Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides

The beneficial effects of food-derived antioxidants in health promotion and disease prevention are being increasingly recognized. Recently, there has been a particular focus on milk-derived peptides; as a source of antioxidants, these peptides are inactive within the sequence of the parent protein but can be released during enzyme hydrolysis. Once released, the peptides have been shown to possess radical scavenging, metal ion chelation properties and the ability to inhibit lipid peroxidation. A variety of methods have been used to evaluate in vitro antioxidant activity, however, there is no standardised methodology, which hinders comparison of data. This review provides an overview on the generation of antioxidative peptides from milk proteins, the proposed mechanisms of protein/peptide induced antioxidant activity, in vitro measurement of antioxidant activity, in vivo evaluation of plasma antioxidant capacity and the bioavailability of antioxidative peptides. The understanding gained from other food proteins is referred to where specific data on milk-derived peptides are limited. The potential applications and health benefits of antioxidant peptides are discussed with a particular focus on the aging population. The regulatory requirements for peptide-based antioxidant functional foods are also considered.     

Improvement of the Antioxidant Activity of Fenugreek Protein Isolates by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Fermentation

This study investigated the antioxidant capacity of fenugreek protein isolate and its improvement by Lc. lactis fermentation through bioactive peptides production and the effect of molecular weight variation on fenugreek fractions antioxidant activity. Fenugreek protein isolate showed a significant increase of antioxidant and radical scavenging activity after 24 h of fermentation, by about 23.7, 42.9 and 40% for respectively antioxidant activity coefficient AAC, DPPH and ABTS radical scavenging activity. FI fermentation led to a hydrolysis of peptide bands with MW?>?35 kDa and a generation of new bands with a MW?<?25 kDa. A significant reduction in molecular-mass distribution of hydrolysates and a great increase of total free amino acids content, especially an increase on isoleucine, leucine, glutamic acid, serine, histidine, glutamine and lysine was noted. The infrared results showed that different reactions may take place after fermentation, and showed an increase of proteins, amides and aromatic compounds. However, fenugreek fraction (F2) with MW 15–50 kDa presented the highest activity instead of fraction (F1) with lower MW. Lc. lactis had the ability to degrade and convert fenugreek proteins into bioactive peptides that contribute positively in the improvement of antioxidant activity of FI and fractions. FI presents a significant antioxidant activity and thus, can be considered as a potential source of high added value natural antioxidants and may be employed as a functional food ingredient with good potential applications in food products.     

Bioactive Peptides from Bovine Milk α-Casein: Isolation,Characterization and Multifunctional properties

α-Casein group of proteins makes up to 65% of the total casein and consists of α_S1- casein_, α_S2- casein and other related proteins. Among all the proteases employed, chymotryptic peptides showed maximum inhibition for angiotensin converting enzyme (ACE). The degree of hydrolysis and release kinetics of the peptides during chymotrypsin hydrolysis was compared with biological activity and the potent peptides fractions were identified. The crude fraction obtained after 110 min of hydrolysis shows multifunctional activities, like ACE inhibition, antioxidant activity, prolyl endopeptidase inhibitory activity and antimicrobial activities. This fraction was further purified by HPLC and sequenced by mass spectra. This fraction constituted peptides with molecular weights of 1,205, 1,718 Da respectively. The sequencing of peptides by MALDI-TOF MS/MS shows sequences QKALNEINQF and TKKTKLTEEEKNRL from α-_S2 casein.     

Hempseed protein hydrolysates’ effects on the proliferation and induced oxidative stress in normal and cancer cell lines

Food proteins from different sources can provide beneficial effects on human health by releasing the bioactive peptides that are integral part of their native structure. In this study, we tested the biological potential of hempseed protein hydrolysates (HPHs) obtained from hempseed cake protein isolate. The HPHs were prepared by enzyme hydrolysis using three different proteases of microbial origin: Alcalase®, Neutrase® and Protamex®. The antioxidant activity of the obtained hydrolysates was determined by oxygen radical absorbance capacity (ORAC) assay, while the proliferative effects on normal (HaCaT) and cancer (HeLa) cells were determined by the CellTiter 96^® AQ_ueous One Solution Reagent (MTS) assay. HPHs showed dose-dependent antiproliferative effects on HeLa cells and stimulatory effects on the proliferation of HaCaT cells. HPH obtained by Neutrase^® (HPH-N) showed the highest antioxidant activity expressed as an ORAC value. The protective effect of HPH-N on H₂O₂-induced oxidative stress in normal and cancer cells was evaluated and 1 mg/mL of HPH-N significantly reduced the formation of intracellular reactive oxygen species (ROS) in both cell lines. The obtained results indicate the benefits of HPHs as potential natural antioxidants for the food industry and contribute to the growing trend of utilizing hempseed by-products.

     

Cleavage of PrePL by Lon promotes growth and pathogenesis in Magnaporthe oryzae

ATP-dependent Lon proteases function in bacterial pathogenesis by regulating the expression of the Type III secretion system; however, little is known about how Lon proteases regulate fungal pathogenesis. We previously investigated Lon-binding proteins involved in fungal pathogenesis that interact with PrePL, the smallest Magnaporthe oryzae Lon-binding protein. Here, we show that Lon cleaves PrePL and produces Pc, an extracellular 11-kDa isoform with catalase and peroxidase activity. The ΔPrePL loss-of-function strain showed stronger sporulation and accelerated disease development, suggesting a temporally specific negative regulatory mechanism controlled by PrePL in disease progression. Neither the truncated Pc, nor the full-length PrePL missing the Lon cleavage site complemented the ΔPrePL phenotype, suggesting that full-length PrePL and Pc both function in fungal development. PrePL targeted to the mitochondria undergoes hydrolysis by Lon to produce Pc, which accumulates in the fungal apoplast. Importantly, recombinant Pc induced plant defence responses and cell death after being infiltrated into selected plant leaves, indicating that it functions as an avirulence factor. This work thus reveals a novel pathogenic factor in the fungal Lon-mediated pathway. Additionally, our results provide new insight into the functions of a full-length protein and its cleaved isoform in fungal pathogenesis.     

Purification and identification of antioxidant peptides from egg white protein hydrolysate

Egg white proteins were hydrolysed separately using five different proteases to obtain antioxidant peptides. The antioxidant activity of egg white protein hydrolysates was influenced by the time of hydrolysis and the type of enzyme. Of the various hydrolysates produced, papain hydrolysate obtained by 3-h hydrolysis (PEWPH) displayed the highest DPPH radical scavenging activity. PEWPH could also quench the superoxide anion and hydroxyl radicals, effectively inhibit lipid peroxidation and exhibit reducing power. Then, PEWPH was purified sequentially by ultrafiltration, gel filtration, RP-HPLC and two fractions with relatively strong antioxidant activity were subsequently subjected to LC-MS/MS for peptide sequence identification. The sequences of the two antioxidant peptides were identified to be Tyr-Leu-Gly-Ala-Lys (551.54?Da) and Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (974.55?Da), and they were identified for the first time from food-derived protein hydrolysates. Last, the two purified peptides were synthesized and they showed 7.48- and 6.02-fold higher DPPH radical scavenging activity compared with the crude PEWPH, respectively. These results indicate that PEWPH and/or its isolated peptides may be useful ingredients in food and nutraceutical applications.     

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production,characterization and application

Abstract

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58?U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91?U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53?U/g) and Aspergillus niger (INCQS 40015)/frit (34.76?U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).     

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

Enzyme production by the nematode-trapping fungus,Duddingtonia flagrans

Duddingtonia flagrans degrades peptides, proteins, starch, pectin, lipase, lecithin and oils when grown on agar medium. Serine proteases with optimal activity at pH 8.5 to 10.5 were produced when it was grown in submerged culture. It also produced phospholipase C with optimal activity at pH 8.5, lipases with high activity at pH 3.5 and at 7.5 to 8.5 and pectin-degrading enzymes with pH optima of 3 and 8. The pH of the culture medium affected the types of lipases and pectin degrading enzymes produced but not the proteases or phospholipase C.     

Turnover of Phospholipids in Bacillus cereus under Various Growth Conditions

As a search for natural antioxidants from plant materials, strong antioxidative activity was observed in leaf waxes extracted from Eucalyptus species. A novel type of antioxidant was isolated from the leaf wax of Eucalyptus globulus and identified as n-tritriacontan-16,18-dione. Antioxidative activities were determined by different methods; a thiocyanate method, a thiobarbituric acid method, a total carbonyl value method and a weighing test. The anti-oxidant showed remarkable antioxidative activity in a water/alcohol system and was more effective than α-tocopherol and BHA; however, it has no antioxidative activity in an oil system.     

In Silico Design,Synthesis, and In Vitro Evaluation of Novel Amphipathic Short Linear Peptides Against Clinically Relevant Bacterial Biofilms

Microbial biofilms are organized communities of cells that are associated with a wide spectrum of resistant and chronic infections that lead to the treatment failure. Accordingly, there is an urgent demand to create novel effective therapeutic drugs that can inhibit biofilm formation with new mechanisms of action to surmount the current escalating resistance. In this study, in silico hybrid model was utilized to develop three novel short linear peptides (4, 5, and 6) with potential biofilm inhibiting activities (scores?>?1.0). The peptides were composed of cationic and hydrophobic residues. They were synthesized using solid-phase strategy. Synthesized peptides were purified and characterized by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization spectroscopy, respectively. They were evaluated using in vitro assay as potential inhibitors of clinically relevant Gram-positive and Gram-negative biofilms. Peptide (4) with five positive charges at physiological pH (4 cationic moieties and W:R?=?1:4) showed activity against biofilms of Gram-positive strains (Staphylococcus epidermidis and Listeria monocytogenes). On the other hand, peptide (5) with six positive charges (5 cationic moieties and W:R?=?2:2) demonstrated activity against Gram-positive (S. epidermidis) and Gram-negative (Escherichia coli) biofilms. Interestingly, peptide (6), with seven positive charges (6 cationic moieties and W:R?=?2:5) revealed higher and broader spectrum of activity against biofilms of Gram-positive (S. epidermidis, S. aureus, L. monocytogenes) and Gram-negative (E. coli).

     

Fungal endophytes from leaves of Avicennia marina growing in semi-arid environment as a promising source for bioactive compounds

Endophytic fungi are broadly dispersed residing inside plant tissues and have been demonstrated as a treasure for bioactive natural products. Unexplored harsh and heavy metal contaminant habitat of Avicennia marina may have diverse and potential fungal association. Therefore, this work aimed to isolate the culturable fungal endophytes associated with leaves of A. marina and to evaluate their medical potentialities. Seventeen isolates of endophyte fungi were isolated from healthy leaves and their antimicrobial activities were evaluated. Results showed that isolates had activity against micro-organisms in addition to their antioxidant activity produced a variety of phenolic compounds, besides exhibited a lowest cytotoxicity against ATCC-CCL-81 cell line. Consequently, selected endophytic fungal isolates were identified genetically as Chaetomium sp., Chaetomium madrasense, Chaetomium sp., Chaetomium globosum, Aspergillus hiratsukae, Aspergillus ochraceus, Alternaria tenuissima and Curvularia lunata with gene bank accession numbers MT089951, MT089952, MT089953, MT089954, MT089955, MT089956, MT089957 and MT089958 respectively. The most potent fungus extract was analysed using Gas chromatography–mass spectrometry which verified the presence of numerous bioactive compounds. These findings confirmed that new endophytic fungal strains derived from A. marina thrive in harsh ecosystem produce bioactive metabolites which can be recommended as a novel source for drug discovery.     

Peroxynitrite-scavenging constituents from the brown alga<Emphasis Type="Italic">Sargassum thunbergii</Emphasis>

Peroxynitrite formationin vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products for their treatment. The three components (1–3) isolated fromSargassum thunbergii as well as the organic solventsoluble fractions and the aqueous layer ofS. thunbergii were evaluated for their potential to scavenge authentic ONOO and ONOO derived from 3-morpholinosydnonimine (SIN-1). The antioxidant activity of the individual fractions was in the order of 85% aquaous (aq.) MeOH>n-BuOH>n-hexane> H₂O. The three known compounds sargahydroquinoic acid (1), sargaquinoic acid (2) and sargachromenol (3) showed peroxynitrite-scavenging activities comparable to those of L-ascorbic acid and penicillamine. These results showed a possible antioxidant activity in major constituents ofS. thunbergii.     

Novel Dipeptidyl Peptidase IV Inhibitory and Antioxidant Peptides Derived from Human Gastrointestinal Endogenous Proteins

Human gastrointestinal endogenous proteins (GEP) include the proteins mucins, serum albumin, digestive enzymes, and proteins from sloughed epithelial and microbial-cells. GEP play a vital role in the digestion of food, but are also simultaneously digested by proteases and peptidases of the gastrointestinal tract (GIT). Recent studies suggest that during gastrointestinal digestion, similar to dietary proteins, GEP may also give rise to bioactive peptides. In the present study, the protein sequences of 11 representative GEP were subjected to simulated in silico GIT (SIGIT) digestion. Following SIGIT digestion, 19 novel GEP-derived peptide sequences were selected using quantitative structure activity relationship rules for chemical synthesis. The peptides were then tested for their in vitro dipeptidyl peptidase IV (DPP-IV) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition, and for their ferric reducing antioxidant power (FRAP). Two novel DPP-IV inhibitory peptides with the amino acid sequences RPCF (IC₅₀ = 800.51 ± 49.00 µM) and MIM (IC₅₀ = 1056.78 ± 61.11 µM), and five novel antioxidant peptides CCK, RPCF, CRPK, QQCP and DCR were identified. The results of this study indicate that GEP are a significant source of bioactive peptides with potential novel bioactive peptide fragments within their sequences.     

Antioxidant Peptides from the Protease Digest of Prawn (Penaeus japonicus) Muscle

The muscle of the prawn Penaeus japonicus was hydrolyzed by various proteases, and antioxidant activity of the hydrolysates was examined. Among the digests, pepsin digest showed the most potent antioxidant activity. Three antioxidant peptides have been isolated from the active peptidic fraction by ion-exchange chromatography, gel filtration, and ODS high-performance liquid chromatography. Their structures were identified as Ile-Lys-Lys, Phe-Lys-Lys, and Phe-Ile-Lys-Lys. Received October 16, 1998; Accepted January 8, 1999.     

Enzymatic generation of peptides from potato proteins by selected proteases and characterization of their structural properties

The use of low grade starting material for the generation of peptides with bioactivity properties is of interest. The proteins from the potato starch industry byproduct is a promising source, as several health benefits may be associated with their hydrolysates. The efficiency of selected proteases (Novo Pro‐D, Alcalase, Flavourzyme, and Papain), exhibiting different substrate specificities and cleavage action modes, to hydrolyze potato protein isolate (patatin and protease inhibitors) was investigated. Novo Pro‐D resulted in the lowest degree of hydrolysis, whereas Alcalase, Flavourzyme, and Papain exhibited a high catalytic efficiency for the hydrolysis of potato proteins. The degree of hydrolysis behaved in a concentration dependent manner. However, the end‐product profile (peptides and free amino acids) was dependent not only on the protease specificity, its cleavage action mode (endo/exo) and the availability of the intermediate substrates but also on the contribution of the protease inhibitors to the reaction kinetics through their inhibitory effects. Indeed, the dependence of the exo‐activity on the catalytic efficiency of the endo‐action of protease was shown to be significant. Papain generated more unique peptide sequences with homology assessment matching several potato proteins when compared with Flavourzyme. This can be attributed to the high exo‐peptidase activity of Flavourzyme resulting in the generation of shorter peptides which were difficult to match. Flavourzyme produced more peptides originated from patatin fraction, whereas Papain resulted in the release of more peptides corresponding to the protease inhibitor fractions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:420–429, 2016     

New Insights into the Antimicrobial Properties of Hydrolysates and Peptide Fractions Derived from Chia Seed (Salvia hispanica L.)

Bioactive peptides derived from chia (Salvia hispanica) seed with antioxidant, antihypertensive, and anti-inflammatory activities have been well documented; however, few studies describe the antimicrobial properties of these peptides, which is of great interest not only in the prevention of food-borne diseases but also food spoilage. The aim of this study was to generate chia seed peptides using microwave-assisted hydrolysis with sequential (alcalase + flavourzyme) enzymes (AF-MW), fractionate them into 3–10 and < 3 kDa fractions, and evaluate their potential antimicrobial activity towards Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Overall, the peptide fraction < 3 kDa showed higher antimicrobial activity than both chia seed hydrolysate and peptide fraction 3–10 kDa. Furthermore, the < 3 kDa fraction showed remarkable increase in membrane permeability of E. coli (71.49% crystal violet uptake) and L. monocytogenes (80.10% crystal violet uptake). These peptides caused a significant extension in the lag phase, decreases in the maximum growth, and growth rate in the bacteria and promoted multiple indentations (transmembrane tunnels), membrane wrinkling, and pronounced deformations in the integrity of the bacterial cell membranes. Finally, a select group of peptides in the AF-MW < 3 kDa fraction contained 16 sequences with cationic and hydrophobic character, with seven of them sharing the exact same sequence (GDVIAIR) and eight of them having the amino acid K as either N- or C-terminal or both. In conclusion, our results indicate that bioactive peptides obtained from chia seed proteins by microwave and enzymatic hydrolysis could be employed as antimicrobial agents in foods and therapeutic applications.

     

Exocellular Peptides from Antarctic PsychrophilePseudoalteromonas Haloplanktis

A novel diketopiperazine, named cyclo-(D-pipecolinyl-L-isoleucine) (DKP 1), and 7 known diketopiperazines were isolated from the cell-free culture supernatant of the Antarctic psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125. Two diketopiperazines containing pipecolinyl moiety were isolated for the first time from a natural source. Two new linear peptides, stable to bacterial proteolytic enzymes, were also characterized. The structures of the isolated compounds were elucidated by means of spectroscopic data (1D-, 2D-NMR, EIMS, FABMS, and ESIMS/MS) and chiral high-performance liquid chromatography. The potential antioxidant activity of the isolated compounds was evaluated by a DPPH free radical scavenging assay.     

Phytochemical and Biological Activities in Limonium Species Collected in Different Biotopes of Tunisia

A particular interest is nowadays given to natural antioxidants occurring in foods which can reduce the risk of several diseases through their protective effect. The genus Limonium is widely distributed in different salt regions of Tunisia and known in traditional medicine for the presence of highly effective viral and bacterial replication inhibitors. Limonium leaves have possible beneficial effects on human health for their antioxidant activities and free radical scavenging abilities. To exploit the potential of plants from extreme environments as new sources of natural antioxidants, we studied the extracts from leaves of eight Limonium species growing in extreme environments in Tunisia. Antioxidant molecules (polyphenols, flavonoids, flavonols, ascorbate, tocopherols), in vitro (DPPH, ORAC) and ex vivo antioxidant potential on human erythrocytes, antioxidant enzymes activities (superoxide dismutase, peroxidases, glutathione reductase) were evaluated to identify the species with the best antioxidant capacity. The results showed variability among the species considered in function of the environmental conditions of their natural biotopes, as for the antioxidants measured. In particular, L. vulgare from Oued Rane biotope, characterized by dryness and high temperatures, was the species with the highest enzymatic activity and antioxidant capacity, making it interesting as possible edible halophyte plant or as food complement.     

Optimization of protease production by endophytic fungus,Alternaria alternata,isolated from an Australian native plant

Endophytes are recognised as potential sources of novel secondary metabolites, including enzymes and drugs, with applications in medicine, agriculture and industry. There is a growing need for new enzymes, including proteases, for use in industry that can function under a variety of conditions. In this study, three fungal endophytes (Alternaria alternata, Phoma herbarum and an unclassified fungus), were isolated from the Australian native plant, Eremophilia longifolia, and assessed for production of proteases. The lyophilised growth media obtained after fungal fermentation were analysed for protease production using enzyme activity assays. Protease production was optimised by assessing the effects of temperature, pH, carbon source and nitrogen source on activity. A. alternata showed the greatest protease activity in a wide range of pH (3–9). The broadest activity between 9 and 50 °C was observed at pH 7, suggesting a neutral protease. Overall, the optimum conditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 BAEE units/mg. The characteristics demonstrated by this fungal endophyte showed that it is a potential source of an enzyme with particular application in the dairy industry. However, further studies of the tolerance to higher temperatures and pH will indicate whether the enzyme is suitable to such applications.