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Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of (Sardinella aurita) by-products proteins
Abstract
In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P1–P8). Fraction P4, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.The results of this study suggest that sardinelle by-products protein hydrolysates are good source of natural antioxidants.
... One study investigated the recombinant synthesis of the BAP, GIISHR (Gly-Ile-Ile-Ser-His-Arg) with notable antioxidant activity from the muscle of the fawless smoothhound (Mustelus griseus) [119]. Antioxidant peptides isolated from spotless smooth-hound exhibited good scavenging activities and protected H 2 O 2 -induced HepG2 cells from oxidative stress by increasing the levels of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase along with decreasing the content of malonaldehyde [4]. ...
... Antioxidant peptides isolated from spotless smooth-hound exhibited good scavenging activities and protected H 2 O 2 -induced HepG2 cells from oxidative stress by increasing the levels of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase along with decreasing the content of malonaldehyde [4]. Te peptide had a strong ability to neutralize hydroxyl, ABTS (2,2′-Azino-bis-(3-ethylbenzotiazoline-6-sulfonic acid)), and superoxide radicals [119]. At the beginning, the strain as host such as E. coli needs to be selected and then the expression vector is designed. ...
Bioactive peptides (BAPs) obtained from plants and microbes have been thoroughly explored and studied due to their prophylactic properties. The use of BAPs seems to be a promising substitute for several currently available antibiotics because of their antimicrobial properties against foodborne pathogens. BAPs have several other useful properties including antitumor, antihypertensive, antioxidant, antiobesity, and antidiabetic activities. Nowadays, scientists have attempted to recombinantly synthesize bioactive peptides to study their characteristics and potential uses, since BAPs are not found in large quantities in nature. Many pathogenic microorganisms including foodborne pathogens are becoming resistant to various antibiotics. To combat these pathogens, scientists are working to find novel, innovative, and safe antimicrobial agents. Plant- and microbe-based BAPs have demonstrated noteworthy antimicrobial activity against a wide range of pathogenic microorganisms, including foodborne pathogens. BAPs can kill pathogenic microorganisms by disrupting membrane integrity, inhibiting DNA and RNA synthesis, preventing protein synthesis, blocking protein activity, or interacting with certain intracellular targets. In addition, the positive effect of BAP consumption extends to gut microbiota modulation and affects the equilibrium of reactive oxygen species in the gut. This article discusses recombinant BAPs, BAPs generated from plants and microbes, and their antimicrobial applications and modes of action for controlling foodborne pathogens.
... As for by-products 'v-xii', visible fat was first removed manually. Then, by-products 'iv-xii' were ground in distilled water (1:1) and thermally treated (90 °C, 20 min) to inactivate endogenous enzymes (Bougatef et al. 2010). After cooling, the material was frozen (-18 °C) to facilitate fat separation and its removal from the samples (Londoño-Zapata et al. 2022), and for improved preservation. ...
... For instance, a protease from Saccharomyces pastorianus was used to produce antioxidant hydrolysates from sardine viscera and sardine muscle by-products (Vieira et al. 2017). Hydrolysates of a mixture of sardinelle heads and viscera produced with crude proteases from Aspergillus clavatus ES1 and Bacillus licheniformis NH1 also displayed antioxidant properties (Bougatef et al. 2010). ...
Protein hydrolysates might display diverse bioactivities with potential relevance to human and animal health and food technology. Enzymatic hydrolysis of agro-industrial by-products is increasingly focused. In this study, a crude protease from Bacillus sp. CL18 was applied to obtain antioxidant protein hydrolysates from porcine, bovine, poultry, and fish by-products. The crude enzyme hydrolyzed all the twelve investigated by-products, as detected by increased soluble protein contents after 4 h of proteolysis. Hydrolysates exhibited higher radical-scavenging, Fe²⁺-chelating and reducing power capacities than non-hydrolyzed by-products. Hydrolysis times (0–8 h) and enzyme-to-substrate (E/S) ratios (384, 860, and 1,400 U/g) were assessed to produce antioxidant bovine lung hydrolysates. The highest E/S ratio accelerated both hydrolysis and increases in antioxidant activities; however, it did not result in bioactivities higher than hydrolysates obtained with the intermediate E/S ratio. Optimal antioxidant activities could be reached after 6 h of hydrolysis using 860 U/g. Animal by-products are interesting sources of bioactive protein hydrolysates, which could be produced with a non-commercial bacterial protease. This might represent a promising strategy for the valorization of animal by-products generated in large amounts by the agri-food sector.
... There are different technologies for extracting bioactive peptides from marine biomass. The traditional methods are enzymatic [37,43,48,51] and fermentation hydrolysis [47]. Enzymatic hydrolysis is mostly preferred and effective due to its high specificity, and this methodology does not require any toxic chemicals. ...
... Enzymatic hydrolysis is mostly preferred and effective due to its high specificity, and this methodology does not require any toxic chemicals. The main commercial enzymes for producing bioactive peptides are alcalase, flavorzyme, neutrase, pepsin, trypsin, and papain [36,37,43,[49][50][51]. Subcritical water hydrolysis has attracted attention as a green extraction methodology for extracting peptides from marine biomass [55,56]. ...
Fishery production is exponentially growing, and its by-products negatively impact industries' economic and environmental status. The large amount of bioactive micro-and macromolecules in fishery by-products, including lipids, proteins, peptides, amino acids, vitamins, carotenoids, enzymes, collagen, gelatin, chitin, chitosan, and fucoidan, need to be utilized through effective strategies and proper management. Due to the bioactive and healthy compounds in fishery discards, these components can be used as functional food ingredients. Fishery discards have inorganic or organic value to add to or implement in various sectors (such as the agriculture, medical, and pharmaceutical industries). However, the best use of these postharvest raw materials for human welfare remains unelucidated in the scientific community. This review article describes the most useful techniques and methods, such as obtaining proteins and peptides, fatty acids, enzymes, minerals , and carotenoids, as well as collagen, gelatin, and polysaccharides such as chitin-chitosan and fucoidan, to ensure the best use of fishery discards. Marine-derived bioactive compounds have biological activities, such as antioxidant, anticancer, antidiabetic, anti-inflammatory, and antimicro-bial activities. These high-value compounds are used in various industrial sectors, such as the food and cosmetic industries, owing to their unique functional and characteristic structures. This study aimed to determine the gap between misused fishery discards and their effects on the environment and create awareness for the complete valorization of fishery discards, targeting a sustainable world.
... Cationic AMP saline derived from salmon milt delayed the growth of Listeria monocytogene on smoked salmon [142]. Previous studies show that proteins, peptides, hydrolysates, and amino acids have significant antioxidant activity [143,144]. Protein's antioxidant activities result from the presence of amino acids in their structure and bioactive peptides from enzymatic hydrolysis. The antioxidant activities of peptides depend on the free radical binding properties and metal ions [144,145]. ...
... Protein's antioxidant activities result from the presence of amino acids in their structure and bioactive peptides from enzymatic hydrolysis. The antioxidant activities of peptides depend on the free radical binding properties and metal ions [144,145]. Hydrolysates from meat and fish have been reported to possess antioxidant activity, and adding hydrolysates to minced meat significantly inhibited lipid peroxidation [143]. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity in the hydrolysates collected from mechanically separated chickens was high [146]. ...
Chemicals used for storage majorly possess insecticidal activities – deterring destructive insect pests and microorganisms from stored agricultural produce. Despite the controversy about their safety, local farmers and agro-wholesalers still predominantly use these chemicals in developing countries, especially Africa, to ensure an all-year supply of agriproducts. These chemicals could have short- or long-term effects. Despite the state-of-the-art knowledge, factors such as poor education and awareness, limited agricultural subventions, quests for cheap chemicals, over-dosage, and many more are the possible reasons for these toxic chemicals' setback and persistent use in developing countries. This paper provides an up-to-date review of the environmental and ecological effects, as well as the health impacts arising from the indiscriminate use of toxic chemicals in agriproducts. Existing data link pesticides to endocrine disruption, genetic mutations, neurological dysfunction, and other metabolic disorders, apart from the myriad of acute effects. Finally, this study recommended several naturally sourced preservatives as viable alternatives to chemical counterparts and emphasized the invaluable role of education and awareness programs in mitigating the use in developing nations for a sustainable society.
... Among these peptides the first tripeptide showed the ability to scavenge DPPH radicals. It is likely that the presence of the amino acid sequence His-Tyr contributes significantly to the activity of these peptides [106]. ...
Bioactive peptides are chains of acids that have the ability to produce specific effects, on the human body. These peptides can be obtained from sources, such, as animal, plant and microbial proteins. Among these marine derived bioactive peptides possess a significant place in literature. In years a lot of research has been conducted on producing, purifying and characterizing peptides because of their potential to promote good health. This chapter aims to explore the methods used in generating marine derived peptides and the techniques employed for their purification and characterization. Moreover it will delve into the range of health benefits associated with consuming bioactive peptides.
... GC-1 and GC-2, the other two active components, had EC 50 values of 3.49 and 5.73 mg protein/mL, respectively [68]. Bioactive peptides from aquatic by-products such as salmon [69], sardinella [70], skipjack tuna [71], and miiuy croaker [72] have been separated using the gel filtering approach. This study's conclusions show that peptide components with smaller average molecular weights have stronger antioxidants and other bioactive characteristics. ...
The synthesis of bioactive peptides demonstrates strong antioxidant, anti-proliferative, anti-hypertensive, and anti-diabetic attributes. This presents a promising path for developing cost-effective pharmaceuticals that have fewer side effects as they are derived from foods. Production of bioactive peptides through enzymatic hydrolysis exhibits greater potential compared to alternative chemical-assisted hydrolysis. The purification of bioactive peptides involves size fractionation techniques such as ultrafiltration and gel filtration. Further separation using reversed-phase high-performance liquid chromatography (RP-HPLC) techniques aids in the production of peptides with different hydrophobicity which may have specific bioactivities. Sequencing of peptides is commonly completed through Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), electrospray ionization (ESI), and Liquid chromatography-tandem mass spectrometry (LC–MS). Generally, smaller peptides with lower molecular weights exhibit higher bioactivity due to higher absorption within the gastrointestinal tract. While most investigations into bioactive peptides have been conducted in vitro only a few studies have confirmed these findings in vivo, particularly regarding the bioavailability and toxicity of fish protein peptides especially in individuals with non-communicable diseases (NCDs) such as cancer, cardiovascular, diabetes and chronic respiratory. Bioactivities of peptides derived from fish show cardioprotective, anti-hypertensive, anti-cancer, anti-diabetic, and anti-oxidative effects, suggesting their promising potential in the treatments and preventive care for NCD. Further research is strongly encouraged to explore these aspects comprehensively.
Graphical Abstract
... Even though some treatments cook the fish sufficiently, there is still a risk of sensitization because of the heatresistant allergen from Anisakis (Moneo et al., 2005;Kochanowski et al., 2020). The Perciformes and Scombriformes are the main fish as food on the table, additionally, the by-products of Scombriformes including organs, are used in the manufacture of fishmeal and fish oil (Guerard et al., 2002;Bougatef et al., 2010). These marine fish are close to the market and consumers, yet the contamination of Anisakis is so serious. ...
Anisakis can cause Anisakiasis in humans if raw or undercooked fish is consumed. Symptoms of infection may include vomiting, acute abdominal symptoms, or allergies. In this study, we collected 187 commercially available marine fish from the Yellow Sea, East China Sea, and South China Sea. Among them, 79 were found positive containing 520 Anisakis worms. The average prevalence rate was found 42% in this investigation. Ninety-two worms from different sea areas were selected and analyzed for identification, revealing the presence of five different species, which are Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium zhoushanense, Hysterothylacium amoyense, and Hysterothylacium sp. In the meta-analysis, three databases: PubMed, CNKI, and BaiduXueshu were searched for surveys on the prevalence of Anisakis in Chinese waters from January 2000 to December 2023. A total of 26 studies were included in this analysis of which 25 publications were retrieved from different databases and one being the present study. The pooled prevalence of Anisakis was 45% among commercially available marine fish. Variances in the prevalence of Anisakis were noted among the four seas, with the highest rates in the East China Sea and the Bohai Sea, reaching 53% [0.38; 0.68] and 49% [0.36; 0.62], respectively. The Prevalence of Anisakis infection was significantly higher in astern parts such as Liaoning, Shanghai, and Zhejiang. Analysis of the host fish subgroups revealed that the orders of Anguilliformes, Scombriformes, and Gadiformes had high rates of infection. These findings suggest a significant prevalence of Anisakis, posing an increasing risk of infection for individuals. This study provides impactful information for implementing preventative measures against Anisakis.
... The bioactivities of FPH or peptides are linked to their structural properties, amino acid composition, and sequences. FPH derived from the enzymatic hydrolysis of fish processing by-products have been demonstrated to be a rich source of bioactive peptides, known for their beneficial effects on consumer health (Bougatef et al., 2010;Ren et al., 2008). Nevertheless, the choice of protease in the enzymatic hydrolysis process plays a crucial role. ...
The fish processing industry generally generates large amounts of byproducts, including skin, bone, scale, or meat residues, etc. These byproducts contain a wide range of nutritional components such as protein, lipid, mineral, etc. These leftovers can be converted to functional ingredients or nutraceuticals. To produce high value-added functional ingredients, the remaining proteins in fish processing leftovers are hydrolyzed, in which hydrolysate containing different peptides and amino acids can be generated. Frame or backbone as well as scale can be converted to fish biocalcium, which serves as a potential source of calcium and minerals. Nevertheless, potential processing or technology must be adopted to obtain desired products with target bioactivitities. This review covers production, characteristics, and bioavailability of fish protein hydrolysate (FPH) and fish biocalcium (BC). This review also focuses on the recent applications of the FPH and BC in drink and foods. Overall, the review points out the better exploitation of fish processing leftovers and simultaneously lower pollution caused by the improper discard or disposal of those wastes to environment. Also, consumers have more choices to consume functional ingredients or nutraceutical from marine resources.
... Subsequently, the activities were then assayed and compared with the control reaction in which no inhibitors or metal ions were added. 50 Production of protein hydrolysates Sardine heads and viscera (HV) hydrolysates (H1, H3, H4) and whole sardine (WM) muscle (H2) were prepared following the procedure outlined by Bougatef et al. 51 with slight modifications. Inactivation of endogenous enzymes was achieved by cooking samples for 20 minutes at 90 °C in distilled water (1:2, w/v). ...
A substantial quantity of solid fish by-products is generated during the fish processing. These by-products offer an intriguing opportunity as a source of high added-value compounds, including digestive proteases. Indeed, fish trypsin is one of the most beneficial and useful biomolecules that can be recovered from fish wastes. Additionally, fish protein hydrolysates have emerged as a valuable and abundant source of high-quality bioactive molecules, which can be efficiently recovered through enzymatic hydrolysis. This study aims to provide valuable insights into the extraction and utilization of fish trypsin, as well as protein hydrolysates derived from fish by-products, specifically those from sardine (Sardina pilchardus).
Firstly, trypsin from the viscera of sardine was purified with an approximate fourteen-fold increase in specific activity using ammonium sulfate precipitation, followed by DEAE-cellulose chromatography.
SDS–PAGE analysis revealed a single band of approximately 27 kDa. Interestingly, the purified enzyme exhibited significant features, including an optimum temperature of 60 °C, and high stability at low temperatures, retaining respectively 100% and 64% of its initial activity at 20 °C and 50 °C. The enzyme also demonstrated excellent stability at pH range 5–10 with an optimum at pH 8. Furthermore, it maintained 40% of its enzymatic activity at the pH 11. The purified enzyme was inhibited by benzamidine and PMSF and partially inhibited by EDTA. Subsequently, four protein hydrolysates (H1, H2, H3, and H4) were prepared from sardine, using purified trypsin and other bacterial proteases.
All hydrolysates exhibited noteworthy antioxidant properties in vitro, indicating their promising potential for application in functional foods as natural preservatives.
Keywords: Sardine by-products; Alkaline trypsin; Purification; Hydrolysates; Antioxidant activity.
... Yellowfin Tuna roe (Thunnus Albacores) trypsin inhibitor was isolated, initiated by column chromatography on, Sephacry S200, DEAE-cellulose, Sephadex G-50, and its apparent molecular weight was determined to be 7 104 Da. Moreover Sephadex is often used in the filtration of aquatic organisms and is suggested for fast group separations, such as exchange of buffers desalination(Bougatef et al., 2010;Hsu, 2010) (Vijaykrishnaraj et al., 2016). Internal organs of Parastromateus niger and the flavor of the mussel were separated in order to conform using Sephadex G-25 (Jai Ganesh et al., 2011). ...
Bioactive peptides from marine species have gained attention due to their promising biological features, and the disciplines of pharmaceutical, cosmeceutical, nutraceutical, and biomedical product development have increased recently. Their molecular mass, immunity, and natural abilities that they evolved are essential for host defense mechanisms. Marine bioactive peptides have extremely complex and diverse structures that vary greatly depending on the sources from which they are obtained. They frequently have secondary structures and can be cyclic in the form of depsi-peptides. Bioactive peptide purification from marine sources can be achieved via chromatography techniques, including reverse-phase high-performance liquid chromatography, gel filtration chromatography, and ion exchange chromatography, which is a current technique to extract biologically active peptides. Studies of marine plants, microbes, and animals over the last several eras have revealed a huge variety of structurally varied and bioactive secondary metabolites. With a particular focus on the conversion of nutraceutical and pharmaceutical research into commercially available products, this review summarizes current findings in marine peptide research as well as emerging patterns, and its promising directions are briefly reviewed. The use of active peptides derived from the sources mentioned earlier their health advantages and bioactivities are also focused. Along with safety concerns, their possible usages in the processed food industry, wound healing, feed, cosmetic, and pharmaceutical industries, for the growth of efficient goods, are highlighted.
... Park et al. [39] purified antioxidant peptides from soy protein containing hydrophobic amino acids including phenylalanine, alanine, and proline, the most abundant of which was phenylalanine. According to Rajapakse et al. [61]; Bougatef et al. [102] hydrophobic amino acids can help to delay or block lipid oxidation reactions by increasing the solubility of peptides in lipid, allowing for better interaction of aliphatic hydrocarbon side chain with fat molecules. Zhang et al. [34] identified four novel peptides from soy protein hydrolysate exhibiting strong radical scavenging capacity, reducing power, and also suppressive effects on the production of intracellular ROS. ...
... Additionally, the presence of amino acids, including threonine, valine, methionine, isoleucine, leucine, tyrosine, and proline in the polypeptide derived from Acer truncatum seed meal increased the inhibitory activity of α-glucosidase [13]. Moreover, Bougatef et al. [14] mentioned that there is an extensive correlation between the bioactivity properties of peptides and their composition, amino acid sequence, molecular weight (MW), and hydrophobic properties caused by various DHs. ...
Bioactive peptides derived from proteins found in various foods provide significant health benefits, including regulating blood sugar levels by inhibiting carbohydrate-hydrolyzing enzymes. Hydrolysates of peanut protein were prepared using alcalase (AH) or trypsin (TH) to generate antidiabetic peptides with high activity against α-amylase (IC50 of 6.46 and 5.71 mg/mL) and α-glucosidase (IC50 of 6.30 and 5.57 mg/mL), as well as antiradical activity to scavenge DPPH• (IC50 of 4.18 and 3.12 mg/mL) and ABTS•+ (IC50 of 2.87 and 2.56 mg/mL), respectively. The bioactivities of hydrolysates were greatest in the ultrafiltration-generated F3 fraction (< 3 kDa). The most active fraction was TH-F3, which was purified by gel filtration chromatography to generate sub-fractions (SF). With IC50 values of 1.05 and 0.69 mg/mL, the F3-SF8 fraction was the most effective at inhibiting the activity of α-amylase and α-glucosidase, respectively. This fraction was further purified using RP-HPLC to generate sub-subfractions (SSF), the most active of which were F3-SF8-SSF9 and SSF10. The peptide sequences F3-SF8-SSF9 and SSF10 were determined using LC-MS/MS. Two novel antidiabetic peptides with the potential to inhibit α-amylase and α-glucosidase were identified, with the sequences Asp-Trp-Arg (476.22 Da, IC50 of 0.78, and 0.35 mg/mL) and Phe-Tyr (329.15 Da, IC50 of 0.91, and 0.41 mg/mL). These results suggest that peptides derived from peanut protein are attractive natural ingredients for diabetes management applications.
... In addition to using commercial enzymes, Je et al. apud Arshad et al. (2019) have also utilized crude mackerel digestion enzymes to obtain Leu-Pro-His-Ser-Gly-Tyr peptides in the skeletal protein hydrolysate of Alaska pollack. Bougatef et al. (2010) apud Arshad et al. (2019) have identified seven antioxidant peptides (Leu-Ala-Arg-Leu, Gly-Gly-Glu, Leu-His-Tyr, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu, and Gly-Ala-Leu-Ala-Aal-His) on protein hydrolysate obtained from sardine processing waste. ...
Mudskipper fish is an endemic amphibian fish that has high potential to be developed into fish protein hydrolysate due to the high protein content (92% per dry weight). The aim of the research was to obtain new peptide compounds and hydrolyze the antioxidant activity of mudskipper fish (Periophthalmodon schlosseri). The design used in this study was a completely randomized design (CRD) with enzyme concentrations of 1.5 and 2% with three repetitions. The data obtained were analyzed using descriptive statistics and ANOVA. The results showed that mudskipper fish has the proximate content in the form of water content of 79.13% (wb), protein of 91.85% (db), fat of 1.50% (db), and ash of 4.54% (db), with glutamate amino acid being the highest (6.78%). The protein content of large molecular weight in fish protein hydrolysate obtained using an enzyme concentration of 1, 1.5, and 2% is relatively small, namely, 24.62, 4.85, and 9.47%, respectively. Several peptide compounds were identified in the hydrolysate using enzyme concentrations of 1, 1.5, and 2%, respectively: L-Arg-L-Pro, Phe-Pro, and L-Leu-L-Leu-L-Glu; L-Leu-L-Pro, L-Arg-L-Pro, and L-Leu-L-lys-L-Pro; and lys-Leu, L-Leu-L-Pro, Leu-Trp-Gln-Thr, L-Tyr-L-Gln-L-Val-L-Pro, L-Tyr-L-Gln-L-Leu-L-Pro, and L-Leu-L-Ser-L-Phe-L-Ala-L-α-Gln-L-Pro-Gly. Furthermore, the hydrolysate obtained effectively scavenged DPPH free radicals with IC50 values of 10.98±0.57%v/v, 4.04±0.79%v/v, and 8.69±0.028%v/v, respectively.
... These peptides have also been reported to be effective in preventing tooth decay, osteoporosis, insomnia, and elevated blood pressure , 2014). Also, the results of the research showed that the antioxidant activity of the protein hydrolysis product of sardine fish waste increases with the increase in the degree of hydrolysis and suggested that short chain peptides are better antioxidants compared to long chain peptides (Bougatef et al., 2010). Further studies showed that the smaller peptide component from 3 kilo Daltons obtained from Saccharomyces cerevisiae yeast protein hydrolysis by trypsin enzyme has more DPPH and ABTS radical inhibitory activity than other peptide components (Mirzaei et al., 2015). ...
Bioactive peptides are protein components which are inactive within the protein structure, and upon release by enzymatic hydrolysis, they exhibit special physiological functions. In the last years, the characteristics of bioactive peptides obtained from various plant, animal and microbial sources have received much attention. Bioactive peptides are produced using hydrolysis by enzymes extracted from plants or microorganisms, or digestive enzymes and fermentation by proteolytic starter cultures. The composition and sequence of the amino acids determines their different functions, including relaxing effects, solute binding properties, strengthening of the immune system, antioxidant, anti-microbial, anti-inflammatory, cholesterol-lowering and anti-hypertensive effects. Bioactive peptides are identified by different methods including membrane separation techniques and chromatography from protein hydrolysis products and using spectrometric techniques. The possibility of using bioactive peptides as health or therapeutic components depends on ensuring their bio stability, bioavailability and safety.
... The free radical scavenging assay using the DPPH radical as a substrate is widely used to assess antioxidant capacity. The results obtained in this study are found to be similar to the results observed from the hydrolysate of oyster (Saccostrea cucullata) [55], hydrolysate of smooth dogfish (Mustelus mustelus), and sardinella (Sardinella aurita) in various concentrations [56], thereby reflecting the strong antioxidant activity of TO. Additionally, several studies reported that antioxidant activities were accompanied by anti-inflammatory activities [57,58]. ...
Ulcerative colitis (UC), an inflammation of the colon lining, represents the main form of inflammatory bowel disease IBD. Nutritional therapy is extremely important in the management of ulcerative colitis. Fish oil contains long-chain omega-3 polyunsaturated fatty acids, which have beneficial effects on health, including anti-inflammatory effects. This study aims to investigate the benefits of bluefin tuna oil extracted by the Soxhlet method in vitro by determining the anti-radical and anti-inflammatory activities and in vivo by evaluating the preventive and curative effects. The experiments were carried out using two doses of oil (100 and 260 mg/kg) and glutamine (400 and 1000 mg/kg) on the acetic acid-induced UC model. UC has been induced in Wistar rats by intrarectal administration of a single dose of 1 mL acetic acid (5% v/v in distilled water). The obtained results indicate that tuna oil and glutamine have a significant anti-free radical effect. Tuna oil has a marked anti-inflammatory power based on membrane stabilization and inhibiting protein denaturation. The reduction of various UC parameters, such as weight loss, disease activity score DAS, and colonic ulceration in rats pre-treated with tuna oil and glutamine, demonstrate that these treatments have a significant effect on UC. Total glutathione GSH, superoxide dismutase SOD, and catalase activities are significantly restored in the tuna oil and glutamine groups, while lipid peroxidation has been markedly reduced.
... Many different antioxidative substances, such as antioxidant enzymes, amino acids and peptides, ascorbic acid, carotenoids, and phenolic compounds, are found in fish species to protect their lipids from damage induced by ROS (Bragadóttir et al., 2001) (Table 13.3). Increased concentrations of hydrophobic amino acids and the amino acid residues histidine, proline, methionine, cysteine, tyrosine, tryptophan, and phenylalanine are typically found in peptides with higher antioxidant activity (Je et al., 2007;Ren et al., 2008;Bougatef et al., 2010;You et al., 2010;Sabeena Farvin et al., 2016). Typically, 2À16 amino acid residues are present in antioxidative peptides extracted from fish proteins (Chalamaiah et al., 2012). ...
... Bougatef et al. [22] with slight modifications. An aliquot of 2 mL sample solution was mixed with 2 mL of DPPH solution (0.2 mmol L -1 in 95% ethanol). ...
... DPPH radical is a stable free radical and is used as a measure for radical scavenging activity of natural antioxidants [23]. Vc was used as the positive control. ...
Loach, rich in nutrients, such as proteins, amino acids, and mineral elements, is being gradually favored by consumers. Therefore, in this study, the antioxidant activity and structural characteristics of loach peptides were comprehensively analyzed. The loach protein (LAP) with a molecular weight between 150 and 3000 Da was graded by ultrafiltration and nanofiltration processes, which exhibited excellent scavenging activity against DPPH radical (IC50 2.91 ± 0.02 mg/mL), hydroxyl radical (IC50 9.95 ± 0.03 mg/mL), and superoxide anion radical (IC50 13.67 ± 0.33 mg/mL). Additionally, LAP was purified by gel filtration chromatography, and two principal components (named as LAP-I and LAP-II) were isolated. A total of 582 and 672 peptides were identified in LAP-I and LAP-II, respectively, through structural analysis. The XRD results revealed that LAP-I and LAP-II had an irregular amorphous structure. The 2D-NMR spectroscopy results suggested that LAP-I had a compact stretch conformation in the D2O solution, while LAP-II had a folded conformation. Overall, the study results suggested that loach peptide could be a potential antioxidant agent and might provide valuable information for chain conformation and antioxidant mechanism research further.
... Hydrolysates from fish proteins are a viable resource in nutritional and pharmaceutical applications because they have amino acids available for different physiological functions (Benhabiles et al., 2012;Rosa Zavareze et al., 2014). Fish protein hydrolysates are a source of bioactive compounds and have shown antioxidant activity (Bougatef et al., 2010(Bougatef et al., , 2016Jang et al., 2016;Je et al., 2015), antihypertensive activity (Aissaoui et al., 2017;Borges-Contreras et al., 2019;Korczek et al., 2018), and antiproliferative activity (Alemán et al., 2011;Gómez et al., 2019;Hsu et al., 2011). The enzymatic hydrolysis of fish proteins has also shown relevance in the food industry because it promotes functional properties such as emulsifying capacity, solubility, water and oil holding capacity, and foaming capacity compared to non-hydrolyzed protein (Alahmad et al., 2022;Vásquez et al., 2022). ...
Fish hydrolysates have become one of the most remarkable sources of bioactive peptides. However, the processing conditions for incorporating hydrolysates into food matrices can affect their bioactive performance. The effect of temperature and pH on the radical scavenging activity of tilapia hydrolysate was determined in the wet hydrolysate. Also, a central composite design was used to study the effect of the drying conditions on moisture, drying ratio, productivity, drying rate, and antioxidant activity in the tilapia hydrolysate. The results showed that the hydrolysate has high activity at acidic and neutral pH; but at pH 10, the activity decreases significantly. In the spray-drying process, the antioxidant activity was higher at 115 °C. Moreover, inlet air temperature and feed flow had a statistically significant effect (p < 0.05) on response variables. High inlet air temperature and fast feed flow decrease the moisture of the powder hydrolysate and increase the drying rate and antioxidant activity. Scanning electron microscopy showed liquid bridges between particles with irregular concavities or pores on the surface and the presence of particle agglomerations due to the hygroscopicity of the hydrolysate. © 2023, Sociedade Brasileira de Ciencia e Tecnologia de Alimentos, SBCTA. All rights reserved.
... However, with the application of two-dimensional, three-dimensional, and four-dimensional NMR, as well as the development of molecular biology and computer processing technology, NMR has gradually become one of the main methods of protein analysis. NMR can be used to determine the amino acid sequence, quantify the composition content of each component in a mixture, and so on [54]. ...
Meat and its products, rich in protein, are one of the important potential sources of antioxidant peptides. However, reviews on the generation and application of meat-derived antioxidant peptides are still limited. To understand the research and application progress of meat-derived antioxidant peptides, the main formation pathways and their commercial applications are exhibited, the research methods for the isolation, purification, and identification are summarized, and the influencing factors, evaluation methods, and intestinal absorption pathways are presented in this work. It is summarized that limited degradation by exogenous and endogenous enzymatic hydrolysis is the main pathway for the production of animal-derived antioxidant peptides. Traditional separation, purification, and identification techniques are also applicable to animal-derived antioxidant peptides. The formation of animal-derived antioxidant peptides is affected by many factors, and the intestinal absorption pathways of antioxidant peptides are different. Finally, insufficient and future development directions are provided.
... Te FRAP method is based on the reduction of the combination of TPTZ (F2, 4, 6-tripyridyl-s-triazine) with iron chloride FeCl 3. 6H2O which is almost colorless and slightly brown. After reducing antioxidants, this chemical substance forms aqueous iron complexes [54]. When antioxidants are present in the environment, the intensity of the blue color in the environment is higher and by measuring the intensity of the color, the antioxidant capacity can be understood [55]. ...
In this study, bioactive peptides were produced by enzymatic hydrolysis of casein protein with trypsin (pH = 8). The hydrolysates were analyzed for the degree of hydrolysis (DH) and antioxidant activities, viz. 2, 2′-diphenyl-1picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay at three temperatures of 40, 50, and 60°C for 4, 5, and 6 hours. Also, the antimicrobial activity of the experimental samples was evaluated using the disk diffusion method. The dilution method was used to evaluate the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). The antimicrobial activity of the resulting peptides was investigated by forming growth inhibitory region at three concentrations of 2, 2.5, and 3 mg/ml−1 on Escherichia coli, Salmonella typhimurium, Bacillus cereus, and Staphylococcus aureus. As the degree of hydrolysis increased, more peptides were produced, and antioxidant activity was increased. All of the experiments were conducted with three replicates. The highest degree of hydrolysis (28.44%) and antioxidant properties (DPPH: 76.62%; FRAP: 55 mM Fe(II); ABTS: 84.05%) was at 60°C and four hours (P
... Antioxidant activity of the WAPI hydrolysate in linoleic acid was detected via the ferric-thiocyanate method (Bougatef et al., 2010). Briefly, 1 mL of hydrolysate (50 μg hydrolysate mL −1 ethanol) along with 1 mL of linoleic acid (2.5%) were mixed in 2 mL phosphate buffer (0.05 M, pH 7). ...
Protein hydrolysates from wild almond protein isolate (WAPI) with different degrees of hydrolysis (DH) were synthesised using free and immobilised ficin on the magnetic layered double hydroxide and characterised. The highest DH values after 3 h of achieving 11.69 ± 0.15 and 11.37 ± 0.12, respectively, and gained hydrolysate were collected for further analysis. The zeta potential for the hydrolysis of free and immobilised ficin over hydrolysing time due to differences in molecular mass distribution decreased. By increasing the hydrolysing time in the case of free and immobilised ficin, the emulsion activity index gradually decreased, whereas the amount of emulsion stability index did not. The immobilised form of ficin‐treated hydrolysate showed a suitable level of DPPH radical scavenging and appeared to be effective in enhancing the oxidative stability of linoleic acid, sunflower oil and O/W emulsion. Thus WAPI hydrolysates that were treated with immobilised ficin could serve as an alternative antioxidant for food applications.
... Protein hydrolysates usually contain peptides or amino acids with hydrogen-donating capabilities; thereby, scavenging free radicals 34 . Studies have shown that many food-derived protein hydrolysates were capable of scavenging DPPH radicals, such as hydrolysates from wheat germ 35 , sardinelle head and viscera 36 , and fish skin 37 . ...
Protein hydrolysates from dietary sources possess many physiological and biological properties. Artocarpus altilis is an evergreen multipurpose plant with many benefits. Therefore, this study evaluates in vitro antioxidant and anti-inflammatory properties of A. altilis protein hydrolysates. Protein was isolated from A. altilis and hydrolysed with pepsin and trypsin separately using different enzyme: substrate ratios (1:8, 1:16, 1:32). Antioxidant properties investigated included Fe²⁺-chelating, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and hydrogen peroxide radical scavenging activities. Anti-inflammatory activities were determined using effects on hypotonic solution-induced cell lysis on red blood cell membrane stabilisation and heat-induced protein denaturation. The degree of hydrolysis of trypsin hydrolysate increased with increasing enzyme–substrate ratio, while pepsin hydrolysate decreased as the enzyme–substrate ratio increased. The dominant amino acids in A. altilis protein and hydrolysates were glutamate, aspartate and leucine. Protein hydrolysates obtained from pepsin and trypsin digestion had DPPH scavenging abilities of 43.0 ± 0.01% and 22.2 ± 0.01%, respectively. However, trypsin-hydrolysed protein had a high Fe²⁺-chelating ability, while pepsin-hydrolysed protein had high hydrogen peroxide scavenging ability. Trypsin-hydrolysed protein showed good membrane stability and inhibition of protein denaturation. The results indicated that A. altilis protein hydrolysates possess significant antioxidant and anti-inflammatory effects and can further lend support to food industries as functional foods.
... The inhibition rate became stable at 80-100 mg/mL, suggesting saturated binding between the substrate and polypeptide. A further increase in the concentration showed no improvement in the inhibitory effect [32]. ...
At present, the incidence rate of diabetes is increasing gradually, and inhibiting α-glucosidase is one of the effective methods used to control blood sugar. This study identified new peptides from rice bran fermentation broth and evaluated their inhibitory activity and mechanism against α-glucosidase. Rice bran was fermented with Bacillus subtilis MK15 and the polypeptides of <3 kDa were isolated by ultrafiltration and chromatographic column, and were then subjected to LC-MS/MS mass spectrometry analysis. The results revealed that the oligopeptide GLLGY showed the greatest inhibitory activity in vitro. Docking studies with GLLGY on human α-glucosidase (PDB ID 5NN8) suggested a binding energy of −7.1 kcal/mol. GLLGY acts as a non-competitive inhibitor and forms five hydrogen bonds with Asp282, Ser523, Asp616, and His674 of α-glucosidase. Moreover, it retained its inhibitory activity even in a simulated digestion environment in vitro. The oligopeptide GLLGY could be developed into a potential anti-diabetic agent.
... As mentioned in the literature review, fish protein hydrolysates generates peptides using various enzyme sources, namely pepsin, trypsin, papain, alcalase, neutrase, pronase E, validase, and flavourzyme, which play a part in the proteolysis process. Some examples of these generated peptides were: sardine fish hydrolysate (L-Q-P-G-Q-G-Q-Q) [48], conger eel hydrolysate (L-G-L-N-G-D-D-V-N) [49], yellowfin sole protein hydrolysate (R-P-D-F-D-L-E-P-P-Y) [50], and pacific hake protein hydrolysate (P-L-F-Q-D-K-L-A-H-A-K and A-E-A-Q-K-Q-L-R) [51]. ...
The development of functional food products is increasingly gaining lots of interest and popularity among stakeholders. The aim of this study was to evaluate the bioaccessibility of three Lactobacillus sp. starter cultures, including Lacticaseibacillus casei KKU-KK1, Lactiplantibacillus pentosus KKU-KK2, and Lactobacillus acidophilus KKU-KK3, in order to enhance the performance of the probiotic potential of Nham protein hydrolysates in Thai fermented sausage using microencapsulation technology. Probiotic microcapsules were created from a novel wall material made up of a combination of glutinous rice flour and inulin through a freeze-drying process. Accordingly, the results of three formulations of Nham probiotic and spontaneous fermentation (control) characterized by their physicochemical and microbiological characteristics displayed a correlation between an increase in the amount of total acidity, the population of lactic acid bacteria, and the generated TCA-soluble peptides, while the pH and total soluble protein gradually decreased under proteolysis during the fermentation time. The fractionation of Nham protein hydrolysates (NPHs) was prepared using a microwave extraction process: NPH-nham1, NPH-nham2, and NPH-nham3 (10 mg/mL with fermentation time 114 h), exhibited the highest DPPH radical-scavenging activity and FRAP-reducing power capacity as well, compared to NPH-nhamcontrol at p < 0.05. Moreover, those NPHs peptides showed dose-dependent inhibiting of selected pathogenic bacteria (E. coli TISTR 073, S. aureus TISTR 029, and Ent. aerogenes TISTR 1540). Anti-microbial properties of NPHs peptides against gram-negative bacteria were higher than against gram-positive bacteria. In conclusion, the bioaccessibility of NPHs peptides was significantly enhanced by micro-encapsulation and showed a potential bioactive characteristic for developing into a probiotic agent.
... Bougatef and his coworkers studied seven peptides from ardinella, Pro-His-Tyr-Leu, Gly-Ala-His, Gly-Ala-Leu-Ala-Ala-His, Leu-His-Tyr, Gly-Gly-Glu, Leu-Ala-Arg-Leu, and Gly-Ala-Trp-Ala. They stated that the Leu-His-Tyr had the highest antioxidant ability (DPPH 63 ± 1.57%) [78]. ...
As one of the oldest plants cultivated by humans, hemp used to be banned in the United States but returned as a legal crop in 2018. Since then, the United States has become the leading hemp producer in the world. Currently, hemp attracts increasing attention from consumers and scientists as hemp products provide a wide spectrum of potential functions. Particularly, bioactive peptides derived from hemp proteins have been proven to be strong antioxidants, which is an extremely hot research topic in recent years. However, some controversial disputes and unknown issues are still underway to be explored and verified in the aspects of technique, methodology, characteristic, mechanism, application, caution, etc. Therefore, this review focusing on the antioxidant properties of hemp proteins is necessary to discuss the multiple critical issues, including in vitro structure-modifying techniques and antioxidant assays, structure-activity relationships of antioxidant peptides, pre-clinical studies on hemp proteins and pathogenesis-related molecular mechanisms, usage and potential hazard, and novel advanced techniques involving bioinformatics methodology (QSAR, PPI, GO, KEGG), proteomic analysis, and genomics analysis, etc. Taken together, the antioxidant potential of hemp proteins may provide both functional food benefits and phytotherapy efficacy to human health.
... The autolysate's DH, PR, protein content, and pH were 17.4%, 19%, 667 g/kg, and 6.59, respectively (Ovissiour et al., 2013). In terms of processing BPs without muscle, Bougatef et al. (2010) showed the feasibility of producing hydrolysates with high biological values from sardinelle (Sardinelle auriata) heads and viscera using autolytic hydrolysis (pH 10.0, 50 • C and 3 h). Autolysate with 8% DH consisted of several active peptides, including Leu-Ala-Arg-Leu (471.3 ...
Autolysis technology has shown potential for protein hydrolysates production from marine and aquaculture byproducts. Viscera are a source of cheap proteolytic enzymes for producing protein hydrolysates from the whole fish or processing byproducts of the most valuable commercial species by applying autolysis technology. The use of autolysis allows economical production of protein hydrolysate and provides an opportunity to valorize downstream fish and shellfish processing byproducts at a lower cost. As a result, production and application of marine byproduct autolysates is increasing in the global protein hydrolysates market. Nevertheless, several restrictions occur with autolysis, including lipid and protein oxidation mediated by the heterogeneous composition of byproducts. The generally poor storage and handling of byproducts may increase the formation of undesirable metabolites during autolysis, which can be harmful. The formation of nitrogenous compounds (i.e., biogenic amines), loss of freshness, and process of autolysis in the byproducts could increase the rate of quality and safety loss and lead to more significant concern about the use of autolysates for human food applications. The current review focuses on the autolysis process, which is applied for the hydrolysis of aquaculture and marine discards to obtain peptides as functional or nutritive ingredients. It further addresses the latest findings on the mechanisms and factors contributing the deterioration of byproducts and possible ways to control oxidation and other food quality and safety issues in raw materials and protein hydrolysates.
Worldwide population growth as well as the accompanying fast development in urbanization and industrialization, have spurred a massive increase in fisheries production driven mainly by new developments in fishing technologies. Current harvesting and processing practices generate a remarkable amount of fish waste globally on a yearly basis. On average, two-thirds of the whole fish catch is thought to be by-products that are abandoned. Fish by-products and processing waste provide significant disposal challenges for the fishing industry. Thus, waste generated during fish processing must now be disposed of and recycled properly in order to sustain industry and save environment. Fish processing wastes, especially digestive organs offer enormous biotechnological potential as enzyme sources. Fish species’ biological variety produces a diverse range of enzymes with distinct characteristics. Despite the wide array of enzymes present in fish, the major ones of economic importance include types commonly used in industrial applications such as proteases, transglutaminases, and lipases. Other major enzymes found in fish viscera include chitinases and collagenases. Fish processing waste-derived enzymes may have distinct features that make them more suitable for industrial uses since they live in a broad range of temperature regimes and have other traits that set them apart from their warm-blooded cousins. Hence, to make the most use of marine resources, those enzymes may be recovered from wastes produced during the processing of fish and employed as value-added products or processing aids in a variety of sectors.
Hypertension is an important risk factor concomitant with cardiovascular disease (CVD) states, which is why we set out to evaluate Californian red worm hydrolysates on antihypertensive activity both in vitro, ex vivo, using rabbit aortic rings and in vivo using hypertensive induced rats. The worms were manually separated, washed with water, purged for 4 h with 4 % sodium bicarbonate, sacrificed with 7 % saline, and finally washed with drinking water. The in vitro antihypertensive capacity was performed by measuring angiotensin-converting enzyme inhibition; for the ex vivo assays, rabbit aorta was used to measure relaxation; for the in vivo assays, rats with induced hypertension were used to perform acute (hypotension) and chronic assays, using captopril as a control in all assays. With respect to angiotensin-converting enzyme (ACE) inhibition, the EC50 value of the worm hydrolysate was found to be 358 ppm; with respect to the analysis in aortic rings, it was found that the mechanisms of action of the hydrolysate are endothelium-dependent, presenting a maximum relaxation of 35 %. With respect to the in vivo assays, the hypotensive test showed that the hydrolysate can reduce blood pressure by up to 32 % in only 2 h, while the chronic analysis showed that the hydrolysate at 150 ppm did not present statistically significant differences with the control (captopril) during the 15 days of analysis. The Red Californian earthworm hydrolysate presents bioactive compounds identified with antihypertensive activities in vitro, ex vivo and in vivo in different isolated and animal models. The study demonstrates the efficacy of the hydrolysate to be used as an alternative in the treatment and prevention of hypertension, and it can be implemented in functional foods or nutraceutical foods. Antihypertensive peptides, particularly those that inhibit angiotensin-converting enzyme (ACE), hold significant importance in medical research, specifically in the context of cardiovascular disease treatment, particularly hypertension. The focus on these peptides and the potential implications of their results in medical research can be summarized through several key points: 1) Mechanisms of Action-Antihypertensive peptides function by inhibiting ACE or renin, crucial enzymes in blood pressure regulation. 2)Alternatives to Synthetic Drugs, 3) Additional Health Benefits, and various other factors.
Bioactive peptides, specific protein fragments with positive health effects, are gaining traction in drug development for advantages like enhanced penetration, low toxicity, and rapid clearance. This comprehensive review navigates the intricate landscape of peptide science, covering discovery to functional characterization. Beginning with a peptidomic exploration of natural sources, the review emphasizes the search for novel peptides. Extraction approaches, including enzymatic hydrolysis, microbial fermentation, and specialized methods for disulfide-linked peptides, are extensively covered. Mass spectrometric analysis techniques for data acquisition and identification, such as liquid chromatography, capillary electrophoresis, untargeted peptide analysis, and bioinformatics, are thoroughly outlined. The exploration of peptide bioactivity incorporates various methodologies, from in vitro assays to in silico techniques, including advanced approaches like phage display and cell-based assays. The review also discusses the structure–activity relationship in the context of antimicrobial peptides (AMPs), ACE-inhibitory peptides (ACEs), and antioxidative peptides (AOPs). Concluding with key findings and future research directions, this interdisciplinary review serves as a comprehensive reference, offering a holistic understanding of peptides and their potential therapeutic applications.
As a heavy metal, copper is toxic to aquatic organisms in water, causing oxidative stress and lipid deposition. However, there is currently no effective dietary strategy to prevent damage caused by copper exposure. Here, copper bioaccumulation, antioxidant enzymes, lipogenic enzymes, lipid metabolism-related gene expression levels and metabolic pathways were synthesized and evaluated in copper-exposed largemouth bass (Micropterus salmoides) after hydrolysis fish peptides (HFP) pretreatment. The results showed that supplementation with 1% (P < 0.05), 3% (P < 0.01) and 5% (P < 0.05) HFP significantly reduced the copper bioaccumulation in largemouth bass. Hydrolysis fish peptides supplementation significantly reduced the activities of total antioxidant capacity (P < 0.01) and catalase (P < 0.01) and the contents of glutathione (P < 0.01) and malondialdehyde (P < 0.05). Fatty acid synthetase concentration was significantly reduced in fish supplemented with 3% (P < 0.05) and 5% HFP (P < 0.05). Similarly, fish fed 3% (P < 0.05) and 5% (P < 0.01) HFP significantly reduced the glucose-6-phosphate dehydrogenase concentration. Serum metabolomics revealed that 85, 144 and 207 differential metabolites were obtained in fish supplemented with 1%, 3% and 5% HFP, respectively. The differential metabolites were mainly lipids and lipid-like molecules, which were associated with the lipid metabolism pathways. The expression levels of fatty acid synthase (P < 0.01), sterol regulatory element binding protein-1c (P < 0.05), liver X receptor (P < 0.001), peroxisome proliferator activated γ (P < 0.01), apolipoprotein B (P < 0.001) and fatty acid-binding protein 1 (P < 0.01) were significantly down-regulated and the expression levels of carnitine palmitoyltransferase 1α (P < 0.01), hormone-sensitive lipase (P < 0.001), apolipoprotein A 1 (P < 0.05) were significantly up-regulated in fish fed with 3% HFP. Additionally, supplementation with 3% (P < 0.01) and 5% (P < 0.001) HFP significantly up-regulated the expression level of B-cell lymphoma-2 with a dose-dependent effect. In conclusion, our study confirmed that HFP supplementation was closely associated with oxidative stress, enzymatic activities and related pathways of lipid metabolism, and apoptosis, and in general alleviated lipid deposition caused by copper exposure in largemouth bass.
Reactive oxygen species (ROS) are produced during oxidative metabolism in aerobic organisms. Under normal conditions, ROS production and elimination are in a relatively balanced state. However, under internal or external environmental stress, such as high glucose levels or UV radiation, ROS production can increase significantly, leading to oxidative stress. Excess ROS production not only damages biomolecules but is also closely associated with the pathogenesis of many diseases, such as skin photoaging, diabetes, and cancer. Antioxidant peptides (AOPs) are naturally occurring or artificially designed peptides that can reduce the levels of ROS and other pro-oxidants, thus showing great potential in the treatment of oxidative stress-related diseases. In this review, we discussed ROS production and its role in inducing oxidative stress-related diseases in humans. Additionally, we discussed the sources, mechanism of action, and evaluation methods of AOPs and provided directions for future studies on AOPs.
Este livro tem por objetivo descrever os avanços ocorridos nas áreas de tecnologia de alimentos e de nutrição humana durante o século XX até os dias de hoje. Novos paradigmas e conceitos foram estabelecidos a partir da segunda metade do século passado, particularmente em nutrição humana, com descobertas nos alimentos de compostos bioativos, até então considerados “não nutrientes”. O primeiro capítulo é uma descrição histórica dos principais avanços ocorridos e da importância das descobertas no período supracitado. Nos capítulos seguintes, é descrito e conceituado o que se convencionou chamar de compostos bioativos e nutrição funcional. Os últimos capítulos tratam de inovações em pesquisas, nas quais os conceitos de dieta funcional, nutrigenômica e nutrição personalizada são introduzidos. Esses conceitos poderão conduzir o leitor à seguinte indagação: Dieta ideal é um desafio atingível ou uma utopia? Para os pesquisadores parece ser mais um desafio inatingível, por se tratar de alcançar uma fronteira que se move constantemente pela pressão evolutiva.
Fish processing wastes are valuable sources of high value bioactive peptides which can be extracted by using useful microorganisms (biological method).
Marine algal carbohydrate and peptide antioxidants
Peptides are short sequences of proteins consisting of two or more amino acids that are linked by peptide bonds. Peptide-based designs and drug deliveries can offer several advantages, such as antioxidant, antimicrobial, antihypertensive activities, along with immunomodulatory and antithrombotic properties, with hormone or drug-like potential. Peptide-based therapeutic formulations are used as drug candidates for the treatment of various diseases. However, there are several concerns associated with the efficacy of peptides in pharmaceutical design and delivery, including rapid degradation, limited solubility, and poor permeability. The nanoformulation of peptides has been identified as a promising approach for improving the stability of peptides and providing metabolic stability and bioavailability. This article provides an overview of the advances in the development of peptides for drug design and formulation applications. It discusses various peptide nanoformulation approaches as well as recent developments in the in vitro and in vivo analyses of nanoformulated peptides for pharmaceutical applications.
Animal aquatic products have high water content, abundant enzyme system and their own diverse microbial flora. These products are severely susceptible to autolysis and degradation after death, resulting in many adverse effects on storage, processing, and transportation. Among them, the endogenous enzyme are the key factor that caused the autolysis and degradation. Autolytic hydrolysis provides an effective way to maximize the use of aquatic by-products and achieve increased protein resources and reduce environmental pollution from by-products. To better acquaintance the autolysis phenomenon and regulation of the autolysis phenomenon. This paper reviews the autolytic mechanism, biochemical changes, influencing factors, and potential applications of animal aquatic products and their by-products to explore autolysis and its effective utilization and regulation. In addition, this study also emphasizes the importance of making full use of aquatic by-products. Furthermore, the research trends and future challenges of autolysis are also discussed. Autolysis can effectively transform aquatic products and by-products into bioactive hydrolysates. The hydrolysates produced by the autolysis of aquatic products and their by-products have attracted attention because of their wide applications in food, healthcare, and animal feed industries. However, the mechanism and regulation (promotion or inhibition) of autolysis should be further studied, and autolysate at the industrial level should be produced to provide high-value-added products for by-product processing and realize the sustainable utilization of resources.
Enzymatic hydrolysis is the most prominent strategy to release bioactive peptides from different food proteins and protein-rich by-products. Unconventional microbial proteases (UMPs) have gaining increased attention for such purposes, particularly from the 2010s. In this review, we present and discuss aspects related to UMPs production, and their use to obtain bioactive protein hydrolysates. Antioxidant and anti-hypertensive potentials, commonly evaluated through in vitro testing, are mainly reported. The in vivo bioactivities of protein hydrolysates and peptides produced through UMPs action are highlighted. In addition to bioactivities, enzymatic hydrolysis acts by modulating the functional properties of proteins for potential food uses. The compiled literature indicates that UMPs are promising biocatalysts to generate bioactive protein hydrolysates, adding up to commercially available enzymes. From the recent interest on this topic, continuous and in-depth research is needed to advance toward the applicability and commercial utility of both UMPs and obtained hydrolysates.
Peptides with strong antioxidant activity have been increasingly extracted from various edible aquatic animals, as natural substitutes for synthetic antioxidants. In this paper, a systematic review of the research progress on the enzymatic hydrolysis strategy and structure-activity relationship of antioxidant peptides from edible aquatic animals, especially marine animals, over the last decade was presented. The selection of enzymes varied markedly among organs and tissues. Tools and indicators used in the purification and identification process were clarified. The similarity and the difference in structure and antioxidant activity between vertebrate-derived peptides and invertebrate-derived peptides were discussed. The stability of antioxidant peptides was reviewed. Most peptides could maintain activity under mild conditions, but they hardly resisted gastrointestinal digestion. The poor ability of peptides to cross the small intestinal epithelium in prototype form brought a challenge for food and pharmaceutical applications.
Protein hydrolysates have the potential to be natural and safer sources of bioactive peptides. In this study, two proteases were used to hydrolyze Chinese sturgeon (Acipenser sinensis) protein, and the hydrolysates were then purified to yield antioxidant peptides. The degree of hydrolysis of 23.56% and 18.14% was obtained using papain and alcalase 2.4L, respectivly, and hydrolysates had 96.80% and 87.24% total amino acid content, respectivly. The papain hydrolysate (PH) and alcalase 2.4L hydrolysate (AH) showed good antioxidant activity against DPPH• (IC50 of 3.64 and 3.15 mg/mL) and ABTS•+ (IC50 of 1.92 and 1.58 mg/mL), respectively. The low-molecular-weight (<1000 Da) fraction of both hydrolysates demonstrated the highest antiradical activity (IC50 of 2.59 and 2.31 mg/mL, DPPH) and (IC50 of 1.54 and 1.36 mg/mL, ABTS), respectively. Nine peptides were separated from both hydrolysates using reverse phase high performance liquid chromatography (RP-HPLC). The IC50 for ABTS•+ scavenging activity of peptide P5 with valine, glycine and asparagine (MW of 282.13 Da) from PH, and peptide P3 with histidine, glycine and alanine (MW of 302.74 Da) from AH was 0.89 and 0.72 mg/mL, respectively. The fractions and purified peptides obtained from Chinese sturgeon hydrolysates could be utilized as natural antioxidant substitutes in pharmaceuticals and food products.
In order to utilize yellowfin sole ( Limanda aspera) frame protein (YFP), which is normally discarded as industrial waste in the process of fish manufacture, yellowfin sole frame protein hydrolysates (YFPHs) were fractionated using an ultrafiltration (UF) membrane system following hydrolysis with pepsin and mackerel intestines crude enzyme (MICE). The YFPHs were separated into five major types, YFPH-I (30–10kDa), YFPH-II (10–5kDa), YFPH-III (5–3kDa), YFPH-IV (3–1kDa), and YFPH-V (below 1kDa) by using UF membranes with molecular weight cut-offs of 30, 10, 5, 3, and 1kDa, respectively. The antioxidative activity of the YFPHs was investigated and compared with that of a natural antioxidant, -tocopherol, used as a reference. Furthermore, the fraction showing strong antioxidative activity was isolated from the YFPHs using consecutive chromatographic methods on an SP-Sephadex C-25 column, on a Sephadex G-75 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. The molecular mass of the antioxidant was identified as 13kDa using HPLC on a gel permeation chromatography (GPC) column, and the antioxidative peptide was composed of 10 N-terminal amino acid residues, RPDFDLEPPY.
The protein extracted from lecithin-free egg yolk, normally discarded by lecithin processing plants, was hydrolyzed with the
aid of Alcalase, a commercial enzyme. The hydrolysate was separated through a series of ultrafiltration membranes with molecular
weight cutoffs of 10, 5, and 1 kDa; and three types of permeates including 10 K (permeate from 10 kDa), 5 K (permeate from
5 kDa), and 1 K (permeate from 1 kDa) were obtained. The antioxidative efficacy of hydrolysates so obtained was investigated
and compared with α-tocopherol. Furthermore, two different peptides showing strong antioxidative activity were isolated from
the hydrolysates by using consecutive chromatographic methods including ion exchange chromatography on a SP-Sephadex C-25
column, gel filtration on a Sephadex G-25 column, and high-performance liquid chromatography on an octadecylsilane column.
The purity of the peptides was identified using capillary electrophoresis. The isolated peptides were composed of 10 and 15
amino acid residues, and both contained a leucine residue at their N-terminal positions.
A bacterium producing thermostable alkaline serine-protease was isolated from an activated sludge reactor treating fishery wastewaters and was identified as Bacillus licheniformis NH1. The most appropriate medium for the growth and protease production is composed of (g/l): casein 5; yeast extract 2–4, KCl 1.5, K2HPO4 0.5 and KH2PO4 0.5. The crude extracellular protease produced by the isolate had optimal activity at 65–70 and 70 °C in the absence or presence of 2 mM CaCl2, respectively. The thermostability of the enzyme was considerably enhanced in the presence of Ca2+ at temperature values above 50 °C. The enzyme retained 62 and 100% of its initial activity after heating for 60 min at 60 °C, in the absence or presence of 2 mM CaCl2, respectively. The protease was highly active and stable from pH 7.0 to 12.0, with an optimum at pH 10.0–11.0. The activity was totally lost in the presence of PMSF, suggesting that the preparation contains serine-protease(s). Furthermore, the enzyme showed excellent stability and compatibility with some commercial laundry detergents. The enzyme retained more than 93% of its initial activity after preincubation 60 min at 40 °C in the presence of 7 mg/ml of Dixan, Axion and New Dex.The aprN gene encoding the alkaline serine-protease was isolated and its DNA sequence was determined. The aprN gene consisted of 1137 bp encoding a protein of 379 amino acids organized into a signal peptide (29 amino acids), a pro-protein (76 amino acids), and mature enzyme (274 amino acids). The deduced amino acid sequence indicates only three amino acid differences between NH1 enzyme and subtilisin Carlsberg from Bacillus licheniformis NCIMB 6816.
This work reports the antioxidant activity of peptides produced by enzymatic hydrolysis of crude egg white with pepsin. Four peptides included in the protein sequence of ovalbumin possessed radical scavenging activity higher than that of Trolox. The hydrolysate of egg white with pepsin for 3 h was previously found to exhibit a strong angiotensin I-converting enzyme (ACE) inhibitory activity in vitro. The combined antioxidant and ACE inhibition properties make it a very useful multifunctional preparation for the control of cardiovascular diseases, particularly hypertension. No correlation was found between antioxidant and ACE inhibitory activities. However, the peptide Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu, which was a strong ACE inhibitor (50% inhibitory concentration, 4.7 microM) also exhibited a high radical scavenging activity (oxygen radical absorbance capacity-fluorescein value, 3.8 micromol of Trolox equivalent per micromol of peptide) and delayed the low-density lipoprotein lipid oxidation induced by Cu2+ at a concentration of approximately 0.16 mg/mg of low-density lipoprotein. Present results support that antioxidant peptides and amino acids not only act individually, but also cooperatively and synergistically.
Peptides derived from tryptic hydrolysate of jumbo squid (Dosidicus gigas) skin gelatin were assessed for their antioxidant properties in different in vitro assay systems. The hydrolysate itself exhibited a strong lipid peroxidation inhibition and it was much higher than that of natural antioxidant, alpha-tocopherol. In addition, it could scavenge highly active free radicals in oxidative systems, in the order of hydroxyl and carbon-centered radicals. Two representative peptides with comparatively higher antioxidant potency were purified and characterized as Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu (880.18 Da) and Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-Pro-Gly-Glu-Arg (1241.59 Da). Furthermore, viability of radical-mediated oxidation-induced human lung fibroblasts was enhanced following the treatment of two peptides. However it did not exhibit substantial ion chelation, and we presumed that the observed radical scavenging potency of these peptides play a vital role for their strong antioxidant activity. Based on our results we suggest that hydrophobic amino acids present in peptide sequences contributed greatly for observed antioxidant activities.
Antioxidative activity of aromatic amino acids and indole compounds for the autoxidation of linoleic acid was found to correlate in some extent with the highest occupied molecular orbital energy which represents the electron donor property of respective molecule. 5-Hydroxytryptophan, one of the best electron donor among the compounds tested, was the most effective antioxidant. However, antioxidative activity of some indole compounds could not be interpreted simply by their highest molecular orbital energies.
Neither the chelating action for the possible metal traces nor the accelerated decomposition of hydroperoxide produced during the course of the reaction explained these actions of indoles. Tryptophan, while preventing the autoxidation of linoleic acid, underwent the ring cleavage at the position of between C2 and C3 or hydroxylation at C5 to yield formylkynurenine, kynurenine, 3-hydroxykynurenine, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, etc. Following mechanisms which were compatible with the experimental results were proposed for the antioxidative action of indoles; indole donates an electron from its π-pool to linoleic acid radical or peroxy radical produced during the autoxidation of linoleic acid to form a loose charge transfer complex through a “local” interaction; an electron transfer occurs within the complex, which brings cleavage of indole rings and an inhibition of autoxidation.
To develop a natural antioxidative peptide, the gelatin was extracted from fish (Yellowfin sole) skin by hot extraction method and hydrolyzed with Alcalase, pronase and collagenase through a continuous 3-step membrane reactor. Each step enzymatic hydrolysates were determined the antioxidative activity and their synergistic effects, compared with and butylated hydroxytoluene (BHT). Also, we tried to investigate the antioxidative disposition of peptide which was successfully separated by gel filtration, ion-exchange chromatography, and HPIC in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Second step enzymatic hydrolysate (SSEH) among all hydrolysates and was showed the strongest antioxidative activity. The optimum concentration of antioxidative activity for SSEH was in linoleic acid. The synergistic effects were increased in using the hydrolysate with tocopherol and BHT. In the presence of the peptide isolated from SSEH, supplemented hepatocytes exposed to TBHP showed that delayed cell killing and decreased significantly the lipid peroxidation, compared with hepatocytes not cultured with isolated peptide.
Alaska pollack frame protein, which is normally discarded as an industrial by-product in the process of fish plant, was hydrolyzed with mackerel intestine crude enzyme (MICE). Alaska pollack frame protein hydrolysate (APH) was fractionated according to the molecular basis into five major types of APH-I (30–10 kDa), APH-II (10–5 kDa), APH-III (5–3 kDa), APH-IV (3–1 kDa), and APH-V (below 1 kDa) using an ultrafiltration (UF) membrane bioreactor system. The antioxidative activity of the APHs was investigated and compared with that of a natural antioxidant, α-tocopherol, used as a reference. The fraction, APH-V, exhibited the highest antioxidative activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. The sequence of the purified peptide was Leu-Pro-His-Ser-Gly-Tyr and molecular weight was 672 Da. In addition, the purified peptide scavenged 35% on hydroxyl radical at 53.6 μM using electron spin resonance (ESR) spectroscopy.
In the course of our search for novel types of natural antioxidants from leaf waxes of Eucalyptus globulus, 4-hydroxytritriacontane-16,18-dione was newly isolated and identified in addition to the known 16-hydroxy-18-tritriacontanone. 4-Hydroxytritriacontane-16,18-dione showed strong antioxidative activity in a water/alcohol system measured by thiocyanate and TBA methods, but had no activity in an oil system. The diketones with long alkyl side chains showed strong activity in comparison with other β-diketone analogues.
Protease hydrolyses of a soybean protein, beta-conglycinin (7S protein), yielded antioxidative activity against the peroxidation of linoleic acid in an aqueous system at pH 7.0. Six antioxidative peptides were isolated from the hydrolysate prepared with protease S by size exclusion chromatography and reversed-phase HPLC. The amino acid sequences of the peptides were determined using a gas-phase protein sequencer and electron spray mass spectrometry. The peptides were composed of 5-16 amino acid residues, including hydrophobic amino acids, valine or leucine, at the N-terminal positions, and proline, histidine, or tyrosine in the sequences.
The antioxidant activity of N-(long-chain-acyl)histidine-containing compounds was investigated. In a homogeneous solution of methyl linoleate with a radical initiator, these compounds suppressed the production of methyl linoleate hydroperoxides. When the oxidation of phosphatidylcoline liposomes was induced by ferrous ion and ascorbic acid, N-(long-chain-acyl)histidine and N-(long-chain-acyl)carnosine could suppress the oxidation more efficiently than histidine and carnosine. The emulsifying activities of these compounds were found to be higher than those of conventional surfactants, that is, casein, Tween 80, and Triton X-100.
The autoxidation of soybean oil in a cyclodextrin emulsion system was studied in the presence of an emulsion stabilizer consisting of polysaccharides such as xanthan, tragacanth gum, and methylcellulose. Xanthan strongly inhibited the peroxidation of soybean oil containing tocopherols but showed no antioxidant activity on soybean oil without tocopherols in the emulsion. Xanthan did not have hydrogen-donating ability but expressed Fe2+-binding activity. The Fe2+-binding activity corresponded to the pyruvate content of xanthan. Depyruvated xanthan did not inhibit effectively the autoxidation of soybean oil. The Fe2+-chelating structure of xanthan is discussed.
Copper(II)/ascorbate-mediated oxidative damage to the peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe was accompanied by the loss of Asp (64%), Arg (49%), Val (35%), and His (52%). In addition, the reaction of copper(II)/ascorbate with the peptide gave three products (AII-1, AII-2, and AII-3). AII-1 was confirmed to be the product of oxidized His, which generated the 2-imidazolone structure. The damage of the N-terminal sequence was reflected by AII-2 and AII-3. Hence, we characterized the reactivity of the N-terminal sequence using Asp-Arg, Arg-Val, Val-Tyr, and Asp-Arg-Val-Tyr. The reactivities of preferred sequences were Asp-Arg-Val-Tyr > Asp-Arg > Arg-Val > Val-Tyr, suggesting that the Asp-Arg-Val-Tyr sequence is important for the reactivity with Cu(II)/ascorbate. On the other hand, the peptide Ile-His-Pro-Phe, corresponding to the C terminus of the native peptide, failed to react with Cu(II)/ascorbate, suggesting that the Asp-Arg-Val-Tyr sequence contributes to the reactivity of His with copper(II)/ascorbate.
Canola protein hydrolysates were prepared using commercial enzymes, namely Alcalase, an endo-peptidase and Flavourzyme with both endo- and exo-peptidase activities. The hydrolysates so prepared were effective as antioxidants in model systems, mainly by scavenging of free radicals and acting as reducing agents. This effect was concentration-dependent and also influenced by the type of enzyme employed in the process. The hydrolysate prepared using flavourzyme showed the highest antioxidant activity among all samples, whereas the hydrolysates prepared by combination of Alcalase and Flavourzyme did not differ significantly (P>0.05) in antioxidant effectiveness from that produced by Alcalase alone. The hydrolysates were also found to be effective in enhancing water-holding capacity and cooking yield in a meat model system. Their capability in improving the cooking yield of meat was in the order of Flavourzyme hydrolysates>combination hydrolysates>Alcalase hydrolysates. These results suggest that canola protein hydrolysates can be useful in terms of their functionality and as functional food ingredients and that their composition determines their functional properties and thus their potential application in the food and feed industries.
Copyright © 2008 Elsevier Ltd. All rights reserved.
Enzymic hydrolysis represents a valid method for recovering proteins from by-products and surplus of the food industry, by protein solubilisation. In view of the economic interest in the recovering of protein from poorly utilised species of fish, screening of some commercially available proteolytic enzymes: alcalase, papain and neutrase, was undertaken in order to test the most suitable one for the substrate employed. From the results, it can be concluded that alcalase and papain, at optimum pH and temperature conditions have significant effects on the nitrogen recovery from sardines, giving hydrolysates of high nutritional value and characterised by high solubility.
Quinoa protein concentrate was hydrolyzed with alcalase to obtain a hydrolysate that was fractionated by ultrafiltration using 10000- and 5000-molecular-weight cutoff membranes. Functional properties of the protein concentrate, protein hydrolysate, and membrane permeates were compared at different pH values. Protein solubility of the hydrolysate and membrane permeates were significantly higher (P= 0.05) than that of the protein concentrate. The protein concentrate had a significantly higher (P= 0.05) emulsifying activity index than the protein hydrolysate and membrane permeates. Membrane fractionation of the protein hydrolysate into lower-molecular-weight pep tides significantly reduced (P= 0.05) foaming properties, but it improved radical scavenging activity and the ability to inhibit the activity of angiotensin-converting enzyme.
The antioxidant efficiency of ascorbic acid, α-tocopherol, Trolox C, and catechin were evaluated for chicken breast meat in a dispersion system at 37 °C. Peroxidation was induced by adding different kinds of initiators. The initiators exhibited the following order of catalytic action: ascorbic acid/Fe2+ > hemoglobin > Cu+/H2O2 > 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH). The antioxidant efficiency of the antioxidants tested depended on their phase distribution and their reactivity to the radicals in various initiation systems. Among the antioxidants tested, catechin was the most effective antioxidant for chicken breast meat oxidation under the experimental conditions.
The radical-combining activity of Maillard reaction products [MRP(aq)], produced by heating d-glucose and l-histidine (3:1) in a 0.1 M phosphate buffer for 10 h at 105°C (final pH 6.53), was estimated directly by means of a diphenylpicrylhydrazyl
radical (DPPH·) method. Additionally, the indirect methods of peroxide values changes (oven test), hexanal formation, and protection factors
(Rancimat method) were determined on a lipid model system that consisted of sunflower seed oil/water (1:2), emulsified with
3% (w/w) Tween 40. Results from the DPPH· method showed a potential antioxidant activity of MRP(aq), which was confirmed by the indirect methods. Surprisingly, histidine in solution alone (heated or not) exhibited an antioxidant
activity greater than or similar to the MRP(aq) activity in the indirect methods with the lipid model system, in contrast to the results from the DPPH· method. The suitability of various solvents for extraction of potential antioxidant compounds from freeze-dried MRP(aq) was examined, and ethanol extracts showed the greatest activity by the DPPH· method. Consequently, the ethanol extract of freeze-dried MRP(aq) was separated by means of preparative reverse-phase high-performance liquid chromatography (HPLC) with a 0.05 M phosphate
buffer (pH 4.4)/water/acetonitrile gradient system. The antioxidant activity of the eluate was measured through the DPPH· method, and a fraction (Fraction A) with antiradical activity was further purified by preparative HPLC. Fraction B was collected,
and its freeze-dried residue exhibited potent antiradical activity, significantly greater than that of the same level of n-propyl gallate.
The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase(®), chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2±1.5% at 2mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P1-P8). Biological functions of all fractions were assayed, and P4 was found to display a high ACE inhibitory activity. The IC50 values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P4 were 1.2±0.09 and 0.81±0.013mg/ml, respectively. Further, P4 showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P4 was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.
Copyright © 2008 Elsevier Ltd. All rights reserved.
The antioxidant activities and total phenolic contents of 30 Chinese medicinal plants were evaluated using the ferric reducing antioxidant power assay and the Folin–Ciocalteu method, respectively. The Chinese medicinal plants were extracted by the traditional method, boiling in water and also in 80% methanol. A significant and linear correlation coefficient between the antioxidant activity and the total phenolic content was found in both aqueous (R2 = 0.7917) and methanol (R2 = 0.7584) extracts. Phenolic compounds are thus a major contributor of antioxidant activity. Comparing the extraction efficiency of the two methods, the boiling water method extracted phenolic compounds more efficiently, and antioxidant activity of the extract was higher. It was found that the Chinese medicinal plants Rhodiola sacra Fu, the stem of Polygonum multiflorum Thunb. and the root of P. multiflorum Thunb. possessed the highest antioxidant activities and thus could be potential rich sources of natural antioxidants.
The antioxidative components of tree nut oils were extracted using a solvent stripping process. Tree nut oil extracts contained phospholipids, sphingolipids, sterols and tocopherols. The chloroform/methanol extracted oils had higher amounts of phenolic compounds than their hexane extracted counterparts. The antioxidant activity of tree nut oil minor component extracts were assessed using the 2,2-azino-bis (3-ethylbenzthiazoline sulphonate) (ABTS) radical scavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity, β-carotene bleaching test, oxygen radical absorbance capacity (ORAC) and photochemiluminescence inhibition assays. Results of these studies demonstrated that extracts of chloroform/methanol extracted oils possessed higher antioxidant activities than extracts of their hexane extracted counterparts. Meanwhile the extract of chloroform/methanol extracted pecan oil possessed the highest antioxidant activity.
Copyright © 2008 Elsevier Ltd. All rights reserved.
The production, stabilization, by enzymatic treatment, physicochemical composition, and biological properties (including the anti-proliferative activity), of a water-soluble rice bran enzymatic extract (RBEE) are described. The main component of RBEE is proteins (38.1%) – in the form of peptide and free amino acids – having a 6% content of sulfur amino acids. The second component is fat (30.0%), with oleic and linoleic acids as the major components, and 1.2 mg/g of γ-oryzanol. Carbohydrates (14.2%) are comprised mainly of slowly absorbed carbohydrates. Preliminary studies on the anti-proliferative effect of RBEE on leukemia tumor cell growth in vitro are also reported. This property makes RBEE potentially useful as a functional food for the treatment and prevention of chronic pathological states associated with abnormal proliferation of cells, as is the case with cancer.
Mackerel (Scomber austriasicus) hydrolysates were prepared by an autolytic process and accelerated hydrolysis with a commercial enzyme, Protease N. Changes in the levels and compositions of free amino acids and small peptides during hydrolysis were investigated to find out their relationships with antioxidant activities. Increased levels of free amino acids, anserine, carnosine and other peptides of the hydrolysates obtained with protease were much higher than those by autolysis. Different antioxidant measurements including the inhibition of linoleic acid autoxidation, the scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical, and the reducing power showed that mackerel hydrolysates possessed noticeable antioxidant activities. A good correlation existed between the amount of peptides and antioxidant activity. Three peptide fractions were separated from the hydrolysate by size exclusion chromatography. Results revealed that the peptide with molecular weight of approximately 1400 Da possessed a stronger in vitro antioxidant activity than that of the 900 and 200 Da peptides.
Soy hydrolysate and the soy-fermented foods, natto and tempeh, were dephosphorylated, deglycosylated and digested with a variety of endoproteases (pronase, trypsin, Glu C protease, plasma proteases and kidney membrane proteases) to generate oligopeptides. The peptides were purified and characterized. They demonstrated a range of biological activities – angiotensin converting enzyme (ACE) inhibitory, anti-thrombotic, surface tension and antioxidant properties. The biologically active peptides were mostly derived from glycinin, a highly expressed soy protein, and were found mainly in the pronase, kidney membrane proteases and plasma proteases digests of the fermented foods. The proteases of lower specificity produced more oligopeptides and a higher percentage of bioactive peptides than the proteases of higher specificity, namely trypsin and Glu C. A peptide with ACE inhibitory activity was found in the pronase digest of natto, which also produced a peptide with surface active properties. The kidney membrane hydrolysate of natto contained an ACE inhibitor, in addition to a peptide with anti-thrombotic activity bearing homology to hirutonin, a previously described synthetic thrombin inhibitor [DiMaio, Gibbs, Lefebvre, & Munn, 1992, J. Med. Chem. 35, 3331]. Synthetic analogs were also evaluated as substrates for some inhibitors.
Enzymatic hydrolysis of tuna stomach proteins by Alcalase was investigated in a batch reactor. The influence of the process variables (enzyme/substrate ratio; effect of intermediate substrate and enzyme addition) was studied with regards to the extent of proteolytic degradation and to the molecular weight distribution of the peptides. A linear correlation was found between the degree of hydrolysis (DH) and the enzyme concentration. After addition of extra substrate during the course of hydrolysis, the final DH obtained was proportional to the substrate added, suggesting that the concentration of hydrolysable bonds was one of the main factors controlling the hydrolysis rate. Preliminary results showed that tuna protein hydrolysates performed effectively as nitrogenous source in microbial growth media.
Enzymatic hydrolysis of proteins from yellowfin tuna (Thunnus albacares) wastes using alcalase. Guérard F., Dufossé L., de la Broise D., Binet A.. Journal of Molecular Catalysis B : Enzymatic, 2001, 11(4-6), 1051-1059.
The antioxidant activities of egg-yolk protein hydrolysates in a linoleic acid system were investigated. Egg-yolk protein hydrolysates were prepared by enzymic hydrolysis of fat-free egg-yolk protein, which led to the main peak of the molecular mass distribution of lower than 1000. Egg-yolk protein hydrolysates showed strong antioxidant activity in a linoleic acid oxidation system as compared with the egg-yolk protein or amino acids mixture in which egg-yolk protein hydrolysates were constituted. The effect of the sample was concentration-dependent. Egg-yolk protein hydrolysates also showed strong antioxidant activities on cookies containing linoleic acid. These results suggest that egg-yolk protein hydrolysates could be a suitable natural antioxidant for preventing the oxidation of polyunsaturated fatty acids and related food ingredients.
Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn2+ and Cu2+.
An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 °C, respectively. The enzyme was stable in the pH range of 7.0–9.0. The protease was activated by divalent cations such as Ca2+ and Mg2+.The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/v) sodium perborate, respectively.The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine-protease.
The importance of functional foods, nutraceuticals and other natural health products has been well recognized in connection with health promotion, disease risk reduction and reduction in health care costs. Whole foods such as whole grains as well as skins and processing by-products of foods often serve as a concentrated source of components with health beneficial effects. In most cases, processing negatively affects the bioactive components of functional foods and nutraceuticals. Therefore, minimally processed products better serve the health conscious consumers.
Antioxidative activity and functional properties of protein hydrolysates from yellow stripe trevally (Selaroides leptolepis) meat, hydrolyzed by Alcalase 2.4L (HA) and Flavourzyme 500L (HF) with different degrees of hydrolysis (DH) were investigated. As the DH increased, DPPH radical-scavenging activity and reducing power of HA decreased (p < 0.05) but no differences were observed for HF (p > 0.05). Metal chelating activity of both HA and HF increased with increasing DH (p < 0.05). HF generally had a higher (p < 0.05) chelating activity than had HA at the same DH tested. At low DH (5%), HA exhibited a better DPPH radical-scavenging activity while, at high DH (25%), HF had a higher (p < 0.05) reducing power. For the functional properties, hydrolysis by both enzymes increased protein solubility to above 85% over a wide pH range (2–12). When the DH increased, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity, foam stability) of hydrolysates decreased (p < 0.05), possibly caused by the shorter peptide chain length. At the same DH, the functionalities of protein hydrolysate depended on the enzyme used. The results reveal that antioxidative activity and functionalities of protein hydrolysates from yellow stripe trevally meat were determined by the DH and by the enzyme type employed.
Four peptide fractions were separated from protein hydrolysates of capelin (Mallotus villosus) using Sephadex G-10 gel filtration column chromatography. Antioxidant activity of each fraction was determined in a β-carotene-linoleate model system. One isolated fraction possessed a notable antioxidant activity, another two had a weak efficacy while the fourth exerted a prooxidant effect. Two-dimensional thin layer chromatography (TLC) of isolated fractions gave spots with both antioxidant and prooxidant activities. Two prooxidant compounds were separated from the hydrolysate by preparative TLC using silica gel plates. One compound exhibited a maximum absorption at 254 nm while the other absorbed at 260 nm.
The total phenolic content and related total antioxidant capacity of 70 medicinal plant infusions was analyzed. Infusions were prepared in common way in which teas are prepared for human consumption. The total phenolics were measured by Folin–Ciocalteau assay. The total antioxidant capacity was estimated by Ferric Reducing/Antioxidant Power (FRAP) assay. To make practical comparison of relative antioxidant potential of phenolics extracted from selected medicinal plants, the phenol antioxidant coefficient (PAC) was calculated for each infusion. The total phenolic content of medicinal plant infusions ranges from 9 to 2218 mg/L. The FRAP range from 0.06 to 25 mM/L. There was significant linear correlation between total phenolic content and FRAP. According to their antioxidant capacity, 70 medicinal plant extracts can be divided in five groups: (a) very low FRAP (<1 mM/L) n = 9; (b) low FRAP (1–5 mM/L), n = 37; (c) good FRAP (5–10 mM/L), n = 15; (d) high FRAP (10–20 mM/L), n = 8; and (e) very high FRAP (>20 mM/L), n = 1 medicinal plant extract. The PAC was ranging from 1.1 to 3.9 (average 2.4). The best results were obtained for Melissae folium infusions: high phenolic concentration, very high FRAP (>20 mM/L) and PAC > 3. The effect of infusion time and infusion temperature on the phenolic content, FRAP, and free radical scavenging ability was tested. DPPH radical scavenging ability of Melissae folium phenolics was similar to (+)-catechin but not as good as for quercetin. Compared to Trolox and vitamin C, Melissae folium phenolics were more efficient free ABTS radical scavengers. The results indicate that Melissae folium infusions could be an important dietary source of phenolic compounds with high antioxidant capacity comparable with red wine or beverages like tea.
We have investigated the antioxidative activity of five hydrolysates from smooth hound (Mustelus mustelus) meat obtained by various gastrointestinal proteases: crude enzyme extract, low molecular weight (LMW) alkaline protease and trypsin-like protease from M. mustelus intestine, pepsin from M. mustelus stomach, and bovine trypsin.The antioxidant activities of the different smooth hound protein hydrolysates (SHPHs) were evaluated using various in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, total antioxidant capacity, lipid peroxidation inhibition in rat liver homogenate and β-carotene bleaching assay. The five hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activity. The hydrolysate produced by the LMW protease generally showed a greater antioxidative activity as indicated by all the methods considered. The IC50 values (the concentration of antioxidant substance removing 50% of DPPH radical) for DPPH and lipid peroxidation were found to be 0.6 ± 0.01 and 1.1 ± 0.06 mg/ml, respectively. Moreover, LMW protease hydrolysate exhibited notable reducing power and strong total antioxidant capacity.The protein hydrolysate produced by the LMW protease was then fractionated by size exclusion chromatography on a Sephadex G-25 into three major fractions (F1–F3). Fraction F3, with molecular weight lower than 3500 Da, was found to display a high antioxidant activity than F1 (12,200 Da) and F2 (molecular weights between 6500 and 12,200 Da).The amino acid analysis by GC/MS showed that F3 was rich in histidine, methionine, tyrosine, leucine, Isoleucine, glycine, and arginine.
Seven human milks were subjected to an in vitro digestion with pepsin and pancreatin to identify the peptides released from human proteins. On the basis of their sequences, 11 of the 23 peptides were synthesised and their angiotensin converting enzyme (ACE)-inhibitory and antioxidant activities were measured. The β-casein peptides HLPLP and WSVPQPK showed potent ACE-inhibitory and antioxidant activity, with a protein concentration needed to inhibit 50% ACE activity (IC50) of 21 μm and a Trolox Equivalent Antioxidant Capacity (TEAC) of 1.297 μmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivs μmol−1 of peptide, respectively. These activities were determined after digestion of eight infant formulas and compared with those found in digested human milk. One of the infant formulas exhibited a low IC50 value (60.11 μg protein mL−1 of reconstituted formula) and a high TEAC value (1.7056 μmol Trolox equivs mg−1 of protein) and was therefore selected to identify the peptides responsible of these activities.
Studies were conducted on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats and hamsters. To obtain information concerning the mechanism of action of BHA on the forestomach, the following areas were examined: the effects of 12 phenolic compounds structurally related to BHA on the hamster forestomach, the effects of combinations of BHA and other antioxidants on the rat forestomach, and the metabolism of BHA in the forestomach. Also examined were the effects of several antioxidants on two-stage carcinogenesis in rats. Squamous-cell carcinomas were induced in the forestomach of rats and hamsters fed BHA. In a limited study, 1 of 13 hamsters developed a squamous-cell carcinoma. The tumorigenic action of crude BHA on the forestomach was largely due to the action of 3-tert-BHA. p-tert-Butylphenol and 2-tert-butyl-4-methylphenol induced pronounced hyperplasia and papillomas in the hamster forestomach. BHA and other antioxidants, particularly propyl gallate and ethoxyquin, showed additive effects in inducing forestomach hyperplasia and cytotoxicity. Neither BHA nor its metabolites were found in the forestomach epithelium, although small amounts of metabolites were detected in the stomach contents. Thus, a direct action on the stomach epithelium may be exerted by BHA itself or by metabolites formed on interaction of BHA with gastric juice. BHA enhanced forestomach carcinogenesis initiated in rats by N-methyl-N'-nitro-N-nitrosoguanidine or N-methylnitrosourea (MNU) and enhanced urinary bladder carcinogenesis initiated by MNU or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In contrast, it inhibited carcinogenesis initiated in the liver by either diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine (EHEN) and mammary carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA). BHT promoted urinary bladder carcinogenesis initiated by BBN or MNU and thyroid carcinogenesis initiated by MNU, but inhibited ear-duct carcinogenesis initiated by DMBA. Ethoxyquin promoted EHEN-initiated kidney carcinogenesis, but inhibited both DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. Sodium ascorbate promoted forestomach and urinary bladder carcinogenesis, and sodium erythorbate also enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear-duct carcinogenesis. No antioxidants tested had any effect on glandular stomach carcinogenesis. Thus antioxidants have independent modifying (promoting or inhibitory) effects in different organs.
Fish viscera silage has been prepared from cod (Gadus morhua) and saithe (Pollachius virens) by mincing the viscera and adding a mixture of formic and propionic acids [1:1 (w/v)] to a final concentration of 1.5% (w/v). Feeding experiments were performed with rats using: (a) freshly prepared silage; (b) fish viscera silage stored for up to 4 days at 30°C; and (c) de-oiled silage prepared after autolysis and subsequent storage for up to 60 days at 15°C. The nutritional value of the silage has been improved by storage and lipid removal, giving an increase of net protein utilisation (NPU) from below 60% for freshly prepared silage to 70% or above after lipid removal. This increase of NPU can be attributed mainly to an increase in the level of lysine.
The proteolytic activity produced by aBacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5–11.
The partially purified enzyme was stable at 60°C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.
The recent explosion of interest in the bioactivity of the flavonoids of higher plants is due, at least in part, to the potential health benefits of these polyphenolic components of major dietary constituents. This review article discusses the biological properties of the flavonoids and focuses on the relationship between their antioxidant activity, as hydrogen donating free radical scavengers, and their chemical structures. This culminates in a proposed hierarchy of antioxidant activity in the aqueous phase. The cumulative findings concerning structure-antioxidant activity relationships in the lipophilic phase derive from studies on fatty acids, liposomes, and low-density lipoproteins; the factors underlying the influence of the different classes of polyphenols in enhancing their resistance to oxidation are discussed and support the contention that the partition coefficients of the flavonoids as well as their rates of reaction with the relevant radicals define the antioxidant activities in the lipophilic phase.
Epidemiologic studies have provided evidence of an inverse relation between coronary artery disease and antioxidant intake, and vitamin E supplementation in particular. The oxidative-modification hypothesis implies that reduced atherosclerosis is a result of the production of LDL that is resistant to oxidation, but linking the reduced oxidation of LDL to a reduction in atherosclerosis has been problematic. Several important additional mechanisms may underlie the role of antioxidants in preventing the clinical manifestations of coronary artery disease (Fig. 2). Specifically, there is evidence that plaque stability, vasomotor function, and the tendency to thrombosis are subject to modification by specific antioxidants. For example, cellular antioxidants inhibit monocyte adhesion, protect against the cytotoxic effects of oxidized LDL, and inhibit platelet activation. Furthermore, cellular antioxidants protect against the endothelial dysfunction associated with atherosclerosis by preserving endothelium-derived nitric oxide activity. We speculate that these mechanisms have an important role in the benefits of antioxidants.
Considerable amounts of fish processing byproducts are discarded each year. By developing enzyme technologies for protein recovery and modification, production of a broad spectrum of food ingredients and industrial products may be possible. Hydrolyzed vegetable and milk proteins are widely used food ingredients. There are few hydrolyzed fish protein foods with the exception of East Asian condiments and sauces. This review describes various manufacturing techniques for fish protein hydrolysates using acid, base, endogenous enzymes, and added bacterial or digestive proteases. The chemical and biochemical characteristics of hydrolyzed fish proteins are discussed. In addition, functional properties of fish protein hydrolysates are described, including solubility, water-holding capacity, emulsification, and foam-forming ability. Possible applications of fish protein hydrolysates in food systems are provided, and comparison with other food protein hydrolysates where pertinent.
The antioxidant activity of the water extract of Tilia argentea Desf ex DC was determined by the thiocyanate method. The antioxidant activity of the water extract increased with the increasing amount of lyophilized extract (50-400 microg) added into the linoleic acid emulsion. Statistically significant effect was determined in 100 microg and higher amounts. Antioxidant activities of water extracts of tilia (Tilia argentea Desf ex DC), sage (Salvia triloba L.), and two Turkish black teas commercially called Rize tea and young shoot tea (Camellia sinensis) were compared. For comparison studies, 100 microg portions of extracts were added into test samples. All samples were able to show statistically significant antioxidant effect. Both of the tea extracts showed highest antioxidant activities, nevertheless, differences between tilia and sage and tilia and tea were not statistically significant (for both cases p > 0.05). Like antioxidant activity, the reducing power of water extract of Tilia argentea Desf ex DC was also concentration dependent. Even in the presence of 50 microg of extract, the reducing power was significantly higher than that of the control (p < 0.05) in which there was no extract. Unlike antioxidant activity, the highest reducing power activity was shown by sage extract. Among the tea extracts, young shoot extract was the most effective one, however, it had significantly lower activity than sage (p < 0.05). Although tea flower had the lowest reducing power activity, it was higher than that of tilia. But this difference was not statistically significant (p > 0.05). From these results, we could suggest that although the reducing power of a substance may be an indicator of its potential antioxidant activity, there may not always be a linear correlation between these two activities. In addition, antimicrobial activities of each of the above extracts were studied by disk diffusion methods on different test microorganisms. None of the extracts showed antibacterial activity on the studied microorganisms.
Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.
The antioxidant activities, reducing powers, 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, amount of total phenolic compounds, and antimicrobial activities of ether, ethanol, and hot water extracts of the leaves and seeds of Rumex crispus L. were studied. The antioxidant activities of extracts increase with increasing amount of extracts (50-150 microg). However, the water extracts of both the leaves and seeds have shown the highest antioxidant activities. Thus, addition of 75 microg of each of the above extracts to the linoleic acid emulsion caused the inhibition of peroxide formation by 96 and 94%, respectively. Although the antioxidant activity of the ethanol extract of seed was lower than the water extract, the difference between these was not statistically significant, P > 0.05. Unlike the other extracts, 75 microg of the ether extract of seeds was unable to show statistically significant antioxidant activity, P > 0.05 (between this extract and control in that there is no extract in the test sample). Among all of the extracts, the highest amount of total phenolic compound was found in the ethanol extract of seeds, whereas the lowest amount was found in the ether extract of seeds. Like phenolic compounds, the highest reducing power and the highest DPPH scavenging activity were found in the ethanol extract of seeds. However, the reducing activity of the ethanol extract of seeds was approximately 40% that of ascorbic acid, whereas in the presence of 400 microg of water and ethanol extracts of seeds scavenging activities were about 85 and 90%, respectively. There were statistically significant correlations between amount of phenolic compounds and reducing power and between amount of phenolic compounds and percent DPPH scavenging activities (r = 0.99, P < 0.01, and r = 0.864, P < 0.05, respectively) and also between reducing powers and percent DPPH scavenging activities (r = 0.892, P < 0.05). The ether extracts of both the leaves and seeds and ethanol extract of leaves had shown antimicrobial activities on Staphylococcus aureus and Bacillus subtilis. However, none of the water extracts showed antimicrobial activity on the studied microorganisms.
Hydrolysates obtained from porcine myofibrillar proteins by protease treatment (papain or actinase E) exhibited high antioxidant activity in a linolenic acid peroxidation system induced by Fe(2+). Hydrolysates produced by both papain and actinase E showed higher activities at pH 7.1 than at pH 5.4. The antioxidant activity of the papain hydrolysate was almost the same as that of vitamin E at pH 7.0. These hydrolysates possessed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and chelating activity toward metal ions. Antioxidant peptides were separated from the papain hydrolysate by ion exchange chromatography. The acidic fraction obtained by this method exhibited higher activity than the neutral or basic fractions. Antioxidant peptides in the acidic fraction were isolated by high-performance liquid chromatography on an ODS column and shown to possess the structures DSGVT, IEAEGE, DAQEKLE, EELDNALN, and VPSIDDQEELM. The DAQEKLE peptide showed the highest activity among these peptides.
Many new in vitro methods have been developed to evaluate antioxidant activity. Unfortunately, these in vitro methods often correlate poorly with the ability of compounds to inhibit oxidative deterioration of foods because the in vitro assays do not account for factors such as the physical location of the antioxidant, its interaction with other food components, and environmental conditions. To accurately evaluate the potential of antioxidants in foods, models must be developed that have the chemical, physical, and environmental conditions expected in food products. This paper outlines model systems of the evaluation of antioxidants in three types of foods: bulk oil, oil-in-water emulsions, and muscle foods. These model systems are not intended to be inclusive of all possible methods to measure lipid oxidation and antioxidant activity. However, use of these models would allow researchers to more easily compare research results from one paper to another.
To extract antioxidant peptide from hoki frame protein hydrolysate (APHPH), we employed six proteases (pepsin, trypsin, papain, alpha-chymotrypsin, Alcalase and Neutrase) for enzymatic hydrolysis, and the antioxidant activities of their hydrolysates were investigated using both lipid peroxidation inhibition assay and free radical scavenging assay by electron spin resonance spin-trapping technique. Among hydrolysates, peptic hydrolysate, having the highest antioxidant activity, further separated into four groups using ultrafiltration membranes and purified consecutive chromatographic methods. Finally, the purified peptide had a molecular mass of 1801 Da, and amino acid sequence was identified as Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn. APHPH inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radical: 1,1-diphenyl-2-pycryl-hydrazyl (IC(50)=41.37 microM), hydroxyl (IC(50)=17.77 microM), peroxyl (IC(50)=18.99 microM) and superoxide radicals (IC(50)=172.10 microM). Furthermore, APHPH decreased t-butylhydroperoxide-induced cytotoxicity on human embryonic lung fibroblasts and efficiently protected free-radical-induced DNA damage.