Healthcare reform and its impact on data collection and dissemination.

Making quality- and outcome-related information available to healthcare consumers and purchasers is a central tenet of the Health Security Act, and few in healthcare oppose this provision of the Act--in concept, says Robert Hartley, MD, MBA, Vice President, Christian Health Services. Consumers and purchasers need such information to make informed choices about their healthcare options. However, the Act does not make clear how or by whom quality measurement data are to be collected and disseminated, nor by what criteria healthcare providers will be held accountable.

Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2.

Two extracellular feruloyl esterases (FAE-I and FAE-II) produced by the anaerobic fungus Neocallimastix strain MC-2 which cleave ferulic acid from O-(5-O-[(E)-feruloyl]-alpha-L- arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) were purified. The molecular masses of FAE-I and FAE-II were 69 and 24 kDa, respectively, under both denaturing and nondenaturing conditions. Apparent Km and maximum rate of hydrolysis with FAXX were 31.9 microM and 2.9 mumol min-1 mg-1 for FAE-I and 9.6 microM and 11.4 mumol min-1 mg-1 for FAE-II. FAE-II was specific for FAXX, but FAE-I hydrolyzed FAXX and PAXX, the equivalent p-coumaroyl ester, at a maximum rate of metabolism ratio of 3:1.

Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.

An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls.

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.

Effect of phenolic acids and phenolics from plant cell walls on rumenlike fermentation in consecutive batch culture.

Information on the interaction between mixed populations in the rumen and plant phenolics is required to fully elucidate the limitations of phenolic compounds on forage digestibility. The objective of this study was to examine the degradation of Italian ryegrass (Lolium multiflorum L.) hay incubated with mixed ruminal populations in consecutive batch culture (CBC) with or without phenolic acids or phenolic compounds extracted from plant cell walls. Each CBC consisted of a series of 10 cultures (3 replicates per culture) inoculated (10%, vol/vol) in sequence at 48-h intervals with microbial suspension from the previous set of cultures. All cultures were grown on a semidefined medium containing Italian ryegrass hay, and each CBC was initiated with an inoculum from the rumen. Rumenlike fermentation characteristics were maintained in control CBCs by repeated inoculum transfer. Treatment CBCs were transferred as described above, but cultures 5, 6, and 7 were incubated in the presence of trans-p-coumaric, cis-p-coumaric, or trans-ferulic acid or phenolics extracted from the cell walls of maize stem or barley straw. Mean apparent dry matter disappearance in control CBC cultures was 495 mg per g of hay, whereas the presence of phenolics reduced the initial dry matter disappearance by 6.3 to 25.6%. trans-p-Coumaric acid and, to a lesser extent, the phenolics from cell walls of maize stem were the most inhibitory compounds for dry matter disappearance and for the production of volatile fatty acids; trans-p-coumaric acid altered the molar ratio of acetate/propionate/butyrate. The CBC further showed variations in the ability of the rumen microbial population to adapt to phenolic compounds.

The lignin fraction of plant cell walls.

Methods are discussed for determining lignin in plant cell walls. The increase in apparent lignin content that may occur as a result of artifacts produced during food preparation is also discussed. The phenolic components, including lignin, of cell walls separated from 12 vegetable, fruit, and cereal foods are determined. Wheat bran, on a fresh weight basis, had a high cell wall content (48.6%) compared with the vegetables and fruit whose contents ranged from 1.0 to 6.0%. Wheat bran and spinach cell walls had the highest lignin contents (11.4 and 4.4%, respectively) and the lowest degradabilities (35.8 and 39.6%, respectively) as determined by a cellulase technique. Cell walls of spinach, beetroot, sweet corn, pineapple, and wheat bran contained the phenolic acids (ferulic, p-coumaric, and diferulic) bound to polysaccharide components. Cell walls of cabbage, Brussels sprout, celery, cauliflower, green bean, carrot, and pea contained only traces of these acids.