Impact of Alcalase Hydrolysis and Simulated Gastrointestinal Digestion on the Release of Bioactive Peptides from Erythrina edulis (Chachafruto) Proteins.

Amidst increasing awareness of diet-health relationships, plant-derived bioactive peptides are recognized for their dual nutritional and health benefits. This study investigates bioactive peptides released after Alcalase hydrolysis of protein from chachafruto (Erythrina edulis), a nutrient-rich South American leguminous plant, focusing on their behavior during simulated gastrointestinal digestion. Evaluating their ability to scavenge radicals, mitigate oxidative stress, and influence immune response biomarkers, this study underscores the importance of understanding peptide interactions in digestion. The greatest contribution to the antioxidant activity was exerted by the low molecular weight peptides with ORAC values for the <3 kDa fraction of HES, GD-HES, and GID-HES of 0.74 ± 0.03, 0.72 ± 0.004, and 0.56 ± 0.01 (µmol TE/mg protein, respectively). GD-HES and GID-HES exhibited immunomodulatory effects, promoting the release of NO up to 18.52 and 8.58 µM, respectively. The findings of this study highlighted the potential of chachafruto bioactive peptides in functional foods and nutraceuticals, supporting human health through dietary interventions.

Distribution and molecular analysis of Subtilase cytotoxin gene (subAB) variants in Shiga toxin-producing Escherichia coli (STEC) isolated from different sources in Iran.

Subtilase exhibits strong cytotoxicity that was first described in O113:H21 strain in Australia as a plasmid- encoded cytotoxin (subAB1). Subsequently, chromosomal variants including subAB2-1, subAB2-2, and subAB2-3 were described. We aimed to investigate the presence of subAB genes in a collection of Shiga toxin-producing Escherichia coli (STEC) strains (n=101) isolated from different sources in Iran. A collection of 101 archived STEC strains isolated from cattle (n=50), goats (n=25), sheep (n=15), wild captive animals (n=8: persian fallow deer, n=3; caspian pony, n=1; Macaca mulatta, n=4), and humans (n=3) during 2007-2016 were analyzed for the detection of different genes encoding the Subtilase variants, plasmidic and chromosomal virulence genes, phylogroups and serogroups. Overall, 57 isolates (56.4%) carried at least one variant of subAB. Most strains from small ruminants including 93% of sheep and 96% of caprine isolates carried at least one chromosomally encoded variant (subAB-2-1 and/or subAb2-2). In contrast, 12 cattle isolates (24%) only harbored the plasmid encoded variant (subAB1). STEC strains from other sources, including deer, pony and humans were positive for subAB-2-1 and/or subAb2-2. Our results reveal the presence of potentially pathogenic genotypes among locus of enterocyte effacement (LEE)-negative isolates, and some host specificity related to Subtilase variants and other virulence markers that may aid in source tracking of STEC during outbreak investigations.

Investigation of Antioxidant Activity of Protein Hydrolysates from New Zealand Commercial Low-Grade Fish Roes.

The objective of this study was to investigate the nutrient composition of low-grade New Zealand commercial fish (Gemfish and Hoki) roe and to investigate the effects of delipidation and freeze-drying processes on roe hydrolysis and antioxidant activities of their protein hydrolysates. Enzymatic hydrolysis of the Hoki and Gemfish roe homogenates was carried out using three commercial proteases: Alcalase, bacterial protease HT, and fungal protease FP-II. The protein and lipid contents of Gemfish and Hoki roes were 23.8% and 7.6%; and 17.9% and 10.1%, respectively. The lipid fraction consisted mainly of monounsaturated fatty acid (MUFA) in both Gemfish roe (41.5%) and Hoki roe (40.2%), and docosahexaenoic (DHA) was the dominant polyunsaturated fatty acid (PUFA) in Gemfish roe (21.4%) and Hoki roe (18.6%). Phosphatidylcholine was the main phospholipid in Gemfish roe (34.6%) and Hoki roe (28.7%). Alcalase achieved the most extensive hydrolysis, and its hydrolysate displayed the highest 2,2-dipheny1-1-picrylhydrazyl (DPPH)˙ and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidant power (FRAP). The combination of defatting and freeze-drying treatments reduced DPPH˙ scavenging activity (by 38%), ABTS˙ scavenging activity (by 40%) and ferric (Fe3+) reducing power by18% (p < 0.05). These findings indicate that pre-processing treatments of delipidation and freeze-drying could negatively impact the effectiveness of enzymatic hydrolysis in extracting valuable compounds from low grade roe.

Construction of highly active and stable recombinant nattokinase by engineered bacteria and computational design.

Nattokinase (NK) is an enzyme that has been recognized as a new potential thrombolytic drug due to its strong thrombolytic activity. However, it is difficult to maintain the enzyme activity of NK during high temperature environment of industrial production. In this study, we constructed six NK mutants with potential for higher thermostability using a rational protein engineering strategy integrating free energy-based methods and molecular dynamics (MD) simulation. Then, wild-type NK and NK mutants were expressed in Escherichia coli (E. coli), and their thermostability and thrombolytic activity were tested. The results showed that, compared with wild-type NK, the mutants Y256P, Q206L and E156F all had improved thermostability. The optimal mutant Y256P showed a higher melting temperature (Tm) of 77.4 °C, an increase of 4 °C in maximum heat-resistant temperature and an increase of 51.8 % in activity at 37 °C compared with wild-type NK. Moreover, we also explored the mechanism of the increased thermostability of these mutants by analysing the MD trajectories under different simulation temperatures.

Ultrasonication assisted enzymatic hydrolysis for generation of pulses protein hydrolysate having antioxidant and ACE-inhibitory activity.

The main objective of this work was to investigate the impact of ultrasonication assisted enzymatic treatment on the physicochemical and bioactive properties of broad bean (BBP), lentil bean (LBP), and mung bean (MBP) protein isolates. The protein was extracted using alkaline acid precipitation method, ultrasonicated at a frequency of 20 kHz, temperature 20-30 °C and then hydrolysed using alcalase enzyme (1 % w/w, pH 8.5, 30 min, 55 οC). The generated hydrolysates were characterized by degree of hydrolysis (DH), SDS, FTIR, surface hydrophobicity, amino acid composition, antioxidant and antihypertensive properties. Results showed that the degree of hydrolysis was found to increase in ultrasonicated protein hydrolysate (18.9 to 40.71 %) in comparison to non- ultrasonicated protein hydrolysate (11 to 16.3 %). SDS-PAGE results showed significant changes in protein molecular weight profiles (100-11kDa) in comparison to their natives. However, no substantial change was found in ultrasonicated and non-ultrasonicated protein hydrolysates. The FTIR spectrum showed structural alterations in ultrasonicated and non-ultrasonicated protein hydrolysates, suggesting modifications in secondary structure such as amide A, amide I and amide II regions. The essential amino acid content varied in the range of 60.09 mg/g to 73.77 mg/g and 28.73 to 50.26 mg/g in case of ultrasonicated and non-ultrasonicated protein hydrolysates, and non-essential content varied in the range of 49.42 to 65.93 mg/g and 43.12 to 47.12 mg/g. Both antioxidant and antihypertensive activities were found to increase significantly in ultrasonicated and non-ultrasonicated protein hydrolysates in comparison to their native counterparts, highlighting their potential as functional ingredients for management of hypertension. It was concluded that ultrasonication assisted enzymatic hydrolysis is an efficient approach for production of bioactive pulse protein hydrolysates with enhanced nutracutical properties, thus offering promising avenues for their utilization in the food industry and beyond.

Comparative Cardioprotective Effectiveness: NOACs vs. Nattokinase-Bridging Basic Research to Clinical Findings.

The use of non-vitamin K antagonist oral anticoagulants (NOACs) has brought a significant progress in the management of cardiovascular diseases, considered clinically superior to vitamin K antagonists (VKAs) particularly in the prevention and treatment of thromboembolic events. In addition, numerous advantages such as fixed dosing, lack of laboratory monitoring, and fewer food and drug-to-drug interactions make the use of NOACs superior to VKAs. While NOACs are synthetic drugs prescribed for specific conditions, nattokinase (NK) is a natural enzyme derived from food that has potential health benefits. Various experimental and clinical studies reported the positive effects of NK on the circulatory system, including the thinning of blood and the dissolution of blood clots. This enzyme showed not only fibrinolytic activity due to its ability to degrade fibrin, but also an affinity as a substrate for plasmin. Recent studies have shown that NK has additional cardioprotective effects, such as antihypertensive and anti-atherosclerotic effects. In this narrative review, we presented the cardioprotective properties of two different approaches that go beyond anticoagulation: NOACs and NK. By combining evidence from basic research with clinical findings, we aim to elucidate the comparative cardioprotective efficacy of these interventions and highlight their respective roles in modern cardiovascular care.

Intravenous injection of nattokinase-heparin electrostatic complex improves the therapeutic effect of advanced tumors by dissolving cancer-related thrombosis.

AIMS: Cancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors. MAIN METHODS: In this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors. KEY FINDINGS: We observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer. SIGNIFICANCE: This study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Peptidic Boronic Acid Plasmodium falciparum SUB1 Inhibitors with Improved Selectivity over Human Proteasome.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Sequential Enzymatic Hydrolysis and Ultrasound Pretreatment of Pork Liver for the Generation of Bioactive and Taste-Related Hydrolyzates.

In the study of protein-rich byproducts, enzymatic hydrolysis stands as a prominent technique, generating bioactive peptides. Combining exo- and endopeptidases could enhance both biological and sensory properties. Ultrasound pretreatment is one of the most promising techniques for the optimization of enzymatic hydrolysis. This research aimed to create tasteful and biologically active pork liver hydrolyzates by using sequential hydrolysis with two types of enzymes and two types of ultrasound pretreatments. Sequential hydrolyzates exhibited a higher degree of hydrolysis than single ones. Protana Prime hydrolyzates yielded the largest amount of taste-related amino acids, enhancing sweet, bittersweet, and umami amino acids according to the Taste Activity Value (TAV). These hydrolyzates also displayed significantly higher antioxidant activity. Among sequential hydrolyzates, Flavourzyme and Protana Prime hydrolyzates pretreated with ultrasound showed the highest ferrous ion chelating activity. Overall, employing both Alcalase and Protana Prime on porcine livers pretreated with ultrasound proved to be highly effective in obtaining potentially tasteful and biologically active hydrolyzates.

Angiotensin-Converting Enzyme and Renin-Inhibitory Activities of Protein Hydrolysates Produced by Alcalase Hydrolysis of Peanut Protein.

Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.

In Vitro Multi-Bioactive Potential of Enzymatic Hydrolysis of a Non-Toxic Jatropha curcas Cake Protein Isolate.

The Jatropha curcas cake, a protein-rich by-product of biofuel production, was the subject of our study. We identified and quantified the ACE inhibitory, antioxidant, and antidiabetic activities of bioactive peptides from a Jatropha curcas L. var Sevangel protein isolate. The protein isolate (20.44% recovered dry matter, 38.75% protein content, and 34.98% protein yield) was subjected to two enzyme systems for hydrolysis: alcalase (PEJA) and flavourzyme (PEJF), recording every 2 h until 8 h had passed. The highest proteolytic capacity in PEJA was reached at 2 h (4041.38 ± 50.89), while in PEJF, it was reached at 6 h (3435.16 ± 59.31). Gel electrophoresis of the PEJA and PEJF samples showed bands corresponding to peptides smaller than 10 kDa in both systems studied. The highest values for the antioxidant capacity (DPPH) were obtained at 4 h for PEJA (56.17 ± 1.14), while they were obtained at 6 h for PEJF (26.64 ± 0.52). The highest values for the antihypertensive capacity were recorded at 6 h (86.46 ± 1.85) in PEJF. The highest antidiabetic capacity obtained for PEJA and PEJF was observed at 6 h, 68.86 ± 8.27 and 52.75 ± 2.23, respectively. This is the first report of their antidiabetic activity. Notably, alcalase hydrolysate outperformed flavourzyme hydrolysate and the cereals reported in other studies, confirming its better multi-bioactivity.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain.

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Effect of enzymatic hydrolysis with Alcalase or Protamex on technological and antioxidant properties of whey protein hydrolysates.

The aim of this study was to evaluate the effect of the enzymatic hydrolysis, performed using Alcalase and Protamex enzymes, on the technological functionalities and the antioxidant capacity of whey protein hydrolysates (WPHs) to identify the conditions allowing to obtain target functionality/ies. Samples were characterized for hydrolysis degree (DH), molecular weight distribution, structural properties, and food-related functionalities. Free sulfhydryl groups and surface hydrophobicity significantly decreased with the increase in DH, regardless of the used enzyme. The foaming and antioxidant properties of Alcalase WPHs were higher as compared to those of WPI, reaching the maximum value at DH = 18-20 %, while higher DH resulted in impaired functionality. Gelling properties were guaranteed when WPI was hydrolysed by Protamex at DH < 15 % while foaming and antioxidant abilities were fostered at 15 < DH < 21 %. These results were well correlated with MW distribution and were rationalized into a road map which represents a useful tool in the selection of proper hydrolysis conditions (time, DH, enzyme type) to obtain WPHs with tailored functionalities. Research outcomes highlighted the possibility to drive protein hydrolysis to optimize the desired functionality/ies.

Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of Nicotiana tabacum phytaspase (NtPhyt) with the free prodomain in Nicotiana benthamiana leaf cells. Using this approach, the generation of the proteolytically active NtPhyt and its transport to the extracellular space at a level comparable to that of the native NtPhyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to NtPhyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized NtPhyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of NtPhyt is dispensable for stress-induced retrograde transport of the enzyme.

Enhanced Thermostability of Nattokinase by Computation-Based Rational Redesign of Flexible Regions.

Nattokinase is a nutrient in healthy food natto that has the function of preventing and treating blood thrombus. However, its low thermostability and fibrinolytic activity limit its application in food and pharmaceuticals. In this study, we used bioinformatics analysis to identify two loops (loop10 and loop12) in the flexible region of nattokinase rAprY. Using this basis, we screened the G131S-S161T variant, which showed a 2.38-fold increase in half-life at 55 °C, and the M3 variant, which showed a 2.01-fold increase in activity, by using a thermostability prediction algorithm. Bioinformatics analysis revealed that the enhanced thermostability of the G131S-S161T variant was due to the increased rigidity and structural shrinkage of the overall structure. Additionally, the increased rigidity of the local region surrounding the active center and its mutated sites helps maintain its normal conformation in high-temperature environments. The increased catalytic activity of the M3 variant may be due to its more efficient substrate binding mechanism. We investigated strategies to improve the thermostability and fibrinolytic activity of nattokinase, and the resulting variants show promise for industrial production and application.

Surface-associated residues in subtilisins contribute to poly-L-lactic acid depolymerization via enzyme adsorption.

Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a biodegradable commodity. Enzymatic recycling is one strategy for ensuring circularity of PLLA, but this approach requires a thorough understanding of enzymatic mechanisms and protein engineering strategies to enhance activity. In this study, we engineer PLLA depolymerizing subtilisin enzymes originating from Bacillus species to elucidate the molecular mechanisms dictating their PLLA depolymerization activity and to improve their function. The surface-associated amino acids of two closely related subtilisin homologues originating from Bacillus subtilis (BsAprE) and Bacillus pumilus (BpAprE) were compared, as they were previously engineered to have nearly identical active sites, but still varied greatly in PLLA depolymerizing activity. Further analysis identified several surface-associated amino acids in BpAprE that lead to enhanced PLLA depolymerization activity when engineered into BsAprE. In silico protein modelling demonstrated increased enzyme surface hydrophobicity in engineered BsAprE variants and revealed a structural motif favoured for PLLA depolymerization. Experimental evidence suggests that increases in activity are associated with enhanced polymer binding as opposed to substrate specificity. These data highlight enzyme adsorption as a key factor in PLLA depolymerization by subtilisins.

Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues.

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 µM, Vmax = 62.91 ± 1.68 µM/s, kcat = 38.45 ± 1.06 s-1, and kcat/Km = 1.49 × 106 M-1 s-1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

The Effect of Whey Protein Isolate Hydrolysate on Digestive Properties of Phytosterol.

Phytosterol (PS) is a steroid, and its bioavailability can be enhanced by interacting with protein in the C-24 hydroxyl group. The interaction between sterols and amino acid residues in proteins can be enhanced by enzymatic hydrolysis. Phytosterol and whey insulation hydrolysates (WPH1-4) fabricated by the Alcalase enzyme at different enzymatic hydrolysis times were selected as delivery systems to simulate sterol C-24 hydroxyl group interaction with protein. Increasing hydrolysis time can promote the production of ß-Lg, which raises the ratio of ß-turn in the secondary structure and promotes the formation of interaction between WPH and PS. The correlation coefficient between hydrogen bonds and encapsulation efficiency (EE) and bioaccessibility is 0.91 and 0.88 (P < 0.05), respectively, indicating that hydrogen bonds of two components significantly influenced the combination by concealing the hydrophobic amino acids and some residues, which improved PS EE and bioavailability by 3.03 and 2.84 times after PS was combined with the WPI hydrolysate. These findings are expected to enhance the absorption of PS and other macromolecules by protein enzymatic hydrolysis to broaden their applications for food.

Green fractionation and hydrolysis of fish meal to improve their techno-functional properties.

A green strategy employing water as solvent has been adopted to obtain protein hydrolysates from fish meal (FM), its water-soluble fraction (WSP), and its non-water-soluble fraction (NSP). The techno-functional properties of the hydrolysates have been investigated and compared to hydrolysates obtained with Alcalase®. In general, SWH hydrolysates presented higher content of free amino acids and higher degree of hydrolysis, which reflected on the molecular size distribution. However, Alcalase® hydrolysates presented better solubility (from 74 ± 4% for NSP at pH = 2 up to 99 ± 1% for WSP at pH = 4-7). According to fluorescence experiments, FM and NSP hydrolysates showed the highest surface hydrophobicity, which has been related to better emulsifying properties and higher emulsion stability. The emulsions stabilized with 2%wt. of SWH-treated NSP showed the smallest particle sizes, with D[4,3] = 155 nm at day 0, and good stability, with D[4,3] = 220 nm at day 7, proving that water fractionation followed by SWH treatment is a good method to improve the techno-functional properties of the hydrolysates.