Fractionation of foam-mat dried rice bean hydrolysates using membrane filtration and solid phase extraction: Peptide- and phenolic-based fractions with bioactive potential.

Flavourzyme was used to hydrolyze the germinated rice bean, and the hydrolysates were separated using membrane ultrafiltration with a molecular weight (MW) cut-off of 3 kDa. The ultrafiltration permeate fraction (UFP), non-fractionated hydrolysate (RH), and ultrafiltration retentate fraction (UFR) were foam-mat dried at two temperatures, 60 and 70 °C. The content of each phenolic composition in dried RH samples decreased with increasing drying temperature particularly gallic acid, p-coumaric acid, vanillin, rutin, and, quercetin dropped by 27, 24, 21, 35 and 33%, however the kind of phenolic compositions identified in dried samples was unaffected by drying temperature. Dried UFR and dried UFP had different chromatograms. When the dried UFP and dried UFR chromatograms were examined, it was discovered that the intensity of the peaks in the dried UFR chromatogram was much lower. The majority of phenolics can pass through ultrafiltration membranes with a molecular weight cut-off of 3 kDa, according to this finding. Individual phenolic compound levels in dried UFP samples were similar to RH, implying that the majority of phenolic components in dried rice bean protein hydrolysate were smaller than 3 kDa. With increasing drying temperature, gallic acid, p-coumaric acid, catechol, epicatechin and naringenin levels in dried UFP samples were decreased. The antioxidant capacity of dried rice bean hydrolysate was discovered to be due to phenolic components (gallic acid, epicatechin, catechol, ferulic acid, and rutin), which were found to be more prevalent than peptide fractions. As a result, rice bean hydrolysates could bring novel health advantages, which could lead to the development of nutraceuticals and food products.

Starch Digestion Enhances Bioaccessibility of Anti-Inflammatory Polyphenols from Borlotti Beans (Phaseolus vulgaris).

The consumption of beans has been associated with chronic disease prevention which may be attributed to the polyphenols present in the seed coat and endosperm. However, their bioaccessibility is likely to be limited by interactions with bean matrix components, including starch, protein and fibre. The aim of this project was to evaluate the effect of domestic processing and enzymatic digestion on the bioaccessibility of polyphenols from Borlotti beans (Phaseolus vulgaris) and to test their anti-inflammatory properties in a macrophage cell model. In vitro digestion of cooked beans released twenty times more polyphenols (40.4 ± 2.5 mg gallic acid equivalents (GAE)/g) than domestic processing (2.22 ± 0.1 mg GAE/g), with starch digestion contributing to the highest release (30.9 ± 0.75 mg GAE/g). Fluorescence microscopy visualization of isolated bean starch suggests that polyphenols are embedded within the granule structure. LC-MS analysis showed that cooked Borlotti bean contain flavonoids, flavones and hydroxycinnamic acids, and cooked bean extracts exerted moderate anti-inflammatory effects by decreasing mRNA levels of IL1ß and iNOS by 25% and 40%, respectively. In conclusion, the bioaccessibility of bean polyphenols is strongly enhanced by starch digestion. These polyphenols may contribute to the health benefits associated with bean consumption.

Effect of acid pretreatment and the germination period on the composition and antioxidant activity of rice bean (Vigna umbellata).

This research evaluated effect of germination period and acid pretreatment on chemical composition and antioxidant activity of rice bean sprouts. Moisture, total phenolics, reducing sugar and B vitamins (thiamine, riboflavin, and niacin) content of steamed sprouts increased with increasing germination time (p⩽0.05). Pretreatment with 1% (w/v) citric acid for 6h significantly increased the total phenolic content. The 18-h-germinated rice beans showed the highest crude protein content, as determined using the Kjeldahl method. During germination, acid pretreatment led to a significant decrease in the intensity of the 76-kDa band. Germination caused a significant increase in radical scavenging activity and ferric reducing antioxidant power, especially in sprouts from citric acid-treated seeds. The antioxidant activities of the ethanolic extracts from both pretreated beans and the control were 1.3-1.6 times higher than those obtained from the water extracts. Major phenolics found in both 0-h and 18-h-germinated rice beans were catechin and rutin.

Transport of trans-tiliroside (kaempferol-3-ß-D-(6"-p-coumaroyl-glucopyranoside) and related flavonoids across Caco-2 cells, as a model of absorption and metabolism in the small intestine.

1. Absorption and metabolism of tiliroside (kaempferol 3-ß-D-(6"-p-coumaroyl)-glucopyranoside) and its related compounds kaempferol, kaempferol-3-glucoside and p-coumaric acid were investigated in the small intestinal Caco-2 cell model. Apparent permeation (Papp) was determined as 0.62 × 10(-6) cm/s, 3.1 × 10(-6) cm/s, 0 and 22.8 × 10(-6) cm/s, respectively. 2. Mechanistic study showed that the transportation of tiliroside, kaempferol-3-glucoside and p-coumaric acid in Caco-2 model were transporter(s) involved, while transportation of kaempferol was solely by passive diffusion mechanism. 3. Efflux transporters, multi-drug-resistance-associated protein-2 (MRP2), were shown to play a role in limiting the uptake of tiliroside. Inhibitors of MRP2, (MK571 and rifampicin) and co-incubation with kaempferol (10 µM), increased transfer from the apical to the basolateral side by three to five fold. 4. Metabolites of kaempferol-3-glucoside and p-coumaric acid were not detected in the current Caco-2 model, while tiliroside was metabolised to a limited extent, with two tiliroside mono-glucuronides identified; and kaempferol was metabolised to a higher extent, with three mono-glucuronides and two mono-sulfates identified. 5. In conclusion, tiliroside was metabolised and transported across Caco-2 cell membrane to a limited extent. Transportation could be increased by applying MRP2 inhibitors or co-incubation with kaempferol. It is proposed that tiliroside can be absorbed by human; future pharmacokinetics studies are warranted in order to determine the usefulness of tiliroside as a bioactive agent.

Determination of amygdalin in apple seeds, fresh apples and processed apple juices.

Cyanogenic glycosides are natural plant toxicants. Action by endogenous plant enzymes can release hydrogen cyanide causing potential toxicity issues for animals including humans. We have quantified amygdalin in seeds from different apple varieties, determined the effects of processing on the amygdalin content of apple juice and quantified amygdalin in commercially-available apple juices. Amygdalin contents of seeds from fifteen varieties of apples ranged from 1 mg g(-1) to 4 mg g(-1). The amygdalin content of commercially-available apple juice was low, ranging from 0.01 to 0.04 mg ml(-1) for pressed apple juice and 0.001-0.007 mg ml(-1) for long-life apple juice. Processing led to juice with low amygdalin content, ranging from 0.01 mg ml(-1) to 0.08 mg ml(-1). The results presented show that the amygdalin contents of commercially-available apple juices are unlikely to present health problems to consumers.

Comparative chemical and biochemical analysis of extracts of Hibiscus sabdariffa.

Hibiscus sabdariffa extracts have attracted attention because of potentially useful bioactivity. However, there have been no systematic studies of extraction efficiencies of H. sabdariffa. The nature of extracts used in different studies has varied considerably, making comparisons difficult. Therefore, a systematic study of extracts of H. sabdariffa made with different solvents was carried out using water, methanol, ethyl acetate and hexane in the presence/absence of formic acid, using different extraction times and temperatures. The extracts were analysed for total polyphenol content, antioxidant capacity using DPPH, FRAP and TEAC assays, and specific anthocyanins were determined using HPLC and LC-MS. The results showed the highest antioxidant capacities were obtained by extracting using water, with or without formic acid, for 10 min at 100°C. These extracts provided the highest concentrations of cyanidin 3-sambubioside and delphinidin 3-sambubioside. It will be important to use extraction conditions giving optimal extraction efficiencies for subsequent bioactivity experiments.

Development and application of an enzyme-linked immunosorbent assay (ELISA) for the quantification of amygdalin, a cyanogenic glycoside, in food.

Amygdalin is a member of the cyanogenic glycoside group of plant secondary metabolites capable of generating hydrogen cyanide under certain conditions. As a consequence, the cyanogenic glycosides have been associated with incidents of acute and subacute food poisoning. Specific antibodies were raised against an amygdalin-bovine serum albumin immunogen synthesized using a novel approach. The antibodies were used in a microtitration plate enzyme-linked immunosorbent assay (ELISA) for the quantification, for the first time, of amygdalin in commercially available foods. Correlation of results with high-performance liquid chromatography was very high (r = 0.983). The limit of detection of the immunoassay was 200 ± 0.05 pg mL(-1), and the 50% inhibitory concentration of amygdalin was 50 ± 0.02 ng mL(-1), making the ELISA particularly sensitive.

Amygdalin content of seeds, kernels and food products commercially-available in the UK.

Cyanogenic glycosides are a large group of secondary metabolites that are widely distributed in the plant kingdom, including many plants that are commonly consumed by humans. The diverse chemical nature of cyanogenic glycosides means that extraction and analysis of individual compounds can be difficult. In addition, degradation can be rapid under appropriate conditions. Amygdalin is one of the cyanogenic glycosides found, for example, in apples, apricots and almonds. We have developed and applied a high performance liquid chromatographic procedure for amygdalin quantification to investigate extraction efficiency and to determine levels in a range of commercially-available foods for the first time. Our results show that seed from Rosaceae species contained relatively high amounts (range 0.1-17.5 mg g(-1)) of amygdalin compared to seed from non-Rosaceae species (range 0.01-0.2 mg g(-1)). The amygdalin content of processed food products was very low.

Effects of pH on the ability of flavonoids to act as Pickering emulsion stabilizers.

The flavonoids tiliroside, rutin and naringin have been investigated as stabilizers of Pickering oil-in-water (O/W) emulsions. The mean droplet size of tetradecane emulsions was considerably smaller at higher pH, especially for rutin. The solubility of flavonoids in the aqueous phase was 4-6 times higher at pH 8 compared to pH 2 for tiliroside and rutin, although all absolute solubilities remained low (<1 mM). This agreed with a slight increase in surface activity of tiliroside and rutin at the O-W interface at pH 8 compared to pH 2. However, improved emulsion stabilization at higher pH is better explained by the significant increase in ζ-potential of the flavonoid particles to more negative values at pH 8, which will improve particle dispersion and increase the charge on the droplets stabilized by them. A buckwheat tea extract, rich in rutin, was also shown to be an effective stabilizer of sunflower O/W emulsions.

Flavanols and methylxanthines in commercially available dark chocolate: a study of the correlation with nonfat cocoa solids.

Intake of flavanols, a subgroup of dietary polyphenols present in many fruits and vegetables, may be associated with health benefits, particularly with reducing the risk of coronary diseases. Cocoa and chocolate products are rich in flavanol monomers, oligomers, and polymers (procyanidins). This study used normal phase HPLC to detect, identify, and quantify epicatechin, catechin, total monomers, procyanidin oligomers and polymers in 14 commercially available chocolate bars. In addition, methylxanthines (theobromine and caffeine) were also quantified. Nonfat cocoa solids (NFCS) were determined both gravimetrically and by calculation from theobromine contents. The flavanol levels of 12 commonly consumed brands of dark chocolate have been quantified and correlated with % theobromine and % NFCS. Epicatechin comprised the largest fraction of total chocolate flavonoids, with the remainder being catechin and procyanidins. Calculated NFCS did not reflect epicatechin (R(2) = 0.41) or total flavanol contents (R(2) = 0.49). Epicatechin (R(2) = 0.96) was a reliable marker of total flavanols, catechin (R(2) = 0.67) to a lesser extent. All dark chocolate tested contained higher levels of total flavanols (93.5-651.1 mg of epicatechin equiv/100 g of product) than a milk or a white "chocolate" (40.6 and 0.0 mg of epicatechin equiv/100 g, respectively). The amount and integrity of procyanidins often suffer in the manufacturing of chocolate, chiefly due to oxidation and alkalinization. In this study, the labeled cocoa content of the chocolate did not always reflect analyzed levels of flavonoids. Increasingly, high % NFCS is being used commercially to reflect chocolate quality. If the flavanol content of chocolate is accepted to be a key determinant of health benefits, then continued monitoring of flavanol levels in commercially available chocolate products may be essential for consumer assurance.

Particle-stabilizing effects of flavonoids at the oil-water interface.

It has been shown that some common food flavonoids can act as excellent stabilizers of oil-in-water emulsions through their adsorption as water-insoluble particles to the surface of the oil droplets, i.e., Pickering emulsions are formed. Flavonoids covering a wide range of octanol-water partition coefficients (P) were screened for emulsification behavior by low shear mixing of flavonoid+n-tetradecane in a vortex mixer. Most flavonoids with very high or very low P values were not good emulsifiers, although there were exceptions, such as tiliroside, which is very insoluble in water. When a high shear jet homogenizer was used with 20 vol% oil in the presence of 1 mM tiliroside, rutin, or naringin, much finer emulsions were produced: the average droplet sizes (d32) were 16, 6, and 5 µm, respectively. These results may be highly significant with respect to the delivery of such insoluble compounds to the gut, as well as their digestion and absorption.

Optical resonance-enhanced absorption-based near-field immunochip biosensor for allergen detection.

An optical immunochip biosensor has been developed as a rapid method for allergen detection in complex food matrixes, and its application evaluated for the detection of the egg white allergens, ovalbumin and ovomucoid. The optical near-field phenomenon underlying the basic principle of the sensor design is called resonance-enhanced absorption (REA), which utilizes gold nanoparticles (Au NPs) as signal transducers in a highly sensitive interferometric setup. Using this approach, a novel, simple, and rapid colorimetric solid-phase immunoassay on a planar chip substrate was realized in direct and sandwich assay formats, with a detection system that does not require any instrumentation for readout. Semiquantitative immunochemical responses are directly visible to the naked eye of the analyst. The biosensor shows concentration-dependent color development by capturing antibody-functionalized Au NPs on allergen-coated chips and has a detection limit of 1 ng/mL. To establish a rapid method, we took advantage of the physicochemical microenvironment of the Au NP-antibody bioconjugate to be bound directly over an interacting poly(styrene-methyl methacrylate) interlayer by an immobilized antigen. In the direct assay format, a coating time with allergen of only 5 min under "soft" nondenaturing conditions was sufficient for accurate reproducibility and sensitivity. In conclusion, the REA-based immunochip sensor is easy to fabricate, is reproducible and selective in its performance, has minimal technical requirements, and will enable high-throughput screening of affinity binding interactions in technological and medical applications.

IgE binding to proteins from sesame and assessment of allergenicity: implications for biotechnology?

Successful prediction of the potential allergenicity of a protein may be a key factor in the development of novel, genetically modified foods. The use of the decision tree approach for the prediction of allergenicity is discussed. The methods currently used for identifying allergenic proteins (including use of IgE from patient sera for recognition of proteins) are reviewed. Finally, a specific review of the literature concerning identification of allergens from sesame leads to the conclusion that in the absence of validated animal models, identification of allergenicity (and, consequently, prediction of allergenicity) may be problematic.

Xanthine oxidase activity in vitro: effects of food extracts and components.

There is significant interest in the direct antioxidant activities of dietary polyphenols, due to associations between consumption of polyphenol-rich foods, such as fruits and vegetables, and decreased incidence of oxidative-stress related disease. However, indirect antioxidant action, such as the inhibition of ROS-producing enzymes, may be equally relevant to health benefits through a general reduction in oxidative stress in vivo. To this end, the effects of food extracts and individual compounds on the in vitro activity of xanthine oxidase (XO) were assessed, many for the first time. Several compounds were shown to be potent inhibitors in vitro, including hesperetin and theaflavin-3,3'-digallate with IC50 values of 39 and 49 microM, respectively. Of the extracts, cranberry juice, purple grape juice, and black tea were the most potent, with IC50 values of 2.4, 3.5, and 5.8% of extracts, respectively. Some samples were shown to promote XO activity over the concentration ranges tested, including orange juice and pink grapefruit juice. Certain "inhibitors", such as purple grape juice and black tea, promoted XO activity at low concentration. The possible role of dietary inhibitors of XO in reducing oxidative stress in vivo is discussed.

Experimental determination of octanol-water partition coefficients of quercetin and related flavonoids.

Octanol-water partition coefficient (log P) values were determined for flavonoids from the flavone, flavonol, flavanone, and isoflavonoid subclasses. Each flavonoid was dissolved in an octanol-water system and allowed to equilibrate, and then both fractions were analyzed by high-performance liquid chromatography. log P was calculated as log[ratio of the concentration in the octanol phase to the concentration in the aqueous phase at pH 7.4]. The aglycons were more lipophilic than any conjugate. The conjugate moiety had a more significant effect on log P than the aglycon moiety. Quercetin was the least lipophilic aglycon (log P = 1.82 +/- 0.32) and, together with kaempferol (log P = 3.11 +/-0.54), gave the most variable results. The isoflavones genistein and daidzein and the isoflavone metabolite equol gave relatively high log P values (3.04 +/- 0.02, 2.51 +/- 0.06, and 3.20 +/- 0.13, respectively), while glycitein had an unexpectedly low value of 1.97 +/- 0.05. The conjugation characteristics and hydroxylation pattern were the most important determinants of log P in general, and log P was highly variable within the flavonoid subclass. The results are discussed in terms of further understanding of the in vivo fate of the flavonoids as important dietary bioactives.

Temperature-dependent binding of monoclonal antibodies to C hordein.

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.