FLUOROMETRIC DETERMINATION OF CYSTEINE IN BEER BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH PRECOLUMN DERIVATISATION

A high-performance liquid chromatographic procedure (HPLC) is described for the fluorometric determination of cysteine (CySH) in beer. Reduced CySH in beer is reacted with a fluorescent reagent, N-(9-acridinyl) maleimide (NAM), at pH 8·8 for 15 h to form a stable fluorescent derivative. The resulting reaction mixture is analysed by reversed-phase gradient elution HPLC. Under the optimised conditions for chromatography, ca 0·1 mg of CySH per litre of beer can be determined. Total CySH is determined by electrolytic reduction at a Hg pool electrode prior to the HPLC analysis. The levels of reduced and total CySH in 28 brands of commercial lager beers ranged from 0·2 to 0·9 mg/litre, and from 2·1 to 13·5 mg/litre, respectively.     

YEAST MUTANTS EXCRETING VITAMIN B1 AND THEIR USE IN THE PRODUCTION OF THIAMINE RICH BEERS*,†

In certain strains of Saccharomyces cerevisiae and Saccharomyces uvarum (carlsbergensis), high doses of ultraviolet radiation induced stable mutants excreting thiamine from their living cells during the ethanol production. In S. cerevisiae, the first mutagenesis step yielded mutants with the production of 0·3–0·5 mg extracellular thiamine. HCL/litre minimum medium without thiamine and the second one increased the production up to 2·0–2·1 mg thiamine. HCL/litre. In S. uvarum (carlsbergensis), mutants producing up to 0·76 mg thiamine. HCL/litre 10% Plato hopped wort were obtained in the first mutagenesis step combined with mitotic recombinations. The increase up to 0·92 mg/litre was achieved here by repeated selections. Both laboratory and pilot plant fermentations in 10 and 4% Plato hopped worts showed the suitability of selected mutants for the production of thiamine rich beers which fulfilled all quality requirements and contained 0·67–0·80 and 0·22–0·33 mg thiamine. HCI/litre, respectively.     

PHENOLIC ACIDS IN BEERS AND WORTS

The total contents of phenolic acids measured by high-performance liquid-chromatography were 5–8 mg/litre in beers brewed in Ireland whereas 16–40 mg/litre were present in four other beers. In all beers the predominant phenolic acids were vanillic, p-coumaric and ferulic acids. Free phenolic acids were extracted from Emma barley grains and malt in very small amounts (15–28 mg/kg) but larger quantities (191 mg/kg) were released on mashing the malt. Little change occurred in the contents of phenolic acids on processing a lager wort through to the finished beer. Treatment with excess Polyclar AT removed astringent flavour and phenolic acids from an experimental ale but this flavour loss could not be accounted for by the adsorption of phenolic acids. The flavour threshold for a nine-component phenolic acid mixture in lager was between 50 mg/litre and 100 mg/litre.     

DETERMINATION OF LOWER BOILING POINT VOLATILE COMPOUNDS IN BEER BY HEADSPACE GAS CHROMATOGRAPHY — COLLABORATIVE TRIAL

The Analysis Committee has collaboratively tested local routine headspace gas chromatographic methods for the determination of the lower boiling point volatile compounds in beer. The repeatability values (r₉₅) were dependent upon mean concentration (m) for acetaldehyde and alcohols but not for esters, whilst reproducibility values (R₉₅) were dependent upon concentration in all cases. The range of values of m and the estimates of r₉₅ and R₉₅ (mg/litre) for each compound were, respectively: acetaldehyde (5–8, 0.21m, 0.60m); propanol (9–23, 0.25m, 2.5+0.57m); isobutanol (5–22, 0.56+0.085m, 1.7+0.15m); methylbutanols (45–105, 0.14m, 0.22m); ethyl acetate (10–54, 3.1, 2.1+0.29m); isoamyl acetate (0.8–4.7, 0.36, 0.20+0.58m); and ethyl hexanoate (0.13–0.36, 0.073, 0.10+0.91m). No advantage was gained by diluting beer samples containing 9% V/V ethanol to 4% ethanol (used for the calibration mixtures) prior to analysis, but use of a standard method of sample preparation decreased most of the R₉₅ values. No recommendation is made in this interim report.     

Evaluation of different Saccharomyces cerevisiae strains for red winemaking. Influence on the anthocyanin,pyranoanthocyanin and non-anthocyanin phenolic content and colour characteristics of wines

The possible industrial use of three previously-selected Saccharomyces cerevisiae strains (1EV, 2EV and 7EV) has been studied in musts derived from Tempranillo and Cabernet Sauvignon. The anthocyanin, pyranoanthocyanin and non-anthocyanin phenolic content, and colour characteristics of the resulting wines have been compared to those of a commercial strain. Anthocyanins were the compounds most influenced by the yeast strain. Independently of the grape variety, wines derived from 2EV presented significantly higher anthocyanin concentrations than those derived from 1EV and 7EV, which presented similar contents. With the exception of hydroxycinnamic acids and derivatives, no particular influence of the yeast strain was observed on the remaining non-anthocyanin phenolic compounds (i.e, hydroxybenzoic acids and flavanols). Pyranoanthocyanins and metabolites resulting from the alcoholic fermentation such as tyrosol and tryptophol, seemed to be more influenced by the must composition and pH, and thus, by the grape variety, than by the yeast strain.     

THE ESTIMATION AND SIGNIFICANCE OF CATECHIN AND DIMERIC CATECHIN IN BEER

A gas-liquid chromatographic method for the estimation of monomeric and dimeric catechin in beer is described. This method has been used to estimate the concentrations of these compounds in a number of beers, which were found to contain 0·5 to 8·0 ppm of monomeric catechin and up to 22·0 ppm of dimeric catechin. Beers bottled with a high level of air in the headspace can have a long shelf-life providing the level of dimeric polyphenols is low. The shelf-lives of beers which contain high concentrations of dimers are very sensitive to the levels of air in the headspace. At the levels found, monomeric catechin has no significant effect on haze whether or not headspace air is present.     

Validation of a Method for Analysis of Aroma Compounds in Red Wine using Liquid–Liquid Extraction and GC–MS

An analytical method for determination of volatile composition of wines using sample preparation by liquid–liquid extraction and gas chromatography coupled to mass spectrometry for separation and detection has been developed and validated. Extraction of volatile compounds was performed in dichloromethane, and 1-octanol was added as an internal standard. Kékfrankos red wine produced in Villány wine region in Hungary was used as a model wine for testing and validation of the method. The developed method allowed satisfactory determination of 33 volatile compounds in the wines. Compounds analyzed include alcohols, esters, lactones, fatty acids, furans, and nitrogen compounds. The calibration curves of the four reference compounds used (2-phenyl ethanol, ethyl nonanoate, butyrolactone, and tyrosol) were linear in all cases with correlation coefficients (R ²) ranging from 0.9951 to 0.9992. The accuracy of the method was checked with a standard addition method (recovery 92.2–103 %), showing good repeatability and reproducibility (RSD?<?10 %).     

Inhibition of the decrease of volatile esters and terpenes during storage of a white wine and a model wine medium by caffeic acid and gallic acid

Caffeic acid and gallic acid were tested as inhibitors of the decrease of volatile acetate esters, ethyl esters and terpenes during storage of a white wine and a model wine medium. Wine and model wine samples were analysed by SPME along with GC–MS. At t = 0, no effect on the concentration of any volatile was observed as a result of adding each phenolic acid in any of wine or model wine samples. Many esters, such as isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, and linalool decreased during storage of Debina-white wine for up to 20 months. Caffeic acid and gallic acid at 60 mg/L inhibited the decrease of these volatiles. Isoamyl acetate, ethyl hexanoate and linalool decreased during storage of the model wine medium containing 0, 20 or 40 mg/L SO₂. Caffeic acid and gallic acid inhibited the decrease of three volatiles; SO₂ inhibited the decrease of isoamyl acetate and ethyl hexanoate. In some cases, SO₂ increased the inhibitory action of two phenolic acids. The inhibitory action of caffeic acid and gallic acid was dose dependent in the range 0–60 mg/L. Caffeic acid was active at 7.5 mg/L while gallic acid at 15 mg/L.Present results indicate that caffeic acid and gallic acid may be taken into account as potent inhibitors of the disappearance of aromatic volatile esters and terpenes in wines at concentrations similar to those existing in wines.     

Free radical scavenging capacity of selected red,rosé and white wines

The free radical scavenging capacity of selected red, rosé and white Spanish wines from different grape varieties was determined by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^·) method using a new methodology developed at our laboratory. The amount of sample necessary to decrease by 50% the initial DPPH^· concentration (EC₅₀), the time needed to reach the steady state at EC₅₀ concentration (T_EC50) and the antiradical efficiency (AE = 1/EC₅₀T_EC50) were determined in the wine samples. Some differences between rosé wines made with Garnacha and Tempranillo grape varieties were observed in the UV‐vis spectra and in the free radical scavenging parameters, those from Garnacha variety having the highest antioxidant activity. The antiradical efficiency of the wines followed a decreasing order: red wines (22.44 × 10⁻⁶) > rosé wines (4.90 × 10⁻⁶) > white wines (1.88 × 10⁻⁶). There was a correlation between antiradical efficiency and total polyphenol (TP): AE = −3.33135 + 0.0180535TP; the correlation coefficient was r = 0.951454. © 1999 Society of Chemical Industry     

Phenolics and related substances in alcohol-free beers

 The present study examines and compares the phenolic compositions of several commercially available alcohol-free and standard beers. In general, the values for the contents of the phenolic components in the alcohol-free beers are lower than the values for the standard beers; this is attributable to differences in the duration of fermentation and the yeast strains employed in brewing alcohol-free beers (e.g., the case of tyrosol) and to losses brought about by the dealcoholization processes employed (e.g., p-coumaric acid, caffeic acid, vanillic acid, etc.). The values for the other low-molecular-weight compounds considered, such as 5-(hydroxymethyl)furfural and tryptophol, are also lower in the alcohol-free than in the standard beers. Received: 16 July 1999     

葡萄酒及葡萄皮渣提取物抑制乙酰胆碱酯酶活性研究

建立乙酰胆碱酯酶抑制剂体外筛选模型,对不同的葡萄酒及葡萄皮渣提取物等19种样品进行了乙酰胆碱酯酶活性抑制作用的研究。结果显示白葡萄酒几乎无乙酰胆碱酯酶抑制活性,红葡萄酒对乙酰胆碱酯酶具有不同的抑制活性,抑制率最高为山葡萄酒,其抑制率为11.12%。葡萄皮渣的水、乙醇、乙酸乙酯提取物,具有抑制乙酰胆碱酯酶活性,其抑制率分别为3.91%、18.25%、20.76%,乙酸乙酯提取物的抑制效果最好。红葡萄酒和葡萄皮渣的乙酸乙酯提取物可作为辅助治疗阿尔茨海默病的先导化合物进行深入研究。     

A NEW COLOURIMETRIC ASSAY FOR FLAVANOIDS IN PILSNER BEERS

A new colourimetric assay (based upon the reaction of their A-rings with the chromogen p-dimethylaminocinna-maldehyde) has been developed for flavanoids in beer. The flavanoid content for 19 different Belgian Pilsner beers by this method varied between 4·8 and 39·5 mg/litre with a mean of 26·3 mg/litre. These results were compared with the analytical data for total polyphenolics (EBC, Singleton), anthocyanogens (Jerumanis), and flavanoids (vanillin procedure). Good correlations existed between the results by the new method and the analytical data for total polyphenolics (EBC) and anthocyanogens (Jerumanis). Analysis by the new assay requires only one reagent and it is clear from the analytical data that the method is highly reproducible and sensitive     

Low‐temperature wine‐making using yeast immobilized on pear pieces

A biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae AXAZ‐1 yeast cells on pear pieces and tested for grape must fermentation in both batch and continuous conditions. The immobilized yeast cells were stable and active even at low temperatures (<10 °C). Wine production under batch fermentation at 8 °C was completed within 15 days while at 3 °C it took 36 days. In continuous fermentation, the bioreactor was operated for 33 days, then stored for 12 days at 10 °C, and re‐run for another 15 days without any diminution of the ethanol productivity. Total acidity of the produced wines remained within the ranges usually observed in dry wines, while volatile acidity was found in rather increased levels. The concentrations of higher alcohols (1‐propanol, isobutyl alcohol and amyl alcohols) were relatively low, while ethyl acetate was detected at up to 118 mg l^?1, contributing to the fruity aroma of the wines produced. Preliminary sensory evaluations carried out in the laboratory indicated the fine quality of the produced wines. Copyright © 2004 Society of Chemical Industry     

ESTIMATION OF ALGINATE,CARRAGEENAN AND FURCELLARAN IN BEER

Quantitative and semi-quantitative methods are given for estimating alginate, carrageenan and furcellaran at low concentrations in beer. A number of commercial beers and experimental beers known to have been brewed with either copper finings or auxiliary finings containing these acidic polysaccharides as active ingredients were analysed for alginate, carrageenan and furcellaran residues. The results show that alginate and carrageenan do not occur in finished beers at levels greater than 1 mg/litre. Using a serological technique which is more sensitive for furcellaran, it was found that levels of furcellaran in most finished beers were below 0.5 mg/litre, though a few beers contained this polysaccharide at about 0.5 mg/litre.     

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts. The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. Lambic and Gueuze are especially rich in caprylic (C₈) and capric (C₁₀) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of lambic and gueuze.     

Nutritional constituents,phytochemical profiles, <Emphasis Type="Italic">in vitro</Emphasis> antioxidant and antimicrobial properties,and gas chromatography–mass spectrometry analysis of various solvent extracts from grape seeds (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.)

The present study revealed that the nutritive value of grape seeds (Vitis vinifera L.) was 383.55±0.13 Kcal/100 g, with magnesium as the most abundant mineral element (70.44±0.88 mg/L). The maximum phenolic (392.58±1.70mg of GAE/g), flavonoid (256.16±1.60 mg of QE/g), and tannin (30.95±0.17mg of CE/g) contents were also found in the ethanol, dichloromethane, and hexane extracts, respectively. The major phytochemical compounds in the ethyl acetate extract were identified via gas chromatography–mass spectrometry (GC–MS) analysis. The ethanol extract has the highest antioxidant activity (IC₅₀=140±1.20 μg/mL for DPPH, 145.28±0.45mg α-tocopherol/g for total antioxidant capacity, and EC₅₀=80±1.41 μg/mL for ferric-reducing power assays). For β-carotene test, the highest antioxidant activity was obtained in the hexane extract. A satisfactory antimicrobial activity was found against a panel of microorganisms with the ethyl acetate extract as the best antimicrobial agent. Additionally, it was found that the bactericidal concentration required for the grape seed extract to kill Listeria monocytogenes should be less than 12.50 mg/mL (minimum inhibitory concentration=4).     

THE INFLUENCE OF WORT GLUCOSE LEVEL ON THE FORMATION OF AROMATIC HIGHER ALCOHOLS

Increasing amounts of glucose either in solid form or in solution were added to all-malt worts, and to worts that contained maize as adjunct. Fermentations were then carried out using either ale or lager yeast. The resultant beers were analysed by gas chromatography for the aromatic higher alcohols tryptophol, tyrosol and phenylethanol; the amyl alcohols were also determined as typical representatives of the aliphatic higher alcohols. Increasing concentration of glucose in the wort resulted in either decreasing levels of tyrosol and phenylethanol (ale yeast) or increasing levels (lager yeast). Tryptophol demonstrated a different pattern both with ale and lager yeasts, by increasing in concentration up to a certain maximum, then decreasing.     

PROPERTIES OF YEAST PROTEINASES

The properties of yeast proteinases, present in the extract of brewer's yeast, were compared with corresponding properties of plant proteinases, pepsin and trypsin in order to find suitable conditions for their activities to be determined independently. The main differences between yeast and plant proteinases were found in their themostability and activity at pH 3·0. In contrast to plant proteinases, yeast proteinases are thermolabile and active at pH 3·0. The proteolytic activity of yeast proteinases in beer is low and may be detected with sensitive methods using radiolabelled protein substrate. Nevertheless, a very wide range of proteolytic activities (67–1082 nkat/litre) was found in samples of unpasteurized beers.     

Gas chromatographic determination of diethylene glycol in wine, grape juice and grape-juice concentrates

A gas chromatographic method for the quantitative determination of diethylene glycol in wine, red and white grape juice, and red and white grape juice concentrates is described. The method includes continuous extraction of the diethylene glycol with ethyl acetate by the aid of a rotation perforator. Using the new generation of chemically bonded stationary phases on fused silica together with an appropriate modern cold on-column injection system coupled with an autoanalyzer and a retention gap, the GLC analysis is shown to provide adequate information for quality control purposes. Linearity, precision and recovery with the described procedure were investigated. The precision of the method was studied using a sample containing approximately 65 mg/l diethylene glycol. Twelve separate determinations were performed by the described method; the coefficient of variation was 3.9%. The detection limit of our method is 5 mg/l diethylene glycol both for wine, grape juice and grape juice concentrates. Recovery data are based on wine, grape juice and grape juice concentrates fortified with diethylene glycol at different levels. The ranges of concentration used in this study were 10 mg/l to 10 g/l for wine and 10 mg/l to 100 mg/l for grape juice and grape juice concentrate. The average recovery of diethylene glycol for wine at all concentrations tested was 98.8%; coefficients of variation were between 1.6 and 8.0%. Recoveries of diethylene glycol added to grape juice at the level of 10 mg/l to 100 mg/l were 96% to 109%.