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The immunoglobulin G (IgG) concentrations of 254 raw milk samples collected from the province of British Columbia (Canada) in 1990 and 1991 ranged from 0.030 to 0.71 mg ml‐1, with mean and median values of 0.28 and 0.27 mg ml‐1 respectively in 1990, and 0.25 and 0.24 mg ml‐1 respectively in 1991. Although significant differences were noted in IgG contents of milks from five geographical areas and 14 dairies for each of the 2 years, there were also significant differences between milks from year to year. All of the milks showed measurable antigen‐binding activity in enzyme‐linked immunosorbent assay (ELISA) against the lipopolysaccharide (LPS) fractions of five bacteria (Shigella flexneri 1A, Escherichia coli O111:B4, Escherichia coli O128:B12, Salmonella typhimurium and Salmonella enteritidis). An immune milk obtained from vaccinated cows showed significantly higher anti‐S. enteritidis LPS activity than any of the raw milk samples. However, levels of specific anti‐LPS activity against the other four bacte...

The authenticity of products labelled as olive oil is of great importance from the standpoints of both commercial value and health aspects. Over the years, a high degree of sophistication has evolved in chromatographic methods for the analysis of both major and minor components of oils and fats. At the same time, spectroscopic methods are emerging as potential tools for rapid screening of samples for the detection of adulteration. However, the complexity and intrinsic variability of biological samples such as olive oil and its potential adulterants demands the application of multivariate calibration or pattern-recognition techniques to aid interpretation of the data obtained using these instrumental methods. Combination of the techniques of analytical chemistry and chemometrics is mandatory for unequivocal identification and quantification of the adulteration of olive oil.

A method was developed to isolate immunoglobulins (Ig) from cheese whey using ultrafiltration (UF) and immobilized metal affinity chromatography (IMAC). Cheddar cheese whey was concentrated up to 30 times using UF prior to applying to an IMAC column. A 50-ml IMAC column was loaded with 17·3 ml of 50 m m CuCl2 for 2 3 Cu saturation of the column bed volume. Using 20 times concentrated whey, 1240 mg of IgG could be isolated with 52% purity using glycine in the eluting buffer. Glycine was found to be a better competing ligand in the eluting buffer than NH4Cl. Approximately 152 mg of lactoferrin and 90 mg of lactoperoxidase were isolated with the IgG. The concentration factor, IMAC matrix support, UF conditions and diafiltration did not affect the recovery or purity of IgG obtained by IMAC. By using concentrated whey rather than unconcentrated whey, an 80% reduction in time for IMAC could be achieved. IMAC was found to give higher recovery and purity of IgG from concentrated whey than ion exchange chromatography. A method was also developed to obtain IgG from raw skim milk using UF and IMAC.

The antioxidative properties of Pacific hake hydrolysates and their peptidic fractions varying in molecular size were assessed. Hydrolysates produced by different proteases (Alcalase, bromelain, Flavourzyme, Protamex, Protease A"Amano"2, Protease N"Amano"K, Protin SD NY10, Umamizyme-K, Validase BNP-L, Validase FPexo) generally possessed good metal ion chelating (33-73% at 3mg/ml), DPPH radical scavenging (18-30% at 1mg/ml), ferric ion reducing power (abs700nm 0.36-0.86 at 3 mg/ml) and ABTS radical scavenging (47-85% at 0.067 mg/ml) activity, as well as a good capability to suppress lipid peroxidation in a linoleic acid model system. Peptide size (

α-Lactalbumin and β-lactoglobulin solutions (in D 2 O) were heated at 50, 70, or 90 o C for 30, 60, or 90 min. Only the samples heated at 90 o C formed transparent gels. The amide I' and amide III' bands of the Raman spectra implicated an increase of β-sheet structure with a simultaneous decrease of helical structure in heard α-lactalbumin, while an increase of β-sheet with a simultaneous decrease of turn structure was suggested in heated β-lactoglobulin. Changes in other regions of the Raman spectra could be interpreted as changes in disulfide conformation as well as in microenmronment around several amino acid residues, i.e., Trp, Tyr, and His

A ten-point augmented simplex-centroid design was used to study the effects of ingredient interactions on properties of food protein mixtures. Hydrophobicity, solubility and functional properties (emulsification and color) of three ingredients(bovine casein and salt-extractable proteins from chicken breast muscle and beef heart) as well as mixtures of the ingredients were tested. Significant deviations were observed between experimentally measured properties of mixtures and values calculated assuming linear addition of individual ingredient properties. Regression models including significant interaction terms were calculated for ingredient-hydrophobicity and ingredient-functionality relationships, and used in computerized optimization of two hypothetical formulations.

A rapid solid-phase particle concentration fluorescence immunoassay (PCFIA) was developed to quantitate the total IgG concentration in bovine milk. The assay is based on agglutination of antibody covalently bound to carboxyl-polystyrene particles with the antigen, which is in turn detected by a fluorescent labeled antibody by epifluorometry. PCFIA is simple to perform, sensitive (5 ng of IgG/mL), accurate, repeatable, and faster than most immunoassays

Raw milk from non‐immunized cows and raw human milk from lactating mothers were examined for specificity and antibody activity against lipopolysaccharides (LPS) of five pathogenic bacteria, i.e. Escherichia coli O111:B4, E. coli O128:B12, Salmonella enteritidis, S. typhimurium and Shigella flexneri 1A in a particle concentration fluorescence immunoassay (PCFIA). Bacterial LPS was covalently coated on submicron polystyrene particles and used in an antibody sandwich technique with commercially available rabbit anti‐bovine fluorescein‐labeled IgG or goat anti‐human fluorescein‐labeled IgA. Comparison was made to a skim milk sample obtained from vaccinated cows. In general, specific activity of milk IgG against bacterial LPS did not parallel the trend of total non‐specific activity. Although the specific anti‐LPS activities were in general high in milk from vaccinated cows, milk from non‐immunized cows also showed significant activity against the bacterial LPS, sometimes higher than the vaccinated cows. IgA i...

Octadecyl polymeric (ODP) vs octadecyl silica (ODS) reversed-phase high-performance liquid chromatography (RP-HPLC) columns were compared for analytical separation of water-soluble fractions extracted from Cheddar cheese. A polystyrene divinylbenzene copolymer-based column did not resolve cheese water-soluble components. Good resolution was achieved on a vinyl alcohol copolymer ODP column as well as two ODS columns under optimized conditions of gradient elution, using increasing concentrations of aqueous acetonitrile containing ion-pair reagent. However, the ODP column had longer useful life characteristics. Use of phosphoric instead of trifluoroacetic acid as ion-pair reagent produced a more stable chromatogram baseline and may prolong column useful life. ODP-RP-HPLC could be useful to monitor ripening of Cheddar cheese samples.

A double sandwich enzyme‐linked immunosorbent assay (ELISA) procedure for the quantification of IgG in bovine milk was developed for detecting the concentration of IgG in various homogenized HTST, UHT, evaporated and raw milk samples as well as skim milk powder. ELISA results were consistently lower than radial immunodiffusion (RID) values for the same sample, suggesting interference by an unknown constituent in the milk matrix. This difficulty was overcome by using a reference milk, quantified previously for IgG by RID. A significant linear relationship (y=2.03x‐0.034, r=0.891, n= 150, p

The potential for producing antihypertensive peptides from oat proteins through enzymatic hydrolysis was assessed in silico and confirmed in vitro. Thermolysin (EC 3.4.24.27) was predicted using BIOPEP database as the enzyme that would theoretically produce the most angiotensin I converting enzyme (ACE) inhibitory peptides from oat. Experimental evidence confirmed that strong ACE-inhibitory activity was produced under various hydrolysis conditions. Hydrolysates produced under high enzyme-to-substrate ratio (3%) short time (20 min) (HEST) and low enzyme-to-substrate ratio (0.1%) long time (120 min) (LELT) conditions had IC(50) values of 30 and 50 microg/mL, respectively. After simulated gastrointestinal digestion, the IC(50) of the HEST hydrolysate was 35 microg/mL whereas the IC(50) of the LELT hydrolysate was higher at 85 microg/mL. Ultrafiltration revealed that potent ACE-inhibitory peptides had molecular weights below 3 kDa. This study demonstrates the usefulness of in silico analysis to select enzymes for hydrolysis of proteins not previously examined as sources of bioactive peptides.

A process was developed to fractionate both water-soluble and water-insoluble components from egg yolk with maximum retention of biological and functional properties. About 60–90% of the immunological activity of yolk was recovered in a supernatant after centrifugation of yolk diluted six- to tenfold with water. The remaining yolk pellet had excellent emulsifying properties for mayonnaise preparation. It was further fractionated by deoxycholate treatment to separate triglycerides (80% recovery). Ultrafiltration of the deoxycholate supernatant yielded protein (74% recovery) in the retentate, and phospholipids and cholesterol in the permeate. Phospholipids were then purified by dich-loromethane:ethanol (3:1) extraction, zinc chloride precipitation and acetone washing.

Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.

Metal chelate interaction chromatography can efficiently recover Ig found in low concentrations in cheese whey. High capacity and purity of recovered Ig resulted from competitive displacement of less tightly bound proteins during whey application. The strength of interaction, ie., order of displacement, of proteins in Cheddar cheese whey was: β-lactoglobulin, β-lactalbumin, bovine serum albumin, lactoperoxidase-lactoferrin, and Ig. Tandem column operation enabled maximal utilization of column capacity for Ig. About 100mg Ig (75 to 95% purity) could be recovered per milliliter of Cu-charged bed volume or 2.5ml of total bed volume. Proteins in the unbound whey fractions from this chromatographic process retained good emulsifying activity.

Natural actomyosin (NAM) from Pacific whiting (PW) showed thermal transition temperatures by circular dichroism at 31.8 and 43.1°C, which were lower than those of threadfin bream (TB) NAM, 35.0 and 49.3°C. Endothermic transitions of PW-NAM by differential scanning calorimetry were at 31.8, 42.1 and 75.3°C, compared to 36.1, 50.9 and 78.4°C for TB-NAM. Based on surface hydrophobicity, α-helical content, and solubility, PW-NAM unfolded to a greater extent than did TB-NAM when incubated at 25°C for 4h and 40°C for 2h, suggesting its lower thermal stability. Transglutaminase generally catalyzed more extensive cross-linking of PW-myosin heavy chain (MHC) than TB-MHC, and the MHC cross-linking mediated by microbial transglutaminase (MTG) was greater than by fish transglutaminase (FTG). Textural properties of PW-NAM gels increased approximately 3.6-6.1-fold and 1.3-1.5-fold in the presence of MTG and FTG, respectively.

A database consisting of 224 di- to tetradecapeptides and five amino acids was compiled to study quantitative structure-activity relationships of bitter peptides. Partial least-squares regression-1 analysis was conducted using the amino acid three z-scores and/or three parameters (total hydrophobicity, residue number, and log mass values) as X-variables and bitterness values (log 1/T where T is the bitterness threshold) as Y-variables. Using the three parameters only, significant models (p0.001) were obtained describing the entire data set as well as data subsets, except that comprised only of octa- to tetradecapeptides. For data sets comprising different peptide lengths, the models were improved by including the three z-scores at the N-terminal and C-terminal positions. Correlation coefficients for bitterness prediction of 48 dipeptides and 12 pentapeptides were 0.75 (RMSEP = 0.53) and 0.90 (RMSEP = 0.48), respectively. Bulky hydrophobic amino acids at the C terminus and bulky basic amino acids at the N terminus were highly correlated to bitterness.

Structural changes of alkali-treated rockfish protein isolate (AKPI) during frozen storage were elucidated using a Raman spectrometer and scanning electron microscope (SEM). The results were compared to conventional surimi (CS). No significant textural difference was noted between AKPI stored at pH 5.5 and 7.0. The strongest texture was found for AKPI frozen with cryoprotectants and CS, while the weakest texture was observed in AKPI frozen without cryoprotectants. SEM revealed the most discontinuity in gels of AKPI with no cryoprotectants and a more aggregated microstructure after storage at pH 5.5 than at neutral pH. Raman spectral analysis demonstrated refolding of AKPI by pH readjustment to 7.0, although the refolded structure was not identical to that before the pH shift. CS showed higher alpha-helix content (approximately 50%) than AKPI (approximately 20-30%). Frozen storage induced a decrease and an increase in the alpha-helix content of CS and AKPI samples, respectively. AKPIs were slightly less stable than CS during frozen storage.

Angiotensin I converting enzyme (ACE) inhibitory activity was determined in the soy protein isolate (SPI) digest produced by in vitro pepsin-pancreatin sequential digestion. The inhibitory activity was highest within the first 20 min of pepsin digestion and decreased upon subsequent digestion with pancreatin. An IC(50) value of 0.28 +/- 0.04 mg/mL was determined after 180 min of digestion, while no ACE inhibitory activity was measured for the undigested SPI at 0.73 mg/mL. Chromatographic fractionation of the SPI digest resulted in IC(50) values of active fractions ranging from 0.13 +/- 0.03 to 0.93 +/- 0.08 mg/mL. Although many of the fractions showed ACE inhibition, peptides with lower molecular masses and higher hydrophobicities were most active. The findings show that many different peptides with ACE inhibitory activities were produced after in vitro pepsin-pancreatin digestion of SPI and lead to the speculation that physiological gastrointestinal digestion could also yield ACE inhibitory peptides from SPI.

A new method, homology similarity analysis (HSA), was developed to investigate homology pattern similarities of selected segments within sequences of peptides. This new approach facilitated elucidation of the structure-function relationships of lactoferricin derivatives. Helix propensity of positions 4-9 in the lactoferricin sequence was the most important in determining the antimicrobial activity of lactoferricin against Escherichia coli, followed by cationic charge pattern at positions 4-9 and 1-3. The pattern similarity of segments within sequences could be a useful tool for representing the distribution attributes of amino acid residue properties to the structure-function relationships of proteins and peptides, especially when used in conjunction with principal component similarity analysis followed by the regression version of artificial neural networks.

A growing recognition of the tremendous potential of vibrational spectroscopic techniques by food scientists over the past few decades has fuelled an exponential growth in the scientific literature describing research that involves the use of near-infrared, mid-infrared and/or Raman spectroscopy for the analysis of food systems. This chapter highlights some of the myriad applications of vibrational spectroscopy conducted to meet a diverse range of analytical needs in food science. For example, vibrational spectroscopic techniques, in conjunction with various chemometric tools, are being applied for the determination of food or beverage composition, authentication, or adulteration, the assessment and prediction of quality and process-induced changes, and the detection of chemical or microbiological contaminants related to food safety. Applications in basic research have contributed to a better understanding of the chemical, functional, sensory, and textural properties of food. With ongoing advances in the technology and an increasing level of sophistication and expertise of users familiar with the potential advantages and challenges of these techniques, the future is promising for emergent innovative applications of vibrational spectroscopy in the areas of quality assurance, process control, and food safety management, and for fundamental research in food science. Keywords: near-infrared; mid-infrared; Raman; food composition; food quality; food safety; process-induced changes; food authenticity; food adulteration; fundamental research

Surimi and natural actomyosin (NAM) from ling cod (Ophiodon elongatus) were subjected to frozen storage in the absence or presence of cryoprotectants (sorbitol, sucrose, lactitol, and Litesse, either individually or in combination). Effects of frozen storage were studied for NAM frozen at -10 degrees C for 10 days and for surimi after eight freeze-thaw cycles. A commercial blend cryoprotectant (4% sucrose and 4% sorbitol), individual cryoprotectants at 8%, and optimal blends at 4, 5.5, 6, and 8%, were effective in maintaining the gel strength of surimi and NAM gels. Surimi or NAM frozen in the absence of cryoprotectants or with only 4% individual cryoprotectants, showed increased percent alpha-helical content by Raman analysis. Increased disulfide content was also observed in the treatment without cryoprotectants by the Raman SS stretching band and by chemical determination. Tyrosine residues were in a buried environment before and after freezing for all treatments, and surface hydrophobicity measured by 1-anilinonaphthalene-8-sulfonate decreased after frozen storage in the absence of cryoprotectants.

Lysozyme (25% in D2O, corn oil, and their emulsions (10% w/w oil/D2O solution) were examined by Fourier transform Raman spectroscopy. Emulsions showed three layers, namely, top oil, middle cream, and bottom aqueous layers. Raman spectral analysis revealed hydrophobic interactions involving both protein and lipid components. Compared to lysozyme in D2O, the difference spectrum obtained after subtraction of oil from the cream layer spectrum showed reduced intensity of tryptophan bands at 760, 1013, 1340, and 1360 cm(-)(1), reduced intensity ratio of the tyrosine doublet at 850 and 830 cm(-)(1), and increased intensity of the C-H bending band at 1455 cm(-)(1). Compared to corn oil, the difference spectrum after subtraction of lysozyme from the cream layer spectrum indicated decreased intensity at 2855 cm(-)(1) (lipid CH(2) symmetric stretch) and 3011 cm(-)(1) (unsaturated fatty acid hydrocarbon chain =C-H stretch) and a higher intensity ratio of the C-H stretching band at 2900 cm(-)(1) to bands at 2885 and 2933 cm(-)(1). Spectra of the top and bottom layers resembled corn oil and lysozyme, respectively, except for changes in the D2O band. Raman spectroscopy can be used to detect structural changes in proteins, lipids, and D2O due to protein-lipid interactions.

The influence of type of fluorescent probe on the surface hydrophobicity values determined for three native and heated proteins was assessed using uncharged [6-propionyl-2-(N, N-dimethylamino)naphthalene or PRODAN] versus anionic aliphatic (cis-parinaric acid or CPA) and aromatic (1-anilinonaphthalene-8-sulfonic acid or ANS) probes. Surface hydrophobicities of whey protein isolate, beta-lactoglobulin, and bovine serum albumin under heated (80 degrees C for 30 min) and unheated conditions and at varying pH values (3.0, 5.0, 7.0, and 9. 0) were measured using ANS, CPA, and PRODAN. ANS and CPA yielded opposing results for the effects of pH and heating on protein hydrophobicity. Hydrophobicity was lower at pH 3.0 than at other pH values for all proteins measured by PRODAN, whereas the values measured by ANS and CPA at pH 3.0 were quite high compared to those at other pH values, suggesting the influence of electrostatic interactions on anionic probe-protein binding. These results suggest that the presence or absence of a permanent charge as well as the aromatic and aliphatic nature of fluorescent probes can affect protein hydrophobicity values measured under various pH conditions.

Raman spectra of amino acids showed complexity in the C-H stretching region (2800-3100 cm(-)(1)) attributed to diversity of CH, CH(2), and CH(3) groups in the side chains, ionization state, and microenvironment. The involvement of specific amino acids in the C-H stretching region of selected proteins, namely, lysozyme, alpha-lactalbumin, beta-lactoglobulin, and their binary mixtures, was investigated by deconvolution using maximum likelihood techniques. The main protein band near 2940 cm(-)(1) was attributed not only to aromatic and aliphatic amino acids but also to many other amino acids. A band near 3065 cm(-)(1) was assigned to aromatic residues, whereas bands near 2880 and 2900 cm(-)(1) corresponded primarily to aliphatic amino acids. Heating at 90 degrees C increased relative intensity near 2940 cm(-)(1) and decreased relative intensity at 2895-2902 cm(-)(1) for lysozyme and its mixtures with alpha-lactalbumin or beta-lactoglobulin. Additional bands at 2812 or 2838 and 3003 cm(-)(1) were observed after heating or in 8 M deuterated urea, reflecting changes upon denaturation.

Immunoaffinity columns were made with specific egg yolk immunoglobulin (Ig) Y against bovine IgG1 and IgG2 and were used to isolate pure IgG1 and IgG2 from Cheddar cheese whey or colostrum. About 10% of the IgY was specific for IgG, and 3% of the IgY was subclass-specific after hyperimmunization of laying hens with either IgG1 or IgG2. Up to 38% of the potential binding capacity of IgY was obtained after immobilization by reductive amination. The IgY columns were stable, and one column could be reused for more than 50 times for over a year with minimal loss in binding capacity. Milk that was free of either IgG subclass was successfully produced by the selective removal of IgG1 or IgG2 subclasses. Double-immunodiffusion analysis confirmed the isolation of subclasses from whey and colostrum and also confirmed that their removal from milk was specific.

Raman spectroscopy can be a useful tool to probe protein structure in solid and liquid food systems. Bands in the Raman spectrum arising from amide I, amide III and skeletal stretching modes of peptides and proteins are useful for characterizing backbone conformation, including the estimation of secondary structure fractions. Bands attributed to various stretching or bending vibrational modes of functional groups of amino-acid residues can be used to monitor the environment around these side-chains. In particular, valuable information may be obtained on SS or SH groups of cystinyl or cysteinyl residues, CH groups of aliphatic residues, and aromatic rings of tryptophanyl, tyrosinyl and phenylalanyl residues. One important parameter distinguishing Raman spectroscopy from many other spectroscopic methods is its applicability to systems containing high concentrations of proteins, which is critical for the investigation of structural changes during processes such as coagulum or gel formation. Thus, changes in both intramolecular and intermolecular interactions can be studied. In this chapter, examples are presented on the application of Raman spectroscopy to investigate protein structure as a function of processing, such as heating, drying, salt addition or homogenization with lipids, which may be important to correlate with protein functionality in food systems.

QSAR (quantitative structure-activity relationship), widely used in chemistry with hydrophobic, electronic, and steric parameters as structural factors, was found to be appropriate for use with food proteins, despite the difficulty, due to the complexity in macromolecular structure, in defining the steric terms. Emulsifying ability was closely related to hydrophobicity, and incorporation of solubility to hydrophobicity as factors improved the R2 of regression analysis. Foaming activity required both hydrophobicity and other factors pertaining to the adsorption of proteins at the interface in order to obtain adequate foam lamella strength. Hydrophobicity as well as other factors relating to the intermolecular interactions, for example, Ca and SH are involved in thermally induced gelation. For breadmaking, although no extensive QSAR work had been carried out, the important function of high molecular glutenin subunits was confirmed, and, notably, the critical function of hydrophobicity in breadmaking also was demonstrated. PLS (partial least-squares regression) and neural networks classify more correctly than other multivariate techniques, thereby yielding higher r2 values in modeling and prediction. However, multiple regression analysis and PCR (principal component regression) also were found to be effective for modeling because the information useful in elucidating the mechanism of protein function could be readily obtained. A characteristic property of unsupervised learning techniques, especially PCS (principal component similarity analysis), in identifying influential factors in the function mechanisms was demonstrated.

Although it is generally agreed that topography is more conserved than sequences, proteins sharing the same fold can have different functions, while there are protein families with low sequence similarity. An alternative method for profile analysis of characteristic conserved positions of the motifs within the 3D structures may be needed for functional annotation of protein sequences. Using the approach of quantitative structure-activity relationships (QSAR), we have proposed a new algorithm for postulating functional mechanisms on the basis of pattern similarity and average of property values of side-chains in segments within sequences. This approach was used to search for functional sites of proteins belonging to the lysozyme and cystatin families. Hydrophobicity and β-turn propensity of reference segments with 3–7 residues were used for the homology similarity search (HSS) for active sites. Hydrogen bonding was used as the side-chain property for searching the binding sites of lysozymes. The profiles of similarity constants and average values of these parameters as functions of their positions in the sequences could identify both active and substrate binding sites of the lysozyme of Streptomyces coelicolor, which has been reported as a new fold enzyme (Cellosyl). The same approach was successfully applied to cystatins, especially for postulating the mechanisms of amyloidosis of human cystatin C as well as human lysozyme. Pattern similarity and average index values of structure-related properties of side chains in short segments of three residues or longer were, for the first time, successfully applied for predicting functional sites in sequences. This new approach may be applicable to studying functional sites in un-annotated proteins, for which complete 3D structures are not yet available.

Properties of proteins in salt extracts from beef top round and rockfish fillet changed upon heating and were dependent on pH, freezing, species and sample variability. Generally, heating to >45°C, freezing or lower pH (5.5) decreased dispersibility, increased surface hydrophobicity and slightly decreased sulfhydryl content. Emulsifying and fat binding properties followed parallel trends, being improved under conditions favoring combinations of high dispersibility, hydrophobicity and sulfhydryl content (i.e., heating at

Tetrahymena pyriformis W was used in a bioassay to evaluate the improvement in protein quality of wheat gluten by enrichment using either simple addition or carbodiimide-mediated attachment of 1% lysine, Ne-acetyl lysine or Ne-benzylidene lysine. Pepsin predjgestion was more effective in solubilizing samples for the assay than papain. All of the enriched samples had relative nutritive values (RNV) approximating that of the casein standard, which had a value of 100, compared to the gluten and acid-solubilized gluten controls which had RNV of 54 and 64, respectively. No difference in the RNV between lysine and the NE-lysine derivatives, or between covalent attachment and free addition was detected.

Three-level fractional factorial experiments were used to study effects of heating conditions (pH, time, temperature, solids content, calcium addition) on whey protein concentrate. Increasing pH and temperature led to lower solubility at pH 4.6 and 7.0, lower sulfhydryl content, higher hydroxymethylfurfural, generally darker color, lower DNBS-available lysine and altered pepsin pancreatin digestion profiles. Mercaptoethanol and SDS demonstrated relative importance of disulfide and hydrophobic bonds on solubility loss. Polyacrylamide gel electrophoresis indicated heat stability of proteose peptones; susceptibility was greatest at pH 8.0, 95°C for β-lactoglobulin and α-lactalbumin, and pH 4.6, 95°C for bovine serum albumin. HPLC gel filtration showed that heating rendered a high molecular weight fraction undissociable by mercaptoethanol.

Nɛ-Protected lysine derivatives and threonine were covalently attached to wheat gluten using 1-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide. Influential reaction factors were studied using fractional factorial experiments. Lysine content increased 4.0-fold and 6.5-fold after reaction of pepsin-hydrolyzed gluten with NE-benzylidene lysine and NE-acetyl lysine respectively, but product yields were low (40–65%). Using unhydrolyzed gluten as the starting material, 1.6-fold and 2.5-fold lysine increases resulted using NC-benzylidene and NE-acetyl lysine, respectively, with 95% yield. At least 4-fold increase in threonine could be obtained from reaction of threonine with pepsin-hydrolyzed gluten, but with only 30–50% product yield. Yields increased to 96% by reacting threonine with unhydrolyzed gluten, with 1.8-fold increase in threonine.

According to the original idea of quantitative structure-activity relationship, electric, hydrophobic, and structural parameters should be taken into consideration for elucidating functionality. Changes in these parameters are reflected in the property of protein solubility upon modification of whey proteins by heating. Although solubility is itself a functional property, it has been utilized to explain other functionalities of proteins. However, better correlations were obtained when hydrophobic parameters of the proteins were used in conjunction with solubility. Various treatments reported in the literature were applied to whey protein concentrate in an attempt to obtain whipping and gelling properties similar to those of egg white. Mapping simplex optimization was used to search for the best results. Improvement in whipping properties by pepsin hydrolysis may have been due to higher protein solubility, and good gelling properties resulting from polyphosphate treatment may have been due to an increase in exposable hydrophobicity. However, the results of angel food cake making were still unsatisfactory.

Cation exchange column chromatography was investigated for simple and efficient separation of lysozyme from homogenized egg white. A macroporus weak acid type resin, Duolite C-464, was selected based on high lysozyme recovery in the90–95% range, retention of whipping and gelling properties in the lysozyme-separated egg white, and ease of column operation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated high purity of the recovered lysozyme and simultaneous recovery of avidin under the eluting conditions used. The process for lysozyme separation from egg white using Duolite C-464 is suitable for operation in an automated, continuous mode.

Physicochemical and functional properties of chicken breast muscle samples and their salt-extractable proteins were investigated as a function of increasing post-mortem storage time at abovefreezing temperature prior to deboning and further processing. Functional properties, including water binding capacity, emulsifying capacity and thermally-induced gelation, were generally poorer after 2 days of storage, compared to no storage (0 day). After 7 days of storage, functionality improved significantly, and in most cases did not differ (P>0.05) from functionality at 0 day. No significant differences (P>0.10) were observed for pH after different storage times, but salt-extractable protein content of the chicken mince was higher at 7 days than at 0 or 2 days. Salt-extracted proteins from 7 day samples had lower solubility and higher hydrophobicity, while dispersibility and sulfhydryl group content did not differ compared to 0 and 2 day samples. On the other hand, the exposed hydrophobicity of proteins after heat treatment was lower at 7 days than at 0 or 2 days. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated some changes in the myosin and troponin components in the extracts from different post-mortem storage times.