@article{sherman_guenther_reade_rochon_sit_smith_2020, title={Near-Atomic-Resolution Cryo-Electron Microscopy Structures of Cucumber Leaf Spot Virus and Red Clover Necrotic Mosaic Virus: Evolutionary Divergence at the Icosahedral Three-Fold Axes}, volume={94}, ISSN={["1098-5514"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85077664748&partnerID=MN8TOARS}, DOI={10.1128/JVI.01439-19}, abstractNote={Members of the Tombusviridae family have nearly identical shells, and yet they package genomes that range from 4.6 kb (monopartite) to 5.3 kb (bipartite) in size. To understand how this genome flexibility occurs within a rigidly conserved shell, we determined the high-resolution cryo-electron microscopy (cryo-EM) structures of cucumber leaf spot virus and red clover necrotic mosaic virus. In response to genomic size differences, it appears that the ssRNA binding (R) domain of the capsid diverged evolutionarily in order to recognize the different genomes. The next region, the “arm,” seems to have also coevolved with the R domain to allow particle assembly via interactions at the icosahedral 3-fold axes. In addition, there are differences at the icosahedral 3-fold axes with regard to metal binding that are likely important for transmission and the viral life cycle. ABSTRACT Members of the Tombusviridae family have highly similar structures, and yet there are important differences among them in host, transmission, and capsid stabilities. Viruses in the Tombusviridae family have single-stranded RNA (ssRNA) genomes with T=3 icosahedral protein shells with a maximum diameter of ∼340 Å. Each capsid protein is comprised of three domains: R (RNA binding), S (shell), and P (protruding). Between the R domain and S domain is the “arm” region that studies have shown to play a critical role in assembly. To better understand how the details of structural differences and similarities influence the Tombusviridae viral life cycles, the structures of cucumber leaf spot virus (CLSV; genus Aureusvirus) and red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) were determined to resolutions of 3.2 Å and 2.9 Å, respectively, with cryo-electron microscopy and image reconstruction methods. While the shell domains had homologous structures, the stabilizing interactions at the icosahedral 3-fold axes and the R domains differed greatly. The heterogeneity in the R domains among the members of the Tombusviridae family is likely correlated with differences in the sizes and characteristics of the corresponding genomes. We propose that the changes in the R domain/RNA interactions evolved different arm domain interactions at the β-annuli. For example, RCNMV has the largest genome and it appears to have created the necessary space in the capsid by evolving the shortest R domain. The resulting loss in RNA/R domain interactions may have been compensated for by increased intersubunit β-strand interactions at the icosahedral 3-fold axes. Therefore, the R and arm domains may have coevolved to package different genomes within the conserved and rigid shell. IMPORTANCE Members of the Tombusviridae family have nearly identical shells, and yet they package genomes that range from 4.6 kb (monopartite) to 5.3 kb (bipartite) in size. To understand how this genome flexibility occurs within a rigidly conserved shell, we determined the high-resolution cryo-electron microscopy (cryo-EM) structures of cucumber leaf spot virus and red clover necrotic mosaic virus. In response to genomic size differences, it appears that the ssRNA binding (R) domain of the capsid diverged evolutionarily in order to recognize the different genomes. The next region, the “arm,” seems to have also coevolved with the R domain to allow particle assembly via interactions at the icosahedral 3-fold axes. In addition, there are differences at the icosahedral 3-fold axes with regard to metal binding that are likely important for transmission and the viral life cycle.}, number={2}, journal={JOURNAL OF VIROLOGY}, publisher={American Society for Microbiology}, author={Sherman, Michael B. and Guenther, Richard and Reade, Ron and Rochon, D'Ann and Sit, Tim and Smith, Thomas J.}, editor={Parrish, Colin R.Editor}, year={2020}, month={Jan} }
 @article{smith_foegeding_drake_2016, title={Flavor and Functional Characteristics of Whey Protein Isolates from Different Whey Sources}, volume={81}, ISSN={["1750-3841"]}, DOI={10.1111/1750-3841.13248}, abstractNote={This study evaluated flavor and functional characteristics of whey protein isolates (WPIs) from Cheddar, Mozzarella, Cottage cheese, and rennet casein whey. WPIs were manufactured in triplicate. Powders were rehydrated and evaluated in duplicate by descriptive sensory analysis. Volatile compounds were extracted by solid-phase microextraction followed by gas chromatography-mass spectrometry. Functional properties were evaluated by measurement of foam stability, heat stability, and protein solubility. WPI from Cheddar and Cottage cheese whey had the highest cardboard flavor, whereas sweet aromatic flavor was highest in Mozzarella WPI, and rennet casein WPI had the lowest overall flavor and aroma. Distinct sour taste and brothy/potato flavor were also noted in WPI from Cottage cheese whey. Consistent with sensory results, aldehyde concentrations were also highest in Cheddar and Cottage cheese WPI. Overrun, yield stress, and foam stability were not different (P > 0.05) among Cheddar, Mozzarella, and rennet casein WPI, but WPI foams from Cottage cheese whey had a lower overrun and air-phase fraction (P < 0.05). Cottage cheese WPI was more heat stable at pH 7 (P < 0.05) than other WPI in 4% protein solutions, and was the only WPI to not gel at 10% protein. Cottage cheese WPI was less soluble at pH 4.6 compared to other WPI (P < 0.05) and also exhibited higher turbidity loss at pH 3 to 7 compared to other WPI (P < 0.05). This study suggests that WPI produced from nontraditional whey sources could be used in new applications due to distinct functional and flavor characteristics.}, number={4}, journal={JOURNAL OF FOOD SCIENCE}, author={Smith, T. J. and Foegeding, E. A. and Drake, M. A.}, year={2016}, month={Apr}, pages={C849–C857} }
 @article{smith_campbell_jo_drake_2016, title={Flavor and stability of milk proteins}, volume={99}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2016-10847}, abstractNote={A greater understanding of the nature and source of dried milk protein ingredient flavor(s) is required to characterize flavor stability and identify the sources of flavors. The objective of this study was to characterize the flavor and flavor chemistry of milk protein concentrates (MPC 70, 80, 85), isolates (MPI), acid and rennet caseins, and micellar casein concentrate (MCC) and to determine the effect of storage on flavor and functionality of milk protein concentrates using instrumental and sensory techniques. Spray-dried milk protein ingredients (MPC, MPI, caseins, MCC) were collected in duplicate from 5 commercial suppliers or manufactured at North Carolina State University. Powders were rehydrated and evaluated in duplicate by descriptive sensory analysis. Volatile compounds were extracted by solid phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry. Compounds were identified by comparison of retention indices, odor properties, and mass spectra against reference standards. A subset of samples was selected for further analysis using direct solvent extraction with solvent-assisted flavor extraction, and aroma extract dilution analysis. External standard curves were created to quantify select volatile compounds. Pilot plant manufactured MPC were stored at 3, 25, and 40°C (44% relative humidity). Solubility, furosine, sensory properties, and volatile compound analyses were performed at 0, 1, 3, 6, and 12 mo. Milk proteins and caseins were diverse in flavor and exhibited sweet aromatic and cooked/milky flavors as well as cardboard, brothy, tortilla, soapy, and fatty flavors. Key aroma active compounds in milk proteins and caseins were 2-aminoacetophenone, nonanal, 1-octen-3-one, dimethyl trisulfide, 2-acetyl-1-pyrroline, heptanal, methional, 1-hexen-3-one, hexanal, dimethyl disulfide, butanoic acid, and acetic acid. Stored milk proteins developed animal and burnt sugar flavors over time. Solubility of MPC decreased and furosine concentration increased with storage time and temperature. Solubility of MPC 80 was reduced more than that of MPC 45, but time and temperature adversely affected solubility of both proteins, with storage temperature having the greatest effect. Flavor and shelf stability of milk proteins provide a foundation of knowledge to improve the flavor and shelf-life of milk proteins.}, number={6}, journal={JOURNAL OF DAIRY SCIENCE}, author={Smith, T. J. and Campbell, R. E. and Jo, Y. and Drake, M. A.}, year={2016}, month={Jun}, pages={4325–4346} }
 @article{smith_smith_drake_2016, title={Short communication: Flavor and flavor stability of cheese, rennet, and acid wheys}, volume={99}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2015-10482}, abstractNote={Dried whey ingredients are valuable food ingredients but potential whey sources are underutilized. Previous work has established flavor and flavor stability differences in Cheddar and Mozzarella wheys, but little work has compared these whey sources to acid or rennet wheys. The objective of this study was to characterize and compare flavor and flavor stability among cheese, rennet, and acid wheys. Full-fat and fat-free Cheddar, rennet and acid casein, cottage cheese, and Greek yogurt fluid wheys were manufactured in triplicate. Wheys were fat separated and pasteurized followed by compositional analyses and storage at 4°C for 48 h. Volatile compound analysis and descriptive sensory analysis were evaluated on all liquid wheys initially and after 24 and 48 h. Greek yogurt whey contained almost no true protein nitrogen (0.02% wt/vol) whereas other wheys contained 0.58%±0.4% (wt/vol) true protein nitrogen. Solids and fat content were not different between wheys, with the exception of Greek yogurt whey, which was also lower in solids content than the other wheys (5.6 vs. 6.5% wt/vol, respectively). Fresh wheys displayed sweet aromatic and cooked milk flavors. Cheddar wheys were distinguished by diacetyl/buttery flavors, and acid wheys (acid casein, cottage cheese, and Greek yogurt) by sour aromatic flavor. Acid casein whey had a distinct soapy flavor, and acid and Greek yogurt wheys had distinct potato flavor. Both cultured acid wheys contained acetaldehyde flavor. Cardboard flavor increased and sweet aromatic and buttery flavors decreased with storage in all wheys. Volatile compound profiles were also distinct among wheys and changed with storage, consistent with sensory results. Lipid oxidation aldehydes increased in all wheys with storage time. Fat-free Cheddar was more stable than full-fat Cheddar over 48h of storage. Uncultured rennet casein whey was the most stable whey, as exhibited by the lowest increase in lipid oxidation products over time. These results provide baseline information for the viability of processing underutilized wheys into value-added ingredients.}, number={5}, journal={JOURNAL OF DAIRY SCIENCE}, author={Smith, S. and Smith, T. J. and Drake, M. A.}, year={2016}, month={May}, pages={3434–3444} }
 @article{smith_gerard_drake_2015, title={Effect of temperature and concentration on benzoyl peroxide bleaching efficacy and benzoic acid levels in whey protein concentrate}, volume={98}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2015-9890}, abstractNote={Much of the fluid whey produced in the United States is a by-product of Cheddar cheese manufacture and must be bleached. Benzoyl peroxide (BP) is currently 1 of only 2 legal chemical bleaching agents for fluid whey in the United States, but benzoic acid is an unavoidable by-product of BP bleaching. Benzoyl peroxide is typically a powder, but new liquid BP dispersions are available. A greater understanding of the bleaching characteristics of BP is necessary. The objective of the study was to compare norbixin destruction, residual benzoic acid, and flavor differences between liquid whey and 80% whey protein concentrates (WPC80) bleached at different temperatures with 2 different benzoyl peroxides (soluble and insoluble). Two experiments were conducted in this study. For experiment 1, 3 factors (temperature, bleach type, bleach concentration) were evaluated for norbixin destruction using a response surface model-central composite design in liquid whey. For experiment 2, norbixin concentration, residual benzoic acid, and flavor differences were explored in WPC80 from whey bleached by the 2 commercially available BP (soluble and insoluble) at 5 mg/kg. In liquid whey, soluble BP bleached more norbixin than insoluble BP, especially at lower concentrations (5 and 10 mg/kg) at both cold (4°C) and hot (50°C) temperatures. The WPC80 from liquid whey bleached with BP at 50°C had lower norbixin concentration, benzoic acid levels, cardboard flavor, and aldehyde levels than WPC80 from liquid whey bleached with BP at 4°C. Regardless of temperature, soluble BP destroyed more norbixin at lower concentrations than insoluble BP. The WPC80 from soluble-BP-bleached wheys had lower cardboard flavor and lower aldehyde levels than WPC80 from insoluble-BP-bleached whey. This study suggests that new, soluble (liquid) BP can be used at lower concentrations than insoluble BP to achieve equivalent bleaching and that less residual benzoic acid remains in WPC80 powder from liquid whey bleached hot (50°C) than cold (4°C), which may provide opportunities to reduce benzoic acid residues in dried whey ingredients, expanding their marketability.}, number={11}, journal={JOURNAL OF DAIRY SCIENCE}, author={Smith, T. J. and Gerard, P. D. and Drake, M. A.}, year={2015}, month={Nov}, pages={7614–7627} }
 @article{jervis_smith_drake_2015, title={Short communication: The influence of solids concentration and bleaching agent on bleaching efficacy and flavor of sweet whey powder}, volume={98}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2014-8804}, abstractNote={Recent studies have demonstrated the effect of bleaching conditions and bleaching agent on flavor and functional properties of whey protein ingredients. Solids concentration at bleaching significantly affected bleaching efficacy and flavor effects of different bleaching agents. It is not known if these parameters influence quality of sweet whey powder (SWP). The purpose of this study was to determine the effects of solids concentration and bleaching agent on the flavor and bleaching efficacy of SWP. Colored cheddar whey was manufactured, fat separated, and pasteurized. Subsequently, the whey (6.7% solids) was bleached, concentrated using reverse osmosis (RO) to 14% solids, and then spray dried, or whey was concentrated before bleaching and then spray dried. Bleaching treatments included a control (no bleaching, 50 °C, 60 min), hydrogen peroxide (HP; 250 mg/kg, 50 °C, 60 min), benzoyl peroxide (50 mg/kg, 50 °C, 60 min), lactoperoxidase (20 mg/kg of HP, 50 °C, 30 min), and external peroxidase (MaxiBright, DSM Food Specialties, Delft, the Netherlands; 2 dairy bleaching units/mL, 50 °C, 30 min). The experiment was repeated in triplicate. Sensory properties and volatile compounds of SWP were evaluated by a trained panel and gas chromatography-mass spectrometry, respectively. Bleaching efficacy (norbixin destruction) and benzoic acid were measured by HPLC. Differences in bleaching efficacy, sensory and volatile compound profiles, and benzoic acid were observed with different bleaching agents, consistent with previous studies. Solids concentration affected bleaching efficacy of HP, but not other bleaching agents. The SWP from whey bleached with HP or lactoperoxidase following RO had increased cardboard and fatty flavors and higher concentrations of lipid oxidation compounds compared with SWP from whey bleached before RO. The SWP bleached with benzoyl peroxide after RO contained less benzoic acid than SWP from whey bleached before RO. These results indicate that solids concentration at bleaching and bleaching agent affect quality of SWP.}, number={4}, journal={JOURNAL OF DAIRY SCIENCE}, author={Jervis, M. G. and Smith, T. J. and Drake, M. A.}, year={2015}, month={Apr}, pages={2294–2302} }
 @article{qiu_smith_foegeding_drake_2015, title={The effect of microfiltration on color, flavor, and functionality of 80% whey protein concentrate}, volume={98}, DOI={10.3168/jds.2014-9174}, abstractNote={The residual annatto colorant in fluid Cheddar cheese whey is bleached to provide a neutral-colored final product. Currently, hydrogen peroxide (HP) and benzoyl peroxide are used for bleaching liquid whey. However, previous studies have shown that chemical bleaching causes off-flavor formation, mainly due to lipid oxidation and protein degradation. The objective of this study was to evaluate the efficacy of microfiltration (MF) on norbixin removal and to compare flavor and functionality of 80% whey protein concentrate (WPC80) from MF whey to WPC80 from whey bleached with HP or lactoperoxidase (LP). Cheddar cheese whey was manufactured from colored, pasteurized milk. The fluid whey was pasteurized and fat separated. Liquid whey was subjected to 4 different treatments: control (no bleaching; 50°C, 1 h), HP (250 mg of HP/kg; 50°C, 1 h), and LP (20 mg of HP/kg; 50°C, 1 h), or MF (microfiltration; 50°C, 1 h). The treated whey was then ultrafiltered, diafiltered, and spray-dried to 80% concentrate. The entire experiment was replicated 3 times. Proximate analyses, color, functionality, descriptive sensory and instrumental volatile analysis were conducted on WPC80. The MF and HP- and LP-bleached WPC80 displayed a 39.5, 40.9, and 92.8% norbixin decrease, respectively. The HP and LP WPC80 had higher cardboard flavors and distinct cabbage flavor compared with the unbleached and MF WPC80. Volatile compound results were consistent with sensory results. The HP and LP WPC80 were higher in lipid oxidation compounds (especially heptanal, hexanal, pentanal, 1-hexen-3-one, 2-pentylfuran, and octanal) compared with unbleached and MF WPC80. All WPC80 had >85% solubility across the pH range of 3 to 7. The microstructure of MF gels determined by confocal laser scanning showed an increased protein particle size in the gel network. MF WPC80 also had larger storage modulus values, indicating higher gel firmness. Based on bleaching efficacy comparable to chemical bleaching with HP, flavor, and functionality results, MF is a viable alternative to chemical or enzymatic bleaching of fluid whey.}, number={9}, journal={Journal of Dairy Science}, author={Qiu, Y. and Smith, T. J. and Foegeding, E. A. and Drake, M. A.}, year={2015}, pages={5862–5873} }
 @article{smith_li_drake_2014, title={Short communication: Norbixin and bixin partitioning in Cheddar cheese and whey}, volume={97}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2013-7614}, abstractNote={The Cheddar cheese colorant annatto is present in whey and must be removed by bleaching. Chemical bleaching negatively affects the flavor of dried whey ingredients, which has established a need for a better understanding of the primary colorant in annatto, norbixin, along with cheese color alternatives. The objective of this study was to determine norbixin partitioning in cheese and whey from full-fat and fat-free Cheddar cheese and to determine the viability of bixin, the nonpolar form of norbixin, as an alternative Cheddar cheese colorant. Full-fat and fat-free Cheddar cheeses and wheys were manufactured from colored pasteurized milk. Three norbixin (4% wt/vol) levels (7.5, 15, and 30 mL of annatto/454 kg of milk) were used for full-fat Cheddar cheese manufacture, and 1 norbixin level was evaluated in fat-free Cheddar cheese (15 mL of annatto/454 kg of milk). For bixin incorporation, pasteurized whole milk was cooled to 55 °C, and then 60 mL of bixin/454 kg of milk (3.8% wt/vol bixin) was added and the milk homogenized (single stage, 8 MPa). Milk with no colorant and milk with norbixin at 15 mL/454 kg of milk were processed analogously as controls. No difference was found between the norbixin partition levels of full-fat and fat-free cheese and whey (cheese mean: 79%, whey: 11.2%). In contrast to norbixin recovery (9.3% in whey, 80% in cheese), 1.3% of added bixin to cheese milk was recovered in the homogenized, unseparated cheese whey, concurrent with higher recoveries of bixin in cheese (94.5%). These results indicate that fat content has no effect on norbixin binding or entrapment in Cheddar cheese and that bixin may be a viable alternative colorant to norbixin in the dairy industry.}, number={6}, journal={JOURNAL OF DAIRY SCIENCE}, author={Smith, T. J. and Li, X. E. and Drake, M. A.}, year={2014}, month={Jun}, pages={3321–3327} }
 @article{fox_smith_gerard_drake_2013, title={The Influence of Bleaching Agent and Temperature on Bleaching Efficacy and Volatile Components of Fluid Whey and Whey Retentate}, volume={78}, ISSN={["1750-3841"]}, DOI={10.1111/1750-3841.12251}, abstractNote={Fluid whey or retentate are often bleached to remove residual annatto Cheddar cheese colorant, and this process causes off-flavors in dried whey proteins. This study determined the impact of temperature and bleaching agent on bleaching efficacy and volatile components in fluid whey and fluid whey retentate. Freshly manufactured liquid whey (6.7% solids) or concentrated whey protein (retentate) (12% solids, 80% protein) were bleached using benzoyl peroxide (BP) at 100 mg/kg (w/w) or hydrogen peroxide (HP) at 250 mg/kg (w/w) at 5 °C for 16 h or 50 °CC for 1 h. Unbleached controls were subjected to a similar temperature profile. The experiment was replicated three times. Annatto destruction (bleaching efficacy) among treatments was compared, and volatile compounds were extracted and separated using solid phase microextraction gas chromatography mass spectrometry (SPME GC-MS). Bleaching efficacy of BP was higher than HP (P < 0.05) for fluid whey at both 5 and 50 °C. HP bleaching efficacy was increased in retentate compared to liquid whey (P < 0.05). In whey retentate, there was no difference between bleaching with HP or BP at 50 or 5 °C (P > 0.05). Retentate bleached with HP at either temperature had higher relative abundances of pentanal, hexanal, heptanal, and octanal than BP bleached retentate (P < 0.05). Liquid wheys generally had lower concentrations of selected volatiles compared to retentates. These results suggest that the highest bleaching efficacy (within the parameters evaluated) in liquid whey is achieved using BP at 5 or 50 °C and at 50 °C with HP or BP in whey protein retentate.}, number={10}, journal={JOURNAL OF FOOD SCIENCE}, author={Fox, A. J. and Smith, T. J. and Gerard, P. D. and Drake, M. A.}, year={2013}, month={Oct}, pages={C1535–C1542} }