Antimicrobial in vitro activities of condensed tannin extracts on avian pathogenic Escherichia coli.

Condensed tannins (CTs), which extracted from yew leaves, tilia flower and black locust leaves, were examined for their antimicrobial in vitro activity against avian pathogenic Escherichia coli (APEC). Past research demonstrated that CTs which contain procyanidins and prodelphinidins that could inhibit the growth of a wide range of bacteria. However, there is no information on how these affect pathogenic bacteria from chickens such as APEC. The high concentration of extracts, 10, 5, 2·5 mg ml-1 , affected the growth curves of APEC, which gave different inhibition values for the three CT extracts. Furthermore, these CTs had significant effects (P ≤ 0·05) on APEC biofilm and motility depending on each CT concentration and composition. However, at low concentration (0·6 mg ml-1 ), the tilia flowers, a high molar percentage of procyanidins, enhanced bacterial cell attachment and improved the swimming motility of APEC. In contrast, yew, an equal molar percentage of procyanidins/prodelphinidins, and black locust, a high molar percentage of prodelphinidins, interrupted and blocked swarming and swimming motility. The data suggested that the antimicrobial activity of the CT extracts was elicited by a positive relationship between anti-biofilm formation and anti-motility capacities. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that condensed tannins (CTs), which were a group of secondary metabolites of many plants and rich in prodelphinidins (PD), had greater antibacterial activity against avian pathogenic Escherichia coli (APEC) than CTs that were rich in procyanidins (PC). The mode of action of the CTs was to inhibit the swimming and swarming motility of APEC, and its ability to form biofilms. The significance of this finding is that the use of PD-rich CTs to control APEC should not encourage the development of antibiotic resistance by APEC because a different mechanism is used. If confirmed in vivo, this could provide the poultry industry with a valuable and novel means of controlling the antibiotic resistance.

Hydrolysable tannin-based diet rich in gallotannins has a minimal impact on pig performance but significantly reduces salivary and bulbourethral gland size.

Tannins have long been considered 'anti-nutritional' factors in monogastric nutrition, shown to reduce feed intake and palatability. However, recent studies revealed that compared with condensed tannins, hydrolysable tannins (HT) appear to have far less impact on growth performance, but may be inhibitory to the total activity of caecal bacteria. This in turn could reduce microbial synthesis of skatole and indole in the hindgut of entire male pigs (EM). Thus, the objective of this study was to determine the impact of a group of dietary HT on growth performance, carcass traits and boar taint compounds of group housed EM. For the study, 36 Swiss Large White boars were assigned within litter to three treatment groups. Boars were offered ad libitum one of three finisher diets supplemented with 0 (C), 15 (T15) or 30 g/kg (T30) of HT from day 105 to 165 of age. Growth performance, carcass characteristics, boar taint compounds in the adipose tissue and cytochrome P450 (CYP) isoenzymes CYP2E1, CYP1A2 and CYP2A19 gene expression in the liver was assessed. Compared with C, feed efficiency but not daily gain and daily feed intake was lower (P<0.05) in T15 and T30 boars. Except for the percentage carcass weight loss during cooling, which tended (P<0.10) to be greater in T30 than C and T15, carcass characteristics were not affected by the diets. In line with the numerically lower androstenone level, bulbourethral and salivary glands of T30 boars were lighter (P<0.05) than of T15 with intermediate values for C. Indole level was lower (P<0.05) in the adipose tissue of T30 than C pigs with intermediate levels in T15. Skatole levels tended (P<0.10) to be lower in T30 and C than T15 pigs. Hepatic gene expression of CYP isoenzymes did not differ between-treatment groups, but was negatively correlated (P<0.05) with androstenone (CYP2E1 and CYP1A2), skatole (CYP2E1, CYP2A) and indole (CYP2A) level. In line with the numerically highest androstenone and skatole concentrations, boar taint odour but not flavour was detected by the panellists in loins from T15 compared with loins from C and T30 boars. These results provide evidence that HT affected metabolism of indolic compounds and androstenone and that they affected the development of accessory sex glands. However, the effects were too small to be detected by sensory evaluation.

Interactions Between Nutrition and Infections With Haemonchus contortus and Related Gastrointestinal Nematodes in Small Ruminants.

Interactions between host nutrition and feeding behaviour are central to understanding the pathophysiological consequences of infections of the digestive tract with parasitic nematodes. The manipulation of host nutrition provides useful options to control gastrointestinal nematodes as a component of an integrated strategy. Focussed mainly on the Haemonchus contortus infection model in small ruminants, this chapter (1) illustrates the relationship between quantitative (macro- and micro-nutrients) and qualitative (plant secondary metabolites) aspects of host nutrition and nematode infection, and (2) shows how basic studies aimed at addressing some generic questions can help to provide solutions, despite the considerable diversity of epidemiological situations and breeding systems.

Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum.

Proanthocyanidins (PA) from shea (Vitellaria paradoxa) meal were investigated by thiolytic degradation with benzyl mercaptan and the reaction products were analysed by high performance liquid chromatography-mass spectrometry. These PA were galloylated (≈40%), contained only B-type linkages and had a high proportion of prodelphinidins (>70%). The mean degree of polymerisation was 8 (i.e. average molecular size was 2384Da) and epigallocatechin gallate (EGCg) was the major flavan-3-ol subunit in PA. Shea meal also proved to be a potentially valuable source for extracting free flavan-3-ol-O-gallates, especially EGCg (575mg/kg meal), which is known for its health and anti-parasitic benefits. Proanthocyanidins were isolated and tested for bioactivity against Ascaris suum, which is an important parasite of pigs. Migration and motility tests revealed that these PA have potent activity against this parasitic nematode.

Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methane production and fermentation characteristics.

Our study investigated the effects of condensed tannins (CT) on rumen in vitro methane (CH4 ) production and fermentation characteristics by incubating lucerne in buffered rumen fluid in combination with different CT extracts at 0 (control), 40, 80 and 120 g CT/kg of substrate DM. Condensed tannins were extracted from four sainfoin accessions: Rees 'A', CPI63763, Cotswold Common and CPI63767. Gas production (GP) was measured using a fully automated GP apparatus with CH4 measured at distinct time points. Condensed tannins differed substantially in terms of polymer size and varied from 13 (Rees 'A') to 73 (CPI63767) mean degree of polymerization, but had relatively similar characteristics in terms of CT content, procyanidin: prodelphinidin (PC: PD) and cis:trans ratios. Compared to control, addition of CT from CPI63767 and CPI63763 at 80 and 120 g CT/kg of substrate DM reduced CH4 by 43% and 65%, and by 23% and 57%, respectively, after 24-h incubation. Similarly, CT from Rees 'A' and Cotswold Common reduced CH4 by 26% and 46%, and by 28% and 46% respectively. Addition of increasing level of CT linearly reduced the maximum rates of GP and CH4 production, and the estimated in vitro organic matter digestibility. There was a negative linear and quadratic (p < 0.01) relation between CT concentration and total volatile fatty acid (VFA) production. Inclusion of 80 and 120 g CT/kg of substrate DM reduced (p < 0.001) branched-chain VFA production and acetate: propionate ratio and was lowest for CPI63767. A decrease in proteolytic activity as indirectly shown by a change in VFA composition favouring a shift towards propionate and reduction in branched-chain VFA production varied with type of CT and was highest for CPI63767. In conclusion, these results suggest that tannin polymer size is an important factor affecting in vitro CH4 production which may be linked to the CT interaction with dietary substrate or microbial cells.

Heat treatment enhances the antimicrobial activity of (+)-Catechin when combined with copper sulphate.

UNLABELLED: The aim of the study was to compare the antimicrobial activities of freshly made, heat-treated (HT) and 14 day stored (+)-Catechin solutions with (+)-catechin flavanol isomers in the presence of copper sulphate. (+)-Catechin activity was investigated when combined with different ratios of Cu(2+) ; 100°C heat treatment; autoclaving; and 14 day storage against Staphylococcus aureus. Cu(2+) -(+)-Catechin complexation, isomer structure-activity relationships, and H2 O2 generation were also investigated. Freshly made, HT, and 14 day stored flavanols showed no activity. While combined Cu(2+) -autoclaved (+)-Catechin and -HT(+)-Catechin activities were similar, HT(+)-Catechin was more active than either freshly made (+)-catechin (generating more H2 O2 ) or (-)-Epicatechin (though it generated less H2 O2 ) or 14 day-(+)-Catechin (which had similar activity to Cu(2+) controls-although it generated more H2 O2 ). When combined with Cu(2+) , in terms of rates of activity, HT(+)-Catechin was lower than (-)-Epigallocatechin gallate and greater than freshly made (+)-Catechin. Freshly made and HT(+)-Catechin formed acidic complexes with Cu(2+) as indicated by pH and UV-vis measurements although pH changes did not account for antimicrobial activity. Freshly made and HT(+)-Catechin both formed Cu(2+) complexes. The HT(+)-Catechin complex generated more H2 O2 which could explain its higher antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural products attract considerable attention in the search for novel antimicrobials, prebiotics and antioxidants. Enhanced biological activity of natural products has been demonstrated with chemical and heat treatment. This article extends the few publications on heat treatments of plant products and combinations with adjuncts, to raise antimicrobial activity against pathogens such as Staphylococcus aureus. We demonstrated that heat treatment could increase the activity of (+)-Catechin, a weak antimicrobial flavanol found commonly in plants in the presence of copper sulphate. Heat treatment of readily available resources merits consideration in the development of more potent substances for use in clinical settings and agriculture.

Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock.

Parasitic infections with gastrointestinal nematodes (GINs) still represent a worldwide major pathological threat associated with the outdoor production of various livestock species. Because of the widespread resistance to synthetic chemical anthelmintics, there is a strong impetus to explore novel approaches for a more integrated management of these infections. The use of nutraceuticals in the control of GINs is one of the alternatives which has been widely studied for 20 years. The objectives of this review are: (i) to define and illustrate the concept of 'nutraceutical' in the context of veterinary parasitology based on data obtained on the most studied models to control GINs in small ruminants, the tannin-containing legumes (Fabaceae); (ii) to illustrate how the 'nutraceutical concept' could be expanded to other plants, other livestock production systems and other GI parasitic diseases, and (iii) to explain how this concept is opening up new research fields for better understanding the interactions between the host, the digestive parasites and the environment.

Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats.

High prevalence of anthelmintic-resistant gastrointestinal nematodes (GIN) in goats has increased pressure to find effective, alternative non-synthetic control methods, one of which is adding forage of the high condensed tannin (CT) legume sericea lespedeza (SL; Lespedeza cuneata) to the animal's diet. Previous work has demonstrated good efficacy of dried SL (hay, pellets) against small ruminant GIN, but information is lacking on consumption of fresh SL, particularly during the late summer-autumn period in the southern USA when perennial warm-season grass pastures are often low in quality. A study was designed to determine the effects of autumn (September-November) consumption of fresh SL forage, grass pasture (predominantly bermudagrass, BG; Cynodon dactylon), or a combination of SL+BG forage by young goats [intact male Spanish kids, 9 months old (20.7 ± 1.1 kg), n = 10/treatment group] on their GIN infection status. Three forage paddocks (0.40 ha) were set up at the Fort Valley State University Agricultural Research Station (Fort Valley, GA) for an 8-week trial. The goats in each paddock were supplemented with a commercial feed pellet at 0.45 kg/head/d for the first 4 weeks of the trial, and 0.27 kg/head/d for the final 4 weeks. Forage samples taken at the start of the trial were analyzed for crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) content, and a separate set of SL samples was analyzed for CT in leaves, stems, and whole plant using the benzyl mercaptan thiolysis method. Animal weights were taken at the start and end of the trial, and fecal and blood samples were collected weekly for determination of fecal egg counts (FEC) and packed cell volume (PCV), respectively. Adult GIN was recovered from the abomasum and small intestines of all goats at the end of the experiment for counting and speciation. The CP levels were highest for SL forage, intermediate for SL+BG, and lowest for BG forage samples, while NDF and ADF values were the opposite, with highest levels in BG and lowest in SL forage samples. Sericea lespedeza leaves had more CT than stems (16.0 g vs. 3.3g/100g dry weight), a slightly higher percentage of PDs (98% vs. 94%, respectively) and polymers of larger mean degrees of polymerization (42 vs. 18, respectively). There were no differences in average daily gain or blood PCV between the treatment groups, but SL goats had lower FEC (P < 0.05) than the BG or SL+BG forage goats throughout most of the trial. The SL+BG goats had lower FEC than the BG forage animals by the end of the trial (week 8, P < 0.05). The SL goats had lower numbers (P < 0.05) of male Haemonchus contortus and tended to have fewer female (P < 0.10) and total (P < 0.07) H. contortus compared with the BG goats. The predominant GIN in all the goats was Trichostrongylus colubriformis (73% of total GIN). As a low-input forage with activity against pathogenic GIN (H. contortus), SL has a potential to reduce producers' dependence upon synthetic anthelmintics and also to fill the autumn 'window' in good-quality fresh forages for goat grazing in the southern USA.

Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats.

Infection with Eimeria spp. (coccidia) can be devastating in goats, particularly for young, recently-weaned kids, resulting in diarrhea, dehydration, and even death. Feeding dried sericea lespedeza [SL; Lespedeza cuneata (Dum.-Cours.) G. Don.] to young goats has been reported to reduce the effects of internal parasites, including gastrointestinal nematodes (GIN) but there have been no reports of the effects of feeding this forage on Eimeria spp. in goats. Two confinement feeding experiments were completed on recently-weaned intact bucks (24 Kiko-cross, Exp. 1; 20 Spanish, Exp. 2) to determine effects of SL pellets on an established infection of GIN and coccidia. The bucks were assigned to 1 of 2 (Exp. 1) or 3 (Exp. 2) treatment groups based upon the number of Eimeria spp. oocysts per gram (OPG) of feces. In Exp. 1, the kids were fed 1 of 2 pelleted rations ad libitum; 90% SL leaf meal+10% of a liquid molasses/lignin binder mix and a commercial pellet with 12% crude protein (CP) and 24% acid detergent fiber (n=12/treatment group, 2 animals/pen). For Exp. 2, treatment groups were fed (1) 90% SL leaf meal pellets from leaves stored 3 years (n=7), (2) 90% SL pellets from leaf meal stored less than 6 months, (n=7), and the commercial pellets (n=6) ad libitum. For both trials, fecal and blood samples were taken from individual animals every 7 days for 28 days to determine OPG and GIN eggs per gram (EPG) and packed cell volume (PCV), respectively. In Exp. 2, feces were scored for consistency (1=solid pellets, 5=slurry) as an indicator of coccidiosis. In Exp. 1, EPG (P<0.001) and OPG (P<0.01) were reduced by 78.7% and 96.9%, respectively, 7 days after initiation of feeding in goats on the SL pellet diet compared with animals fed the control pellets. The OPG and EPG remained lower in treatment than control animals until the end of the trial. In Exp. 2, goats fed new and old SL leaf meal pellets had 66.2% and 79.2% lower (P<0.05) EPG and 92.2% and 91.2% lower (P<0.05) OPG, respectively, than control animals within 7 days, and these differences were maintained or increased throughout the trial. After 4 weeks of pellet feeding in Exp. 2, fecal scores were lower (P<0.01) in both SL-fed groups compared with control animals, indicating fewer signs of coccidiosis. There was no effect of diet on PCV values throughout either experiment. Dried, pelleted SL has excellent potential as a natural anti-coccidial feed for weaned goats.

Potential of legume-based grassland-livestock systems in Europe: a review.

European grassland-based livestock production systems face the challenge of producing more meat and milk to meet increasing world demands and to achieve this using fewer resources. Legumes offer great potential for achieving these objectives. They have numerous features that can act together at different stages in the soil-plant-animal-atmosphere system, and these are most effective in mixed swards with a legume proportion of 30-50%. The resulting benefits include reduced dependence on fossil energy and industrial N-fertilizer, lower quantities of harmful emissions to the environment (greenhouse gases and nitrate), lower production costs, higher productivity and increased protein self-sufficiency. Some legume species offer opportunities for improving animal health with less medication, due to the presence of bioactive secondary metabolites. In addition, legumes may offer an adaptation option to rising atmospheric CO2 concentrations and climate change. Legumes generate these benefits at the level of the managed land-area unit and also at the level of the final product unit. However, legumes suffer from some limitations, and suggestions are made for future research to exploit more fully the opportunities that legumes can offer. In conclusion, the development of legume-based grassland-livestock systems undoubtedly constitutes one of the pillars for more sustainable and competitive ruminant production systems, and it can be expected that forage legumes will become more important in the future.

Antioxidant effects of ryegrass phenolics in lamb liver and plasma.

Sixteen lambs were divided into two groups and fed two different diets. Eight lambs were stall-fed with a concentrate-based diet (C), and the remaining eight lambs were allowed to graze on Lolium perenne (G). The antioxidant status was measured in the liver and plasma samples before and after solid-phase extraction (SPE) to probe the antioxidant effects that grass phenolic compounds may have conferred onto the animal tissues. The liver and plasma samples from grass-fed lambs displayed a greater antioxidant capacity than the tissues from C lamb group, but only if samples had not been passed through SPE cartridges. Finally, the feed and animal tissues, which had been purified by SPE, were analysed by liquid chromatography combined with mass spectrometry (LC���MS) to identify phenolic compounds present in L. perenne and to evaluate the results from the antioxidant assays. It would appear that the improvement of the antioxidant capacity of lamb liver and plasma from lambs fed ryegrass was not related to the direct transfer of phenolic compounds from grass to the animal tissues.

In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes.

Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40 µg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10 µg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin.

Investigation of sainfoin (Onobrychis viciifolia) cultivar differences on nitrogen balance and fecal egg count in artificially infected lambs.

Research in ruminant nutrition and helminth control with forages, which contain condensed tannins (CT), suggests that varying responses may depend not only on CT concentration but also on CT composition. An experiment was designed to test this by feeding 2 dried sainfoin cultivars (Visnovsky and Perly), which differed in CT properties, to lambs that were artificially infected with the abomasal blood-sucking nematode Haemonchus contortus. Twenty-four infected lambs received 1 of these 2 cultivars; the feeds were either untreated or treated with the CT-binding polyethylene glycol over 4 wk (n = 6). The 2 cultivars were also fed to 2 × 6 uninfected lambs. Nutrient digestibility, N balance, ADG, plasma urea, together with indicators of infection [fecal egg count (FEC), abomasal worm count, per capita female fecundity, erythrocytic indices, and serum protein], were determined. The specific effects of sainfoin cultivar, CT, and infection were evaluated by contrast analysis. Digestibility of both NDF and ADF were less (P < 0.001) with Perly compared with Visnovsky. The apparent nutrient digestibility was reduced (P < 0.001) by CT. However, no clear cultivar effects were evident on N excretion and retention. Condensed tannins reduced (P = 0.05) body N retention and shifted (P < 0.001) N excretion from urine to feces. Unlike cultivar and CT, infection decreased (P = 0.002) ADG. Plasma urea concentration was decreased (P = 0.007) in Perly- compared with Visnovsky-fed lambs and was decreased (P < 0.001) by CT. Plasma concentrations of essential and semiessential AA were increased (P < 0.001) by CT. The groups of infected lambs did not clearly differ in abomasal worm counts and erythrocytic indicators. In the last 2 to 3 wk of the experiment, FEC was decreased (P ≤ 0.01) when feeding CT. The lack of substantial cultivar effects suggests that the differences in CT properties may have been too small to result in nutritional and anthelmintic effects. The present results indicate that sainfoin CT had a mitigating effect on FEC and, consequently, pasture infectivity. However, the reduction was too small to expect any significant benefits in an Haemonchus-dominated system. Therefore, the use of sainfoin for controlling H. contortus should only be one component within an integrated worm control system.

Simple and surprisingly effective one-step extraction-cleanup by soxflo for DDT and its metabolites from environmental samples.

A pilot study found that DDT breakdown at the GC inlet was extensive in extracts from some--but not all--samples with high organic carbon contents. However, DDT losses could be prevented with a one-step extraction-cleanup in the Soxflo instrument with dichloromethane and charcoal. This dry-column procedure took 1 h at room temperature. It was tested on spiked soil and peat samples and validated with certified soil and sediment reference materials. Spike recoveries from freshly spiked samples ranged from 79 to 111% at 20-4000 microg/kg concentrations. Recoveries from the real-world CRMs were 99.7-100.2% of DDT, 89.7-90.4% of DDD and 89.6-107.9% of DDE. It was concluded that charcoal cleanups should be used routinely during surveys for environmental DDX pollution in order to mitigate against unpredictable matrix-enhanced breakdown in the GC.

Advantages of soxflo extractions for phytochemical analysis and bioassay screening. 1. Terpenoids.

The Soxflo technique was evaluated for the rapid extraction of plant materials (<90 min) at room temperature. Qualitatively similar chromatograms were obtained by gas chromatography and thin layer chromatography (TLC) with Soxflo (SoF) and Soxhlet (SoL) extracts. Sequential solvent extractions by SoF gave slightly higher yields (132%) of five major sesquiterpenoids. TLC revealed that SoF extractions at room temperature were more selective as extracts contained compounds with a narrower range of R(f)() values. This means that the SoF technique offers the potential for one-step extractions and partial fractionation. This study also showed that there were large differences in the volatile composition of dried and fresh Piper fruits: dried fruits had predominantly sesquiterpenoids while fresh fruits had considerable quantities of both mono- and sesquiterpenoids. This is the first report of alpha-guaiene and alpha- and beta-selinene in Piper guineense fruits. It is suggested that the SoF technique can be useful for the screening of large numbers of plants for phytochemicals or for the preparation of plant extracts for subsequent bioassay studies.

Investigations into the biochemical basis for nematode resistance in roots of three musa cultivars in response to Radopholus similis infection.

The Musa cultivars, Dwarf Cavendish, Yangambi Km5 and Kunnan, exhibit considerable differences in resistance to Radopholus similis. Infection resulted in significant increases in condensed tannins and flavan-3,4-diols in roots (P < 0.001). The highly resistant cultivar Kunnan had the highest levels of condensed tannins before and after infection. The preinfection levels were similar to the postinfection levels of the two other cultivars. Tannins had mostly procyanidin character, but Kunnan also contained propelargonidins; these compounds may be involved in the resistance mechanism. It is suggested that the butanol/HCl assay be used as a rapid test in screening for resistance to R. similis.

Polyphenols, condensed tannins, and other natural products in Onobrychis viciifolia (Sainfoin).

An acetone/water extract of the fodder legume Onobrychis viciifolia afforded arbutin, kaempferol, quercetin, rutin, afzelin, the branched quercetin-3-(2(G)-rhamnosylrutinoside), the amino acid L-tryptophan, the inositol (+)-pinitol, and relatively high concentrations of sucrose (ca. 35% of extractable material). Acid-catalyzed cleavage of the condensed tannins with phloroglucinol afforded catechin, epicatechin and gallocatechin as the terminal and extender units, but epigallocatechin was only present in extender units. The condensed tannins in O. viciifolia presumably consist of hetero- and homopolymers containing both procyanidin and prodelphinidin units. Comparison of data from the present study and the literature suggests that sainfoin tannins have a highly variable composition with cis:trans ratios ranging from 47:53 to 90:10 and delphinidin:cyanidin ratios from 36:64 to 93:7. The composition of terminal and extender units in sainfoin tannins seems to be cultivar specific.

Evaluation of the novel Soxflo technique for rapid extraction of crude fat in foods and animal feeds.

The new Soxflo instrument was evaluated for the determination of crude fat in foods and animal feeds. Samples are packed into small columns and extracted with petroleum ether at room temperature. The Soxflo yielded accurate data from foods, ranging from 0.4 to 73.2% crude fat, compared with Soxhlet extractions and Certified Reference Materials, for which recoveries averaged 99.7 and 100.7%, respectively. Relative standard deviations (1.81%) were approximately half those of Soxhlet extractions (3.68%). Regression analysis of the data suggested that there was no proportional bias. A small but acceptable constant bias was measured. Soxflo extractions are easy to perform and take approximately 1 h to complete. The main difference between the Soxflo and Soxhlet techniques lies in the extraction procedure. Estimated savings during extractions are in time (85% reduction), energy (95%), cooling water (100%), and solvents (50%). Soxflo extractions are, therefore, more environmentally friendly than Soxhlet extractions.

The activity of porcine pancreatic phospholipase A2 in 20% alcohol/aqueous solvent, by experiment and electrostatics calculations.

The activity of porcine pancreatic phospholipase A2 (pla2), measured at pH 8, is reduced when methanol or ethanol is added to the aqueous solution. Finite difference electrostatics calculations were used to study the effect of modelling mixed solvents on the pKas of histidine 48 and the amino-terminal group, both of which influence the pH-dependence of catalysis. Calculations and experiment indicate that these pKa values cannot account for the activity reduction. Charge separation in the transition state is destabilized in 20% alcohol solvent relative to 100% aqueous solvent. The calculated values, which are combinations of stabilizing and destabilizing contributions, are in qualitative agreement with experiment. Saturating dielectric theory is used to model solvent water ordering in a high electric field, and water dielectric structure is assumed to dominate at the 20% alcohol level. The observed agreement demonstrates the utility of transition state stabilization theory and continuum solvent modelling. It is further suggested that electrostatic effects on kcat contribute to the pH-dependence of activity around pH 7, and to previously reported activity changes for charge mutants.

Modification of the head-group selectivity of porcine pancreatic phospholipase A2 by protein engineering.

On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A2 (pla2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla2. To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis. The E46L mutant, expressed in Escherichia coli, was purified and characterized. The mutation did not affect the activity toward the mixed micellar substrate, but the activity of E46L toward DiC12-P, which has two negative charges on the head group, was three times higher than that of DiC12-PC, which carries no net charge in the head group. The native pla2 was inhibited by the product(s) released from DiC12-P but not the mutant enzyme. Kinetic analysis revealed that the E46L mutant and the native pla2 had comparable affinities (Km) toward monomeric and micellar phospholipids of zwitterionic type while the activity (kcat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla2. When micellar DiC12-P was used as a substrate, the Kmapp value for E46L was four times lower and the kcatapp/kmapp was 5-fold higher than those of native pla2. However, the kinetic parameters of mutant and native pla2s remained unchanged for monomeric HEPG, with one negative charge in the head group. Thus, we have modified the head-group selectivity of porcine pancreatic pla2 by protein engineering.