Purification and Partial Characterization of Caprine Milk Lipoprotein Lipase

Lipoprotein lipases of caprine and bovine milks were isolated, purified, and partially characterized from raw whole milk. Fractions with enzyme activity were two for caprine and one for bovine lipase. The milk lipoprotein lipase enzyme fractions were purified further by hydroxylapatite and intervent dilution chromatography. Caprine enzyme fractions were purified 142,000 and 87,000-fold from the isolates compared to the bovine enzyme, which was purified 5500-fold. For the two caprine enzyme fractions were three distinct electrophoretic bands of molecular weights 66,100; 58,900; and 55,000. Electrophoretic patterns of the two fractions were similar. Isoelectric focusing of the highly purified caprine fractions also revealed heterogeneity with minor differences in isoelectric points, pH 5.02 and 5.14. Bovine enzyme had two distinct electrophoretic bands with molecular weights of 74,100 and 66,100, but poly aery lamide gel electrophoresis in the absence of sodium dodecylsulfate or β-mercaptoethanol revealed only one major band indicating a quaternary structure. The pH optimum of the caprine milk lipoprotein lipase was 8.7 compared to bovine enzyme of 8.5. Increase of yield of enzyme from both species during purification was attributed to several factors, the most important being the removal of an inhibitor. If in milk, this inhibitor may play a role in susceptibility of milk to hydrolytic rancidity.     

Electrophoretic analysis of wheat and rye mixtures in meal,flour and baked goods

Starch gel electrophoresis of endosperm prolamines extracted with 2 M urea, provides distinction between varieties of either wheat or rye, but the major differences in general pattern type between all rye samples and all wheat samples offers a basis for distinction between the two cereals. The proportions of wheat and rye in a mixture can be determined by comparing relative areas of densitometric scans for standard mixtures with that of a sample for analysis, or by direct calculation from area ratios for pure wheat and rye. For baked products, extraction with urea containing a reducing agent is recommended. Electrophoretic patterns are altered both as a result of baking and of using the reducing agent, but distinction between wheat and rye is still possible.     

Application of PhastSystem to the resolution of bovine milk proteins on urea-polyacrylamide gel electrophoresis.

Optimal conditions were established for alkaline urea-PAGE using modified precast, ultrathin gradient gels on the automated PhastSystem. Profiles of milk proteins showed that the caseins and whey proteins resolved extremely well. Major bands were observed for alpha s1-casein and beta-casein, and alpha s2-casein appeared as a well-resolved doublet. In contrast, kappa-casein separated from other caseins as a faint doublet, and purified kappa-casein appeared as one major and one minor band. Whey proteins (serum albumin, alpha-lactalbumin, beta-lactoglobulin) separated into broad bands resolved from each other and from the caseins. Partially (40%) dephosphorylated whole casein showed multiple bands for alpha s1-casein and beta-casein at different levels of phosphorylation. Separation of genetic phenotypes was observed for beta-lactoglobulin A and B; alpha s1-casein A, B, and C; and beta-casein A, B, and C. Electrophoretic patterns of milk proteins extracted from cheese samples varied among the different types of cheeses. Our modified procedure provides researchers with a rapid technique to separate both caseins and whey proteins on the same urea gel according to their charge to mass ratios.     

Physical and Molecular Properties of Egg-white Lipid Films

Polyethylene glycol-plasticized films were cast from alkaline (pH 11.25), heated (45 °C for 20 min), aqueous egg white (EW) solutions, with or without (10% or 20% w/w of EW) milkfat (two fractions), oleic acid, or egg yolk lysophospho-lipid (LPL). The lipids did not reduce (P > 0.05) film water vapor permeability. Oleic acid increased (P < 0.05) tensile strength and elongation, and surface sulfhydryl group (SH) concentrations in EW solutions. Oleic acid probably increased negative charges on EW proteins, unfolding protein chains, and exposing SH groups. LPL also slightly increased (P < 0.05) surface SH concentrations in non-heated mixtures. Electrophoretic patterns suggested oleic acid interactions with ovalbumin, ovotransferrin, and lysozyme. No lysinoalanine was in film-forming mixtures based on lysine measurements.     

Complex coacervation of chitosan and soy globulins in aqueous solution: a electrophoretic mobility and light scattering study

The effect of pHs and heating on the protein–polysaccharide complexation between the 0.5 wt% soy globulin (7S or 11S) and 0.1 wt% chitosan was studied. Electrophoretic and light scattering techniques were used to examine the electrical charge and aggregation of the individual biopolymers and complexes. At pH 3.0–6.5, 7S (or 11S) globulin in the presence of chitosan had significantly higher ζ‐potentials and lower particles size than 7S (or 11S) globulin alone did (e.g. 600–6000 nm at pH 5.5), indicating the formation of complexes. After heating 7S (or 11S)–chitosan mixtures had higher positive value of ζ‐potential. 7S (or 11S)–chitosan mixtures exhibited a significant increase in positive value of ζ‐potential and stability after heating at lower pH values (pH 3.3 instead of pH 4.5). Compared with other mixtures, at pH 2.5–6.0, the most remarkable decrease in aggregation was obtained for 11S–chitosan mixtures after heating at pH 3.3.     

Characterisation of the casein fraction of Ibérico cheese by electrophoretic techniques

The physicochemical characteristics of Ibérico cheese, a semi‐hard Spanish variety manufactured from mixtures of cow's, ewe's and goat's milk, were studied. The casein fraction and breakdown products of 6‐month‐old cheeses were characterised by various electrophoretic techniques: urea polyacrylamide gel electrophoresis (urea‐PAGE) at alkaline pH, isoelectric focusing (IEF) and two‐dimensional polyacrylamide gel electrophoresis (2DE). Proteins were separated in 2DE according to their charge/mass ratio by urea‐PAGE at alkaline pH in the first dimension and according to their isoelectric point by IEF in the second dimension. Some individual bands considered homogeneous by urea‐PAGE at alkaline pH (ie different grades of phosphorylation of α_s1‐casein and α_s2‐casein) or by IEF (ie overlapping of several bands of α_s2‐casein with γ‐casein bands) were found to be complex mixtures of casein components by 2DE. The two‐dimensional electrophoretic pattern was characteristic of the milk of each animal species included in Ibérico cheese. Capillary electrophoresis (CE) was also used to study the Ibérico cheeses. The high resolution of this technique allowed the identification of the main caseins of the different species (ie para‐κ‐casein, β‐casein, γ₂‐casein and γ₃‐casein). © 2002 Society of Chemical Industry     

Improved emulsion stabilizing properties of whey protein isolate by conjugation with pectins

Functional properties of glyco-protein conjugates of the anionic polysaccharide pectin with whey protein isolate, obtained by dry heat treatment at 60 °C for 14 days, have been investigated in O/W emulsions containing 20% (w/w) soybean oil and 0.4% (w/w) protein both at pH 4.0 and 5.5. Emulsion stabilizing properties of mixtures and conjugates were compared at five protein to pectin weight ratios by determining changes in droplet size distribution and extent of serum separation with time. The results indicated that the dry heat-induced covalent binding of low methoxyl pectin to whey protein, as shown by SDS-PAGE, led to a substantial improvement in the emulsifying behaviour at pH 5.5, which is near the isoelectric pH of the main protein β-lactoglobulin. At pH 4.0, however, a deterioration of the emulsifying properties of whey protein was observed using either mixtures of protein and pectin or conjugates.The observed effects could be explained by protein solubility and electrophoretic mobility measurements. The protein solubility at pH 5.5 was hardly changed using mixtures of protein and low methoxyl pectin or conjugates, whereas at pH 4.0 it was decreased considerably. Electrophoretic mobility measurements at pH 5.5 revealed a much more pronounced negative charge on the emulsion droplets in the case of protein–pectin conjugates, which clearly indicated that conjugated pectin did adsorb at the interface even at pH conditions above the protein's iso-electric point. Hence, the improved emulsifying properties of whey protein isolate at pH 5.5 upon conjugation with low methoxyl pectin may be explained by enhanced electrosteric stabilization.Comparing two different commercial pectin samples, it was clearly shown that the dextrose content during dry heat treatment of protein–pectin mixtures should be as low as possible since protein–sugar conjugates not only resulted in increased brown colour development, but also gave raise to a largely decreased protein solubility which very badly affected the emulsifying properties.     

USE OF A MODIFIED UREA GEL ISOELECTRIC FOCUSING METHOD FOR SPECIES IDENTIFICATION OF RAW OR BOILED WHITE,PINK, AND ROCK SHRIMP1

Isoelectric focusing (IEF) polyacrylamide gel containing an 80% pH 4–6.5 and 20% pH 3–10 ampholyte mixture greatly improved protein banding pattern for species identification of water extracts of raw pink, white and rock shrimp compared with the system using only the pH 3–10 range ampholyte. Identification of a specific species in mixture samples was achieved by the detection of water-extractable shrimp specific protein bands present in the gel. Sodium dodecyl sulfate (SDS) was a better protein extractant than water for cooked shrimp. Both water and SDS extracts of cooked shrimp showed specific protein banding patterns and improved resolution for species identification.     

Analysis of Raw Beef Samples for Adulterant Meat Species by Enzyme-Staining of Isoelectric Focusing Gels

Electrophoretic methods for detecting small proportions of contaminating species in fresh or frozen beef samples were investigated. By staining isoelectric focusing gels for particular enzymes, rather than for proteins in general, lower proportions of contaminants could be detected, because the band patterns were less complex. Buffalo, horse and pig muscles possess phosphoglucomutase isozymes of higher isoelectric point that beef, and this enabled detection of these species at contaminant:beef ratios as low as 1:99. By staining for adenylate kinase, kangaroo and horse were detected in beef samples at similar concentrations. Enzyme stains also proved useful for distinguishing some species not differentiated by general-protein stains, e.g. phosphogluconate dehydrogenase distinguishes sheep from goat.     

PCR identification of beef, sheep, goat, and pork in raw and heat-treated meat mixtures

A PCR assay has been developed for the specific and qualitative detection of pork (Sus scrofa domesticus), beef (Bos taurus), sheep (Ovis aries), and goat (Capra hircus) in raw and heat-treated meat mixtures. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear species identification. Analysis of experimental meat mixtures demonstrated that the detection limit of the assay was 1% (wt/wt) for each species analyzed. This assay can be useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in meat mixtures.     

Association between electrophoretic patterns of gliadin proteins and quality characteristics of wheat cultivars

Electrophoretic patterns for cereal-grain prolamins were coded in numerical form to permit computer-based comparison, either for facilitating the identification of cultivars or for the study of genotypic relationships. In a group of 80 cultivars, including wheats grown in countries other than Australia, gliadin patterns reflected both pedigree and taxonomic relationships, particularly in the separate grouping of a rye, and of durum and club wheats. Certain gliadin bands were also found to be significantly associated with the quality characteristics of hardness and dough strength.     

Moving Boundary Electrophoresis of Food Stabilizers

SUMMARY— The electrophoretic characteristics of locust bean gum, guar gum, arabic gum, carboxymethylcellulose, K- and h-carrageenan, alginate and the non-dialyzable fraction of corn syrup solids were studied by moving boundary electrophoresis. The physical properties of these colloids require the use of modified techniques in order to eliminate boundary disturbances which otherwise might be interpreted as heterogeneity of composition.
A high degree of homogeneity was observed for carboxymethylcellulose, arabic gum, alginate and X-carrageenan. In contrast, K-carrageenan was composed of a mixture of migrating components. The colloidal fractions of guar gum, locust bean gum, and corn syrup solids did not migrate in the electrical field.
Mixtures of carboxymethylcellulose with guar gum or gum arabic could be separated at pH 7. Mixtures of carboxymethylcellulose with carrageenan could be effectively separated at pH 2. Electrophoretic separation at 23°C of different stabilizer mixtures was unsatisfactory and the patterns showed evidence of interactions.     

Identification of fish species by thin-slab polyacrylamide gel electrophoresis of the muscle myogens

Electrophoretic band patterns for twenty commercially important British fish species have been obtained by electrophoresis of their muscle myogens on thin slabs of polyacrylamide gel. Since each band pattern is characteristic of one species and is practically unaltered as a result of ice storage or frozen storage of the fish, it is possible to identify any fresh and frozen fillet belonging to these twenty species by comparing its band pattern with the standards obtained. The method can also be used to identify two species of fresh fish in admixture. Cooked fish cannot be identified by this method.     

Muscadine Grape Polyphenoloxidase: Partial Purification by High Pressure Liquid Chromatography and Some Properties

A rapid method of isolating grape polyphenoloxidase (PPO) was developed, using two muscadine cultivars, “Noble” and “Welder”. PPO extracts from acetone powders were partially purified followed by filtration through 0.2μ membrane filter by HPLC. Reactivity of the active fraction per unit protein, increased 10-fold for Noble and 33-fold for Welder cultivars over values for the corresponding crude extracts. Electrophoretic analysis and activity stain showed that the enzyme displayed a single band in Welder and two bands for Noble. Optimum pH for PPO activity in Welder was pH 4.5 while that for Noble was pH 4.5–5.5. The enzyme in the Noble cultivar was relatively more active at alkaline pH. Activites of the enzymes were stable at temperatures up to 30°C for both cultivars.     

Properties of Biopolymers from Cross-linking Whey Protein Isolate and Soybean 11S Globulin

Biopolymers were prepared by cross-linking whey protein isolate (WPI) and soybean 11S using transglutaminase. Electrophoretic pattern, solubility, emulsification, hydrophobicity and foaming properties of the biopolymers were determined. SDS-PAGE showed bands corresponding to high-molecular-weight components (MW>200 kDa). Biopolymer solubility was > 90% at pH 3 and below, and at pH 7 and above. Emulsifying properties of biopolymers were lower than those of WPI. The foaming capacity of the biopolymers (23.6 mL) and WPI/11S mixture (22.7 mL) were similar to that of egg albumin (20.3 mL). The foaming stability of the biopolymers (122 min) was higher than that of WPI/11S mixture (33.7 min), and was similar to that of egg albumin (132 min).     

Phase behaviour,rheology and microstructure of mixture of meat proteins and kappa and iota carrageenans

The phase behaviour of mixtures of salt soluble meat proteins, kappa (κ) and iota (ι) carrageenan in non-gelling conditions (45 °C) were determined at pH 5.6, 6.2 and 7.1. The concentration of meat proteins ranged from 0.1 to 1.0 percent and that of κ-carrageenan and ι-carrageenan from 0.02 to 0.3 percent in the mixtures. Mixtures separated under gravity to form soluble/liquid and gelled/complex phases. For meat proteins- κ-carrageenan mixtures, phase separations at all meat protein/carrageenan ratios were observed. For meat protein-ι-carrageenan mixtures, soluble complexes were formed at low meat protein to ι-carrageenan ratios and gels at higher ratios. The yield of the complex/gels increased with the increase in the concentration of the meat proteins and carrageenans and decreased with increase in the pH of the initial mix. The complex/gels formed became stronger with the increase in carrageenan in the mix and with κ-carrageenan compared to ι-carrageenan. Chemical analyses and scanning electron and phase contrast microscopy indicated that in phase separated mixtures, the bulk of the meat proteins and carrageenan were found in the gel compared to the liquid phase; and that meat protein interacted with carrageenan in the gel and formed soluble complexes with carrageenan in the liquid phase. SDS-PAGE showed that the meat proteins that interacted to form the complex/gels with carrageenan included myosin heavy chain, α-actinin, actin, myosin light chains and proteins with molecular weights around 150 and 50 kD. The outcomes of the present study could be used in the formulation of multi-component foods with a range of consistencies containing meat proteins.     

Complex coacervation of pea protein isolate and alginate polysaccharides

Complex coacervation between pea protein isolate (PPI) and alginate (AL) was investigated as a function of pH (1.50–7.00) and mixing ratio (1:1–20:1 PPI:AL) by turbidimetric analysis and electrophoretic mobility during an acid titration. Conformational changes to the secondary structures during coacervation were also studied by Raman spectroscopy. Critical structure-forming events associated with the formation of soluble (pH 5.00) and insoluble (pH 2.98) complexes were found for a 1:1 PPI–AL mixture, with optimal biopolymer interactions occurring at pH 2.10 (pH_opt). As mixing ratios increased between 4:1 and 8:1, critical pHs shifted towards higher pH. Maximum coacervate formation at pH_opt occurred at a mixing ratio of 4:1. Electrophoretic mobility measurements showed a shift in net neutrality from pH 4.00 in homogenous PPI solutions, to pH 1.55 for the 1:1 mixture. As biopolymer ratios increased towards 8:1 PPI:AL, net neutrality shifted to higher pHs (～3.80). Raman spectroscopy revealed minimal complexation-induced conformational changes. Findings could aid in the design of pH-sensitive biopolymer carriers for use in functional food and bio-material applications.     

On the specificity of tuna-directed primers in PCR-SSCP analysis of fish and meat

Single strand conformation polymorphism (SSCP) of an amplicon (148 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrom b gene used to identify tuna species was studied with other fish and animal species. Single-stranded DNA (ssDNA) patterns of two to four strong bands were obtained with blue ling, carp, haddock, mackerel, mackerel shark, saithe, catfish, Alaska pollack, and skipjack which, however, differed from those obtained with tuna samples. Other fish species resulted in weak (cod, spined dogfish) or no ssDNA bands (Atlantic salmon, halibut, herring, pike-perch, plaice, redfish, sprat, trout). Samples from animals other than fish resulted in strong ssDNA bands differing from those of tuna and from each other (crayfish; cattle, European rabbit, fallow deer, hare, horse, red deer, roe deer; goose, turkey), in bands differing from tuna but not from each other (domestic goat/sheep, domestic pig/wild boar), or in weak bands (octopus, shrimp; chicken, duck). Increasing the stringency of PCR caused a more pronounced difference between strong and weak ssDNA bands. Inter-laboratory reproducibility of the method was good.     

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.