Cottonseed Storage Protein Research Articles

Dr. Leon S. Dure, III (1931-2014).

with a bachelor’s degree in philosophy, and then served as a Marine Corps officer in the mountains of Korea. After returning home and leaving formal duty in the Marines (because of Leon’s loyalty and love for the Marine Corps as well as his patriotism, he remained an active reserve Officer, reaching the rank of Colonel before retiring from the Marine Reserves), Leon returned to the University of Virginia where he received an M. S. degree in plant embryology. He then continued his graduate education at the University of Texas where he received his Ph.D. in plant physiology. After this, Leon joined the developing faculty of biochemistry of the UGA in 1960 as a postdoctoral researcher working on bioluminescence—a pioneering step as he was in fact the first postdoctoral researcher at the UGA! He continued on the faculty of biochemistry and retired in 2003 as Franklin Professor of Biochemistry after a distinguished research career. Leon was also greatly respected as a masterful teacher, which the Franklin professorship validated as one of the best. In the mid-1960s, Dr. Dure developed his independent research laboratory where he focused on the molecular bases of seed germination. During this early period, one of the important findings was discovery and characterization of long-lived messenger or mRNA that coded for specialized proteins in germinating cotton seed. This was one of the earliest reports on the long-term stability of at least some eukaryotic mRNAs. Leon’s group did one of the most thorough and excellent studies at the time on PolyA + and Poly A– mRNAs in a plant system. Dr. Dure’s research continued to focus on many aspects of cotton seed embryogenesis and germination, including additional work on the seed mRNAs and studies on cotton seed storage proteins from general characterization of their complexity and abundance to biosynthetic pathways. The role of abscisic acid in these processes represents another major group of contributions. Dr. Leon S. Dure III passed away on May 11, 2014. He was Franklin Professor of Biochemistry at the time of his retirement from the University of Georgia (UGA), Athens, Georgia, where he spent his entire faculty career from 1960 to 2003. Born in January 1931 in Macon, Georgia, Leon grew up at his family home in East Belmont Farm, Kenswick, Virginia. He graduated from the University of Virginia

Influence of lipids with and without other cottonseed reserve materials on aflatoxin B(1) production by Aspergillus flavus.

Cottonseed storage lipids (primarily triglycerides), in either crude or refined form, were found to support growth and aflatoxin B(1) production by Aspergillus flavus. When lipids were removed from ground whole cottonseed by petroleum ether extraction, aflatoxin production dropped by more than 800-fold. Reconstitution of the lipid-extracted ground whole seed with a crude preparation of cottonseed lipids restored aflatoxin production to the previous levels. Fungal utilization of the three major cottonseed reserve materials, raffinose, triglycerides (refined cottonseed oil), and cottonseed storage protein, was monitored in vitro over a 7 day fermentation period. The fermentation medium contained the reserve compounds in proportions approximating those found in mature cottonseed. A. flavus rapidly converted raffinose to fructose and melibiose, presumably by action of invertase, and then hydrolyzed the melibiose. These simple sugars apparently supported initial growth and aflatoxin B(1) production. Raffinose and the resulting melibiose were nearly exhausted by day 2. Fungal hydrolysis of triglycerides began as exhaustion of carbohydrate approached. After day 2, rapid catabolism of the released fatty acids began and coincided with glucose regeneration through gluconeogenesis, which peaked on day 6. The fungus did not preferentially utilize specific fatty acids. A. flavus also produced a number of storage metabolites, including arabitol, erythritol, mannitol, and trehalose. Mannitol was produced in much higher concentrations than the other storage metabolites. Selective use of simple carbohydrates by A. flavus to drive aflatoxin production may suggest strategies for reducing vulnerability of cottonseed to aflatoxin contamination.

Raffinose content may influence cottonseed susceptibility to aflatoxin contamination

AbstractThe effect of the cotton storage trisaccharide raffinose and cottonseed storage protein (CSP) in combination on aflatoxin production by Aspergillus flavus was investigated. The ability of ground whole cottonseed and water‐extracted cottonseed meal to support fungal biosynthesis of aflatoxin also was assessed in vitro. Dose response data showed that utilization of raffinose as a sole carbon source supported growth and aflatoxin production by A. flavus. When raffinose was a carbon source and CSP was the sole nitrogen source, aflatoxin levels were stimulated up to fourfold above those in raffinose reference media. Results with ground whole cottonseed as a sole carbon/nitrogen source demonstrated the capacity for aflatoxin production in A. flavus cultures. Lipid extraction of ground whole seed severely reduced aflatoxigenesis potential; however, lipid extraction followed by water extraction of ground whole seed restored much of the aflatoxin biosynthetic potential, suggesting the presence of a water soluble inhibitory factor. Accessible carbon appeared to be the limiting resource in water‐extracted meal, as a raffinose supplement stimulated aflatoxin production. Either cottonseed storage lipid or raffinose was capable of providing accessible carbon to A. flavus. Raffinose and CSP in developing and mature cottonseed may predispose seed to potentially high levels of aflatoxin contamination by A. flavus upon seed infection.

Effects of oilseed storage proteins on aflatoxin production by Aspergillus flavus

AbstractPotential involvement of seed storage proteins in susceptibility to aflatoxin contamination was assessed with in vitro tests. Initially, two oilseed storage proteins [cottonseed storage protein (CSP) and zein] were compared with bovine serum albumin (BSA) and collagen. Supplementation of a complete defined medium with either oilseed storage protein resulted in significantly more aflatoxin production by Aspergillus flavus than supplementation with either BSA or collagen. Little or no aflatoxin was produced when either BSA, CSP, or zein was employed (at 0.5%) as both the sole carbon and the sole nitrogen source. Media with collagen (0.5%) as the sole nitrogen and carbon source supported aflatoxin production similar to the complete defined medium. Although lower than levels observed with defined medium, aflatoxin production increased with both increasing CSP concentration (0 to 2.0%) and increasing zein concentration (0 to 6.0%) when these proteins served as both the sole carbon and sole nitrogen source. Denaturing polyacrylamide gel electrophoresis and protease activity assays indicated that fungal acquisition of protein carbon was probably via hydrolysis mediated by the 35 kD metalloprotease of A. flavus. Media lacking nitrogen but containing sucrose (5.0%) and supplemented with either zein (1.7%) or CSP (2.0%) supported three‐ to eightfold more aflatoxin production than the complete defined medium. The results suggest seed storage proteins, when present with an accessible carbon source, may predispose oilseed crops to support production of high levels of aflatoxins by A. flavus during seed infection.

Effects of oilseed storage proteins on aflatoxin production by aspergillus flavus

AbstractPotential involvement of seed storage proteins in susceptibility to aflatoxin contamination was assessed with in vitro tests. Initially, two oilseed storage proteins [cottonseed storage protein (CSP) and zein] were compared with bovine serum albumin (BSA) and collagen. Supplementation of a complete defined medium with either oilseed storage protein resulted in significantly more aflatoxin production by Aspergillus flavus than supplementation with either BSA or collagen. Little or no aflatoxin was produced when either BSA, CSP, or zein was employed (at 0.5%) as both the sole carbon and the sole nitrogen source. Media with collagen (0.5%) as the sole nitrogen and carbon source supported aflatoxin production similar to the complete defined medium. Although lower than levels observed with defined medium, aflatoxin production increased with both increasing CSP concentration (0 to 2.0%) and increasing zein concentration (0 to 6.0%) when these proteins served as both the sole carbon and sole nitrogen source. Denaturing polyacrylamide gel electrophoresis and protease activity assays indicated that fungal acquisition of protein carbon was probably via hydrolysis mediated by the 35 kD metalloprotease of A. flavus. Media lacking nitrogen but containing sucrose (5.0%) and supplemented with either zein (1.7%) or CSP (2.0%) supported three‐ to eightfold more aflatoxin production than the complete defined medium. The results suggest seed storage proteins, when present with an accessible carbon source, may predispose oilseed crops to support production of high levels of aflatoxins by A. flavus during seed infection.

Effect of Heat Treatment Upon the Chemical Composition of Cottonseed Meal and Upon the Reactivity of Cottonseed Condensed Tannins

The effects of heat treatment on the chemical composition of cottonseed meal (CSM), with or without the addition of cottonseed hulls (containing condensed tannins; CT), and upon reactivity of the CT were studied. Heat was applied in a forced draught oven at 100°C for 2 h. Fluorodinitrobenzene (FDNB)-available lysine, free gossypol, extractable- and bound-CT concentrations, in vitrototal nitrogen (N) solubility and thein vitrorumen degradation of the two major seed proteins (52 and 48 kDa) present in cottonseed kernel (which does not contain CT) were determined. The reactivity of CT was assessed by determining N solubility and rumen degradation of cottonseed kernel proteins in the presence or absence of polyethylene glycol (PEG; molecular weight (MW) 3500), which binds and inactivates CT. Heat treatment reduced the concentrations of free gossypol and FDNB-available lysine by small amounts, reduced measurable total CT content by 13%, reduced the solubility of total N, and reduced potential degradability of the 52 and 48 kDa cottonseed storage proteins by mixed rumen microorganisms. Addition of hulls further depressed solubility of total N and ruminal degradation of the two major storage proteins in cottonseed kernel. The action of PEGin vitroindicated that only part of the depression caused by hull addition could be explained by the presence of CT in the hulls, and that the effects of CT upon N solubility and potential degradability in heated CSM were similar to that in unheated CSM. Addition of hulls also substantially reduced FDNB-available lysine. In commercially produced materials, CSM from the Brisbane mill had a lower total CT content, lower N solubility and lower ruminal protein degradation rate than CSM from the Narrabri mill, but a similar level of FDNB-available lysine. Although application of heat inactivated 13% of the total CT, such that it could no longer be extracted and detected with butanol/HCl, it did not seem to change the overall effects produced by CT in reducing N solubility and protein degradation. The effect of hull addition in reducing available lysine has considerable relevance for feeding CSM to monogastric livestock. Interactions involving heat, hulls and CT need to be further studied.

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates.

A SIMPLE, RAPID AND SIMULTANEOUS PREPARATION OF GLANDLESS COTTENSEED 7S AND 11S PROTEIN FRACTIONS AND CHARACTERIZATION OF SOME PHYSICOCHEMICAL PROPERTIES

A simple and rapid method for simultaneous preparation of individual protein fractions from glandless cottonseed storage proteins was developed. With gel filtration chromatography, using Sephacryl S-300 HR and 10% NaCl solution as an eluent, 3 fractions, FI, FII, FIII were obtained. FII and FIII produced single peaks by gel filtration rechromatography and anion exchange chromatography. FII and FIII showed both a single major and a barely noticeable minor band, respectively, on polyacrylamide gel electrophoresis. Analytical ultracentrifuge patterns showed impurites less than 2% in FII and a single symmetrical peak for FIII. Sedimentation coefficients were computed to be 10.7S and 6.8S for FII and FIII, respectively; molecular weights by osmometry were 2.39 × 105 and 1.40 × 105 g/mole for FII and FIII, respectively. Both fractions were high in acidic and low in sulfur-containing amino acids, and contained phytate and gossypol at less than 10 and 2 ppm, respectively.

Developmental biochemistry of cottonseed embryogenesis and germination

The developmental time period and the magnitude of expression of the genes for the principal cottonseed storage proteins have been measured by several means. RNA was extracted from cotton cotyledons at stages during embryogenesis and the relative amounts of the mRNAS for these proteins were determined by cell-free translation in the wheat germ system and by dot and northern hybridization of the RNA with cloned cDNA probes representing the three subfamilies of the major storage protein genes. The rates of reassociation in solution of some of the RNAs with one of the cDNA clones were also determined. Data from all four procedures show that the storage protein mRNAs are demonstrable in very small embryo cotyledons, rapidly reach a high abundance level that is maintained during most of embryo growth, and then fall precipitously in amount in the last days of embryogenesis. The expression of all three gene subfamilies appears coordinate.Further, cDNA reverse transcribed from the poly(A)(+) mRNA from a stage of maximum storage protein synthesis was hybridized to saturation with cDNA clones representing each of the subfamilies. These data indicate that the mRNAs for two of the families reach the same relative level in the total mRNA population which is about 15% of the total mRNA mass. The mRNA of the third subfamily comprises only 5% of the total mRNA mass at this stage. This apparent 3∶3∶1 ratio of mRNAs does not change during the period of storage protein synthesis. Based on the amounts of the storage protein species in the mature seed, the mRNAs of each subfamily appear to be translated to the same extent during embryogenesis.

Developmental Biochemistry of Cottonseed Embryogenesis and Germination

The synthesis of the principal cottonseed storage proteins during embryogenesis has been followed by analyses of stained protein gels and of fluorographs of protein synthesized in vivo and from purified RNA in vitro in the wheat germ system. The kinetics of in vivo labeling as well as immunochemical cross-reactivity indicate that the 52- and 48-kilodalton mature storage protein sets are derived from 70- and 67-kilodalton precursor protein sets that are abundant proteins in embryonic cotyledons and disappear in late embryogenesis. Identification of the initial translation products of the storage protein mRNA has not been clearly established although products of apparent molecular weights of 69,000 and 60,000 are the likely storage protein precursors.Storage protein synthesis falls off markedly in late embryogenesis simultaneously with the loss of a superabundant class of mRNAs (shown by cDNA:RNA reassociation) that are presumed to be those for the storage proteins. The synthesis of these proteins ceases abruptly when immature embryos are removed from the boll and allowed to germinate precociously or when this precocious germination is prevented by incubation in abscisic acid. Thus, abscisic acid is not implicated in the expression of the storage protein genes.A scheme involving co-translational processing into vesicles, glycosylation, and slow in situ cleavage to produce the mature storage proteins is proposed.

Cottonseed Proteins in Frankfurters: Effect on pH, Cured Color, and Sensory and Physical Properties

ABSTRACTFrankfurter‐type sausages were made in which 5%, 10%, or 15% of the meat was replaced with the following cottonseed‐derived protein products: genetically glandless cottonseed flour (CF); liquid cyclone processed deglanded cottonseed flour (LCPC); genetically glandless cottonseed storage protein isolate (CI). All‐meat controls (0% cottonseed protein additive) were formulated at each replacement level with fat contents equivalent to those of the cottonseed protein‐added frankfurters. Compared to all‐meat controls, frankfurters made with increasing levels of cottonseed proteins generally had higher pH values, less cured color, less firmness of skin, softer texture, and were less desirable as judged by sensory panels. At the 10% and 15% replacement levels, frankfurters containing LCPC, had significantly (P < 0.05) lower scores for external and internal visual color, overall satisfaction and bioyield and rupture force values than did Cl‐added frankfurters. Simple correlation coefficients suggest that significantly (P < 0.05) correlated responses exist within and across replacement levels between pH and sensory panel scores, Instron values and visual color scores.

COTTONSEED AND SOY PROTEIN INGREDIENTS IN SOFT‐SERVE FROZEN DESSERTS

ABSTRACTSix vegetable protein ingredients (glandless cottonseed flour, liquid cyclone process deglanded cottonseed flour, glandless cottonseed storage protein isolate, soy flour, soy protein concentrate, and shy protein isolate) were evaluated as partial replacements for milk solids‐not‐fat (MSNF) in soft‐serve frozen desserts. Physical tests and sensory evaluations were conducted with the experimental desserts. All ingredients tested produced satisfactory results at low levels of substitution for MSNF. At higher levels of substitution, glandless cottonseed storage protein isolate appeared to be the best of the materials tested.

PRODUCTION OF PROTEIN ISOLATES AND CONCENTRATES FROM OILSEED FLOUR EXTRACTS USING INDUSTRIAL ULTRAFILTRATION AND REVERSE OSMOSIS SYSTEMS

ABSTRACTThe consumption of protein isolates from oilseed flours is expected to continue its upward trend as meat analogs from vegetable proteins are more widely marketed. However, conventional protein isolation procedures are lengthy and somewhat expensive. These processes also result in whey‐like liquid by‐products which constitute a water pollution threat unless properly processed. Preparation of protein isolates and concentrates from glandless cottonseed and soy flours by extracting the protein and ultrafiltering the solubilized protein directly from the liquid extract was investigated. In the process devised, proteins normally precipitated to produce isolates were recovered together with the conventional whey protein by ultrafiltration (UF). The UF permeate was further processed with a reverse osmosis (RO) membrane. Different industrial UF and RO systems were utilized in 22–60 lb extractions. UF membranes were found which gave desirably high permeation rates and satisfactory constituent separations, especially with cottonseed storage protein (SP) extract. With SP extract, initial LJF permeate flux achieved was in excess of 1.50 gal of permeate per square foot of membrane area per day (gfd). The flux declined to 96 gfd over a period of 8 hr while the volume of original extract in the feed solution was being reduced by a ratio of 12.5 to 1 in the final UF concentrate. As expected, nonstorage protein (NSP) and SP extracts from cottonseed were found to require membranes having different molecular weight cutoff points. Noncellulose‐based membranes performed better for this application because of their broader pH and temperature operating ranges.