Yeasts in malting,with special emphasis on <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> (synonym <Emphasis Type="Italic">Pichia anomala</Emphasis>)

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.     

Development of DNA markers associated with beer foam stability for barley breeding

Traits conferring brewing quality are important objectives in malting barley breeding. Beer foam stability is one of the more difficult traits to evaluate due to the requirement for a relatively large amount of grain to be malted and then the experimental costs for subsequent brewing trials. Consequently, foam stability tends to be evaluated with only advanced lines in the final stages of the breeding process. To simplify the evaluation and selection for this trait, efficient DNA makers were developed in this study. Previous studies have suggested that the level of both of the foam-associated proteins Z4 and Z7 were possible factors that influenced beer foam stability. To confirm the relationship between levels of these proteins in beer and foam stability, 24 beer samples prepared from malt made from 10 barley cultivars, were examined. Regression analyses suggested that beer proteins Z4 and Z7 could be positive and negative markers for beer foam stability, respectively. To develop DNA markers associated with contents of proteins Z4 and Z7 in barley grain, nucleotide sequence polymorphisms in barley cultivars in the upstream region of the translation initiation codon, where the promoter region might be located were compared. As a result, 5 and 23 nucleotide sequence polymorphisms were detected in protein Z4 and protein Z7, respectively. By using these polymorphisms, cleaved amplified polymorphic sequence (CAPS) markers were developed. The CAPS markers for proteins Z4 and Z7 were applied to classify the barley grain content of 23 barley cultivars into two protein Z4 (pZ4-H and pZ4-L) and three protein Z7 (the pZ7-H, pZ7-L and pZ7-L2) haplotypes, respectively. Barley cultivars with pZ4-H showed significantly higher levels of protein Z4 in grain, and those with pZ7-L and pZ7-L2 showed significantly lower levels of protein Z7 in grain. Beer foam stability in the cultivars with pZ4-H and pZ7-L was significantly higher than that with pZ4-L and pZ7-H, respectively. Our results indicate that these CAPS markers provide an efficient selection tool for beer foam stability in barley breeding programs.     

Yeasts isolated from industrial maltings can suppress <Emphasis Type="Italic">Fusarium</Emphasis> 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.     

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called "shot-gun" approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

The impact of Fusarium culmorum infection on the protein fractions of raw barley and malted grains

Contaminating fungi, such as Fusarium species, produce metabolites that may interfere with normal barley grain proteolysis pattern and consequently, affect malt and beer quality. Protein compositional changes of an initial mixture of 20 % Fusarium culmorum infected and 80 % noninfected mature barley grains and respective malt are reported here. Proteolytic activity of infected barley grains (IBG) and respective malt, with controls (uninfected grains), were characterized using protease inhibitors from each class of this enzyme, including metallo-, cysteine, serine, and aspartic proteases, as well as uninhibited protease fractions. The proteins were extracted according to the Osborne fractionation and separated by size exclusion chromatography. Additionally, two-dimensional (2D) gel electrophoresis (GE) was used to analyze hydrophobic storage proteins isolated from the control and IBG. Analyses revealed that F. culmorum IBG had a twofold increase of proteolytic activity compared to the control sample, which showed an increase in all protease classes with aspartic proteases dominating. Infected and control malt grains were comparable with cysteine proteases representing almost 50 % of all proteolytic enzymes detected. Protein extractability was 31 % higher in IBG compared to the control barley. The albumin fraction showed that several metabolic proteins decreased and increased at different rates during infection and malting, thus showing a complex F. culmorum infection interdependence. Prolamin storage proteins were more hydrophobic during barley fungal infection. F. culmorum interfered with the grain hydrolytic protein profile, thereby altering the grain's protein content and quality.     

Genetic analysis of grain and malt quality in an elite barley population

Quantitative trait loci (QTLs) associated with grain weight, grain width, kernel hardness and malting quality were mapped in a doubled haploid population derived from two elite Australian malting barley varieties, Navigator and Admiral. A total of 30 QTLs for grain weight, grain width and kernel hardness were identified in three environments, and 63 QTLs were identified for ten malting quality traits in two environments. Three malting quality traits, namely β-amylase, diastatic power and apparent attenuation limit, were mainly controlled by a QTL linked to the Bmy1 gene at the distal end of chromosome 4H encoding a β-amylase enzyme. Six other malting quality traits, namely α-amylase, soluble protein, Kolbach index, free amino-acid nitrogen, wort β-glucan and viscosity, had coincident QTL clustered on chromosomes 1HS, 4HS, 7HS and 7HL, which demonstrated the interdependence of these traits. There was a strong association between these malt quality QTL clusters on chromosomes 1HS and 7HL and the major QTL for kernel hardness, suggesting that the use of this trait to enable early selection for malting quality in breeding programs would be feasible. In contrast, the majority of QTLs for hot-water extract were not coincident with those identified for other malt quality traits, which suggested differences in the mechanism controlling this trait. Novel QTLs have been identified for kernel hardness on chromosomes 2HL and 7HL, hot-water extract on 7HL and wort β-glucan on 6HL, and the resulting markers may be useful for marker-assisted selection in breeding programs.     

Fungi,Aflatoxins, Fumonisin B<Subscript>l</Subscript> and Zearalenone Contaminating Sorghum-based Traditional Malt,Wort and Beer in Botswana

Brewing and consumption of traditional beer have social–economic significance in most African countries including Botswana. Traditional sorghum malt, wort, and beer samples were collected from three villages around Gaborone, Botswana. Forty-six malt samples were analyzed for fungi on three different media and developing colonies were subcultured for identification. Rhizopus, Fusarium, Mucor, and Aspergillus were the most common genera isolated. Out of the 46 malt samples, 72% contained Rhizopus stolonifer, 63% Fusarium verticillioides (syn. Fusarium moniliforme), and 37% Aspergillus flavus. Although Aspergillus flavus was isolated from malt samples, aflatoxins (B₁, B₂, G₁, and G₂) were not detected in any of the samples analyzed. When the malt, wort, and beer samples were analyzed for fumonisin B_l and zearalenone, fumonisin B₁ was detected in 3 malt samples, with concentrations ranging from 47 to 1316 μg/kg, while zearalenone was detected in 56%, 48% and 48% of the malt, wort and beer samples, respectively. Zearalenone concentration in samples ranged from 102 to 2213 μg/kg in malt, 26 to 285 μg/l in wort and 20 to 201 μg/l, in beer. Zearalenone carry-over from wort to beer ranged from 23 to 403%. Therefore, although aflatoxins and fumonisin B₁ do not appear to be major contaminants, zearalenone is common and could pose a potential problem in traditional beer in Botswana.     

Microflora of Barley Kernels

Numbers and kinds of microflora were determined in 160 samples of barley grown in different regions of the United States; microflora were more abundant in the grains grown in the central states than in those grown in the western states. During steeping and germination in micromalting equipment, the number of colonies of filamentous fungi increased from two to five times, colonies of yeasts from five to ten times, and bacteria from 50 to more than 100 times the numbers present in the grain before malting. Kiln drying according to a commercial schedule reduced the number of all types of microflora below the number present before kilning, but all were present in larger numbers in the kilned malt than in the original grain. In barley stored at room temperature and at a moisture content of 15 to 18%, members of the Aspergillus glaucus group increased with increasing time and increasing moisture content, and germination percentage of the seeds decreased. Stored free of storage fungi at room temperature, barley with a moisture content just over 15% retained a high germination percentage for 5 months, but at a moisture content of 16% the germination decreased to zero.     

Microflora of Barley Kernels

Numbers and kinds of microflora were determined in 160 samples of barley grown in different regions of the United States; microflora were more abundant in the grains grown in the central states than in those grown in the western states. During steeping and germination in micromalting equipment, the number of colonies of filamentous fungi increased from two to five times, colonies of yeasts from five to ten times, and bacteria from 50 to more than 100 times the numbers present in the grain before malting. Kiln drying according to a commercial schedule reduced the number of all types of microflora below the number present before kilning, but all were present in larger numbers in the kilned malt than in the original grain. In barley stored at room temperature and at a moisture content of 15 to 18%, members of the Aspergillus glaucus group increased with increasing time and increasing moisture content, and germination percentage of the seeds decreased. Stored free of storage fungi at room temperature, barley with a moisture content just over 15% retained a high germination percentage for 5 months, but at a moisture content of 16% the germination decreased to zero.     

Chromosomal loci associated with endosperm hardness in a malting barley cross

A breeding objective for the malting barley industry is to produce lines with softer, plumper grain containing moderate protein content (9–12%) as they are more likely to imbibe water readily and contain more starch per grain, which in turn produces higher levels of malt extract. In a malting barley mapping population, ‘Arapiles’ × ‘Franklin’, the most significant and robust quantitative trait locus (QTL) for endosperm hardness was observed on the short arm of chromosome 1H, across three environments over two growing seasons. This accounted for 22.6% (Horsham 2000), 26.8% (Esperance 2001), and 12.0% (Tarranyurk 2001) of the genetic variance and significantly increased endosperm hardness by 2.06–3.03 SKCS hardness units. Interestingly, Arapiles and Franklin do not vary in Ha locus alleles. Therefore, this region, near the centromere on chromosome 1H, may be of great importance when aiming to manipulate endosperm hardness and malting quality. Interestingly, this region, close to the centromere on chromosome 1H, in our study, aligns with the region of the genome that includes the HvCslF9 and the HvGlb1 genes. Potentially, one or both of these genes could be considered to be candidate genes that influence endosperm hardness in the barley grain. Additional QTLs for endosperm hardness were detected on chromosomes 2H, 3H, 6H and 7H, confirming that the hardness trait in barley is complex and multigenic, similar to many malting quality traits of interest.     

Dissection of a malting quality QTL region on chromosome 1 (7H) of barley

Malting and brewing are major uses of barley (Hordeum vulgare L.) worldwide, utilizing 30–40% of the crop each year. A set of complex traits determines the quality of malted barley and its subsequent use for beer. Molecular genetics technology has increased our understanding of genetic control of the many malting and brewing quality traits, most of which are quantitatively inherited. The objective of this study was to further dissect and evaluate a known major malting quality quantitative trait locus (QTL) region of about 28 cM on chromosome 1 (7H). Molecular marker-assisted backcrossing was used to develop 39 isolines originating from a Steptoe / Morex cross. Morex, a 6–row malting type, was the donor parent and Steptoe, a 6–row feed type, was the recurrent parent. The isolines and parents were grown in four environments, and the grain was micro-malted and analyzed for malting quality traits. The effect of each Morex chromosome segment in the QTL target region was determined by composite interval mapping (CIM) and confirmed and refined by multiple interval mapping (MIM). One QTL was resolved for malt extract content, and two QTLs each were resolved for -amylase activity, diastatic power, and malt -glucan content. One additional putative malt extract QTL was detected at the plus border of the target region by CIM, but not confirmed by MIM. All QTLs were resolved to intervals of 2.0 to 6.4 cM by CIM, and to intervals of 2.0 cM or less by MIM. These results should facilitate marker-assisted selection in breeding improved malting barley cultivars.     

Structural Changes in Starch Molecules during the Malting of Barley

Barley was made into a normal and an over-modified malt, and the loss in starch was 14.6% and 67.7%, respectively. Starch granules, isolated from the barley and malts, were observed by scanning electron and light microscopes. In normal malt, 14% of the large granules were eroded and the small granules remained almost intact. In the case of over-modified malt, 38% of the large granules were eroded, and a marked reduction was found in the population of the small granules. Iodine affinities and blue values of the starches increased as malting proceeded. The malting of barley resulted in an apparent increase in the amylose component of the starch but hardly affected its molecular size distribution when examined by Bio-Gel A-50m column chromatography. The fine structures of the barley and malt amylopectins were compared by Shephadex G-50 and Bio-Gel P-2 column chromatographies after debranching with pullulanase. No change was observed during malting in spite of a significant reduction in the amylopectin component of the starch.     

Protein modification in malting sorghum

Steeping time, moisture content and germination times were deployed in assessing protein modification in sorghum varieties: ICSV400, SK5912 and KSV8. Grains were steeped for 45 h using 6 h wet and 3 h dry cycles and germinated for 8 days. Moisture contents and their effects on protein modification were monitored at various intervals. Optimum moisture contents of 37–43% and out-of-steep values of 32–35% were recorded. Significant positive correlations existed between moisture content and free alpha amino nitrogen (FAN), total non-protein nitrogen (TNPN) and cold water soluble protein (CWS-P), all key protein modification indicators, during steeping. Maximum values for FAN, TNPN and CWS-P were recorded in both ICSV400 and SK5912 after 40 h of steeping, suggesting a similarity in the physiology of the grains in both varieties while those of KSV8 occurred after 45 h. Variety and steeping time significantly affected moisture content at P < 0.01 and P < 0.001, respectively as well as the development of FAN, TNPN and CWS-P during steeping. Optimum values for the above parameters occurred on day 5 of germination in all the sorghum varieties. Variety and germination time highly significantly (P < 0.001) affected protein modification during germination.     

高温下干旱和渍水对冬小麦花后旗叶光合特性和物质转运的影响

以蛋白质含量不同的两个冬小麦品种扬麦9号和豫麦34为材料,研究了不同温度和水分条件下小麦花后旗叶光合特性的变化、营养器官花前贮藏干物质和氮素转运特征及其与籽粒产量和品质形成的关系.结果表明,高温及干旱和渍水均明显降低了旗叶光合速率和叶绿素含量（SPAD值）,但高温下干旱和渍水对光合作用的影响加重.小麦营养器官花前贮藏干物质、氮素转运量和转运率在适温下表现为干旱>对照>渍水,高温下则表现为对照>干旱>渍水.适温下花后同化物积累量表现为对照>渍水>干旱,高温下则表现为对照>干旱>渍水.花后氮素积累量在适温和高温下均表现为对照>渍水>干旱.籽粒淀粉含量以适温适宜水分处理最高,高温渍水下最低;蛋白质含量以高温干旱下最高,适温渍水下最低.温度和水分逆境下小麦粒质量和淀粉含量的降低与花后较低的光合能力及干物质积累有关,而蛋白质含量则与花前贮藏氮素的转运量和转运率有关.     

Moisture Sorption Isotherms of Various Tobaccos

The equilibrium moisture contents of sun-cured (Kroumougrad), flue-cured (Bright Yellow—4) and air-cured (Burley-21 and Matsukawa) tobaccos were measured over a relative humidity range from 5 to 80% at 20°C. The moisture sorption isotherms of tobaccos were of sigmoid type, and classified into two groups. In a lower humidity range below ca. 40% RH, the A group (Kroumougrad and BY-4) had a smaller moisture sorption capacity than B group (Burley-21 and Matsukawa), while in a higher humidity range above ca. 50% RH the former had a larger moisture sorption capacity than the latter. By extracting with water, the moisture content of BY-4 was increased in the lower humidity range, while it decreased in the higher humidity range. However, the moisture content of Matsukawa was scarecely changed by extracting it with water. These results suggest that the differences in equilibrium moisture content with the type of curing were due to the differences in contents of water soluble com- ponents. To control the hygroscopic properties of a tobacco, therefore, the influences of the addition of sucrose and glycerol on the equilibrium moisture content were quantitatively analysed. The moisture sorption capacity of tobacco was greatly different from its nitrogen sorption capacity. The specific surface area of tobacco calculated from moisture sorption isotherm was ca. 110 times larger than the specific surface area calculated from the nitrogen sorption isotherm. Both the nitrogen and moisture sorption data should be necessary for better understanding of the complicated sorption-desorption phenomena in tobaccos.     

Phytase production and decrease of phytic acid content in canola meal byAspergillus carbonarius in solid-state fermentation

Solid-state fermentation (SSF) usingAspergillus carbonarius with canola meal as a substrate showed that production of phytase was associated with growth; maximum activity was achieved after 72 h. Apparent 25% and 10% increases in the protein content of the canola meal were noticed after 48 h and 72 h, respectively but total carbohydrate concentration had fallen by 25% by the end of fermentation. The rate of decrease of phytic acid content was optimum with a moisture content between 53% and 60%; homogenization of the inoculum for 120 s led to the greatest biomass and lowest phytic acid content. Inoculation of sterile meal led to lower phytic acid contents than inoculation of non-sterile meal.The authors are with the Department of Chemical Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada     

Sorghum: a cereal with lager beer brewing potential

The potential of sorghum as an alternative substrate for lager beer brewing was recognized over five decades ago. Factors which appear to influence brewing with sorghum include: the variety of sorghum, storage time, steep period, germination time, duration and levels of temperature-time sequence of the kilning cycle and temperature-time regimes during mashing. Malts from sorghum varieties that have high diastatic power, amylase and starch contents are desirable. Soluble and insoluble amylases in grain sorghum contribute towards the hydrolysis of grain constituents during mashing. Optimizing conditions for malting, mashing and fermentation are therefore necessary for the production of acceptable sorghum lager beer. This review aims to update research results on lager beer brewing with sorghum.     

Protein enrichment of brewery spent grain from <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> by solid-state fermentation

Brewery spent grain represents approximately 85 % of total by-products generated in a brewery. Consisting of carbohydrates, fiber, minerals and low amounts of protein, the use of brewery spent grain is limited to the feeding of ruminants; however, its potential use should be investigated. The reuse of this by-product using microorganisms by solid-state fermentation process as the case of protein enrichment by single-cell protein incorporation is an alternative to ensure sustainability and generate commercially interesting products. In this context, the aim of this study was to grow Rhizopus oligosporus in brewery spent grain under different initial moisture contents and nitrogen sources to increase the protein content of the fermented material. After 7 days of fermentation, increase of 2–4 times in the crude protein and soluble protein content was verified, respectively, compared to unfermented brewery spent grain. The kinetics of protein enrichment demonstrated the possibility of application of this technique, which can be a great alternative for use in diets for animals.     

Probing heat-stable water-soluble proteins from barley to malt and beer

Proteins determine the quality of barley in malting and brewing end-uses. In this regard, water-soluble barley proteins play a major role in the formation, stability, and texture of head foams. Our objective was to survey the barley seed proteins that could be involved in the foaming properties of beer. Therefore, two-dimensional (2-D) electrophoresis and mass spectrometry were combined to highlight the barley proteins that could resist the heating treatments occurring during malting and brewing processes. As expected, from barley to malt and to beer, most of the heat-stable proteins are disulfide-rich proteins, implicated in the defense of plants against their bio-aggressors, e.g., serpin-like chymotrypsin inhibitors (protein Z), amylase and amylase-protease inhibitors, and lipid transfer proteins (LTP1 and LTP2). For LTP1s, the complex pattern displayed in 2-D electrophoresis could be related to some chemical modifications already described elsewhere, such as acylation or glycation through Maillard reactions, which occur on malting. Our proteomics approach allowed the identification of the numerous proteins present in beer in addition to the major ones already described. The involvement of these proteins in the quality of beer foam can now be evaluated.