Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe, hydrolysed by Alcalase 2.4 L (RPH) with different degrees of hydrolysis (DH) at various concentrations were examined. As DH increased, the reduction of DPPH, ABTS radicals scavenging activities and reducing power were noticeable (p < 0.05). The increases in metal chelating activity and superoxide scavenging activity were attained with increasing DH (p < 0.05). However, chelating activity gradually decreased at DH above 30%. All activities except superoxide anion radical scavenging activity increased as the concentration of hydrolysate increased (p < 0.05). Hydrolysis using Alcalase could increase protein solubility to above 80% over a wide pH range (2–10). The highest emulsion ability index (EAI) and foam stability (FS) of hydrolysates were observed at low DH (5%) (p < 0.05). Concentrations of hydrolysates determined interfacial properties differently, depending on DH. The molecular weight distribution of RPH with 5%DH (RPH5) was determined using Sephadex G-75 column. Two major peaks with the molecular weight of 57.8 and 5.5 kDa were obtained. Fraction with MW of 5.5 had the strongest metal chelating activity and ABTS radical scavenging activity. The results reveal that protein hydrolysates from defatted skipjack roe could be used as food additives possessing both antioxidant activity and functional properties.     

Protein hydrolysates from meriga (Cirrhinus mrigala) egg and evaluation of their functional properties

Protein hydrolysates from underutilised meriga (Cirrhinus mrigala) fish egg were prepared by using commercial Alcalase and papain enzymes. The degree of hydrolysis was 62% for Alcalase and 17.1% for papain, after 90 min digestion at 50–55 and 60–65 °C, respectively. The protein content of Alcalase-produced hydrolysate was higher (85%) than that of papain hydrolysate (70%) (p < 0.05). Hydrolysis by both enzymes increased protein solubility of fish egg protein hydrolysates to above 72.4% over a wide pH range (2–12). Results showed that the hydrolysates had good fat absorption capacity (0.9 and 1.0 g/g sample), foam capacity (70% and 25%) and emulsifying capacity (4.25 and 5.98 ml/g hydrolysate), respectively for Alcalase and papain protein hydrolysates. Gel filtration chromatograms and SDS–PAGE analysis indicated the distribution of smaller peptides. These results suggested that fish egg protein hydrolysates could be useful in the food industry.     

Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides derived therefrom

Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.     

Antioxidant and biochemical properties of protein hydrolysates prepared from Silver carp (Hypophthalmichthys molitrix)

The antioxidant and biochemical properties of enzymatically hydrolyzed silver carp (Hypophthalmichthys molitrix) protein were studied. The molecular weight of the main peaks of the hydrolysates by both Alcalase and Flavourzyme was lower than 5000 Da. The hydrolysates treated by Alcalase for ?1.5 h (hydrolysis time) showed that the relative proportion of <1000 Da fraction was more than 60%. For the biochemical properties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range; and when the hydrolysis time was prolonged (>3 h), the colour of the hydrolysates turned yellowish. The protein hydrolysates exhibited significant hydroxyl radical-scavenging activity and inhibited linoleic acid peroxidation. For Alcalase treatment, the hydroxyl radical-scavenging activity and the inhibition of linoleic acid peroxidation of hydrolysates appeared to reach a maximum level for 1.5, 2.0 h of hydrolysis, respectively; and their antioxidant activity was close to that of α-tocopherol in a linoleic acid emulsion system, and carnosine in the 2-deoxyribose oxidation system. The hydrolysate with lower molecular weight distribution possessed stronger Fe²⁺ chelation ability at a sample concentration of 5.0 mg/mL. The results suggested that the antioxidant activity of silver carp protein hydrolysates were related to its degree of hydrolysis (DH), hydrolysis time and molecular weight.     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

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.     

Characteristics of Bellamya purificata snail foot protein and enzymatic hydrolysates

The foot muscle protein of Bellamya purificata (mud snail, named Luosi in Chinese) was investigated. Its conformation change and increase in solubility were researched during enzymatic hydrolysis. The protein conformation was looser following an increase in pH (from 10 to 12), while the β-sheet was the main conformation at pH 12. Blending, ultrasonic extraction, ultradispersing and alkaline treatment increased the solubility of the foot muscle protein. The effects of several proteases on its hydrolysis were compared and Proleather FG-F was chosen. The relative molecular mass distribution, the free amino acids (FAA) content and the angiotensin-I converting enzyme (ACE) inhibitory activity of the hydrolysates were quantitatively analyzed and compared. In the Proleather FG-F hydrolysates, the percentage of the peptides with molecular weight between 150 and 2000 Da were 84.65%, much more than that in the Alcalase 2.4L hydrolysates (68.44%). Proleather FG-F released much less FAA (5.80%), than Alcalase 2.4L (17.01%). The IC₅₀ of the Proleather FG-F hydrolysate was 0.69 mg/ml, whereas for the Alcalase 2.4L hydrolysate the value was 3.30 mg/ml. Finally, response surface methodology (RSM) was used to optimize the factors (pH, enzyme: substrate ratio- E/S- and temperature) affecting Proleather FG-F hydrolysis.     

Hypocholesterolaemic effect of Bifidobacterium animalis subsp. lactis (Bb12) and trypsin casein hydrolysate

Hypercholesterolaemia is one of the most important risk factors in the development of cardiovascular disease. In this study, we report the in vitro potential of Bifidobacterium animalis subsp. lactis (Bb12) cultures and bovine casein hydrolysates formed by trypsin and Bb12 culture to reduce cholesterols levels. Cholesterol levels in vitro were reduced by up to 48% after incubation with Bb12 and up to 87% after incubation with trypsin hydrolysates, whereas unhydrolysed bovine casein did not affect cholesterol levels. Individual peptide fractions, obtained from size-exclusion chromatography, from casein hydrolysates formed by trypsin after a 48 h hydrolysis, reduced cholesterol levels by 2.7–50%. The molecular masses of these fractions, containing hypocholesterolaemic peptides, were below 1200 Da, as determined by LC-MS.     

Common bean (Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macrophages through suppression of NF-κB pathways

The objectives of this study were to evaluate the antioxidant capacity of protein hydrolysates of the common bean (Phaseolus vulgaris L.) varieties Negro 8025 and Pinto Durango and determine their effect on the markers of inflammation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Cell viability was determined and the percentage of viable cells was calculated and concentrations that allowed >80% cell viability were used to determine the markers of inflammation. Alcalase hydrolysates and pepsin–pancreatin hydrolysates showed the highest antioxidant capacity after 80 and 120 min of hydrolysis, respectively. Alcalase hydrolysates of the common bean Pinto Durango at 120 min inhibited inflammation, with IC₅₀ values of 34.9 ± 0.3, 13.9 ± 0.3, 5.0 ± 0.1 and 3.7 ± 0.2 μM, while var. Negro needed 43.6 ± 0.2, 61.3 ± 0.3, 14.2 ± 0.3 and 48.2 ± 0.1 μM for the inhibition of cyclooxygenase-2 expression, prostaglandin E₂ production, inducible nitric oxide synthase expression and nitric oxide production, respectively. Also, hydrolysates significantly inhibited the transactivation of NF-κB and the nuclear translocation of the NF-κB p65 subunit. In conclusion, hydrolysates from the common bean can be used to combat inflammatory and oxidative-associated diseases.     

Comparison of in vitro digestion characteristics and antioxidant activity of hot- and cold-pressed peanut meals

Due to the poor protein solubility, hot-pressed peanut meal (HPM) has less value than cold-pressed peanut meal (CPM) in the food industry. The objective of this study was to determine whether the denatured proteins in HPM were suitable for hydrolysis by digestive enzymes. The hydrolysis characteristics and antioxidant activity of HPM and CPM during in vitro digestion were compared. The results showed that HPM was hydrolysed more extensively than CPM. There were more free amino acids and small peptides with MW < 5 kDa in HPM hydrolysates. In addition, HPM hydrolysates displayed stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical-scavenging activities, ORAC value and reducing power than CPM hydrolysates, which could be due to the higher content of small peptides, antioxidative amino acids and melanoidins in HPM hydrolysates. The above results indicated that HPM was a potential source of protein supplement for human consumption.     

Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates

Enzymatic hydrolysis of hemp protein isolate (HPI) by six proteases (alcalase, flavourzyme, neutrase, protamex, pepsin and trypsin) and antioxidant activities of the resulting hydrolysates, obtained for 2 and 4 h were investigated. The yield of trichloroacetic acid (TCA)-soluble peptides (Y_sp), protein composition and surface hydrophobicity (H_o) of the hydrolysates were evaluated. The results showed that the hydrolysates exhibited varying DPPH radical scavenging (with lowest IC₅₀, ∼2.3 mg/mL) and Fe²⁺⁺ chelating (with lowest IC₅₀ of 1.6–1.7 mg/mL) abilities and reducing power (with highest absorbance at 700 nm of 0.31–0.35), depending on their Y_sp and H_o values. The DPPH radical scavenging and Fe²⁺⁺ chelating abilities of the hydrolysates were positively correlated with their Y_sp or H_o values. The results suggest that enzymatic hydrolysis can be used as an effective technique to produce high value-added products of hemp proteins.     

Short-wave ultraviolet-C light effect on pitaya (Stenocereus griseus) juice inoculated with Zygosaccharomyces bailii

Fresh pitaya (Stenocereus griseus) juice was inoculated with Zygosaccharomyces bailii to be processed using a continuous ultraviolet-C light (UV-C) (57 μW/cm²) system. Inoculated and uninoculated juices were processed at selected flow rates (16.49, 23.78, and 30.33 mL/s) and treatment times (5, 10, 15, 20, 25, and 30 min). Untreated, inoculated, and uninoculated pitaya juices were stored at 4 °C during 25 days. Microbiological (yeasts plus molds and total counts) and physicochemical (pH, total soluble solids, color, phenolic compounds, betalains, and antioxidant activity) characteristics were evaluated in fresh and processed juices. The net change in color increased as treatment time increased, reaching a maximum value of 3.9. A substantial reduction of phenolic compounds (11.6%), betalains (14.6%), and antioxidant activity (37.0%) were observed in juice treated at 30.33 mL/s during 30 min. A reduction of 1.8 log cycles of Z. bailii was observed at the highest UV-C light treatment.     

In vitro and in vivo protein hydrolysis of beans (Phaseolus vulgaris) genetically modified to express different phaseolin types

Experiments were conducted to study whether phaseolin type could influence proteolysis susceptibility and nutritional value of total bean protein. The DOR-390 bean cultivar was genetically modified to express different phaseolin types (S, T or I). Beans were soaked and autoclaved. A sequential hydrolysis was carried out in vitro with pepsin and pancreatin. Differences in the degree of protein hydrolysis among bean lines started at 30 min and remained until 240 min, with the S bean proteins presenting lower values (P < 0.05). Subsequently, rats were fed with diets containing beans expressing different phaseolin types as the only source of protein for N digestibility and nutritional value determination. No differences (P > 0.05) in ileal protein digestibility and rat growth were observed. In conclusion, the differences in in vitro hydrolysis between bean lines expressing different phaseolin types had no consequences on growth and N retention in rats.     

Comparison of hydrolysis characteristics on defatted peanut meal proteins between a protease extract from Aspergillus oryzae and commercial proteases

A crude protease extract (CPE) was prepared from Aspergillus oryzae HN 3.042 in this work. Three commercial proteases (Alcalase 2.4L, Protamex and papain) and CPE were employed to hydrolyse defatted peanut meal (DPM). CPE was found to be the most effective protease with protein recovery of 80.6%. Moreover, CPE produced a higher degree of hydrolysis (DH, 43.4%) than the tested commercial proteases. The test of molecular weight distribution indicated that DPM proteins were mainly consisted of >10 KDa fraction (86.6%), whereas 3–6 KDa fraction was observed to be the main fraction of all the hydrolysates. CPE hydrolysate possessed a higher nutritional quality than DPM and other hydrolysates on the basis of FAO/WHO (1991) reference pattern. The sensory taste evaluation showed that CPE hydrolysate had better taste than other hydrolysates.     

Lipid oxidation and fishy odour development in protein hydrolysate from Nile tilapia (Oreochromis niloticus) muscle as affected by freshness and antioxidants

Lipid oxidation and fishy odour development in protein hydrolysate from fresh and ice-stored Nile tilapia (Oreochromis niloticus) were investigated. During iced storage of 18 days, heme iron content decreased with a concomitant increase in non-heme iron content (P < 0.05). Peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) values increased. Phospholipid content decreased with a corresponding increase in free fatty acid content. The results suggested that lipid hydrolysis and oxidation took place during storage. When protein hydrolysates were produced from fresh and 18 days ice-stored Nile tilapia muscle, higher lipid oxidation and fishy odour/flavour along with higher amount volatile compounds were obtained in hydrolysate for unfresh sample (P < 0.05). However, the addition of mixed antioxidants during hydrolysis process markedly lowered lipid oxidation, b^∗, ΔC^∗, ΔE^∗ values, fishy odour/flavour as well as the formation of volatile compounds in the resulting hydrolysates prepared from both fresh and unfresh samples. Therefore, hydrolysate from Nile tilapia muscle with reduced fishy odour and lighter colour could be prepared by using fresh fish and incorporation of mixed antioxidants during hydrolysis.     

Optimization of the production of Momordica charantia L. Var. abbreviata Ser. protein hydrolysates with hypoglycemic effect using Alcalase

Momordica charantia L. Var. abbreviata Ser. protein was hydrolyzed using six different proteases. The results showed Alcalase 2.4L to have the best hydrolyzing capacity, followed by Pancreatin. In addition, Alcalase hydrolysate had stronger hypoglycemic effect than that of Pancreatin hydrolysate at the same dose. Alcalase was chosen to produce M. charantia L. Var. abbreviata Ser. protein hydrolysates (MCPHs) with hypoglycemic effect. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions using Alcalase. Model equation was proposed with regard to the effect of enzyme/substrate ratio, pH and temperature. The optimum values for enzyme/substrate ratio, pH and temperature were found to be 2.37%, 9.2 and 57 °C respectively.     

Antioxidant potentials and rosmarinic acid levels of the methanolic extracts of Salvia verticillata (L.) subsp. verticillata and S. verticillata (L.) subsp. amasiaca (Freyn & Bornm.) Bornm

This study was designed to examine the in vitro antioxidant activities and rosmarinic acid levels of the methanol extracts of Salvia verticillata subsp. verticillata and S. verticillata subsp. amasiaca. The extracts were screened for their possible antioxidant activity by two complementary test systems, namely DPPH free radical-scavenging and β-carotene/linoleic acid systems. In the first case, S. verticillata subsp. verticillata was superior to the subsp. amasiaca with an IC₅₀ value of 14.5 ± 1.21 μg mg⁻¹. In the β-carotene/linoleic acid test system, inhibition capacity of S. verticillata subsp. verticillata was 74.4 ± 1.29%. Antioxidant activities of BHT, ascorbic acid, curcumin and α-tocopherol were determined in parallel experiments. Activity of rosmarinic acid was also screened for better establishing the relationship between rosmarinic acid level and antioxidant activity for the plant extracts. S. verticillata subsp. verticillata had the highest rosmarinic acid level with a value of 28.7 ± 0.89 μg mg⁻¹. There is a strong correlation between the rosmarinic acid level and antioxidant activity potential. Our results showed that rosmarinic acid and its derivatives are more likely to be responsible for most of the observed antioxidant activities of Salvia species.     

Use of viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus) for the production of protein hydrolysate from toothed ponyfish (Gazza minuta) muscle

Proteolytic activity of viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was studied. The optimal pH and temperature were 9.0 and 50 °C, respectively, when toothed ponyfish (Gazza minuta) muscle was used as a substrate. When viscera extract from hybrid catfish was used for the production of protein hydrolysate from toothed ponyfish muscle, extract concentration, reaction time, and fish muscle/buffer ratio affected the hydrolysis and nitrogen recovery (NR) (p < 0.05). Optimum conditions for toothed ponyfish muscle hydrolysis were 3.5% hybrid catfish viscera extract, 15 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). High correlation between the degree of hydrolysis (DH) and NR (R² = 0.974) was observed. Freeze-dried hydrolysate had a high protein content (89.02%, dry weight basis) and it was brownish yellow in colour (L^∗ = 63.67, a^∗ = 6.33, b^∗ = 22.41). The protein hydrolysate contained a high amount of essential amino acids (48.22%) and had arginine and lysine as the dominant amino acids.     

The effect of enzymatic hydrolysis time and temperature on the properties of protein hydrolysates from Persian sturgeon (Acipenser persicus) viscera

Protein hydrolysate was prepared from the viscera of Persian sturgeon (Acipenser persicus), a major sturgeon species in the Caspian Sea. Hydrolysis was performed at three different temperatures (35, 45 and 55 °C), pH 8.5, using commercially available Alcalase^® and an enzyme to substrate ratio of 0.1 AU/g viscera protein over a 205 min incubation period. Protein and lipid content of the hydrolysate were 65.82%, and 0.18%, respectively. Protein recovery and degree of hydrolysis ranged from 34.97% to 61.96% and 13.32% to 46.13%, respectively. The highest degree of hydrolysis was observed at 55 °C after 205 min (p < 0.05). The amino acid score of the hydrolysates was similar to that of the FAO/WHO reference protein. It is revealed that Persian sturgeon visceral protein hydrolysate amino acid fulfils adult human requirements. Based on National Research Council guidelines, phenylalanine is the first limiting amino acid in the hydrolysate. However, the hydrolysate has the potential for application as an ingredient in formulated diets.     

Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna

Functional properties and antioxidant activities of protein hydrolysates prepared from ornate threadfin bream (Nemipterus hexodon) muscle, using skipjack tuna pepsin, with different degrees of hydrolysis (DH: 10%, 20% and 30%), were evaluated. Emulsifying and foaming properties of hydrolysates were governed by their DH and concentrations used. Hydrolysates with 20% DH had the highest scavenging activities for ABTS and DPPH radicals. However, chelating activity of hydrolysates for ferrous ion increased as DH increased. Size exclusion chromatography of the hydrolysate with 20% DH using Sephadex G-25 revealed that antioxidative peptides with molecular weight of approximately 1.3 kDa exhibited the highest ABTS radical-scavenging activity. In vitro simulated gastrointestinal digestion indicated that ABTS radical-scavenging activity of the antioxidative peptides was not affected by pepsin hydrolysis, whilst further digestion by pancreatin enhanced the activity. Therefore, protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties.     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

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 2 mg/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 ± 0.013 mg/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.