Improvement of sleep duration and quality through GABAA receptor by whey protein hydrolysate containing DIQK as the active main compound.

This study characterized the sleep activity, sleep mechanism, and active peptides of whey protein hydrolysates selected through behavioral analysis of fruit-flies (Drosophila melanogaster). Sleep-inducing whey protein (WP) hydrolysate was selected through fruit fly behavior analysis, and sleep activity was measured using a pentobarbital model and electroencephalographic analysis. The mechanism of action was confirmed using a γ-aminobutyric acid (GABA) receptor antagonist, and the active peptide was identified using liquid chromatography-mass spectroscopy. Whey protein hydrolysate, prepared using Alcalase and Prozyme (WP-AP), increased sleep time in a dose-dependent manner. WP-AP significantly increased not only sleep time but also slow-wave sleep and showed an insomnia-alleviating effect in a caffeine-induced insomnia mouse model. In addition, the gene and protein expression levels of GABA sub-type A (GABAA) receptors increased in the brains of mice orally administered with WP-AP. Through peptide analysis, the mixture of DIQK, VPPF peptide, and GABA contained in WP-AP was estimated to exhibit sleep activity, and due to its high content, DIQK was speculated to be the main sleep -inducing ingredient. These results indicate that WP-AP has the potential to be used as a new ingredient to improve sleep quality.

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.

Protein hydrolysates prepared by Alcalase using ultrasound and microwave pretreated almond meal and their characterization.

The study aimed to optimize ultrasonic (US: 40 kHz/200 W for 10, 20, 30, 40, and 50 min), and microwave (MW: 160 W for 45, 90, 125, 180, and 225 s) pretreatment conditions on protein extraction yield and degree of protein hydrolysis (DH) from almond de-oiled meal, an industrial by-product. First order model was used to describe the kinetics of almond protein hydrolysates obtained with Alcalase. The highest DH, 10.95% was recorded for the US-50 min and 8.87% for MW-45 s; while it was 5.76% for the untreated/control sample. At these optimized pretreatment conditions, a 1.16- and 1.18-fold increment in protein recovery was observed for the US and MW pretreatments, respectively in comparison to the conventional alkaline extraction. The molecular weight distribution recorded for pretreated samples disclosed a significant reduction in the band thickness in comparison with control. Both the pretreatments resulted in a significant increase (P < 0.05) in the antioxidant activity, and TCA solubility index when compared with the control. Results evinced that US and/or MW pretreatments before enzymatic hydrolysis can be a promising approach for the valorization of almond meal for its subsequent use as an ingredient for functional foods/nutraceuticals which otherwise fetches low value as an animal feed.

Alcalase-Assisted Mytilus edulis Hydrolysate: A Nutritional Approach for Recovery from Muscle Atrophy.

Muscle atrophy is a complex physiological condition caused by a variety of reasons, including muscle disuse, aging, malnutrition, chronic diseases, immobilization, and hormonal imbalance. Beyond its effect on physical appearance, this condition significantly reduces the quality of human life, thus warranting the development of preventive strategies. Although exercising is effective in managing this condition, it is applicable only for individuals who can engage in physical activities and are not bedridden. A combination of exercise and nutritional supplementation has emerged as a more advantageous approach. Here, we evaluated the effects of enzyme-assisted hydrolysates of Mytilus edulis prepared using Protamex (PMH), Alcalase (AMH), or Flavourzyme (FMH) in protecting against muscle atrophy in a dexamethasone (Dex)-induced muscular atrophy model in vitro and in vitro. Alcalase-assisted M. edulis hydrolysate (AMH) was the most efficient among the tested treatments and resulted in higher protein recovery (57.06 ± 0.42%) and abundant amino acid composition (43,158 mg/100 g; 43.16%). AMH treatment also escalated the proliferation of C2C12 cells while increasing the total number of nuclei, myotube coverage, and myotube diameter. These results were corroborated by a successful reduction in the levels of proteins responsible for muscle atrophy, including E3 ubiquitin ligases, and an increase in the expression of proteins associated with muscle hypertrophy, including myogenin and MyHC. These results were further solidified by the successful enhancement of locomotor ability and body weight in zebrafish following AMH treatment. Thus, these findings highlight the potential of AMH in recovery from muscle atrophy.

The Preparation and Physiochemical Characterization of Tenebrio molitor Chitin Using Alcalase.

Chitin is mostly produced from crustaceans, but it is difficult to supply raw materials due to marine pollution, and the commonly used chemical chitin extraction method is not environmentally friendly. Therefore, this study aims to establish a chitin extraction process using enzymes and to develop edible insect-derived chitin as an eco-friendly new material. The response surface methodology (RSM) was used to determine the optimal conditions for enzymatic hydrolysis. The optimal conditions for enzymatic hydrolysis by RSM were determined to be the substrate concentration (7.5%), enzyme concentration (80 µL/g), and reaction time (24 h). The solubility and DDA of the mealworm chitosan were 45% and 37%, respectively, and those of the commercial chitosan were 61% and 57%, respectively. In regard to the thermodynamic properties, the exothermic peak of mealworm chitin was similar to that of commercial chitin. In the FT-IR spectrum, a band was observed in mealworm chitin corresponding to the C=O of the NHCOCH3 group at 1645 cm-1, but this band showed low-intensity C=O in the mealworm chitosan due to deacetylation. Collectively, mealworm chitosan shows almost similar physical and chemical properties to commercial chitosan. Therefore, it is shown that an eco-friendly process can be introduced into chitosan production by using enzyme-extracted mealworms for chitin/chitosan production.

Protective effects of tree peony seed protein hydrolysate on Cd-induced oxidative damage, inflammation and apoptosis in zebrafish embryos.

The objective of this study was to investigate protective effects of tree peony seed protein hydrolysate by Alcalase (AL-TPSPH) on oxidative damage, inflammation and apoptosis using Cd-induced zebrafish embryos. Zebrafish embryos were treated with either Cd (2 µg/L) or AL-TPSPH (25, 50 and 75 µg/mL) alone or in combination of both from 4 to 144 h post fertilization (hpf). The effects of these treatments on developments, antioxidant parameters and mRNA expression of genes related to oxidative damage, inflammation and apoptosis were examined. The results showed that co-treatment with Cd and AL-TPSPH significantly increased hatching and survival rates and decreased malformation rates of zebrafish embryos compared with Cd treatment alone group (P < 0.05). Cd-induced increase of MDA content, decreases of T-AOC content, GSH/GSSG ratio and activities of SOD, CAT and GPx in zebrafish embryos were modified upon treatment with AL-TPSPH. AL-TPSPH treatment significantly suppressed Cd-induced down-regulations of the antioxidant gene expressions (Mn-sod, Cat and GPx1a) in zebrafish embryos (P < 0.05). AL-TPSPH also prevented Cd-induced up-regulations of pro-inflammatory cytokine (TNF-α, IL-1ß and IFN-γ) expressions. Moreover, AL-TPSPH inhibited Cd-induced up-regulations of pro-apoptotic genes (C-jun, Caspase-3 and Caspase-9) in zebrafish embryos. Collectively, these results indicated that AL-TPSPH could reduce Cd-induced oxidative damage, inflammation and apoptosis in zebrafish embryos, suggesting its future applications as functional food or pharmaceutical ingredient.

Recycled Sericin Hydrolysates Modified by Alcalase® Suppress Melanogenesis in Human Melanin-Producing Cells via Modulating MITF.

Because available depigmenting agents exhibit short efficacy and serious side effects, sericin, a waste protein from the silk industry, was hydrolyzed using Alcalase® to evaluate its anti-melanogenic activity in human melanin-producing cells. Sericin hydrolysates consisted of sericin-related peptides in differing amounts and smaller sizes compared with unhydrolyzed sericin, as respectively demonstrated by peptidomic and SDS-PAGE analysis. The lower half-maximum inhibitory concentration (9.05 ± 0.66 mg/mL) compared with unhydrolyzed sericin indicated a potent effect of sericin hydrolysates on the diminution of melanin content in human melanoma MNT1 cells. Not only inhibiting enzymatic activity but also a downregulated expression level of tyrosinase was evident in MNT1 cells incubated with 20 mg/mL sericin hydrolysates. Quantitative RT-PCR revealed the decreased mRNA level of microphthalmia-associated transcription factor (MITF), a tyrosinase transcription factor, which correlated with the reduction of pCREB/CREB, an upstream cascade, as assessed by Western blot analysis in MNT1 cells cultured with 20 mg/mL sericin hydrolysates for 12 h. Interestingly, treatment with sericin hydrolysates for 6-24 h also upregulated pERK, a molecule that triggers MITF degradation, in human melanin-producing cells. These results warrant the recycling of wastewater from the silk industry for further development as a safe and effective treatment of hyperpigmentation disorders.

Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers.

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO2-G0Si-NH2 and SiO2-G1Si-NH2), a CBS dendrimer with a polyphenoxo core (SiO2-G1O3-NH2), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO2-G0PAMAM-NH2 and SiO2-G1PAMAM-NH2). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO2 ratio, and presence of Ca2+ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO2-G0PAMAM-NH2. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.

Response surface optimization of enzymatic hydrolysis and ROS scavenging activity of silk sericin hydrolysates.

CONTEXT: Sericin, a protein found in wastewater from the silk industry, was shown to contain a variety of biological activities, including antioxidant. The enzymatic conditions have been continuously modified to improve antioxidant effect and scavenging capacity against various free radicals of silk sericin protein. OBJECTIVE: Variables in enzymatic reactions, including pH, temperature and enzyme/substrate ratio were analysed to discover the optimum conditions for antioxidant activity of sericin hydrolysates. MATERIALS AND METHODS: Hydrolysis reaction catalysed by Alcalase® was optimized through response surface methodology (RSM) in order to generate sericin hydrolysates possessing potency for % inhibition on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, ferric-reducing power and peroxyl scavenging capacity. Flow cytometry was performed to evaluate cellular ROS level in human HaCaT keratinocytes and melanin-generating MNT1 cells pre-treated either with 20 mg/mL RSM-optimized sericin hydrolysates or 5 mM N-acetyl cysteine (NAC) for 60 min prior exposure with 1 mM hydrogen peroxide (H2O2). RESULTS: Among these three variables, response surface plots demonstrate the major role of temperature on scavenging capacity of sericin hydrolysates. Sericin hydrolysates prepared by using Alcalase® at RSM-optimized condition (enzyme/substrate ratio: 1.5, pH: 7.5, temperature: 70 °C) possessed % inhibition against H2O2 at 99.11 ± 0.54% and 73.25 ± 8.32% in HaCaT and MNT1 cells, respectively, while pre-treatment with NAC indicated the % inhibition only at 30.26 ± 7.62% in HaCaT and 51.05 ± 7.14% in MNT1 cells. DISCUSSION AND CONCLUSIONS: The acquired RSM information would be of benefit for further developing antioxidant peptide from diverse resources, especially the recycling of waste products from silk industry.

Significance of whey protein hydrolysate on anti-oxidative, ACE-inhibitory and anti-inflammatory activities and release of peptides with biofunctionality: an in vitro and in silico approach.

The study aimed to investigate potent antioxidant activities (ABTS assay, Hydroxyl free radical scavenging assay, and Superoxide free radical assay), ACE inhibitory activity, and anti-inflammatory activity in the WPC (whey protein concentrate) hydrolysate using Alcalase. The hydrolysis conditions (addition rate and incubation times) for peptide synthesis were also optimized using proteolytic activity. The generation of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages was reduced when the protein hydrolysate concentration was low. In comparison to unhydrolyzed WPC, SDS-PAGE examination revealed no protein bands in WPC hydrolysates. Two-Dimensional (2D) gel electrophoresis did not show any protein spots. Using the 'In-solution trypsin digestion' approach, the trypsin digested protein samples were put into RPLC/MS for amino acid sequencing. Peptides were also identified using RPLC/MS on fractions of 3 and 10 kDa permeates and retentates. The MASCOT database was used to look up the raw masses of LC/MS. By comparing hydrolyzed whey protein to the BLASTp (NCBI), PIR, BIOPEP, and AHTPDB databases, novel antioxidative and ACE inhibitory peptides were reported. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05282-3.

Comparative functional and spectroscopic analysis of spent hen meat hydrolysate by individual and combined treatment of microbial proteases.

Simultaneous (Sm) and sequential (Sq) use of microbial proteases for the hydrolysis of spent hen/chicken meat from antioxidant potential perspective is relatively unexplored and requires attention. In this work, meat was hydrolyzed using Flavourzyme (Fz) and Alcalase (Ac), each at 1, 2, and 3% for 6 h as well as using both enzymes (at 2% each) in Sm and Sq treatment. Maximum attained %DPPH-RSA (Fz:68.25; Ac:77.18; Sm:59.82; and Sq:65.97) and FRAP (mM TEAC/g) values (Fz:3.77; Ac:2.56; Sm:2.54; and Sq:3.37) were measured as a function of hydrolysis time. The highest (23.38%) and lowest (10.68%) degree of hydrolysis (DH) was obtained with 3% Ac and 1% Fz, respectively. FTIR spectroscopy clearly revealed changes in the secondary structure of proteins. SDS PAGE profiling of hydrolysates showed that Fz produces low molecular weight peptides (2-75 kDa) as compared to Ac or its combination with Ac. As per the results of this study, Sq enzyme treatment is recommended for preparing spent hen meat hydrolysate with higher functional attributes for possible use as functional food/nutraceutical.

Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysates from visceral waste of Labeo rohita.

The study aimed to evaluate the extent of hydrolysis on the functional properties of fish protein hydrolysates from fish wastes. Fish protein hydrolysate (FPH) was prepared from the visceral waste of Rohu (Labeo rohita) using Alcalase®. Independent variables of the hydrolysis conditions, namely, time, temperature and enzyme-substrate ratio were optimized through response surface methodology using a completely randomized factorial design. Functional properties of FPH, such as antioxidant activity, metal chelating activity, emulsifying and foaming capacity were studied. Pre-heating of the raw viscera resulted 83% reduction of lipid content in the FPH. The DH was found to be significantly influenced by the hydrolysis conditions studied. The gel electrophoretic study showed that FPH contained peptides in the range of < 10-25 kDa. Optimum degree of hydrolysis (DH) was obtained as 34.7% at 87.5 min, 48.64 °C and enzyme concentration of 0.99%. The results demonstrated that the antioxidant and metal chelating properties of FPH increased with the increase of DH. The surface active properties like emulsifying and foaming capacity were found to be decreased with the increased levels of hydrolysis. These results suggest that hydrolysates from visceral waste of Rohu could find potential use as supplement in animal feed.

Effect of proteases and alcohols used for debittering on characteristics and antioxidative activity of protein hydrolysate from salmon frames.

Protein hydrolysates were obtained from salmon frame using Alcalase or Flavourzyme at 3% (w/w protein) for 180 min. Protein hydrolysates prepared using Alcalase (HA) and Flavourzyme (HF) had DH and yield of 25.1-26.9% and 28.5-32.3 g/100 g sample, respectively. HF showed lower bitterness score (5.78) than that of HA (8.68) (P < 0.05). When HA and HF were further subjected to debittering with 2-butanol or isopropanol, the recovery of 77.88-81.60% was obtained (P < 0.05). HF and HA debittered with 2-butanol possessed less bitterness score, 3.60 and 3.77, respectively (P < 0.05). Surface hydrophobicity of 81.4 and 124.8 was attained when HF and HA were debittered with 2-butanol (P < 0.05). Selected debittered hydrolysates, produced using Flavourzyme, followed by fractionation using 2-butanol (HF-B) contained glutamic acid/glutamine (15.14 g/100 g), aspartic acid/asparagine (10.07 g/100 g) and glycine (9.30 g/100 g) as the predominant amino acids. HF-B had the decreased ABTS radical scavenging activity and metal chelating activity. A280 of peptides separated by gel filtration was lowered to some extent and coincided with the lower bitterness score and surface hydrophobicity. Thus, debittered protein hydrolysate from salmon frame could serve as a nutritive ingredient at high levels in health promoting foods.

Enzymatically excised oligopeptides from Bellamya bengalensis shows potent antioxidative and anti-hypertensive activity.

ABSTRACT: Bellamya bengalensis, an edible mollusca, serves as a protein rich food source for the tribes in India. The objective of the present study was to isolate the protein fraction of the edible foot part of B. bengalensis for hydrolysis with three proteases, namely papain, pepsin, and alcalase. B. bengalensis protein isolates and hydrolysates were characterised for the functional properties like protein solubility index, emulsifying property, foaming property. The proximate composition of the protein isolate was determined along with nutritional value that included biological value, protein efficiency ratio, amino acid score, nutritional index, essential amino acid index. The molecular weight distribution of the protein isolate and the three hydrolysates were analyzed by SDS-PAGE. The hydrolysates were fractionated by ultrafiltration and the in vitro antioxidative properties were measured. The antihypertensive property of the in vitro angiotensin converting enzyme inhibitory activity of the hydrolysates was compared with the standard drug lisinopril. Thus, the results indicated that the hydrolyzed peptides had potent antioxidative and antihypertensive activity. The enzyme pepsin and papain produced partially hydrolyzed peptides suitable for use in the bakery industry while alcalase hydrolysis resulted in shorter peptides with the antihypertensive activity that may be used as a promising nutraceutical.

Purification and the secondary structure of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the alcalase hydrolysate of seahorse protein.

Bioactive peptides with blood pressure-lowering functions have received increasing attention. In recent years, many ACE-inhibiting peptides have been widely purified from various food-derived proteins and have received considerable interest owing to their potential role in cardiovascular diseases and in the reduction of side effects. In this study, we hydrolyzed a three-spot seahorse (Hippocampus trimaculatus Leach) protein by alcalase to obtain a hydrolysate containing angiotensin I-converting enzyme (ACE) inhibitory peptide. Then, the hydrolysate was fractionated by dialysis, Sephadex G-25 gel filtration chromatography, and reverse-phase high performance liquid chromatography. After consecutive purification, a potent ACE-inhibiting peptide composed of 8 amino acids (Pro-Ala-Gly-Pro-Arg-Gly-Pro-Ala; MW: 721.39 Da; IC50 value: 7.90 µM) was successfully isolated from three-spot seahorse protein. For the first time, a novel ACE-inhibiting peptide (PAGPRGPA) was isolated from the seahorse. Circular dichroism (CD) analyses suggested that the secondary structure of the purified peptide was mainly composed of random coil. Therefore, the peptide from seahorse protein may be used as a favorable ingredient in nutraceuticals, medicines, and functional foods against antihypertensive and related diseases.

Impact of sequential enzymatic hydrolysis on antioxidant activity and peptide profile of casein hydrolysate.

ABSTRACT: This paper shows the potential of dual enzyme approach on antioxidant activity of casein hydrolysates. Casein was hydrolysed using the proteolytic enzymes alcalase, flavourzyme in isolation and in sequential order. Casein hydrolysates were evaluated for the degree of hydrolysis, antioxidant activity, molecular weight distribution patterns and peptide sequence. Casein hydrolysate produced by the sequential hydrolysis of alcalase and flavourzyme showed higher degree of hydrolysis and antioxidant activity as compared to hydrolysate obtained by individual enzymes. In size exclusion chromatograph of casein hydrolysate S3, peptides with molecular weight of 0.57 kDa share 12% area in total area of chromatogram which was 10 times higher than that of hydrolysate S1 and nearly half of that of hydrolysate S2. On subjecting to HPLC-TOF-ESI separation potential antioxidant peptides were identified. The peptide sequence VLPVPQ along with potential fragments was identified in hydrolysate S1 and S2 and HPHPHLS along with its potential sequence was identified in hydrolysate S1, S2 and S3. Sequential hydrolysis of casein showed better antioxidant activity and peptide profile in less duration as compared to the casein hydrolysate obtained by individual enzyme.

Protective Effects of Novel Antioxidant Peptide Purified from Alcalase Hydrolysate of Velvet Antler Against Oxidative Stress in Chang Liver Cells in Vitro and in a Zebrafish Model In Vivo.

Velvet antler has a long history in traditional medicine. It is also an important healthy ingredient in food as it is rich in protein. However, there has been no report about antioxidant peptides extracted from velvet antler by enzymatic hydrolysis. Thus, the objective of this study was to hydrolyze velvet antler using different commercial proteases (Acalase, Neutrase, trypsin, pepsin, and α-chymotrypsin). Antioxidant activities of different hydrolysates were investigated using peroxyl radical scavenging assay by electron spin resonance spectrometry. Among all enzymatic hydrolysates, Alcalase hydrolysate exhibited the highest peroxyl radical scavenging activity. Alcalase hydrolysate was then purified using ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. The purified peptide was identified to be Trp-Asp-Val-Lys (tetrapeptide) with molecular weight of 547.29 Da by Q-TOF ESI mass spectroscopy. This purified peptide exhibited strong scavenging activity against peroxyl radical (IC50 value, 0.028 mg/mL). In addition, this tetrapeptide showed significant protection ability against AAPH-induced oxidative stress by inhibiting of reactive oxygen species (ROS) generation in Chang liver cells in vitro and in a zebrafish model in vivo. This research suggests that the tetrapeptide derived from Alcalase-proteolytic hydrolysate of velvet antler are excellent antioxidants and could be effectively applied as functional food ingredients and pharmaceuticals.

Sequence Identification of Bioactive Peptides from Amaranth Seed Proteins (Amaranthus hypochondriacus spp.).

Amaranthus hypochondriacus spp. is a commonly grown cereal in Latin America, known for its high protein content. The objective of this study was to separate and identify bioactive peptides found in amaranth seeds through enzymatically-assisted hydrolysis using alcalase and flavourzyme. Hydrolysis was carried out for each enzyme separately and compared to two-step continuous process where both enzymes were combined. The biological activity of the resulting three hydrolysates was analyzed, finding, in general, higher bioactive potential of the hydrolysate obtained in a continuous process (combined enzymes). Its fractions were separated by RP-HPLC, and their bioactivity was analyzed. In particular, two fractions showed the highest biological activity as ACE inhibitors with IC50 at 0.158 and 0.134, thrombin inhibitors with IC50 of 167 and 155, and antioxidants in ABTS assay with SC50 at 1.375 and 0.992 mg/L, respectively. Further sequence analysis of the bioactive peptides was carried out using MALDI-TOF, which identified amino acid chains that have not been reported as bioactive so far. Bibliographic survey allowed identification of similarities between peptides reported in amaranth and other proteins. In conclusion, amaranth proteins are a potential source of peptides with multifunctional activity.

Housefly Pupae-Derived Antioxidant Peptides Exerting Neuroprotective Effects on Hydrogen Peroxide-Induced Oxidative Damage in PC12 Cells.

In this study, two antioxidant peptides were identified and characterized from the alcalase-hydrolysate of housefly (Musca domestica L.) pupae guided by ABTS cation radical scavenging activity. Peptides sequences were identified as DFTPVCTTELGR (DR12, 1338.48 Da) and ARFEELCSDLFR (AR12, 1485.66 Da) using nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both DR12 and AR12 exert strong ABTS cation radical scavenging ability with EC50 values of 0.39 and 0.35 mM, respectively. Moreover, AR12 can effectively protect PC12 cells from oxidative damage induced by hydrogen peroxide (H2O2) by decreasing intracellular reactive oxygen species (ROS) and malonaldehyde (MDA), recovering cellular mitochondrial membrane potential (MMP), and increasing the activity of intracellular superoxide dismutase (SOD). Stability tests suggest that AR12 is competent for the challenge of heating, acid, alkali or simulated gastrointestinal (GI) digestion and exhibits great activity to remove ABTS cation radical. DR12 shows a great stability against heating, but its antioxidative ability declines after being treated with acid, alkali or simulated GI digestion. In general, both DR12 and AR12 identified from housefly pupae hydrolysate stand a chance of being potential antioxidants or precursors to antioxidants and AR12 might be applied in the field of neuroprotection.