Formation of styrene dependent on fermentation management during wheat beer production

Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reactions proceed in parallel to the decarboxylation of ferulic- and p-cumaric acid to 4-vinylguaiacol and 4-vinylphenol by the same decarboxylase enzyme. However, the formation of styrene occurs much faster and all available cinnamic acid in wort was converted completely within a few hours. Moreover, the comparison of various manufacturing parameters shows that a higher fermentation temperature of 25 °C compared to 16 °C and an open fermentation management lead to a rapid decrease of styrene. This allows minimising the content of styrene in beer while maintaining the typical wheat beer flavours.     

Efficient synthesis of hydroxystyrenes via biocatalytic decarboxylation/deacetylation of substituted cinnamic acids by newly isolated Pantoea agglomerans strains

BACKGROUND: Decarboxylation of substituted cinnamic acids is a predominantly followed pathway for obtaining hydroxystyrenes—one of the most extensively explored bioactive compounds in the food and flavor industry (e.g. FEMA GRAS approved 4‐vinylguaiacol). For this, mild and green strategies providing good yields with high product selectivity are needed. RESULTS: Two newly isolated bacterial strains, i.e. Pantoea agglomerans KJLPB4 and P. agglomerans KJPB2, are reported for mild and effective decarboxylation of substituted cinnamic acids into corresponding hydroxystyrenes. Key operational parameters for the process, such as incubation temperature, incubation time, substrate concentration and effect of co‐solvent, were optimized using ferulic acid as a model substrate. With strain KJLPB4, 1.51 g L^?1 4‐vinyl guaiacol (98% yield) was selectively obtained from 2 g L^?1 ferulic acid at 28 °C after 48 h incubation. However, KJPB2 provided vanillic acid in 85% yield after 72 h following the oxidative decarboxylation pathway. In addition, KJLPB4 was effectively exploited for the deacetylation of acetylated α‐phenylcinnamic acids, providing corresponding compounds in 65–95% yields. CONCLUSION: Two newly isolated microbial strains are reported for the mild and selective decarboxylation of substituted cinnamic acids into hydroxystyrenes. Preparative‐scale synthesis of vinyl guaiacol and utilization of renewable feedstock (ferulic acid extracted from maize bran) have been demonstrated to enhance the practical utility of the process. Copyright © 2011 Society of Chemical Industry     

Ferulic Acid Release and 4‐Vinylguaiacol Formation during Chinese Rice Wine Brewing and Fermentation

The release of ferulic acid (FA) and the subsequent enzymatic decarboxylation to 4‐vinylguaiacol (4‐VG) were nearly ubiquitous during the production of Chinese rice wine. To evaluate the release of FA and the transformation to 4‐VG, the levels of FA and 4‐VG were determined by high performance liquid chromatography (HPLC) and gas chromatography‐mass spectrum (GC‐MS), respectively. During rice wine brewing, the concentrations of FA and 4‐VG increased as fermentation time prolonged, with a rapid rise in days 1 to 5 of the main fermentation and followed by a slower rise from the 6th day post inoculation. FA and 4‐VG levels in mashes, fermented by yeast with wheat Qu, were significantly higher than those in mashes fermented by yeast using commercial enzyme additions. The release of FA from rice by wheat Qu involves an enzymatic release mechanism. The levels of FA and 4‐VG from rice flour by wheat Qu rose gradually during the first 24 h and then tended to stabilize. The capacity of yeasts to decarboxylate phenolic acids (Pof⁺ phenotype) was absent in most rice wine brewing yeasts. These results suggest that the production of FA and 4‐VG originated from materials in the wheat Qu rather than by the yeast during Chinese rice wine brewing and fermentation.     

Possibilities to improve the antioxidative capacity of beer by optimized hopping regimes

Different hopping regimes were evaluated to investigate the effect on the oxidative stability of wort and beer. Compared with a single hop dosage at the beginning of wort boil, it was possible to increase the concentration of α‐acids in pitching wort and beer by applying incremental hop dosage, dry hopping or the use of a pre‐isomerized hop product in combination with an α‐acid extract, which concomitantly resulted in lower iron concentrations and an enhanced flavour stability as indicated by standard wort and beer analyses, atomic absorption spectroscopy, electron spin resonance spectroscopy and sensory analysis of fresh and force‐aged beers. The functional principle of hop dosage variations is explained by saving of α‐acids throughout the wort production process, which yields an increased formation and precipitation of pro‐oxidative acting transition metal ions (e.g. Fe) in α‐acid‐complexes during the whirlpool rest and fermentation. Consequently, fewer reactive oxygen species are generated. Additional laboratory trials simulating wort cooling and beer storage in buffered model solutions proved that un‐isomerized α‐acids are strong iron chelators and confirmed the functional principle of the applied hopping regimes. Negative effects of higher α‐acid contents on fermentation performance and depletion of the zinc concentration, which is an essential nutrient for yeast, could be excluded. Copyright © 2014 The Institute of Brewing & Distilling     

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     

Transformation of substituted cinnamic acids using l-cysteine metal complexes in aqueous media

4-Hydroxycinnamic, 4-methoxycinnamic, ferulic and cinnamic acids were both non-oxidatively and oxidatively decarboxylated in alkaline aqueous media in the presence of l-cysteine–Fe(II) and l-cysteine–Co(II) heterogeneous catalysts using hydrogen peroxide or molecular oxygen. GC/MS analysis of diethylether extracts of reaction mixtures confirmed that the addition of hydrogen peroxide resulted predominantly in oxidative decarboxylation of substituted cinnamic acids, producing the corresponding carbonyl compounds (4-hydroxybenzaldehyde, 4-methoxybenzaldehyde, vanillin, benzaldehyde). On the other hand, saturation of this heterogeneous reaction system with molecular oxygen led to the formation of a variety of products, probably via peroxoacid anions or peroxoradical intermediates, e.g., ferulic acid was transformed to vinylguaiacol and vanillin with yields of 22% and 0.7%, respectively.     

Metabolism of ferulic acid during growth of Lactobacillus plantarum and Lactobacillus collinoides

BACKGROUND: Food‐isolated lactic acid bacteria can transform ferulic acid (FA) into several products. Since quantification of these metabolites during the different bacterial growth phases is lacking, the aim of this study was to identify and quantify conversion products of FA and to follow the kinetics of FA metabolism during growth of Lactobacillus plantarum and Lactobacillus collinoides. RESULTS: Lactobacillus plantarum and Lactobacillus collinoides were incubated in MRS broth, to which different amounts of FA were added (final concentrations of 0, 0.5, 1.5 and 3 mmol L^?1), at 30 °C until the late stationary phase. Lactobacillus plantarum metabolised FA into 4‐vinylguaiacol (4‐VG) and hydroferulic acid (HFA). Conversion to 4‐VG started simultaneously with the degradation of FA, while formation of HFA started in the mid‐exponential phase. Lactobacillus collinoides only formed 4‐VG, mainly in the stationary phase. No significant effect of the different amounts of FA was seen on the growth and fermentation characteristics of both bacteria. CONCLUSION: The results demonstrate that both bacteria are able to convert FA. However, start of conversion differs between the two strains. The different amounts of FA had no influence on the growth and fermentation characteristics of both bacteria. Copyright © 2012 Society of Chemical Industry     

Effect of thermal sterilization on ferulic, coumaric and cinnamic acids: dimerization and antioxidant activity

BACKGROUND: Some phenolic compounds, such as ferulic acid and p‐coumaric acid, exist in the form of free acids, in fruits, rice, corn and other grains. Thermal treatment (121 °C at 15–17 psi) for different times on ferulic, p‐coumaric and cinnamic acids as well as equimolar mixtures of these acids was investigated. RESULTS: Ferulic and p‐coumaric acids underwent decarboxylation, yielding dimeric products formed through their corresponding radical intermediates, while cinnamic acid was recovered unreacted. High‐performance liquid chromatography analysis showed no cross‐dimerization when equimolar mixtures of pairs of hydroxycinnamic acids were treated under the same conditions. Dimers were characterized as (E)‐4′,4″‐(but‐1‐ene‐1,3‐diyl)bis(2′‐methoxyphenol)) (dimer of 4‐vinylguaiacol) and (E)‐4,4′‐(but‐1‐ene‐1,3‐diyl)diphenol) (dimer of 4‐vinylphenol) by nuclear magnetic resonance and mass spectrometry. CONCLUSION: Sterilization by thermal processing produced dimers of ferulic and coumaric acid. The antioxidant activity of these dimers was greater than that of the respective hydroxycinnamic acids. These results may be relevant for fruits and grains that contain hydroxycinnamic acids and undergo sterilization processes such as canning. Copyright © 2012 Society of Chemical Industry     

THE STRUCTURAL AND STORAGE CARBOHYDRATES OF SACCHAROMYCES CEREVISIAE: CHANGES DURING FERMENTATION OF WORT AND A ROLE FOR GLYCOGEN CATABOLISM IN LIPID BIOSYNTHESIS

Detailed analysis of the structural and storage carbohydrates of Saccharomyces cerevisiae (NCYC 240) during wort fermentation showed that there were no significant changes in the amounts of trehalose or alkali-soluble glycogen. However, glucan and mannan individually increased from ca4% of the yeast dry weight at pitching to ca6% during the first 3–17 h of fermentation, butthen declined to the former level. In the first 2 h of fermentation, prior to yeast multiplication, acid-soluble glycogen was rapidly dissimilated from ca 40% to ca 6% of the yeast dry weight. During this period of oxygen uptake, wort sugars were not removed by the yeast. Glycogen, therefore, was the sole source of metabolic energy for lipid resynthesis and hexose transport appeared to require the formation of a component membrane. During the latter phase of fermentation when the yeast was not growing but expending energy for maintenance of cellular functions, glycogen reserves were slowly depleted; after a period of prolonged anaerobic storage, the content of glycogen fell well below that which was initially present in the pitching yeast.     

Determinants of flocculence of brewer's yeast during fermentation in wort

Ability of Saccharomyces cerevisiae MPY3 cells to flocculate during fermentation in wort was found to be triggered after growth limitation by oxygen shortage and to coincide with a sharp increase in cell surface hydrophobicity of the cells. Presence of oxygen in the pitching wort influenced final cell number, flocculence of the cells and cell surface hydrophobicity. Flocculation ability of cells grown in air-depleted pitching wort was hampered, concomitant with a decrease in final cell number and in final cell surface hydrophobicity. Addition of ergosterol and Tween 80 to air-depleted wort restored normal growth of the cells as well as flocculation ability and the increase in cell surface hydrophobicity. The same parameters increased in value after addition of ergosterol and Tween 80 to a fermentation with air-saturated pitching wort. Hydrophobicity of a non-flocculent mutant of S. cerevisiae strain MPY3, fermenting in air-saturated pitching wort, did not increase at cell division arrest. These results support the hypothesis that cell surface hydrophobicity is a major determinant for yeast cells to become flocculent, and suggest that shortage of sterols and unsaturated fatty acids precedes flocculence under brewing conditions.     

PITCHING RATE AND YEAST ACTIVITY DURING FERMENTATION OF BREWER'S WORT

Departure from a normal pitching rate during batch fermentation of brewer's wort by strains of Sacchoromyces cerevisiae results in disproportionate changes in the time needed for fermentation. Variations in pitching rate alter the ability of the yeast to utilize maltose and this mainly determines the rate of fermentation of the wort. These changes in yeast activity modify the effect of altered yeast concentration due to pitching rate and are responsible for the disproportionate changes in fermentation time.     

Comparative evaluation of the formations of gamma‐aminobutyric acid and other bioactive amines during unhopped wort fermentation

This study aimed to investigate the effect of Saccharomyces cerevisiae on the formation of gamma‐aminobutyric acid (GABA) and the other bioactive amines during wort fermentation. Within 8 days of fermentation, GABA concentration increased to 182.80 and 534.10 mg/L in unspoiled and spoiled worts, respectively. Although formation of tyramine and histamine did not occur in unspoiled wort, 142 mg/L of tyramine and 130 mg/L of histamine were found in spoiled wort at the end of fermentation. Decreased concentrations of tyrosine and histidine were associated with increased concentrations of tyramine and histamine, respectively, in spoiled wort. The results indicated that S. cerevisiae is a causative agent for the accumulation of GABA in wort during fermentation. Therefore, occurrence of GABA in beers should not be considered as one of the indicators of microbial contamination differently from tyramine and histamine.

Practical applications

Bioactive amines have important metabolic and physiological roles in the body. Their formation in foods is generally related to microorganisms having decarboxylase activity. It was found in this study that spoilage microorganisms produced tyramine and histamine while both spoilage microorganisms and Saccharomyces cerevisiae are responsible for the formation of gamma‐aminobutyric acid during unhopped wort fermentation.     

Impact of buffering capacity on the acidification of wort by brewing‐relevant lactic acid bacteria

Acidified wort produced biologically using lactic acid bacteria (LAB) has application during sour beer production and in breweries adhering to the German purity law (Reinheitsgebot ). LAB cultures, however, suffer from end product inhibition and low pH, leading to inefficient lactic acid (LA) yields. Three brewing‐relevant LAB (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7) were examined during batch fermentation of wort possessing increasing buffering capacities (BC). Bacterial growth was progressively impaired when exposed to higher LA concentrations, ceasing in the pH range of 2.9–3.4. The proteolytic rest (50°C) during mashing was found to be a major factor improving the BC of wort. Both a longer mashing profile and the addition of an external protease increased the BC (1.21 and 1.24, respectively) compared with a control wort (1.18), and a positive, linear correlation (R ² = 0.957) between free amino nitrogen and BC was established. Higher levels of BC led to significant greater LA concentration (up to +24%) after 48 h of fermentation, reaching a maximal value of 11.3 g/L. Even higher LA (maximum 12.8 g/L) could be obtained when external buffers were added to wort, while depletion of micronutrient(s) (monosaccharides, amino acids and/or other unidentified compounds) was suggested as the cause of LAB growth cessation. Overall, a significant improvement in LA production during batch fermentation of wort is possible when BC is improved through mashing and/or inclusion of additives (protease and/or external buffers), with further potential for optimization when strain‐dependent nutritional requirements, e.g. sugar and amino acids, are considered. Copyright © 2017 The Institute of Brewing & Distilling     

INFLUENCE OF NITRATE AND BACTERIAL CONTAMINATION ON THE FORMATION OF APPARENT TOTAL N-NITROSO COMPOUNDS (ATNC) DURING FERMENTATION

Formation of Apparent Total N-Nitroso Compounds (ATNC) was monitored throughout fermentations of all-malt ale worts supplemented with nitrate (0–100 mg Litre^?1 (0–100 ppm)). The pitching yeasts were obtained from commercial breweries and contained different levels of the contaminant bacterium Obesumbacterium proteus (0–2.1% by number). Levels of ATNC present at the end of fermentation were dependent on both initial wort nitrate levels and the initial level of bacterial contamination of the pitching yeast. Only relatively low nitrate levels were required to produce ATNC levels greater than the Brewers' Society recommended limit of 20 μg Litre^?1 (ppb), provided that the bacteria were present. Indeed, the use of whole hops alone would contribute sufficient nitrate to the wort to produce excessive amounts of ATNC, provided O.proteus was present. The only feasible solution to ATNC production during fermentation is to remove the contaminating bacteria from both the pitching yeast and brewing plant. Effective removal of O.proteus can be achieved by acid washing the pitching yeast under carefully controlled conditions, prior to fermentation.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

FERMENTATIVE CAPACITY OF YEASTS WHICH METABOLIZE MALTOTRIOSE RAPIDLY

A strain of Saccharomyces carlsbergensis which removes maltotriose particularly rapidly from wort exhibits a very high rate of fermentation of this sugar in isolation. This rate is quickly established after pitching and is maintained throughout the greater part of a batch fermentation despite a rapid decline in the ability of the cells to ferment glucose. This same strain and certain others of the same species which also remove maltotriose readily from wort are able to ferment the trisaccharide as fast as or even faster than maltose after growth on the disaccharide as sole carbon source; this property is usually accompanied by repression of glucose fermentation. Evidence is presented which indicates the presence, in these strains, of two or more α-glucosidases having different relative affinities for the oligosaccharides.     

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－）·     

The effect of pitching rate on fermentation,maturation and flavour compounds of beer produced on an industrial scale

The aim of the study was to determine the effect of the initial number of yeast cells in the wort on the process of fermentation, maturation and the content of the volatile components of beer, as well as the viability and vitality of the yeast biomass. The experiments were performed on an industrial scale, with fermentation and maturation in cylindro‐conical fermentation tanks with a capacity of 3800 hL. Yeast for pitching was collected after secondary fermentation (third passage) and wort pitching levels were 5 × 10⁶, 7 × 10⁶ and 9 × 10⁶ cells/mL. During fermentation and maturation, the changes in the content of the extract, yeast growth, yeast vitality and selected volatile components were investigated. Experiments showed that the yeast inoculum had a significant impact on the course of the fermentation and metabolic changes. With increasing numbers of cells introduced into the wort, the content of the esters and fusel alcohols increased, while the acetaldehyde concentration decreased. These changes affected the final quality of the beer. Copyright © 2015 The Institute of Brewing & Distilling     

Part II: the influence of the serial repitching of Saccharomyces pastorianus on the uptake dynamics of amino acids during the fermentation of barley and gluten‐free buckwheat and quinoa wort

The present paper is part of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of the barley, buckwheat and quinoa fermentation medium. In particular, it focuses on the uptake dynamics of amino acids during 11 successive fermentations. Samples were taken every 20 h after pitching, analysed for the particular amino acid content and statistically evaluated. The term ‘completion time’ (t₉₅), here defined as the percentage attenuation time necessary for ~95% of the total assimilation, has been introduced. In addition, ‘the serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Amino acids that were essentially affected by serial repitching were glutamine, arginine, alanine and tryptophan in barley, aspartate, glutamate and tryptophan in buckwheat, and all in the quinoa wort fermentation. As opposed to buckwheat and quinoa, in barley the amino acids behaved more or less independently from each other, which for buckwheat and quinoa indicates a more general systemic change in the yeast. From the amino acids point of view, buckwheat can be fully regarded as a suitable gluten‐free substitute for barley beer since the amino acid assimilation was very consistent and hardly influenced by the serial repitching, especially regarding the final amino acid assimilation. In the case of quinoa, the assimilation of all amino acids became significantly affected after the sixth fermentation and quinoa is probably unsuitable for the production of beer‐like beverages. Results suggest no substitutional potential of quinoa for barley beer, but if a nutrient‐rich beverage of choice from quinoa malt is intended to be prepared, it seems that the serial repitching is limited to six fermentations at most. Copyright © 2015 The Institute of Brewing & Distilling