Antioxidative Properties of Squid Protein Hydrolysates Prepared Using Seer Fish Visceral Enzymes in Comparison with Commercial Enzymes

ABSTRACT

In the present study, antioxidative potential of squid protein hydrolysates (SPH) prepared using endogenous pepsin and trypsin prepared from the stomach and pyloric ceca of seer fish respectively was evaluated, in comparison with commercial enzymes. Degree of hydrolysis (DH) of the pepsin SPH ranged between 8–11% and trypsin SPH between 12–14% after 150 min of hydrolysis. SPH prepared with endogenous enzymes had significantly higher DH than commercial enzymes. Pepsinogen SPH expressed more DH than pepsin SPH. Trypsin SPH exhibited good metal chelating ability (98%), which was equivalent to EDTA at 130 ppm. Pepsin SPH showed higher super oxide anion scavenging activity (75%). Trypsin and pepsin SPH also had high DPPH radical scavenging (85%), ABTS radical scavenging (93%) and ferric reducing (Abs700 = 1.2) abilities. Thus, metal chelating and superoxide anion radical scavenging activities of SPH are dependent on enzymes rather than DH.     

大眼金枪鱼头蛋白酶解物1 ku超滤组分的抗氧化活性及其理化性质

研究大眼金枪鱼头蛋白酶解物1 ku超滤组分体外的还原力、自由基清除能力及对衰老小鼠体内抗氧化能力的影响,分析1 ku超滤组分的一般成分、氨基酸组成及分子量分布,为进一步分离纯化金枪鱼头抗氧化肽提供基础。体外结果显示,1 ku超滤组分对羟基自由基、超氧阴离子和DPPH自由基的清除活性随浓度的增加而增强,IC50分别为1.38、0.73与0.93mg/mL,还原力也随浓度的增加而增大,在浓度为12.5 mg/mL时为0.763;体内结果显示,灌胃30 mg/kg的1 ku超滤组分连续42 d,D-半乳糖致衰老小鼠肝组织的超氧化物歧化酶(SOD)活性、肝组织和血清的谷胱甘肽过氧化物酶(GSH-PX)活性显著提高(P<0.05),血清丙二醛(MDA)含量显著降低(P<0.01);理化分析结果显示,1 ku超滤组分(干基计)蛋白质含量为96.40%,脂肪0.11%,灰分4.86%,疏水性氨基酸占氨基酸总量的35.8%,活性组分分子量在1 802~2 519 u和422~922 u。     

Physicochemical Properties and Angiotensin I Converting Enzyme Inhibitory Peptides of Freshwater Fish Skin Collagens

ABSTRACT

The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptides from extracted TS and HS collagen using pepsin were also determined. HS collagen had a higher amount of imino acid than TS collagen, while TS contained higher amounts of tyrosine (Tyr) and lysine (Lys). Fourier-transform infrared spectra of both collagens showed predominant helix structure. The HS collagen hydrolysate prepared by pepsin was fractionated using sequential ultrafiltration membranes, and the fraction with molecular weight (MW) <5 kDa showed the highest ACE inhibitory activity (p < .05). After cation exchange and two steps size exclusion chromatography, peptides showed ACE inhibitory activity of 72.06%. This study revealed that ACE inhibitory peptides derived from HS collagen could be developed as a functional food with potential antihypertensive properties.     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.     

Optimization of Hydrolysis Conditions for Production of Angiotensin-Converting Enzyme Inhibitory Peptides from Basa Fish Skin Using Response Surface Methodology

ABSTRACT

Basa fish skin free from fat and noncollagenous proteins was hydrolyzed by different proteinases to produce angiotensin-converting enzyme (ACE) inhibitory peptides. Protamex hydrolysates were found to have the highest ACE inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions of Protamex—including enzyme-to-substrate ratio, hydrolysis time, and solid (fish skin)-to-liquid (water for hydrolysis)—at a temperature of 50°C and a pH of 7.0. The mathematical model demonstrated a good fit with the experimental data. An enzyme-to-substrate ratio of 6,108 U/g, a hydrolysis time of 1.24 h, and a solid-to-liquid ratio of 1:8.05 g/mL were found to be the optimal conditions to achieve the highest value of ACE inhibitory activity. The IC₅₀ of the hydrolysates for ACE inhibitory activity was 1.417 mg/mL under these conditions.     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

Anti-Inflammatory,Antioxidant, and Antimicrobial Effects of Underutilized Fish Protein Hydrolysate

Antimicrobial, anti-inflammatory, and antioxidant activities of protein hydrolysates from Argentine croaker (Umbrina canosai) protein isolate (CPI) or Argentine croaker myofibrillar protein with different degrees of hydrolysis (DH: 10–20%) prepared using Alcalase or Protamex were determined. Results showed that an increase in the DH resulted in higher content of hydrophobic and aromatic amino acids (AAAs) and in a decrease in molecular weight (MW) distribution for all hydrolysates obtained. Furthermore, the enzyme and raw material used influenced the amino acid content and MW determined. Hydrolysates from CPI with a 20% DH by Alcalase had higher 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity, metal chelation, and ferric-reducing antioxidant power (p < 0.05). All hydrolysate samples decreased the pro-inflammatory capacity. In all the evaluated microorganisms, only seven were inhibited, most being Gram-positive. Alcalase was found to exert a considerable influence on antibacterial activity. These hydrolysates are an alternative as natural antimicrobials, anti-inflammatory, and antioxidant compounds.     

Recovery of Proteins from Squid By-Products with Enzymatic Hydrolysis and Increasing the Hydrolysate’s Bioactivity by Maillard Reaction

ABSTRACT

The Maillard reaction is an effective method for enhancing the activity of bioactive peptides. In this study, squid by-product hydrolytic peptides (SPHPs) were obtained. The effects of the Maillard reaction on the antioxidant and antibacterial activities of the SPHPs were explored. The results showed most of the peptides in the < 500 Da fraction of SPHPs reacted with D-arabinose to form the 500–1000 Da fraction (89.60%). The Maillard reaction products (MRPs) possessed more potent antioxidant activities, including free radical scavenging activity, ferrous chelating capacity, and reducing power, than the SPHPs. Although both the SPHPs and MRPs had broad-spectrum antibiotic activities and could inhibit the growth of both gram-negative and gram-positive bacteria, the antibacterial activities of the MRPs were higher than those of the SPHPs. Furthermore, the efficacy of the MRPs lasted significantly longer than that of ampicillin and could last for more than 30 days. In addition, both the SPHPs and MRPs had good functional properties and many potential applications. The results suggested that the Maillard reaction has potential to be used to improve the activities and physicochemical properties of bioactive peptides.     

Antioxidative Status of a Nutrient Enriched Formulation of Perna viridis in a Time Series Shelf-Life Study

ABSTRACT

The freeze-dried concentrate of green mussel, Perna viridis, was added with naturally derived antioxidative oleoresins of rosemary and turmeric along with trace amounts of other natural additives to prepare different treatments and was subjected to accelerated shelf life study (50°C) to evaluate its oxidative stability. The total oxyradical scavenging activities of samples with higher concentrations of turmeric over rosemary demonstrated the prominent role of the turmeric extracts to arrest free radical chain reactions. Time dependent antioxidative status in relation to the nutritional compositions was evaluated to identify the oxidative changes for different treatments. The addition of natural additives with polyphenolic moieties in the treatments shielded the mussel’s unsaturated fatty acids against oxidation as evident by higher polyunsaturated fatty acids in the two treatments as compared to control after 90 days of accelerated shelf-life study. Higher n-3/n-6 ratios after 90 days’ storage were also observed in these treatments. A threshold level of combined turmeric (0.8 g/100 g) and rosemary (0.4 g/100 g) was sufficient for optimum antioxidant activity and to maintain nutritional composition of the green mussel extract at desired levels.     

Purification and Characterization of a New DPPH Radical Scavenging Peptide from Shrimp Processing By-products Hydrolysate

A peptide with high antioxidant activity was isolated and identified from shrimp processing by-products hydrolysate. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity was used to evaluate the antioxidant activity of fractions. The purified antioxidant peptide was identified as Ser-Val-Ala-Met-Leu-Phe-His (804.4 Da) by electrospray ionization tandem mass spectrometry. The purified peptide at 50 μg/mL showed antioxidative activity of 65.7 ± 0.9%, which was 3.18-fold higher compared with the first step separation by ion-exchange chromatography. It is possible to produce natural antioxidative peptides from shrimp processing by-products hydrolysate. The high antioxidant activity may be due to the presence of Phe-His segment at the C-terminus of the peptide.     

Effect of Degree of Hydrolysis and Protease Type on the Antioxidant Activity of Protein Hydrolysates From Cuttlefish (Sepia officinalis) By-Products

We have investigated the antioxidant activities of eight hydrolysates from cuttlefish by-products obtained by treatment with various gastrointestinal proteases (chymotrypsin, trypsin, and crude alkaline enzyme extracts from cuttlefish and sardinelle) and bacterial proteases (Alcalase and crude enzymes from Bacillus pumilus A1, Bacillus mojavensis A21, and Bacillus cereus BG1). The antioxidant activities of the cuttlefish by-products protein hydrolysates (CPHs) were evaluated using various in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, and total antioxidant capacity. All hydrolysates showed different degrees of hydrolysis (DH) and varying degrees of antioxidant activity. Among the different hydrolysates, cuttlefish crude enzyme hydrolysate exhibited the highest antioxidant activity, followed by sardinelle crude enzyme and Alcalase hydrolysates. Further, CPHs with different degrees of hydrolysis were prepared by treatment with proteolytic enzymes from cuttlefish, sardinelle, and B. mojavensis A21. All hydrolysates showed a greater antioxidative activity as indicated by all the methods considered. In addition, antioxidant activity in hydrolysates was positively correlated with the increase of DH. The results of this study indicated that CPHs might be a good candidate for further investigation in developing new antioxidants.     

Antioxidant,ACE-Inhibitory,and Antimicrobial Activities of Peptide Fractions Obtained From Dried Giant Squid Tunics

ABSTRACT

Squid tunics were divided in two batches: freeze-dried and air-dried. Dried squid tunics were directly hydrolyzed with pepsin, Alcalase, and Esperase. Freeze-dried tunics showed better aptitude for hydrolysis than air-dried tunics. Pepsin showed the lowest efficiency in protein breakdown and gave hydrolysates with low antioxidant activity, whereas Alcalase and Esperase peptide fractions showed strong radical scavenging ability, ACE-inhibitory activity, and noticeable antimicrobial properties. B. cereus, B. coagulans, and D. hansenii were found to be the most sensitive bacteria. Dried squid tunics proved to be an alternative to a previously extracted gelatin for obtaining bioactive peptides.     

Recovery of Bioactive Peptides and Omega-3 Fatty Acids-Containing Phospholipids from Squid Processing By-Product Hydrolysate

ABSTRACT

The present study examined whether bioactive peptides and omega-3 fatty acids-containing phopholipids could be recovered from squid processing by-product (SPB) hydrolysate. The hydrolysate was produced at 55°C for varying times with endogenous proteases and centrifuged to yield peptides-containing supernatant and phospholipids-containing precipitate. The supernatant showed angiotensin I-converting enzyme (ACE) inhibitory activity with half maximal inhibitory concentration (IC₅₀) values decreasing with hydrolysis time from 2.11 mg (0 h) to 1.71 mg (1 h), 1.38 mg (1.5 h), and 1.34 mg (2 h). Two-hour squid hydrolysate was further fractionated to isolate the most active fraction whose molecular weight was found to be below 10 kDa with IC₅₀ of 0.32 mg. The phospholipids-containing precipitate (45.6 g/100 g oil) was analyzed for a fatty acid profile, with the levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) being 16.86 and 29.24 g/100 g oil, respectively. The ACE inhibition by hydrolysate peptides and omega-3 fatty acid recovery support the nutraceutical potential of the SPB hydrolysate.     

Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny

Digestive enzymes of freshwater fish Labeo rohita (family Cyprinidae; class Actinopterygii; infraclass Teleostei; order Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationship with age of rohu, whereas trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS‐PAGE of crude enzyme extract revealed that the proteases of higher molecular weight (MW) appeared during early ontogeny, whereas low MW proteases were observed at a later stage. Substrate SDS‐PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12 and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor (SBTI) (58.6–81.2%), phenyl methyl sulphonyl fluoride (PMSF) (55.6–70%), N‐α‐p‐tosyl‐l ‐lysine chloromethyl ketone (TLCK) (41.1–52.3%) and N‐tosyl‐l ‐phenylalanine chloromethyl ketone (TPCK) (27.9–44.5%) indicated the presence of serine proteases, trypsin‐ and chymotrypsin‐like enzymes in rohu larvae. Inhibition with SBTI and TPCK showed power, TLCK and ethylenediaminetetraacetic acid showed exponential, whereas PMSF showed polynomial relationships during the study period.     

Impact of microwave-assisted enzymatic hydrolysis on functional and antioxidant properties of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> by-products

Fishery by-products can be better utilized following enzymatic hydrolysis treatment to produce fish protein hydrolysates (FPH) with potentially enhanced interface-stabilizing properties (e.g. functionality). The production of FPH could be accelerated through the application of rapid heating methods [e.g. microwave-assisted heating (MW)] rather than slower conventional heating (CH) treatments. The objective of this study was to investigate the effects of microwave heating during enzymatic hydrolysis on the functionality and antioxidant properties of FPH. Trout by-products were hydrolyzed with Alcalase at an enzyme substrate ratio (E:S) of 0.5, 1.7, and 3.0% (w/v), respectively, for 3, 5 and 15 min using a microwave system (1200 W, 20% power with 50% duty cycle at 50–55 °C) and a conventional heating method (water bath at 50 °C). The degree of hydrolysis and protein solubility was higher (P < 0.05) for the MW-FPH than for the CH-FPH. MW-FPH at 5 min (0.5% E:S) demonstrated higher (P < 0.05) emulsifying activity and emulsion stability than CH-FPH with the same treatment. Foam capacity and stability were also greater (P < 0.05) for MW-FPH samples that were treated 15 min by microwave-assisted heating (0.5% E:S) when compared to CH. Overall, MW-FPH exhibited higher (P < 0.05) 2,2-diphenyl-1-picryhydrazyl and ferric ion reducing capacity than CH-FPH. We therefore conclude that microwave-assisted hydrolysis is an alternative method to produce FPH with improved solubility, emulsifying activity, foaming properties and antioxidant activity.     

Inhibitory Activities of Protein Hydrolysates from Spotted Babylon Snails on Tyrosinase and Melanogenesis

The optimal conditions for protein hydrolysates preparation from spotted babylon Babylonia areolata exhibiting tyrosinase inhibitory activity and antioxidant activity using alkaline protease (Protease G6) were undiluted Protease G6 (5.8 × 10⁵ DU/g) at 60 min of hydrolysis time and eightfold diluted Protease G6 (7.25 × 10⁴ DU/g) at 240 min of hydrolysis time. These two conditions were fractioned using molecular weight (MW) cutoff values of 10, 5, and 3 kDa membranes, and their anti-melanogenic and antioxidant properties were further analyzed. Among the fractions, the MW < 3 kDa fraction exhibited high levels of inhibitory activity toward the mono- and diphenolase activities of tyrosinase, with IC₅₀ values of 1.758 and 8.995 μg/mL, respectively. Kinetic studies revealed that this fraction behaved as an uncompetitive inhibitor. The results demonstrated that the MW < 3 kDa fraction suppressed melanin synthesis and decreased cellular tyrosinase activity with no cytotoxicity to B16F10 melanoma cells.     

Biochemical Properties and Stability of Antioxidative Activity of Half-Fin Anchovy (Setipinna taty) Fermented Product

The aim of this work was to investigate the biochemical properties of fermented product of half-fin anchovy (HAFP; Setipinna taty) made by Natto bacillus fermentation—including molecular weight distribution, amino acid composition, solubility, foaming, and emulsifying profiles. Furthermore, the influences of thermal processing, pH alteration, and in vitro simulated gastric and intestinal digestion on the antioxidative stability of HAFP in terms of the scavenging 1,1-diphenyl-2-picrylhydrazayl (DPPH) radical were evaluated. Results showed that HAFP contained small peptides (< 1,500 Da) and free amino acids. The amounts of essential amino acids and flavor amino acids were 155.219 and 178.636 mg/g of dry HAFP, respectively. Moreover, HAFP contained high levels of antioxidative and hydrophobic amino acids. In suitable pH values, HAFP exhibited good solubility, foaming, and emulsifying properties. The antioxidative activity of HAFP on the scavenging DPPH radical increased after thermal processing. Furthermore, the DPPH radical scavenging activity of HAFP was enhanced slightly or dramatically after being treated under pH values from 3 to 6, or in vitro simulated gastric and intestinal digestion. It can be concluded that HAFP could have potential application in foods as a natural antioxidative ingredient.     

Enzymatic Hydrolysis of Alaska Pollock Proteins Based on Kinetics Model and Lysine Biosensor–Neural Network Model

Controlled proteolysis is important for target hydrolysates. The hydrolysis kinetics of pollock bone protein (PBP) was obtained by mathematic deduction and experimental analysis. The equation of degree of hydrolysis was as follows: DH = 2.801 ln|1 + (0.06044E₀/S₀?0.1005)t|, which could predict the hydrolysis process of PBP when hydrolysis condition was strictly controlled. However, the hydrolysis reaction was affected by several factors, and the predicted value may be against the experimental result. Therefore, online monitoring was necessary for obtaining the target peptides. Biosensor and artificial neural network (ANN) were employed for monitoring hydrolysis process online. Free lysine level was chosen as the monitoring factor determined by immobilized lysine oxidase electrodes. Based on the lysine biosensor and ANN, the hydrolysis monitoring model LYS-ANN was built, including 3 input layer nodes (E₀, S₀, and LYS), 1 output layer node (DH), and 11 hidden layer nodes. R² value between sample value and simulation value was 0.9964. Simulation error was in the range of 0–4.56%, and the average relative error was 0.94%. The verification tests of PBP hydrolysis showed that the LYS-ANN model could forecast hydrolysis process successfully with high efficiency and accuracy even if the hydrolysis conditions varied.