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Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins.

W.F. FETT, C. WIJEY, R.A. MOREAU AND S.F. OSMAN. 1999.Ten strains belonging to various Thermomonospora species were tested for their ability to hydrolyse the insoluble plant polyester cutin. One strain, the thermophile T. fusca ATCC 27730, was found to produce a highly inducible cutinase when grown in broth medium containing purified apple cv. Golden Delicious cutin. Apple pomace, tomato peel, potato suberin and commercial cork were also shown to induce cutinase production. Addition of glucose to the culture medium either at the beginning of fermentation or after 2 days of incubation in the presence of apple cutin led to repression of cutinase production. The cutinase was active against a wide range of cutins, including those isolated from other apple cultivars as well as tomato, cucumber, grapefruit, and green pepper. Cutinase activity in the induced culture supernatant fluids exhibited a half-life of over 60 min at 70 °C and a pH optimum of 11·0. Some potential applications for cutinases are discussed.

Capacitation is an important process in bovine sperm maturation and is an obligatory step prior to fertilization. Two capacitating agents, namely heparin and high-density lipoprotein (HDL), have been shown to induce sperm capacitation. A family of major proteins of bovine seminal plasma designated BSP-A1/A2, BSP-A3, and BSP-30 kDa (collectively called BSP proteins) bind to the sperm surface upon ejaculation via their membrane choline phospholipids. Our previous studies with bovine epididymal sperm showed that BSP proteins potentiate sperm capacitation induced by heparin and HDL. This study was undertaken to clarify the mechanism of capacitation induced by heparin and HDL in the presence of BSP proteins. Washed bovine ejaculated sperm were incubated with heparin (12 microg/ml) or HDL (10-160 microg/ml) in the presence of polyclonal antibodies against purified BSP proteins (anti-BSP proteins). The percentage of capacitated sperm was evaluated after the induction of the acrosome reaction (AR) with lysophosphatidylcholine. When sperm were incubated for 5 h with heparin and anti-BSP proteins (40 microg/ml), the AR level was not significantly different from control levels (16. 8 +/- 0.9% vs. 12.9 +/- 0.9%). In contrast, incubation of sperm for 8 h with HDL and anti-BSP proteins did not inhibit the AR (42.4 +/- 1.1% vs. 17.1 +/- 1.6 for the control samples). We also investigated the effect of heparin and HDL on protein tyrosine phosphorylation associated with capacitation. The tyrosine phosphorylation of a group of proteins was increased in the presence of heparin. However, HDL did not significantly stimulate protein phosphorylation. The increase in phosphorylation was correlated with an increase in the AR after the incubation with heparin but not with HDL. These results indicate that heparin and HDL mediate capacitation via different mechanisms.

Since 1970, AOCS has been a regular host to the sterol symposia. Table 1 summarizes the history of the AOCS Sterol Symposium Series. The pioneers who established the AOCS Sterol Symposium during its first decade include Dr. Henry W. Kircher (University of Arizona, an expert on natural product chemistry and insect-sterol ecology), Dr. James W. Hendrix (University of Kentucky, an expert on fungal sterols), Dr. John L. Laseter (University of New Orleans, an expert on the analysis of sterols by gas chromatography), Dr. William R. Nes (Drexel University, a bioorganic chemist and an expert on the structure, function and evolution of sterols, and whose research accomplishments were commemorated in Steroids 53:261–648, 1989), and Dr. Erich Heftmann (Western Regional Research Center, USDA, ARS, a biochemist and an expert on the chromatography and biosynthesis of plant sterols, and whose research accomplishments were commemorated in J Chromatogr A 452, 1–634, 1988). Throughout the years the sterol symposia have focused on current research in the areas of sterol structure, biosynthesis, chemistry, regulation, and function. The 2008 Sterol Symposium, ‘‘Recent Advances in Sterol Research,’’ was held at the AOCS Annual Meeting in Seattle, Washington. This year the symposium held special significance, for it hosted the presentation of the fourth G. J. Schroepfer Jr. Award for sterol research. The Award was established to honor the memory of Dr. George J. Schroepfer Jr., a prominent sterol biochemist and chemist who made major and lasting contributions to the sterol field. Much of his research dealt with the biosynthesis of cholesterol and its regulation. In addition, he maintained a strong organic synthesis program to support his biochemical studies. A biography describing many of Dr. Schroepfer’s contributions can be found in this journal (Wilson, W. K. Lipids 35, 242, 2000). Dr. Schroepfer was scheduled to be the keynote speaker at the sterol symposium in Orlando, Florida, in 1999, but unfortunately, he passed away on 11 December 1998. The three previous recipients of the Schroepfer Award were Professor Geoffrey Gibbons (2002), Professor Jan Sjovall (2004) and Professor Ingemar Bjorkhem (2006). The fourth recipient of the G. J. Schroepfer Jr. Award for sterol research was Professor Michel Rohmer from the Institute of Chemistry at the University Louis Pasteur/ CNRS in Strasbourg, France. Professor Rohmer, a member of the French Academy of Sciences, has made major contributions to the sterol field and we were pleased when we learned that he had been chosen to receive this R. A. Moreau (&) Eastern Regional Research Center, USDA, ARS, Wyndmoor, PA 19038, USA e-mail: robert.moreau@ars.usda.gov

One of the hypotheses to explain the mechanism of capacitation involves the loss of sperm membrane cholesterol. Here, we studied whether or not the major proteins of bovine seminal plasma designated as BSP-A1, -A2, -A3, and -30-kDa (collectively called BSP proteins), which are implicated in sperm capacitation, induce cholesterol efflux. When epididymal sperm were labeled with [3H]cholesterol and incubated with bovine seminal plasma (0.05-2%) or BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [3H]cholesterol (3.6-fold and 3-fold, respectively). The same results in the presence of BSP-A1/-A2 were obtained (3.5-fold) by direct determination of cholesterol on unlabeled epididymal sperm. Analysis of efflux particles by ultracentrifugation on a sucrose gradient revealed a single symmetrical peak of radioactivity at 1.14 g/ml. Immunoblotting of the fractions obtained from size-exclusion chromatography of the efflux particles showed that a portion of the BSP proteins were associated with [3H]cholesterol. Heparin (12 microg/ml) alone did not stimulate cholesterol efflux. In contrast, high-density lipoprotein (HDL, 100 microg/ml) alone stimulated cholesterol efflux up to 3.1-fold after 8 h. When labeled epididymal sperm were preincubated for 20 min with BSP-A1/-A2 (120 microg/ml), washed, and incubated with HDL (100 microg/ml) for 8 h, the total cholesterol efflux of the sperm suspension was 51.8 +/- 5.0% compared to 39.3 +/- 1.2% when HDL alone was used. These results indicate that BSP proteins and HDL play an important role in the sperm sterol efflux that occurs during capacitation. Furthermore, the heparin-induced sperm capacitation did not involve the efflux of sperm membrane cholesterol.

Total lipid extracts were prepared from the leaves of potatoes, tomatoes, grapes and spinach, and each was analyzed by normal-phase high performance liquid chromatography (HPLC). When these total lipid extracts were subjected to mild alkaline hydrolysis and the hydrolysates analyzed by normal-phase HPLC, distinct peaks were detected in the region where common phospholipids typically elute (i.e. at 48, 50, and 68 min). The ultra violet-visible spectra of these peak fractions revealed that each exhibited absorption maxima at 400 and 660 nm, suggesting that the peaks were porphyrins, most likely derived from chlorophylls. Mild alkaline hydrolysis apparently cleaved the ester bond of the chlorophylls and released the porphyrin and phytol components. This explanation was verified when commercially prepared chlorophylls a and b were subjected to the same alkaline hydrolysis conditions and identical peaks at 48 and 68 min were observed. Experiments with buffered (pH 6.0) aqueous homogenates of potato and tomato revealed that similar chlorophyll-derived porphyrins were generated by endogenous enzymes. With the increasing popularity of HPLC as a tool for plant lipid analysis, users of this methodology should be cautioned as to the occurence of these non-phospholipid peaks in the retention time region where phospholipids commonly elute. © 1998 John Wiley & Sons, Ltd.

The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short- or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp + phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp + phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp + phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp + phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 A of the active site by the double mutation leu146lys + ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe112 largely eliminated catalytic activity of the lipase.

Candida bombicola is well-studied for the production of a biosurfactant, the sophorolipids. In this paper, the cloning of a glucosyltransferase gene using polymerase-chain-reaction (PCR) technique is described. Degenerative primer-pairs were first designed based on the highly conserved amino-acid sequences of several selected yeast glucosyltransferases. Using these primers, an amplified sequence (amplicon) of 700 base-pair from C. bombicola was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional target-specific PCR primers were designed for use in subsequent rounds of 3′- and 5′-extension using DNA walking technique to eventually obtain a C. bombicola genomic sequence containing an open-reading-frame putatively identified as a glucosyltransferase (gtf-1). The gene was subcloned in Saccharomyces cerevisiae for expression and functional characterization. Quantitative RT-PCR confirmed the expression of gtf-1 in the recombinant S. cerevisiae. In vitro assay with the sonicated cells of the recombinant yeast confirms the presence of glucosylation activity on sterol and hydroxy fatty acid substrates. This study reports for the first time the cloning and characterization of a broad-specificity lipid glucosylation gene from C. bombicola, and the functional activity of its gene product.

Extraction of commercial corn fiber with hexane or supercritical CO2 yielded an oil that comprised from 0.54 to 3.68 wt % of the fiber. An HPLC method with sensitive evaporative light-scattering de...

Zein is an alcohol-soluble protein isolated from corn. The effect of ground cover fi lms prepared from zein on the growth of tomato plants and corresponding evaporative water loss was investigated in greenhouse experiments. Results indicated that there was a decrease in water loss from the growth media for pots treated with zein fi lms compared to the control (no fi lm). There was an 11% increase height and 65% increase in dry weight of the treated plants relative to the control. In a second experiment, tomato plants mulched with zein isolates, low in free fatty acids (LFFA), exhibited an 18% increase in height and a 28% increase in dry weight compared to the control. Tomato plants treated with black polyethylene sheathing mulch were the tallest of the plants tested and had the greatest dry weight. Adding corn gluten meal directly to the soil surface resulted in tomato plants that were 26% taller and 29% heavier than those grown in untreated soil. Zein isolate fi lms appear to be a viable ground cover replacement for polyethylene sheathing. Nonbiodegradable, polymeric materials presently used as ground cover to control weeds and contain or reduce water evaporation are inexpensive to manufacture but expensive to

A series of oligomeric fragments can be obtained by partial alkaline hydrolysis of cutin. The oligomers obtained from tomato cutin were analyzed by high-performance liquid chromatographymass spectrometry and the major products were characterized as linear dimeric esters of w,8-, w,9-, and w,lO-dihydroxyhexadecanoic acids, the major monomers of tomato cutin. Analysis of the products obtained from tomato cutin by treatment with cutinase suggests an exo mode of depolymerization for this enzyme.

A recently developed HPLC technique has been used to identify 10 major monomers from potassium hydroxide hydrolysis and transesterification of lime cutin, revealing 16-hydroxy-10-oxo-hexadecanoic and 10,16-dihydroxyhexadecanoic acids as the major constituents. Solid-state 13 CNMR spectroscopy has also been used to examine the unreacted residues following hydrolytic treatment. For transesterification with methanolic boron trifluoride, an alternative protocol using MS has been developed. Using HPTLC to separate epoxy and hydroxy fatty esters, and making a series of trimethylsilyl ether derivatives, the monomeric products have been subjected to analysis by GC-CI-mass spectrometry. Although GC cannot discriminate between positional isomers of oxo and dihydroxy fatty acids, the parent ions and fragmentation patterns obtained with CI-mass spectrometry allow definitive identification of each isomer and reveal five new constituents of the cutin biopolymer. Both the methodology and the monomeric structures are compared with prior reports for citrus fruit cuticle; implications for the molecular architecture and biosynthesis of the lime cutin polymer are also discussed.

This research focuses on the overall extraction process of alkylresorcinols (ARs) from uncooked grains and baked products that have been processed with wheat, corn, rice, and white flour. Previously established extraction methods developed by Ross and colleagues, as well as a semiautomated method involving accelerated solvent extraction (ASE), were applied to extract ARs within freshly ground samples. For extraction of alkylresorcinols, nonpolar solvents such as ethyl acetate have been recommended for the extraction of uncooked foods, and polar solvents such as 1-propanol:water (3:1 v/v) have been recommended for the extraction of baked foods that contain rye, wheat, or other starch-rich grains. A comparison of AR extraction methods has been investigated with the application of gas chromatography and a flame ionization detector (GC-FID) to quantify the AR content. The goal of this research was to compare the rapid accelerated solvent extraction of the alkylresorcinols (ASE-AR) method to the previous manual AR extraction methods. Results for this study as well as the investigation of the overall efficiency of ASE-AR extraction with the use of a spiking study indicated that it can be comparable to current extraction methods but with less time required. Furthermore, the extraction time for ASE (approximately 40 min) is much more convenient and less tedious and time-consuming than previously established methods, which range from 5 h for processed foods to 24 h for raw grains.

Treatment of tobacco suspension cells (Nicotiana tabacum cv. KY 14) with a purified β-1,4-endoxylanase from Trichoderma viride [1 μg enzyme (ml cells)−1] caused a 13-fold increase in the levels of acylated sterol glycosides and elicited the synthesis of phytoalexins. A commercial preparation of xylanase from Trichoderma viride caused an identical shift in sterols. In contrast, a commerical xylanase from Aureobasidium pullaulans had no effect on the levels of acylated sterol glycosides, but did elevate the levels of sterol esters. Treatment of the cells with Cu2+ or Ag+ also evoked a severalfold increase in the levels of acylated sterol glycosides. Analysis of the various sterol lipid classes revealed that the large xylanase-induced increase in acylated sterol glycosides occurred at the expense of sterol esters, free sterols and sterol glycosides. Further analyses revealed that the most abundant phytosterol in each of the four classes of sterol lipids was β-sitosterol. Linoleic acid was the most abundant fatty acid in the sterol esters, and palmitic and linoleic acids were the most abundant fatty acids in the acylated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acvlated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acylated sterol glycoside fractions. The results of the present study demonstrate that xylanase from Trichoderma viride induces a dramatic shift in the level of acylated sterol glycosides, indicating that endoxylanase was probably the active component in the cellulase enzyme preparations used in our previous study.

Tobacco cell suspensions produce sesquiterpene phytoalexins when treated with cellulase. This response is enhanced by CaCl 2 , MgCl 2 or CdCl 2 . For CaCl 2 and MgCl 2 , enhancement extends for 8 hr after elicitor treatment. Several classes of signal transduction antagonists were tested on this plant system. EGTA inhibits sesquiterpene accumulation with an effective concentration range suggesting that Ca 2 + is the divalent cation being affected. Mg 2 + is as effective as Ca 2 + in relieving the inhibition by EGTA. This type of shared role by Ca 2 + and Mg 2 + has not been described previously and is yet to be characterized. The timing of the EGTA effect suggests a role of Ca 2 + in signal transduction. Calcium ion influx across the plasma membrane is one possibility since inhibitors of Ca 2 + influx inhibit sesquiterpene accumulation induced by cellulase. Verapamil is an inhibitor and its action is relieved by the Ca 2 + ionophore A23187. Among the cations which inhibit Ca 2 + influx, La 3 + is the most effective inhibitor of sesquiterpene accumulation. Calmodulin, or a Ca 2 + -dependent protein kinase, is also implicated in the signal transduction sequence, based on the results with W7, W5 and staurosporine. Neomycin and LiCl, inhibitors of phosphoinositide cycle steps, enhance cellulase-elicited sesquiterpene accumulation, suggesting a complex interaction of this cycle in the phytoalexin response. The results indicate that Ca 2 + and Mg 2 + play several roles in sesquiterpene biosynthesis. The involvement of a multicomponent elicitor signal transduction pathway, with calcium ion functioning at more than one step, is suggested.

Phytosteryl glycosides occur in natural foods but little is known about their metabolism and bioactivity. Purified acylated steryl glycosides (ASG) were compared with phytosteryl esters (PSE) in mice. Animals on a phytosterol-free diet received ASG or PSE by gavage in purified soybean oil along with tracers cholesterol-d(7) and sitostanol-d(4). In a three-day fecal recovery study, ASG reduced cholesterol absorption efficiency by 45 ± 6% compared with 40 ± 6% observed with PSE. Four hours after gavage, plasma and liver cholesterol-d(7) levels were reduced 86% or more when ASG was present. Liver total phytosterols were unchanged after ASG administration but were significantly increased after PSE. After ASG treatment both ASG and deacylated steryl glycosides (SG) were found in the gut mucosa and lumen. ASG was quantitatively recovered from stool samples as SG. These results demonstrate that ASG reduces cholesterol absorption in mice as efficiently as PSE while having little systemic absorption itself. Cleavage of the glycosidic linkage is not required for biological activity of ASG. Phytosteryl glycosides should be included in measurements of bioactive phytosterols.

The composition and structure of dewaxed cuticular membranes of three genotypes of pear (Pyrus spp.) leaves differing in resistance to attack by pear psylla (Cacopsylla pyricola Forester) were studied by chemical and enzymatic hydrolysis. The amount of chloroform/methanol-soluble material obtained after hydrolysis by methanolic KOH (indicative of leaf cuticle thickness) was inversely proportional to the relative degree of insect resistance. HPLC and GUMS analyses were used to identify and quantify the fatty acids and phenolics present in the extracts. The major cutin monomer of all three genotypes was 10,16-dihydroxyhexadecanoic acid. Enzymatic hydrolysis with three commercial lipases and a cutinase isolated from Nectria haematococca indicated that there were structural differences among the dewaxed cuticular membranes of the pear genotypes.

As a part of our study of microbial cutimases, a mixture of oligomers of 16-hydroxyhexadecanoic acid has been prepared by partial polymerization to serve as defined substrates. The mixture has been characterized by spectroscopic and chromatographic methods. Enzyme-catalyzed hydrolysis of the oligomers using fungal lipase, fungal cutinase or bacterial lipase required the addition of β-cyclodextrin to the reaction medium, whereas apple and other naturally occurring cutins can be enzymatically hydrolyzed without the aid of this cyclic oligosaccharide. The difference in reactivity may be due to the greater hydrophobicity of the synthetic oligomers compared with natural cutin polymers.

The ability of a commercial immobilized lipase preparation (Lipozyme) to hydrolyze the fatty acyl ester bonds of soybean phosphatidylcholine in organic media was investigated. Response surface methodology, based on a Modified Central Composite design, was employed to examine the effects on hydrolysis of solvent polarity, water, pH, duration and temperature of incubation, and the amounts of substrate and catalyst. A second-order regression model was developed, which allows evaluation of the effects of these parameters, alone or in combination. Hydrolysis increased in a relatively straightforward manner in response to increases in incubation time and the amount of catalyst and was approximately constant over the range of substrate amounts studied. Solvent polarity had a profound effect on the degree of hydrolysis, and the qualitative and quantitative degrees of this effect were dependent upon the values of the other parameters studied. Conditions were identified where enzyme activity was strong in either nonpolar or polar solvents, with activity increasing as the polarity of the medium increased. Enzyme activity was minimum at about 37°C, increasing below and above this temperature. Activity was not affected by the presence of acid or base in the reactions. Increasing amounts of water stimulated enzyme activity in solvents more polar than hexane, while in less polar solvents water inhibited activity.

Tobacco (Nicotians tabacum) KY14 cell cultures have previously been reported to produce capsidiol and other stress metabolites when treated with fungal elicitor or cellulase. Using a new high performance liquid chromatographic technique, we have measured the changes in sesquiterpene phytoalexins and membrane lipid classes thai occur upon elicitation of tobacco cell cultures with cellulase. Measurable levels of capsidiol and debneyol were found in the tobacco cells and in the culture medium after 8 h of elicitor treatment, with levels continuing to increase for up to 24 h. For the duration of the experiments, the levels of most of the galactolipids and phospholipids were found to decrease in elicited cells and increase in control cells. The most striking change was a rapid decrease in the level of digalactosyldiacylglycerol in elicited cells, to less than 10% of the level in control cells. Among the sterol lipid classes, the most notable changes occurred in the levels of sterol esters and acylated sterol glycosides, which increased significantly in elicited cells within 2 to 4 h after addition of cellulase, but remained unchanged in control cells. Free sterols and sterol glycosides declined slightly, while free fatty acids dropped to low levels 24 h after treatment of cells with cellulase. The present results and those of previous studies indicate that esterification of phytosterols may be a widespread response to environmental or chemical stress.

Several methods have been developed to obtain oil from corn germ, oilseeds, and other oil-rich plant materials using aqueous enzymatic methods. Unlike traditional oil extraction methods, these new processes are performed without the use of presses and without organic solvents. Beginning with olives in ancient times, oil has been obtained from oil-rich plant materials. The large variations in cell wall ultrastructure and chemical composition (varying proportions of cellulose, hemicellulose and pectin) of oil-rich plant materials have created a challenge for the development of aqueous enzymatic oil extraction strategies. For most oil-rich plant materials, three types of enzymes (cellulases, proteases and pectinases) have proven to be most effective for the aqueous enzymatic oil extraction. Although the high cost of enzymes is a major hurdle to the commercialization of aqueous enzymatic oil extraction methods, recent advances in enzyme production technology are gradually reducing enzyme costs and bringing these technologies closer to becoming economically feasible.

Forty-fiveStreptomyces strains, including representatives of the plant pathogensS. acidiscabies, S. scabies, andS. ipomoea, were screened for ability to produce enzymes (cutinases) capable of hydrolyzing the insoluble plant biopolyester cutin. Initially, all strains were tested for production of extracellular esterase in liquid shake (250 rpm) cultures at room temperature in defined (glycerol-asparagine) or complex (tryptone-yeast extract with or without addition of mannitol) broth media supplemented with either tomato or apple cutin. Esterase activity was determined by a spectrophotometric assay utilizing the model substratep-nitrophenyl butyrate. Of the five strains exhibiting highest esterase activity, four (S. acidiscabies ATCC 49003,S. “scabies” ATCC 15485 and IMRU 3018, andS. badius ATCC 19888) were confirmed to produce enzymes with cutin-degrading activity (cutinases). Confirmation of extracellular cutinase production was accomplished by use of a new high-performance liquid chromatography method for separation and quantification of released cutin monomers. Monomer identification was confirmed by GC/MS analyses. Cutinase production was induced 2- to 17-fold by inclusion of cutin in the media. To our knowledge this constitutes the first report of cutinase production byStreptomyces spp. other thanS. scabies.

Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases). Initially, esterase activity of culture filtrates of strains grown in nutrient broth-yeast extract medium supplemented with 0.4% apple or tomato cutin was determined by a spectrophotometric assay utilizing the model substrate p -nitrophenyl butyrate. The culture filtrates of the 10 Pseudomonas aeruginosa strains tested exhibited the highest esterase activity, with values of >500 nmol/min/ml. Of these 10 strains, 3 (K799, 1499A, and DAR41352) demonstrated significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium. The ability of culture filtrates of the three strains to cause release of apple cutin monomers was confirmed by a novel high-performance liquid chromatography technique. Monomer identification was confirmed by gas chromatography-mass spectroscopy analyses. Addition of the nonionic detergent n -octylglucoside stimulated cutinase activity of culture filtrates from strains K799 and DAR41352, but not that of filtrates from strain 1499A. Time course studies in nutrient broth-yeast extract medium supplemented with apple cutin indicated maximal levels of cutinase in the culture fluids after cultures entered stationary phase. Incubation temperatures below the optimal temperature for growth (37°C) led to maximal production of cutinase.

A silicic acid high performance liquid chromatographic method which utilizes an evaporative light scattering detector has been developed for the separation of cutin monomers. This binary gradient system was successfully applied to separate underivatized monomers of several fruit cutins including those from apple cvs. Golden Delicious and Red Delicious (Malus pumila), tomato (Lycopersicon esculentum), grapefruit (Citrus paradisi) green pepper (Capsidum annum) and pumpkin (Cucurbita pepo). All cutin monomers, from the least polar, monohydroxy saturated and unsaturated fatty acids, to the most polar, trihydroxy saturated and unsaturated fatty acids, were separated. Monomer identity was confirmed by gas chromatography-mass spectromety of collected peaks. The gradient was also able to separate positional isomers of dihydroxy fatty acids, a separation not yet achieved by gas chromatography. The relationship between detector response and the mass of five common cutin monomers was obtained.

A new high performance liquid chromatographic technique has been developed for the rapid analysis of sesquiterpene phytoalexins such as capsidiol, rishitin, lubimin and phytuberol. This method employs a cyanopropyl-bonded phase column with an isocractic mixture of hexane and isopropanol. When used with a flame ionization detector the lower limits of detection are about 0.1 μg and the response of the detector is linear in the range 0.1–30 μg. With the UV detector at 205 nm, the lower limits of detection are about 0.1 μg for capsidiol and rishitin and 0.5 μg for lubimin, phytuberol and debneyol. The response of the UV detector is linear in the range 0.5–30 μg. Although both detectors proved to be useful, the signal response with the flame ionization detector was proportional to the mass of each of the phytoalexins, while the signal with the UV detector was proportional to the number of carbon-carbon double bonds in each of the compounds.

A new high-performance liquid chromatography technique with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of hydroxy and epoxy fatty acids. This method employs a gradual binary gradient (hexane/isopropanol) and ELSD detection. The minimum limit of detection is about 1 µg and ratio of mass to signal is essentially linear in the range of 10 to 200 µg. This high-performance liquid chromatography (HPLC) technique is able to separate various positional isomers of mono-hydroxy and dihydroxy fatty acids and can also discriminate between monohydroxy, epoxy, epoxyhydroxy, dihydroxy and trihydroxy fatty acids.

Assigned from data sets measured in water at 2, 25, and 60 degrees C containing (13)C=O NMR chemical shifts and [theta](222) ellipticities, helical propensities are reported for the 20 genetically coded amino acids, as well as for norvaline and norleucine. These have been introduced by chemical synthesis at central sites within length-optimized, spaced, solubilized Ala(19) hosts. The resulting polyalanine-derived, quantitative propensity sets express for each residue its temperature-dependent but context-independent tendency to forego a coil state and join a preexisting helical conformation. At 2 degrees C their rank ordering is: PGHC, T, NSY, F, WV, DKQIR, MLEA; at 60 degrees C the rank becomes: H, PGCR, KT, Y, FN, VSQW, DI, MEAL. The DeltaDeltaG values, kcal/mol, relative to alanine, for the cluster T, N, S, Y, F, W, V, D, Q, imply that at 2 degrees C all are strong breakers: DeltaDeltaG(mean) = +0.63 +/- 0.11, but at 60 degrees C their breaking tendencies are dramatically attenuated and converge toward the mean: DeltaDeltaG(mean) = +0.25 +/- 0.07. Accurate modeling of helix-rich proteins found in thermophiles, mesophiles, and organisms that flourish near 0 degrees C thus requires appropriately matched propensity sets. Comparisons are offered between the temperature-dependent propensity assignments of this study and those previously assigned by the Scheraga group; the special problems that attend propensity assignments for charged residues are illustrated by lysine guest data; and comparisons of errors in helicity assignments from shifts and ellipticity data show that the former provide superior precision and accuracy.

Publisher Summary This chapter discusses the two lesser-known types of corn oil that have received much attention in recent years, corn kernel oil and corn fiber oil. The sequential extraction process was developed for the production of corn kernel oil and it involves the flaking of corn before ethanol extraction. The grinding (milling) of corn fiber is recommended for the production of corn fiber oil to a particle size of 1 micron (20 mesh) or smaller before beginning hexane extraction to achieve optimal oil yields. Both corn kernel oil and corn fiber oil contain unique health-promoting components that are lacking in commercial corn oil, which is obtained by extracting corn germ. Corn kernel oil contains very high levels of lutein and zeaxanthin; a regular consumption of it may prevent the onset of age-related macular degeneration. Corn fiber oil contains the highest levels of phytosterols of any known natural oil or extract and a regular consumption of it may reduce the levels of serum LDL- and total cholesterol.

Publisher Summary Barley is an ancient domesticated grain. As the oil content of most hulled and hulless barley cultivars is low, obtaining oil from whole barley grain is not easy or economical. However, when barley is milled by various methods, some of its milling fractions are enriched in oil and the fractions are used to produce barley oil. Almost all of the studies of barley oil and its lipid composition were conducted by extracting the oil and lipids with hexane. Barley oil is high-linoleate oil with a fatty-acid composition similar to corn oil and several other commodity plant oils. The levels of phytosterols in barley oil are higher than those in most other edible-plant oils. The most unique feature about barley oil is the very high levels of tocotrienols.

Publisher Summary The major tree nuts include almonds, Brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecans, pine nuts, pistachio nuts, and walnuts. Tree nuts belong to several plant families; however, their common features are that they are rich in oil and the nuts are larger in size than the seeds of oilseed species. Tree nut oils are appreciated in food applications because of their flavors and are generally more expensive than other gourmet oils. Before oil extraction, the nuts first need to be removed from the shell and then from the husk. The nut oils differ in their oxidative stabilities, which dictates the processing and storage conditions that are applied to each nut. Nut oils are also extracted from the nuts by Supercritical Fluid Extraction (SFE) using compressed carbon dioxide in wide temperature and pressure ranges. Nut-kernel oils are used as: salad oils, in cooking and other food applications, in massages and as lubricants, as emollients in pharmaceuticals, and in cosmetics, soaps, shampoos and hair conditioning/repair products, skin lotions, and other cosmetic products. Some tree nut oils, such as hazelnut oil, may serve as cooking oils in specialty dishes because of their unique flavors.

An unusual class of lipid with amphiphilic properties has been detected in nodule tissue of Alnus and Ceanothus. High levels of the same lipid (20-50% of total cell lipids) were detected in solvent extracts of Frankia spp. cells. However, the lipid was absent in host roots. The lipid was purified and quantified by high-performance liquid chromatography/flame ionization detector. Phenol-sulfuric acid determinations and proton nuclear magnetic resonance indicated that the purified lipid is not a glycolipid. Mass spectra of the predominant species are consistent with published spectra for bacteriohopanetetrol (C(35)H(62)O(4)), a pentacyclic triterpenoid, or hopanoid.

Five plant tissues were extracted and found to contain enzymes which could hydrolyze p- nitrophenylphorylcholine (PNP-PC), an artificial substrate which has been reported to be a convenient substrate for measuring the activity of phospholipase C. The leaves and tubers of Solanum tuberosum L. were then examined in more detail and found to contain enzymes which were capable of hydrolyzing several phosphorylcholine-containing metabolites. These metabolites included phosphatidylcholine (PC), glycerol phosphorylcholine, and cytodine diphosphocholine. The PNP-PC hydrolase from potato leaves was partially purified (a 229-fold increase in specific activity was achieved) and was found to have a molecular weight of about 28,000 and an isoelectric pH of approximately 6.8. When PNP-PC was used as a substrate, the enzyme was found to exhibit optimal activity at pH 6.0, was inhibited by Zn 2+ , and was found to have a K m of about 10 mM for PNP-PC. This hydrolytic enzyme activity was then tested for activity with naturally-occurring phosphorylcholine-containing metabolites and was found to hydrolyze CDP-choline but not PC or glycerol phosphorylcholine. This report indicates that although several plants contain enzymes that are capable of hydrolyzing PNP-PC, this substrate appears to be hydrolyzed by enzymes other than C-type phospholipases.

We recently reported that corn fiber oil contains high levels of three potential cholesterol-lowering phytosterol components: ferulate-phytosterol esters (FPE) (3–6 wt%), free phytosterols (1–2 wt%), and phytosterol-fatty acyl esters (7–9 wt%). A previous study also indicated that corn bran oil contained less phytosterol components than corn fiber oil. The current study was undertaken to attempt to confirm this preliminary observation using more defined conditions. Accordingly, oil was extracted from corn fiber and corn bran prepared under controlled laboratory conditions, using the same sample of corn hybrid kernels for each, and using recognized bench-scale wet-milling, and dry-milling procedures, respectively. After extraction, the chemical composition of the phytosterol components in the oil were measured. This study confirmed our previous observation—that FPE levels were higher in corn fiber oil than in corn bran oil. During industrial wet-milling, almost all of the FPE are recovered in the ...

Bioprocesses were developed to enhance the value of proteins from deoiled corn germ. Proteins were hydrolyzed with trypsin, thermolysin, GC 106, or Flavourzyme to generate the bioactive peptide sequences. At an enzyme to substrate ratio of 1:100, protein hydrolysis of wet-milled germ was greatest using thermolysin followed by trypsin, GC 106, and Flavourzyme. For the dry-milled corn germ, protein hydrolysis was greatest for GC 106 and least for Flavourzyme. Electrophoretic patterns indicated that the hydrolysis conditions used were adequate for generating low molecular weight peptides for both germs. Unhydrolyzed dry- and wet-milled corn germ did not appear to contain angiotensin I converting enzyme (ACE)-inhibitory peptides. After hydrolysis with trypsin, thermolysin, and GC 106 but not Flavourzyme, ACE inhibition was observed. ACE inhibition was greatest for the GC 106 hydrolysate for both wet- and dry-milled corn germ. Denaturing the protein with urea before hydrolysis, in general, increased the amount of ACE-inhibitory peptides found in the hydrolysate. Membrane fractionations of both the wet- and dry-milled hydrolysates indicated that most of the ACE-inhibitory peptides were in the

Although HPLC has been successfully used to separate most types of biomolecules, the lack of a quantitative detection technique has prevented the application of this powerful separation tool for the direct analysis of classes of plant lipids. The commercial availability of a reliable flame ionization detector (FID) has now opened up this field. We have developed a ternary gradient system (isooctane-isopropanol-water) which will separate all of the common classes of nonpolar lipids, galactolipids and phospholipids present in most plant tissues. The lower limits of detection utilizing this system are ca 1 μg of each lipid component per injection. Standards of 23 common plant lipids could be resolved with this new technique. The HPLC-FID system was used to provide a quantitative analysis of the lipid classes in potato leaves, potato tubers, corn roots, tobacco leaves and spinach leaves.

Hominy feed is a low value ($83.7/metric ton) coproduct of the corn dry milling process that accounts for nearly 35% of the starting corn quantity. The average composition of hominy feed on a dry basis is 56.9% starch, 25.2% neutral detergent fiber, 11.1% protein, and 5.3% fat. Starch in hominy feed can be fermented to ethanol thus increasing its levels of protein and fat. The increase in protein and fat percentages may increase the market competitiveness and price of hominy feed. Hydrolysis and fermentation were performed on nine hominy feed samples collected from three corn dry milling plants in the USA. The original hominy feed samples and postfermentation solids were analyzed for starch, protein, fat, and fiber content. Compared to the original hominy feed, the percentage increase in protein, fat and fiber in postfermentation solids of nine samples ranged from 10.4 to 21.3, 6.78 to 10.6, and 12.6 to 28.7% (dry basis), respectively. Ethanol yields varied from 271.7 to 380.2 l/metric ton for the nine hominy feed samples. These results indicate that the value of hominy feed as an animal feedstock can potentially be increased with fermentation and can produce more profit per metric ton than currently being derived by its sale as a low protein feed ingredient.

Corn fiber gum (CFG) is a hemicellulose (arabinoxylan)-enriched fraction obtained by the extraction of corn bran/fiber using a proprietary alkaline hydrogen peroxide process. When purified CFG prepared by this process was hydrolyzed with more concentrated base (1.5 N methanolic KOH at 70 degrees C for 1 hour), considerable amounts of hydroxycinnamic acids (up to 0.015% of mainly ferulic acid) and lipids (up to 0.43%) were released. The released phenolic acids and lipids were identified and quantified using high-performance liquid chromatography (HPLC) with detection by both UV and evaporative light-scattering detection (ELSD). During the wet milling of corn, two types of corn fiber are produced: coarse fiber, which is primarily from pericarp, and fine fiber, which is from the endosperm. The total phenolic acid content in CFGs purified from coarse corn fiber (pericarp fiber) is comparatively higher than that purified from fine corn fiber (endosperm fiber). It was also determined that the purified CFG samples contained significant amounts of strongly associated proteins, from 2 to 5% by weight. The presence of these phenolic acids, lipids, and proteins strongly associated or bound to CFG may contribute to its excellent ability to emulsify oil-in-water emulsions.

The aim of this study was to investigate the cholesterol-lowering mechanisms of corn fiber oil (CFO), ferulate phytostanyl esters (FPEs) and parent compounds of FPE, including sitostanol and ferulic acid, in hamsters.Seventy male Golden Syrian hamsters were randomly assigned to six experimental diets for 4 weeks: (1) cornstarch-casein-sucrose-based control diet (control); and (2) control diet plus 0.1% (wt/wt) cholesterol (cholesterol-control). The remaining four groups were given cholesterol-control diet with: (3) 10% (wt/wt) CFO; (4) 0.5% (wt/wt) sitostanol; (5) 0.23% (wt/wt) ferulic acid; and (6) 0.73% (wt/wt) FPE. At the end of dietary intervention, total plasma cholesterol, high-density lipoprotein cholesterol and triglyceride concentrations were determined. Parameters of cholesterol kinetics, including cholesterol absorption and synthesis, as well as mRNA expression of sterol transporters such as Niemann-Pick C1 like 1 (NPC1L1), ATP-binding cassette G5 (ABCG5) and ABCG8, were assessed.Supplementation with CFO decreased (P.0001) plasma total cholesterol levels by 29% as compared with the cholesterol-control group, while FPE and sitostanol reduced (P.02) cholesterolemia by 15% and 14%, respectively. CFO and sitostanol decreased (P.05) cholesterol absorption by 24% compared to the cholesterol-control group. Dietary intervention did not alter the intestinal gene expression of ABCG5, ABCG8 and NPC1L1.The present results show that the CFO-induced and sitostanol-induced decrease in cholesterol absorption is independent of intestinal enterocyte sterol transporters such as ABCG5, ABCG8 and NPC1L1 in hamsters.

The lipid profiles of cotton fiber cells were determined from total lipid extracts of elongating and maturing cotton fiber cells to see whether the membrane lipid composition changed during the phases of rapid cell elongation or secondary cell wall thickening. Total FA content was highest or increased during elongation and was lower or decreased thereafter, likely reflecting the assembly of the expanding cell membranes during elongation and the shift to membrane maintenance (and increase in secondary cell wall content) in maturing fibers. Analysis of lipid extracts by electrospray ionization and tandem MS (ESI-MS/MS) revealed that in elongating fiber cells (7-10 d post-anthesis), the polar lipids-PC, PE, PI, PA, phosphatidylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylglycerol-were most abundant. These same glycerolipids were found in similar proportions in maturing fiber cells (21 dpa). Detailed molecular species profiles were determined by ESI-MS/MS for all glycerolipid classes, and ESI-MS/MS results were consistent with lipid profiles determined by HPLC and ELSD. The predominant molecular species of PC, PE, PI, and PA was 34:3 (16:0, 18:3), but 36:6 (18:3,18:3) also was prevalent. Total FA analysis of cotton lipids confirmed that indeed linolenic (18:3) and palmitic (16:0) acids were the most abundant FA in these cell types. Bioinformatics data were mined from cotton fiber expressed sequence tag databases in an attempt to reconcile expression of lipid metabolic enzymes with lipid metabolite data. Together, these data form a foundation for future studies of the functional contribution of lipid metabolism to the development of this unusual and economically important cell type.

Because of the positive health effects of phytosterols, phytosterol-enriched foods and foods containing elevated levels of natural phytosterols are being developed. Phytosterol contents in cereals are moderate, whereas their levels in the outer layers of the kernels are higher. The phytosterols in cereals are currently underutilized; thus, there is a need to create or identify processing fractions that are enriched in phytosterols. In this study, pearling of hulless barley and rye was investigated as a potential process to make fractions with higher levels of phytosterols. The grains were pearled with a laboratory-scale pearler to produce pearling fines and pearled grains. Lipids were extracted by accelerated solvent extraction, and nonpolar lipids were analyzed by normal-phase HPLC with ELSD and UV detection. Total sterol analyses were performed by GC. After a 90-s pearling, the amounts of pearling fines from hulless barley and rye were 14.6 and 20.1%, respectively, of the original kernel weights. During pearling, higher levels of phytosterols and other lipids were fractionated into the fines. The contents of free sterols and sterols esterified with FA in the fines were at least double those in the whole grains. Pearling fines of hulless barley and rye contained >2 mg/g phytosterol compounds, which makes them a good source of phytosterols and thus valuable raw materials for health-promoting foods.

All fruits, vegetables, and grains contain phytosterols. Numerous clinical studies have documented that phytosterols lower LDL-cholesterol levels and thereby reduce the risk of cardiovascular disease. Most experts believe that the cholesterol-lowering mechanism of phytosterols requires that they be in their "free" form. In addition to their occurrence in the free form, phytosterols also occur as four common phytosterol conjugates: (i) fatty acyl esters, (ii) hydroxycinnamate esters, (iii) steryl glycosides, and (iv) fatty acylated steryl glycosides. This study was undertaken to investigate the extent of hydrolysis of four common phytosterol conjugates by mammalian digestive enzymes (cholesterol esterase and pancreatin, a mixture of pancreatic enzymes) and for comparison purposes, by KOH. Two types of purified hydroxycinnamate esters (sitostanyl ferulate and oryzanol, a mixture of hydroxycinnamate esters purified from rice bran oil) were hydrolyzed by cholesterol esterase and by pancreatin. Both cholesterol esterase and pancreatin hydrolyzed the phytosteryl esters in two functional food matrices, and they hydrolyzed the hydroxycinnamate esters in corn fiber oil. This is the first report to demonstrate that phytostanyl ferulate esters (which are present at levels of 3-6% in corn fiber oil) are hydrolyzed by pancreatic cholesterol esterase. It is also the first report that pancreatin contains enzymes that hydrolyze the fatty acyl moiety of fatty acylated steryl glycoside, converting it to steryl glycoside. Pancreatin had no effect on steryl glycosides. The ability of pancreatin to hydrolyze three other types of lipid conjugates was also evaluated. Phospholipids were completely hydrolyzed. About half of the galactolipids were hydrolyzed, and less than 10% of the polyamine conjugates were hydrolyzed. The extents of hydrolysis of phytosteryl esters by base (saponification) were also studied, and conditions commonly used for the saponification of acyl lipids (1.5 N methanolic KOH, 30 min at 70 degrees C), were found to result in a nearly 100% hydrolysis of TAG but only about 35-45% hydrolysis of the phytosteryl fatty acyl esters or phytosteryl hydroxycinnamate esters.

A new process was developed to separate fiber from distillers dried grains with solubles (DDGS) in a dry grind corn process. Separation of fiber from DDGS would provide two valuable coproducts: 1) DDGS with reduced fiber, increased fat and increased protein contents and 2) fiber. The process, called Elusieve process, used two separation methods, sieving and elutriation, to separate the fiber. Material carried by air to the top of the elutriation column was called the “lighter fraction” and material that settled to the bottom of the column was called the “heavier fraction”. We evaluated the compositions of fractions produced from sieving and elutriation. Two commercial samples of DDGS were obtained from two dry grind corn plants. Sieving over four screens (869, 582, 447 and 234 µm openings) created five size categories. The two smallest size categories contained > 40% (w/w) of the original DDGS and had reduced fiber and increased protein and fat contents relative to the original DDGS. Elutriation of the remaining three size categories increased protein and fat contents and reduced fiber contents in the heavier fractions. Elutriation at air velocities between 1.59 and 5.24 m/s increased the protein content of the heavier fraction by 13 to 41% and increased the fat content of the heavier fraction by 4 to 127% compared to the bulk fractions of each size category. This process was effective in separating fiber from both DDGS samples evaluated. Elusieve process does not require changes in the existing dry grind process and can be implemented at the end of the dry grind process.