Improved method for the synthesis of trans-feruloyl-beta-sitostanol.

Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels in humans by up to 15%, and at least two products, Benecol and Take Control, are now on the market as naturally derived fatty acid esters of phytostanols (stanol esters) and phytosterols (sterol esters), respectively. A synthetic process was developed to synthesize gram quantities of trans-feruloyl-beta-sitostanol from ferulic acid and beta-sitostanol, with high purity and yields of approximately 60%. The process involves (a) condensation of trans-4-O-acetylferulic acid with the appropriate phytostanol or phytostanol mixture in the presence of N,N-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, (b) separation of the trans-4-O-acetylferuloyl products by preparative liquid chromatography, (c) selective deacetylation of the feruloyl acetate, and (d) chromatographic purification of the feruloylated phytostanols. The process was successfully applied to synthesize stanol trans-feruloyl esters from "Vegetable Stanols", a mixture of approximately 70:30 beta-sitostanol and beta-campestanol, in comparable purity and yield.

Comparison of oil and phytosterol levels in germplasm accessions of corn, teosinte, and Job's tears.

Seeds of 49 accessions of corn (Zea mays ssp. mays), 9 accessions of teosinte (Zea species that are thought to be ancestors and probable progenitors to corn), and 3 accessions of Job's tears (Coix lacryma), obtained from a germplasm repository, were ground and extracted with hexane. Whole kernel oil yields and levels of four phytonutrients (free phytosterols, fatty acyl phytosterol esters, ferulate phytosterol esters, and gamma-tocopherol) in the oils were measured. Among the seeds tested, oil yields ranged from 2.19 to 4.83 wt %, the levels of ferulate phytosterol esters in the oil ranged from 0.047 to 0.839 wt %, the levels of free phytosterols in the oil ranged from 0.54 to 1.28 wt %, the levels of phytosterol fatty acyl esters in the oil ranged from 0.76 to 3.09 wt %, the levels of total phytosterols in the oil ranged from 1.40 to 4.38 wt %, and the levels of gamma-tocopherol in the oil ranged from 0.023 to 0.127 wt %. In general, higher levels of all three phytosterol classes were observed in seed oils from accessions of Zea mays ssp. mays than in seed oils from accessions of the other taxonomic groups. The highest levels of gamma-tocopherol were observed in teosinte accessions.

Isolation of oligogalacturonic acids up to DP 20 by preparative high-performance anion-exchange chromatography and pulsed amperometric detection.

Milligram quantities of oligogalacturonic acids up to a degree of polymerization (DP) of 20 were purified by high-performance anion-exchange chromatography utilizing a preparative-scale (21-mm i.d.) CarboPac PA1 column and a nonlinear potassium acetate (pH 7.5) gradient. Detection was accomplished by pulsed amperometry without post-column addition of hydroxide. Pulsed amperometry at near-neutral pH is an excellent detection method for preparative separations of carbohydrates because it avoids base-catalyzed degradation reactions that can occur at high pH. This method was simpler, faster, had higher sample loading capacity and allowed for the isolation of higher DP oligogalacturonic acids than other methods reported previously. With this improved method, multi-milligram quantities of valuable oligogalacturonic acids (up to DP 20) can be readily isolated.

Hexose oxidase from Chondrus crispus: improved purification using perfusion chromatography(*).

1An improved method for purifying hexose oxidase (D-hexose: O(2) 1-oxidoreductase, EC 1.1.3.5) from the marine red alga Chondrus crispus is described for obtaining enzyme suitable for structural characterization and use in bioconversion of lactose to lactobionic acid. This involved extracting enzyme from finely ground lyophilized tissue in sodium phosphate buffer (pH 7) containing 20% ammonium sulfate, eliminating the previously used solvent extraction and protease treatments, and by applying Poros perfusion chromatography media to achieve rapid separations of high resolution. Primary separation of contaminating phycobiliproteins and carrageenans was achieved using Poros DEAE-50. Sequential HPLC purification steps using Poros HP2 and Poros HQ were followed by Sephacryl S200 h chromatography. Enzyme activity was determined with a peroxidase-coupled assay using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) substrate. A final specific activity of 69 U/mg was obtained, representing a 100-fold purification with an activity recovery of about 10%. A native size of approximately 117,000 Da was determined by size exclusion chromatography, and SDS-PAGE revealed the presence of 38,000 and 29,000 Da polypeptides that appear to be derived from a 65,000 Da subunit. Further properties of the enzyme are described.

Phytosterols in the aleurone layer of corn kernels.

Corn hulls are composed of two major layers: the outer layer, the pericarp, is made up of non-living cell walls, and an inner layer, the aleurone, consists of a single layer of living cells, surrounded by thick cell walls. Dissected pure pericarp and aleurone fractions were ground and extracted with hexane and the yields and compositions of the resulting oils were examined. This study revealed that the high levels of ferulate-phytosterol esters and the high concentration of sitostanol previously reported in corn-fibre oil actually originate in the aleurone cells.

Effect of heat pretreatment on the yield and composition of oil extracted from corn fiber.

Previously, hexane extraction of corn fiber was reported to produce a unique and potentially valuable oil that contained high levels of several phytosterols (which have been noted for their cholesterol-lowering properties). Current studies revealed that heat treatment (over the range of 100-175 degrees C) of corn fiber in either a convection oven or a vacuum oven caused only a modest reduction in the levels of the phytosterol components. However, these same heat pretreatments caused a considerable increase (up to 10-fold) in the levels (increasing from 0.34 wt % to a maximum of 3.64 wt % gamma-tocopherol in the oil) and yields (increasing from 5.4 mg of gamma-tocopherol/100 g of corn fiber to a maximum of 52.1 mg of gamma-tocopherol/100 g of corn fiber) of gamma-tocopherol in corn fiber oil. The main differences between the convection oven and vacuum oven pretreatments were associated with the disappearance of free fatty acids and free phytosterols at the higher temperature pretreatments in the vacuum oven, probably due to the lower boiling points of these lipids. Microwave pretreatment was also effective but caused a much smaller increase in the levels of gamma-tocopherol.

Modulation of lipoxygenase activity by bacterial hopanoids.

Tetrahydroxybacteriohopane (1), a bacterial hopanoid, inhibited soybean 15-lipoxygenase with an IC50 of about 10 microM. After per-O-acetylation of 1 no inhibition of the 15-lipoxygenase was observed. Two other bacterial hopanoids, tetrahydroxybacteriohopane glucosamine (2) and tetrahydroxybacteriohopane ether (3), stimulated the activity of soybean 15-lipoxygenase. The activities of two other arachidonic acid-metabolizing enzymes, human 5-lipoxygenase and prostaglandin H synthase, were unaffected by 1.

Polymerized cyclomaltoheptaose (beta-cyclodextrin, beta-CDn) inclusion complex formation with chlorogenic acid: solvent effects on thermochemistry and enthalpy-entropy compensation.

The inclusion of chlorogenic acid (CA) by epichlorohydrin-polymerized cyclomaltoheptaose (beta-cyclodextrin, beta-CDn) was studied with regard to temperature, solvent, and water activity aH2O approximately mole fraction = XH2O = 0.8-1 using MeOH as the diluent; 0.1 M sodium phosphate buffer). We discovered that the extreme convex curvature in K (the apparent stability constant) as a function of temperature was nearly eliminated at the lowest XH2O. The latter finding argues that this unusual CD behavior in aqueous media was due to perturbations in beta-CD's spatial organization in the polymeric matrix with temperature. Related to this we found, from the dependence of K on XH2O (K = K'XzH2O), that the beta-CDn.CA complex's stoichiometric coefficient, z, for water, varied between 5 and 8, depending on the temperature of the solution (K' = 400-800 M-1; T approximately 295-315 K). Our determinations of z were similar to those reported previously for beta-CD.(+)-limonene (z approximately 7), soluble beta-CD.CA (z approximately 6) or obtained by molecular dynamics calculations for beta-CD.CA reported herein (z approximately 5). However, beta-CDn.CA's z values did show a significant positive correlation with temperature not evident in equivalent solution experiments. Calculations of delta H and delta S at various XH2O values show linear enthalpy-entropy compensation (delta H plotted against delta S) but with a slope (Tc = theta delta H/theta delta S approximately 228 K) significantly less than Tc values determined from either standard aqueous thermodynamic experiments (Tc approximately 305 K on either beta-CD or beta-CDn) or variable XH2O (Tc approximately 272 K) experiments. To the best of our knowledge, this is the smallest Tc value detected in a multitude of CD.guest studies. This evident solvent effect on Tc strongly argues that the chemical part process of inclusion complex formation involves changes in the solvation of the beta-CDn's binding site.

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.

Cloning and characterization of a pectate lyase gene from the soft-rotting bacterium Pseudomonas viridiflava.

Pseudomonas viridiflava is a soft-rotting pathogen of harvested vegetables that produces an extracellular pectate lyase (PL) responsible for maceration of plant tissue. A pel gene encoding PL was cloned from the genome of strain SJ074 and efficiently expressed in Escherichia coli. After a series of deletion subclonings and analysis by transposon mutagenesis, the pel gene was located in a 1.2-kb PstI-BglII genomic fragment. This fragment appears to contain a promoter at the PstI end required for pel gene expression. The PL produced by pectolytic E. coli clones is identical to those produced by strain SJ074 and by other strains of P. viridiflava in terms of molecular weight (42 kDa) and pI (9.7). A mutant of strain SJ074, designated MEI, which had Tn5 specifically inserted in the pel locus was constructed by site-directed mutagenesis. The MEI mutant produced 70- to 100-fold less PL than the wild type and failed to cause tissue maceration in plants. PL production and soft-rot pathogenicity in MEI and in a Pel- mutant previously isolated from strain SF312 were restored to the wild-type level by complementation in trans with the cloned pel gene. By using the 1.2-kb fragment as a probe, pel homologs were detected in four bacteria that are pathologically unrelated to P. viridiflava. These include three pathovars of P. syringae (pv. lachrymans, pv. phaseolicola, and pv. tabaci) and Xanthomonas campestris pv. malvacearum. No DNA fragments showing homology to pel of P. viridiflava were detected in genomic digests prepared from two strains of soft-rot erwinias.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure and n.m.r. analysis of lactulose trihydrate.

The 13C CPMAS n.m.r. spectrum of 4-O-beta-D-galactopyranosyl-D-fructose (lactulose) trihydrate, C12H22O11.3 H2O, identifies the isomer in the crystals as the beta-furanose. This is confirmed by a crystal structure analysis, using CuK alpha X-ray data at room temperature. The space group is P212121, with Z = 4 and cell dimensions a = 9.6251(3), b = 12.8096(3), c = 17.7563(4) A. The structure was refined to R = 0.031 and Rw 0.025 for 1929 observed structure amplitudes. All the hydrogen atoms were unambigously located on difference syntheses. The conformation of the pyranose ring is the normal 4C1 chair and that of the furanose ring is 4T3. The 1----4 linkage torsion angles are O-5'-C-1'-O-1'-C-4 = 79.9(2) degrees and C-1'-O-1'-C-4-C-5 = -170.3(2) degrees. All hydroxyls, ring and glycosidic oxygens, and water molecules are involved in the hydrogen bonding, which consists of infinite chains linked together by water molecules to form a three-dimensional network. There is a three-centered intramolecular, interresidue hydrogen bond from O-3-H to O-5' and O-6'. The n.m.r. spectrum of the amorphous, dehydrated trihydrate suggests the occurrence of a solid-state reaction forming the same isomeric mixture as was observed in crystalline anhydrous lactulose, although the mutarotation of the trihydrate when dissolved in Me2SO is very slow.

Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection.

Underivatized oligogalacturonic acids with a degree of polymerization (DP) ranging from 2 to 50 have been separated for the first time on a high-performance CarboPac PA1 pellicular anion-exchange stationary phase column. Baseline separation of these pectic fragments was accomplished using a nonlinear gradient of pH 6 potassium oxalate buffer as the mobile phase. Acetate buffer linear gradients were also useful as mobile phases, but only for separations of oligogalacturonic acids that were soluble in this solvent (DP less than 20). Additionally, oligogalacturonic acid separations were accomplished on a lower capacity AS4A stationary phase column. Triple pulse amperometric detection was selective, sensitive, and reproducible, nevertheless, oligogalacturonic acid response factors were affected by DP and compositional changes in the mobile phase.

Preparative liquid chromatography of carbohydrates: mono- and di-saccharides, uronic acids, and related derivatives.

The general principles and practical aspects of preparative high-performance liquid chromatography (l.c.) of mono- and di-saccharides, sugars acids, lactones, and N-acetylated amino sugar derivatives are described. Milligram to gram quantities of these carbohydrates were isolated on semi-preparative (0.78 X 30 cm) or preparative (approximately 2.0 X 30 cm) columns packed with aminopropyl silica gel provided better resolution of individual mono- and di-saccharides, but columns of cation-exchange resin had higher capacity and were more durable and economical to use. Preparative, cation-exchange columns were operated at flow rates of less than 5 mL/min and pressures of approximately 1-2 MPa, allowing them to be used on unmodified analytical l.c. systems. Details are given for the efficient packing, use, and care of these columns, and on the effects of column selectivity, packing technique, and sample size on chromatographic resolution. Isolation of naturally occurring sugars from biological sources on a laboratory-packed column is described.

Effect of Storage of Raw and Pasteurized Goats' Milk on Flavor Acceptability, Psychrotrophic Bacterial Count, and Content of Organic Acids.

Pasteurization of raw goats' milk either at 63°C for 30 min or 72°C for 15 s within 1 d of milking ensures a better tasting product both initially and during storage at 4.5°C for 6 weeks than if the raw milk is aged for several days at 4.5°C before being pasteurized. Pasteurized milks processed from high-count raw milks aged 1 to 2 weeks had lower acceptability ratings (on a 9-point hedonic scale), which decreased further in cold storage and were independent of bacterial increases in the log phase of growth. Pasteurized milks processed from raw milk 7 or more days old were subject to rapid increases in bacterial numbers in storage if they were trace-contaminated during pasteurization even though initial counts were <100 psychrotrophs/ml. For all raw and pasteurized milks, three peaks were consistently observed from an HPLC analysis designed to monitor some organic acids. Two of the components decreased and the third appeared and increased during storage. Disappearance of one component coincided with appearance of another. These compounds may be associated with loss of flavor quality of the milk since in some instances these changes significantly correlated with the decrease in hedonic ratings of the stored milks.

Inhibition of bacteria by lactulose preparations.

Lactulose syrups were similar to sucrose syrups in water activity-lowering effects but were more inhibitory toward test microorganisms. Heat-treated commercial lactulose syrups were most inhibitory, whereas non-heat-treated pure lactulose was only slightly more inhibitory than sucrose.