ESTIMATION OF FREE FATTY ACIDS (C8-C18) IN WORT AND BEER

A procedure is described for estimating the free fatty acids in wort and beer by gas liquid chromatography. The free fatty acids with carbon number 8 to 18 are estimated simultaneously from one injection, and by including two internal standards the reproducibility of the method is maintained over the whole range. It has been demonstrated that other lipid materials such as glycerides and esters of fatty acids do not interfere with the assay. Compared with other methods, this procedure is fairly rapid and is suitable for routine purposes. The reproducibility of the method has been assessed by analysing replicate samples of wort and beer and the precision in estimating individual fatty acids is given.     

METHODS FOR THE ANALYSIS OF C4 TO C10 FATTY ACIDS IN BEER,WORT AND CARBOHYDRATE SYRUPS

Methods have been developed for the analysis of C4 to C10 fatty acids in beer, wort and carbohydrate syrups. The methods are based on the adsorption of the aqueous samples on to solid supports followed by removal of the fatty acids with organic solvents. The methods are suitable for routine batchwise analysis of fatty acids and the reproducibility of the analysis and recoveries of the acids are generally good. Problems often encountered with analysis of C4 to C10 fatty acids and possible solutions are also discussed.     

RAPID METHODS FOR THE ANALYSIS OF THE FATTY ACIDS OF FERMENTING WORT AND BEER (C6-C10) AND YEAST (C6-C18)

Relatively rapid and simple methods for measuring the content of the free C₆-C₁₀ fatty acids in fermenting wort and beer and of the total C₆-C₁₈ fatty acids in yeast are described. The acids are extracted, after saponification in the case of yeast, using a small volume of chloroform and analysed by gas chromatography as free fatty acids. The precision of the procedures is reasonable.     

THE EFFECT OF CARBOHYDRATE ADJUNCTS ON BREWER'S WORT FERMENTATION BY SACCHAROMYCES UVARUM (CARLSBERGENSIS)*

The use of carbohydrate adjuncts such as sucrose, fructose and glucose in brewer's wort significantly modifies the initial wort sugar spectrum and also the pattern of sugar uptake during fermentation by a strain of Saccharomyces uvarum (carlsbergensis). Under these conditions, the concentration of glucose and fructose in the wort was observed to increase when compared to worts in which corn starch was employed as an adjunct and glucose was taken up at a faster rate than fructose. The increase in glucose concentration in the wort also resulted in severe repression of maltose and maltotriose utilization with significant levels of these sugars remaining in the beer produced.     

THE IDENTITY OF AN INHIBITOR IN WORT OF DIMETHYL SULPHOXIDE REDUCTASE FROM YEAST

Wort contains a substance which inhibits the reduction of dimethyl sulphoxide by yeast. The inhibitor has been purified by dialysis followed by chromatography on Sephadex G-15 and Dowex 50. It has been identified as methionine sulphoxide.     

不饱和脂肪酸分离技术研究概况

不饱和脂肪酸作为重要的膳食脂肪酸,具有特殊的理化性质和独特的生理功能。对目前采用的不饱和脂肪酸主要分离技术进行了综述,并分析了各自的优缺点。     

AN UPDATE OF ZINC ION AS AN EFFECTOR OF FLOCCULATION IN BREWER'S YEAST STRAINS

Zn⁺⁺ ion was observed to be a strong effector of the flocculation—deflocculation process in an in vitro system for strains of Saccharomyces cerevisiae at ion concentration ranges that are normal in conventional substrates, such as wort. All strains of Saccharomyces uvarum (carlsbergensis) examined in this study did not exhibit any flocculation response to Zn⁺⁺ ion. This test could be employed to distinguish between ale and lager flocculating yeast strains.     

YEAST GROWTH IN RELATION TO THE DISSOLVED OXYGEN AND STEROL CONTENT OF WORT

The extent of the requirement for oxygen in cells of brewing yeast is determined by the availability of oxygen during propagation. Cells with no oxygen requirement ferment satisfactorily when added to either air-saturated or de-aerated wort. Cells produced during fermentation develop an oxygen-requirement and ferment poorly when added to de-aerated wort because of restriction of both rate and extent of exponential growth. The quantity of dissolved oxygen needed to ensure satisfactory growth varies greatly with yeast strain. In all cases examined, the oxygen requirement can be eliminated by addition to the growth medium of ergosterol and Tween 80 However Tween 80 alone is without effect. It seems likely that oxygen is required because it is essential for biosynthesis of sterols.     

MINIMUM REQUIREMENTS FOR ZINC AND MANGANESE IN BREWER'S WORT

Five procedures for the extraction of metals from wort are described. Using worts with defined metal content the essential nature of zinc and manganese in fermentation has been confirmed. Despite this, zinc appeared to prevent yeast growth and fermentation when present at 0.6 ppm in a wort containing less than 0.01 ppm of manganese. This inhibition was released on addition of 0.6 ppm of manganese to the wort and it seems that the desirable concentration of any one metal ion in a wort depends to some extent on the concentration of the other related metals present.     

THE ORIGIN OF THE MEDIUM CHAIN LENGTH FATTY ACIDS PRESENT IN BEER

The medium chain length fatty acids that are excreted during fermentation are produced by synthesis and not by degradation. The fermentation of a wort supplemented with propionic acid (C₃) or valeric acid (C₅) leads to the excretion of nonanoic acid (C₉) in addition to the usual even chain acids. C₉ acid was not detected in the beer when the inoculated yeasts contained a high proportion of pentadecanoic acid (C₁₆) and heptadecanoic acid (C₁₇) or when the C₁₇ acid was added to the wort, demonstrating that a degradative route is unimportant. The content of the medium chain length fatty acids in beer varies directly with their content in yeast; thus the fatty acid composition of the beer reflects changes in the content of these acids in yeast brought about by alteration in the supply of oxygen or by the addition of C3 acid to wort.     

EFFECT OF YEAST ADAPTATION TO MALTOSE UTILIZATION ON SUGAR UPTAKE DURING THE FERMENTATION OF BREWER'S WORT

Yeast adaptation to maltose utilization diminishes the repressing effect of glucose on maltose uptake. Furthermore, it affects both the rates and profiles of maltose, glucose and maltotriose uptake during high cell density brewer's wort fermentation. Yeast cells pre-grown in maltose, as sole carbon source, and harvested while the sugar is still present in the growth medium, are better adapted to utilize maltose. The adapted cells are less sensitive to glucose inhibition, and the uptake of glucose is inhibited in the early stages of fermentation. Cells grown for longer periods and harvested following maltose depletion, lose their ability to preferentially utilize maltose. In addition, they become more sensitive to glucose repression, and are able to utilize glucose faster than the cells harvested when maltose is still present in the medium. During the process of adaptation, maltose controls the induction of its own transport systems and appears to affect the biosynthesis of the glucose transport systems.     

LIPID COMPOSITION OF AEROBICALLY AND ANAEROBICALLY PROPAGATED BREWER'S BOTTOM YEAST

Aerobically grown pitching yeast is very rich in unsaturated fatty acids and sterol esters compared to traditional, anaerobic yeast. The principal fatty acids in aerobic yeast cells are unsaturated palmitoleic and oleic acids, whereas in anaerobic cells saturated palmitic acid predominates. The difference in fatty acid distribution between aerobic and anaerobic cells is most marked in the sterol esters. The fatty acids of phospho-lipids are more stable, although remarkable differences are observed. The sterols of aerobic cells are almost entirely in esterified form and zymosterol is the principal sterol. During the first hours of fermentation a rapid synthesis of palmitoleic acid is observed when anaerobic yeast is used for pitching and the wort is aerated. The synthesis of oleic acid requires more oxygen and time than is available under normal brewing conditions. When aerobic pitching yeast is used no more unsaturated fatty acids are synthesised and the lipid stores of pitching yeast are distributed among the daughter cells. The decrease in acetate ester production by aerobic pitching yeast is concluded to be due to a decrease in acetyl CoA synthesis, which may be caused by the high proportion of unsaturated fatty acids in membrane lipids.     

THE INFLUENCE OF LIPIDS DERIVED FROM MALT SPENT GRAINS ON YEAST METABOLISM AND FERMENTATION

The addition to wort of lipids derived from malt spent grains had a pronounced effect on yeast metabolism. The lipids allowed the fermentation of de-oxygenated wort and also stimulated yeast growth and the corresponding rate and extent of fermentation of air-saturated wort by yeast strains having a high oxygen requirement. The lipids increased the fusel alcohols content of beer and decreased the content of esters and medium chain-length fatty acids. The yeast incorporated sitosterol and unsaturated fatty acids from the spent grain lipids and the unsaturated fatty acids changed the pattern of fatty acids and sterols synthesized by the yeast. The fatty acids were present in the spent grain lipids mainly as triglycerides, free fatty acids and phospholipids. Using pure lipid compounds it was shown that the triglycerides were inactive and that the spent grain lipids exerted their effect on fermentation through the synergistic action of free unsaturated fatty acids, sitosterol and phospholipid. Phospholipid could be replaced by the detergent, Triton X-100. The effect of the lipids on the synthesis of esters, fusel alcohols and medium chain fatty acids could be explained solely by their content of unsaturated fatty acids.     

MALT FLAVOUR — TRANSFORMATION OF CARBONYL COMPOUNDS BY YEAST DURING FERMENTATION

Laboratory and pilot scale fermentations have been carried out using worts spiked with a range of carbonyl compounds, many of which are derived from malt. Aldehydes and vinyl ketones were chemically reduced during fermentation and could not be detected in the resulting beers; on the other hand certain saturated and non-conjugated unsaturated ketones were only partially reduced. The corresponding saturated alcohols and acetate esters were usually detected as transformation products.     

THE CONTROL OF VOLATILE ESTER SYNTHESIS DURING THE FERMENTATION OF WORT OF HIGH SPECIFIC GRAVITY

Some effects have been studied of fermenting wort of unusually high specific gravity, followed by dilution with water to give beer of normal original gravity. This procedure permits increased overall rates of beer production, but matching of flavours requires control of the level of flavour determinants. If not controlled, the concentration of volatile esters may be disproportionately increased so that, after dilution, beer flavour is markedly different. Higher alcohol production is not affected in this way. The concentration of esters can be adjusted to appropriate levels by increasing the production of yeast mass during fermentation. Unsaturated fatty acids, which increase yeast dry matter production without altering the rate of fermentation, are particularly effective in reducing the extent of ester synthesis.     

STRAIN SPECIFIC RESPONSE OF BREWER'S YEAST STRAINS TO ZINC CONCENTRATIONS IN CONVENTIONAL AND HIGH GRAVITY WORTS*

The effect of zinc on a variety of yeast strains is extensively documented in the literature. However, due to the varied experimental protocols employed in each study there is little opportunity to directly compare the strain specificity of this ion. In the present study, the response of six yeast strains (three Saccharomyces cerevisiae (ale type) and three S.cerevisiae (lager type)) to altered zinc concentrations, in both high (1080 OG) and conventional (1048 OG) gravity worts, was investigated. Varying the initial wort zinc concentration in both gravities had an effect on the ethanol production, rate of fermentation, cell number and sugar uptake of all six strains studied. The extent of the response was found to be dependent upon the zinc concentration, the strain employed and wort gravity employed.     

METHODS FOR THE DETERMINATION OF VOLATILE NITROSAMINES IN MALT,WORT AND BEER

Methods for the analysis of beer, wort and malt for NDMA at the one in 10⁹ level using the thermal energy analyzer have been evaluated. The beer and wort procedure using Preptube extraction has been shown to give excellent results. For malt, the preparation of an aqueous extract, followed by analysis by the beer procedure, also gives excellent results. Vacuum distillation from mineral oil has also been examined for the analysis of malt. Providing sufficient aqueous phase is initially present, this procedure yields results similar to those from aqueous extraction. The methods will also detect NDEA, although no NDEA has been observed in any of the beer, wort or malt samples examined. NDMA has been observed to be present in bound forms in malt and Preptubes and is released by the addition of water     

THE INFLUENCE OF ASSIMILABLE NITROGEN COMPOUNDS IN WORT ON THE ABILITY OF YEAST TO REDUCE DIMETHYL SULPHOXIDE

Dimethyl sulphoxide (DMSO) reductase activity increased at the end of the growth cycle of Saccharomyces cerevisiae NCYC 240 on complex medium. On defined medium, DMSO reductase and thioredoxin reductase activities and thioredoxin were all elevated in cultures limiting in nitrogen, but not in nitrogen-sufficient cultures. In nitrogen limiting cultures it was also shown that the reduction of DMSO (measured as disappearance of [¹⁴C] from the medium) was much increased compared with conditions of nitrogen excess. This applied whether ammonium or glutamate was the limiting nitrogen source. When methionine was used as the limiting nitrogen source, DMSO reduction was higher still. Methionine-limited cells accumulated [¹⁷C] methionine much more rapidly than did cells from methionine-sufficient cultures, and [¹⁴C] methionine sulphoxide could be identified in the cells after 1 min incubation with labelled methionine. It is concluded that DMSO reductase activity is controlled by nitrogen catabolite repression and may be involved in methionine uptake by yeast. The amino nitrogen content of wort will in consequence have an important effect on levels of dimethylsulphide (DMS) produced during fermentation.     

CONSTRUCTION OF FLOCCULENT BREWER'S YEAST BY CHROMOSOMAL INTEGRATION OF THE YEAST FLOCCULATION GENE FLO1

Yeast flocculation gene FLO1, located on chromosome I of Saccharomyces cerevisiae, has been cloned previously¹⁶. However, it has recently been found that the gene was an in-frame deletion derivative of the chromosomal intact FLO1 gene¹⁹. When introduced into non-flocculent industrial strains, including brewer's yeast, the latter gene, FLO1L, containing an open reading frame of 4,611 bp, conferred stronger flocculation than the former gene, FLO1S, containing an open reading frame of 2,586. By chromosomal integration of the ADH1-controlled FLO1L gene, “gene therapy” of the flocculation behaviour of the parent non-flocculent brewer's yeast was successfully achieved.     

DISPOSAL OF EXCESS BREWER'S YEAST BY RECYCLING TO THE BREWHOUSE

Waste streams high in B.O.D. are produced by the brewing process itself. These wastes are wholesome but unwanted materials that were once part of or were in intimate contact with the brewing milieu. They are not inherently foreign to the process or the product, and disposal could be accomplished by recycling to the process. We have studied the effect on brewing processes and beer flavour of recycling waste yeast slurry to the mash mixer. Beer flavour is essentially unaffected by recycling, and of the brewhouse processes only lautering is slowed at higher recycle rates. Extract (up to 1% of the total) and soluble nitrogen can be recovered from the recycled yeast, but is strongly influenced by temperature during yeast storage and mashing. Worts made with recycled yeast tend to ferment more rapidly than normal worts and tend to have a lower end gravity.