Whey protein isolate polydispersity affects enzymatic hydrolysis outcomes

The effects of heat-induced denaturation of whey protein isolate (WPI) on the enzymatic breakdown of α-La, caseinomacropeptide (CMP), β-Lg A and β-Lg B were observed as hydrolysis proceeded to a 5% degree of hydrolysis (DH) in both unheated and heat-treated (80 °C, 10 min) WPI dispersions (100 g L⁻¹). Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. LC–MS/MS identified 23 distinct peptides which were identified in the denatured WPI hydrolysate – the majority of which were derived from β-Lg. The mapping of the detected regions in α-La, β-Lg, and CMP enabled specific cleavage points to be associated with certain serine endo-protease activities. The outcomes of the study emphasise how a combined approach of substrate heat pre-treatment and enzymology may be used to influence proteolysis with attendant opportunities for targeting unique peptide production and amino acid release.     

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.     

Improved emulsifying properties of soy proteins by acylation with saturated fatty acids

Effects of acylation on emulsifying properties of soy proteins were investigated using a variety of saturated fatty acids. Beta conglycinin (7S), glycinin (11S), and acid-precipitated protein (APP) were acylated with activated fatty acid esters (6C–18C) to form covalent linkage between the carboxyl group of the fatty acid and the free amino groups of the protein. Reduction in the free amino groups of acylated 7S, 11S and APP resulted into the dissociation of the protein, indicating a structural change, as evidenced by the fluorescence spectra and the degree of modification. It was shown that the emulsifying activity (EAI) and emulsion stability (ES) of 7S and 11S were significantly improved (p < 0.05) upon acylation with all saturated fatty acids, whereas no change in EAI and ES for the acylated APP was observed upon attachment of short and long chain fatty acids. The fluorescence intensity was also remarkably affected by acylation showing significant changes in protein structure. Covalent attachment of fatty acids resulted into 1.4–2.2 and 1.1–1.8-fold increase in the oil binding capacity (OBC) of 7S and 11S respectively, however no changes in acylated APP. Acylated 7S showed 3.0–9.4-fold increase in the water binding capacity (WBC), with no change in acylated 11S, while acylated APP with longest chains showed low WBC. The surface hydrophobicity of 7S was significantly improved (p < 0.05) by acylation; no changes were observed in the acylated 11S. Furthermore, acylation decreased the surface hydrophobicity of APP. Thus, it was demonstrated that saturated fatty acids with adequate chain length are suitable candidates for the preparation of functional lipoproteins from soy proteins.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

Improvement of the antioxidant properties of squid skin gelatin films by the addition of hydrolysates from squid gelatin

Gelatin hydrolysates (HG1 and HG2) were obtained from giant squid (Dosidicus gigas) gelatins (G1 and G2) by hydrolysis with Alcalase. Antioxidant properties of both gelatins were highly increased by hydrolysis, especially ABTS radical scavenging capacity, whereas no significant differences were found between HG1 and HG2. The amino acid composition of HG1 and HG2 closely resembled the amino acid composition of the parent proteins, gelatins G1 and G2. Both, HG1 and HG2 were composed by peptides below 30 kDa, although no clear protein bands were observed in HG2. Edible gelatin films with increasing percentages of HG1 (0–10%) were made from G1, giving rise to increasing values of FRAP and ABTS, as well as changes in mechanical properties (decrease puncture force and increase puncture deformation) and water vapour permeability (increase). HG1 gelatin hydrolysate showed lower antioxidant capacity in the gelatin films than in the free form at the same amount added into the filmogenic solution, probably due to interactions with protein matrix.     

Amino acid composition of three species of Nigerian fish: Clarias anguillaris,Oreochromis niloticus and Cynoglossus senegalensis

An investigation into the concentrations of amino acids in three species of Nigerian fish: Clarias anguillaris, Oreochromis niloticus and Cynoglossus senegalensis was carried out. The most abundant amino acid was Glu (108–118 mg/g crude protein) and Leu was the most abundant essential amino acid (58.0–64.7 mg/g crude protein). While total amino acid content was 618–637 mg/g crude protein, the total essential amino acid content was 300–317 mg/g or 48.6–50.0% with His but 283–299 mg/g crude protein or 45.8–47.0% without His. The limiting amino acid was Thr for C. anguillaries and C. senegalensis but Val for O. niloticus whereas Phe + Tyr scores were greater than 1.0 (1.06–1.21) in all the fish samples. Significant differences existed between the contents of essential amino acids and non-essential amino acids at p < 0.05 in all the fish samples. Percentage contents of total neutral amino acids ranged from 52.2–54.9%; for total acidic amino acids the range was 26.9–29.6% and for total basic amino acids the range was 17.7–18.2%.     

Influence of oxidative browning inhibitors and isolation techniques on sweet potato protein recovery and composition

Effects of oxidative browning inhibitors on sweet potato protein (SPP) recovery and quality were studied. Oxidative browning inhibitors successfully decreased sweet potato oxidative browning, but reduced both SPP extractability and recovery. Ultrafiltration/diafiltration processed sweet potato (UDSP) protein (at pH 4, 6 and 7) showed significantly (p < 0.05) higher yield, purity, solubility, thermal stability and amino acid constituents than that of isoelectrically precipitated sweet potato (IPSP) protein (at pH 4). The yield of UDSP proteins was more than twice that of IPSP protein. Denaturation temperature (T_d), enthalpy change (ΔH) and solubility (at pH 3 and 8) of UDSP proteins were in the ranges 82.89–90.29 °C, 6.34–11.35 (J/g) and 71.4–94.2%, respectively, while that of IPSP protein were 85.27 °C, 2.35 (J/g) 31.2% and 55.5%, respectively. Ratio of SPP essential amino acid to the total amino acid ratio ranged from 0.49 to 0.51. SPP in vitro digestibility and digestibility-corrected amino acid score (PDCAAS) ranged 70–80.7% and 44.79–51.08%, respectively.     

Influence of temperature and degree of hydrolysis on the peptide composition of trypsin hydrolysates of β-lactoglobulin: Analysis by LC–ESI-TOF/MS

Enzymatic hydrolysis of proteins is influenced, either positively or negatively, by the hydrolysis conditions, temperature, enzyme concentration and pH, as well as substrate pre-treatments, e.g. heat-denaturing, glyco-conjugation and/or cross-linking. Purified bovine β-lactoglobulin (96.0% nitrogen) was hydrolysed using trypsin (EC 3.4.21.4, bovine pancrease) at between 30 and 50 °C to degrees of hydrolysis (DHs) between 1 and ∼9.0%. The time taken to reach the desired DH varied greatly, being shortest at 45 and 50 °C and longest at 30 °C. The hydrolysates were analysed by tandem liquid chromatography–electrospray ionisation time-of-flight mass spectra (LC–ESI-TOF/MS) and results showed that the detectable peptides, at both 30 °C and 35 °C, were similar at DH 1%. However, not only were the detectable peptides produced at 40–50 °C different from those produced at lower temperatures, but the trypsin released peptides due to non-specific hydrolysis of β-Lg. The pattern resembled a shift of trypsinolysis towards chymotrypsinolysis, probably due to steric ‘stretching’ and increase of the catalytic pocket, thus allowing bulky amino acids to be processed. Hydrolysis at 30 °C to DH 5% and 10% also led to the release of peptides due to non-specific cleavage by trypsin. These results indicate that trypsin could only release peptides in a predictable manner at temperatures near, but lower than, the declared optimum of 37 °C. Above this temperature and above DH 5–10% at 30 °C, hydrolysis followed a mixed trypsin- and chymotrypsin-like activity. Lys–Pro, Lys–Ile(–Pro) and Lys–Phe bonds remained stable to trypsin at all temperatures. Some peptides with a high content of hydrophobic amino acids were undetected by ESI-TOF/MS, probably due to their poor ionisation.     

Inhibition of polyphenol oxidase and peroxidase activities on fresh-cut apple by simultaneous treatment of ultrasound and ascorbic acid

The effects of ultrasound and ascorbic acid on activity changes of polyphenol oxidase and peroxidase, of fresh-cut apple during storage, were investigated. The combined treatment of ultrasound and ascorbic acid inactivated monophenolase, diphenolase, and peroxidase, whilst the individual treatment of ultrasound or ascorbic acid had inverse and limited inhibitory effect on the enzymes. The main protein bands had a molecular weight of approximately 63 kDa. A diffuse band, lacking the electrophoretic mobility of proteins, was observed after combined treatment. This investigation revealed that simultaneous treatment with ultrasound and ascorbic acid had synergistic inhibitory effects on several enzymes related to enzymatic browning.     

Folic acid complexes with human and bovine serum albumins

The interaction of folic acid with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various folic acid contents was investigated. FTIR, UV–visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse folic acid binding sites, the binding constant and the effect on HSA and BSA stability and conformations. Structural analysis showed that folic acid binds HSA and BSA via both hydrophilic and hydrophobic contacts with overall binding constants of K_{folic acid–HSA} = 8.1 (±0.5) × 10⁴ M⁻¹ and K_{folic acid–BSA} = 1.0 (±0.3) × 10⁵ M⁻¹. The number of bound acid molecules per protein was 1.7 (±0.4) for HSA and 1.5 (±0.3) for BSA complexes. Molecular modelling showed participation of several amino acids in folic acid–protein complexes stabilised by hydrogen bonding network. Folic acid complexation altered protein secondary structure by major reduction of α-helix from 59% (free HSA) to 35% (acid-complex) and 62% (free BSA) to 25% (acid-complex) with an increase in random coil, turn and β-sheet structures indicating protein unfolding. The results suggest that serum albumins might act as carrier proteins for folic acid in delivering it to target molecules.     

Optimisation,characterisation and quantification of phenolic compounds in olive cake

The phenolic compounds in extracts from pressed olive cake were investigated. Free phenolic compounds were extracted from olive cake using methanol. To liberate bound phenolic compounds, the olive cake was subjected to basic and acidic hydrolysis followed by methanol extraction. The individual phenolic compounds and antioxidant activity of the extracts were determined. The highest total phenolic content and antioxidant activity were obtained using methanol extraction for 12 h at 70 °C. The RP-HPLC profiles for full-fat and defatted olive cake showed that protocatechuic acid, hydroxybenzoic acid, sinapic acid, p-coumaric acid, rutin and hesperidin were the predominant free phenolic compounds. Meanwhile, syringic acid, sinapic acid, caffeic acid and protocatechuic acid were the predominant bound phenolic acids. A positive correlation was observed between total phenolic content and antioxidant activity. The results indicated that most of the phenolic compounds in olive products were present in their free forms (75–90% of total phenolic content), while bound phenolic compounds were only a small proportion (10–25%) of total phenolic content.     

Kinetic of the hydrolysis of pectin galacturonic acid chains and quantification by ionic chromatography

These experiments were designed to develop a rapid, repeatable and accurate analysis method for the quantification of galacturonic acid of pectins. Different pectin hydrolysis procedures (chemical and enzymatic) were carried out with H₂SO₄, TFA and HCl at different acid concentrations (0.2, 1 and 2 M) and temperatures (80 and 100 °C). Enzymatic and combined chemical and enzymatic hydrolysis of pectin were also studied. A acid hydrolysis under drastic conditions (100 °C) is insufficient for complete hydrolysis and results in low recovery of galacturonic acid residues. Mild chemical hydrolysis (0.2, 1 and 2 M H₂SO₄ 72 h at 80 °C) is also insufficient for complete depolymerization. Its main advantage is cleavage of the galacturonic acid chains into oligomeric forms without any degradation within 72 h of hydrolysis. However, enzymatic hydrolysis with VL9 for 2 h at 50 °C and combined chemical and enzymatic hydrolysis (0.2 M TFA at 80 °C for 72 h and VL9) give high recovery of this acid. Analysis of the liberated sugar residue by HPAEC allows us to determine the galacturonic acid composition of this polysaccharide accurately with high selectivity and sensitivity in one assay without the need for derivatization.     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

Trypsin (EC 3.4.21.4) hydrolysis of food proteins are done at the optimum pH (7.8) and temperature (37 °C). Little information is available on the effect of sub-optimal conditions on hydrolysis. Bovine β-lactoglobulin (β-Lg) was hydrolysed by trypsin under acidic pH (pH 4–7) between 20 and 60 °C and the substrate concentration from 2.5% to 15% (w/v) and compared with hydrolysis at pH 7.8 and 37 °C. Aliquots were taken at different times (t = 0 up to 10 min). Samples were analysed using matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI–TOF–MS/MS) with α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxyacetophenone (DHAP) matrices. Hydrolysis patterns of β-Lg were generally similar at pH 7.8, 7, 6 and 5 while at pH 4 fewer peptides were detected except a unique fragment f(136–141). The different cleavage sites of β-Lg showed low resistance to trypsin at optimum conditions and pH 7 while being random and simultaneous. At lower pH, some cleavage sites showed increased resistance, while hydrolysis was relatively slow and ordered. Initial attack by trypsin occurred at Arg⁴⁰–Val⁴¹, Lys¹⁴¹–Ala¹⁴² and Arg¹⁴⁸–Leu¹⁴⁹ resistance was at Lys⁶⁰–Trp⁶¹, Arg¹²⁴–Thr¹²⁵ and Lys¹³⁵–Phe¹³⁶. Five domains were identified based on β-Lg resistance to trypsin in the order f(1–40) < f(41–75) < f(76–91) > f(92–138) > f(139–162). Results suggest that hydrolysis away from trypsin optimum offer better hydrolysis process control and different peptides. This strategy may be used to protect target bioactive or precursor peptides, or avoid the production of unwanted peptides.     

Comparison of free amino acid,carbohydrates concentrations in Korean edible and medicinal mushrooms

Ten popular species of both edible and medicinal Korean mushrooms were analysed for their free amino acids and disaccharides. The average total free amino acid concentration was 120.79 mg g⁻¹ in edible mushrooms and 61.47 mg g⁻¹ in medicinal mushrooms, respectively. The average total of free amino acids for all mushrooms, edible mushrooms and medicinal mushrooms was 91.13 mg g⁻¹. Agaricus blazei (227.00 mg g⁻¹) showed the highest concentration of total free amino acids; on the other hand, Inonotus obliquus (2.00 mg g⁻¹) showed the lowest concentration among the 10 species of mushrooms. The average total carbohydrates concentration was 46.67 mg g⁻¹ in the 10 species of mushrooms, where the edible mushrooms contained 66.68 mg g⁻¹ and the medicinal mushrooms contained 26.65 mg g⁻¹. The carbohydrates constituents of the 10 mushroom species were mainly mannose (36.23%), glucose (34.70%), and xylose (16.83%).     

Chemical compositions and nutritional value of Asian hard clam (Meretrix lusoria) from the coast of Andaman Sea

Chemical compositions and nutritive value of the edible portions including foot, mantle and viscera of Asian hard clam (Meretrix lusoria) harvested from the coast of Andaman Sea were determined. Proximate compositions varied with portions tested. Edible portions had moisture (76.23–84.22%) and protein (9.09–12.75%) as the major components. Carbohydrate (0.32–7.89%), fat (1.58–6.58%) and ash (1.23–2.58%) were also found at various levels, dependent upon portions. Myofibrillar proteins were observed as the major fraction in foot (40.54%) and mantle (31.65%), whilst non-protein nitrogen constituents were dominant in the viscera (36.85%). All portions contained a large amount of essential amino acids (167.66–187.63 mg/g sample), in which leucine (30.91–36.96 mg/g sample) and lysine (35.24–36.03 mg/g sample) were predominant. They were rich in polyunsaturated fatty acids (46.84–49.18% of total fatty acid) with high level of DHA (13.33–16.47 % of total fatty acids) and EPA (4.75–7.11% of total fatty acids). Cholesterol of 0.07–0.21% wet weight was detected. All portions were also rich in macro- (Na, K, Ca and Mg) and micro- (Fe, Zn, Cu and Cr) minerals. Therefore, Asian hard clam is an excellent source of several nutrients, which could be beneficial for the health of the consumers.     

Immunomodulatory effects of feruloylated oligosaccharides from rice bran

Feruloylated oligosaccharides (FOs), the ferulic acid ester of oligosaccharides, can be released either by the enzymatic or mild acid hydrolysis of arabinoxylans present in cereal bran, and are usually considered as natural antioxidants. However, no related research is available to explain their immunomodulatory effects. This report elucidated their immunomodulatory effects through the variations of pro-inflammatory mediators in vitro. FOs were obtained from the mild acid hydrolysis of rice bran. We found that FOs (0.1–100 μg/ml) induced tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, nitric oxide (NO) and PGE₂ production in unstimulated macrophages, RAW264.7 cells. Furthermore, pre- and post-treated FOs (0.1–100 μg/ml) dose-dependently suppressed TNF-α, IL-1β, IL-6 and NO production, and induced IL-10 production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells without exerting cytotoxicity. As a result anti-inflammatory and therapeutic activities were revealed. It is noteworthy that prostaglandin E₂ (PGE₂) production was significantly suppressed at an FO level of 100 μg/ml. The in vitro assessment of inflammatory mediators should be useful in further characterising the effects of FOs on immunomodulation. Moreover, it will create the economical value of rice bran, which has long been considered as conventional agricultural wastes.     

Chemical changes in the volatile fractions of Brazilian honeys during storage under tropical conditions

The chemical transformations of the volatile fractions of two different Brazilian honeys (cashew and marmeleiro) were monitored during storage under tropical conditions. Five systems, namely: 1 – fresh samples; 2 and 3 – samples heated for 3 and 6 months at 35 – 40 °C; 4 and 5 – samples kept under similar conditions to systems 2 and 3, but containing sodium metabisulphite, were tested. The transformations noted in the volatile fractions of these honeys could be mainly associated to acid or enzymatic hydrolysis of glycosides, volatilization, oxidation or esterification processes. The formation of furan derivatives, linalool derivatives and esters appears to be quite affected by the sodium metabisulphite. The concentrations of most powerful odorants increased during the storage (e.g.: benzenemethanol and isovaleric acid) or remained unchangeable (e.g.: vanillin, furfuryl mercaptan, 2-methoxyphenol).     

Cellulase-assisted release of secoisolariciresinol from extracts of flax (Linum usitatissimum) hulls and whole seeds

Extraction of secoisolariciresinol from seed hulls and whole seeds of flax was improved using an enzymatic step with cellulase R10 from Trichoderma reesei that allowed better yield as compared to β-glucosidase. The cellulase assisted extraction process was further optimised for different parameters such as duration and concentration of hydromethanolic extraction, duration of alkaline hydrolysis, pH, duration and incubation temperature as well as enzyme concentration. Best results were obtained using a method including the following successive steps: 16 h of 70% hydromethanolic extraction, 6 h of 0.1 M sodium hydroxide hydrolysis followed by a 6 h incubation with 1 unit ml⁻¹ of cellulase R10 in 0.1 M citrate–phosphate buffer pH 2.8 at 40 °C. Under these conditions, all forms of the main flax lignan were recovered as the aglycone form, i.e. secoisolariciresinol. Highest yields in secoisolariciresinol diglucoside (SDG) equivalent reached 7.72% of flaxseed hull (cv. Baladin) dry weight and 2.88% of whole seed (cv. Barbara) weight, thus allowing a significant improvement in comparison with published methods.     

Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga)

Pepsinogen (PG) from the stomach of albacore tuna (Thunnus alalunga) was purified to homogeneity by using a series of chromatographies involving Sephacryl S-200HR, Sephadex G-50 and DEAE-cellulose with a 658-fold increase in purity. Based on the native-PAGE and zymography, PG showed a single band with pepsin activity. Molecular weights (MW) of PG and active pepsin were estimated to be 39.9 and 32.7 kDa as determined by SDS–PAGE, respectively. PG was converted to the corresponding pepsin through an intermediate form (MW ≈ 36.8 kDa) and the complete activation was observed after 30–60 min. The N-terminal amino acid sequence of the first 15 amino acids of activation segment of pepsinogen was FHKLPLIKGKTAREE. The optimal pH and temperature for pepsin activity were 2.0 and 50 °C, respectively. The activity was stable in the pH range of 2–5. Residual activity more than 85% was found after heating at temperatures up to 50 °C for 30 min. Pepsin activity was strongly inhibited by pepstatin A, whilst E-64, ethylenediaminetetraacetic acid (EDTA) and soybean trypsin inhibitor exhibited the negligible effect. SDS and cysteine also showed inhibitory effects, whilst ATP, molybdate, NaCl and CaCl₂ had no impact on pepsin activity.