Effects of Fermentation with Tetragenococcus halophilus and Zygosaccharomyces rouxii on the Volatile Profiles of Soybean Protein Hydrolysates.

The effects of fermentation with lactic acid bacteria (LAB) and yeast on the aroma of samples were analyzed in this work. The volatile features of different soybean hydrolysates were investigated using both GC-MS and GC-IMS. Only 47 volatile flavor compounds (VFCs) were detected when using GC-IMS, while a combination of GC-MS and GC-IMS resulted in the identification of 150 compounds. LAB-yeast fermentation could significantly increase the diversity and concentrations of VFCs (p < 0.05), including alcohols, acids, esters, and sulfurs, while reduce the contents of aldehydes and ketones. Hierarchical clustering and orthogonal partial least squares analyses confirmed the impact of fermentation on the VFCs of the hydrolysates. Seven compounds were identified as significant compounds distinguishing the aromas of different groups. The partial least squares regression analysis of the 25 key VFCs (ROAV > 1) and sensory results revealed that the treatment groups positively correlated with aromatic, caramel, sour, overall aroma, and most of the key VFCs. In summary, fermentation effectively reduced the fatty and bean-like flavors of soybean hydrolysates, enhancing the overall flavor quality, with sequential inoculation proving to be more effective than simultaneous inoculation. These findings provided a theoretical basis for improving and assessing the flavor of soybean protein hydrolysates.

Exploring the Release of Elastin Peptides Generated from Enzymatic Hydrolysis of Bovine Elastin via Peptide Mapping.

To enhance the understanding of enzymatic hydrolysis and to accelerate the discovery of key bioactive peptides within enzymatic products, this research focused on elastin as the substrate and investigated the variations in peptide profiles and the production of key bioactive peptides (those exceeding 5% of the total) and their impacts on the biological activity of the hydrolysates. Through the application of advanced analytical techniques, such as stop-flow two-dimensional liquid chromatography and ultra-high-performance liquid chromatography-tandem mass spectrometry, the research tracks the release and profiles of peptides within elastin hydrolysates (EHs). Despite uniform peptide compositions, significant disparities in peptide concentrations were detected across the hydrolysates, hinting at varying levels of bioactive efficacy. A comprehensive identification process pinpointed 403 peptides within the EHs, with 18 peptides surpassing 5% in theoretical maximum content, signaling their crucial role in the hydrolysate's bioactivity. Of particular interest, certain peptides containing sequences of alanine, valine, and glycine were released in higher quantities, suggesting Alcalase® 2.4L's preference for these residues. The analysis not only confirms the peptides' dose-responsive elastase inhibitory potential but also underscores the nuanced interplay between peptide content, biological function, and their collective synergy. The study sets the stage for future research aimed at refining enzymatic treatments to fully exploit the bioactive properties of elastin.

Impact of ternary NADES prepared from proline, glucose and water on the Maillard reaction: Reaction activity, Amadori compound yield, and taste-enhancing ability.

This study employed proline, glucose, and water to prepare natural deep eutectic solvents (NADES) through heating and stirring. The Maillard reaction was then performed, producing a high yield of Amadori rearrangement product (ARP) and physicochemical properties of NADES were examined for impacts on the reaction. Water had a dual function by promoting the formation of hydrogen bonding networks within the NADES when present at less than 15%, and also working as a diluting agent that could potentially disturb its structure when exceed 15%. These changes further affected the subsequent Maillard reaction, especially the ARP accumulation (reached the highest when water content was 15%). Correlation analysis shows strong positive viscosity-ARP and negative water activity-ARP correlations within a range. Moreover, the product (rich in ARP) remarkably enhanced umami and saltiness. This finding provides insights into modulating the Maillard reaction by adjusting NADES properties, demonstrating feasibility of this approach for flavor enhancer development.

Application of electronic tongue in umami detection and soy sauce refining process.

The article systematically investigated the response behaviors of lipid-film equipped umami taste sensor to various umami compounds, including typical umami substances (umami amino acids, GMP, IMP, disodium succinate) and novel umami chemicals (umami peptide and Amadori rearrangement product of umami amino acid). The umami taste sensor has great specificity to all umami substances. Relationships between output values and concentrations of umami substances in certain ranges were consistent with Weber-Fechner law. The umami synergistic effect detected by the sensor was in great agreement with human sensory results as well, fitting logarithm model. Moreover, the taste profile mixing model of raw soy sauce was established using five different taste sensors and principal component analysis, realizing the simplification of soy sauce blending and acceleration of the soy sauce refining process. Thus, flexible design of the experimental procedure and multi-analysis of the sensor data is essential.

Effects of Jiuqu inoculating Rhizopus oryzae Q303 and Saccharomyces cerevisiae on chemical components and microbiota during black glutinous rice wine fermentation.

In this study, the physicochemical properties, metabolites, sensory characteristics, and microbiota of black glutinous rice wine (BGRW) during traditional fermentation (TF) and inoculated fermentation (IF) were investigated, and their correlation relationships were revealed. Results indicated that IF promoted total sugar utilization and ethanol and esters synthesis in BGRW. Altogether 67 reliable metabolites were identified, 36 of which were labeled components, primarily esters (OAV > 1). Meanwhile, the concentrations of ethanol, esters, and total flavor on the 11th day of IF were close to those on the 22nd day of TF. Combined with the sensory description indicated that the IF could improve the quality of BGRW. Besides, TF contains vast unfavorable microorganisms, such as Chaetomium, Penicillium, Stachybotrys, and Trichocladium. Therefore, IF can accelerate the fermentation and enhance the flavor of BGRW, and inhibit the growth of pathogenic and spoilage organisms. Finally, Pearson's correlation analysis demonstrated that Saccharomyces, Rhizopus, Lactobacillus, Aspergillus, and Bacillus were the pivotal functional microorganisms in BGRW. The study provided scientific and effective basis for improving the quality, shortening the fermentation cycle, and controlling pathogenic and spoilage organisms of BGRW.

A fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry and its application in food-derived protein hydrolysates.

Food-derived bioactive peptides have many outstanding features like high safety, easy absorption, etc. However, explorations of the peptides are suffering from the limited knowledge of sample composition and low efficiency of separation techniques. In this work, a fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS) was designed and constructed in-house. For chromatographic system optimization, the effects of column pairs and fraction transfer volumes on separation performance were studied. The pair of Protein BEH SEC and HSS T3 columns was found of high orthogonality. The peak capacity detected by the optimized 2DLC reached 1165 (for corn protein hydrolysates), indicating high resolving power. Moreover, the number of peptides identified from corn, soybean and casein protein hydrolysates reached as high as 8330, 8925 and 7215, respectively, demonstrating the high potential of the system. This would help reveal the peptide composition and facilitate the research on exploring bioactive peptides from food-derived protein hydrolysates.

Effects of Pediococcus acidilactici and Rhizopus Oryzae on microbiota and metabolomic profiling in fermented dry-cure mutton sausages.

Traditional fermentation of dry mutton sausages (DMSs) commonly causes some quality problems. In this study, high-throughput sequencing (HTS) and metabolomics were used to study the microorganisms and metabolites of DMSs produced by mixed strains of Pediococcus acidilactici and Rhizopus Oryzae, to discover the effect of inoculated fermentation (IF) on the metabolites of DMSs. A total of 92 volatile metabolites and 58 non-volatile significantly different metabolites were identified in DMSs. After inoculated fermentation, the volatile types increased, and the total contents of aldehydes and esters were enhanced at the end of fermentation. In addition, the levels of amino acids, fatty acids, palmitoyl carnitine, acetyl-l-carnitine, and betaine were also improved. The correlation analysis showed that the P. acidilactici and R. Oryzae were highly correlated with a variety of metabolites. Together, these findings provide excellent strain resources for improving the quality of mutton sausages.

Arginine-Containing Peptides Derived from Walnut Protein Against Cognitive and Memory Impairment in Scopolamine-Induced Zebrafish: Design, Release, and Neuroprotection.

The purpose of this study was to investigate the neuroprotective effect of Arg-containing peptides from walnut storage protein sequences in scopolamine-induced zebrafish and further to validate the potential neuroprotection of Arg-containing peptide enriched walnut hydrolysates prepared by in silico hydrolysis and controlled enzymatic release. Results showed that walnut derived Arg-containing peptides with high abundance and great bioactivity predicted by bioinformatics displayed potent neuroprotection in scopolamine-induced zebrafish, and regulation of neurotransmitter level and antioxidant enzyme activity might be the main target for Arg-containing peptides to exert neuroprotection. Notably, Arg-containing peptides (not free arginine) contributed greater neuroprotection, and the positive charge and cell-penetrating properties also affected their neuroprotection. Subsequently, Arg-containing peptides could be released efficiently from walnut protein following hydrolysis by trypsin, pepsin, papain, and thermolysin (bound arginine content: ranging from 110.43 ± 1.58 to 121.82 ± 1.02 mg/g). Among them, trypsin had excellent potential for releasing Arg-containing peptides in silico hydrolysis, and its hydrolysate was confirmed to have neuroprotective capacity, indicating that the combination of in silico hydrolysis and controlled enzymatic release might be an effective approach to obtain Arg-containing neuroprotective peptides.

Preparation, Sensory Characterization, and Umami-Enhancing Mechanism of Novel Peptide Glycoconjugates.

Previous studies supposed that Amadori rearrangement products (ARPs) of peptides might have better umami-enhancing abilities. To confirm this, five ARPs (EP-ARP, AH-ARP, EE-ARP, ß-AH-ARP, RFPHADF-ARP) were synthesized using a food-grade preparation method, and their chemical structures were clearly demonstrated by mass spectrometry and 1D/2D NMR. Sensory experiments showed that ARPs had better umami-enhancing abilities than the corresponding peptides in this research, though their enhancing performance varied. ARPs showed a synergistic effect with multiple umami substances (MSG and GMP), while their corresponding peptides did not. RFPHADF-ARP had good umami-enhancing capacity, despite that RFPHADF was a bitter peptide without any umami/umami-enhancing property. RFPHADF-ARP could bind to the T1R3, which is beneficial to the stability of the active conformation of the umami receptor. The introduction of glucose via the Maillard reaction increased the binding force of RFPHADF with the umami receptor by influencing the electron density distribution and offering more binding groups (hydroxide group).

Expression of a Salt-Tolerant Pseudolysin in Yeast for Efficient Protein Hydrolysis under High-Salt Conditions.

Protease biocatalysis in a high-salt environment is very attractive for applications in the detergent industry, the production of diagnostic kits, and traditional food fermentation. However, high-salt conditions can reduce protease activity or even inactivate enzymes. Herein, in order to explore new protease sources, we expressed a salt-tolerant pseudolysin of Pseudomonas aeruginosa SWJSS3 isolated from deep-sea mud in Saccharomyces cerevisiae. After optimizing the concentration of ion cofactors in yeast peptone dextrose (YPD) medium, the proteolytic activity in the supernatant was 2.41 times more than that in the control group when supplemented with 5 mM CaCl2 and 0.4 mM ZnCl2. The extracellular proteolytic activity of pseudolysin reached 258.95 U/mL with optimized expression cassettes. In addition, the S. cerevisiae expression system increased the salt tolerance of pseudolysin to sodium chloride (NaCl)and sodium dodecyl sulfate (SDS) and the recombinant pseudolysin retained 15.19% activity when stored in 3 M NaCl for 7 days. The recombinant pseudolysin was able to efficiently degrade the ß-conglycinin from low-denatured soy protein isolates and glycinin from high-denatured soy protein isolates under high temperatures (60 °C) and high-salt (3 M NaCl) conditions. Our study provides a salt-tolerant recombinant protease with promising applications in protein hydrolysis under high-salt conditions.

Round Scad-Derived Octapeptide WCPFSRSF Confers Neuroprotection by Regulating Akt/Nrf2/NFκB Signaling.

We previously identified peptides derived from round scad as potential Nrf2 activators. However, the neuroprotection of these peptides is still unclear. In this study, we aimed to investigate the neuroprotective effect of WCPFSRSF against glutamate-induced neurotoxicity, and the memory-improving effects of WCPFSRSF in mice were also explored. Results showed that WCPFSRSF ameliorated oxidative stress by improving the activities of antioxidant enzymes and promoting the Nrf2-mediated endogenous defense system. Moreover, there is an interaction between the up-regulation of Nrf2 and the down-regulation of NFκB induced by the peptide, which was related to the generation of reactive oxygen species (ROS) and could be abolished by the Akt inhibitor LY294002. Further analysis demonstrated that WCPFSRSF may act as a radical scavenger and Nrf2 activator. The antioxidant and anti-inflammatory effects might be related to the Cys and Trp in WCPFSRSF. Moreover, WCPFSRSF could improve spatial memory impairment in sleep-deprived mice. Thus, this work provided evidence for WCPFSRSF as a potential candidate against neurotoxicity and memory deficits.

Identification and Screening of Potential Bioactive Peptides with Sleep-Enhancing Effects in Bovine Milk Casein Hydrolysate.

Casein tryptic hydrolysate (CTH) has been proven to possess stress-relieving and sleep-enhancing effects, but only one decapeptide YLGYLEQLLR (α-CZP) in CTH was reported to exhibit affinity for the benzodiazepine site of a GABAA receptor (GABAAR). This study aimed to compare the sleep-enhancing effects between CTH and α-CZP and to explore novel sleep-enhancing peptides. Our results showed that CTH significantly prolonged sleep duration in mice, which was almost 2-fold longer than that of α-CZP. The α-CZP in CTH was degraded more slowly than the synthetic α-CZP; meanwhile, CTH could release other potential sleep-enhancing peptides during gastrointestinal digestion. Additionally, two peptides YPVEPF and YFYPEL with strong sleep-enhancing activity were explored by virtual screening. Especially, YPVEPF could significantly prolong the sleep duration from 559.00 ± 272.24 to 2501.63 ± 1021.21 s and increase the sleep rate from 58.33 to 83.33% in mice. Moreover, YPVEPF and YFYPEL could bind with the Ser-205 and Phe-77 residues of GABAAR via hydrogen bonds and lipid contacts. They were largely released after digestion with 11.19 ± 0.15 and 1.78 ± 0.01 g/kg, respectively.

Fabrication and characterization of anchovy protein hydrolysates-polyphenol conjugates with stabilizing effects on fish oil emulsion.

Conjugation between peptides and polyphenols could improve their bioactive and functional properties. The improvement effects of anchovy protein hydrolysates (APH) -polyphenol (catechin (CA), gallic acid (GA), tannic acid (TA)) conjugates were investigated. The content of protein and polyphenols and ratio of polyphenols/peptides in conjugates increased as the number of OH group increased with TA > CA > GA. Results showed that APH-CA and APH-GA exhibited the highest ORAC and ABTS+ scavenging capacity, respectively. Mass spectrometry analysis suggested the highest number of bioactive peptides were identified in APH-CA 5:1 (APH/polyphenols). The physical stability of fish oil emulsions during storage was significantly enhanced by TA 5:1 conjugate followed by CA 5:1 conjugate. The oxidative stability was remarkably elevated by APH-GA 10:1. This was due to the antioxidant capacity and the peptides adsorbed at the interfacial. This study demonstrated that APH-polyphenol conjugates could bring the possibility of utilizing peptides-polyphenols in the nutraceutical and functional food ingredient fields.

Characterization and Exploration of Potential Neuroprotective Peptides in Walnut (Juglans regia) Protein Hydrolysate against Cholinergic System Damage and Oxidative Stress in Scopolamine-Induced Cognitive and Memory Impairment Mice and Zebrafish.

The purpose of this study was to determine the neuroprotective effect and mechanism of walnut protein hydrolysates (WPH) against memory deficits induced by scopolamine in mice and further to validate the potent neuroprotective peptides identified by integrated approaches of in silico analysis and peptidomics in scopolamine-induced zebrafish. Results showed that a remarkable amelioration on behavioral performance was observed for oral administration of WPH, and disorders of cholinergic system and oxidative stress were normalized in the brains of mice also. Unfortunately, no obvious inflammatory response and anti-inflammatory effect were observed. Additionally, WPH significantly upregulated the expressions of antioxidant defense-related protein (Nrf2) and neurotrophic-related protein (BDNF and CREB). Furthermore, 20 peptides with relatively higher abundance and PeptideRanker scores were predicted by docking to AchE and Keap1. Among them, FY and SGFDAE with the highest binding affinities, -9.8 and -8.0 kcal/mol, were considered as the promising AchE and Keap1 inhibitors, respectively. They were further validated to have neuroprotective capacity in scopolamine-induced zebrafish, indicating that peptidomics and in silico prediction might be the effective approaches to screen neuroprotective peptides.

Insight into the advantages of premixing yeast-wheat gluten and combining ultrasound and transglutaminase pretreatments in producing umami enzymatic protein hydrolysates.

This study aimed to utilize effectively industrial byproducts, yeast suspension (Y) and wheat gluten (W), to produce umami protein hydrolysates as seasonings. Y and W were mixed to yield YW, followed by a pretreatment (ultrasound, transglutaminase (TG), or their combination) and then proteolysis with a yeast extract enzyme and trypsin. Premixing Y and W promoted their dispersibility, and suppressed gluten aggregation and hydrolysate's bitterness. All pretreatments increased protein recovery. Ultrasound alone or ultrasound with TG increased the embedding of yeasts in W, umami and salty tastes, hydrolysis degree and proportion of molecules < 3 kDa of the YW hydrolysate. For the first time, premixing Y and W, and pretreating YW (by ultrasound then TG-catalyzed protein crosslinking), were found to increase the ß-sheet and random coil contents and decreased the ß-turn content and surface hydrophobicity, leading to a low-cost umami and non-bitter protein hydrolysate with 56% of species < 1 kDa.

Protein solubility, secondary structure and microstructure changes in two types of undenatured type II collagen under different gastrointestinal digestion conditions.

Sodium dodecyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism spectroscopy (CD), transmission electron microscope (TEM) and atomic force microscope (AFM) were used to analyze the structural properties and gastrointestinal digestive characteristics of natural insoluble undenatured type II collagen (IC II) and pepsin soluble undenatured type II collagen (SC II). Results showed that, after gastric digestion, especially under low pH conditions, some thick and short collagen fibers were dissolved from IC II, which was accompanied by the release of soluble protein with triple helix structure. As to SC II, when gastric digestion was performed under higher pH conditions, collagen fibers were mildly thinned and curved, and the triple helix structure was slightly destroyed. However, those changes hardly occurred during the intestinal digestion. Moreover, the undenatured type II collagen existing in SC II digestive supernatant was 1.2-12.4 times higher than that of IC II depending on the pH conditions. These results suggested that, it is more likely for SC II to exert its activity of relieving arthritis, and it should be taken when the pH of gastric environment is high.

Two-stage selective enzymatic hydrolysis generates protein hydrolysates rich in Asn-Pro and Ala-His for enhancing taste attributes of soy sauce.

This study demonstrated the contribution of peptides to umami soy sauce taste. Asn-Pro and Ala-His with remarkable umami taste and umami-enhancing capacity were found in original soy sauce, possessing umami thresholds of 175 and 160 mg/L and umami-enhancing thresholds of 10 and 13 mg/L, respectively. Firstly, an industrially viable two-stage hydrolysis at 55 °C (a 12-h hydrolysis with the neutral protease, then a 12-h hydrolysis with the aminopeptidase) was established to produce protein hydrolysates rich in umami-tasting and umami-enhancing peptides (e.g. Asn-Pro and Ala-His) from non-soy sauce protein preparations (soy protein isolate, rice proteins, wheat proteins, peanut proteins or pea proteins). The soy protein isolate hydrolysate produced via the two-stage hydrolysis had Asn-Pro and Ala-His contents 3.32 and 1.15 times higher than those produced via the one-stage hydrolysis using the neutral protease only. Adding the hydrolysate to original soy sauce at 5% w/v significantly increased umami and reduced bitterness.

Identification of post-digestion angiotensin-I converting enzyme (ACE) inhibitory peptides from soybean protein Isolate: Their production conditions and in silico molecular docking with ACE.

This study attempts to investigate natural angiotensin-I converting enzyme (ACE) inhibitors. Soybean protein isolated (SPI) hydrolysate (SPIH) was prepared by Alcalase from inexpensive SPI, and their ACE inhibitory peptides were obtained via membrane separation, ethanol precipitation and adsorption chromatography enrichment. Activated carbon was more suitable for peptide enrichment than eight macroporous resins. The peptide fraction yielded under optimal conditions (protein-active carbon mass ratio 2:1; adsorption pH 3.0 and time 2 h; desorption time 2 h) exhibited a 10.4 times higher ACE-inhibitory activity than SPIH. Novel peptides IY, YVVF, LVF, WMY, LVLL and FF (hydrophobicity values 10.51-12.87; activity scores 0.2373-0.999) might be the main contributors to SPIH's ACE inhibition. IY had the lowest IC50 (0.53 ± 0.02 µM). YVVF had the greatest affinity (-9.8 kcal/mol) for 2OC2 (ACE's C-domain receptor) via H-bonds. IY and WMY could be potent ACE inhibitors, and their ACE-inhibitory activities unaltered and increased after in vitro gastrointestinal digestion.

Physicochemical and Structural Characteristics of Soybean Protein Isolates Induced by Lipoxygenase-Catalyzed Linoleic Acid Oxidation during In Vitro Gastric Digestion.

The effects of oxidation on the gastric digestion properties of soybean protein isolates (SPIs) in a model of lipoxygenase (LOX)-catalyzed linoleic acid (LA) oxidation system and the multiscale structural characterization of SPI hydrolysate were investigated. Results indicated that the feature of SPI hydrolysate is dependent upon the degree of oxidation. Pepsin hydrolysis caused a red shift in fluorescence intensity and a reduction in surface hydrophobicity and diminished the particle size of SPI hydrolysate during gastric digestion. Compared with the control, mild oxidation was beneficial to protein unfolding and gastric digestibility, as manifested by minimal molecular weight (MW) distribution >50 kDa (32.34%) and smaller peptide fragments under scanning electron microscopy. However, severe oxidation brought about 39.47% loss of free amino acids. It was interesting to find that glycinin was more vulnerable to pepsin hydrolysis after oxidation as compared to the native SPI. Overall, the moderately oxidized SPI appeared to be digested to a greater extent.

Chicken breast-derived alcohol dehydrogenase-activating peptides in response to physicochemical changes and digestion simulation: The vital role of hydrophobicity.

Chicken breast muscle hydrolysates (CBMHs) could promisingly activate alcohol dehydrogenase (ADH) and ameliorate alcohol-induced liver injury. The aim of this work was to investigate the stability of CBMHs against physicochemical treatments and gastrointestinal digestion simulation. Results indicated that CBMHs showed good stability towards heating (25-121 °C), pH treatment (pH 2-12) and remained stable in the presence of NaCl (0.01-2 M) and low concentration of metal ions (0.1 mM Zn2+, Ca2+, Fe2+, and Fe3+). Results from in vitro digestion implied a retained activity of CBMHs after gastric tract, but marked decrease (33.42%) after intestinal tract. UPLC-ESI-Q-ToF-MS/MS analysis together with in silico assessments then revealed that the degradation of hydrophobic peptides (i.e., VAPEEHPTLL, YPGIADRM, ADGPLKGIL, and KDLFDPVIQ) during simulated intestinal digestion may be account for the decreased activity. Conformational changes of ADH upon hydrophobic interaction with synthetic peptides were further confirmed by fluorescence quenching study, possibly responsible for the enhanced ADH activity. Hence, CBMHs noticeably showed good stability against physicochemical treatments and digestion simulation, while attempt establishing the structure-activity relationship of peptides is also fundamental before applying CBMHs as functional ingredient.