Enzymatic formation of styrene during wheat beer fermentation is dependent on pitching rate and cinnamic acid content

Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reaction processes simultaneously to the decarboxylation of ferulic‐ and p‐cumaric acid to clove‐like 4‐vinylguaiacol and phenolic 4‐vinylphenol by the same PAD1 and FDC1 decarboxylase enzymes. However, the formation of styrene occurs much faster within the first hours of fermentation. In addition, the conversion of cinnamic acid starts immediately after pitching without an adaption of yeast on the new medium. Only after 120 min does the level of transposition decrease. Moreover, high cinnamic acid content in pitching wort, in combination with an open fermentation management, causes faster and higher styrene formation during this period. In contrast to the formation of 4‐vinylguaiacol, a correlation between pitching rate and styrene formation during open fermentation could be shown. The resulting time interval between styrene and 4‐vinylguaiacol formation provides scope for minimization strategies for styrene, while maintaining the typical wheat beer flavours. Copyright © 2012 The Institute of Brewing & Distilling     

Kinetic of the hydrolysis of pectin galacturonic acid chains and quantification by ionic chromatography

These experiments were designed to develop a rapid, repeatable and accurate analysis method for the quantification of galacturonic acid of pectins. Different pectin hydrolysis procedures (chemical and enzymatic) were carried out with H₂SO₄, TFA and HCl at different acid concentrations (0.2, 1 and 2 M) and temperatures (80 and 100 °C). Enzymatic and combined chemical and enzymatic hydrolysis of pectin were also studied. A acid hydrolysis under drastic conditions (100 °C) is insufficient for complete hydrolysis and results in low recovery of galacturonic acid residues. Mild chemical hydrolysis (0.2, 1 and 2 M H₂SO₄ 72 h at 80 °C) is also insufficient for complete depolymerization. Its main advantage is cleavage of the galacturonic acid chains into oligomeric forms without any degradation within 72 h of hydrolysis. However, enzymatic hydrolysis with VL9 for 2 h at 50 °C and combined chemical and enzymatic hydrolysis (0.2 M TFA at 80 °C for 72 h and VL9) give high recovery of this acid. Analysis of the liberated sugar residue by HPAEC allows us to determine the galacturonic acid composition of this polysaccharide accurately with high selectivity and sensitivity in one assay without the need for derivatization.     

糖化酵母在干啤酒生产中应用的研究（Ⅰ）──具有糖化酶活性和去除POF1基因的糖化酵母的选育

比较了多株单、双倍体糖化酵母，选出糖化酶活性及发酵度较高的糖化酵母单倍体──Ｓｏｃ．ｄｉａｓｔａｔｉｃｕｓ２６０６７－４４。同时发现糖化酵母单倍体菌株的酶活性高于二倍体菌株。含有ＰＯＦ１基因的糖化酵母（ｐｏｆ＋表型）可脱羧肉桂酸形成苯乙烯，ｐｏｆ－菌株无此能力。通过气相色谱检测肉桂酸－酒花麦汁发酵液中的苯乙烯含量，可以鉴别出去除ＰＯＦｌ基因的菌株。本文利用酵母菌的群体杂交法，通过Ｓ．ｄｉａｓｔａｔｉｃｕｓ２６０６７－４４与酿酒酵母单倍体杂交，获得１株糖化酶活性及发酵度较高，并且去除了ＰＯＦ１基因的单倍体菌株Ｈ－３（２）－２（ａ，ＳＴＡ，ｐｏｆ－）·     

Release of phenolic flavour precursors during wort production: Influence of process parameters and grist composition on ferulic acid release during brewing

In this study, the effects of mashing variables such as mashing-in temperature, time and pH, mash thickness, grist coarseness and composition, and stirring regime on the release of ferulic acid were examined. Ferulic acid is a precursor for the formation of flavour-active volatile phenols and a potent natural antioxidant in beer. Given one barley malt variety, the multitude of choice in setting various process parameters and adding brewery adjuncts during brewhouse operations can give rise to worts with widely varying ferulic acid levels. A clear difference in temperature- and pH-dependence between the release of the water-extracted and the enzymatically hydrolyzed fraction was found. The T,t-dependencies of arabinoxylan-degrading enzyme activities were correlated with ferulic acid release during mashing. Results from laboratory-scale mashing experiments were validated with those from a pilot-scale (5 h) wort production process. Enhancing the enzymatic release of phenolic flavour precursors from bound forms during mashing can greatly enhance the phenolic aroma potential of wort. Optimising this precursor release during mashing may be a means for controlling final volatile phenol levels in beer.     

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.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.     

CONTROL OF FERULIC ACID AND 4-VINYL GUAIACOL IN BREWING

Phenolic acids in beer are important because they can be decarboxylated to phenols, which usually impart off-flavours. An improved high performance liquid chromatographic system was used to monitor phenolic acids and phenols during the brewing process. Ferulic acid was the most significant phenolic acid found in beers prepared from malted barley. Extraction of ferulic acid from malt involved an enzymatic release mechanism with an optimum temperature about 45°C. Mashing-in at 65°C significantly decreased the release of free ferulic acid into the wort. Wort boiling produced 4-vinyl guaiacol by thermal decarboxylation, in amounts (0.3 mg/L) close to its taste threshold, from worts that contained high contents of free ferulic acid (> 6 mg/L). The capacity of yeasts to decarboxylate phenolic acids (Pof⁺ phenotype) was strong in wild strains of Saccharomyces and absent in all lager brewing yeast and most ale brewing yeasts. Some top-fermenting strains, especially those used in wheat beer production, possessed a weak decarboxylating activity (i.e. Pof^δ). During storage of beers there were appreciable temperature-dependent losses of 4-vinyl guaiacol. These results indicated that the production of 4-vinyl guaiacol is amenable to close technological control.     

125th Anniversary Review: Diacetyl and its control during brewery fermentation

Diacetyl is a butter‐tasting vicinal diketone produced as a by‐product of yeast valine metabolism during fermentation. Concentration is dependent on a number of factors including rate of formation of the precursor α‐acetolactate by yeast, spontaneous decarboxylation of this acetohydroxy acid to diacetyl and removal of diacetyl by yeast via the action of various reductase enzymes. Lowering concentrations of diacetyl in green beer represents an expensive and time‐consuming part of the brewing process and strategies to minimize diacetyl formation or hasten its reduction have potential for improving overall efficiency of the lager brewing system. Here we review the processes that determine diacetyl levels in green beer as well as the various ways in which diacetyl levels can be controlled. The amount of diacetyl produced during fermentation can be affected by modifying process conditions, wort composition or fermentation technique, or by yeast strain development through genetic engineering or adaptive evolution. The process of diacetyl reduction by yeast is not as well understood as the process of formation, but is dependent on factors such as physiological condition, cell membrane composition, temperature and pH. The process of diacetyl removal is typically rate‐limited by the reaction rate for the spontaneous decarboxylation of α‐acetolactate to diacetyl. Copyright © 2013 The Institute of Brewing & Distilling     

Apple polyphenoloxidase inactivation during heating in the presence of ascorbic acid and chlorogenic acid

It was recently reported that during osmotic dehydration of ascorbic acid (AA)-treated apple cubes, losses in AA and phenolics could partly arise from enzymatic oxidation, provided polyphenoloxidase (PPO) was still active under the processing conditions. To determine the impact of dehydration temperatures on PPO action, as well as chemical and enzymatic oxidation reactions, apple PPO inactivation alone or with AA (1 mM) and/or chlorogenic acid (CG, 3 mM), as well as AA and CG levels evolution, during heating of the model solutions at 45 and 60 °C were investigated. At pH 3.8, PPO was still functional, keeping 61 and 4% residual activity after 2 h of heating at 45 and 60 °C, respectively. The combined treatment of heating and AA was more effective in reducing PPO activity, while incubation at 60 °C with AA and CG minimised the losses of PPO activity. CG remained stable during heating, even in the presence of AA which, in turn, was more affected by heating. Thus, during heating, provided PPO remained active with enough available O₂ in the model systems, CG oxidation and coupled oxidoreduction with AA could readily develop.     

Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

Partial characterization of lipases produced by a newly isolated Penicillium sp. in solid state and submerged fermentation: A comparative study

The aim of this research was the partial characterization of enzymatic extracts produced by a newly isolated Penicillium sp. in submerged (SmF) and solid state fermentation (SSF). The partial characterization of the crude enzymatic extract obtained by SSF and SmF systems showed optimum activity at pH 5.5 and 47 °C, and pH 7.0 and 37 °C, respectively (15.17 U/mL and 11.28 U/mL). The crude enzymatic extracts obtained by SmF and SSF presented the best stability at pH from 4.9 to 8.5 and temperature from 25 °C to 35 °C and pH 7.0 and 25 °C, respectively. These results confirm the interesting potential of SSF, because, besides the higher activities obtained in this system, the half-life time at 25 °C was higher than that observed for the lipase extract obtained in the SmF system.     

An approximate shelf life prediction of elaborated lager beer in terms of degradation of its iso-α-acids

A 3-l laboratory-scale beer production protocol was designed and developed to produce beer. Concentrations of cis-/trans-iso-α-acids were quantified by HPLC. The trans-/cis-ratio varied from 0.62 to 0.49 after accelerated aging from 4 °C to 50 °C. The degradation of trans-iso-α-acids in elaborated pale lager beers followed first order reaction and this degradation conforms to the Arrhenius equation. The activation energy (E_a) and frequency factor (A) for trans-iso-α-acids in elaborated pale lager beer ranged from 69.2 kJ mol⁻¹ and 1.88 × 10¹⁰ days⁻¹ for trans-isocohumulone to 74.4 kJ mol⁻¹ and 1.3642 × 10¹¹ days⁻¹ for trans-isoadhumulone. The higher value of E_a demonstrated greater temperature sensitivity of trans-iso-α-acids during accelerated storage. The average half life of trans-iso-α-acids in elaborated pale lager beer was found to decrease from 471 days to 12 days when temperature increased from 4 °C to 40 °C. Using the activation energy of trans-α-acid degradation and the temperature profile of the accelerated aging, a mathematical model was employed to predict the loss of iso-α-acids, when the initial concentration of iso-α-acids in the product is known. The results obtained in the investigation can be of great importance to the industry in predicting the alteration of beer bitterness during warm periods and in tropical countries where summer temperature can reach 40 °C.     

Predicting acetic acid content in the final beer using neural networks and support vector machine

Most acetic acid found in beer is produced by yeast during fermentation. It contributes significantly to beer taste, especially when its content is higher than the taste threshold in beer. Therefore, the control of its content is very important to maintain consistent beer quality. In this study, artificial neural networks and support vector machine (SVM) were applied to predict acetic acid content at the end of a commercial‐scale beer fermentation. Relationships between beer fermentation process parameters and the acetic acid level in the fermented wort (beer) were modelled by partial least squares (PLS) regression, back‐propagation neural network (BP‐NN), radial basis function neural network (RBF‐NN) and least squares‐support vector machine (LS‐SVM). The data used in this study were collected from 146 production batches of the same beer brand. For predicting acetic acid content, LS‐SVM and RBF‐NN were found to be better than BP‐NN and PLS. For the comparison of RBF‐NN and LS‐SVM, RBF‐NN had a better reliability of model, but lower reliability of prediction. SVM had better generalization, but lower reliability of model. In summary, LS‐SVM was better than RBF‐NN modelling for the prediction of acetic acid content during the commercial beer fermentation in this study. Copyright © 2013 The Institute of Brewing & Distilling     

Effect of sterol biosynthesis-inhibiting (SBI) fungicides on the fermentation rate and quality of young ale beer

The influence of five sterol biosynthesis-inhibiting (SBI) fungicides (cyproconazole, diniconazole, epoxiconazole, flutriafol, and tebuconazole) on the fermentation and quality of young ale beer was assessed. Fungicides were individually supplied to the brewer wort to obtain a concentration of 0.2 μg/ml. A noticeable influence of the fungicide residues on the fermentation rate was observed in all cases. From the third day onwards, the fermentation rate was low in all treated samples. At the end of fermentation (8 days), statistically significant differences (p < 0.05) were found for the extract and attenuation values, which were higher for all the samples treated with fungicides. As a consequence, a higher amount of residual sugars (mainly maltose and maltotriose) was found in the beer. No significant differences were found for the pH and the polyphenol content after fermentation among the blank and the treated samples, while the values of colour intensity (lower) and tint (higher) in the beer were statistically different.     

Monitoring beer during storage by fluorescence spectroscopy

The present study demonstrates the use of fluorescence spectroscopy and multivariate data analysis for monitoring changes in beer during storage. Total luminescence spectra and synchronous scanning fluorescence spectra were recorded for fresh beer and beer samples stored in clear glass vials for three weeks in darkness at 4 °C and at 22 °C, and exposed to light at 22 °C, respectively. A pronounced decrease of fluorescence features ascribed to riboflavin was observed in samples exposed to light as compared to those kept in the dark. Principal component analysis of synchronous scanning fluorescence spectra revealed clear clustering of samples according to storage conditions. Successful classification of differently stored samples was accomplished using both the nearest neighbour method (kNN) and linear discriminant analysis (LDA).     

Apple pieces as immobilization support of various microorganisms

The biocatalyst prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on apple pieces was used for fermentation of grape must both in batch and continuous system. The immobilized yeast showed an important operational stability without any decrease of its activity. In continuous process, the bioreactor was operated for 95 days without any diminution of the ethanol productivity. Concentrations of higher and amyl alcohols were low indicating an improved quality product.Apple pieces were also used as support for immobilizing Kluyveromyces marxianus IMB3 for high-temperature (45 °C) wine-making. The fermented grape must contained 3-4% alcohol and semi-sweet wines were produced by the addition of potable alcohol.Preliminary sensory evaluation of the produced wines ascertained the fruity aroma and the overall improved quality compared to other commercially available products.Lactobaccillus casei cells immobilized on apple pieces were used for probiotic, fermented milk and lactic acid production. Apple-supported L. casei used for successive fermentation batches of whey proved to be very effective and suitable for food-grade lactic acid production. The immobilized biocatalyst was also used for milk fermentation and was able to ferment after storage for 15, 98 and 129 days at 4 °C, while no infection was reported during storage periods.     

Vitamins in brewing: presence and influence of thiamine and riboflavin on wort fermentation

Thiamine and riboflavin vitamers are present in a wide range of foods including beer. These vitamers play critical roles in a variety of enzymatic complexes and can promote and maintain metabolism. Currently, the presence and role of these vitamers in the malting and brewing industry have not been widely explored. This research investigated the effects of various fermentation conditions that may lead to the variations in the vitamin content in beer observed by previous researchers. The present research found that during fermentation, the thiamine content of wort is quickly utilized within the first 6 h of a standard fermentation and the uptake of this vitamin is not affected by increases in wort gravity. While no significant changes were observed in extracellular phosphorylated vitamers of thiamine, both free thiamine and thiamine diphosphate accumulated intracellularly during the wort fermentation. Meanwhile extracellular riboflavin vitamers were only poorly utilized during beer fermentations, however flavin mononucleotide rapidly accumulated intracellularly and more so under aerobic conditions. When yeast was exposed to an all‐malt high‐gravity wort, the thiamine or riboflavin utilization was not affected. However, thiamine utilization was reduced in adjunct‐driven high‐gravity worts. Notwithstanding the lowered thiamine uptake under high‐gravity conditions; there were some minor improvements in fermentation performance and yeast viability. The addition of thiamine to an all‐malt wort did appear to enhance yeast viability, both under normal and high‐gravity conditions. Copyright © 2016 The Institute of Brewing & Distilling     

Study on the buffering capacity of wort

The effect of the buffering capacity of wort on a beer fermentation and the contribution of pH, phosphate, amino acids and organic acids to the buffering capacity of wort were studied. The beer pH depended on the buffering capacity of the corresponding wort and an increase in the buffering capacity of the wort could prolong the time of diacetyl conversion. The higher the wort pH was, the larger the wort buffering capacity. Phosphate was not an effective buffer at the pH of wort. Glutamic acid, aspartic acid and histidine gave weak contributions to the buffering capacity of wort, and the total contribution of these three amino acids was <0.1. Organic acids contributed substantially to the buffering capacity of the wort and the total contribution of organic acids to the buffering capacity of the wort was estimated to be about 0.31. The buffering capacity of lactic acid, citric acid, succinic acid, fumaric acid and pyruvic acid was 30, 50, 77, 15 and 9% of that of acetic acid, respectively, at the same mass concentration. Copyright © 2015 The Institute of Brewing & Distilling     

The influence of wort aeration and yeast preoxygenation on beer staling processes

The influence of oxygen on the beer staling process is considered to be of major importance. Therefore, the impact of wort aeration, which is thought to cause wort oxidation processes, on beer ageing, has been examined. Pilsner and ale beers were produced with the classical wort aeration technique or by the use of the yeast preoxygenation process, in which yeast cells are exposed to oxygen before fermentation. The staling of these beers was studied using P&T GC–MS and sensory analysis. GC–MS analyses of the natural and forced aged beers showed no significant differences between the two treatments. Sensory evaluation of natural and forced beers confirmed these results. Thus, normal wort aeration (8 mg/l) does not appear to determine flavour stability in a direct manner. This is probably due to the short contact of the wort with oxygen at low temperatures before the onset of fermentation.     

控制啤酒中残留草酸含量的探讨

通过对酿造原料（包括麦芽、大米、酒花）的考察，发现不同的麦芽品种中草酸含量各不相同，酒花对麦汁中的草酸含量影响较大，添加辅料大米有助于降低草酸，酵母发酵对草酸含量影响很小。当麦汁中的钙离子含量达到80mg／L时，啤酒中的钙离子浓度在60mg／L，啤酒中的草酸15mg／L左右。