ACE inhibitory effect and saltiness-enhancing properties of chicken-derived umami peptides: Digestive stability, inhibition kinetics, multiple ligand docking and central composite design.

Angiotensin-I-converting enzyme (ACE) inhibitory activity and saltiness-enhancing properties of chicken-derived umami peptides were investigated. DGGRYY and NEFGYSNR were screened and the IC50 values were 28.71 µM and 283.24 µM, indicating their potential as novel ACE inhibitors. DGGRYY and NEFGYSNR have good pH and thermal stability. After gastrointestinal digestion, the ACE-inhibitory activity of DGGRYY retained about 53 %, whereas NEFGYSNR retained about 57 %. The inhibition pattern of both peptides was determined to be uncompetitive, consisting with the result of multiple ligand docking that the binding sites were outside the ACE active pocket. Trp59, Tyr62, Asp121, Arg124, and Ser516 were the key binding sites that contributed to the total binding energy. In addition, saltiness and palatability models were established according to sensory analysis and central composite design. In 0.1 % âˆ¼ 0.3 % NaCl solutions, the addition of DGGRYY and NEFGYSNR could enhance the salty intensity and compensate for the palatability loss caused by salt reduction.

Combined Peptidomics and Metabolomics Analyses to Characterize the Digestion Properties and Activity of Stropharia rugosoannulata Protein-Peptide-Based Materials.

Protein-peptide-based materials typically possess high nutritional value and various physiological regulatory activities. This study evaluated the digestion, metabolism, and activity of Stropharia rugosoannulata protein-peptide-based materials. After the S. rugosoannulata protein-peptide-based materials were digested (simulated) orally, in the stomach, and in the intestines, the proportions of >10,000 Da, 5000~10,000 Da, and <180 Da in the digestion products increased, and the peptide content was maintained at more than 120 mg/g dry weight. The digestion products of eight test groups with different oral-gastrointestinal digestion-level settings all had suitable ACE inhibitory activity (IC50 range 0.004~0.096 mg/mL). The main metabolite groups were lipid-like molecules, fatty acids, carboxylic acids, their derivatives, amino acids, peptides, and analogs. Bile and glycosylated amino acids were the main compounds that caused differences between groups. KEGG pathways enriched in differentially expressed metabolites included eight significantly upregulated pathways, including valine, leucine, and isoleucine biosynthesis, etc., and six significantly downregulated pathways, including the citric acid cycle (tricarboxylic acid cycle), etc. The arginine and proline metabolism pathways and the aminoacyl-tRNA biosynthesis pathways were upregulation and downregulation pathways that enriched multiple differentially expressed metabolites. Twenty-six metabolites, including bile acids, total bile acids, and the essential amino acids L-isoleucine and L-leucine, were differentially expressed metabolite markers of the protein-peptide-based material oral-gastrointestinal digestion products.

Phytochemical Investigation of Carex praecox Schreb. and ACE-Inhibitory Activity of Oligomer Stilbenes of the Plant.

Phenolic compounds are the main special metabolites of Cyperaceae species from phytochemical, pharmacological, and chemotaxonomical points of view. The present study focused on the isolation, structure determination, and pharmacological investigation of constituents from Carex praecox. Twenty-six compounds, including lignans, stilbenes, flavonoids, megastigmanes, chromenes, and phenylpropanoids, were identified from the methanol extract of the plant. Five of these compounds, namely, carexines A-E, are previously undescribed natural products. All compounds were isolated for the first time from C. praecox. The ACE-inhibitory activity of seven stilbenoid compounds was tested, and (-)-hopeaphenol proved to be the most active (IC50 7.7 ± 0.9 µM). The enzyme-kinetic studies revealed a mixed-type inhibition; therefore, domain-specific studies were also conducted. The in silico docking of (-)-hopeaphenol to the ACE affirmed some favorable interactions. In addition, the antiproliferative and antibacterial effects of some compounds were also evaluated.

Production and characterization of anti-hypertensive and anti-diabetic peptides from fermented sheep milk with anti-inflammatory activity: in vitro and molecular docking studies.

BACKGROUND: The present study aimed to evaluate the anti-hypertensive and anti-diabetic activities from biologically active peptides produced by fermented sheep milk with Lacticaseibacillus paracasei M11 (MG027695), as well as to purify and characterize the angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic peptides produced from fermented sheep milk. RESULTS: After 48 h of fermentation at 37 °C, sheep milk demonstrated significant changes in anti-diabetic effects and ACE-I effects, with inhibition percentages observed for ACE inhibition (76.32%), α-amylase (70.13%), α-glucosidase (70.11%) and lipase inhibition (68.22%). The highest level of peptides (9.77 mg mL-1) was produced by optimizing the growth conditions, which included an inoculation rate of 2.5% and a 48 h of incubation period. The comparison of molecular weight distributions among protein fractions was conducted through sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis, whereas spots were separated using 2D gel electrophoresis according to both the molecular weight and pH. Peptide characterization with ultra-filtration membranes at 3 and 10 kDa allowed the study to assess molecular weight-based separation. Nitric oxide generated by lipopolysaccharide and the secretion of pro-inflammatory cytokines in RAW 264.7 immune cells were both inhibited by sheep milk fermented with M11. Fourier-transform infrared spectroscopy was employed to assess changes in functional groups after fermentation, providing insights into the structural changes occurring during fermentation. CONCLUSION: The present study demonstrates that fermentation with L. paracasei (M11) led to significant changes in fermented sheep milk, enhancing its bioactive properties, notably in terms of ACE inhibition and anti-diabetic activities, and the generation of peptides with bioactive properties has potential health benefits. © 2024 Society of Chemical Industry.

Purification of angiotensin converting enzyme inhibitory peptides and antihypertensive effect generated from Indonesian traditional fermented beef (Cangkuk).

OBJECTIVE: Traditional fermented meat products can be considered a source of bioactive peptides. Cangkuk, a traditional Indonesian fermented beef product is one source of angiotensin converting enzyme (ACE) inhibitory peptides. This study aimed to identify ACE-inhibitory peptides from Cangkuk and analyze their antihypertensive effects. METHODS: The water-soluble fraction of Cangkuk was fractionated to obtain ACE-inhibitory peptides using an ethanol solvent at several concentrations and solid-phase extraction with an OASIS HLB cartridge followed by purification with reversed-phase high-performance liquid chromatography (RP-HPLC). HPLC-MS was used to identify target peptides, followed by automatic protein sequencer analysis to detect peptide sequences. Antihypertensive effects were analyzed on the water-soluble fraction and synthesized peptides. The animal model comprised 14-16-week-old male spontaneously hypertensive rats (SHRs) (~320 g average body weight) with mean systolic blood pressures (SBPs) higher than 190 mmHg. All oral doses of peptides were 1 mL in volume. Distilled water was used as a control. The antihypertensive activities of the sample and control were observed by measuring the SBP at 0, 2, 4, 6, 8 and 24 h after oral administration. RESULTS: Two sequences of ACE inhibitory peptides were found, EAPLNPKANR (IC50 value of 44.6 µmol/L) and IVG (IC50 value of 97.3 µmol/L). The water-soluble fraction demonstrated an antihypertensive effect on SHRs after oral administration at 100 mg/kg body weight, maximally lowering the SBP by 14.9 mmHg 8 h after administration. The tripeptide IVG showed the highest reduction of SBP, 24.76±2.1 mmHg 8 h after administration. The decapeptide EAPLNPKANR showed the highest reduction of SBP, 21.0±1.9 mmHg, 8 h after administration. All the samples differed significantly from the control (p<0.01). CONCLUSION: Cangkuk has potential as a functional food ingredient acting as an antihypertensive agent.

Production of Antioxidant, Angiotensin-Converting Enzyme Inhibitory and Osteogenic Gelatin Hydrolysate from Labeo rohita Swim Bladder.

Optimization of antioxidants and angiotensin-converting enzyme (ACE) inhibitory potential gelatin hydrolysate production from Labeo rohita (rohu) swim bladder (SBGH) by alcalase using central composite design (CCD) of response surface methodology (RSM) was investigated. The maximum degree of hydrolysis (DH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), total antioxidants (TAO), and ACE inhibitory activity were achieved at 0.1:1.0 (w/w) enzyme to substrate ratio, 61 °C hydrolysis temperature, and 94-min hydrolysis time. The resulting SBGH obtained at 19.92% DH exhibited the DPPH (24.28 µM TE/mg protein), ABTS (34.47 µM TE/mg protein), TAO (12.01 µg AAE/mg protein), and ACE inhibitory (4.91 µg/mg protein) activity. Furthermore, SBGH at 100 µg/ml displayed osteogenic property without any toxic effects on MC3T3-E1 cells. Besides, the protein content of rohu swim bladder gelatin (SBG) and SBGH was 93.68% and 94.98%, respectively. Both SBG and SBGH were rich in glycine, proline, glutamic acid, alanine, arginine, and hydroxyproline amino acids. Therefore, SBGH could be an effective nutraceutical in functional food development.

Synchronous reducing anti-nutritional factors and enhancing biological activity of soybean by the fermentation of edible fungus Auricularia auricula.

Auricularia auricula fermentation was performed to reduce anti-nutritional factors, improve nutritional components, and enhance biological activity of soybean. Results showed that the contents of raffinose, stachyose, and trypsin inhibitor were significantly decreased from initial 1.65 g L-1, 1.60 g L-1, and 284.67 µg g-1 to 0.14 g L-1, 0.35 g L-1, and 4.52 µg g-1 after 144 h of fermentation, respectively. Simultaneously, the contents of polysaccharide, total phenolics, and total flavonoids were increased, and melanin was secreted. The isoflavone glycosides were converted to their aglycones, and the contents of glyctin and genistin were decreased from initial 1107.99 µg g-1 and 2852.26 µg g-1 to non-detection after 72 h of fermentation, respectively. After 96 h of fermentation, the IC50 values of samples against DPPH and ABTS radicals scavenging were decreased from 17.61 mg mL-1 and 3.43 mg mL-1 to 4.63 mg mL-1 and 0.89 mg mL-1, and those of samples inhibiting α-glucosidase and angiotensin I-converting enzyme were decreased from 53.89 mg mL-1 and 11.27 mg mL-1 to 18.24 mg mL-1 and 6.78 mg mL-1, respectively, indicating the significant increase in these bioactivities. These results suggested A. auricula fermentation can enhance the nutritional quality and biological activity of soybean, and the fermented soybean products have the potential to be processed into health foods/food additives.

Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze ß-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

Targeted Affinity Purification and Mechanism of Action of Angiotensin-Converting Enzyme (ACE) Inhibitory Peptides from Sea Cucumber Gonads.

Protein hydrolysates from sea cucumber (Apostichopus japonicus) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC50 value of 333.5 µmol·L-1, HDWWKER with an IC50 value of 583.6 µmol·L-1, and THDWWKER with an IC50 value of 1291.8 µmol·L-1) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔGtotal) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol-1, -71.6 Kcal·mol-1, and -69.1 Kcal·mol-1, respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.

Effect of Conventional Humid-Dry Heating through the Maillard Reaction on Chemical Changes and Enhancement of In Vitro Bioactivities from Soy Protein Isolate Hydrolysate-Yeast Cell Extract Conjugates.

This study investigated the formation of soy protein isolate hydrolysate-yeast cell extract (SPIH-YCE) conjugates through a humid-dry heating process and their impact on bioactivity. The incubation of SPIH-YCE samples at 60 °C and ~75% humidity for varying durations (0, 5, 10, 15, and 20 days) resulted in a significant decrease in reducing sugars and free amino acids, while the degree of glycation increased by approximately 65.72% after 10 days. SDS-PAGE analysis and size exclusion chromatography revealed the presence of peptides and glycoprotein molecules, with an increase in the distribution of larger peptide size chains. The conjugated SPIH-YCE (10 days) exhibited the highest antioxidant capacity compared to the other samples at different incubation times. A comparative study between SPIH-YCE (day 0) and SPIH-YCE after 10 days of incubation showed significantly higher anti-inflammatory and ACE inhibitory activities for the conjugates subjected to the humid-dry heating process. This suggests that SPIH-YCE conjugates could serve as an alternative substance with the potential to provide health benefits by mitigating or preventing non-communicable diseases (NCDs). This research highlights the importance of the Maillard reaction in enhancing bioactivity and offers insights into the alterations of the chemical structure of these conjugates.

Characterization of fermented pomegranate juice: ACE inhibitory activity under in vitro digestion, antioxidant capacity, phenolics composition, chemical properties and sensory evaluation.

Consuming pomegranate juice (PJ) is beneficial for hypertensive regulation because of the phenolic compounds in PJ and their inhibitory activity on angiotensin-I-converting enzyme (ACE). To better utilize bioactive function of food, microorganism fermentation has been adopted to alter phenolic metabolism. This study confirms that even under in vitro digestion, fermented PJ (FPJ) maintains higher ACE inhibitory activity than that of PJ. The main phenolic compounds in PJ were compared either under fermentation or in vitro digestion. This study finds that fermentation promotes antioxidant capacity of PJ. The chemical properties of FPJ are evaluated and the corresponding relationship with bioactivities is analyzed. A sensory evaluation comparison is conducted between FPJ and PJ, furnishing interesting information for consumers. This study highlights the relationship between ACE inhibitory activity of PJ and phenolic composition under fermentation and in vitro digestion, providing novel insights for diet regulation of phenolic-rich FPJ in ACE inhibition therapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01388-w.

Lactic acid fermentation of kamaboko, a heated Alaska pollock surimi, enhances angiotensin I-converting enzyme inhibitory activity via fish protein hydrolysis.

To enhance the value of surimi, efforts have been made to develop a fermentation method with lactic acid bacteria (LAB) to proteolyze fish protein. However, fermenting unheated surimi poses a spoilage risk due to its high bacterial content. Surimi heat treatment can prevent spoilage, but gel formation induced by heating introduces another technical issue: it hinders uniform fermentation. Thus, this study aims to observe the proteolysis and enhance the functionality of seafood product through lactic acid fermentation of kamaboko, a heated surimi. Upon analyzing the kamaboko fermented with Lactobacillus helveticus JCM1004, we observed that LAB produced protease, resulting in the degradation of myosin heavy chain and actin during fermentation. Lactic acid fermentation significantly augmented the peptide content of kamaboko, subsequently elevating the angiotensin Ⅰ-converting enzyme (ACE) inhibitory activity in 200-fold diluted extract of fermented kamaboko to approximately 70% and higher. Notably, our investigation revealed that proteolysis was confined to the surface of kamaboko, as evidenced by SDS-PAGE analysis. This observation implies that the surface area of kamaboko influences the ACE inhibitory activity. Through a comparative analysis of various bacterial strains, we demonstrated that the increase in ACE inhibitory activity is contingent on the protease generated by LAB. These results suggest that LAB-mediated proteolysis of fish proteins liberates bioactive peptides, thereby manifesting in the ACE inhibitory activity. In summary, this study underscores that the fermentation of kamaboko employing proteolytic LAB holds promise in the development of novel functional seafood products.

Antihypertensive Effect, ACE Inhibitory Activity, and Stability of Umami Peptides from Yeast Extract.

Bioactive peptides from foods have garnered considerable attention as viable supplements for hypertensive patients. Herein, the antihypertensive effect and mechanism of umami peptides from yeast extract were investigated based on the pharmacophore model, simulated digestion, spontaneously hypertensive rat (SHR) model, and molecular docking. Notably, umami peptide LLLLPKP exhibited favorable angiotensin I-converting enzyme (ACE) inhibitory activity (IC50 = 10.22 µM) in vitro and regulated blood pressure in the SHR model with excellent durability. Remarkably, LLLLPKP showed the highest Fitvalue (4.022) of the pharmacophore model, indicating its similar pharmacological effects as ACE inhibitors. During the simulated gastrointestinal digestion, the ACE inhibition rate of LLLLPKP was merely reduced by 5.89%, but it was enzymatically cleaved into 14 peptide segments. The C-terminal sequence comprising L (4), P (5), K (6), and P (7) exhibited robust stability and a notable presence within the peptide segments postdigestion. Meanwhile, according to molecular docking, these four residues within LLLLPKP were responsible for all interactions with key sites within active pockets S1 and S2 and the active pocket of Zn2+. In light of these findings, LLLLPKP is a highly promising antihypertensive peptide. Developing this umami peptide with antihypertensive effects holds substantial importance for the long-term treatment of hypertension.

Affinity Purification and Molecular Characterization of Angiotensin-Converting Enzyme (ACE)-Inhibitory Peptides from Takifugu flavidus.

An affinity chromatography filler of CNBr-activated Sepharose 4B-immobilized ACE was used to purify ACE-inhibitory peptides from Takifugu flavidus protein hydrolysate (<1 kDa). Twenty-four peptides with an average local confidence score (ALC) ≥ 80% from bounded components (eluted by 1 M NaCl) were identified by LC-MS/MS. Among them, a novel peptide, TLRFALHGME, with ACE-inhibitory activity (IC50 = 93.5 µmol·L-1) was selected. Molecular docking revealed that TLRFALHGME may interact with the active site of ACE through H-bond, hydrophobic, and electrostatic interactions. The total binding energy (ΔGbinding) of TLRFALHGME was estimated to be -82.7382 kJ·mol-1 by MD simulations, indicating the favorable binding of peptides with ACE. Furthermore, the binding affinity of TLRFALHGME to ACE was determined by surface plasmon resonance (SPR) with a Kd of 80.9 µmol, indicating that there was a direct molecular interaction between them. TLRFALHGME has great potential for the treatment of hypertension.

Investigation of the Elemental Contents, Functional and Nutraceutical Properties of Kefirs Enriched with Spirulina platensis, an Eco-friendly and Alternative Protein Source.

In this study, the effect of the use of S. platensis, which is presented as an eco-friendly and alternative protein source, in the production of kefir, a probiotic dairy product, on various physicochemical properties as well as FAA, ACE inhibitory activity, proteolysis, TPC, DPPH, ABTS+, and mineral values was investigated. It was observed that the addition of S. platensis at different ratios to the kefir samples had a statistically very significant (p < 0.01) effect on all physicochemical analyses; L. mesenteroides count; all amino acids except isoleucine, aspartic acid, and glutamic acid; ACE inhibitory activity, TN, TCAN, TCAN/TN, mM Gly, TPC, DPPH, ABTS+, Na, Mg, K, and Fe. In plain kefir samples, mineral contents were determined by order of K > P > Na > Ca > Mg > Zn >> Fe > Cr > Cr > Mn. Furthermore, a general increase was observed in FAA, ACE inhibitory activity, TPC, DPPH, ABTS+, and mineral values, as well as in the counts of Lactococcus spp. and L. mesenteroides in the samples, depending on the proportion of S. platensis added, compared to plain kefir samples. In this context, it was concluded that the addition of S. platensis to kefir at different rates could meet various components required by the body on a daily basis and result in a nutraceutical product.

Comparison of Amino Acid Profile, ACE Inhibitory Activity, and Organic Acid Profile of Cow and Goat Yogurts Produced with Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12, and Classical Yogurt Culture.

In this study, we aimed to produce a standard, more functional, and nutritious yogurt by using 5 different combinations of cow milk and goat milk and 2 types of starter cultures (classical yogurt culture and commercial probiotic culture). It was determined that the use of different milk types and different starter cultures in yogurt production had a statistically very significant effect (P < 0.01) on all physicochemical, microbiological, and biochemical properties. In addition, the storage period was effective on all parameters examined at varying rates. In the context, the use of goat milk in the experimental yogurt samples caused an increase in the ACE inhibitory activity values and the count of S. thermophilus, while the use of cow milk caused an increase in serum separation and pH values. On the other hand, serum separation, pH values, and ACE inhibitory activity and phenylalanine and leucine levels were found to be higher in the yogurts produced by using ABT-2 probiotic culture. It was observed that an increase in the levels of asparagine, aspartic acid, proline, and serine, as well as lactic acid, orotic acid, and citric acid, is higher in the yogurts produced by using classical yogurt culture. It has been concluded that the combination of goat milk and cow milk at different proportions and the use of probiotic culture together in yogurt production can produce yogurt that is more functional and richer in terms of organic compounds and essential amino acids.

Impact of Harvest Month and Drying Process on the Nutritional and Bioactive Properties of Wild Palmaria palmata from Atlantic Canada.

The macroalga Palmaria palmata could be a sustainable and nutritional food resource. However, its composition may vary according to its environment and to processing methods used. To investigate these variations, wild P. palmata from Quebec were harvested in October 2019 and June 2020, and dried (40 °C, ≃5 h) or stored as frozen controls (-80 °C). The chemical (lipids, proteins, ash, carbohydrates, fibers), mineral (I, K, Na, Ca, Mg, Fe), potential bioactive compound (carotenoids, polyphenols, ß-carotene, α-tocopherol) compositions, and the in vitro antioxidant activity and angiotensin-converting enzyme (ACE) inhibition potential of water-soluble extracts were determined. The results suggested a more favorable macroalgae composition in June with a higher content of most nutrients, minerals, and bioactive compounds. October specimens were richer only in carbohydrates and carotenoids. No significant differences in antioxidant or anti-ACE inhibitory activities were found between the two harvest months. The drying process did not significantly impact the chemical and mineral compositions, resulting in only small variations. However, drying had negative impacts on polyphenols and anti-ACE activities in June, and on carotenoids in October. In addition, a concentration effect was observed for carotenoids, ß-carotene and α-tocopherol in June. To provide macroalgae of the highest nutritional quality, the drying process for June specimens should be selected.

ACE inhibitory activity and salt-reduction properties of umami peptides from chicken soup.

Angiotensin-I-converting enzyme (ACE) inhibitory activity and salt-reduction properties of umami peptides identified in chicken soup were investigated. The ACE inhibition rate of TPLVDR (91.22%) and AEINKILGN (81.26%) was significantly higher than other umami peptides, and their semi-inhibitory concentration was 0.017 mM and 0.034 mM, respectively. After in vitro digestion, the inhibitory activity of AEINKILGN and TPLVDR decreased, but the original sequences were still detected. The docking results showed that AEINKILGN and TPLVDR mainly interacted with Zn2+ and key sites (His353, Lys511and Glu411) in the active pockets of ACE through hydrogen bonds, which was crucial to the ACE inhibitory activity. Based on response surface methodology and sensory analysis, saltiness and palatability models were established to investigate the salt-reduction effect. The optimal level of AEINKILGN was about 1.16 mg/mL in 0.44% salt solution. And the TPLVDR was applicable to the low salt solution (0.1-0.2%) at a concentration from 0.23 to 0.29 mg/mL.

Identification and Characterization of Novel ACE Inhibitory and Antioxidant Peptides from Sardina pilchardus Hydrolysate.

Sardina pilchardus is a valuable source of bioactive peptides with potential applications in functional foods. In this study, we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of Sardina pilchardus protein hydrolysate (SPH) produced using dispase and alkaline protease. Our results showed that the low molecular mass fractions (<3 kDa) obtained through ultrafiltration exhibited more effective ACE inhibition, as indicated by screening with ACE inhibitory activity. We further identified the low molecular mass fractions (<3 kDa) using an LC-MS/MS rapid screening strategy. A total of 37 peptides with potential ACE inhibitory activity were identified based on high biological activity scores, non-toxicity, good solubility, and novelty. Molecular docking was used to screen for peptides with ACE inhibitory activity, resulting in the identification of 11 peptides with higher -CDOCKER ENERGY and -CDOCKER INTERACTION ENERGY scores than lisinopril. The sequences FIGR, FILR, FQRL, FRAL, KFL, and KLF were obtained by synthesizing and validating these 11 peptides in vitro, all of which had ACE inhibitory activity, as well as zinc-chelating capacity. All six peptides were found to bind to the three active pockets (S1, S2, and S1') of ACE during molecular docking, indicating that their inhibition patterns were competitive. Further analysis of the structural characteristics of these peptides indicated that all six peptides contain phenylalanine, which suggests that they may possess antioxidant activities. After experimental verification, it was found that all six of these peptides have antioxidant activities, and we also found that the SPH and ultrafiltration fractions of SPH had antioxidant activities. These findings suggest that Sardina pilchardus may be a potential source of natural antioxidants and ACE inhibitors for the development of functional foods, and using LC-MS/MS in combination with an online database and molecular docking represents a promising, effective, and accurate approach for the discovery of novel ACE inhibitory peptides.

Investigating structure, biological activity, peptide composition and emulsifying properties of pea protein hydrolysates obtained by cell envelope proteinase from Lactobacillus delbrueckii subsp. bulgaricus.

We present the structure, biological activity, peptide composition, and emulsifying properties of pea protein isolate (PPI) after hydrolysis by cell envelope proteinase (CEP) from Lactobacillus delbrueckii subsp. bulgaricus. Hydrolysis resulted in the unfolding of the PPI structure, characterized by an increase in fluorescence and UV absorption, which was related to thermal stability as demonstrated by a significant increase in ΔH and the thermal denaturation temperature (from 77.25 ± 0.05 to 84.45 ± 0.04 °C). The hydrophobic amino acid of PPI significantly increased from 218.26 ± 0.04 to 620.77 ± 0.04 followed by 557.18 ± 0.05 mg/100 g, which was related to their emulsifying properties, with the maximum emulsifying activity index (88.62 ± 0.83 m2/g, after 6 h hydrolysis) and emulsifying stability index (130.77 ± 1.12 min, after 2 h hydrolysis). Further, the results of LC-MS/MS analysis demonstrated that the CEP tended to hydrolyze peptides with an N-terminus dominated by Ser and a C-terminus dominated by Leu, which enhanced the biological activity of pea protein hydrolysates, as supported by their relatively high antioxidant (ABTS+ and DPPH radical scavenging rates were 82.31 ± 0.32% and 88.95 ± 0.31%) and ACE inhibitory (83.56 ± 1.70%) activities after 6 h of hydrolysis. 15 peptide sequences (score > 0.5) possessed both antioxidant and ACE inhibitory activity potential according to the BIOPEP database. This study provides theoretical guidance for the development of CEP-hydrolyzed peptides with antioxidant and ACE inhibitory activity that can be used as emulsifiers in functional foods.