@article{panneerselvam_sharma-shivappa_kolar_clare_ranney_2013, title={Hydrolysis of ozone pretreated energy grasses for optimal fermentable sugar production}, volume={148}, ISSN={["1873-2976"]}, DOI={10.1016/j.biortech.2013.08.119}, abstractNote={Ozonated energy grass varieties were enzymatically hydrolyzed to establish process parameters for maximum fermentable sugar production. Conditions for ozonolysis were selected on the basis of maximum delignification and glucan retention after pretreatment. To study the effect of lignin degradation products generated during ozonolysis on cellulolytic enzymes, hydrolysis was carried out for washed and unwashed pretreated solids. Washing the solids significantly (p < 0.05) enhanced glucan conversion from 34.3% to 100% while delivering glucose yields of 146.2–431.9 mg/g biomass. Highest fermentable sugars were produced when grasses were ozonated for maximum delignification and washed solids were hydrolyzed using 0.1 g/g Cellic® CTec2. In a comparative study on alkaline pretreatment with 1% NaOH for 60 min, Saccharum arundinaceum exhibited the highest glucan conversion with maximum sugar production of 467.9 mg/g. Although ozonolysis is an effective and environmentally friendly technique for cellulosic sugar production, process optimization is needed to ascertain economic feasibility of the process.}, journal={BIORESOURCE TECHNOLOGY}, author={Panneerselvam, Anushadevi and Sharma-Shivappa, Ratna R. and Kolar, Praveen and Clare, Debra A. and Ranney, Thomas}, year={2013}, month={Nov}, pages={97–104} }
 @article{mudgal_daubert_clare_foegeding_2011, title={Effect of Disulfide Interactions and Hydrolysis on the Thermal Aggregation of beta-Lactoglobulin}, volume={59}, ISSN={["1520-5118"]}, DOI={10.1021/jf101893v}, abstractNote={The roles of sulfhydryl/disulfide interactions and acid/pepsin hydrolysis on β-lactoglobulin (β-lg) thermal aggregation at acidic pH 3.35 and 2 were studied using rheology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM), and Western blotting. Pepsin promoted additional hydrolysis compared to the acid-hydrolyzed control sample based on a 12% increase in free amino groups. Hydrolysis with pepsin also resulted in an increase in the apparent viscosity by 2 logs upon heating 8% β-lg solutions at pH 3.35. Seemingly, hydrolysis promoted thermal aggregation of β-lg, correlating well with viscosity increases. Large microgels were observed in heated pepsin hydrolysates using TEM, supporting the increased viscosities of these dispersions. During thermal aggregation (85 °C, 3 h) of β-lg at pH 3.35, beyond the existence of limited disulfide interactions, acid hydrolysis and noncovalent interactions more likely play a crucial role in defining the functionality of acidified powdered modified whey ingredients.}, number={5}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Mudgal, Prashant and Daubert, Christopher R. and Clare, Debra A. and Foegeding, E. Allen}, year={2011}, month={Mar}, pages={1491–1497} }
 @article{clare_daubert_2011, title={Expanded Functionality of Modified Whey Protein Dispersions after Transglutaminase Catalysis}, volume={76}, ISSN={["1750-3841"]}, DOI={10.1111/j.1750-3841.2011.02128.x}, abstractNote={Abstract:  The functionality of whey dispersions, prepared with a modified whey protein concentrate (mWPC) ingredient, was significantly altered after cross‐linking with microbial transglutaminase (TGase) upon pH adjustment to 8. Test TGase–mWPC solutions, pH 8, gelled faster than control mWPC dispersions, as measured in real time; whereas, the gelling temperature of pretreated TGase–mWPC samples (37 °C, 2.5 h) increased from 67.8 to 74.8 °C with a minimal change in gel strength. Prolonged prior incubation with the enzyme (37 °C, 20 h) raised the gel strength in both control mWPC and TGase–mWPC dispersions, though these values were approximately 2.7 times lower in TGase–mWPC samples. Furthermore, the gelling temperature was raised by 9 °C after extensive polymerization. The water holding capacity was not impacted by enzymatic processing while emulsions prepared with TGase–mWPC dispersions proved very stable with no evidence of phase separation during storage at room temperature for 1 mo. Moreover, the apparent viscosity of TGase–mWPC emulsions exhibited a 10‐fold increase compared to nonenzyme‐treated mWPC samples. The particle size was nearly 11 μm in covalently linked TGase–mWPC test fractions compared with 8 μm in nonpolymerized mWPC dispersions. Ultimately, the functional characteristics of TGase–mWPC ingredients may be designed to deliver superior performance, especially with regard to improving heat and emulsion stability.}, number={4}, journal={JOURNAL OF FOOD SCIENCE}, author={Clare, Debra A. and Daubert, Christopher R.}, year={2011}, month={May}, pages={C576–C584} }
 @article{clare_daubert_2010, title={Transglutaminase Catalysis of Modified Whey Protein Dispersions}, volume={75}, ISSN={["0022-1147"]}, DOI={10.1111/j.1750-3841.2010.01605.x}, abstractNote={ABSTRACT:  Transglutaminase (TGase) cross‐linking reactions were accomplished using a heat‐modified whey protein concentrate (mWPC) substrate after pH adjustment to 8. Based on earlier reports, the degree of lactosylation with respect to β‐lactoglobulin was lower in mWPC dispersions than measured in commercial whey concentrate (cWPC) protein solutions. In this study, a higher concentration of free sulfhydryl groups was detected in soluble supernatant fractions. Both factors potentially impact the availability of reactive lysine/glutaminyl residues required for TGase reactivity. The addition of 10 mM dithiothreitol (DTT) to the substrate mix, CBZ‐glutaminyl glycine and hydroxylamine, revealed a 3.6‐fold increase in TGase activity, likely due in part to maintenance of the catalytic cysteine residue in a reduced state. Furthermore, inclusion of DTT to mWPC dispersions significantly raised the apparent viscosity, independently of enzyme modification, while the rate of polymerization increased 2‐fold based on OPA assay measurements. Limited cross‐linking slightly increased the apparent viscosity, whereas extensive coupling lowered these values compared to equivalent nonenzyme‐treated mWPC samples. Carbohydrate‐staining revealed formation of glyco‐polymers due to covalent linkages between glucosamine and mWPC proteins after TGase processing. Again, the apparent viscosity decreased after extensive enzymatic modification. Larger particles, sized 11.28 μm, were observed in the structural matrix of TGase‐mWPC‐fixed samples compared to 8 μm particles in control mWPC samples as viewed in scanning electron micrographs. Ultimately, the functional characteristics of TGase‐mWPC ingredients may be custom‐designed to deliver alternative functional attributes by adjusting the experimental reaction conditions under which catalysis is achieved.}, number={4}, journal={JOURNAL OF FOOD SCIENCE}, author={Clare, Debra A. and Daubert, Christopher R.}, year={2010}, month={May}, pages={C369–C377} }
 @article{lillard_clare_daubert_2009, title={Glycosylation and expanded utility of a modified whey protein ingredient via carbohydrate conjugation at low pH}, volume={92}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2008-1263}, abstractNote={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.}, number={1}, journal={JOURNAL OF DAIRY SCIENCE}, author={Lillard, J. S. and Clare, D. A. and Daubert, C. R.}, year={2009}, month={Jan}, pages={35–48} }
 @article{clare_zheng_hassan_swaisgood_catignani_2008, title={Antimicrobial properties of milkfat globule membrane fractions}, volume={71}, ISSN={["1944-9097"]}, DOI={10.4315/0362-028X-71.1.126}, abstractNote={Milkfat globule membranes (MFGMs) were prepared from bovine cream according to standard procedures. These membranes and peptide hydrolysates, which were generated by proteolysis with immobilized digestive enzymes, were screened for antibacterial activity against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Typhimurium, Pseudomonas fluorescens, Bacillus cereus, Lactobacillus acidophilus, and Lactobacillus gasseri. Assays were first performed on beef heart infusion (BHI) plates spotted with test protein-peptide fractions and then seeded with lawns of indicator cells to monitor the zone of growth inhibition. Under these experimental conditions, MFGMs were most active against Salmonella Typhimurium and P. fluorescens. However, antibacterial activity was not seen after plating on Luria-Bertani (LB) medium. We determined that the antimicrobial effects observed on BHI plates were due to the generation of H2O2 by xanthine oxidase, a major protein constituent of the MFGMs, as a result of purine catalysis. This substrate is present in BHI but lacking in LB medium. Evaluation of purified xanthine oxidase alone resulted in analogous data trends. The growth of probiotic Lactobacillus strains were affected only marginally when grown on lactobacilli deMan Rogosa Sharpe plates, suggesting the decreased sensitivity of these bacteria to H2O2. In this study, several MFGM hydrolysates exhibited variable antibacterial activity against test food pathogens on agar plates prepared with M9 minimal media, and this variation was not attributable to xanthine oxidase enzymatic activity. The probiotic microorganisms were mostly resilient to these antibacterial fractions. Bovine MFGM fractions may represent an excellent resource material from which to generate native, naturally occurring biodefensive proteins and/or peptides.}, number={1}, journal={JOURNAL OF FOOD PROTECTION}, author={Clare, Debra A. and Zheng, Zuoxing and Hassan, Hosni M. and Swaisgood, Harold E. and Catignani, George L.}, year={2008}, month={Jan}, pages={126–133} }
 @article{clare_gharst_maleki_sanders_2008, title={Effects of Transglutaminase Catalysis on the Functional and Immunoglobulin Binding Properties of Peanut Flour Dispersions Containing Casein}, volume={56}, ISSN={["1520-5118"]}, DOI={10.1021/jf801641d}, abstractNote={The functionality of light roasted peanut flour (PF) dispersions containing supplemental casein (CN) was altered after polymerization with microbial transglutaminase (TGase). The formation of high molecular weight covalent cross-links was observed with likely development of PF-PF, PF-CN, and CN-CN polymers based on Western blotting patterns visualized using antiserum directed against Ara h 1, Ara h 2, Ara h 3, or casein. The gelling temperature of TGase-treated PF dispersions containing 2.5% CN was significantly raised compared to the nontreated PF-CN control solutions. Furthermore, the gel strength and water holding capacity of cross-linked PF-CN test samples containing 5% CN was increased, while the yield stress and apparent viscosity were lowered compared to control dispersions. The immunological staining patterns were also changed where, in some cases, IgE binding to TGase-treated PF-CN fractions appeared less reactive compared to equivalent polymeric PF dispersions lacking supplemental CN and non-cross-linked PF-CN samples. Perhaps, covalent modification masked IgE peanut protein binding epitopes, at least to some degree, on an individual patient basis. Casein proved to be an effective cosubstrate with PF for creating Tgase modified PF-CN dispersions for use as a novel high protein food ingredient.}, number={22}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Clare, Debra A. and Gharst, Greg and Maleki, Sohelia J. and Sanders, Timothy H.}, year={2008}, month={Nov}, pages={10913–10921} }
 @article{clare_lillard_ramsey_amato_daubert_2007, title={Calcium effects on the functionality of a modified whey protein ingredient}, volume={55}, ISSN={["1520-5118"]}, DOI={10.1021/jf071613z}, abstractNote={The primary objective for this study addressed the effects of supplemental calcium on the functional properties of a modified whey protein ingredient (mWPC), prepared by acidification to pH 3.35, followed by extended heat treatment, gelation, and spray drying. In the presence of added calcium (mWPC-Ca2+), protein solutions showed increased thickening capacity, especially under refrigeration temperatures, compared to dispersions made with mWPC alone. A rheological assessment included the determination of (i) power law parameters, (ii) viscoelastic properties, and (iii) the effects of heating and cooling on these protein systems. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding profile suggested that various disulfide-linked molecular forms of beta-lactoglobulin, bovine serum albumin, and immunoglobulin were likely formed during manufacturing of the mWPC ingredient based on the patterns obtained when electrophoresis was performed in the absence of beta-mercaptoethanol compared to those observed with commercial WPC samples. An enhanced water-holding capacity was measured in mWPC-Ca2+ dispersions. Differential scanning calorimetry established that the addition of calcium salts caused a 2-fold increase in the amount of bound or unfreezeable water compared to mWPC controls. The physical appearance of the network structure varied significantly upon visualization with scanning electron microscopy, in which case the formation of large, rounded, spherical structures was noted in mWPC-Ca2+ samples, ascribed to an increased surface tension caused by the higher salt content. Ultimately, such attributes may afford distinct advantages for whey-based ingredients intended for application within food systems, especially under cold processing conditions.}, number={26}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Clare, Debra A. and Lillard, S. John and Ramsey, Sharon R. and Amato, Penny M. and Daubert, Christopher R.}, year={2007}, month={Dec}, pages={10932–10940} }
 @article{gharst_clare_davis_sanders_2007, title={The effect of transglutaminase crosslinking on the rheological characteristics of heated peanut flour dispersions}, volume={72}, ISSN={["0022-1147"]}, DOI={10.1111/j.1750-3841.2007.00442.x}, abstractNote={ABSTRACT:  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 °C using PF dispersions treated with TGase compared to untreated dispersions devoid of the enzyme (about 68 °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.}, number={7}, journal={JOURNAL OF FOOD SCIENCE}, author={Gharst, G. and Clare, D. A. and Davis, J. P. and Sanders, T. H.}, year={2007}, month={Sep}, pages={C369–C375} }
 @article{clare_gharst_sanders_2007, title={Transglutaminase polymerization of peanut proteins}, volume={55}, ISSN={["1520-5118"]}, DOI={10.1021/jf062309t}, abstractNote={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.}, number={2}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Clare, D. A. and Gharst, G. and Sanders, T. H.}, year={2007}, month={Jan}, pages={432–438} }
 @article{ahlborn_clare_sheldon_kelly_2006, title={Identification of Eggshell Membrane Proteins and Purification of Ovotransferrin and β-NAGase from Hen Egg White}, volume={25}, ISSN={1572-3887 1573-4943}, url={http://dx.doi.org/10.1007/s10930-006-0010-8}, DOI={10.1007/s10930-006-0010-8}, abstractNote={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.}, number={1}, journal={The Protein Journal}, publisher={Springer Science and Business Media LLC}, author={Ahlborn, G. J. and Clare, D. A. and Sheldon, B. W. and Kelly, R. W.}, year={2006}, month={Jan}, pages={71–81} }
 @article{clare_bang_cartwright_drake_coronel_simunovic_2005, title={Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage}, volume={88}, ISSN={["1525-3198"]}, DOI={10.3168/jds.S0022-0302(05)73103-9}, abstractNote={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.}, number={12}, journal={JOURNAL OF DAIRY SCIENCE}, author={Clare, DA and Bang, WS and Cartwright, G and Drake, MA and Coronel, P and Simunovic, J}, year={2005}, month={Dec}, pages={4172–4182} }
 @article{truong_clare_catignani_swaisgood_2004, title={Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase}, volume={52}, ISSN={["1520-5118"]}, DOI={10.1021/jf034397c}, abstractNote={Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.}, number={5}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Truong, VD and Clare, DA and Catignani, GL and Swaisgood, HE}, year={2004}, month={Mar}, pages={1170–1176} }
 @misc{clare_catignani_swaisgood_2003, title={Biodefense properties of milk: The role of antimicrobial proteins and peptides}, volume={9}, ISSN={["1873-4286"]}, DOI={10.2174/1381612033454874}, abstractNote={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.}, number={16}, journal={CURRENT PHARMACEUTICAL DESIGN}, author={Clare, DA and Catignani, GL and Swaisgood, HE}, year={2003}, pages={1239–1255} }
 @article{fasina_classen_garlich_swaisgood_clare_2003, title={Investigating the possibility of monitoring lectin levels in commercial soybean meals intended for poultry feeding using steam-heated soybean meal as a model}, volume={82}, ISSN={["0032-5791"]}, DOI={10.1093/ps/82.4.648}, abstractNote={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.}, number={4}, journal={POULTRY SCIENCE}, author={Fasina, YO and Classen, HL and Garlich, JD and Swaisgood, HE and Clare, DA}, year={2003}, month={Apr}, pages={648–656} }
 @article{wilcox_clare_valentine_swaisgood_2002, title={Immobilization and utilization of the recombinant fusion proteins trypsin-streptavidin and streptavidin-transglutaminase for modification of whey protein isolate functionality}, volume={50}, ISSN={["1520-5118"]}, DOI={10.1021/jf011603c}, abstractNote={A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.}, number={13}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Wilcox, CP and Clare, DA and Valentine, VW and Swaisgood, HE}, year={2002}, month={Jun}, pages={3723–3730} }
 @article{zhao_clare_catignani_swaisgood_2002, title={Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli}, volume={21}, ISSN={["0277-8033"]}, DOI={10.1023/A:1021134617137}, abstractNote={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.}, number={6}, journal={JOURNAL OF PROTEIN CHEMISTRY}, author={Zhao, F and Clare, DA and Catignani, GL and Swaisgood, HE}, year={2002}, month={Aug}, pages={413–418} }
 @article{clare_valentine_catignani_swaisgood_2001, title={Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein}, volume={28}, ISSN={["1879-0909"]}, DOI={10.1016/S0141-0229(00)00361-6}, abstractNote={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.}, number={6}, journal={ENZYME AND MICROBIAL TECHNOLOGY}, author={Clare, DA and Valentine, VW and Catignani, GL and Swaisgood, HE}, year={2001}, month={Apr}, pages={483–491} }
 @misc{clare_swaisgood_2000, title={Bioactive milk peptides: A prospectus}, volume={83}, ISSN={["1525-3198"]}, DOI={10.3168/jds.S0022-0302(00)74983-6}, abstractNote={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.}, number={6}, journal={JOURNAL OF DAIRY SCIENCE}, author={Clare, DA and Swaisgood, HE}, year={2000}, month={Jun}, pages={1187–1195} }
 @article{clare_lecce_1991, title={COPURIFICATION OF BOVINE-MILK XANTHINE-OXIDASE AND IMMUNOGLOBULIN}, volume={286}, ISSN={["1096-0384"]}, DOI={10.1016/0003-9861(91)90034-G}, abstractNote={Xanthine oxidase, isolated from bovine milk, exhibited an A280:A450nm 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, β-lactoglobulin, α-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.}, number={1}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, author={CLARE, DA and LECCE, JG}, year={1991}, month={Apr}, pages={233–237} }