Production Of Antioxidant Peptides Research Articles

Investigation of optimal conditions for production of antioxidant peptides from duck blood plasma: response surface methodology

Duck blood is rich in protein. It is one of the main by-products in the slaughter industry. The objective of this research was to optimize and establish a method for producing duck plasma antioxidant peptides. The composition of duck plasma powder was analyzed. Protease selection experiment (Alcalase, Protamex, and Flavourzyme) and single-factor experiment were performed, and response surface methodology was used to determine the optimal hydrolysis conditions for duck plasma. Among the proteases, Alcalase hydrolysate exhibited the strongest 1,1-diphenyl-2-picrylhydrazyl scavenging rate. The optimum enzymatic hydrolysis conditions were hydrolysis time of 6 h, temperature of 65.5°C, pH 10.0, and enzyme-to-substrate ratio of 0.3%. The 1,1-diphenyl-2-picrylhydrazyl scavenging rate reached 64.84%, and the ratio of essential amino acids was 38.76%. Briefly, the duck plasma hydrolysate exhibited strong antioxidant properties and reasonable composition of amino acids. Thus, it may be used as a nutritional or functional ingredient in foods or medicines. This research provides a theoretical basis for comprehensive processing and high value utilization of duck plasma.

The optimization of production and characterization of antioxidant peptides from protein hydrolysates of Agrocybe aegerita

Oxidative stress caused by the excess of radical oxygen species (ROS) could lead to many kinds of human diseases. Antioxidant peptides, a kind of natural antioxidants, have attracted much attention for their low toxicities and strong antioxidant activities. Food proteins can be regarded as good materials for the production of antioxidant peptides by enzymatic hydrolysis. In this work, we aimed to find novel natural antioxidant peptides derived from Agrocybe aegerita proteins by enzymatic hydrolysis. The method of response surface methodology (RSM) was adopted for optimizing the hydrolysis parameters. Under the optimal condition, antioxidant peptides of Agrocybe aegerita were separated and sequenced. Finally, twenty-five novel antioxidant peptides with high antioxidant scores were identified. These results suggest that the hydrolysates of Agrocybe aegerita proteins could be used as natural antioxidants in functional foods and food processing.

The optimization of peptides - producing conditions for antioxidant peptides in goat milk by Lactobacillus casei L61.

The chemical synthesis of antioxidants has exposed more and more of their disadvantages. Therefore, the development of safe, healthy and efficient natural antioxidants has become a research hotspot. At present, antioxidant peptides are gradually becoming more popular due to their strong antioxidant activity and high safety. The aim of this experiment was to obtain the optimum fermentation conditions for producing antioxidant peptides from Lactobacillus casei L61. The effects of various factors (fermentation temperature, fermentation time, concentration of regenerated milk and inoculum) on the production of antioxidant peptides by Lactobacillus casei L61 were studied using a single factor test. Four methods (DPPH radical scavenging activity, the ability to chelate iron, hydroxyl radical scavenging rate, total reduction force) were used to determine antioxidant activity in vitro. The optimum fermentation conditions were determined by orthogonal experiment. The results showed that the optimal fermentation conditions for the production of antioxidant peptides by L61 were 11% reconstituted milk, 5% inoculated milk, fermentation at 41℃ and 16 hours of fermentation time. Under these conditions, the results of the orthogonal test showed that the DPPH radical scavenging rate of the whey sample was 59.97 ±0.87%, which was higher than those of the other 9 groups, and significantly higher than that of the control group (41.97 ±1.37%). The concentration of reconstituted milk and the amount of inoculated milk had a significant effect on the production of antioxidant peptides by Lactobacillus casei L61. It provides a reference for the optimization of the fermentation nutritional composition of antioxidant peptides produced by Lactobacillus casei L61.

Antioxidant and Antimicrobial Activity of Porcine Liver Hydrolysates Using Flavourzyme

Oxidative stress is implicated in human diseases including cancer or neurodegenerative diseases. On the other hand, lipid and microbial spoilage are the main issues of food degradation. Bioactive peptides with antioxidant and antimicrobial activity could solve both problems and create an opportunity to improve the sustainability of the meat industry. Recently, meat by-products are subject of numerous studies to produce antioxidant peptides, highlighting pork liver as a potential source of hydrolysates. To achieve this purpose, pork liver was digested with Flavourzyme at four reaction times (4, 6, 8, and 10 h) and filtered with cut-offs of 5, 10, and 30-kDa molecular weight. Monitoring hydrolysis with SDS-PAGE showed that the reaction was almost complete. Free amino acid profile exhibited that aliphatic and aromatic amino acids were released in a higher amount at longer reaction times. Heat map analysis demonstrated that a hydrolysis time beyond 6 h, displayed a differential amino acid pattern enabling us to optimize the enzymatic reaction. Antioxidant activity was assessed using ABTS, DPPH, FRAP, and ORAC tests, while antimicrobial assay was carried out against Gram-positive and Gram-negative. ABTS and DPPH values revealed that hydrolysates showed a high antioxidant capacity, as well as an inhibition of growth of Brochothrix thermosphata particularly 30 kDa hydrolysates.

Preparation, antioxidant activity evaluation, and identification of antioxidant peptide from black soldier fly (Hermetia illucens L.) larvae.

Black soldier fly larvae protein (BLP) was hydrolyzed using alcalase, neutrase, trypsin, and papain. The BLP hydrolysates (BLPHs) were fractionated by ultrafiltration into three peptide fractions of molecular weight (<3kDa, 3-10kDa and>10kDa). Their antioxidant activities in vitro and the amino acid composition were determined. Results showed that the alcalase was more efficient in hydrolyzing the BLP into oligopeptides. BLPHs-I presented the best scavenging activity to superoxide radicals, hydroxyl radicals, DPPH, and ABTS radicals. The best scavenging activities were found in BLPHs-I containing high levels of aromatic and hydrophobic amino acids. Seventeen novel sequences with typical features of well-known antioxidant proteins were identified by LC-MS/MS. Results demonstrated that BLPHs-I possesses a great capacity as antioxidant peptides applied in functional foods. PRACTICAL APPLICATIONS: Black soldier fly larvae protein (BLP) can also be hydrolyzed to produce antioxidant peptides and their sequences were identified. It can be used in pharmaceutical products and functional foods.

Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin

Whey protein is one of the most relevant co-products manufactured by the dairy industry and it is a powerful environmental pollutant. Therefore, the enzymatic hydrolysis of whey protein concentrate (WPC 35) to produce antioxidant peptides is an innovative approach which can provide added value to whey. The WPC 35 hydrolysis with trypsin was carried out for 4.31 h at 41.1 °C with an enzyme/substrate ratio of 0.017. Under such hydrolysis conditions, the peptides produced have the highest radical scavenging activity and cytoprotector effect. The WPC hydrolysate and a permeate ≤3 kDa were characterized by SDS-page, RP-HPLC and MALDI-TOF-MS. Furthermore, O2•− and HO• scavenging activity and the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18) were determined. In this study, strong antioxidant and cytoprotective peptides were obtained from a low-cost dairy industry product, which could improve consumers’ health when used as functional ingredients.

Quantitative analysis of the relationship between structure and antioxidant activity of tripeptides.

Peptides from enzymatic hydrolysates of food proteins exhibit significant antioxidant activity. Several studies have attempted to determine the factors contributing to the antioxidant activity of peptides; however, the physicochemical properties and factors essential for the antioxidant activity of peptides are still unclear. In this study, in order to clarify the factors important for peptide antioxidant activity based on the properties of component amino acids, 55 tripeptides were synthesized from 20 natural amino acids and their antioxidant activity was measured using the Trolox equivalent antioxidant capacity (TEAC) assay system. The tripeptides were divided into two data sets: a training set comprising 50 compounds and a validated set comprising five compounds. The structure-activity relationship of the training set was then analyzed using classical quantitative structure-activity relationship (QSAR) analysis. The study findings demonstrate that the presence of a cysteine residue at any position, an aromatic amino acid at the C-terminus, higher hydrophobicity of the N-terminal residue, and smaller HOMO-LUMO energy gap of the middle residue can significantly enhance the antioxidant activity. The activities of the five validated compounds were predicted using the constructed QSAR model, and a good correlation between measured and predicted activities was observed. The information obtained from the QSAR model could be useful for effective production of antioxidant peptides from food proteins such as egg white proteins.

Doubleenzyme hydrolysis for producing antioxidant peptide from egg white: Optimization, evaluation, and potential allergenicity.

Egg white is a good source of high-quality proteins in food products and an excellent source of antioxidant active peptides through hydrolysis. The hydrolysis conditions for the preparation of potent antioxidant peptides from egg white with chymotrypsin and pepsin were optimized by the response surface methodology. The antioxidant activity and potential allergenicity of the prepared peptides were evaluated by the oxidative damage model and IgE-binding capability, respectively. After ultrafiltration, the peptide produced using the optimized parameters (preheating time of 3.16hr, hydrolysis time of 3hr, a sample/solution ratio of 10%, multiple enzymes ratio (E1/E2) of 1.7:1, and E/S of 0.4%) showed antioxidant activity of was 30.86μmol AAE/g DW and with low potential for allergenicity. The optimized method is efficient and economical and may be applied to the industrial production of antioxidant peptides to obtain nutraceutical and pharmaceutical agents with low sensitivity. However, further in vivo studies must be conducted. PRACTICAL APPLICATIONS: Egg is consumed as an excellent source of protein. Antioxidant peptides released from egg white is considered to be used in food preservation and human health. Few researches on the optimization of egg peptides were aimed to obtain practical techniques used in the food industry. In this paper, egg white hydrolysis peptide with high antioxidant property and low potential allergenicity was prepared after the optimization of doubleenzyme hydrolysis. The products could be a natural health care products derived from a dietary source and considered using in additive during food production and health food. And the methods used are economical and energy-saving and could be developed to utilize in the food industry.

Separation and Purification of Antioxidant Peptides from Enzymatically Prepared Scorpion (Buthus martensii Karsch) Protein Hydrolysates

The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein hydrolysates (SPHs). Scorpion protein (SP) was first hydrolyzed by trypsin, papain, and alcalase, respectively. Results from hydrolysis tests revealed that peptides hydrolyzed with papain showed the highest degree of hydrolysis (DH), yield and antioxidant activity. The effect of papain hydrolysis on scorpion protein was optimized using the response surface methodology. The highest DH (31.31%) and yield (52.02%) of SPHs were obtained under the following conditions: hydrolysis time, 4.0 h; hydrolysis temperature, 50 °C; and enzyme/substrate ratio, 2.43%. Ultrafiltration, gel filtration and reversed-phase high-performance liquid chromatography was used and two novel antioxidant peptides were obtained. The sequences of the peptides determined by MALDI–TOF–MS/MS were LPTETLH (MW: 810.43 Da, P4-1) and IEEDLER (MW: 903.44 Da, P4-2), respectively. The results revealed SPHs as a potential valuable bioresource for production of antioxidant peptides in the food system.

Antioxidative activities of hazelnut protein hydrolyzates as predicted by in silico analysis techniques

Recently, a variety of studies have been carried out on the production and analysis of bioactive peptides. Here, based on in silico methods, antioxidative behavior of hazelnut (Corylus avellana L.) peptides, which could be prepared by enzymatic treatments with 3 gastrointestinal (GI) and 3 non-GI enzymes were evaluated. On 10/03/2017, UniProt database listed 469 hazelnut proteins. In the current study, a subset (23 ribosomal proteins) of these proteins were examined and the activity of the GI proteases (trypsin, pepsin, chymotrypsin) for the production of antioxidative peptides were compared to non-GI proteases (thermolysin, papain and bromelain). Firstly, potential antioxidative peptide sequences were determined. GI proteases were less effective compared to non-GI proteases in the manufacture of antioxidative peptides. Antioxidative property of peptides, which were obtained by thermolysin or papain treatments, were significantly higher compared to GI proteases. When all 23 proteins were treated 37 antioxidative peptides were formed by thermolysin, while 10 antioxidative peptides were predicted for trypsin. Of the 138 cases studied (23 proteins x 6 proteases), 44 antioxidative peptides were detected (i.e., 1 peptide in approx. 32% of all cases). Based on current findings, hazelnut proteins can be considered a valuable resource for antioxidative peptide manufacture.

Antioxidant Peptides from the Protein Hydrolysate of Spanish Mackerel (Scomberomorous niphonius) Muscle by in Vitro Gastrointestinal Digestion and Their In Vitro Activities.

For the full use of Spanish mackerel (Scomberomorous niphonius) muscle to produce antioxidant peptides, the proteins of Spanish mackerel muscle were separately hydrolyzed under five kinds of enzymes and in vitro gastrointestinal digestion, and antioxidant peptides were isolated from the protein hydrolysate using ultrafiltration and multiple chromatography methods. The results showed that the hydrolysate (SMPH) prepared using in vitro GI digestion showed the highest degree of hydrolysis (27.45 ± 1.76%) and DPPH radical scavenging activity (52.58 ± 2.68%) at the concentration of 10 mg protein/mL among the six protein hydrolysates, and 12 peptides (SMP-1 to SMP-12) were prepared from SMPH. Among them, SMP-3, SMP-7, SMP-10, and SMP-11 showed the higher DPPH radical scavenging activities and were identified as Pro-Glu-Leu-Asp-Trp (PELDW), Trp-Pro-Asp-His-Trp (WPDHW), and Phe-Gly-Tyr-Asp-Trp-Trp (FGYDWW), and Tyr-Leu-His-Phe-Trp (YLHFW), respectively. PELDW, WPDHW, FGYDWW, and YLHFW showed high scavenging activities on DPPH radical (EC50 1.53, 0.70, 0.53, and 0.97 mg/mL, respectively), hydroxyl radical (EC50 1.12, 0.38, 0.26, and 0.67 mg/mL, respectively), and superoxide anion radical (EC50 0.85, 0.49, 0.34, and 1.37 mg/mL, respectively). Moreover, PELDW, WPDHW, FGYDWW, and YLHFW could dose-dependently inhibit lipid peroxidation in the linoleic acid model system and protect plasmid DNA (pBR322DNA) against oxidative damage induced by H2O2 in the tested model systems. In addition, PELDW, WPDHW, FGYDWW, and YLHFW could retain their high activities when they were treated under a low temperature (<60 °C) and a moderate pH environment (pH 5–9). These present results indicate that the protein hydrolysate, fractions, and isolated peptides from Spanish mackerel muscle have strong antioxidant activity and might have the potential to be used in health food products.

Production of antioxidant peptides through hydrolysis of medicinal pumpkin seed protein using pepsin enzyme and the evaluation of their functional and nutritional properties.

BACKGROUNDA hydrolyzed protein composition is a mixture of peptide and amino acids that have been achieved through hydrolysis by the enzyme from different sources, acid or caustic soda. These peptides show important health improving properties including anti-oxidation, antimicrobial, anti-cancer, anti-diabetic, anti-hypertensive activity.METHODSThe aim of the present study was to hydrolyze the protein extracted from medicinal pumpkin seed (Cucurbita Pepo Con. Pepo Var Styriaca) seed meal by pepsin enzyme to obtain bioactive peptides with the highest antioxidant capacity. For this, response surface method (RSM) and central composite design were used at different enzyme concentrations (1%-2%), hydrolysis times (2-5 hours), and temperatures (30-40 °C) as independent variables. Then, the functional properties (emulsifying capacity, foaming capacity, water absorption capacity, and oil absorption capacity), heat and pH stability, and amino acid analysis were measured for the optimum treatment.RESULTS2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging capacity of peptides achieved in optimum conditions (82.07%) was highly similar to the results predicted by the software (80.31%) and their functional properties were significantly different from the initial protein (P > 0.050). Amino acid profile showed that the antioxidant capacity of the hydrolysates could be due to the total hydrophobic amino acid content that accounts for 39.85% of total amino acids in pumpkin seed meal.CONCLUSIONAccording to the results, pumpkin seed meal hydrolysates, with outstanding functional properties, can be used in different food formulation to improve their physical and chemical properties and extend their shelf life, and as antihypertensive and antioxidant agents in the prevention of cardiovascular disease.

Identification and Active Evaluation of Antioxidant Peptides from Protein Hydrolysates of Skipjack Tuna (Katsuwonus pelamis) Head.

For the full use of fish by-products to produce antioxidant peptides, skipjack tuna (Katsuwonus pelamis) heads generated during can processing were defatted and hydrolyzed using the in vitro gastrointestinal (GI) digestion (pepsin–trypsin system) method and six antioxidant peptides (P1 to P6) were purified from the head hydrolysate (KPH) using ultrafiltration and serial chromatography methods. Six isolated peptides (P1 to P6) were identified as Val-Glu-Glu (VEE, P1), Trp-Met-Phe-Asp-Trp (WMFDW, P2), Asp-Ala-Gly-Pro-Tyr-Gly-Pro-Ile (DAGPYGPI, P3), Trp-Met-Gly-Pro-Tyr (WMGPY, P4), Glu-Arg-Gly-Pro-Leu-Gly-Pro-His (ERGPLGPH, P5), and Glu-Met- Gly-Pro-Ala (EMGPA, P6), respectively, using a protein sequencer and electrospray ionization-mass spectrometer. Among skipjack tuna head hydrolysates, fractions, and six isolated peptides (P1 to P6), WMFDW (P2), WMGPY (P4), and EMGPA (P6) showed the highest radical scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH) (EC50 values of 0.31, 0.33, and 0.46 mg/mL for WMFDW, WMGPY, and EMGPA, respectively), hydroxyl (EC50 values of 0.30, 0.43, and 0.52 mg/mL for WMFDW, WMGPY, and EMGPA, respectively), and superoxide anion (EC50 values of 0.56, 0.38, and 0.71 mg/mL for WMFDW, WMGPY, and EMGPA, respectively). Moreover, WMFDW, WMGPY, and EMGPA showed strong capability in reducing power and lipd peroxidation inhibition in the linoleic acid system. In addition, WMFDW, WMGPY, and EMGPA can retain strong antioxidant activity at temperatures lower than 60 °C and pH values ranged from 5 to 9. The results showed that six isolated peptides (P1 to P6) from skipjack tuna heads, especially WMFDW, WMGPY, and EMGPA, might be applied in health care products acting as powerful antioxidant agents.

Antioxidation and memory protection effects of solid‐state‐fermented rapeseed meal peptides on D‐galactose‐induced memory impairment in aging‐mice

AbstractThis study aims to explore the bioactivity of peptides from solid‐state‐fermented rapeseed meal. Three fragments (RP1, RP2, and RP3) were separated by ultrafiltration after solid‐state fermentation; RP1 (MW ≤3 kDa) showed the highest DPPH scavenging rate (EC50 0.27 mg/mL). Then the protective effect of RP1 contra oxidative stress in aging‐mice induced by d‐galactose was investigated by Morris water maze and biochemical analysis (superoxide dismutase [SOD], glutathione peroxidase [GSH‐Px], and malondialdehyde [MDA]) in mouse brain tissue and blood serum. RP1 suppressed in brain and serum of mice the lipid peroxidation by increasing SOD (127.09 ± 8.05 U/mL) and GSH‐Px (20.04 ± 0.71 U/mL). MDA levels showed decreased values (2.94 ± 0.57 U/mL) with the presence of RP1. Also, RP1 avoided mitochondria dysfunction as shown in the increased amounts of ATP. As a result, it suppressed aging, an adverse effect of d‐galactose in mice. The results suggest that the rapeseed peptides from solid‐state fermentation have a potential for the development of therapeutic products.Practical applicationsRapeseed meal is the protein‐rich by‐product after oil is removed from the rapeseed. This meal is an important source of proteins which can be used to develop value‐added products and at the same time reducing food waste. However, the presence of anti‐nutritional factors such as glucosinolate, tannin, and phytic acid has limited its utilization. The present study provides a practical application of solid‐state fermentation. In this process microbial fermentation produces proteolytic enzymes able to hydrolyze proteins into peptides and at the same time degrade toxic substances in rapeseed meal. Solid‐state fermentation was able to produce antioxidant peptides from rapeseed meal. These peptides showed in vivo antioxidant and protective effects against memory impairment on d‐galactose‐induced aging‐mice. The results show that solid‐state fermentation may potentially produce healthy value‐added products from rapeseed peptides.

Improvement of thermal, microwave and ultrasonication pretreatment on the production of antioxidant peptides from sweet potato protein via in vitro gastrointestinal digestion

SummaryEffects of thermal (boiling, steaming and autoclaving), microwave and ultrasonication pretreatments on the production of sweet potato protein hydrolysates (SPPH) through in vitro gastrointestinal digestion (GID) were investigated. All pretreatments significantly increased the degree of hydrolysis (DH), antioxidant activities and molecular weight (MW) &lt;3 kDa peptide fractions contents of SPPH in the order of autoclaving &gt; microwave, steaming &gt; boiling &gt; ultrasonication (P &lt; 0.05). Correlation analysis between peptides content and antioxidant activity suggested that antioxidant activity of SPPH mainly attributed to MW &lt;3 kDa peptides. Diverse peptides ranged from 487.24 to 1477.74 Da with 7–13 amino acids were identified in the MW &lt;3 kDa peptides fraction with autoclaving pretreatment and matched sporamins A, A precursor and B sequences from LC–QTOF–MS/MS analysis. Conformational structures of nine peptides were predicted with well‐known antioxidant amino acids. There is a high potential for SPPH used as a functional supplement in food system.

Antioxidant Peptides from Goat Milk Fermented by Lactobacillus casei L61: Preparation, Optimization, and Stability Evaluation in Simulated Gastrointestinal Fluid.

Antioxidant peptides are currently the focus of many studies, since they eliminate free radicals in the human body without harmful effects. In the present study, Lactobacillus casei L61 was used as a starter culture to ferment goat milk because of its high capacity to produce antioxidant peptides. An optimal nutrients formula (casein, casein peptone, glucose, soybean peptone, inulin, calcium lactate, and cysteine) was investigated by Plackett–Burman (P–B) and Box–Behnken (B–B) designs for response surface methodology (RSM). Antioxidant peptides were successively isolated and purified from the fermented goat milk. Furthermore, the stability of the antioxidant peptides was evaluated in a simulated gastrointestinal tract at 37 °C. The results showed that calcium lactate, glucose, and casein peptone significantly affected the antioxidant activity of goat milk. The optimal additive amounts were 0.99% (w/v) calcium lactate, 0.21% (w/v) glucose, and 0.29% (w/v) casein peptone. The hydroxyl free radical scavenging rate increased significantly (p < 0.001) from 56.50 ± 0.57% to 88.01 ± 0.69%; the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate increased up to 63.48 ± 1.22% under the optimal conditions (n = 3). Our research provides a fitted mathematical model for antioxidant peptides production. Besides, these antioxidant peptides had great stability during simulated gastrointestinal digestion.

Purification and characterization of antioxidant peptides from cooked eggs using a dynamic in vitro gastrointestinal model in vascular smooth muscle A7r5 cells

Antioxidant peptides derived from food sources are considered as safer alternatives to commercially available antioxidant drugs. As one of the most abundant protein sources, hen’s egg proteins were extensively used to produce antioxidant peptides by enzymatic hydrolysis. Our previous work indicated that gastrointestinal digestion of cooked eggs significantly increased the antioxidant activity due to hydrolysis of egg proteins. To characterize the responsible antioxidant peptides, cooked eggs were digested in a simulated in vitro model of human gastro-intestinal digestion. Prepared digests were fractionated with FPLC (Fast Protein Liquid Chromatography) and RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) and the antioxidant activity was determined in A7r5 cells (vascular smooth muscle cell line). Further identification of peptides from peptide fractions with the highest antioxidant activity was carried out using LC-MS/MS. Four peptides derived from ovalbumin, DSTRTQ (48–53), DKLPG (61–65), DVYSF (96–100), and ESKPV (205–209), were identified; of which DKLPG did not show antioxidant activity in cells. Enzyme cleave analysis suggested that these four peptides were likely released from ovalbumin only by pepsin non-specific cleaves. It is postulated that egg consumption may exert protection against oxidative stress on human health due to release of antioxidant peptides during digestion.

The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides.

A psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060U/L). The native enzyme was purified to homogeneity by cation exchange chromatography followed by a size exclusion step, resulting in a 103-fold increase in specific activity (660U/mg) with 25% recovery. The enzyme displayed 10%-30% of its maximum activity at 0-25°C, with the optimum temperature being 50°C. Protease G8 was strongly inactivated by pepstatin A, an aspartic protease inhibitor. The enzyme was used to hydrolyze four natural substrates, and their antioxidant activities were evaluated against 1,1-diphenyl-2-picrylhydrazyl. The highest antioxidant activity (69%) was recorded for beef casein.

Optimization of Collagenase Production by Pseudoalteromonas sp. SJN2 and Application of Collagenases in the Preparation of Antioxidative Hydrolysates.

Collagenases are the most important group of commercially-produced enzymes. However, even though biological resources are abundant in the sea, very few of these commercially popular enzymes are from marine sources, especially from marine bacteria. We optimized the production of marine collagenases by Pseudoalteromonas sp. SJN2 and investigated the antioxidant activities of the hydrolysates. Media components and culture conditions associated with marine collagenase production by Pseudoalteromonas sp. SJN2 were optimized by statistical methods, namely Plackett–Burman design and response surface methodology (RSM). Furthermore, the marine collagenases produced by Pseudoalteromonas sp. SJN2 were seen to efficiently hydrolyze marine collagens extracted from fish by-products, and remarkable antioxidant capacities of the enzymatic hydrolysates were shown by DPPH radical scavenging and oxygen radical absorbance capacity (ORAC) tests. The final optimized fermentation conditions were as follows: soybean powder, 34.23 g·L−1; culture time, 3.72 d; and temperature, 17.32 °C. Under the optimal fermentation conditions, the experimental collagenase yield obtained was 322.58 ± 9.61 U·mL−1, which was in agreement with the predicted yield of 306.68 U·mL−1. Collagen from Spanish mackerel bone, seabream scale and octopus flesh also showed higher DPPH radical scavenging rates and ORAC values after hydrolysis by the collagenase. This study may have implications for the development and use of marine collagenases. Moreover, seafood waste containing beneficial collagen could be used to produce antioxidant peptides by proteolysis.

Hydrolysis conditions for antioxidant peptides derived from enzymatic hydrolysates of sandfish (Arctoscopus japonicus).

Sandfish (Arctoscopus japonicus) meat and roe were used as natural materials for the preparation of antioxidant peptides using enzymatic hydrolysis. Meat and roe were hydrolyzed using Alcalase 2.4L and Collupulin MG, respectively. Optimal hydrolysis conditions were determined through the effects of pH, temperature, enzyme concentration, and hydrolysis time on the radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH). The optimal hydrolysis conditions for meat hydrolysate (MHA) obtained via Alcalase 2.4L treatment were a pH of 6.0, temperature of 70°C, enzyme concentration of 5% (w/w), and a hydrolysis time of 3h. The optimal hydrolysis conditions for roe hydrolysate (RHC) obtained via Collupulin MG treatment were pH 9.0, 60°C temperature, 5% (w/w) enzyme concentration, and 1h hydrolysis time. Under the optimal conditions, the DPPH radical scavenging activities of MHA and RHC were 60.04 and 79.65%, respectively. These results provide fundamental data for the production of antioxidant peptides derived from sandfish hydrolysates.