Application of plant-based antimicrobials for the growth inhibition of clostridia in pressed beet pulp silage

BACKGROUND: In this study the inhibition of hop beta acids on the growth of clostridia in soil‐contaminated pressed sugar beet pulp silages was investigated. Hop beta acids are natural substances which display their effect at low concentrations. Fresh pressed beet pulp material was mixed with soil to artificially contaminate it with clostridia. Laboratory silos were filled with the substrate, stored at 25 °C and opened for sampling at 0, 2, 8, 15, 30, 60, and 90 days. The impact on clostridial growth during silage fermentation was monitored by determination of the pH value and dry matter content, as well as chemical analysis of the fermentation products. Throughout the experiments, the effect of a commercial silage inoculant based on lactic acid bacteria (LAB) and hop‐resistant LAB were examined with and without the combination of plant‐based antimicrobials. RESULTS: Results indicate that in contaminated silage samples without any additives high butyric acid contents occurred due to clostridial growth. This spoilage could not be suppressed by the application of LAB, whereas the combined application of LAB and hop beta acids significantly improved silage quality, which was reflected by favourable organic acid composition (P < 0.05). CONCLUSION: The experimental data indicate that the application of hop beta acids improves the preservation effect of LAB in suppressing clostridial growth in silages and thus demonstrates some potential for the combined use of plant‐based antimicrobials and LAB. Copyright © 2011 Society of Chemical Industry     

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     

CAMBRIDGE PRIZE LECTURE. STUDIES ON THE SENSITIVITY OF LACTIC ACID BACTERIA TO HOP BITTER ACIDS

Hop bitter acids act as mobile-carrier ionophores. They inhibit the growth of beer-spoilage bacteria by dissipating the transmembrane pH gradient. Their activity is pH dependent. Low pH favours antibacterial activity but high pH reduces it. Resistance to hop bitter acids is a stable character, associated only with beer-spoilage lactic acid bacteria. Hop-resistant organisms can maintain a larger transmembrane pH gradient and ATP pool than can hop-sensitive organisms. Prior exposure of bacteria to trans-isohumulone does not influence the degree of resistance to hop bitter acids. However, in some strains, exposure to trans-isohumulone does induce the ability to spoil beer. The chemistry of these compounds is more complex than previously thought. In aqueous solutions, such as beer, hop acids bind to metal ions and may be covalently hydrated.     

An improved HPLC method for single‐run analysis of the spectrum of hop bittering compounds usually encountered in beers

A single‐run reverse phase‐high performance liquid chromatography method for the quantification of humulinones, α‐acids, iso‐α‐acids and reduced iso‐α‐acids (where present) in commercial beer samples is presented. The method utilizes a binary solvent system consisting of (A) 1% v/v acetic acid and (B) 0.1% v/v orthophosphoric acid in acetonitrile. Separation was achieved on a Purospher® star RP‐18 column (250 × 46 mm, 3 µm) with a flow rate of 0.5 mL/min. The compounds of interest eluted within 32 min. The method was fully validated according to International Conference on Harmonization guidelines and subsequently applied to monitor degradation of hop acids in a storage trial where four lager beers were aged at 28 and 38 °C for 70 and 60 days, respectively. Results confirmed the widely reported degradation through storage of trans‐iso‐α‐acids whilst demonstrating that the HLPC method was sufficiently sensitive to monitor and model this degradation. One beer exhibited a significantly lower (P < 0.05) rate of trans‐iso‐α‐acid degradation than the other conventionally hopped beers in the study, which might have been linked to its higher pH (4.71 vs 4.36). The relative stability of reduced iso‐α‐acids during ageing was also confirmed.     

Assessment of changes in hop resins and polyphenols during long‐term storage

Changes in the content and composition of hop secondary metabolites during storage are reflected in beer quality and in the economics of beer production. A 12‐month storage experiment with T90 pellets of four hop varieties showed different dynamics of hop aging in relation to both storage conditions and hop variety. Negligible effects on the α‐ and β‐acids were detected during storage without air access at +2°C. Storage at +20°C resulted in a final loss of 20–25% α‐acids, but the content of β‐acids did not change significantly. Large decreases in α‐acids (64–88%) and in β‐acids (51–83%) were found in hops stored with access to air at +20°C. The rate of decline accelerated markedly after 6 months of storage. In terms of hop resin changes, Premiant and Sládek were the most and the least stable varieties, respectively. After 12 months, the content of the total polyphenols and flavonoids decreased by 30–40% and by 20–30%, respectively, irrespective of storage conditions. The rate of decline accelerated strongly after 6 months. The DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) antiradical potential decrease was significant only in hops stored under aerobic conditions. The depletion was 9–25% after 1 year; Saaz was the most stable variety. Copyright © 2012 The Institute of Brewing & Distilling     

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     

Effects of hop varieties on ruminal fermentation and bacterial community in an artificial rumen (rusitec)

BACKGROUND: There is a growing interest in the use of hops (Humulus lupulus) as an alternative to antibiotics to manipulate ruminal fermentation. However, the effects of different hop varieties on ruminal fermentation and bacterial populations have not been studied. Here the effects of three hop varieties, Cascade (CAS), Millennium (MIL) and Teamaker (TM), at a level of 800 µg mL^?1 inoculum on ruminal fermentation and microbial populations in an artificial rumen system (rusitec) fed a barley silage‐based total mixed ration were investigated. Bacterial populations were assessed using real‐time polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies. RESULTS: All hops reduced (P < 0.001) total gas, methane and the acetate:propionate ratio. Liquid‐associated Fibrobacter succinogenes, Ruminococcus albus and Streptococcus bovis were reduced (P < 0.05) by MIL and TM. Feed particle‐associated S. bovis was reduced (P < 0.01) by MIL and TM, but TM and CAS increased (P < 0.01) Ruminobacter amylophilus and Prevotella bryantii respectively. Methanogens were decreased (P < 0.05) by MIL in both liquid and solid fractions and by CAS in the solid fraction. The total amount of α‐ and β‐acids in hops affected the ruminal fermentation. CONCLUSION: Hop‐induced changes in fermentation and microbial populations may improve energy efficiency use in the rumen. Further research is needed to determine the effects of hops on in vivo ruminal fermentation, microbial populations and animal performance. Copyright © 2012 Society of Chemical Industry     

A Review of Hop Resistance in Beer Spoilage Lactic Acid Bacteria

Hop bitter acids play a major role in enhancing the microbiological stability of beer. However, beer spoilage lactic acid bacteria (LAB) are able to grow in beer by exhibiting strong hop resistance. Recently two hop resistance genes, horA and horC, have been identified in beer spoilage Lactobacillus brevis ABBC45. The horA gene was shown to encode an ATP dependent multidrug transporter that extrudes hop bitter acids out of bacterial cells. In contrast, the product of the horC gene confers hop resistance by presumably acting as a proton motive force (PMF)‐dependent multidrug transporter. Strikingly, the homologs of horA and horC genes were found to be widely and almost exclusively distributed in various species of beer spoilage LAB strains, indicating these two hop resistance genes are excellent species‐independent genetic markers for differentiating the beer spoilage ability of LAB. Furthermore the nucleotide sequence analysis of horA and horC homologs revealed that both genes are essentially identical among distinct beer spoilage species, indicating horA and horC have been acquired by beer spoilage LAB through horizontal gene transfer. Taken collectively, these insights provide a basis for applying horA and horC to the species‐independent determination of beer spoilage LAB, including yet uncharacterized species. In addition to the hop resistance mechanisms mediated by multidrug transporters, proton translocating ATPase and the ATP production system were shown to contribute to the hop resistance mechanisms in beer spoilage LAB by generating PMF and ATP that are necessary for survival in beer.     

Humulus lupulus – a story that begs to be told. A review

The hop cones of the female plant of the common hop species Humulus lupulus L. are grown almost exclusively for the brewing industry. Only the cones of the female plants are able to secrete the fine yellow resinous powder (i.e. lupulin glands). It is in these lupulin glands that the main brewing principles of hops, the resins and essential oils, are synthesized and accumulated. Hops are of interest to the brewer since they impart the typical bitter taste and aroma to beer and are responsible for the perceived hop character. In addition to the comfortable bitterness and the refreshing hoppy aroma delivered by hops, the hop acids also contribute to the overall microbial stability of beer. Another benefit of the hop resins is that they help enhance and stabilize beer foam and promote foam lacing. In an attempt to understand these contributions, the very complex nature of the chemical composition of hops is reviewed. First, a general overview of the hop chemistry and nomenclature is presented. Then, the different hop resins found in the lupulin glands of the hop cones are discussed in detail. The major hop bitter acids (α‐ and β‐acids) and the latest findings on the absolute configuration of the cis and trans iso‐α‐acids are discussed. Special attention is given to the hard resins; the known δ‐resin is reviewed and the ε‐resin is introduced. Recent data on the bittering potential and the antimicrobial properties of both hard resin fractions are disclosed. Attention is also given to the numerous essential oil constituents as well as their contributions to beer aroma. In addition to the aroma contribution of the well‐known essential oil compounds, a number of recently identified sulfur compounds and their impact on beer aroma are reviewed. The hop polyphenols and their potential health benefits are also addressed. Subsequently, the importance of hops in brewing is examined and the contributions of hops to beer quality are explained. Finally, the beer and hop market of the last century, as well as the new trends in brewing, are discussed in detail. Hop research is an ever growing field of central importance to the brewing industry, even in areas that are not traditionally associated with hops and brewing. This article attempts to give a general overview of the different areas of hop research while assessing the latest advances in hop science and their impact on brewing. Copyright © 2014 The Institute of Brewing & Distilling     

Effects of Mentha longifolia L. essential oil and Lactobacillus casei on the organoleptic properties and on the growth of Staphylococcus aureus and Listeria monocytogenes during manufacturing,ripening and storage of Iranian white‐brined cheese

This research evaluated the effects of Mentha longifolia L. essential oil (EO) in concentrations 0, 50, 150 and 300 ppm and Lactobacillus casei (10⁸–10⁹ CFU/mL) on the growth of Staphylococcus aureus and Listeria monocytogenes during the manufacturing, ripening and storage of Iranian white‐brined cheese. The growth of the two pathogens was significantly reduced (P < 0.01) by both EO concentrations ≥50 ppm and probiotic and their combination in the standard manufacturing and storage process conditions of the cheese. Furthermore, the treatment containing 150 ppm of this EO combined with probiotic had a favourable inhibitory effect on the growth of two pathogenic micro‐organisms and also was the most appropriate treatment in sensory assessment. The synergistic effects of the above‐mentioned concentration level between the essential oil and probiotic were significant compared to other treatments, including essential oil and probiotic only. Thus, a lower concentration of this EO can be used when it is combined with this probiotic.     

Thymol and modified atmosphere packaging to control microbiological spoilage in packed fresh cod hamburgers

A study on the use of mild technologies to produce packaged fish hamburgers was presented. In particular, the antimicrobial effect of some natural compounds (carvacrol, eugenol, thymol, green tea extract, rosemary extract, grapefruit seed extract and lemon extract), at various concentrations (500–10 000 ppm), was screened in vitro against the main fish spoilage micro‐organisms (Shewanella putrefaciens and Photobacterium phosphoreum). Lemon extract and thymol, in combination with modified atmosphere packaging, showed the greatest inhibition activity, therefore, thymol was subsequently used as an ingredient for producing fish hamburgers. Results pointed out that this combination is effective in controlling the growth of microbial species mainly involved in fresh fish spoilage; in particular, it significantly (P < 0.05) reduced the growth rate of bacterial population, performing about 4.8 log CFU g^?1 and 6.5 log CFU g^?1 reduction of the hydrogen sulphide producing bacteria and psychrotrophic aerobic specific spoilage organisms cell load, respectively, if compared with the control.     

Impact of Different Hop Compounds on the Overfoaming Volume of Beer Caused by Primary Gushing

When weather conditions favour the growth of moulds on barley, beers brewed from the resulting malts often tend to gush. Certain Fusarium species (e.g., F. graminearum and F. culmorum) may cause this problem. Supersaturated with CO₂, a primary gushing beer contains an overcritical concentration of microbubbles; these are reputed to be stabilised by Fusarium‐derived hydrophobins. Research with varying brewhouse parameters has been performed to investigate the factors of primary gushing. As hops are known to contribute to a wide range of both gushing positive and negative substances in beer, the hopping regime has emerged as an important aspect. This paper examines the impact of different hop varieties on gushing. Hop oils and unsaturated fatty acids are reputed to be gushing‐suppressors. Compounds such as dehydrated humulinic acid can intensify the effect. Hop pellets, with a prevalent range of conductometric values (5–10% α‐acid), commonly employed in breweries to adjust bitterness and aroma were selected. By working with the same “gushing malt”, the spectrum of compounds in the finished beer only differed through the hop product used. The overfoaming volumes of different samples were determined according to MEBAK guidelines. Respective hop oil and fatty acid concentrations (by GC) and iso‐α‐acid contents (by HPLC) were compared and a chronological sequence of the changing percentages of beer loss is shown.     

Biogenic amines and their producers in Akawi white cheese

The aim of this work was to study the biogenic amine content of brine‐ripened cheeses after one year of storage and then to investigate possible contaminating micro‐organisms with decarboxylase activity. The biogenic amine production of isolates was tested in vitro. The most frequent biogenic amines were putrescine, histamine and tyramine. The biogenic amine content detected in one cheese sample was above 120 mg/kg; this can be considered toxicologically relevant. Decarboxylase activity was found for 33 contaminating micro‐organisms. Isolates belonging to Bacillus licheniformis, Debaryomyces hansenii, Staphylococcus equorum and Serratia marcescens produced significant amounts of putrescine and cadaverine.     

Ripening of salted anchovy (Engraulis anchoita): development of lipid oxidation, colour and other sensorial characteristics

BACKGROUND: Changes in lipids are in general associated with quality deterioration, though earlier studies in anchovy indicate that the volatile compounds of importance to the characteristic flavour could be generated by lipid oxidation. The aim of the present paper was to study the changes in the fatty acid composition and the lipid oxidation of Engraulis anchoita during ripening and their relationship with the development of the typical sensorial characteristics. RESULTS: As a result of ripening, polyunsaturated fatty acids decrease from 4.27 to 2.42 and 2.00 g kg^?1 fatty acids in gutted fish and fillet, respectively. The most affected class of fatty acids was the n‐3 fatty acids, especially docosahexaenoic acid. Moreover, saturated fatty acids were the most stable to the process. 2‐Thiobarbituric acid reactive substances (TBARS) increased throughout ripening, and the use of fillets instead of gutted fish reduced this value. The a* value was the parameter most modified, showing a marked increase which correlates with the acquisition of a uniform pink colour. Sensory analysis did not show development of off‐odour or undesirable changes in colour during ripening. CONCLUSION: As a result of ripening, the fatty acid profile was modified and an increment of TBARS was observed. However, these changes did not lead to deterioration in the quality of the product. Copyright © 2010 Society of Chemical Industry     

Detection and quantification of Escherichia coli and Pseudomonas aeruginosa in cow milk by near‐infrared spectroscopy

This work investigated the potential of NIR technology to be applied in the dairy industry for the detection of micro‐organisms. To this end, two types of cow milk samples were studied, one in which only bacterial biomass was considered and the other in which bacteria were cultured and grown in milk for 24 h. The study was carried out using two micro‐organisms Escherichia coli and Pseudomonas aeruginosa. Both types of samples with different counts of both micro‐organisms were analysed by a NIR analyser in the range 10 000–4000 cm^?1 based on transmittance measurements. Multivariate models indicated that a better discrimination between micro‐organisms was attained in those milk samples in which micro‐organisms have been grown.     

Characterisation of four popular Polish hop cultivars

Polish cultivars of hop (Humulus lupulus L.) are well known as aroma and bitter raw material in the brewing industry. To characterise four popular Polish hop cultivars, Lubelski (Lublin), Marynka, Sybilla and Iunga (also known as Junga), the essential oil profiles were determined by gas chromatography/flame ionisation detector (GC‐FID) and gas chromatography/chemical ionisation mass spectrometry (GC‐CIMS) and the alpha/beta acids amounts by high‐performance liquid chromatography/diode array detector (HPLC‐DAD) analysis. In addition, the multivariate statistical analysis of the volatile profiles was performed. The results obtained evidenced qualitative and quantitative differences between the four cultivars studied and the foreign hops described in the literature. Lubelski and Iunga showed the typical chemical profile of the fine‐aroma and high‐alpha cultivars, respectively. In Marynka and Sybilla, the average amounts of alpha acids were similar to the bitter hops, but with a volatile profile characteristics of the fine‐aroma and aroma cultivars respectively.     

Alterations of the Profiles of Iso‐α‐Acids During Beer Ageing,Marked Instability of Trans‐Iso‐α‐Acids and Implications for Beer Bitterness Consistency in Relation to Tetrahydroiso‐α‐Acids

Age‐induced decomposition of iso‐α‐acids, the main bittering principles of beer, determines the consistency of the beer bitter taste. In this study, the profiles of iso‐α‐acids in selected high‐quality top‐fermented and lager beers were monitored by quantitative high‐performance liquid chromatography at various time intervals during ageing. The degradation of the iso‐α‐acids as a function of time is represented by the ratio, in percentage, of the sum of the concentrations of trans‐isocohumulone and trans‐isohumulone to the sum of the concentrations of cis‐isocohumulone and cis‐isohumulone. This parameter is relevant with respect to the evaluation of bitterness deterioration in aged beers. Trans‐iso‐α‐acids having a shelf half‐life of less than one year proved to be significantly less stable than cis‐iso‐α‐acids, but it appears feasible to counteract degradation if a suitable beer matrix is available. The fate of the trans‐iso‐α‐acids in particular adversely affects beer bitterness consistency. In addition to using hop products containing low amounts of trans‐iso‐α‐acids, brewers may profit of the remarkable stability of tetrahydroiso‐α‐acids, even on prolonged storage, for the production of consistently bitter beers.     

Survival of dairy-associated yeasts in yoghurt and yoghurt-related products

The poor survival of probiotic bacteria added to yoghurts is mainly attributed to the low pH of the product environment. Since yeasts have the ability to metabolize organic acids, resulting in a decrease in acidity, the inclusion of yeasts as part of the normal microflora, in association with probiotic bacteria has been suggested with the intention to assure better survival of the probiotic organisms in bio-yoghurt. Furthermore, a commensalistic association between yeasts and lactic acid bacteria exists. In order to understand the potential impact of yeast on probiotic bacteria, it was firstly important to assess the ability of yeast isolates to grow and survive in yoghurt.Accordingly, four dairy-associated yeasts, Debaryomyces hansenii, Kluyveromyces marxianus, Yarrowia lipolytica and Issatchenkia orientalis, associated commonly with yoghurt were isolated and inoculated subsequently into yoghurt and related dairy products during processing. The survival and growth of the yeasts were monitored over a 4-week storage period, the normal time accepted as the shelf-life of yoghurts. pH, sugar utilization and the production of organic acids were determined on a regular basis during the shelf-life to evaluate the possible contribution of the yeasts towards the products. The yeast species were able to survive in bio-yoghurt reaching maximum counts exceeding 10⁷ cfu g⁻¹. Despite the inability of some species to utilize lactose, the yeast species utilized available organic acids, galactose and glucose derived from bacterial metabolism of the milk lactose, as well as possible free fatty acids or free amino acids present in the dairy products. Excessive gas and ethanol production initiated by some yeast species proved, however, to be major constraints.     

The effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Number and size of lupulin glands,glandular trichomes of hop (Humulus lupulus L.), play a key role in contents of bitter acids and polyphenols in hop cone

Hop (Humulus lupulus L.) inflorescence, commonly known as ‘hop cone’, is valuable for contents of bitter acids, essential oils and polyphenols, used in brewing industry and biomedical application. These compounds are predominantly formed in glandular trichomes, known as ‘lupulin glands’. In this study, we investigated chemical and morphological characteristics of lupulin glands by microscopy and HPLC analyses. Even though no significant correlations were found between lupulin glands and hop cones for contents of alpha and beta bitter acids, polyphenols, all ratios between individual metabolites (alpha/beta acids, X/alpha acids, X/DMX) were highly and significantly correlated. The average number of lupulin glands on bract/bracteole ranged from 115 to 713 with 28.5% of variability. Linear regression analyses confirmed that total volume of lupulin glands and total lupulin glands area on bract/bracteole surface highly correlated with contents of bitter acids and polyphenols in hop cone. Therefore, lupulin glands are unique and exclusive organs responsible for contents of bitter acids and polyphenols in hop cone.