THE INFLUENCE OF 2-ACETOHYDROXY ACIDS ON THE DETERMINATION OF VICINAL DIKETONES IN BEER AND DURING FERMENTATION

During sampling and determination of diacetyl, 2-acetohydroxy acids are easily converted to vicinal diketones. A simple procedure for gas chromatographic determination of diacetyl, 2-acetolactate, acetoin and the homologous compounds is given. By careful sampling, less than 0·01 ppm of diacetyl was detected during the main fermentation in one brewery, whereas another strain of brewer's yeast yielded a maximum of 1·7 ppm of diacetyl. When samples of fermenting liquids are exposed to air at 60°C, complete conversion of 2-acetohydroxy acids takes place in less than one hour. The possibility that part of 2-acetolactate may be converted to acetoin, however, cannot be excluded. In finished beer 2-acetolactate levels of 0·2–0·5 ppm were observed. During the main fermentation the values ranged from 0·5–2·5 ppm.     

THE EFFECT OF LOW CO2 PRESSURES ON THE ABSORPTION OF AMINO ACIDS AND PRODUCTION OF FLAVOUR-ACTIVE VOLATILES BY YEAST

Laboratory fermentation of 51 batches of wort under CO₂ pressures of 0·5 and 1·0 atm have shown that whilst the speed of fermentation was affected only slightly, absorption of amino acids, production of several flavour-active compounds and some yeast features were affected. The CO₂ pressure led to reduced absorption of Group B amino acids although Group A compounds were unaffected; fusel oil and ester concentration was reduced whereas that of the vicinal diketones and acetaldehyde was increased. Yeast crop and cell viability fell with increase in pressure and at 1·0 atm the mean cell volume was increased. The results suggest that CO₂ could have significant effects on yeast and beer quality within the range of concentration which can be expected to occur in unpressurized commercial fermentations.     

THE RELATIONSHIP OF DIMETHYL SULPHIDE LEVELS IN MALT,WORT AND BEER

The half-life for the conversion of malt dimethyl sulphide (DMS) precursor to free DMS has been determined at various temperatures and pH values. At pH 5·2 the half-life of the precursor in wort (S.G. 1·060) at its boiling point is 38 min, and is doubled for each 6°C fall in temperature. At pH 5·5 the half-life at the boiling point is 32·5 min. Knowing the stability of the precursor at the various temperatures in the brewing process, the extent of conversion to free DMS in wort at pitching can be predicted for malt of a given precursor content and for a given set of process conditions. The results of DMS analyses of samples taken during brewery trials are in reasonable agreement with the predicted values. This work involved infusion mashing only, but the same principles apply to decoction mashing. The fate of precursor and free DMS during fermentation and conditioning has been followed on a production scale. With some brews, where high levels of free DMS were present at pitching, much free DMS was lost during fermentation. Also, precursor DMS reappeared in the beer after a few days and there was some increase in the level of free DMS. The DMS precursor in green malt has been isolated by ion-exchange and gel-filtration chromatography. A preparation has been obtained which has 0·6 mol potential DMS per mol amino nitrogen. Thin layer chromatography showed that the preparation and its hydrolysis product had the same R_f values as S-methylmethionine and homoserine respectively.     

Leuconostoc fallax,an acid and ethanol tolerant lactic acid bacterium

Leuconostoc fallax, known to be present in sauerkraut, was reisolated from exudates of Gerbera jamesonii. The identity of the isolates with L fallax was demonstrated by sequence analysis of the first 600 bases of the 16S rRNA. L fallax utilised a small number of sugars and showed a remarkable resistance to lactic acid. The final pH in glucose broth was 3·9. Moreover it was able to grow in the presence of ethanol (9·0% v/v) and salt (5·5%), but it was unable to carry out a malo-lactic fermentation.     

ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

Post-harvest pod storage: A method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in Malaysia

In Malaysia, pulp preconditioning by post-harvest storage of cocoa pods leads to the reduction of nib acidification during subsequent fermentation, reduction of the acid note and an increase in cocoa flavour in the resulting raw cocoa. Data from several shallow-box fermentations, with material from unstored and stored pods, are compared and interpreted, obtained in the years 1984 to 1987 in a cooperational investigation of the Malaysian Agricultural Research and Development Institute (MARDI), Malaysia, and the Botanical Institute, Technical University of Braunschweig (TUBS), FRG. Prior to and during fermentation, pulp volume and pulp sugars; pH value, acetic acid and lactic acid content in the pulp and nibs; and oxygen concentration and temperature in the mass were determined. Some flavour assessments from selected samples are given. The great reduction in pulp volume per seed rather than the decrease of pulp sugars per seed during pod storage was found to be of the most importance. Pulp-volume reduction enhances mass aeration and increases the ratio of respiration to ethanol fermentation and its subsequent oxidation to acetic acid. As a consequence, the acidification of the seeds during the formative stages of flavour precursors (after the death of the seeds) is strongly reduced. With effectively dry stored pods (pulp volume per gram of seed ≤0·6 ml) the anaerobic phase during the initial stages of fermentation which is common with unstored pods is suppressed. Under these conditions the nib pH value does not fall below 5·0 and no drastic acid (and flavour) degradation at the end of fermentation is necessary to reduce the acidity in the seeds.     

FERMENTATION OF MINOR WORT CARBOHYDRATES BY BREWING YEASTS

Typical worts prepared from all-malt grists and from those using a proportion of adjuncts contain maltulose, maltotriulose, isomaltose, panose and isopanose at concentrations between 0·01 and 0·12%, and maltotetraose between 0·2 and 0·7%. Maltulose and maltotriulose are fermented by all brewing strains of Saccharomyces cerevisiae so far examined, soon after utilization of maltose and maltotriose respectively begins. Fast-fermenting strains absorb small amounts of panoses and certain of them remove some maltotetraose whereas slow fermenting strains fail to utilize these three sugars. The fast-fermenting strains utilize more isomaltose than do slow fermenting yeasts. Of the total (2–4 g/litre) minor sugars of wort, fast fermenting strains remove about 90% whilst slow fermenting strains absorb 70–80%.     

Human Colonic Bacterial Degradability of Dietary Fibres from Sea-Lettuce (Ulva sp)

Sea-lettuce (Ulva sp) is one of the commonly consumed seaweeds which contains 16·5% of water-soluble and 13·3% insoluble dietary fibres. Since physiological effects of fibres are partly related to their colonic bacteria fermentability, Ulva sp and its constitutive soluble and insoluble fibres were incubated with faecal bacteria in an in vitro batch fermenter system. After 24 h of incubation, 32·0±0·4%, 25·9±0·4% and 50·9±7·4% of Ulva, soluble and insoluble fibres constitutive sugars, respectively, were degraded. Consequently, Ulva and its soluble fibre, ulvan, are poorly fermented by colonic bacteria. The constitutive sugars, rhamnose and glucuronate and the aldobiouronate β-D -glucuronosyluronate-(1,4)-L -rhamnose of the glucuronoxylorhamnan sulphate present in the soluble fibre are highly fermented. Chemical desulphation and/or carboxyl group reduction did not modify this fermentation behaviour. Thus, the particular chemical structure of ulvan is responsible for the resistance of this polysaccharide and of Ulva to colonic bacterial fermentation. As a physiological consequence of this particular behaviour, consumption of dietary fibres from sea-lettuce could be expected to act mainly as bulking agents with little effect on nutrient metabolism due to colonic bacterial fermentation products (short-chain fatty acids). © 1997 SCI.     

Conservation of wilted and unwilted grass ensiled in air-tight metal containers with and without the addition of molasses

Italian ryegrass was ensiled in gas-tight silos in unwilted (21% dry matter) and wilted (42% dry matter) conditions without an additive and with the addition of molasses at a mean level of 8% in the dry matter. The total dry matter losses were 8·7, 7·2, 4·5 and 5·0% for unwilted, molassed unwilted, wilted, and molassed wilted grass silages respectively. The silages were all well preserved but the extent of fermentation had been reduced in the wilted silages. The losses of individual sugars during the silage process and the products of fermentation were used to assess the pathways of fermentation. The dominant pathway of fructose utilisation was by heterolactic fermentation. The addition of molasses reduced the ratio of fructose: glucose in the ensiled material and reduced the amount of mannitol produced during the ensilage process.     

METABOLIC QUOTIENTS OF RESPIRATION-DEFICIENT MUTANTS OF BREWER'S YEAST AND THE APPEARANCE OF A NEGATIVE PASTEUR EFFECT

Four industrial strains of bottom brewer's yeast and a group of their spontaneous respiration deficient (RD) mutants were tested for rates of metabolism of glucose and maltose under aerobic and anaerobic conditions. Q_o2 of all the RD mutants tested (26 isolates) ranged from 1·9 to 6·8 μl/mg yeast dry weight on glucose and was lowered to about one-half on maltose although the original strains had the same Q_o2 values on both sugars. No isolate showed any increase in glucose fermentation in aerobic conditions as compared with the original strains and the decrease of Pasteur effect found in certain isolates was always accompanied by a strong decrease of glycolysis in both aerobic and anaerobic conditions. Two mutants showed a strongly negative Pasteur effect, for their fermentation rates were higher in aerobic conditions than in anaerobic ones. Two other mutants showed a strong negative Pasteur effect only on maltose. The ratio of Q^N_2CO2 on glucose in most mutants was significantly lower than in their parental strains.     

Modelling mould spoilage in cold-filled ready-to-drink beverages by Aspergillus niger and Penicillium spinulosum

Mathematical models have been developed to predict the probability of growth of spoilage moulds in response to various preservative systems in ready to drink beverages. A Box-Behnken experimental design included five variables, each at three levels: pH (2·8, 3·3, 3·8), titratable acidity (0·20%, 0·40%, 0·60%), sugar content (8·0, 12·0, 16·0 °Brix), and preservative concentrations (sodium benzoate and potassium sorbate, each 100, 225, 350 ppm). Duplicate samples were inoculated with a mould cocktail consisting of equal proportions of Aspergillus niger and Penicillium spinulosum spores (5·0×10⁴spores/ml). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create predictive models. The pH, titratable acidity, sugar content, sodium benzoate, and potassium sorbate levels were all found to be significant factors in predicting the probability of mould growth over time. Interactions between pH and sodium benzoate, pH and potassium sorbate, and pH and sugar content were also statistically significant. This logistic model was validated against 14 new conditions and predicted the growth of mould after 8 weeks with over 96% accuracy. Product developers can use these models to predict mould growth in ready to drink beverages.     

THE INSTITUTE OF BREWING ANALYSIS COMMITTEE REVISED METHODS OF EXTRACT DETERMINATION

Following a Brewer's Society request to consider a change from a standard Miag Disc Mill setting of 0·5 mm to one of 0·7 mm the Analysis Committee has re-evaluated the determination of hot water extract. It is recommended that from 1st December 1979–1. A Miag Disc Mill setting of 0·7 mm be adopted for grinding malt for the determination of hot water extract. 2. Where an estimate of the fine/coarse grind hot water extract difference is required, mill settings of 0·2 mm and 0·7 mm respectively should be used. The reproducibility of this procedure is adequate to separate malts into classes with high and low values but analytical tolerances cannot be recommended for commercial transactions. Revised methods for setting the Miag Disc Mill and for determining the hot water extract of fine and coarse ground malts are given in Appendix I. It is proposed that all mashes should be made in stainless steel beakers, the use of the standard brewers' mash flask being limited to making the mash to the correct volume at the end of the determination. It is recommended that, from 1st December 1979, the 0·7 mm Miag Disc Mill setting should replace also the 0·5 mm setting in all Recommended Methods for coloured malts, dark malts, roasted barley and unmalted grain. It is further recommended that from 1st December 1979 all specific gravity determinations for laboratory extracts of malt, coloured malt, dark malt, roasted barley, unmalted grain, sugars, syrup, caramel and spent grains should be made at 20°C.     

pH IN MALTING AND BREWING—A REVIEW

Variations in pH during malting, kilning, mashing, lautering, wort boiling and fermentation are reviewed. The fragmentary information available suggests that the pH of cold water extracts of barley falls during steeping, rises during germination and falls again during kilning. Brewing liquor, mashing method, addition of acid, and the nature of the malt influence the pH of unhopped wort, which falls by about 0·3 of a unit during boiling. Fermentation reduces the pH by a further unit yielding ales with pH values of about 3·8-4·2, or lagers of pH 4·2–4·8. Literature evidence does not suggest any obvious reason why ales and lagers should differ in pH.     

KINETIC STUDIES OF THE DECONTAMINATION OF YEAST SLURRIES WITH PHOSPHORIC ACID AND ACIDIFIED AMMONIUM PERSULPHATE AND A METHOD FOR THE DETECTION OF SURVIVING BACTERIA INVOLVING SOLID MEDIUM REPAIR IN THE PRESENCE OF CATALASE

Destruction of Obesumbacterium proteus in pitching yeast slurries is more efficiently accomplished using an acidified ammonium persulphate wash (0·75% w/v; pH 2·8) than by using phosphoric acid at pH 2·1. The effectiveness of both methods against the lactic acid bacteria, acetic acid bacteria and Enterobacter agglomerans is similar. Material cost savings of up to 60% can be achieved through the use of the acidified ammonium persulphatewash. Economy in the use of materials and optimum process efficiency is achieved by determination of the quantity of acid required to adjust the pH of the yeast slurry before washing. Bacteria surviving the yeast wash can be detected reliably using a plating technique involving solid medium repair in the presence of catalase. A buffered diluent must be used to prepare the inoculum.     

Osmoregulatory active sodium-glycerol co-transport in the halotolerant yeast Debaryomyces hansenii

Several authors have shown that the halotolerant yeast Debaryomyces hansenii, when growing exponentially in glucose medium in the presence of sodium chloride, maintains osmotic balance by establishing sodium and glycerol gradients of opposite signs across the plasma membrane. Evidence is presented here that the two gradients are linked through a sodium-glycerol symport that uses the sodium gradient as a driving force for maintaining the glycerol gradient. The symporter also accepts potassium ions as co-substrate. The kinetic parameters at 25°C, pH 5·0 were the following: V_max, decreasing from over 500 to less than 40 μmol g^?1 per h over a concentration range of 0–3 M extracellular sodium chloride; K_m (glycerol) 0·40–0·6 mM over the same range; K_m (sodium ions) 16·0 ± 3·21μM ; K_m, (potassium ions) 10·4 ± 3·6μM . Furthermore, it was observed that glycerol uptake was accompanied by proton uptake when extracellular sodium chloride was present and that the protonophore carbonylcyanide-M-chlorophenylhydrazone induced collapse of the glycerol gradient, supporting earlier proposals by others that the sodium gradient is maintained by an active sodium-proton exchange mechanism.     

Influence of Fermentation Time and an ‘Indigenous Tenderiser’ (Kanwa) on the Microbial Profile,Chemical Attributes and Shelf-Life of Rice Masa (a Nigerian Fermented Product)

Investigations on the influence of fermentation time, ‘Kanwa’ (an indigenous tenderiser and a flavouring agent) and potassium sorbate (0·5, 1·0 or 1·5 g kg⁻¹) were conducted by examining the microbial profile, chemical attributes and shelf-life of rice ‘Masa’ (a Nigerian fermented product). Various types of microorganisms occurred at the initial stages of rice slurry fermentation and these included fungi (Aspergillus spp, Penicillium spp, Saccharomyces spp, Rhizopus spp) and bacteria (Lactic acid bacteria, Bacillus spp, Enterobacter spp and Acetobacter spp). But beyond 8 h fermentation time, fewer types of micro-organisms (Lactobacillus spp, Saccharomyces spp and Rhizopus spp) were isolated.Total aerobic counts increased significantly (P=0·05) as fermentation progressed (0, 6 or 12 h), with lactic acid bacteria (LAB) and yeasts dominating and probably contributed to the sour–sweet spongy characteristic of the rice Masa. But after fermentation and baking process, the rice Masa became dominatedand spoilt by Bacillus spp, Lactobacillus spp and Saccharomyces spp withstorage time. Longer fermentation time resulted in a less acceptable product,being more acidic. Rice Masa produced from a 6 h fermentation showedbetter sensory qualities (aroma and visual appearance). Use of Kanwa(Na₂CO₃.NaHCO₃.2H₂O) improved aroma but was detrimental to visualappearance and spoilage was accelerated, probably due to higher pH. Potassium sorbate at higher concentrations (1·0 and 1·5 g kg⁻¹) induced beneficial antimicrobial effects and reduced the microbial load thereby prolonging the shelf life of the rice Masa (treated with 1·5 g kg⁻¹) by about 3 days.     

MALT DIASTATIC ACTIVITY. PART II. A MODIFIED EBC DIASTATIC POWER ASSAY FOR THE SELECTIVE ESTIMATION OF BET A-AMYLASE ACTIVITY,TIME AND TEMPERATURE DEPENDENCE OF THE RELEASE OF REDUCING SUGARS

Ammoniumcitrate, sodium EDTA and ammonium oxalate were tested as possible inhibitors of α-amylase in the EBC diastatic power assay. A contact time of 90 min at a concentration of 0·1 M ammonium oxalate was shown to inactivate α-amylase completely in the enzymic malt extract while only very slightly enhancing β-amylase activity. The selective release of reducing sugars by β-amylase in the diastatic power assay was evaluated as a function of time using a p-hydroxybenzoic acid hydrazide assay and compared with that of the diastatic power procedure with the same colourimetric technique. The diastatic power results of eight different malts are well correlated with those of the β-amylase activity (r² = 0·98). In a linear regression model the diastatic power results at 20°C of the same malts correlated better with results obtained at 55°C (r² = 0·77) than with those obtained at 60°C (r² = 0·63). For the results of β-amylase activities we found r² = 0·86 for the relation between the activities at 20°C and at 55°C and r² = 0·92 for the relation between the activities at 20°C and 60°C. Since the temperature conditions (20°C) of the EBC-diastatic power assay do not correspond to those of brewing practice it is suggested that the activity of β-amylase as well as the malt diastatic power be evaluated at 55°C.     

Effect of mass and turning time on free amino acid,peptide-N,sugar and pyrazine concentration during cocoa fermentation

A response surface methodology (RSM) was used to determine the optimum condition for mass and turning time during cocoa fermentation. Mass and turning time were used as independent variables; concentrations of free amino acids, peptide-N, sugars and pyrazines were the dependent variables. The R² values for peptide-N, tetramethylpyrazine and total pyrazines were greater than 0·9. Both lower (10 kg) and higher (100 kg) cocoa mass together with lower (0 min) and higher (10 min) turning time gave products containing low concentrations of hydrophobic, total and other free amino acids, peptide-N, fructose, glucose and total reducing sugars; in contrast, those of acidic free amino acids gave higher concentrations. Trimethyl-, tetramethyl- and total pyrazines increased significantly (P<0·05) at higher mass (100 kg) and higher turning time (10 min). From the highest concentration of the important flavour precursors ie hydrophobic free amino acids, total reducing sugars and peptide-N, the recommended mass of cocoa beans and turning time for an optimum cocoa fermentation condition was 60 kg and 5 min, respectively. © 1998 Society of Chemical Industry.     

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.     

CONTROLLED PRODUCTION OF CIDER BY INDUCTION OF ALCOHOLIC FERMENTATION AND MALOLACTIC CONVERSION

Characterization experiments involving lactic bacteria allowed a strain of Leuconostoc oenos to be selected in terms of growth capacity at variable pH, temperature, ethanol and sulphite concentrations, malic to lactic conversion yield, acetic acid uptake and dextran production capacity. Cider was produced under controlled conditions where the effect of Kloeckera apiculata yeasts on the development of Saccharomyces cerevisiae and production of major non-volatile compounds influencing end product quality was studied. The apiculate yeast was found to produce large amounts of acetic acid and use the other organic acids; also, it hindered fermentation to a certain extent. A study of the effect of the inoculation time with L oenos as an inducer of malolactic fermentation revealed sequential inoculation of the lactic bacterium once most major sugars in the must had been depleted to be the most favourable. Using yeast cell walls boosted fermentation development, as well as degradation of malic acid and synthesis of succinic and acetic acid.