic nature. A nonpathogenic soil isolate identified as Bacillus licheniformis (BL1) was thus investigated for 2,3-BDO production. Soy flakes, soy flour, defatted soy, and soybean meal-based hydrolysates replaced yeast extract and peptone as nitrogen sources. Defatted soy flakes and soybean meal hydrolysate led to an equivalent 2,3-BDO yield and productivity as compared to that of Yeast Extract and peptone. The pH and oxygen variation influenced the proportion of various products of the mixed acid-butanediol pathway. Further, the batch mode fermentation with soy hydrolysate and optimized process parameter resulted in 2,3-BDO titer, yield and productivity of 11.06 g/L, 0.43 g/g and 0.48 g/L h respectively. Glucose concentration above 5% was inhibitory and led to reduction in the specific growth rate of BL1 in batch cultivation. Intermittent glucose feeding in fed-batch mode overcame this substrate limitation resulting in increased titers (49.8 g/L) and productivity (0.62 g/L h). Modified medium containing soy hydrolysate as nitrogen source with fermentation process optimization resulted in 67% decrease in medium cost for 2,3-BDO production.

Enzymatic hydrolysis of Carioca bean (Phaseolus vulgaris L.) protein as an alternative to commercially rejected grains

This study on Carioca bean protein aims to improve its functional properties by enzymatic hydrolysis at 6 and 9% degree of hydrolysis using the pH-stat method. The production of protein fractions of lower molar masses, evidenced by gel electrophoresis (SDS-PAGE), allowed an increased solubility at basic pH values, reduction in hydrophobicity, improvement in emulsion properties (emulsification capacity, emulsification activity, and emulsion stability), and foaming properties (foaming ability, rates of liquid incorporated into the foam and drainage). These parameters were studied for hydrolyzed and control samples at pH 3.0, 5.0, 7.0, and 9.0. Carioca bean protein hydrolysates presented higher values of solubility, hydrophobicity, emulsification capacity, and emulsifying activity compared to soy hydrolysates. It was also observed that foam formation and collapse behaviors were similar between hydrolyzed Carioca bean protein and soybean protein. It was concluded that Carioca bean protein has a potential to be used in food formulations when emulsion and foaming characteristics are desirable.

Production and characterization of liposomes with encapsulated bioactive soy protein hydrolysate

Soy proteins known for their high nutritional value and pronounced techno-functional properties, can be hydrolyzed by using proteolytic enzymes and thus converted into hydrolysates rich in di-, tri- and oligopeptides. The resulting peptides are carriers of valuable biological activities, which make the soy hydrolysates very important in functional food applications as techno-functional and bioactive ingredients. However, commercial incorporation and application of soy protein hydrolysates can be hinderedby their low bioavailability and instability, bitter taste, hygroscopicity and possibility to interact with the food matrix. The aim of this research is encapsulation of the soy protein hydrolysate in liposomes in order to overcome the stated shortcomings, while preserving the biological activities that protein hydrolysates exhibit. The soy hydrolysate was prepared by a two-step enzymatic hydrolysis of a soy protein concentrate using commercial food-grade proteases, endoprotease from Bacillus amyloliquefaciens (Neutrase?) and egzo- and endoprotease from Aspergillus oryzae (Flavourzyme?) and encapsulated within liposomes. The liposomes were produced by a thin film method using a commercial lipid mixture (Phospolipon? 90G) containing mainly phosphatidylcholine. Next, the obtained multilamellar vesicles (MLV) with the soy protein hydrolysate were treated by high-intensity ultrasound waves generated by using (1) an ultrasonic probe at a frequency of 20 kHz and (2) an ultrasonic bath with a frequency 40 kHz. The smallest (310 nm) and uniform (unimodal size distribution) liposomes with the highest efficiency of peptide encapsulation (19 %) were obtained by the probe sonication. The presented results showed that incorporation of the soy protein hydrolysates was achieved within the liposome membrane and caused an increase in the liposome size in all tested formulations, namely: from 297 to 310 nm by using the ultrasonic probe, from 722 to 850 nm by using the ultrasonic bath, while in formulations without the ultrasonic treatments the increase from 2818 to 3464 nm was recorded. The entrapped peptides caused enlargement of all liposomes and the increase in negative charge of zeta potential values, which in the case of MLV liposomes was below -30 mV, indicating high stability of these liposomes. Significant antioxidant activity of the probe-sonicated liposomal formulation was confirmed by the ABTS scavenging ability and iron-chelating activity. Release studies conducted under simulated gastrointestinal conditions confirmed that liposomes provide prolonged release of encapsulated soy protein hydrolysates as compared to diffusion of the free hydrolysate. In the first 75 min, only 20 % of liposome encapsulated soy peptides diffused, which is 2.2-fold lower as compared to the diffusion of the non-encapsulated soy hydrolysate. Liposome encapsulated soy protein hydrolysates may provide the possibility for application in the areas such as food science and technology, with the aim to enhance the nutritional value and shelf life of food products, and develop functional foods.

Soy hydrolysate enhances the isoproterenol-stimulated lipolytic pathway through an increase in β-adrenergic receptor expression in adipocytes.

Activation of the adipose lipolytic pathway during lipid metabolism is mediated by protein kinase A (PKA), which responds to β-adrenergic stimulation, leading to increased lipolysis. Soy is well known as a functional food and it is able to affect lipolysis in adipocytes. However, the mechanism by which soy components contribute to the lipolytic pathway remains to be fully elucidated. Here, we show that hydrolyzed soy enhances isoproterenol-stimulated lipolysis and activation of PKA in 3T3-L1 adipocytes. We also found that the expression of β-adrenergic receptors, which coordinate the activation of PKA, is elevated in adipocytes differentiated in the presence of soy hydrolysate. The activity of the soy hydrolysate towards β-adrenergic receptor expression was detected in its hydrophilic fraction. Our results suggest that the soy hydrolysate enhances the PKA pathway through the upregulation of β-adrenergic receptor expression and thereby, increase lipolysis in adipocytes.

A LC–MS/MS platform for the identification of productivity markers in industrial mammalian cell culture media

Abstract Cell culture media formulations supplemented with soy hydrolysates for commercial scale manufacturing of therapeutic proteins were characterized by high-resolution LC–MS/MS to identify specific media components that contributed to cellular bioprocess variability. Initial untargeted screening of media formulations resulted in the combined quantification of >2500 features upon analysis by reversed-phase and hydrophilic interaction liquid chromatography. Chemometric evaluation of quantified features by Principal Component Analysis and Hierarchical Clustering revealed clear correlation between media lots and the corresponding hydrolysates implemented during formulation development stages, suggesting that the observed variation in culture performance could be influenced by components present within hydrolysates. Through multivariate data analysis, a small number of dipeptides, identified as Tyrosyl-Leucine, Phenylalanyl-Valine and LL-Cyclo(Leucylprolyl) were determined in media as potential contributors to bioprocess variability due to their statistically significant higher abundance in formulations which yielded low cellular productivity. Subsequent quantification of these features was performed through targeted LC-QQQ-MS/MS which however, revealed discrepancies between dipeptide concentration levels in media and corresponding hydrolysates presumably due to their potential instability upon interaction with other chemically defined components such as metals or chelating agents. The nutritional impact of the aforementioned features of interest on CHO cell growth performance was assessed through spiking studies in representative shake-flask cultures which however, failed to demonstrate clear correlation between dipeptide concentrations and achieved product yields further indicating the presence of a plausible synergetic effect between the identified dipeptides and alternative media components.

Bioactive Peptide VHVV Upregulates the Long-Term Memory-Related Biomarkers in Adult Spontaneously Hypertensive Rats.

Hypertension is one of the growing risk factors for the progression of long-term memory loss. Hypertension-mediated memory loss and treatment remain not thoroughly elucidated to date. Plant-based natural compounds are an alternative solution to treating human diseases without side effects associated with commercial drugs. This study reveals that bioactive peptides extracted from soy hydrolysates mimic hypertension-mediated memory loss and neuronal degeneration and alters the memory molecular pathway in spontaneously hypertensive rats (SHR). The SHR animal model was treated with bioactive peptide VHVV (10 mg/kg/oral administration) and angiotensin-converting-enzyme (ACE) inhibitors (5 mg/kg/oral administration) for 24 weeks. We evaluated molecular level expression of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and survival markers phospho-protein kinase B (P-AKT) and phosphoinositide 3-kinase (PI3K) after 24 weeks of treatment for SHR in this study. Western blotting, hematoxylin and eosin (H&E) staining, and immunohistochemistry showed long-term memory loss and neuronal degeneration in SHR animals. Bioactive peptide VHVV-treated animals upregulated the expression of long-term memory-relate proteins and neuronal survival. Spontaneously hypertensive rats treated with oral administration of bioactive peptide VHVV had activated CREB-mediated downstream proteins which may reduce hypertension-mediated long-term memory loss and maintain neuronal survival.

Soy hydrolysate mimic autocrine growth factors effect of conditioned media to promote single CHO-K1 cell proliferation

The increasingly competitive biopharmaceutical industry requires companies to focus on rapid and low-cost cell line development. Single-cell cloning (SCC) is a critical and high-value process for cell line development, and typically problematic because single cell proliferates slowly when cultivated at low cell densities. Conditioned media (CM) provide autocrine growth factors to facilitate single cell proliferation, thus improve SCC efficiency. However, conditioned media (CM) are not a feasible solution for industrial cell line development due to variation and cross-contamination concerns. Here, we have found an improvement in the SCC efficiency similar to CM when soy hydrolysate was supplemented in SCC media. Therefore, we concluded that hydrolysate can mimic the autocrine growth factor(s) effect to improve cloning efficiency observed in CM.

Development of a versatile high-temperature short-time (HTST) pasteurization device for small-scale processing of cell culture medium formulations.

The compatibility of CHO cell culture medium formulations with all stages of the bioprocess must be evaluated through small-scale studies prior to scale-up for commercial manufacturing operations. Here, we describe the development of a bespoke small-scale device for assessing the compatibility of culture media with a widely implemented upstream viral clearance strategy, high-temperature short-time (HTST) treatment. The thermal stability of undefined medium formulations supplemented with soy hydrolysates was evaluated upon variations in critical HTST processing parameters, namely, holding times and temperatures. Prolonged holding times of 43s at temperatures of 110°C did not adversely impact medium quality while significant degradation was observed upon treatment at elevated temperatures (200°C) for shorter time periods (11s). The performance of the device was benchmarked against a commercially available mini-pilot HTST system upon treatment of identical formulations on both platforms. Processed medium samples were analyzed by untargeted LC-MS/MS for compositional profiling followed by chemometric evaluation, which confirmed the observed degradation effects caused by elevated holding temperatures but revealed comparable performance of our developed device with the commercial mini-pilot setup. The developed device can assist medium optimization activities by reducing volume requirements relative to commercially available mini-pilot instrumentation and by facilitating fast throughput evaluation of heat-induced effects on multiple medium lots.

Antioxidant capacity of egg, milk and soy protein hydrolysates and biopeptides produced by Bromelia pinguin and Bromelia karatas-derived proteases

Given the lack of knowledge in the generation and biological activity of biopeptides (BP) produced by Bromelia pinguin and Bromelia karatas-derived proteases, the objective of this work was to evaluate the antioxidant capacity (AOX) of protein hydrolysates and peptide fractions from hydrolysis of egg, milk and soy proteins with enzymes extracted from Bromelia pinguin and Bromelia karatas fruits. The degree of hydrolysis (DH) of the mentioned proteins was measured. The hydrolysates were sequentially fractionated by ultrafiltration using different membrane pore sizes (≤30, ≤10, ≤5 and ≤1 kDa) to obtain biopeptide fractions (BPF). The antioxidant capacity (AOX) of protein hydrolysates and BPF were evaluated. AOX was performed using 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. after the enzymatic action of B. pinguin and B. karatas proteases, the highest DH values for egg, milk and soy protein hydrolysates ranged from 50-61%, 55-68% and 73-81%, respectively. The AOX was higher for soy hydrolysates than for egg and milk hydrolysates. Additionally, the highest AOX from ABTS and FRAP assays was observed for BPF ≤30 kDa, PF ≤5 kDa and ≤1 kDa, respectively. These results highlight the potential of B. pinguin and B. karatas proteases to hydrolyze food proteins and release bioactive peptides. Moreover, it has been shown that the hydrolysis products are a mixture of BP of different molecular weights, which could present AOX through different mechanisms.

Immunomodulating protein aggregates in soy and whey hydrolysates and their resistance to digestion in an in vitro infant gastrointestinal model: new insights in the mechanism of immunomodulatory hydrolysates.

Hydrolysates, which are used in hypoallergenic infant formulas, have been found to possess immune modulating effects. For an optimal utilization of hydrolysates, the working mechanisms and responsible proteins underlying the effects should be elucidated. In this study, the immunomodulating activity of whey and soy hydrolysates was studied by quantifying TLR activation and assessing cytokine production in hydrolysate stimulated dendritic cells. The responsible protein fraction was identified and characterized by gel electrophoresis. The immune effects under gastrointestinal conditions were studied by digesting the hydrolysates in an in vitro infant digestion model, after which the digests were analyzed. In both soy and whey hydrolysates, TLR activation and cytokine production in dendritic cells were induced by a fraction containing protein aggregates larger than 1000 kDa, which were formed by electrostatic interactions and disulfide bonds. Only soy aggregates remained intact during duodenal digestion, and maintained the TLR activating capacity. Soy and whey protein aggregates larger than 1000 kDa possess immunomodulatory properties, but only soy aggregates remain under intestinal digestion conditions. This knowledge is important for a better understanding of the effects of hydrolysates.

The epithelial barrier-protecting properties of a soy hydrolysate.

Enhancing the epithelial barrier function could be a possible strategy to prevent food allergy or reduce its symptoms. Soy hydrolysates containing bioactive peptides could be instrumental in this. In this study, the protective effects of pretreatment with 6 soy hydrolysates on calcium ionophore A23187-induced TEER reduction were studied in T84 cells. The effects of the most potent soy hydrolysate on tight junction gene expression were studied. In order to identify the underlying pathways involved, the barrier disruptor specificity of the effect was studied by comparing the protective effects on TEER and Lucifer Yellow flux after the exposure to barrier disruptors that work via different intracellular pathways, i.e. the disruptors A23187, mellitin, and deoxynivalenol (DON). Preincubation with one of the six hydrolysates protected the epithelial cells from a decrease in TEER induced by A23187 (restored to 105% of the starting point, while A23187 alone decreased to 53% of the starting value) and mellitin (restored to 11% of the starting point, while mellitin alone decreased to 3.8% of the starting value). This soy hydrolysate was found to increase claudin-1 and decrease claudin-2 expression. The protective effect of the hydrolysate on TEER was specific for the barrier disruptors A23187 and mellitin, but was not observed for DON. This observation suggests that the soy hydrolysate may act via PKC isoforms, which are known to lead to changes in the expression of claudin-1 and 2. Our data suggest that specific soy hydrolysates may be designed to strengthen the epithelial barrier which might be instrumental in the management of the barrier function in individuals at risk of developing food allergy.

Protein-rich beverage developed using non-GM soybean (R08-4004) and evaluated for sensory acceptance and shelf-life.

Protein beverages have been in demand due to an increasing consumers' interest in healthy eating habit. However, there is an increased concern on the use of genetic modified (GM) ingredient in the food product. This study aimed to develop protein hydrolysate beverages using a non-GM soybean (R08-4004/high protein line) grown in Arkansas. Protein isolate was prepared from the soybean using alkaline method (pH 9.5). Due to its poor solubility in acidic condition, alcalase 2.4L (food grade protease) hydrolyzed soy protein was used to develop a beverage containing 20g protein per serving (500mL). Three flavored beverages: Chai tea (C), tangerine (T), and mixed berries (MB) were prepared using bitter blocker, masking agent, and citric acid to minimize an unpleasant bitter taste developed in the soy hydrolysates. Protein solubility, pH, microbial growth, instrumental color parameters, and turbidity were measured to evaluate the shelf-life stability of the beverages at refrigerated storage (5°C) for 42days. Beverages T and MB received overall highest scores from the sensory panel. Citric acid alone or in combination with bitter blocker or masking agent lowered the bitterness. Pasteurization (90-95°C for 5min) was effective in preventing microbial growth. Although pH remained constant, decrease in protein solubility and color changes were observed over the storage time in all the three flavored beverages. Cloudiness in beverage C increased over the storage period while beverages T and MB were very stable. Overall, T and MB flavored beverages have the potential for commercial application.