Isoflavone during protease hydrolysis of defatted soybean meal

The study examined the effects of proteases on the isoflavones during enzymatic hydrolysis of soybean flour. Protease itself did not affect the isoflavones during hydrolysis, whereas the applied conditions and contaminated β-glucosidase in the enzyme could greatly affect the content and composition of isoflavones. Soybean flour hydrolysis by ENZECO Alkaline Protease L-FG at high pH (10) resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates in the hydrolysate. However, these conjugates such as 6″-O-malonylgenistin and 6″-O-malonyldaidzin remained as the principal compounds accounting for 66.2, 58.3 and 70.5% of the total isoflavones in the Protease M “Amano”, Alcalase 2.4L, and neutral enzyme ENZECO Neutral Protease-NBP-L hydrolysates, respectively, compared to that of 57.8% in the original soybean flour. The residue prepared by Protease M contained 10 times higher aglycones than that of soy flour, which was due to the contaminated β-glucosidase activity in the enzyme preparation. Our result showed that β-glucosidase contaminated in Protease M has a unique selectivity compared to that of the purified almond β-glucosidase. Results from the study indicated that hydrolysis of soybean flour may provide another alternative approach to enrich aglycone isoflavones in soybean-containing products.     

Antioxidant activities and functional properties of soy protein isolate hydrolysates obtained using microbial proteases

Soy protein isolate (SPI) hydrolysates were prepared using microbial proteases to produce peptides with antioxidant activity. The process parameters (substrate and enzyme concentrations), hydrolysis time, functional properties and the effects of ultrafiltration were further investigated. The results showed that the soy protein isolate exhibited a 7.0‐fold increase in antioxidant activity after hydrolysis. The hydrolysis parameters, defined by the experimental design, were a substrate concentration of 90 mg mL^?1 and the addition of 70.0 U of protease per mL of reaction. The maximum antioxidant activities were observed between 120 and 180 min of hydrolysis, where the degree of hydrolysis was approximately 20.0%. The hydrolysis increased solubility of the soy protein isolate; however, the hydrolysates exhibited a tendency to decrease in the interfacial activities and the heat stability. The SPI hydrolysates fractions obtained by ultrafiltration showed that the enzymatic hydrolysis resulted in samples with homogenous size and strong antioxidant activity.     

Effects of microwave and ultrasound pretreatments on enzymolysis of milk protein concentrate with different enzymes

Milk protein concentrate was pretreated either by microwave irradiation or by ultrasound before initiation of 3‐h enzymatic hydrolysis. The duration of pretreatment ranged from 1 to 8 min at a power level of 800 W, with the control not being subjected to any pretreatment, and five enzymes (Alcalase, Trypsin, Neutrase, Alkaline Protease and Flavourzyme) were employed. The effects of microwave and ultrasound pretreatments on the kinetics and degree of hydrolysis, protein solubility, bitterness and angiotensin‐converting enzyme inhibitory activity were evaluated. Pretreatments increased the degree of hydrolysis and stabilised the solubility of the hydrolysates but could not significantly reduce bitterness of the hydrolysates The angiotensin‐converting enzyme inhibitory activity of the hydrolysates were improved with 5‐min ultrasound‐pretreated Neutrase hydrolysates giving IC₅₀ value of 0.23 mg mL^?1. Kinetic parameters showed improved catalytic efficiencies. Pretreatments of milk protein concentrates with either microwave or ultrasound significantly improve the bioactivity and functional characteristics of the resulting hydrolysates.     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Physicochemical and sensory characteristics of soya protein isolate hydrolysates with added substrate‐like amino acids

Soya protein isolate (SPI) with or without added substrate‐like amino acid was subject to enzymatic hydrolysis catalysed by commercial proteases (Alcalase 2.4 L, flavourzyme and pancreatin). Addition of a small amount of amino acids (amino acid: SPI = 1: 2500, mol g^?1) during hydrolysis would cause a significantly (P < 0.05) reduced protein recovery, increased degree of hydrolysis, and altered amino acid composition and antioxidant activities of SPI hydrolysates. The SPI hydrolysates prepared with added Asp, Arg or Lys exhibited a higher antioxidant activity than the control. The bitterness of SPI hydrolysates was largely reduced upon addition of Met, Asp or Glu during hydrolysis, whilst the umami taste and mouthfeel‐liking were remarkably increased. Therefore, adding amino acid during hydrolysis is a feasible and beneficial approach to improve both the functional and sensory properties of SPI hydrolysate.     

Effect of limited enzymatic hydrolysis on physico‐chemical properties of soybean protein isolate‐maltodextrin conjugates

The effect of limited hydrolysis was investigated on the physico‐chemical properties of soy protein isolate–maltodextrin (SPI‐Md) conjugate. The hydrolysates at a degree of hydrolysis (DH) of 1.8% showed much higher surface hydrophobicity (H₀; 71.39 ± 3.60) than that of the SPI control (42.09 ± 2.17) and SPI‐Md conjugates (53.46 ± 2.74). Intrinsic fluorescence analysis demonstrated the unfolding of protein molecule and exposure of hydrophobic groups of SPI‐Md conjugate hydrolysates. As evidenced by far‐UV circular dichroism (CD) spectroscopy, the limited hydrolysis increased the unordered secondary structures of SPI‐Md conjugates. The denaturation temperature (T_d) of SPI‐Md conjugate was significantly increased by subsequent limited hydrolysis from 102.53 ± 0.60 °C to 108.11 ± 0.61 °C at DH 1.8%. In particular, the emulsifying activity index (EAI) was improved notably after limited hydrolysis of DH 1.8% (147.76 ± 4.39 m² g^?1) compared with that of native SPI (88.90 ± 1.44 m² g^?1) and SPI‐Md conjugate (108.97 ± 1.45 m² g^?1).     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Functional properties of soy protein hydrolysate produced from a continuous membrane reactor system

Two types of soy protein hydrolysates were produced by hydrolysis of isolated soy protein (ISP) with a combination of Alcalase and Flavourzyme in a continuous membrane system with 3000 and 30,000 daltons molecular-weight-cut-off (MWCO) membranes, respectively. Both hydrolysates were completely soluble over a pH range of 2–9. Their water-binding capacity increased 1.8–3.4 times at a water activity of 0.6–0.95 as compared to intact ISP. The antioxidant activity of ISP was remarkably enhanced by enzyme hydrolysis. The hydrolysate from the 30,000 daltons MWCO membrane had a much higher antioxidant activity than that from the 3000 daltons MWCO membrane. The results suggested that both selected enzymes and MWCO membrane governed the functional properties of protein hydrolysates. Long-term operation study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate over 16 h.     

Antioxidant function of tea dregs protein hydrolysates in liposome–meat system and its possible action mechanism

Tea dregs possess abundant proteins, and the objective of this study was to investigate the antioxidant activity of tea dregs protein hydrolysate with limited hydrolysis by protamex and its possible action mechanism. Tea dregs protein was hydrolysed by alcalase, protamex or neutrase. The hydrolysis condition was optimised, and the hydrolysate was characterised for 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) radical‐scavenging activity, hydroxyl radical‐scavenging activity and antioxidant activity in linoleic acid (LA) system and in chicken products. Tea dregs protein hydrolysate (TDPH) was formulated (0.1%, 0.5%, 1.0%, w/w) into chicken products to determine in situ antioxidant efficacy. Thiobarbituric acid‐reactive substances (TBARS) and peroxide value (POV) formed in chicken products during storage (4 °C, 0–7 days) were analysed. Results showed that the optimum hydrolysis condition was at 50 °C, pH 7.0 for 20 min, and the concentration of tea dregs protein was 1.5%; ratio of protamex to substrate was 6000 U g^?1. The radical‐scavenging ratio of TDPH to 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) was 90.30% at the concentration of 0.1 mg mL^?1 and that to hydroxyl radical was 65.18% at the concentration of 1.0 mg mL^?1. Moreover, it also showed strong antioxidant activity both in linoleic acid (LA) system and in chicken products. The molecular weight distribution of tea dregs hydrolysates was determined by nanofiltration tubular membrane, and the protein hydrolysates with molecular weight above 8000 Da had more effective antioxidant activity. The radical‐scavenging activities to DPPH and hydroxyl radical were 85.72% at 0.1 mg mL^?1 and 71.52% at 1.0 mg mL^?1, respectively. These findings suggest that the enzymatic hydrolysate of tea dregs protein probably possesses the specific peptides/amino acids which could stabilise or terminate the radicals through donating hydrogen. In addition, the hydrolysate could form a physical barrier around the fat droplets.     

Purification and characterization of antioxidative peptides derived from rice bran protein hydrolysates

Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular masses ranging from 670–3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.     

Production of bioactive proteins and peptides from the diatom Nitzschia laevis and comparison of their in vitro antioxidant activities with those from Spirulina platensis and Chlorella vulgaris

This research focuses on green production of bioactive proteins and hydrolysates from Nitzschia. A comparison of antioxidant activities was established between protein extracts and hydrolysates from Nitzschia and two other well‐known microalgae, chlorella and spirulina. Protein hydrolysates from these microalgae were produced using Alcalase^®, Flavourzyme^® and Trypsin. The hydrolysis process enhanced the antioxidant activities in general, especially those obtained using Alcalase^®. Nitzschia showed the highest (P < 0.05) total phenolic content/reducing capacity (2.4 ± 0.02 mg GAE/100 g) after 90 min of hydrolysis with Alcalase^®. The ABTS [2,2′‐Azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid)] radical scavenging activity (66.77 ± 0.00%) was highest (P < 0.05) after 120 min of hydrolysis, but DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl radical) was low (29.59 ± 0.02%). A correlation between ABTS activity and total phenolic contents was the highest (P < 0.05) for protein hydrolysates from all three organisms using Alcalase^®, but superoxide anion radical scavenging activity was intermediate for Nitzschia. Therefore, Nitzschia protein hydrolysates have the potential to be used as antioxidants.     

Limited hydrolysis of β‐casein by cell wall proteinase and its effect on hydrolysates's conformational and structural properties

Optimisation of enzymatic hydrolysis of β‐casein with cell envelope proteinase (CEP) from Lactobacillus acidophilus JQ‐1 to produce the angiotensin‐I‐converting enzyme (ACE) inhibitory peptides using response surface methodology (RSM). Under optimal conditions (enzyme‐to‐substrate ([E]/[S]) ratio (w/w) of 0.132 and pH of 8.00 at 38.8 °C), the ACE inhibitory activity of hydrolysates was 72.06% and the total peptides was 11.75 mg mL^?1. Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β‐casein surface structure was gradually weakened and small holes appeared after enzymatic treatment, while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in the chemical composition and macromolecular conformation of β‐casein after enzymatic hydrolysis. Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates had higher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfhydryl content of β‐casein hydrolysates compared with raw β‐casein, which led to the increase in the antioxidant activity of β‐casein hydrolysates.     

Antioxidant and anti‐acetylcholinesterase activities of anchovy (Coilia mystus) protein hydrolysates and their memory‐improving effects on scopolamine‐induced amnesia mice

Anchovy protein hydrolysates (APHs) were prepared through hydrolysis for 2, 4 or 8 h (APH‐2, APH‐4 and APH‐8, respectively). The chemical analyses, in vitro assessments [antioxidant activity and acetylcholinesterase (AchE) inhibitory activity] and in vivo mice tests were evaluated. Results revealed that APH‐8 exhibited the strongest reducing power and AchE inhibitory capacity (IC₅₀ = 159.76 ± 0.03 mg mL^?1), which may be due to its specific amino acid composition and newly formed peptides. In addition, AchE inhibitory kinetics of amino acids suggested that lysine was featured of both competitive and noncompetitive inhibitors. Furthermore, the results of in vivo study showed that all APHs exhibited memory‐improving action on scopolamine‐induced amnesia mice especially, APH‐8, indicating that anchovy protein is a potential source for health‐promoting peptides.     

Effects of combined high-pressure homogenization and enzymatic treatment on extraction yield,hydrolysis and function properties of peanut proteins

Peanut protein isolate (PPI) was extracted by high-pressure homogenization (HPH) under 0.1 MPa (atmospheric pressure) and 40 or 80 MPa (high pressure). Effects of Alcalase (a proteolytic enzyme) on the enzymatic hydrolysis of PPI and the antioxidant activity of the PPI hydrolysates were investigated. The molecular weight distributions of the PPI hydrolysates were analyzed using Sephadex G-25 gel filtration chromatography while the antioxidant activities, including reducing power, 1,1-dipheny-2-picrylhydrazyl (DPPH) radical-scavenging activity and hydroxyl free radical-scavenging activity of the PPI hydrolysates were evaluated. The extraction yields of PPI by HPH under 0.1, 40 and 80 MPa were 16.84, 30.65 and 39.86%, respectively, which showed that HPH treatment improved the PPI extraction. The HPH treatment increased the degree of hydrolysis of PPI and significantly increased the reducing power and hydroxyl radical­scavenging activity. Furthermore, the molecular weight distributions of the PPI hydrolysates appeared principally over the range of 1000–5000 Da, while the HPH treatment enhanced the production of small peptides, which was in agreement with the high PPI hydrolysis degree. These results suggest that HPH treatment in combination with enzymatic hydrolysis could modify PPI properties and increase the antioxidant activities of the PPI hydrolysates.Industrial relevanceThis study was focused to evaluate the effects of high-pressure homogenization (HPH) in combination with enzymatic hydrolysis on extraction yield and enzymatic hydrolysis of PPI and antioxidant activity of the PPI hydrolysates. This study indicated the possibility of improving the availability of PPI by HPH treatment via increasing extraction yield and enzymatic hydrolysis of the PPI, which can provide a better utilization of the peanut by-product.     

The effect of lactic acid bacteria fermentation on the antioxidant activity of wheat gluten pancreatin hydrolysates

The antioxidant activities of the fermented wheat gluten hydrolysates with different fermentation times were investigated to elucidate the impact of lactic acid bacteria (LAB) fermentation on the wheat gluten hydrolysates. Prior to LAB fermentation, wheat gluten was deamidated by hydrochloric acid and then hydrolysed by pancreatin to 12 and 24 h, respectively. Results showed that LAB fermentation had significant impacts on the enzymatic efficiency and antioxidant activities of wheat gluten. The degree of hydrolysis and protein recovery of hydrolysates gradually increased and then reached maximum values, respectively, when fermenting with LAB for 36 h. The hydrolysis degree and protein recovery of fermented pancreatin 24‐h hydrolysates were larger than that of the fermented pancreatin 12‐h hydrolysates during the whole fermentation. The antioxidant activity analysis revealed a marked increase and improvement in the scavenging activities of 1,1‐Diphenyl‐2‐picrylhydrazyl·radicals, hydroxyl radicals and oxygen radical absorbance capacity, while the scavenging activities of ABTS⁺ radical decreased as the fermentation time extended. The antioxidant activities of pancreatin 24‐h hydrolysates were higher than that of the pancreatin 12‐h hydrolysates during the whole LAB fermentation.     

Determination of reaction kinetics of hydrolysis of tilapia (Oreochromis niloticus) protein for manipulating production of bioactive peptides with antioxidant activity,angiotensin‐I‐converting enzyme inhibitory activity and Ca‐binding properties

To manipulate enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein for production of bioactive peptides, its reaction kinetics was intensively studied. The study showed that the production of peptides with different bioactive properties including antioxidant activity, angiotensin‐I‐converting enzyme (ACE) inhibition and Ca‐binding property and their kinetics were affected by the degree of hydrolysis and substrate concentration. A comparative study on reaction kinetics found that the kinetic parameters for the production of each bioactive peptide are unique, that is, the maximum initial velocity, V_max, for hydrolysis of protein was as high as 1.07 mg mL^?1 min^?1, but that for the production of peptides with antioxidant activity and Ca‐binding property were very low, range of 7.14–66.7 μg mL^?1 min^?1, and that for the production of peptides with ACE inhibitory activity was the lowest, at 2.57 μg mL^?1 min^?1. This knowledge of reaction kinetics of protein hydrolysis would be useful for manipulating and optimising the production of peptides with desired bioactive properties.     

Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

Purification of a histidine-containing peptide with calcium binding activity from shrimp processing byproducts hydrolysate

The shrimp processing byproducts were hydrolyzed by various proteases, and calcium binding activity of the hydrolysates was examined. Among the digests, trypsin digest showed the most potent calcium binding activity (0.294 mmol/g-protein) and highest degree of hydrolysis (18.4%). The trypsin hydrolysate was fractionated according to the molecular weights using ultrafiltration membrane system. The lowest molecular weight fraction (<1 kDa) showed the highest calcium binding activity. Then, the lowest molecular weight fraction was isolated and purified by ion-exchange chromatography, gel filtration, and ODS reversed high-performance liquid chromatography. The purified peptide showed the highest calcium binding activity of 2.70 mmol/g-protein, and its structure was identified as Thr-Cys-His by ESI/MS/MS. Therefore, these results suggested that the peptide derived from shrimp processing byproducts protein hydrolysates is responsible for higher calcium binding properties and may be as natural functional additive in food industry.     

Effects of washing and membrane removal pretreatments on the antioxidant properties of grass carp (Ctenopharyngodon idella) protein hydrolysates produced by in vitro digestion

The effects of washing and membrane removal pretreatments on the antioxidant properties of grass carp protein hydrolysates prepared through in vitro digestion were investigated. Furthermore, antioxidant hydrolysate was fractionated using ultrafiltration membranes (10, 5, 3 and 1 kDa). Oxygen radical antioxidant capacity (ORAC), DPPH and ABTS‐scavenging activity in a gastrointestinal digest produced from pretreated minced grass carp was increased 1.74‐fold, 1.08‐fold and 1.72‐fold, respectively, compared to untreated minced carp. Compared to the alkaline protease hydrolysate, ORAC, ferric reducing antioxidant power, ABTS‐ and DPPH‐scavenging activity in a gastrointestinal digest prepared from pretreated minced carp were reduced by 11.5%, 60.9%, 16.3% and 78.4%, respectively. The ultrafiltration fraction (<1 kDa) displayed the highest antioxidant activity. The size of molecular weight and the amount of hydrophobic and aromatic residues in hydrolysates played an important role in antioxidant activity. Low‐molecular‐weight fish hydrolysates could serve as a potential source of functional ingredients for promoting health.     

Production and characterisation of antioxidant peptides from sweet potato protein by enzymatic hydrolysis with radio frequency pretreatment

Effect of radio frequency (RF at 70, 80 and 90 °C) pretreatment on production and characterisation of sweet potato protein hydrolysates (SPPH) prepared using Alcalase (ALC), Protease (PRO) and the combination of ALC + PRO was investigated. RF highly improved degree of hydrolysis (DH) and enhanced antioxidant activity of all SPPH produced by ALC, PRO and the combination as compared to traditional water bath (WB) heating pretreatment (P < 0.05). RF significantly increased molecular weight (MW) <3 kDa peptide fraction from SPPH produced by ALC, and MW <3 kDa peptide fraction from RF80- and RF90-pretreated samples has higher antioxidant activity. Diverse peptides in MW <3 kDa fractions with RF80 and RF90 pretreatments were identified using LC–QTOF–MS/MS, which matched the sequences of sporamins and contained antioxidant amino acids Trp, Tyr, Met, Phe and/or His. There is a great potential application of using SPPH in functional foods as a novel ingredient.