Comparison of milk‐derived whey protein concentrates containing various levels of casein

Milk permeate was obtained from microfiltration (MF) and concentrated to produce milk‐derived whey protein concentrate (MWPC); MF at low temperatures yielded permeate with caseins (MWPC‐HC), and at higher MF temperatures, low concentrations of caseins were present (MWPC‐LC). MWPC samples were compared to whey protein concentrates (WPCs). Solutions of MWPC were less turbid and produced larger foam overruns and more stable foams than WPC. MWPC‐HC solutions produced the most stable foams. MWPC contained fewer types and lower relative quantities of volatile compounds than WPC before and after storage. Compared with WPC, MWPC have superior sensory, foaming and storage properties.     

大鲵胃蛋白酶分离纯化及其性质的研究

通过研究大鲵胃蛋白酶,可以了解大鲵消化机能,同时为食品加工提供新的材料。经80%硫酸铵沉淀、DEAE-52离子交换层析、SephadexG-100凝胶过滤层析以及HPLC,得到大鲵胃蛋白酶电泳纯制品。纯化倍数为239.87,回收率为4.4%,经SDS-PAGE测得该胃蛋白酶分子量为31ku,大鲵胃蛋白酶的最适温度是40℃,低于40℃稳定,当温度上升,活性迅速下降;最适pH为2,在pH2～6有很好的稳定性。EDTA、BrAc、NBS使该酶活性下降。以酪蛋白为底物,在pH2.0、40℃时测得米氏常数Km值为7.3×103mg/L,Vmax是2.674μg/min。      

Antioxidant activities of pepsin hydrolysates of water‐ and salt‐soluble protein extracted from pork hams

The objective of this study was to evaluate the antioxidant activities of pepsin‐digested water‐soluble protein (WSP) and salt‐soluble protein (SSP) extracted from pork ham. WSP and SSP were hydrolysed by pepsin for 2–10 h with 2‐h increments. The protein hydrolysates by pepsin were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and reducing power of the hydrolysates was measured. In addition, antioxidant activity of the hydrolysates was determined using linoleic acid emulsion system. Protein bands with molecular weight higher than 7 kDa in WSP and SSP were completely hydrolysed by pepsin after 2 h of digestion time. WSP hydrolysates (WSPH) and SSP hydrolysates (SSPH) had higher ferric reducing power than controls (WSP and SSP without pepsin digestion). Reducing powers of WSPH were higher than those of SSPH, regardless of digestion time. The oxidative activity of linoleic acid was predominantly inhibited by the addition of WSPH and SSPH, especially 0.5% protein hydrolysate. These results indicate that several functional peptides of pork protein digested by pepsin might have antioxidant activity, and thus they may be used as an antioxidant agent in muscle food system.     

Enzymatic hydrolysis of by‐products from the fish‐filleting industry; chemical characterisation and nutritional evaluation

Fish frames without heads from Atlantic cod and Atlantic salmon were proteolysed with the industrial enzymes neutrase®, alcalase® and pepsin for 1, 15, 30, 45, 60, 90 and 120 min. After 120 min of hydrolysis, salmon treated with alcalase and cod treated with pepsin yielded significantly (p < 0.05) higher protein recoveries (67.6 and 64% respectively) as compared to salmon treated with neutrase or pepsin and cod treated with neutrase or alcalase (53–62%). To minimise bitterness in the fish hydrolysates, kojizyme™ was added after 120 min of pre‐hydrolysis with alcalase, and the hydrolysis was run for additional times of 120, 240, 360, 480, 600 and 720 min. Protein recovery did not change significantly during the hydrolysis with kojizyme, but the degree of hydrolysis increased significantly (p < 0.01) in both the cod and salmon hydrolysates. A hydrolysate from cod treated with alcalase (150 min) followed by treatment with kojizyme (510 min) was produced. The final hydrolysate was freeze‐dried to a fish protein hydrolysate (FPH) and chemically characterised. The nutritional value of the FPH was established in an experiment with rats. Inclusion of 10% FPH‐N showed significantly (p < 0.05) higher nutritional value as compared to rats fed higher inclusion levels of FPH. © 2000 Society of Chemical Industry     

Purification of a novel pepsin inhibitor from Coriolus versicolor and its biochemical properties

A novel pepsin inhibitor was isolated from Coriolus versicolor. The purification was carried out by a 2-step ultrafiltration followed by DEAE-52 and Mono Q ion-exchange chromatography. SDS-PAGE and gel filtration chromatography analysis showed that the isolated inhibitor was a 22.3 kDa protein with a single subunit. Heat stability of this inhibitor was estimated and only 7% of its inhibitory activity lost after treatment at 98 °C. The inhibitor was more specific against pepsin than several other proteases. The dissociation constant (K(i)) and concentration required for 50% pepsin inhibition (IC50) were 5.84 × 10(-5) M and 26.26 μg/mL, respectively. Apparent decrease of α-helix and increase of random coil were observed in the circular dichroism spectra of pepsin when an equimolar amount of the inhibitor was added. The inhibition mechanism of this inhibitor differs from the reported aspartic protease inhibitors, according to the secondary structure and the kinetic studies of this inhibitor.     

Rennet coagulation properties of UHT‐treated phosphocasein dispersions as a function of casein and NaCl concentrations

The renneting properties of whey protein‐free, UHT‐heated (140 °C/10 s) casein dispersions were investigated as a function of casein and NaCl concentration. It was found that the rennet coagulation time and gel firmness can be optimised when the whey protein‐free casein concentration is increased, while the added NaCl concentration is kept low. The strongest gel firmness occurs at 0.05 and 0.08 m NaCl addition and at a micellar casein concentration between 6.0 and 6.6 g/100 mL. Weak rennet gels were formed at 3.0–3.6 g/100 mL casein at all NaCl concentrations tested.     

Development and characterisation of a sugar‐free milk‐based dessert formulation with fig (Ficus carica L.) and carboxymethylcellulose

The objective of this study was to develop an optimum formula for ‘Shir Anjir’, an Iranian dessert containing milk, dried fig (13, 16.5, 20%) and carboxymethylcellulose (CMC) (0, 0.35, 0.7%) with no added sugar. Apparent viscosity, flow behaviour indices, pH, acidity (% lactic acid), total solid and colour parameters were compared. Sensory tests were performed to evaluate degree of liking of colour, appearance, taste, texture, mouthfeel and general acceptance. Figs and CMC influenced the dessert's characteristics. The most popular formula contained milk, 16.5% figs and 0.7% CMC and had an apparent viscosity of 3.78 Pa·s at 5 °C, exhibiting a shear‐thinning behaviour.     

Effects of pepsin hydrolysis on the soy β‐conglycinin aggregates formed by heat treatment at different pH

The effects of pepsin hydrolysis on the β‐conglycinin aggregates formed by heat treatment at different pH were investigated. Results showed that fibrils were still observed, whereas the random aggregates were easily to be digested in the simulated gastric fluid. Electrophoresis and molecular weight analysis indicated that large aggregates still existed after pepsin treatment for fibrils. Hydrolysis resulted in changes in the apparent viscosity (ηapp) of 6% fibril solutions. The ηapp at the shear rate range (0–30 s^?1) increased in the order of fibrils < fibrils with pepsin for 60 min < fibrils with pepsin for 30 min. Smaller peptide/fibril fragments were generated, and additional aggregates were reformed during the hydrolysis process, as evidenced by thioflavin T and atomic force microscopy images. The native β‐conglycinin hydrolysates comprised a mixture of polypeptides enriched in about 47 kDa. These findings would provide valuable information about effects of enzymatic hydrolysis on plant oligometric globulin aggregates.     

Improvement of the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates through pepsin pretreatment

This study aimed to investigate the effect of pepsin pretreatment on the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates prepared with alcalase and protamex. The protein recovery, TCA‐soluble peptide content, surface hydrophobicity, particle size and zeta potential were evaluated. Results showed that the hydrolysates exhibited varying ACE‐inhibitory (i.e. the highest value 72.6% by alcalase and 84.3% by protamex) and DPPH radical scavenging activities (i.e. the highest value 51.9% by alcalase and 51.7% by protamex). Pepsin pretreatment could make soya proteins more susceptible for hydrolysis, and the results showed that the ACE‐inhibitory and DPPH radical scavenging activities of the resultant hydrolysates were improved. A highly significant positive correlation between ACE‐inhibitory and DPPH radical scavenging activities was observed in the alcalase hydrolysates, while no significant correlation among other treatments. The physical properties of hydrolysates, that is surface hydrophobicity, particle size and zeta potential, could influence their ACE‐inhibitory and DPPH radical scavenging activities.     

Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus)

BACKGROUND: Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by‐product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin‐solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS: PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of β‐chain but a lower content of α‐chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189–193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6–31.3 °C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple‐helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15–7.46 and 5.97–6.44 respectively. CONCLUSION: Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found. Copyright © 2009 Society of Chemical Industry     

Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake

Palm kernel cake (PKC), the most useful by-product resulted from palm kernel oil production. In this study, PKC-derived protein product was found suitable for use as an antimicrobial agent with potent antibacterial activity, particularly against Bacillus species, after enzymatic hydrolysis with alcalase. The hydrolysate was further purified by gel filtration chromatography. The purified fraction was found to have 14.63 ± 0.70% (w/w) protein, a molecular mass of 2.4 kDa and low hemolytic activity (<50% hemolysis of human erythrocytes at concentration of 1000 μg/ml). The presence of lysine and the major component lauric acid derivative, as indicated by electrospray ionisation-mass spectrometry (ESI-MS) direct infusion and nuclear magnetic resonance (NMR) spectroscopy, may have contributed to the antibacterial effect of purified PKC fraction. This study suggests that the antibacterial PKC compound may be not a pure peptide but instead a peptide-containing compound high in lauric acid derivative.     

Comparison of physicochemical properties,antioxidant and metal‐chelating activities of protein hydrolysates from Phaseolus lunatus and hard‐to‐cook Phaseolus vulgaris

Limited and extensive hydrolysates were obtained from Phaseolus lunatus (LHl and EHl) and hard‐to‐cook Phaseolus vulgaris (LHv and EHv) using the enzymes Flavourzyme^®, Alcalase^®, Pancreatin^® and a sequential Pepsin^®–Pancreatin^® system. Degrees of hydrolysis varied from 8.32% to 31.60%. SDS‐PAGE of extensive hydrolysates showed molecular weights smaller than limited hydrolysates. Differential scanning calorimeter (DSC) analysis of LHl and EHl revealed the presence of two endothermic transitions; LHv and EHv had only one. LHv presented a higher content of hydrophobic amino acids whose surface hydrophobicity was 12.17. Functional properties such as nitrogen solubility, foaming capacity and emulsifying activity index in LHv were better than LHl at different pH evaluated. However, the latter showed better foaming stabilities. Amino acids such as His, Tyr, Trp and Arg were observed in greater amounts in both extensive hydrolysates. 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging and metal‐chelating activities in EHv and EHl increased significantly compared to the source material.     

Purification and characterisation of an antioxidative peptide from enzymatic hydrolysates of duck processing by-products

Duck processing by-products were hydrolysed using eight proteases to produce an antioxidative peptide. Of the various hydrolysates produced, the pepsin extract exhibited the highest hydroxyl radical scavenging activity. The derived peptide was purified using high-performance liquid chromatography (HPLC) to identify any potent radical scavenging activity. The sequence of the antioxidative peptide obtained was identified as Asp-Val-Cys-Gly-Arg-Asp-Val-Asn-Gly-Tyr, with a molecular weight of 1096 Da. The IC₅₀ value of purified peptide for hydroxyl radical scavenging activity was 75 μg/ml as the measurement by an electron spin resonance (ESR) spectrometer. In addition, the purified peptide exhibited a protective effect on H₂O₂-induced DNA damage. These results indicate that the purified peptide possesses a potent antioxidative activity and protective effect of DNA against H₂O₂-induced oxidative damage.     

Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K⁺ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN 11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 °C and was lost at 15 °C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 °C.     

Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06

Three exoinulinases (Exo-I, Exo-II, and Exo-III) and two endoinulinases (Endo-I and Endo-II) were purified from the culture broth of Aspergillus ficuum JNSP5-06 by ammonium sulphate precipitation, DEAE-cellulose column chromatography, Sepharose CL-6B column chromatography and preparative electrophoresis. The molecular weights of Exo-I, Exo-II, Exo-III, Endo-I, and Endo-II were determined to be 70 kDa, 40 kDa, 46 kDa, 34 kDa, and 31 kDa, respectively. Using inulin as the substrate, their K_m values were 43.1 mg/ml, 31.5 mg/ml, 25.3 mg/ml, 14.8 mg/ml, and 25.6 mg/ml, respectively. These five inulinases were stable below 50 °C with optimum activity at 45 °C, and were stable at a pH range of 4–8 with an optimum pH at 4.5 for exoinulinase and at 5.0 for endoinulinase. The inulinase activity was completely inhibited by Ag⁺ and strongly inhibited by Fe²⁺ and Al³⁺, whereas K⁺, Ca²⁺, Li²⁺, EDTA and urea had no significant influence on the inulinase activity.     

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.     

Purification and characterisation of transglutaminase from four fish species: Effect of added transglutaminase on the viscoelastic behaviour of fish mince

The transglutaminase (TGase) enzyme from four different fish species, namely bigeye snapper, Indian oil sardine, tilapia and common carp have been isolated and characterised. The specific activity of the enzyme was higher in tilapia followed by oil sardine, common carp and bigeye snapper. The molecular weight of pure TGase was found to be in the range of 73–95 kD for four different fish species. The temperature optima for maximum activity of TGase varied amongst four species studied. The effect of activator (calcium chloride) and inhibitors (ethylenediaminetetraacetic acid (EDTA), ammonium chloride and free lysine–hydrochloride) at different concentrations on the TGase enzyme activity have been evaluated. The addition of isolated TGase to fish mince from Cynoglossus sp. was attempted in order to evaluate the setting and gelling ability. The setting and gelling ability of fish mince in presence of TGase improved considerably as revealed by small strain and large strain test.