Landis W. Doner

Partitioning of Intermediary Carbon Metabolism in Vesicular-Arbuscular Mycorrhizal Leek.

Vesicular-arbuscular mycorrhizal fungi are symbionts for a large variety of crop plants; however, the form in which they take up carbon from the host is not established. To trace the course of carbon metabolism, we have used nuclear magnetic resonance spectroscopy with [13C]glucose labeling in vivo and in extracts to examine leek (Allium porrum) roots colonized by Glomus etunicatum (and uncolonized controls) as well as germinating spores. These studies implicate glucose as a likely substrate for vesicular-arbuscular mycorrhizal fungi in the symbiotic state. Root feeding of 0.6 mM 1-[13C]glucose labeled only the fungal metabolites trehalose and glycogen. The time course of this labeling was dependent on the status of the host. Incubation with 50 mM 1-[13C]glucose caused labeling of sucrose (in addition to fungal metabolites) with twice as much labeling in uncolonized plants. There was no detectable scrambling of the label from C1 glucose to the C6 position of glucose moieties in trehalose or glycogen. Labeling of mannitol C1,6 in the colonized root tissue was much less than in axenically germinating spores. Thus, carbohydrate metabolism of host and fungus are significantly altered in the symbiotic state.

SOLUBILIZATION OF GELLAN GELS BY CHELATION OF CATIONS

Chelation solubilization of gellan under mild conditions has been accomplished for the first time by exposure to either 10 mM sodium citrate buffer (pH 6.0) or to 1 mM sodium hexametaphosphate (pH 6.6). The citrate system was preferred for most applications since its is a ubiquitous cellular component, its solutions are autoclavable, and because viable plant tissues, fungi, and bacteria could be recovered from culture. Such recovery is not possible from more commonly used media such as agar.

Isolation of Hemicellulose from Corn Fiber by Alkaline Hydrogen Peroxide Extraction

ABSTRACT For the first time, alkaline hydrogen peroxide (AHP) extraction conditions were used to isolate hemicellulose (arabinoxylan) from destarched corn fiber. Yields of the water-soluble hemicellulose B ranged from 35% (24 hr extraction at 25°C) to 42% (2 hr extraction at 60°C). The hemicellulose B resulting from the 2 hr extraction (pH 11.5) was off-white in color, and a very low proportion (1.7%) of water-insoluble hemicellulose A was extracted. AHP treatment caused delignification and facilitated the alkaline extraction of hemicellulose from the lignocellulosic fiber matrix. In the absence of H2O2, yields were reduced by more than one-third when using otherwise identical extraction conditions of time, temperature and pH. In the standard protocol, corn fiber, NaOH solution, and H2O2 were mixed in a 1:25:0.25 (w/v/w) ratio. Extractions were conducted at pH 11.5 at 25°C or 60°C. The pH was adjusted to 11.5 by addition of NaOH at ambient and elevated temperatures. The optimum hemicellulose yield (51.3%;...

High-performance liquid chromatographic separation of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid

High-performance liquid chromatography on a Zorbax NH2 analytical column, with acetonitrile: 0.05 m KH2PO4 (75:25, ww) used as eluant, has allowed the separation, in less than 14 min, of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid. Ultraviolet monitoring at 268 nm allows ascorbic acid and erythorbic acid to be detected at the 25-ng level, while refractive index detection monitors the elution of all six compounds. Tyrosine is a good internal standard, being well separated from the other compounds and having an adequate ultraviolet absorption at 268 nm. We have found dithiothreitol to be effective in rapidly reducing dehydroascorbic acid to ascorbic acid, providing the basis for indirectly determining dehydroascorbic acid after its reduction. The potential of this high-performance liquid chromatographic procedure for evaluating the levels of these compounds in orange juice and urine is demonstrated.

Recovery of Fiber in the Corn Dry-Grind Ethanol Process: A Feedstock for Valuable Coproducts

ABSTRACT A new process was developed to recover corn fiber from the mash before fermentation in dry-grind ethanol production. In this process, corn is soaked in water (no chemicals) for a short period of time and then degermed using conventional degermination mills. In the remaining slurry, corn coarse fiber is floated by increasing the density of the slurry and then separated using density differences. The fiber recovered is called quick fiber to distinguish it from the conventional wet-milled fiber. This study evaluated the percent of quick fiber recovery for a normal yellow dent and high oil corn hybrid. The quick fiber was analyzed for levels of corn fiber oil, levels of ferulate phytosterol esters (FPE) and other valuable phytosterol components in the oil and compared with conventional wet-milled corn coarse and fine fiber samples. Fiber samples were also analyzed and compared for yields of potentially valuable corn fiber gum (CFG, hemicellulose B). Comparisons were made between the quick fiber sampl...

An Improved Process for Isolation of Corn Fiber Gum

ABSTRACT Sequential alkaline extraction and alkaline hydrogen peroxide (AHP) bleaching have been used to prepare corn fiber gum in yields ranging from 21 to 40%, depending on the pH of the extraction medium. The pH was adjusted by using different ratios of NaOH and Ca(OH)2 The whitest product was obtained after AHP bleaching of the extract obtained using the lowest pH value. In order for the product gum to give its characteristic clear and low viscosity solutions, it was necessary to remove starch from the corn fiber substrate using α-amylase. The water-insoluble hemicellulose A fraction, a minor component, was removed by neutralizing AHP-treated extracts before ethanol precipitation of the useful hemicellulose B (corn fiber gum) fraction. At ambient temperature, AHP bleaching was near optimal after ≈2 hr under the processing conditions used. High ratios of arabinose (39%) to xylose (50%) were present in the corn fiber gum extracted under various alkaline conditions, and the H2O2 processing step did not s...

Purification of commercial gellan to monovalent cation salts results in acute modification of solution and gel-forming properties

Lithium, sodium, potassium, and ammonium salts of the industrial polysaccharide gellan were prepared. The salts were freely soluble in water at room temperature (25 degrees C). The opinion had been generally held that heating to 100 degrees C was necessary for gellan to achieve complete solubility in the presence of mono- or multivalent cations. Then, upon cooling, the solutions would form gels. These conclusions were based on the properties imposed upon gellan samples by the presence of contaminating divalent cations. Commercial gellan samples contain calcium and magnesium at levels exceeding 0.9%, sufficient for counterion formation with over one-third of gellans carboxyl groups. Purification was rapid and included sequential treatments with a cation-exchange (H+) resin, LiOH, NaOH, KOH, or NH4OH, and an anion-exchange (Cl-) resin. About 95% of the divalent cations and nearly 90% of the phosphate that contaminated commercial gellan were removed. The purified monovalent salts of gellan set in the presence of divalent cations and provide well-defined agents for gelling media used for propagation of microbes and plants. In a manner analogous to sodium alginate, solutions of lithium, sodium, potassium, or ammonium gellanate form beads when dropped into solutions of divalent cations. This property was exploited for entrapment of enzymes and cells in beads.

Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase

The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs.

Isolation of oligogalacturonic acids in gram quantities by preparative h.p.l.c.

Abstract Oligogalacturonic acids up to a degree of polymerization of 7 (d.p. 7) were isolated in gram quantities by preparative h.p.l.c. from endo-polygalacturonase- and pectate lyase-depolymerized polygalacturonic acid. A Dynamax-60A NH 2 (21.4 × 250 mm) 1-aminopropyl silica gel column was used with an isocratic acetate buffer (ca. 0.9 m , pH 5) mobile phase. Automated operation of the preparative h.p.l.c. system allowed for rapid, high-resolution separation and collection of oligogalacturonic acids that typically were 95–99% pure on a chromatographic peak area basis. The chromatographic system described represents an advance in oligogalacturonic acid isolation and purification methodology since it is faster, less labor intensive, and it provides higher isolation rates (over 300 mg/h of total oligosaccharides) than the traditionally used ambient pressure strong anion-exchange chromatography.

Rapid purification of commercial gellan gum to highly soluble and gellable monovalent cation salts

Abstract Commercial gellan gum contains divalent cation contaminants (mainly Ca 2+ and Mg 2+ ) in levels sufficient to neutralize over one-third of its car☐yl groups. Consequently, in order to dissolve gellan gum in water, the mixtures must be heated to more than 90°C. This has prevented applications of gellan gum to such uses as the immobilization of viable enzymes and cells in beads. A rapid two-step method is described here to purify commercial gellan gum to the monovalent cation salts in an overall yield of 85%, through the intermediate free acid form. The gellan monovalent cation salts were highly soluble at temperatures as low as 5°C, and readily gelled upon exposure to solutions of divalent cations. Laboratory-scale preparations of 100 g of gellan monovalent cation salts were readily achieved in a day.