Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysate from grass carp (Ctenopharyngodon idella) skin

Protein hydrolysates from grass carp skin were obtained by enzymatic hydrolysis using Alcalase^®. Hydrolysis was performed using the pH-stat method. The hydrolysis reaction was terminated by heating the mixture to 95 °C for 15 min. At 5.02%, 10.4%, and 14.9% degree of hydrolysis (DH), the hydrolysates were analyzed for functional properties. The protein hydrolysates had desirable essential amino acid profiles. Results demonstrated that the hydrolysates had better oil holding and emulsifying capacity at low DH. The water holding capacity increased with increased levels of hydrolysis. Enzymatic modification was responsible for the changes in protein functionality. These results suggest that grass carp fish skin hydrolysates could find potential use as functional food ingredients as emulsifiers and binder agents.     

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.     

Effects of skipjack roe protein hydrolysate on properties and oxidative stability of fish emulsion sausage

Effects of skipjack roe protein hydrolysate (SRPH) at various levels (0–3 g/100 g) on properties and oxidative stability of emulsion sausage from broadhead catfish (Clarias macrocephalus) fortified with skipjack tuna roe lipids were investigated. The addition of SRPH increased hardness, cohesiveness and resilience of sausage (p < 0.05). Finer fat globules were visualised in the sample added with SRPH at higher amounts. Nevertheless, the incorporation of SRPH at all levels had no impact on likeness of sausages. SRPH was shown to retard lipid oxidation of sausage during extended storage of 12 days as evidenced by the lower peroxide value (PV) and thiobarbituric acid-reactive substances (TBARS), in comparison with the control. After 12 days, the sausage with 3 g/100 g SRPH had the retained docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), accounting more than 80%. Addition of SRPH had no effect on the organoleptic properties but could prevent the development of rancidity. Nevertheless, it showed no pronounced impact on microbial growth. SRPH could therefore be used as a natural antioxidative emulsifier in cooked fish emulsion sausage.     

Isolation of antioxidative and ACE inhibitory peptides from protein hydrolysate of skipjack (Katsuwana pelamis) roe

Bioactive peptides from protein hydrolysate of defatted skipjack (Katsuwonus pelamis) roe with 5% degree of hydrolysis (DH) prepared by Alcalase digestion were isolated and characterised. Two active fractions with ABTS radical scavenging activity (973.01–1497.53 μmol TE/mg sample) and chelating activity (0.05–0.07 μmol EE/mg sample) from consecutive purification steps including ultrafiltration, cation exchange column chromatography and reverse phase high performance liquid chromatography (RP-HPLC), were subjected to analysis of amino acid sequence by LC–MS/MS. Seven dominant peptides with 6–11 amino acid residues were identified as DWMKGQ, MLVFAV, MCYPAST, FVSACSVAG, LADGVAAPA, YVNDAATLLPR and DLDLRKDLYAN. These peptides were synthesised and analysed for ACE-inhibitory activity and antioxidative activities. MLVFAV exhibited the highest ACE inhibitory activity (IC₅₀ = 3.07 μM) (p < 0.05) with no antioxidative property, whilst DLDLRKDLYAN showed the highest metal chelating activity, ABTS radical and singlet oxygen scavenging activities. Therefore, peptides prepared from skipjack roe could be further employed as a functional food ingredient.     

Physico-chemical, amino acid composition, functional and antioxidant properties of roe protein concentrates obtained from Channa striatus and Lates calcarifer

Roe protein concentrates prepared from Channa striatus (CRPC) and Lates calcarifer (LRPC) were investigated for physico-chemical characteristics, amino acid composition, functional properties and antioxidant activity. Channa and Lates roes yielded 20.7% and 22.5% of protein concentrates possessing 90.2% and 82.5% protein, respectively. Major differences were not observed in each of the amino acids except leucine in CRPC and LRPC. The solubility of protein was 3.93-54.6% and 1.6-55.5% over a pH range of 2-12 in CRPC and LRPC, respectively. Water absorption, oil absorption, foam capacity, stability and emulsifying capacity were found to be higher in CRPC than in LRPC. Antioxidant activity determined by the radical scavenging activity and ferric reducing power was higher in CRPC. SDS-PAGE of both roe protein concentrates showed protein bands of 170, 95 and 55 kDa. Moisture sorption isotherms of protein concentrates indicated their hygroscopic nature.     

Physicochemical and functional properties of the protein isolate and major fractions prepared from Akebia trifoliata var. australis seed

The physicochemical and functional properties of protein isolate (API) and major protein fractions prepared from Akebia trifoliata var. australis seed were investigated. The seed contained 38.83% of oil and 17.23% of protein. Albumin (51.65%) and glutelin (46.40%) were the predominant fractions in the protein component of the seed. The major amino acids were found to be glutamic acid and aspartic acid, while the contents of sulphur-containing amino acids and threonine were very low. One to eight distinct bands with molecular weight (MW) ranging from 12.0 to 50.0 kDa were displayed by SDS–PAGE. The solubilities of API, albumin and glutelin from seeds of the A. trifoliata var. australis were the lowest at pH 4.0–5.0. The high surface hydrophobicity indices of these three proteins were observed at pH 7.0, while the excellent emulsifying properties were displayed at pH 9.0. Circular dichroism measurements indicated that API, albumin and glutelin were rich in β-strand and random coil structures.     

Effects of limited enzymatic hydrolysis with trypsin on the functional properties of hemp (Cannabis sativa L.) protein isolate

Effects of limited enzymatic hydrolysis induced by trypsin on the physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate (HPI) were investigated. The enzymatic hydrolysis was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC). SEC and differential scanning calorimetry (DSC) analyses confirmed the presence of aggregates in the corresponding hydrolysates (with the degree of hydrolysis of 2.3–6.7%). Functional properties, including protein solubility (PS), thermal properties, emulsifying and foaming properties, and water holding and fat adsorption capacities (WHC and FAC) were evaluated. The PS was remarkably improved by the limited enzymatic hydrolysis at all tested pH values. However, the enzymatic hydrolysis led to the marked decreases in emulsifying activity index, foaming capacity and foam stability, WHC and FAC. These decreases were to a great extent related to the presence of aggregates in the hydrolysates.     

Protein isolates from two Mediterranean legumes: Lathyrus clymenum and Lathyrus annuus. Chemical composition,functional properties and protein characterisation

Protein isolates were analysed from two Mediterranean legumes, Lathyrus clymenum and L. annuus. Protein isolates were prepared by alkaline extraction, including sodium sulphite and acid precipitation of Lathyrus proteins at their isoelectric point (pH 4.5). The percentage of proteins recovered from L. annuus and L. clymenum flours during the preparation of the protein isolates was around 60%. Chemical composition, nutritional parameters, main functional properties and protein composition of Lathyrus protein isolates were studied. L. annuus and L. clymenum protein isolates contained 81.07% and 82.4% of proteins, respectively, and they have a balanced content of essential amino acids, except for sulphur amino acids, with respect to the FAO pattern. The in vitro protein digestibility increased in the protein isolates to 93% and 95% in L. annuus and L. clymenum, respectively. Functional properties were similar to those observed in other legumes protein isolates. These results confirm the interest of local crops as sources of high value protein products obtained after convenient protein extraction procedures and the removal of antinutritional components. These high added value protein isolates are of interest for the food industry and for the revalorisation of L. annuus and L. clymenum favouring the bioconservation of Lathyrus.     

Physicochemical and functional properties of makal (Xanthosoma yucatanensis) starch

Physicochemical and functional properties of makal (Xanthosoma yucatanensis) starch were determined. Granules were oval in shape and 12.4 μm average diameter. Starch purity was high (96.7%) with low protein (0.1%), fat (0.2%), fibre (0.4%) and ash (0.1%) contents. Amylose content was 22.4%. The gelatinization temperature was 78.5 °C and transition enthalpy was 15 J/g. At 90 °C, solubility was 32.9%, swelling power was 28.6 g water/g starch and water absorption capacity was 19.2 g water/g starch. Pasting characteristics were: temperature 75 °C, maximum viscosity 280 BU, breakdown −8 BU, setback 180 BU and consistency 172 BU. Clarity, expressed as transmittance, was 35.8%. Gel deformation was 20.8% with a 0.03 kgf maximum load. Makal starch’s high gelification temperature and firmness make it appropriate for use in high temperature food systems, but its low stability in refrigeration and freezing cycles make it inadequate for use in foods subject to those conditions.     

Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent K_m and K_cat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S⁻¹, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.     

Antioxidative properties of protein hydrolysate from defatted peanut kernels treated with esperase

The aim of this study was to facilitate development of natural antioxidants from defatted peanut kernels. Protein hydrolysates obtained from defatted peanut kernels with esperase treatment for 2 h exhibited higher antioxidative activity toward linolenic acid peroxidation than other proteases (including neutrase, pepsin, protease A and protease N). The esperase hydrolysate of peanut protein (EHPP) was further separated with 3 and 5 kDa molecular cut-off membranes to determine the influence of molecular weight. EHPP with molecular weight 3∼5 kDa showed higher relative antioxidative activity, DPPH radical scavenging activity and metal chelating activity than that with molecular weight lower than 3 kDa or higher than 5 kDa. The fraction was further purified by ion exchange chromatography and high-performance liquid chromatography; the final peanut peptides exhibited higher relative antioxidative activity (27.5) than ascorbic acid (9.5). This study reported for the first time that protein hydrolysates from defatted peanut kernels possess antioxidative activity.     

Nitrogen extractability and functional properties of defatted Erythrina variegata flour

Defatted Erythrina variegata flour was prepared from dehusked seed meal by hexane extraction of residual oil. The resulting flour had 403 g kg⁻¹ of protein as compared to 282 g kg⁻¹ in the whole seed-defatted meal. Nitrogen extractability of the defatted flour in water was not much influenced by the length of extraction period above 40 min, but an increased extraction was observed at a higher liquid to solid ratio up to a studied limit of 1:60; the optimal ratio was found to be 1:30. The minimum of 26.9% nitrogen was extracted in the pH range 3.0–4.0 and maximum of 94.8% at pH 12. Addition of sodium chloride (0.1 or 0.5 M) broadened the pH range of minimum nitrogen extractability and shifted it toward a lower pH region. At both concentrations of sodium chloride, a marked increase in nitrogen extractability, in the pH range 3.0–7.0, was observed. Precipitation of protein from an extract of pH 10.0 was maximum (85.3%) at pH 4.75. A higher buffer capacity of the flour was observed in the acidic medium (0.195 mmol HCl g⁻¹ flour) than in alkaline medium (0.093 mmol NaOH g⁻¹). Water absorption and oil absorption values for the whole E. variegata seed flour and the dehusked, defatted flour were 1.81, 1.43 and 1.02, 1.52 kg kg⁻¹, respectively.     

Functional properties and in vitro antioxidant activity of roe protein hydrolysates of Channa striatus and Labeo rohita

Bioactive roe protein hydrolysates were prepared from Channa striatus (CRPH) and Labeo rohita (LRPH) and their functional and in vitro antioxidant properties evaluated. The degree of hydrolysis was 28.41% at 60min in channa and 18.85% in labeo roe concentrates at 90min. The yields of protein hydrolysates were 24.15% and 12.45% for channa and labeo roe protein concentrates, respectively. The protein content was identical (58%) in both roe protein hydrolysates. Protein solubility in channa was higher (90.48%) when compared to labeo (50.6%) at pH 12. Higher oil absorption capacity and foam stability were observed in CRPH and higher emulsifying capacity was found in LRPH. Smaller peptides of 12kDa were noted in both CRPH and LRPH. In vitro antioxidant activity was higher in CRPH than in LRPH as seen from DPPH radical scavenging and ferric reducing power.     

Chemical composition and functional properties of Gleditsia triacanthos gum

The seeds of Gleditsia triacanthos (Fabaceae) are a source of galactomannans with a variable mannose:galactose (Man:Gal) ratio that depends on the isolation and purification methods. In this study, three processes were used: (1)—treatment with boiling water (M1); (2)—concentration with boiling 2 N NaOH (M2) and (3)—swelling with water and later manual removal of the endosperm (M3). The functional properties of the hydrocolloids obtained by the three isolating methods were compared with those exhibited by xanthan and guar gums, which are widely used as food additives. The product obtained by boiling water (M1) had the lowest yield, although that hydrocolloid showed high water absorption capacity and solubility, high emulsifying and foaming capacities, and imparted high stability to the dispersions (foams and emulsions). These properties could be influenced by its high Man:Gal ratio and protein content.     

Amino acid composition,antioxidant and functional properties of protein hydrolysates from the roe of rainbow trout (Oncorhynchus mykiss)

A fish roe protein hydrolysate from rainbow trout (Oncorhynchus mykiss) trout roe protein hydrolysates (TRH) was produced by pepsin and Alcalase. Proximate, amino acid compositions, protein digestibility and molecular mass distribution of the hydrolysates were determined. The degree of hydrolysis was found to be 44.08% and 27.62% (pepsin and Alcalase, respectively). The two hydrolysates contained a high amount of essential amino acids (33.53% Alcalase–29.39% pepsin). The results showed that TRH by different enzymes is a good source of the leucine and lysine amino acids. The pepsin produced a white powder with higher brightness (L* = 89.50). Alcalase hydrolysate was brownish yellow in colour (L* = 52.85, a* = 10.30, b* = 26.25). The hydrolysates represented excellent antioxidant activities in various concentrations. TRHs showed a good foaming and emulsification properties. The results thus revealed that protein hydrolysates from rainbow trout roe could be used as food additives possessing essential amino acids and antioxidant activity.     

Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type

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.     

Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis)

Three trypsin isoforms, trypsins A, B and C, from the spleen of skipjack tuna (Katsuwonus pelamis) were purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl-cellulose to obtain a single band on native-PAGE and SDS–PAGE. The molecular mass of all the trypsin isoforms was estimated to be 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature of the three isoforms for the hydrolysis of N-p-tosyl-l-arginine methyl ester hydrochloride were 8.5 and 60 °C, respectively. Trypsins were stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. All isoforms were stabilised by calcium ions. The trypsin activities were effectively inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid, while E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibitory effect. Activities decreased continuously as NaCl concentration (0–30%) increased. Trypsins A, B and C showed K_m of 0.11–0.29 mM and K_cat of 57.1–114 s⁻¹. The N-terminal amino acid sequence of 20 residues of three trypsin isoforms was IVGGYECQAHSQPHQVSLNS and had high homology to those of other fish trypsins.     

Seasonal effects on the physicochemical characteristics of fish sauce made from capelin (Mallotus villosus)

Fresh capelin (Mallotus villosus) was harvested from the North Atlantic during both summer and winter fishing seasons. Reaction conditions for fish sauce processing were optimized with respect to temperature, salt concentration and reaction time, using a response surface methodology (RSM) experimental design. Whole capelin was minced and samples were ground with increasing salt concentrations. RSM optimizations were conducted, ranging from 5% to 30% salt, and incubating at 5° intervals from 0 to 65 °C. Autolytic activity was estimated by extracting the liquid formed by the mixture with trichloroacetic acid and estimating protein content by the Lowry method. Samples for fish sauce production were then prepared under optimized conditions by mixing ground capelin with 10% salt and incubating at 50 °C for up to 270 days for the summer capelin and up to 360 days for the winter capelin. Samples were collected at regular intervals and analyzed for liquid yield, moisture, protein, soluble solids, specific gravity, pH, colour and amino acid content. Kjeldahl protein content in the fish sauce from summer capelin was 2.03% after 250 days of fermentation and twice as high as that in winter capelin fish sauce. Moisture content and pH were lower in the summer capelin fish sauce, but Brix and density were higher than those in fish sauce from winter capelin. Brown colour formation was very rapid in the summer capelin fish sauce but slow in the winter capelin fish sauce. Summer capelin may be successfully utilized for the production of fish sauce without added enzymes.     

Functional Properties of Grass Carp (Ctenopharyngodon Idella), Nile Perch (Lates Niloticus) and Nile Tilapia (Oreochromis Niloticus) Skin Hydrolysates

The influence of using an endo-peptidase (alcalase) on the functional properties of hydrolysis products from Nile perch, Grass carp, and Nile tilapia skin was studied. Reaction conditions were controlled at pH 8.25, 60°C, and the enzyme was added on the basis of standard activity units at an enzyme to substrate ratio of 1.7 g/100 g protein. The reaction was allowed to proceed for 85 min and enzyme was inactivated by heat. The soluble protein fractions were recovered and lyophilized. All freeze-dried fish skin hydrolysates powders had a light yellow color and contained up to 90% protein. Nitrogen solubility varied from 95.93 to 98.72% and was not significantly different at 5% probability level. The water and oil holding capacities of the skin hydrolysates were good in the range of 2.8 to 3.2 mL/g and 3.4 to 3.8 mL/g, respectively. Emulsification capacity varied from 11.3 to 21 mL/0.5 g with Nile perch skin hydrolysate having the highest score while Nile tilapia skin hydrolysate was the lowest. Grass carp skin hydrolysate was not able to form stable foam, unlike the Nile perch and Nile tilapia skin hydrolysate. Alcalase treated freshwater fish skin exhibited satisfactory functional properties hence may play an important role as an ingredient in the food and pharmaceutical industry.