The impact of different hop compounds on the growth of selected beer spoilage bacteria in beer

Beer spoiling lactic acid bacteria are a major reason for quality complaints in breweries around the world. Spoilage by a variety of these bacteria can result in haze, sediment, slime, off-flavours and acidity. As these bacteria occur frequently in the brewing environment, using certain hop products that inhibit the growth of these spoilers could be a solution to prevent problems. To investigate the impact of seven different hop compounds (α-acids, iso-α-acids, tetrahydro-iso-α-acids, rho-iso-α-acids, xanthohumol, iso-xanthohumol and humulinones) on the growth of six major beer spoilage bacteria (Lactobacillus brevis. L. backi, L. coryniformis, L. lindneri, L. buchneri, Pediococcus damnosous), two concentrations (10 and 25 mg/L) of each hop substance were added to unhopped beer. The potential growth of the spoilage bacteria was investigated over 56 consecutive days. A comparison of the results shows a strong inhibition of growth of all spoilage bacteria at 25 mg/L of tetrahydro-iso-α-acids closely followed by α-acids as the second most inhibitory substance. The results showed a high resistance of L. brevis to all hop compounds as well as an inhibition of L. coryniformis and L. buchneri at low concentrations of most hop components. In comparison with the control sample, L. lindneri showed increased growth in the presence of some hop compounds (rho-iso-α-acids, xanthohumol, iso-xanthohumol, humulinones). © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

LACTIC ACID PRODUCTION IN SWEET WORTS

The production of D-(-)-lactic acid in sweet wort was due to growth of Lactobacillus delbrueckii whereas L-(+)-lactic acid was produced by Bacillus coagulans. Incubation of sweet worts at 45°C favoured the production of D-(-)-lactic acid whereas L-(+)-lactic acid was produced in greatest amounts at 55°C. Low concentrations of hop iso-α-acids (4–12 ppm) inhibited the growth of both B. coagulans and L. delbrueckii at 55 °C.     

ESTIMATION OF α-, β- AND ISO-α-ACIDS IN HOPS,HOP PRODUCTS AND BEER USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Methods are described in which high performance liquid chromatography (HPLC) is used to estimate α-, β- and iso-α-acids in hops, hop products and beer. The chromatography relies on an isocratic elution of components from a polythene ‘cartridge’ column, and the method is calibrated with the pure substances as primary standards. Using such a column over 1000 analyses have been carried out without any significant loss in resolution or precision. The procedures are sufficiently rapid for use in commercial transactions and for quality control purposes. For hops and hop extracts coefficients of variation (%) of 2·5 and 0·8 were obtained respectively for α-acids. Values of 0·9 and 0·3 were obtained for iso-α-acids in isomerised extracts and beers respectively. For some isomerised extracts it has been observed that peaks in addition to those given by the iso-α-acids are present on the chromatogram. The current method recommended by the EBC over estimates the iso-α-acid content since these other constituents are included in the analysis.     

Analysis of hop acids by capillary electrophoresis

Micellar electrokinetic chromatography (MEKC) was used to separate components of hop extracts. The separation of a sample of iso-α-acids by MEKC was better and faster than by an established HPLC method, giving <0.8% RSD on migration times and 5–10% RSD on peak areas. MEKC was also successfully used to separate the oxidation products of the α- and β-acids and thus to monitor the stability of hop products containing them. Furthermore, MEKC distinguished among samples of reduced iso-α-acids (rho-, tetrahydro- and hexahydro- derivatives).     

ON THE SUNSTRUCK FLAVOUR OF BEER

It has been confirmed that sunstruck flavour is due to the formation of 3-methyl-2-butene-1-thiol formed by photolysis of iso-α-acids in the presence of sulphur-containing amino acids.     

酒花制品中异α-酸浓度的测定方法

采用分光光度法探索测定异α 酸、四氢异α 酸异构体、六氢异α 酸异构体等酒花制品中异α 酸的浓度 ,利用该方法对已知标准样品中异α 酸浓度进行检测 ,其重复性与精密度的结果表明 ,该方法的变异系数≤ 1 %,回收率在 99 8%～ 10 0 5 %之间 ;方法操作简单 ,可作为啤酒企业衡量评价异α 酸、四氢异α 酸异构体、六氢异α 酸异构体等酒花制品中异α 酸浓度的检测方法     

HUMULINIC ACID IN ISOMERIZED HOP EXTRACTS

Humulinic acid, which is not bitter, behaves similarly to iso-α-acids in some analytical estimations of beer bitterness. The humulinic acid content of a number of isomerized hop extracts was estimated using thin layer chromatography.     

PREPARATION,PURIFICATION AND SEPARATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF HUMULINIC ACIDS,DEHYDROHUMULINIC ACIDS,AND HULUPONES

Modification of published procedures are described for the preparation of humulinic acids, dehydrohumulinic acids and hulupone, using iso-α-acids as starting material. These procedures provide facile routes for preparation of these compounds in higher yields than was hitherto reported, and also in sufficient purity for their use as standards in quantitative High Performance Liquid Chromatography. An alternative and more feasible route to hulupones, using β-acids as starting material, is also outlined.     

Developmental changes of carbohydrates,organic acids,amino acids,and phenolic compounds in ‘Honeycrisp’ apple flesh

The developmental changes of carbohydrates, organic acids, amino acids and phenolic compounds in ‘Honeycrisp’ apple flesh were investigated using GC–MS and HPLC. A total of 12 carbohydrates, 8 organic acids, 20 amino acids, and 18 phenolic compounds were identified and quantified. Each metabolite showed characteristic changes during fruit development, but in general, concentrations of most sugars and sugar alcohols either increased or remained unchanged whereas concentrations of most organic acids, amino acids and phenolic compounds decreased with fruit development, indicating that most sugars and sugar alcohols are synthesised and/or accumulate at a faster or similar rate relative to fruit growth whereas organic acids, amino acids and phenolics are synthesised and/or accumulate at a slower rate relative to fruit growth. On a whole fruit basis, the content of most metabolites increased with fruit development. In the flesh of mature ‘Honeycrisp’ apple, fructose and sucrose and sorbitol are the major sugars and sugar alcohol; malic acid is the major organic acid; aspartic acid, asparagine, glutamic acid, proline, threonine and γ-aminobutyric acid are the major amino acids; and procyanidin B1, procyanidin B2, chlorogenic acid, catechin and epicatechin are the major phenolic compounds, respectively.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Potential impact of medium characteristics on the isomerisation of hop α-acids in wort and buffer model systems

Many brewing variables, e.g., pH, boiling time, temperature, and the presence of metal catalysts, are essential in view of the isomerisation of hop α-acids into iso-α-acids. In this work, a comparative study on the impact of several selected factors on α-acids isomerisation was performed in both buffer model solutions and wort, in order to find out enhanced boiling conditions for higher isomerisation yields.     

Practical implications of lactate and pyruvate metabolism by lactic acid bacteria in food and beverage fermentations

This article reviews the metabolism of pyruvate and lactate by lactic acid bacteria (LAB) involved in food and beverage fermentations with an emphasis on practical implications. First, the formation of pyruvate and lactate from a range of substrates, including carbohydrates, organic acids and amino acids, is briefly described. The catabolism of pyruvate and lactate by LAB is then reviewed. This is followed by a discussion of lactate degradation and racemisation by LAB from specific fermented foods and beverages. Finally, the impact of environmental factors and metabolic engineering on pyruvate and lactate metabolism by LAB is evaluated with regard to practical significance.     

Pediococcus damnosus strains isolated from a brewery environment carry the horA gene

Beer is recognized as a safe beverage, owing to its excellent microbiological stability provided by its components, especially iso‐α‐acids from hop and ethanol which have antimicrobial activity. Despite these unfavourable conditions for bacteria, some lactic acid bacteria (LAB) can cause beer spoilage. Resistance to hop compounds is caused, in part, by the product of genes like horA . Understanding how LAB adapts to hop compounds as well as quick detection of these microorganisms is necessary to ensure high‐quality beverages produced by the brewing industry. In this work, we searched for the presence of two main hop‐resistance genes, horA and ORF5, and determined the capacity of four strains of Pediococcus damnosus isolated from a brewery environment to grow in the presence of increasing concentrations of iso‐α‐acids. All strains were able to grow in increasing concentrations of iso‐α‐acids up to 150 μg mL⁻¹. This amount is 10 times greater than the concentration in average beer. Genetic amplification of genes associated with hop‐resistance, demonstrated the presence of horA , but not ORF5 in all tested strains. This communication represents the first report of the presence of horA gene in bacteria isolated from breweries in our country. Copyright © 2017 The Institute of Brewing & Distilling     

EFFECT OF STORAGE ON HOP EXTRACTS

Representative commercial hop extracts suffered no appreciable loss either of α- or β-acids or of bittering value when kept for 2 to 21 months in closed containers. Though there was loss of α- and β-acids on prolonged exposure of the extracts to air, it did not lead to a corresponding loss of bittering value. Of a range of commercial isomerized extracts, most proved satsifactorily stable on storage. However, when large losses of iso-α-acids did occur they were accompanied by corresponding reductions in bittering value. With reference to bittering compounds, there appeared to be no significant differences in the storage stability of beers bittered using hops or extracts of various types.     

Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, mainly formed by the microbial decarboxylation of amino acids. The consumption of food containing large amounts of some BAs can have toxicological consequences. Histamine is the most active BA and the most frequently involved in food-borne intoxications. This article reports the concentrations of histamine found in 80 cheeses made from different types of milk and subjected to different ripening periods. Histamine was detected in 41.25% of the samples, with values ranging from 20 to over 1000 mg per Kg of cheese. The highest histamine concentrations were recorded in long ripening cheeses made from raw milk. The presence and quantification of histamine-producing lactic acid bacteria was determined by RT-qPCR, and a good association was obtained between the C_t values and histamine concentrations determined by HPLC.     

GAS CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF SOFT RESINS AND RELATED COMPOUNDS

The α-acids, β-acids, iso-α-acids, hulupones and humulinic acid have been separated by gas chromatography utilising the cool on-column injection technique on various bonded phase capillary columns. The α-acids were separated into cohumulone and humulone; the β-acids into colupulone and lupulone; and the hulupones into cohulupone and hulupone. Iso-α-acids were separated into four peaks; cis-isocohumulone, trans-isocohumulone, cis-isohumulone and trans-isohumulone. Flame ionization detection (FID) and chemical ionization mass spectrometry (CIMS) were used for chromatographic detection. Best results were achieved by employing a DB-1 fused silica capillary column with isooctane as the sample solvent .     

Lactic acid bacteria decarboxylation reactions in cheese

Fermentation in cheese comprises oxidation-reduction of carbohydrates to yield organic acids, alcohols and carbon dioxide. Furthermore, organic acid and amino acid metabolism produces a series of compounds that positively or negatively affect final cheese quality. Under the strong selective pressure of the acidic environment of cheese ripening, lactic acid bacteria have developed multiple stress-resistant strategies, including decarboxylase and deiminase reactions that play a main physiological role during the ripening process of cheese production. The control of the expression and activity of these enzymes is one active strategy for intracellular acid-base homeostasis. This review covers relevant pathways and aspects related to gene regulation of gene clusters present in starter or non-starter lactic acid bacteria that are involved in sensory changes such as flavour development. From the point of view of food safety the main decarboxylation pathways that lead to the formation of biogenic amines are described.     

QUANTITATIVE ANALYSIS OF HOPS AND HOP EXTRACTS BY HIGH PRESSURE ION EXCHANGE CHROMATOGRAPHY

A new analysis for hop acids in hops and hop extracts is described. It is based on recent developments in high pressure liquid chromatography using pellicular anion exchange column filling material. Quantitative evaluation of α-acids, β-acids and oxidation products in hops and hop extracts is carried out by standard addition of pure humulone. The α-acids are completely separated from other hop substances before quantitation. The results of α-acids determinations must therefore be more accurate than was formerly possible and they are compared with conductometric titration results which are systematically higher. This is to be expected as it becomes more and more obvious that paper strip and conductometric analysis are not selective enough and determine fractions as “α-acids” which are in fact oxidation products of the hop acids.     

Structure determination and sensory evaluation of novel bitter compounds formed from β-acids of hop (Humulus lupulus L.) upon wort boiling

In order to screen for the bitter compounds generated from hop-derived precursors during the wort boiling process, an ethanolic hop extract was fractionated; the fractions obtained were thermally treated under model wort boiling conditions and, then, sensorially evaluated for their bitterness. Besides the isomerisation of the α-acids into the intensely bitter iso-α-acids, the bitterness of the fraction containing the β-acids was also found to be enhanced after wort boiling. To gain first insights into the β-acid-derived bitter compounds, the β-acid colupulone was isolated, thermally treated under wort boiling conditions and, then, investigated for bitter tasting degradation products by means of a taste dilution analysis (TDA). Besides the cohulupone, five previously unreported bitter-tasting colupulone degradation products, all of which exhibited a lingering, β-acid-like bitter taste with low recognition thresholds between 37.9 and 90.3 μmol/l, were isolated and their structures determined as two tricyclocolupone epimers, two dehydrotricyclocolupone epimers, and nortricyclocolupone, respectively, by means of LC–TOF–MS and 1D/2D-NMR spectroscopy.