Glycosylation and expanded utility of a modified whey protein ingredient via carbohydrate conjugation at low pH.

Whey protein, at one time considered a by-product of the cheese-making process, is now commonly used in foods for its thickening and emulsifying properties. Currently, approximately 30% of these proteinaceous resources remain under-utilized. Previously, an acidified, thermally treated whey protein concentrate (mWPC) was developed to produce a cold-set thickening ingredient. Mass spectroscopy revealed an approximate 2.5-fold decrease in the lactosylation of beta-lactoglobulin in mWPC starting materials compared with commercial whey protein concentrates, manufactured at a higher pH. Potentially, this should increase the number of reactive sites that remain available for carbohydrate attachment. With this study, the formation of glycoprotein complexes was demonstrated between the mWPC ingredient and lactose, naturally occurring in mWPC powders, or between mWPC protein components with dextran (35 to 45 and 100 to 200 kDa) materials at low pH. In fact, additional dry heating of mWPC powders showed a 3-fold increase in the amount of lactosylated beta-lactoglobulin. Evidence of Maillard reactivity was suggested using colorimetry, o-phthaldialdehyde assays, and sodium dodecyl sulfate PAGE followed by glycoprotein staining. Resultant glycoprotein dispersions exhibited altered functionality, in which case steady shear and small amplitude oscillatory rheology parameters were shown to be dependent on the specific reducing sugar present. Furthermore, the emulsion stability of mWPC-dextran fractions was 2 to 3 times greater than either mWPC or commercial WPC dispersions based on creaming index values. The water-holding capacity of all test samples decreased with additional heating steps; however, mWPC-dextran powders still retained nearly 6 times their weight of water. Scanning electron microscopy revealed that mWPC-dextran conjugates formed a porous network that differed significantly from the dense network observed with mWPC samples. This porosity likely affected both the rheological and water-binding properties of mWPC-dextran complexes. Taken together, these results suggest that the functionality of mWPC ingredients can be enhanced by conjugation with carbohydrate materials at low pH, especially with regard to improving the emulsifying attributes.

The effect of transglutaminase crosslinking on the rheological characteristics of heated peanut flour dispersions.

Peanut flour (PF) is a high-protein ingredient prepared after the partial extraction of oil from roasted peanut seed. Microbial transglutaminase (TGase) catalyzes protein crosslinking via acyl-transfer reactions, resulting in the modification of functional properties such as viscosity, gelation, solubility, and water holding capacity. This work was conducted to observe changes in rheological properties of PF dispersions in the presence and the absence of TGase and amidated pectin (AP). Dispersions were characterized across a range of conditions, including controlled heating and cooling rates under both large- and small-strain deformations. Gelation occurred at temperatures above 78 degrees C using PF dispersions treated with TGase compared to untreated dispersions devoid of the enzyme (about 68 degrees C). The addition of AP (0.5%) resulted in a general increase in viscoelasticity for all dispersions. AP addition also minimized the shift in gel point temperature caused by TGase polymerization reactions. High-molecular-weight polymers were formed in TGase-treated PF dispersions in both the presence and the absence of AP; however, polymer formation was more rapid in PF dispersions without AP. Ortho-phthaldialdehyde assays indicated about 40% protein coupling in PF dispersions treated with TGase compared to about 20% in those containing both AP and TGase. Collectively, these data suggest potential applications of TGase-treated PF dispersions, both in the presence and the absence of AP, for use in peanut-base food products, including protein bars, shakes, and value-added baked goods.

Transglutaminase polymerization of peanut proteins.

Transglutaminase promotes protein cross-linking reactions through an acyl transferase mechanism involving protein-bound glutaminyl residues and primary amines including the epsilon-amino group of lysine residues in soy, myosin, gluten, oat globulin, casein, and whey. Herein, we present a first report of exogenous transglutaminase catalysis of several peanut protein fractions, including purified Ara h 1. In most cases, SDS-PAGE banding patterns revealed the formation of high molecular weight polymers while catalysis of Ara h 1 resulted in distinct dimer formation. Cross-linking effects were accomplished in the presence and absence of the reducing reagent, dithiothreitol. Ortho-phthaldialdehyde assays, used to quantify the degree of polymerization, indicated approximately 21% and approximately 30% coupling over a similar time interval, using either cold hexane extracted peanut protein fractions or lightly roasted flour dispersions, respectively. Rheological measurements established that transglutaminase-modified peanut extracts exhibited lowered viscosity readings compared to nontreated dispersions. Peanut protein polymers and glycoprotein conjugates, created by covalent linkage between protein substrates and monosaccharide amino sugars, exhibited similar IgE binding activity, compared to control solutions. These results suggested that potential allergic responses were not enhanced after enzymatic modification. Ultimately, these approaches may provide novel peanut-based food ingredients with unique functional characteristics for expanded applications within the world marketplace.

Identification of eggshell membrane proteins and purification of ovotransferrin and beta-NAGase from hen egg white.

Exposure of selected Gram-positive and Gram-negative bacterial pathogens to egg shell membranes (ESM) significantly reduced their thermal resistance and/or inactivated cells. Although the components responsible for this antibacterial activity have not been conclusively identified, several proteins associated with the ESM activity have been identified including beta-N-acetylglucosaminidase, lysozyme and ovotransferrin, with each displaying varying degrees of antibacterial activity. Numerous attempts to purify active fractions of beta-N-acetylglucosaminidase, lysozyme and ovotransferrin from the ESM proved somewhat limited; however, hen egg white (HEW) beta-N-acetylglucosaminidase was purified using a two-step chromatographic procedure, isoelectric focusing followed by cation exchange chromatography. Pure fractions of ovotransferrin were also obtained in the process. SDS-PAGE electrophoresis and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry were then used to partially characterize the individual protein components. Purified protein fractions such as these will be required in order to fully elucidate the mechanism responsible for the antimicrobial properties associated with the ESM.

Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage.

Shelf-stable milk could benefit from sensory quality improvement. Current methods of heating cause flavor and nutrient degradation through exposure to overheated thermal exchange surfaces. Rapid heating with microwaves followed by sudden cooling could reduce or eliminate this problem. The objectives for this study were focused on designing and implementing continuous microwave thermal processing of skim fluid milks (white and chocolate) to compare sensory, microbiological, and biochemical parameters with conventionally prepared, indirect UHT milks. All test products were aseptically packaged and stored at ambient temperature for 12 mo. Every 3 mo, samples were taken for microbiological testing, reactive sulfhydryl determinations, active enzyme analysis, instrumental viscosity readings, color measurements, and descriptive sensory evaluation. Microbiological plate counts were negative on all milks at each time point. Enzymatic assays showed that plasmin was inactivated by both heat treatments. 5,5'-dithio-bis(2-nitrobenzoic acid) analysis, a measure of reactive sulfhydryl (-SH-) groups, showed that the initial thiol content was not significantly different between the microwave-processed and UHT-treated milks. However, both heating methods resulted in an increased thiol level compared with conventionally pasteurized milk samples due to the higher temperatures attained. Sulfhydryl oxidase, a milk enzyme that catalyzes disulfide bond formation using a variety of protein substrates, retained activity following microwave processing, and decreased during storage. Viscosity values were essentially equivalent in microwave- and UHT-heated white skim milks. Sensory analyses established that UHT-treated milks were visibly darker, and exhibited higher caramelized and stale/fatty flavors with increased astringency compared with the microwave samples. Sweet aromatic flavor and sweet taste decreased during storage in both UHT and microwave milk products, whereas stale/fatty flavors increased over time. Sensory effects were more apparent in white milks than in chocolate varieties. These studies suggest that microwave technology may provide a useful alternative processing method for delivery of aseptic milk products that retain a long shelf life.

Biodefense properties of milk: the role of antimicrobial proteins and peptides.

Mammary fluids, colostrum and milk, deliver nature's first host defense systems upon birth, and these essential liquids are critical for survival of the neonate. The identification and characterization of anti-infectious proteins were among the early scientific discoveries and this group of proteins has long been recognized for promoting health benefits in both newborns and adults. Among the more widely studied are the immunoglobulins, lactoperoxidase, lysozyme, and lactoferrin. Recently, it was shown that alpha--lactalbumin may also function in a protective capacity dependent upon its folding state. Some of these, especially lactoferrin, also display an immunomodulatory role in which case a totally separate cascade of host defense responses is initiated. It was noted that the mechanism of action for this cluster of sentry proteins does vary; thus, this protective strategy provides for a broad range of responsive reactions to infection. Presently, there is a major focus on the discovery of novel peptides that can be generated from existing milk proteins via proteolytic reactions. To date, this substrate list includes alpha--lactalbumin, beta-lactoglobulin, all casein fractions, and lactoferrin. Again, the immunoregulatory effects prompted as a result of the appearance of these peptides are currently being defined. Herein, we review the principal biological properties associated with each of these contributing milk components with a special emphasis on the role of biodefensive milk peptides. We envision future contributions emerging from this research field as an opportunity to develop effective new therapies to be used in treating infectious diseases and promoting health benefits in vivo.

Investigating the possibility of monitoring lectin levels in commercial soybean meals intended for poultry feeding using steam-heated soybean meal as a model.

Native soybean lectins (SBL) could potentially have deleterious effects on young animals. The objectives of this study were to determine the optimum processing temperature and time at which SBL is inactivated and to investigate the possibility of using urease activity (UA) to predict residual lectin levels in soybean meal (SBM). Raw defatted SBM was steam-heated at incremental temperatures between 90 and 120 degrees C for 5 to 20 min in an autoclave. The processed meals were subjected to native-PAGE and measurement of total carbohydrate-binding lectin (TCBL), agglutinating lectin (AL), UA, and trypsin inhibitor (TI). Processing severity was evaluated by determining protein solubility in 0.2% potassium hydroxide. Results indicated that levels of all antinutrients (TCBL, AL, UA, and TI) decreased with increasing processing temperature (P < 0.05). The intensity of the lectin band on the electrophoresis gel was considerably reduced when meal was heated at 100 degrees C for 5 min. This result implied that lectin inactivation occurred at 100 degrees C. More than 90% of all the original antinutrient levels in the raw meal were destroyed when meals were heated at 100 degrees C for 5 min. Meals processed at 100 degrees C for 5 to 20 min had protein solubility values (80 to 85%) indicative of adequate processing. The denaturation pattern of UA was highly correlated with that of SBL (r > or = 0.73), indicating that UA could be used for monitoring lectin levels in commercial meals. We concluded that UA of 0.03 to 0.09 units of pH change are indicative of adequately processed meals that contain negligible lectin levels.

Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli.

Expression of fusion protein trypsin-streptavidin (TRYPSA) in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio-beta-D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-L-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria-Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30 degrees C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39-40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation.

Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein*(1).

A trypsin-streptavidin (TRYPSA) fusion protein was designed and its expression in Escherichia coli was evaluated. The streptavidin gene was PCR modified and cloned into the pET expression vector. The trypsin gene was subsequently inserted into this plasmid, thus generating a colinear fusion of trypsin and streptavidin genes (pTRYPSA). This engineering strategy was verified, and TRYPSA was expressed after IPTG induction using the E. coli strains, BL21(DE3) and BL21(DE3)pLysS. Standard protein fractions of the cell lysate were prepared and trypsin activity was primarily detected in the periplasmic and inclusion body fractions. Immunoblotting showed a single Western-positive band exhibiting a molecular weight of 39,000 Da. A biotinylated porous glass affinity matrix was prepared and selective adsorption resulted in a one-step purification and immobilization of TRYPSA from crude cell lysate. Trypsin activity was verified using a synthetic substrate. This enzyme bioreactor should serve as an excellent prototype for future studies that will examine the effect of limited proteolysis on functional characteristics of milk proteins, including gelling, emulsifying and foaming properties.

Bioactive milk peptides: a prospectus.

Bioactive peptides have been identified within the amino acid sequences of native milk proteins. Hydrolytic reactions, such as those catalyzed by digestive enzymes, result in their release. These peptides directly influence numerous biological processes evoking behavioral, gastrointestinal, hormonal, immunological, neurological, and nutritional responses. The specific bioreactions associated with each physiological class have been well characterized. Herein, we review the scientific literature and attempt to stimulate consideration of the continued use of bioactive peptides and their expanded development as a commercial product. Several applications have already evolved. For example, phosphopeptides derived from casein fractions are currently used as both dietary and pharmaceutical supplements. Potentially, the addition of bioactive peptides to food products could improve consumer safety as a result of their antimicrobial properties. Lastly, bioactive peptides may function as health care products, providing therapeutic value for either treatment of infection or prevention of disease.

The effect of organizational type and hierarchical level on nurses' perceptions of hospital issues.

Managers in four acute care hospitals in southeast Florida described the resource, management, outcome, and external issues confronting their organizations. Eight major issues and four contributing and resulting subissues associated with each were identified from the 746 separate issue statements the managers made. Nurses in the same four hospitals were asked to evaluate the impact of the eight issues on their organization and their work, as well as the extent to which the subissues had either contributed to or resulted from the main ones. Overall perceptual patterns are discussed, as well as the differential effects of both hospital type and level of employment on perceptions. It is suggested that differential perceptions among such a key component of the care delivery system as nurses will need to be better understood as hospitals seek to survive in a turbulent environment.

Copurification of bovine milk xanthine oxidase and immunoglobulin.

Xanthine oxidase, isolated from bovine milk, exhibited an A280:A450 nm ratio of 5.0. This ratio is reported to be indicative of highly purified enzyme preparations. Serum from a rabbit hyperimmunized against this enzyme fraction exhibited two precipitation lines when incubated with the protein in agarose double diffusion plates. Serum albumin, beta-lactoglobulin, alpha-lactalbumin, lactoferrin, casein, chymosin, and immunoglobulin were tested for reactivity. The second antigen was identified as bovine immunoglobulin. Commercial preparations of xanthine oxidase also contained immunoglobulin as a contaminant. IgG and IgA were present in Sigma (Grade III) fractions and IgM was identified in Boehringer Mannheim preparations. Immunofluorescent studies indicated that xanthine oxidase antiserum reacted with the capillary endothelium of bovine heart. Absorption of this antiserum with bovine IgG abrogated this reaction. These findings may explain apparent discrepancies between reported immunohistological association of xanthine oxidase in heart capillary endothelial cells and the absence of detectable enzymatic activity.

Purification and properties of sulfhydryl oxidase from bovine pancreas.

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.

Development of resistance of enterocytes to rotavirus in neonatal, agammaglobulinemic piglets.

The first part of this report describes the development of a technique for evaluating the growth of rotavirus under controlled conditions that approximate a natural infection. A standard dose of rotavirus (approximately 10(9) viral particles) was injected into ligated segments in the small intestine of newborn, agammaglobulinemic, colostrum-deprived piglets. After various periods postinoculation, the segments were retrieved and the enterocytes were evaluated for the presence of rotaviral antigens by immunofluorescence and rotaviral particles by transmission electron microscopy. Peak immunofluorescence in enterocytes was detected at 8 h postinoculation in the upper and middle jejunum and ileum. Transmission electron microscopy at this time revealed fully formed virions which were not seen in sections examined before this 8-h period. The second part of our study describes the use of ligated segments in determining the susceptibility to rotavirus of enterocytes in piglets ranging in age from newborn to 2 weeks. By the time piglets were 2 days old, enterocytes in the upper half of the small intestines appeared to be resistant to rotavirus, whereas those in the lower half seemed partially resistant. Between 4 and 8 days of age, enterocytes in the lower half also became resistant. Resistance paralleled the loss in capacity of piglets to transport macromolecules through enterocytes and was not correlated with the loss in capacity to internalize macromolecules.

Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase.

The usual method of staining polyacrylamide gel electropherograms for superoxide dismutase activity utilizes a photochemical flux of O2- to reduce nitroblue tetrazolium. Superoxide dismutases intercept O2-, preventing formazan production and thus causing achromatic bands. In the presence of H2O2, catalases also yield achromatic bands during this staining procedure. This is due to local elevation of pO2 by the catalatic decomposition of H2O2. O2, in turn, inhibits the reduction of the tetrazolium by O2-. This phenomenon provides a new activity stain for catalase. A previously described activity stain for catalase has also been reexamined and significantly improved.

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase.

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

A hybrid superoxide dismutase containing both functional iron and manganese.

A hybrid superoxide dismutase containing functional Mn and Fe has been isolated from Escherichia coli. Streptomycin, which binds tightly to both the Mn- and the Fe-containing superoxide dismutases, had the expected effect on the electrophoretic and chromatographic behavior of the hybrid. Treatment of the hybrid with H2O2, which selectively inactivates the Fe-containing enzyme, resulted in partial inactivation accompanied by a resegregation of subunits, with the formation of active Mn-enzyme and inactive Fe-enzyme. A similar resegregation of subunits was observed when the hybrid was exposed to 2.5 M guanidinium chloride. Hybrids containing Mn or Fe could be generated in vitro by mixing the Mn-enzyme with the Fe-enzyme, removing metals with 8-hydroxyquinoline in the presence of 2.5 M guanidinium chloride, and then dialyzing against Mn(II) or Fe(II) salts. Ten per cent of the activity of the Fe-superoxide dismutases is resistant to H2O2, which correlates with its content of Mn. Since the activity remaining after exhaustive treatment with H2O2 exhibited the electrophoretic mobility of the Fe-enzyme, we concluded that some of the active sites of the Fe-enzyme were actually occupied by Mn. It should be noted, however, that for purposes of metal reconstitution experiments, a definite specificity was demonstrated. The Mn-enzyme was reconstituted with Mn(II), whereas the Fe-enzyme activity was recovered using only Fe(II). We propose that the Fe-superoxide dismutase may be heterogeneous and that 10% of its activity is actually due to a Mn-containing variant with the same electrophoretic mobility. Only the apohybrid enzyme regained enzymatic activity using both Mn(II) and Fe(II).