CARRAGEENAN MEDIATED CLARIFICATION OF DIALYSED WORT SYSTEMS

Fining trials with kappa-carrageenan in dialysed wort demonstrated that potassium or calcium cations were essential for the flocculation of non-microbiological particulate material. The relative ability of metal cations to promote flocculation followed identical trends as established for carrageenan helix stabilising action and gelation (K⁺ > Ca²⁺ > Na⁺). Carrageenan also exhibited interaction with soluble wort polypeptides, in particular fractions of relative molecular mass (Mr) 70 000 and 40 000. This interaction occurred in the absence of added metal cations and was suppressed by increasing concentrations of potassium or calcium. Addition of sodium ions to systems containing potassium had no effect on either interaction with soluble polypeptides or the flocculation of particulate material. Decreasing the pH of dialysed wort impaired flocculation reactions but enhanced removal of soluble wort polypeptides. These results suggest that reaction between carrageenan and soluble polypeptides occurs via electrostatic interactions whereas flocculation of particulate material requires the presence of carrageenan in a helical conformation.     

Investigation of foam‐active polypeptides during beer fermentation

During ale fermentation there was an accumulation of total and hydrophobic polypeptides in the foam relative to the wort. Comparisons were made not only of the total and hydrophobic polypeptide contents but also of the molecular weights of these polypeptides present in wort, partially fermented wort and its concomitant foam. Wort, fermented wort and foam fractions had very similar polypeptide compositions with a major group having molecular weights of 40–43 kDa. Material of molecular weight in the range of 5–17 kDa and at 66 kDa was also detected. The polypeptides accumulated in foam displayed both hydrophobic and non‐hydrophobic character. The presence of yeast polypeptides in foam was confirmed. Comparison was also made between the fermentations of 10°Plato and 15°Plato wort. The results of the work may contribute to a better understanding of the mechanism of foam formation during beer fermentation, leading to reduced foaming and enabling an increase in the working capacities of fermenters. Copyright © 2004 Society of Chemical Industry     

APPLICATIONS OF FAST PROTEIN LIQUID CHROMATOGRAPHY (FPLC) TO THE ANALYSIS OF THE NITROGENOUS CONSTITUENTS OF BEER

The nitrogenous constituents of beer were investigated by several Fast Protein Liquid Chromatography (FPLC) techniques. Size exclusion chromatography of dialysed beer material using columns of Superose 6 and Superose 12 suggested that beer polypeptide material was distributed across a wide relative molecular mass (Mr) range with discrete fractions of high Mr (Mr 300 000, Mr 500 000), Mr c60 000, Mr c40 000 and relatively low Mr (Mr 5 000–20 000). The composition of fractions Mr >40 000 and Mr 40 000–60 000 was investigated by ion exchange chromatography. Differences were detected in the elution profiles of fractions prepared from beers brewed from grists comprising 100% malt, 80% malt plus 20% torrfied wheat and 100% malted wheat consistent with differences in the polypeptide composition of these fractions. The Superose 12 column, although designed for the fractionation of high molecular weight components, also provided a method of fractionating low molecular weight nitrogenous materials directly from beer (for example fractions containing purine nucleosides). Reverse phase chromatography was employed in the analysis of beer peptides and demonstrated the complex composition of beer peptide fractions.     

SOME REASONS WHY HIGH GRAVITY BREWING HAS A NEGATIVE EFFECT ON HEAD RETENTION*

Our aim was to examine the effect of high gravity brewing on head retention with respect particularly to the effect of high gravity brewing on hydrophobic polypeptide levels. High gravity brewed beer had poorer head retention values when compared to a similarly brewed low gravity beer. Analysis of hydrophobic polypeptide levels in both high gravity wort (20° Plato) and low gravity wort (10° Plato) produced using a lauter tun, revealed that the high gravity wort contained 8% less hydrophobic polypeptide than the low gravity wort (undiluted basis). Analysis of hydrophobic polypeptides throughout the brewing process for these 10°P and 20°P brews demonstrated that the hydrophobic polypeptide content decreased, especially during the kettle boil and fermentation. Furthermore, the high gravity brewed beer suffered the greatest loss, leaving the final beer with approximately 40% less hydrophobic polypeptides than the low gravity beer. Brewing at 10°P and 20°P using a mash filter demonstrated that these filters can improve the head formation and stability of the resultant beers at sales gravity. However, the low gravity beer still produced a more stable foam (Rudin value 93 s) when compared to the high gravity beer (Rudin value 83 s). The mash filter slightly increased the hydrophobic polypeptide extraction. It is concluded that the mash filter produced higher hydrophobic polypeptide levels in the final beers, as well as having a positive effect on reducing the levels of foam negative compounds such as fatty acids in the wort, and therefore slightly improved head retention values .     

HYDROPHOBIC POLYPEPTIDE EXTRACTION DURING HIGH GRAVITY MASHING— EXPERIMENTAL APPROACHES FOR ITS IMPROVEMENT

The aim was to establish if a substantial increase in hydrophobic polypeptides could be achieved during high gravity mashing. When worts with gravities ranging from 5–20°P were analysed for hydrophobic polypeptide content it was found that there was no appreciable increase in hydrophobic polypeptide levels. Remashing of the spent grains from low and high gravity mashes demonstrated that this resulted from inefficient extraction of hydrophobic polypeptide levels during the mashing process. For example, wort produced from remashed high gravity spent grains contained 150 mg/L hydrophobic polypeptides compared to only 10 mg/L in the low gravity remashed spent grains. Experiments were conducted, employing standard mashing techniques, in an attempt to increase the extraction of hydrophobic polypeptides during high gravity mashing. Thus the use of gypsum, proteolytic stands, varying liquor to grist ratios and wheat malt addition were all investigated for their effect on hydrophobic polypeptide extraction during high and low gravity mashing. Wort analysis demonstrated that none of the techniques employed had a significant effect on hydrophobic polypeptide extraction. When wort from remashed spent grains was used as mashing in liquor for a fresh mash and the resultant worts analysed for hydrophobic polypeptides it was observed that no increase in hydrophobic polypeptide extraction was achieved. For example, wort from the remashed high gravity spent grains, containing 140 mg/L hydrophobic polypeptides, when used as mashing-in liquor, produced no increase in hydrophobic polypeptide levels in the resultant high gravity wort (230 mg/L) when compared to a high gravity wort produced using distilled water as mashing-in liquor (255 mg/L). It is therefore concluded that a saturation point has been reached and no more hydrophobic polypeptides can be extracted during mashing regardless of the procedures employed.     

The Loss of Hydrophobic Polypeptides during Fermentation and Conditioning of High Gravity and Low Gravity Brewed Beer

The object of this study was to investigate the loss of hydrophobic polypeptides, which are important for foam quality and stability in finished beer. Loss of hydrophobic polypeptide due to fermenter foaming occurs during transfer of fermented wort since a gradient of hydrophobic polypeptides towards the surface is created during fermentation. Due to higher polyphenol levels in high gravity (20°Plato) wort, more hydrophobic polypeptides are lost due to cold break (cold trub) precipitation compared to low gravity (12°Plato) wort. Another important factor affecting the loss of hydrophobic polypeptides could be proteinase A activity during fermentation, especially in high gravity fermentation where the yeast is exposed the higher stress. During high gravity fermentation, where osmotic pressures are higher, ethanol levels become greater, and nitrogen‐carbohydrate ratios are lower, more proteinase A is released by the yeast. This release of proteinase A into fermenting wort could have implications for the foam stability of the finished product.     

A quantitative comparison of the extraction of protein fractions from wheat grain by different solvents,and of the polypeptide and amino acid composition of the alcohol-soluble proteins

Using several cultivars, whole milled wheat grain was first extracted with 0.5M NaCl to remove non-protein N, albumins and globulins: the amount of protein subsequently extractable by either 70% ethanol, 55% propan-2-ol, or 50% propan-1-ol (with or without the addition of acetic acid), was then compared. All solvents were tested at 4, 20 and 60°C both with and without the addition of 2-mercaptoethanol (2-ME) as a reducing agent. Raising the temperature, including a reducing agent, and repeated extractions, all combined to maximise protein extractability; least was extracted by 70% ethanol at 4°C and most by 50% propan-1-ol containing acetic acid and 2-ME at 60°C. The differing polypeptide compositions of these alcohol-soluble fractions illustrated why N solubility alone is not a sufficient guide to protein extraction, e.g. 35% of grain N was extractable by either 70% ethanol at 60°C or 70% ethanol containing 2-ME at 4°C, but high mol. wt polypeptides (>60000) were found only in the 60°C extract. In contrast, the complete range of alcohol-soluble polypeptides was extracted by all solvent mixtures based on propan-1-ol, even at 4°C. Amino acid analyses of these alcohol-soluble fractions confirmed the low levels of lysine in fractions free from high mol. wt polypeptides. More glycine was found in all fractions containing high mol. wt polypeptides, but only those extracted by propan-1-ol mixtures had an increased lysine content, the amount of which increased linearly as the extraction temperature was raised. Defatting the milled grain did not affect the extractability of alcohol-soluble proteins, but the amount of protein soluble in 0.5M NaCl decreased. The protein rendered NaCl-insoluble was not extracted by any of the alcoholic solvents tested, hence the N content of the residual material was increased. The inclusion of this denatured metabolic fraction in the residue will affect its amino composition. The problems arising from the retention of the classical nomenclature for Osborne-type fractions are discussed.     

AMINO ACID ANALYSIS OF BEER POLYPEPTIDES

The principles of amino acid analysis of proteins and polypeptides are reviewed. Analysis of the amino acid composition of dialysed beer material prepared from a wide variety of commercial and pilot brewery beers showed that the principal amino acids comprised glutamic acid/glutamine, proline, glycine and aspartic acid/asparagine. The results from the analysis of a series of pilot brewery beers brewed under standardised conditions showed that the composition of the grist may influence the amino acid composition of beer polypeptide fractions. Dialysed beer material prepared from beer brewed from grists containing torrified wheat, wheat flour and malted wheat contained greater proportions of glutamic acid/glutamine compared to material prepared from all malt beers. Further fractionation and analysis of dialysed beer material prepared from pilot brewery beers suggested that fractions MW>60000 contained polypeptide material derived from yeast mannan-protein. In addition fractions MW>60000 prepared from beer brewed from grists containing torrified wheat, wheat flour or all malted wheat may contain high molecular weight polypeptide material derived from wheat proteins. The results from the analysis of fraction MW 40,000–60000 prepared from beers brewed from grists containing all malt, 80% malt and 20% torrified wheat and 50% malt and 50% malted wheat are consistent with the presence of polypeptide material derived from cereal albumins and globulins whereas fractions MW 40,000–60000 prepared from beers brewed from 80% malt and 20% wheat flour and 100% malted wheat may contain polypeptide material derived from wheat prolamins and glutelins. The amino acid composition of fraction MW 20,000–40,000 from all pilot brewery beers investigated is consistent with the presence of polypeptide material derived from cereal prolamins and glutelins. The amino acid composition of beer polypeptide fractions may be used to detect the use of wheat adjuncts in beer brewing.     

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP™, an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.     

THE INFLUENCE OF β-GLUCANASE ON EFFICIENCY OF WORT SEPARATION

Treatment of mixed grists of malt and either barley flakes or barley flour with β-glucanase during infusion mashing at 65°C improves both the rate of run-off of wort and the total yield of extract. The total amount of non-starchy polysaccharide in solution is increased but the high molecular weight β-glucan fraction is decreased by the addition of β-glucanase. An improvement in wort separation is associated with a lowering of wort viscosity but it is concluded that the major factor influencing wort separation is the structure of the mash bed and the presence of inert material to which protein, glucan and pentosans can bind. A micro-mashing unit is described which simulates the problems of wort separation encountered with certain grists in deep infusion mash tuns.     

IDENTIFICATION AND CHARACTERISATION OF BEER POLYPEPTIDES DERIVED FROM BARLEY HORDEINS

Two groups off hordein-reactive antibodies (cross-reactive in barley with B/C monomers or subunits from the B/D-hordein aggregates) were used for the analysis of malt, wort and beer. A number of additional lower molecular weight hordein-derived polypeptides were present in water extracts of malts and worts, and protease inhibitors or higher mash-in temperature had a stabilising effect on certain hordein-derived polypeptides . The solubilisation profiles of hordein polypeptides monitored by specific immunological methods were clearly different from total wort polypeptides monitored by the Bradford method. There were also differences between the profiles for the two groups of hordein-derived polypeptides with the solubilisation of polypeptides recognised by antibody with specificity for the subunits from the B/D aggregates being especially temperature dependent . Beer samples mainly contained hordein-derived polypeptides of lower molecular weight than barley hordeins with polypeptides in the B and C molecular weight regions being lost during the brewing process. Beer polypeptides (Mr > 50,000) were recognised by antibodies cross-reactive with the subunits from the B/D aggregates in barley. A number of lower molecular weight polypeptides (Mr < 30,000) were recognised by antibodies with specificity for B and C hordeins; a subset of the latter antibody group bound to a specific Mr 23,000 beer polypeptide .     

Partial characterization of polypeptide components of sunflower (Helianthus annuus L.) seed albumin fraction

The albumin fraction of sunflower seed (Helianthus annuus, cv. Mirasol) is a family of water soluble basic polypeptides which constitutes about 20% of the seed proteins. This fraction, isolated by selective isoelectric precipitation of globulins, has been studied in detail by sodium dodecyl sulphate gel electrophoresis, non equilibrium pH gradient electrophoresis and combination of these techniques using non reducing and reducing conditions. The molecular mass of the main polypeptides was markedly different between unreduced (12,000 to 16,500 g · mol^?1) and reduced form (10,000 to 18,000 g · mol^?1). As shown by NEPHGE mobility of these polypeptides was also altered by reduction. From these results and other observations is concluded that the stability of the globular structure of some polypeptides is dependent on the presence of intact disulphide crosslink(s). By multidimensional gel electrophoresis it was shown that the polypeptide components of each molecular mass classes displayed a high heterogeneity in net charge. Thus ion exchange chromatography procedures allowed only partial separation of different polypeptidic groups. Their amino acid composition shows that some of these fractions are, on the basis of their lysine and sulphur containing amino acids, of nutritional interest.     

The Effect of Proteinase A on Foam‐Active Polypeptides During High and Low Gravity Fermentation

The ability of beer to produce good foam is influenced by the level of foam‐active polypeptides. Specific polypeptides with hydrophobic domains, such as Lipid Transfer Protein (LTP1), are important components of beer foam. Although, high gravity brewing is a commercially viable technique, it has the disadvantage of producing beer with less foam stability compared to lower gravity brewed counterparts. It is thought that proteinase A plays a key role in the degradation of these hydrophobic polypeptides responsible the beer foam stability. The object of this study was to compare and quantify the loss of hydrophobic polypeptides and specifically foam‐LTP1 during high gravity (20°Plato) and low gravity (12°Plato) wort fermentations and to evaluate the effect of proteinase A on these polypeptides. The losses of hydrophobic polypeptides and foam‐LTP1 were generally greater in high gravity brews. Furthermore, the results obtained suggest that proteinase A alters the hydrophobicity of these polypeptides rather than their molecular size. Approximately 20% of hydrophobic polypeptides and approximately 57% of foam‐LTP1 appeared to be proteinase A resistant. These differential losses of hydrophobic polypeptide and foam‐LTP1 could have implications for the foam stability of the finished product.     

EFFECT OF ENVIRONMENTAL CONDITIONS ON FLOCCULATION AND IMMOBILISATION OF BREWER'S YEAST DURING PRODUCTION OF ALCOHOL-FREE BEER

A method using immobilised yeasts has been developed and successfully applied for production of alcohol-free beer. The influence of environmental conditions present during alcohol-free beer production on the flocculation and immobilisation of the yeast Saccharomyces cerevisiae var. uvarum was investigated in the present study. In wort, the cells developed flocculation at the end of exponential growth, according to the NewFlo phenotype. In defined medium, the flocculation capacity appeared to be temporary and was lost rapidly during the stationary phase. No increase in cell wall hydrophobicity at the onset of flocculation was observed in either medium. Low growth temperatures increased flocculation capacity approximately four-fold, compared to growth at high temperatures. The optimum temperature for flocculation was at 25°C with cells grown at low or high temperature . A novel method using carboxyfluorescein-stained cells was developed to analyse the initial adhesion of cells to carrier. This method also allowed rapid analysis of the effects of immobilisation to DEAE-cellulose carrier during alcohol-free beer production process. It appeared that a high flocculation capacity stimulated adhesion to the DEAE-carrier .     

LOW MOLECULAR WEIGHT NITROGENOUS COMPONENTS AND THEIR INFLUENCE ON THE STABILITY OF BEER FOAM

Addition of low molecular weight nitrogenous components, nucleoside (adenosine), nucleotide (AMP), amino acids (glutamic acid, glycine, proline and asparagine) and a tripeptide (glycylglycylglycine) had no effect on the head retention value of beer. Under such conditions of artificial manipulation of the content of low molecular weight beer nitrogenous components no correlation between the nitrogen molecular weight ratio of beer and beer head retention value was observed. Collapsed beer foam was collected and separated into soluble and precipitated fractions. Analysis of collapsed beer foam demonstrated that high molecular weight polypeptide material and isomerised α acids are concentrated in beer foam and are constituents of beer foam precipitate material. Beer foam precipitate material may be stabilised by ionic interactions between polypeptide material and isomerised α acids. Further analysis of beer and collapsed beer foam demonstrated that purine nucleosides are not concentrated in beer foam. Similarly, under conditions simulating beer dispense, amino acids are not concentrated in beer foam. Under strongly selective foaming conditions, such as those encountered in a foam tower, the hydrophobic amino acids, valine, isoleucine, leucine and phenylalanine are concentrated in beer foam.     

EXTRUDED SORGHUM AS A BREWING RAW MATERIAL

Small scale mashes (50 g total grist) with grists containing up to 50% by weight of extruded whole sorghum produced worts of high extract yield and low viscosity. Increasing the proportion of extruded sorghum in the grist resulted in decreasing wort filtration volume, total nitrogen and free amino nitrogen content. The wort filtration behaviour of mashes containing sorghum extruded at 175°C was superior to that of mashes containing sorghum extruded at 165°C or 185°C. The results from such small scale mashing experiments suggested that extruded sorghum compared favourably to extruded barley and extruded wheat as a brewing adjunct. Worts and beers were produced on a pilot brewery scale (100 1) from grists comprising 70% malt + 30% extruded sorghum and 100% malt under isothermal infusion mashing conditions. Mashes containing sorghum extruded at 175°C showed comparable wort filtration behaviour to that of the all malt control mash whereas mashes containing sorghum extruded at 165°C or 185°C showed poor wort filtration behaviour. Worts produced from grists containing extruded sorghum fermented more quickly than the control wort and attained lower values of final gravity. The resulting beers were filtered without difficulty. Beers produced from grists containing extruded sorghum contained lower levels of total nitrogen and free amino nitrogen compared to the control beer consistent with extruded sorghum contributing little or no nitrogenous material to the wort and beer. Beers brewed from grists containing extruded sorghum were of sound flavour and showed reasonable foam stability behaviour.     

Lipid and protein deterioration during the chilled storage of minced sea salmon (Pseudopercis semifasciata)

Sea salmon is a very appreciated seafood. The aim of this work was to analyze changes in lipid and protein fractions of minced muscle during chilled storage (1 ± 1 °C). Lipid oxidation was important during the first 6 days of storage according to 2‐thiobarbituric acid (TBA) values determined, decreasing mainly ω3 22:6 fatty acid content. Lipid hydrolysis was evident after 9 days of storage. Interaction compounds between oxidation products and other cellular components were analyzed by fluorescence measurements. The results obtained showed evidence of the formation of interaction products involved, mainly polar components such as proteins. Decreases in myosin and actin thermal stability and myosin denaturation were recorded by differential scanning calorimetry. Solubility of total and myofibrillar proteins decreased after 6 days of storage. The electrophoretic profile of soluble fractions showed an increase in the intensity of bands corresponding to low‐molecular‐weight polypeptides and a decrease in high‐molecular‐weight species. Available lysine content did not change during chilled storage. Copyright © 2007 Society of Chemical Industry     

FRACTIONATION OF HIGH AND LOW MOLECULAR WEIGHT COMPONENTS FROM WORT AND BEER BY ADSORPTION CHROMATOGRAPHY USING THE GEL SEPHADEX LH20

Column chromatography using the hydroxypropylated dextran gel Sephadex LH20 was used to fractionate the components of brewers' wort and beer. The elution profile of beer was investigated in detail and components identified. Fractions containing high molecular weight polypeptide material, small peptide material, free proline, phenylalanine, tyrosine and adenosine have been identified. These fractions are common to all beers investigated. This use of LH20 provides a simple and rapid technique for monitoring high and low molecular weight materials in beer production and may be used in diagnosis of beer problems. An example of the application of the LH20 elution profile to the analysis of an out of specification beer is described.     

The Gel Filtration Chromatographic‐Profiles of Proteins and Peptides of Wort and Beer: Effects of Processing — Malting,Mashing, Kettle Boiling,Fermentation and Filtering

Barley and malt proteins, of infusion (IoB) and decoction (EBC) mashing worts as well as commercial wort and beer, obtained from the Castlemaine Perkins brewery, Brisbane, were gel filtered, with or without further treatments. A general, similar pattern of protein and peptide profiles emerged from barley malt and beer. This confirmed the widely assumed fact that beer proteins descend from barley, some transformed and others perhaps mostly unchanged by processing. In the gel‐filtrate profiles, a maximum of 8 or 9 fractions were discerned. These fractions were collected and quantified for protein contents and amino acid compositions. The first four fractions contained the proteins and polypeptides of molecular weight higher than 14,000. Consequently, the remaining fractions contain the smaller peptides (<14,000), that were completely removed by dialysis. The effects of processing on proteins and peptides varied contingent upon the type of processing step considered and the pre‐chromatographic treatment. Malting was the most effective process remarkably increasing the soluble protein contents, especially the smaller peptide fractions and the colour development. This is the first report, as far as we are aware of, on the gel filtration profiles of wort and beer low molecular weight peptides including those of barley wort. The importance of the smaller peptides in foam formation and retention cannot be overemphasised. The amino acid composition of the fractions revealed much more diversity than was observed in the comparison of the profiles. Proline content of fraction 1 resembled that of barley soluble proteins while fractions F2, F3 and F4 that of glutelin and only fraction 8 that of hordein. The latter, suggests that hordeins or, at least the peptide products rich in proline, are likely to be completely digested to amino acids, during malting.     

Determination of the molecular weight distribution of non‐enzymatic browning products formed by roasting of glucose and glycine and studies on their effects on NADPH‐cytochrome c‐reductase and glutathione‐S‐transferase in Caco‐2 cells

After thermal treatment of a mixture of glucose and glycine for 2 h at 125°C, about 60% of the starting material was converted into non‐soluble, black pigments, whereas 40% of the mixture was still water‐soluble. Dialysis of the latter fraction revealed 30.4% of low molecular weight compounds (LMWs; MW < 10 000 Da) and 10.0% high‐molecular weight products (HMWs; MW ⪈ 10 000 Da). The water‐soluble Maillard reaction products (MRPs) were separated by gel permeation chromatography and ultrafiltration, revealing that 60% of the water‐soluble products of the total carbohydrate/amino acid mixture had MWs < 1 000 Da and consisted mainly of non‐coloured reaction products. MRPs with MWs between 1 000 and 30 000 Da were found in comparatively low yields (about 1.3%). In contrast, about 31.1% of the MRPs exhibited MWs > 30 000 Da, amongst which 14.5% showed MWs > 100 000 Da, thus indicating an oligomerisation of LMWs to melanoidins under roasting conditions. To investigate the physiological effects of these MRPs, xenobiotic enzyme activities were analysed in intestinal Caco‐2 cells. For Phase‐I NADPH‐cytochrome c‐reductase, the activity in the presence of the LMW and HMW fraction was decreased by 13% and 22%, respectively. Phase‐II glutathione‐S‐transferase activity decreased by 15% and 18%, respectively, after incubation with the LMW and the HMW fractions. Considering the different yields, 30% and 10%, respectively, of the LMW and the HMW fractions, the total amount of the LMW fraction present in the glucose‐glycine mixture is more active in modulating these enzyme activities than that of the HMW fraction.