Analysis of beers from an 1840s' shipwreck.

Two bottles of beer from an about 170-year-old shipwreck (M1 Fö 403.3) near the Åland Islands in the Baltic Sea were analyzed. Hop components and their degradation compounds showed that the bottles contained two different beers, one more strongly hopped than the other. The hops used contained higher levels of ß-acids than modern varieties and were added before the worts were boiled, converting α-acids to iso-α-acids and ß-acids to hulupones. High levels of organic acids, carbonyl compounds, and glucose indicated extensive bacterial and enzyme activity during aging. However, concentrations of yeast-derived flavor compounds were similar to those of modern beers, except that 3-methylbutyl acetate was unusually low in both beers and 2-phenylethanol and possibly 2-phenylethyl acetate were unusually high in one beer. Concentrations of phenolic compounds were similar to those in modern lagers and ales.

Milling, water uptake, and modification properties of different barley (Hordeum vulgare L.) lots in relation to grain composition and structure.

Milling properties, water uptake, and modification in malting were studied in 14 barley (Hordeum vulgare L.) lots from two consecutive crop years. In all barley lots studied, grains with lower ß-glucan and protein content and higher starch content produced finer flours upon milling. Grains with lower ß-glucan content also hydrated more rapidly during steeping. A detailed study of two cultivars from two crop years indicated that similar environmental conditions could induce a higher ß-glucan content and concentration of aggregated B hordein in the peripheral endosperm and a lower proportion of C hordein entrapped among aggregated hordeins deeper within the endosperm. These characteristics were associated with production of coarser flours during milling as well as with slower water uptake and lower modification. However, the data do not distinguish between the effect of ß-glucan content and that of hordein localization. Distribution of ß-glucan or total protein within the kernel was not linked to hydration or modification.

Day-length effects on protein localisation affect water absorption in barley (Hordeum vulgare) grains.

BACKGROUND: Hordeins are major storage proteins of barley (Hordeum vulgare L.) grains and are considered to influence malting and brewing by forming a matrix surrounding the starch granules which affects the release of fermentable sugars. However, the extent to which environmental factors affect hordein location, and the impact of this on malting performance, have not so far been studied. Therefore the relationship of hordein location to water uptake and malting quality were studied by growing barley cv. Barke under different daylengths (14 h and 18 h of light) in controlled environment conditions. RESULTS: Differences in the locations of hordein storage proteins were observed, with C hordein being located more deeply within the endosperm of both developing grains at 35 days after anthesis and in mature grains under long-day conditions. This deeper location of C hordein was correlated positively with water uptake during the steeping phase of malting. CONCLUSION: An effect of environment (daylength) on the localisation of C hordein was demonstrated. This difference in hordein localisation was also associated with differences in malting quality with water uptake in the steeping phase being associated positively with the deeper location of C hordein. These results indicate that environmental effects on protein location may affect malting performance of barley grains.

Yeasts in malting, with special emphasis on Wickerhamomyces anomalus (synonym Pichia anomala).

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.

Ultrastructure of biofilms formed on barley kernels during malting with and without starter culture.

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. In the malting process, dry barley kernels are steeped in water which initiates germination and invigorates microbial growth on the kernels. In the present study, field emission scanning electron microscopy (FESEM) was used to visualize the microbial community within the tissues of barley kernels before and after the steeping, with and without Lactobacillus plantarum E76 added as a starter culture. The results show that the community of 10(8)cfu g(-1) on dry, stored barley kernels increased 5-10 fold during the steeping forming a dense biofilm of bacteria and fungi with slimy exopolymeric matrix. FESEM revealed that crevices between the outer epidermis and the testa of sound barley kernels were heavily colonized with microbes, whereas there were only few microbes on the outer surface of the husks, in the aleurone layer or in the endosperm underneath an intact testa layer. The microbes frequently possessed appendages forming bridging them to the kernel and the individual microbial cells to each other. The L. plantarum added to the steeping water reduced the amount of exopolymeric matrix in the biofilm and improved the wort filterability.

Yeasts isolated from industrial maltings can suppress Fusarium growth and formation of gushing factors.

Fusarium infection of barley and malt can cause severe problems in the malting and brewing industry. In addition to being potential mycotoxin producers, Fusarium fungi are known to cause beer gushing (spontaneous overfoaming of beer). Cereal-derived bacteria and yeasts are potential biocontrol agents. In this study, the antifungal potential of selected yeasts (12 strains) derived from the industrial malting ecosystem was studied in vitro with a plate-screening assay. Several ascomycetous yeast strains showed antagonistic activity against field and storage moulds, Pichia anomala being the most effective strain. The effects of P. anomala VTT C-04565 (C565) were examined in laboratory scale malting with naturally contaminated barley exhibiting gushing potential. P. anomala C565 restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. Grain germination was not disturbed by the presence of yeast. Addition of P. anomala C565 into the steeping seemed to retard wort filtration, but the filtration performance was recovered when yeast culture was combined with Lactobacillus plantarum VTT E-78076. Well-characterized microbial cultures could be used as food-grade biocontrol agents and they offer a natural tool for tailoring of malt properties.

Heterologous expression of Vitreoscilla haemoglobin in barley (Hordeum vulgare).

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots.

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.

Oxygen deficiency in barley (Hordeum vulgare) grain during malting.

The steep water is generally aerated in industrial barley malting. However, it is questionable whether oxygen actually reaches the embryo, which remains entrapped under the husk, testa, and pericarp until chitting occurs. The aim of our study was to investigate whether barley embryos experience oxygen deficiency during steeping, and whether various steeping conditions affect the oxygen deficiency. Alcohol dehydrogenase Adh2 was induced in all steeping conditions studied. Therefore, oxygen deficiency occurred regardless of the steeping conditions. However, steeping conditions affected the rate of recovery from oxygen deficiency, germination rate, and onset of alpha-amylase production. When barley was subjected to oxygen deficiency by applying N(2) gas during steeping, the timing of the treatment determined its effects. The importance of aeration increased as the process proceeded. Oxygen deprivation at the beginning of the process had little effect on malt quality. Therefore, the timing of aeration is important in the optimization of germination during the steeping stage of malting.

Endosperm structure affects the malting quality of barley (Hordeum vulgare L.).

Twenty-seven barley (Hordeum vulgare L.) samples collected from growing sites in Scandinavia in 2001 and 2002 were examined to study the effect of endosperm structure on malting behavior. Samples were micromalted, and several malt characteristics were measured. Samples were classified as having a mealier or steelier endosperm on the basis of light transflectance (LTm). Because endosperm structure is greatly dependent on protein content, three barley sample pairs with similar protein contents were chosen for further analysis. During malting, the steelier barley samples produced less root mass, but showed higher respiration losses and higher activities of starch-hydrolyzing enzymes. Malts made from steelier barley had a less friable structure, with more urea-soluble D hordein and more free amino nitrogen and soluble protein. The reason for these differences may lie in the structure or localization of the hordeins as well as the possible effects of endosperm packing on water uptake and movement of enzymes.

Expression of Vitreoscilla hemoglobin enhances growth of Hyoscyamus muticus hairy root cultures.

The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines.