Luís F. Guido

Isolation of phenolic compounds from hop extracts using polyvinylpolypyrrolidone: characterization by high-performance liquid chromatography-diode array detection-electrospray tandem mass spectrometry.

The aim of the present work was the development of a suitable methodology for the separation and determination of phenolic compounds in the hop plant. The developed methodology was based on the sample purification by adsorption of phenolic compounds from the matrix to polyvinylpolypyrrolidone (PVPP) and subsequent desorption of the adsorbed polyphenols with acetone/water (70:30, v/v). At last, the extract was analyzed by HPLC-DAD and HPLC-ESI-MS/MS. The first phase of this work consisted of the study of the adsorption behavior of several classes of phenolic compounds (e.g. phenolic acids, flavonols, and flavanols) by PVPP in model solutions. It has been observed that the process of adsorption of the different phenolic compounds to PVPP (at low concentrations) is differentiated, depending on the structure of the compound (number of OH groups, aromatic rings, and stereochemistry hindrance). For example, within the phenolic acids class (benzoic, p-hydroxybenzoic, protocatechuic and gallic acids) the PVPP adsorption increases with the number of OH groups of the phenolic compound. On the other hand, the derivatization of OH groups (methylation and glycosylation) resulted in a greatly diminished binding. The use of PVPP revealed to be very efficient for adsorption of several phenolic compounds such as catechin, epicatechin, xanthohumol and quercetin, since high adsorption and recovery values were obtained. The methodology was further applied for the extraction and isolation of phenolic compounds from hops. With this methodology, it was possible to obtain high adsorption values (>or=80%) and recovery yield values (>or=70%) for the most important phenolic compounds from hops such as xanthohumol, catechin, epicatechin, quercetin and kaempferol glycosides, and in addition it allows the identification of about 30 phenolic compounds by HPLC-DAD and HPLC-ESI-MS/MS.

Determination of E-2-nonenal by high-performance liquid chromatography with UV detection: Assay for the evaluation of beer ageing

The analysis of E-2-nonenal is of considerable interest for the brewery industry as this compound is claimed to be responsible for a paper/cardboard unpleasant flavour. Usually, the presence of E-2-nonenal can be noticed in aged beers at levels higher than 0.1 microg/l. In this work, an analytical method was developed to determine E-2-nonenal in beer involving steam distillation of beer followed by an extraction/concentration step using solid-phase extraction and determination of E-2-nonenal by HPLC with UV detection. Fastness and simplicity are the main advantages of the proposed method, when compared with other existing methodologies for the determination of E-2-nonenal in beer. Using the developed conditions, the interference of E-2-nonenal formed by degradation of its precursors during steam distillation is almost negligible. The presence of sulphur dioxide at legal levels does not interfere with the assay. The method was used in a comparative study of fresh and either naturally or forced aged beers. A much larger chromatographic peak was found near the peak of E-2-nonenal that correlates well with the peak of E-2-nonenal. Identification of the corresponding compound is currently under investigation, considering its future application on the evaluation of beer ageing.

Transthyretin Stabilization by Iododiflunisal Promotes Amyloid-β Peptide Clearance, Decreases its Deposition, and Ameliorates Cognitive Deficits in an Alzheimer's Disease Mouse Model

Alzheimers disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-β (Aβ) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aβ interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aβ interaction, iododiflunisal (IDIF), in Aβ deposition and other AD features, using AβPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aβ levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aβ levels were reduced in plasma suggesting TTR promoted Aβ clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.

Determination of Phenolic Content in Different Barley Varieties and Corresponding Malts by Liquid Chromatography-diode Array Detection-Electrospray Ionization Tandem Mass Spectrometry.

A simple and reliable method for the simultaneous determination of nine phenolic compounds in barley and malted barley was established, using liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS). The phenolic compounds can be easily detected with both systems, despite significant differences in sensitivity. Concentrations approximately 180-fold lower could be achieved by mass spectrometry analysis compared to diode array detection, especially for the flavan-3-ols (+)-catechin and (−)-epicatechin, which have poor absorptivity in the UV region. Malt samples were characterized by higher phenolic content comparing to corresponding barley varieties, revealing a significant increase of the levels of (+)-catechin and (−)-epicatechin during the malting process. Moreover, the industrial malting is responsible for modification on the phenolic profile from barley to malt, namely on the synthesis or release of sinapinic acid and epicatechin. Accordingly, the selection of the malting parameters, as well as the barley variety plays an important role when considering the quality and antioxidant stability of beer.

Sulfites in beer: reviewing regulation, analysis and role

Beer is an extremely complex mixture of more than 3,000 different compounds in an aqueous environment. Thus, it is perhaps not surprising that the maintenance of beer quality throughout its lifetime has been a considerable challenge for brewers. Whilst it is inevitable that chemical changes will occur in beer with the passage of time, it is the formation of flavor-active components which is of immediate concern to an overview of beer shelf life stability. Sulfur dioxide has long been recognized by brewers as the most important factor in delaying flavor staling, and prolonging the shelf life of beer. However, nowadays, sulfur dioxide and sulfites are considered allergens and concerns about the safety of their use as food additives have been on the increase. The present review is structured into three main parts. Firstly, the chemical properties of sulfur dioxide are presented, along with the toxic effects and maximum legal levels permitted according to U.S. and EU legislation. As the accurate determination of the free, bound and total sulfur dioxide in beer is essential to ensuring regulatory compliance, several methods have been developed for analyzing sulfur dioxide in beer. Thus, secondly, various types of methods are reported and compared with the officially recommended ones. Finally, the crucial role of sulfite in the control of flavor instability of beer is discussed in light of the current data. Two courses of action have been proposed, which are elucidated in detail relating firstly to the fact that sulfite inhibits beer oxidation during storage by acting as an antioxidant and, secondly, sulfite reacts with the carbonyl staling compounds in beer, and thereby masks stale flavors.

Further insights into the role of melanoidins on the antioxidant potential of barley malt

The role of Maillard reaction products isolated from barley malt by gel permeation chromatography and ultrafiltration on the antioxidant potential of pale, melano80 and black malts was evaluated. The roasting process is responsible for the polymerisation of early formed lower molecular weight compounds (<10 kDa) into high molecular weight melanoidins (>300 kDa). Melanoidins showed 3-fold higher capacity to scavenge radicals than the lower molecular weight colorants by the metmyoglobin assay. However, a significant decrease of the capacity of black malt and high molecular weight melanoidins to inhibit Fenton induced hydroxyl degradation of deoxyribose was observed. As the high molecular weight fraction, isolated from the black malt extract, exhibited 4-fold higher reducing power than the lower molecular weight fraction, our results support a pro-oxidant effect due to the catalytic formation of hydroxyl radicals in the presence of ferric ions.

Novel application of square-wave adsorptive-stripping voltammetry for the determination of xanthohumol in spent hops.

This paper reports the development of a novel electrochemical assay for xanthohumol (XN) by square-wave adsorptive-stripping voltammetry (SWAdSV) with a hanging mercury drop electrode. The method showed good repeatability (CV < 2%) and linearity (between 10 and 250 μg L(-1)), as well as suitable limits of detection (2.6 μg L(-1)) and quantification (8.8 μg L(-1)). The method was applied for the quantification of this compound in spent hops, and the results obtained were compared with the HPLC-UV method. XN contents determined by the SWAdSV method were 16 ± 1 and 100 ± 4 μg L(-1) for aqueous and methanolic extracts, respectively. The developed new methodology considerably reduces the analysis time, approximately from 25 min (HPLC-UV method) to 7 min, enabling a high sample throughput. In addition, the detection and quantification limits were approximately 5-fold lower than those obtained with the chromatographic method.

Techniques for Extraction of Brewer’s Spent Grain Polyphenols: a Review

Million tons of brewer’s spent grain (BSG) are annually produced worldwide as brewing industry by-products. BSG represents a valuable source of phenolic compounds, which have attracted much attention due to their diverse health benefits. Relevant strategies have been developed for their efficient extraction, in order to commercially exploit these resources. This review focuses on the current extraction methods used to obtain phenolic compounds from BSG, ranging from more traditional to advanced techniques. The commonly used methods are the conventional solid–liquid extractions, employing organic solvents, alkaline, and enzymatic reactions. However, the inherent difficulties in screening and obtaining these compounds have led to the development of advanced extraction techniques. Pressurized fluid extraction, supercritical extractions, and microwave-assisted and ultrasound-assisted extractions are some of the novel extraction techniques that have been recently explored. These techniques have been mostly applied for phenolic recovery from barley and malt, as well as other types of cereals. In this review, it is shown that these novel techniques may provide an innovative approach to extract phenolics from BSG or related products, following an in-depth discussion on the major strengths and weaknesses identified in each technique.

Overall Antioxidant Properties of Malt and How They Are Influenced by the Individual Constituents of Barley and the Malting Process

In the past several years researchers have focused on the study of the antioxidant properties of barley and barley malt as well as their influence on beer quality. Some malt constituents have been reported as potent antioxidants due to their radical-scavenging and reducing properties, with a positive effect on beer oxidative stability. However, barley and malt can suffer some serious modifications during malting and roasting, namely on the levels of phenolic compounds and the development of Maillard reaction products, which may have a great impact on the overall antioxidant properties of malt. Although some studies have reported an increase of the antioxidant capacity during malting, others have mentioned an opposite effect. Recently, researchers have shown that compounds developed in malt during heat treatment at high temperature and long periods of time, as result of the Maillard reaction, can also exhibit pro-oxidant properties involving the metal-catalyzed Fenton reaction due to its reductive properties. This paper reviews important information and recent data regarding the chemical changes malting and roasting undergo along with their influence on the different anti- and pro-oxidant properties described for barley and malt. The contribution of individual components to the overall antioxidant capacity of malt is also discussed.

Composition of pectic polysaccharides in a Portuguese apple (Malus domestica Borkh. cv Bravo de Esmolfe)

Malus domestica Borkh. cv Bravo de Esmolfe is a typical Portuguese apple cultivar classified as Protected Designation and Origin (PDO). It is a traditional product produced under strict conditions and labelled with a specific law protected designation. This cultivar presents quite good sweetness and flavor. The monosaccharide composition of the pectic polysaccharides from this traditional apple is herein reported for the first time. Based on the molar ratios obtained from the sugar composition, the presumable pectin structure could be inferred. The cell-wall polysaccharides present in the alcohol-insoluble residue (AIR) of unpeeled BE apple were sequentially fractionated. In addition, pectic material was also extracted by citric acid treatment prior to heat extraction at acidic pH. The water soluble pectin, imidazole soluble pectin and sodium carbonate soluble pectin account for 44, 16 and 40 % of the AIR, respectively. The pectic polysaccharides extracted in the presence of citric acid had lower galacturonic acid content and higher neutral sugars content. The homogalacturonan (HG) and less-substituted rhamnogalacturonan (RG) domains are extracted first. Pectin treated with citric acid has been shown to contain more substituted polymers, especially RG-I. In addition, the relatively higher Xylose/Galacturonic acid ratio found in the citric acid extract demonstrates that the xylogalacturonan (XG) domain presumably is present in the pectic material of the unpeeled BE apple.