Effect of ozone treatment on the safety and quality of malting barley

Molds and their mycotoxins are an expensive problem for the malting and brewing industries. Deoxynivalenol (DON) is a mycotoxin that is associated with Fusarium spp. These fungi frequently cause Fusarium head blight in wheat and barley in the midwestern region of the United States; Manitoba, Canada; Europe; and China. Barley growers and malt producers would benefit from a postharvest control method for mold growth and DON production. We evaluated the use of gaseous ozone (O(3)) for preventing Fusarium growth and mycotoxin production while maintaining malt quality characteristics. Micromalting was performed in three replications under standard conditions. Ozone treatment was applied to malting barley during steeping via a submerged gas sparger. Ozone treatment conditions were 26 mg/cm(3) for 120 min after 2 and 6 h of steeping. The effects of gaseous ozone on DON, aerobic plate counts, Fusarium infection, and mold and yeast counts of barley throughout the malting process were measured. Various quality parameters of the malt were measured after kilning. Statistical tools were used to determine the significance of all results. Ozonation of malting barley during steeping did not lead to significant reductions in aerobic plate counts but did lead to a 1.5-log reduction in mold and yeast counts in the final malt. The influence of gaseous ozone on DON concentration was inconclusive because of the low initial concentrations of DON in the barley. Ozone significantly reduced Fusarium infection in germinated barley. Gaseous ozone did not negatively influence any aspect of malt quality and may have subtle beneficial effects on diastatic power and β-glucan concentrations.     

Fungal Hydrophobins in the Barley‐to‐Beer Chain

Fungal hydrophobins have been shown to induce gushing of beer. In order to study the occurrence and fate of hydrophobins at different stages of the production chain of beer, barley samples artificially infected in the field with Fusarium culmorum, F. graminearum and F. poae were collected during the growing period as well as during various stages of the malting process. In addition, naturally infected malt was brewed in pilot scale and samples were collected throughout the process. The samples were assayed for hydrophobin content using an ELISA method. The results showed that fungi produced hydrophobins that accumulated during barley grain development in the field, but that production was more pronounced during malting. Prolonged storage of barley tended to reduce the ability of fungi to produce hydrophobins in malting. Studies on the fate of hydrophobins during the brewing process revealed that mashing released hydrophobins from the malt into the wort. Some loss of hydrophobins occurred throughout the brewing process with spent grains, cold break (wort boiling) and surplus yeast. In addition, the beer filtration step reduced hydrophobin levels. Despite the substantial loss of hydrophobins during brewing, the level was high enough to induce the gushing detected in the final beer.     

Effect of hot water treatments on the safety and quality of Fusarium-infected malting barley

Utilization of Fusarium-infected barley for malting may lead to mycotoxin contamination of malt and decreased malt quality. Hot water treatments may prevent or reduce safety and quality defects and allow use of otherwise good quality barley. We evaluated hot water treatments for preventing Fusarium growth and mycotoxin production while maintaining barley-malt characteristics. Four barley lots with varying deoxynivalenol (DON) concentrations were hot water-treated at 45 or 50 degrees C for 0, 1, 5, 12, and 20 min. Treated barley was malted in a pilot-scale malting unit. Barley and malt were analyzed for Fusarium infection (FI), germinative energy (GE), aerobic plate count (APC), mold and yeast count (MYC), and DON. Malt quality parameters included malt extract, soluble protein, wort color, wort viscosity, free amino nitrogen, alpha-amylase, and diastatic power. Significant decreases in FI occurred within 1 min at both 45 degrees C (41-66%) and 50 degrees C (51-69%) in all barley samples. Significant reductions in APC (1.0-1.8 log) and MYC (1.7-1.8 log) in barley were observed after 5 min at both temperatures. The largest reductions for DON were observed in malts prepared from barley treated with hot water at 45 degrees C (79-93%) and 50 degrees C (84-88%) for 20 min. GE and most of the malt quality parameters were only affected when barley was treated at 50 degrees C for 12 and 20 min. The results suggest that hot water treatments may offer the potential for treating mildly FHB infected malting barley.     

Effect of electron-beam irradiation on the safety and quality of Fusarium-infected malting barley

Utilization of Fusarium-infected barley for malting may lead to mycotoxin production during malting and decreased malt quality. Electron-beam irradiation may prevent safety and quality defects and allow use of otherwise good quality barley. We evaluated electron-beam irradiation for preventing Fusarium growth and mycotoxin production while maintaining barley-malt quality characteristics. Four barley lots with varying deoxynivalenol (DON) concentrations were irradiated at 0, 2, 4, 6, 8, and 10 kGy. Treated barley was malted in a pilot-scale malting unit. Barley and malt were analyzed for Fusarium infection (FI), germinative energy (GE), aerobic plate counts (APC), mold and yeast counts (MYC), and DON. Malt quality parameters included malt extract, soluble protein, wort color, wort viscosity, free amino nitrogen, alpha-amylase, and diastatic power. FI, APC, and MYC decreased in barley with an increase in dosage. The APC and MYC for malts from barley exposed to 8–10 kGy were slightly higher than in other malted samples indicating that irradiation-resistant microflora could flourish during malting. Barley GE significantly decreased (3–15%) at 8–10 kGy. Although irradiation had no effect on DON in raw barley, DON decreased significantly (60–100%) in finished malts prepared from treated barley (6–10 kGy). Malt quality parameters were slightly affected by electron-beam radiation. The results suggest 6–8 kGy may be effective for reducing FI in barley and DON in malt with minimal effects on malt quality.     

Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay

Fusarium graminearum and the closely related F. culmorum were found to be associated with over foaming of bottled beer (gushing) when contaminated brewing malt is used. The presence of highly surface active hydrophobins produced by these fungi upon growth on wheat or barley in the field or during malting may affect bubble formation and stability in gushing beers and other carbonated beverages. Aiming for a method for the rapid and user friendly analysis of unmalted and malted cereals during quality control in the brewing industry, a loop-mediated isothermal amplification (LAMP) assay for the detection of Fusarium spp. capable of producing the gushing inducing hydrophobin Hyd5p was set up. A set of primers was designed towards a 221 bp region within the hyd5 gene of F. culmorum. The LAMP product was verified by sequencing a 150 bp portion. Testing specificity with purified DNA from 99 different fungal species as well as barley and wheat showed that DNA synthesis only occurred during LAMP when DNA of the closely related species F. graminearum, F. culmorum, F. cerealis and F. lunulosporum were used as template. In-tube indirect detection of DNA amplification was applied using manganese-quenched calcein as fluorescence indicator for pyrophosphate produced during DNA synthesis. The assay had a detection limit of 0.74 pg of purified target DNA which corresponds 20 copy numbers per reaction within 30 minutes using a simple heating block. Analysis of Fusarium infected cereals revealed that the assay was able to detect F. graminearum at a level of 0.5% of infected grains in uninfected barley by analysis of surface washings without further sample preparation. Results show that the hyd5 based LAMP assay can be a rapid, useful and sensitive tool for quality control in the brewing and malting industry.     

Transfer of Fusarium mycotoxins and 'masked' deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins--deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes--was investigated. In addition to these 'free' mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of 'free' Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5-10 microg kg(-1)), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this 'masked' DON in final beers exceeded 'free' DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).     

Changes in β-glucan and other carbohydrate components of barley during malting

Changes in total (1→3), (1→4)-β-glucan content were followed during the micro-malting of nine varieties of barley with a wide range of malting qualities. These changes were related to estimates of endosperm modification based upon staining with Calcofluor. β-Glucan content declined from an average of 3.54% in the barley to 0.75% in the malt. Pentosan and total starch (including starch-derived oligosaccharides) levels showed comparatively little change during malting. β-Glucan composition of the barley was a poor indicator of malting performance. However, the β-glucan, starch and xylose contents of the malt all showed significant correlations with malt extract. Estimation of malt β-glucan content gave the best indication of malt quality. Direct determination of β-glucan may be of more value in assessing malt quality than indirect techniques based upon assessing modification of stained grains.     

制麦和酿造过程中脱氧雪腐镰刀菌烯醇的初步研究

2008年江苏某农场的赤霉病感染相对严重的KA-4B大麦中,脱氧雪腐镰刀茵烯醇（DON）的含量为1．91mg/kg。以此大麦为原料,实验室规模下进行制麦和酿造实验,结果表明,浸麦可以洗去大麦本身含有的绝大部分DON;而大麦内部没有被洗掉的镰孢霉属真菌孢子在发芽阶段重新萌发、生长代谢,形成并积累大量的DON;焙燥阶段不能破坏DON,成品麦芽和麦根中DON的含量分别是原大麦中DON含量的51％和89％。麦芽中的DON可以经过糖化和发酵过程流入到啤酒中,啤酒中DON的总含量是麦芽粉中DON总量的86％。而同一年份相邻农场的基本未感染赤霉病的KA-4B大麦中,DON含量低于0．1mg/kg,绿麦芽和麦根中均检测到低于0．1mg／kg的DON,而成品麦芽、麦汁和啤酒中均未检测到DON。制麦及酿造实验表明,与基本未感染赤霉病的KA-4B大麦相比,赤霉病感染程度严重的KA-4B大麦微生物污染严重,DON含量相对高,大麦品质较差,发芽率较低,麦芽浸出率低,所制得麦汁的过滤速度快,麦汁和啤酒的色度均较高。     

THE CHROMATOGRAPHIC PROPERTIES OF BARLEY AND MALT β-AMYLASES

Barley β-amlyase occurs as a heterogeneous, polydisperse enzyme in thiol-free extracts of Conquest barley. During malting, the polydisperse enzyme is altered, resulting in the formation of four distinct enzyme components which increase in activity as germination progresses. Addition of thioglycerol to a thiol-free extract of barley, or initial extraction with thioglycerol, produces extracts containing two discrete β-amylase enzymes. β-amylase I is the major component of the extract; β-amylase II occurs as a minor component. Similarly, malt extracts containing thioglycerol have two β-amylase enzymes, β-amylase III and IV. Barley β-amylase II and malt β-amylase III have similar chromatographic properties on CM-cellulose but it is not known whether these enzymes are identical. During the early stages of germination, barley β-amylase I disappears and cannot be detected in extracts of 1-day malt; β-amylase III is the major β-amylase enzyme in this extract. Malt β-amylase IV cannot be detected in barley extracts. It develops during germination until it becomes the major β-amylase in malt extracts.     

Fungal Hydrophobins as Predictors of the Gushing Activity of Malt

Fungal infection of barley and malt, particularly by strains of the genus Fusarium, is known to be a direct cause of beer gushing. We have shown previously that small fungal proteins, hydrophobins, isolated from strains of the genera Fusarium, Nigrospora and Trichoderma act as gushing factors in beer. A hydrophobin concentration as low as 0.003 ppm was sufficient to induce gushing. The gushing‐inducing abilities of the isolated hydrophobins varied probably due to their structural differences. The hydrophobins did not affect beer foam stability. A correlation was observed between the hydrophobin level analyzed by the hydrophobin ELISA developed and the gushing potential of malt. The risk of gushing was found to increase with hydrophobin concentrations above 250 μg/g malt. The levels of hydrophobin and the Fusarium mycotoxin deoxynivalenol (DON) in malts were not correlated which indicated that the formation of those two fungal metabolites may not be linked. Furthermore, we did not observe a correlation between the DON content and the gushing potential of the malt studied. Our observations suggest that the accuracy of predicting gushing could be improved by measuring the amount of the actual gushing factors, hydrophobins, in barley or malt.     

Mold and mycotoxin problems encountered during malting and brewing

Fusarium infections in grains can have severe effects on malt and beer. While some degree of Fusarium mycotoxins, such as deoxynivalenol, present in infected barley may be lost during steeping, the Fusarium mold is still capable of growth and mycotoxin production during steeping, germination and kilning. Therefore, detoxification of grain before malting may not be practical unless further growth of the mold is also prevented. Methods to reduce the amount of mold growth during malting are needed. Physical, chemical and biological methods are reviewed. Irradiation looks very promising as a means to prevent Fusarium growth during malting, but the effect on the surviving mold to produce mycotoxins and the effect on malt quality needs further study. Chemical treatments such as ozonation, which would not leave residual chemical in the beer also appear to be promising. Although biological control methods may be desirable, due to the use of "natural" inhibition, the effects of these inhibitors on malt and beer quality requires further investigation. It may also be possible to incorporate detoxifying genes into fermentation yeasts, which would result in detoxification of the wort when mold growth is no longer a problem. Development of these types of technological interventions should help improve the safety of products, such as beer, made from Fusarium infected grain.     

Occurrence of bound deoxynivalenol in Fusarium head blight-infected barley (Hordeum vulgare L.) and malt as determined by solvolysis with trifluoroacetic acid

Fusarium head blight occurs worldwide in barley production, and the Fusarium mycotoxins, particularly deoxynivalenol (DON), have become a major concern for barley products, such as beer. This study investigated the content of bound DON in barley samples that were naturally infected with Fusarium head blight. Free DON was determined by gas chromatography-electron capture detection after standard acetonitrile:water extraction, while total DON was determined using treatment with trifluoroacetic acid. Bound DON is the difference between the total and free values. Bound DON was detected in approximately 40% of the samples analyzed, and represented 6 to 21% of free DON. A preliminary study indicated that both free and bound DON decrease significantly during the steeping phase of malting. These results suggest that bound DON, like the free form, may be water-soluble.     

微生物对麦芽品质的影响

大麦作为啤酒酿造的主要原料，其表面或外源的微生物在制麦过程中会影响麦芽的品质，并最终影响成品啤酒的质量。近年来研究发现在制麦过程中添加启动子培养物，既能作为微生物控制剂，抑制有害微生糖的生长；同时又能利用微生物分泌的水解酶系来提高麦芽的品质和安全性。本文介绍了麦芽中的微生物种类及其对麦芽质量的影响，并对添加不同启动子培养物对麦芽品质的改善及其工业化应用的可行性进行了探讨。     

Carbohydrate content and structure during malting and brewing: a mass balance study

A holistic view of the fate of barley starch, arabinoxylan and β-glucan throughout malting and brewing is largely missing. Here, an industrial scale malting trial and pilot brewing trial were performed, and the concentration and structural characteristics of carbohydrates were analysed at 28 key points in the process. The barley starch content decreased during malting from 75.0% to 69.7%. During mashing, malt starch was converted to fermentable sugars (75.3%), dextrin (22.8%) or was retained in spent grains (1.8%). Arabinoxylan was partially hydrolysed during malting. Despite mashing-in at 45°C, no further solubilisation of arabinoxylan was observed during mashing. However, the average degree of polymerisation of the soluble arabinoxylan fraction decreased slightly. During fermentation, the arabinoxylan content decreased to 2.5 g/L. The amount of barley β-glucan decreased gradually in time during malting. Of the solubilised β-glucan, 31% was retained in the spent grains during wort filtration, slightly lowering the β-glucan content in the wort. The β-glucan content remained at 0.5 g/L during fermentation. Sucrose was hydrolysed during mashing, probably by barley invertases. From the total amount of malt used, 41.0% was converted to fermentable sugars. This mashing yield could have been improved by the full hydrolysis to fermentable sugars of the present β-glucan (to 41.1%), the remaining starch in spent grains (to 42.0%) and dextrin in wort (to 50.3%). These results provide more insight into the carbohydrate conversions during malting and brewing and can act as a baseline measurement for future work. © 2020 The Institute of Brewing & Distilling     

THE VISCOSITY OF WORTS IN RELATION TO THEIR CONTENT OF β-GLUCAN

A procedure is given for assessing that proportion of wort viscosity which is attributable to β-glucan. Worts obtained from unkilned samples of malt which have been processed for 54 or 72 h show enhanced viscosity. This is principally due to β-glucan although the contribution of other constituents, absent from the wort of fully modified malt, is of significance. Barley variety is shown to have a pronounced effect on wort viscosity. Insoluble β-glucan is brought into solution in mashes at 65° C. The β-glucan isolated from malt which has been inactivated using aqueous ethanol prior to extraction at 65° C, is of higher specific viscosity than that isolated from control worts prepared at the same temperature. The introduction of a rest by mashing initially at 40° C results in the production of wort of lower viscosity, a decrease in the β-glucan content of the wort and a reduction in the specific viscosity of the β-glucan. There is no apparent relationship between the endo-β-glucanase content of the malts and either the viscosity of derived worts or the degree of breakdown of β-glucan which occurred during malting and mashing. Abrasion of barley, which is a factor assisting the distribution of enzymes during malting, acts to reduce wort viscosity.     

法麦Vanessa制麦特性的探讨

法麦Vanessa是优质的啤酒大麦,但该品种大麦不同于国麦和其它进口大麦,制成的麦芽通常粘度和葡聚糖较高,因此,制麦时要根据大麦特性适时制定相应工艺,才能获得优质麦芽。     

Fate of enniatins and beauvericin during the malting and brewing process determined by stable isotope dilution assays

The fate of enniatins A, A1, B, B1 and beauvericin during the malting and brewing process was investigated. Three batches of barley grains were used as starting material, one was naturally contaminated, two were artificially inoculated with Fusarium fungi. Samples were taken from each key step of the malting and brewing procedure, the levels of the toxins were determined with stable isotope dilution assays using liquid chromatography–tandem mass spectrometry detection. Significant increases of the toxins were found during germination of two batches of barley grains, resulting in green malts contamination up to a factor of 3.5 compared to grains before germination. Quantitative PCR analyses of fungal DNA revealed in all batches growth of Fusarium avenaceum during germination. After kilning, only 41–72% of the total amounts of the toxins in green malts remained in kilned malts. In subsequent mashing stage, the toxins in kilned malts predominantly were removed with spent grains. In the final beer, only one batch still contained 74 and 14 μg/kg of enniatin B and B1, respectively. Therefore, the carryover of these enniatins from the initial barley to final beer was less than 0.2% with the main amounts remaining in the spent grains and the malt rootlets.     

Residue levels of malathion and fenitrothion and their metabolites in postharvest treated barley during storage and malting

In order to investigate residue levels of malathion and fenitrothion and their metabolites (malaoxon, isomalathion and fenitrooxon) during storage and malting, pesticide-free barley was treated with these insecticides. Barley was placed in a sealed plastic container and treated with a dust of malathion (2%). Fenitrothion emulsion (41.6% wettable powder) was applied onto the walls of a small-scale storage vessel. Residues were determined in barley at about 1-month intervals during storage and in malt produced from the barley stored at various times. The analysis of the residues was carried out by GC equipped with a nitrogen-phosphorus detector (NPD). Although the approved doses of insecticides for stored grain were used, the residue levels exceeded the maximum residue limits (MRLs) at the beginning of storage. While the degradation of malathion and isomalathion in barley was observed to be about 65–72% during the storage period, the malaoxon was degraded extensively (85%). A significant percentage of fenitrothion residues (80%) were dissipated from grains for the short-term storage probably because of hot weather conditions. In malt, rates of degradation and volatilisation of the residues increased by the heat involved in malting. The carryover of the residues from barley into malt was also found to be dependent on the log P_ow (partition coefficient between n-octanol and water) values of the insecticides.     

Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins — deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes — was investigated. In addition to these ‘free’ mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of ‘free’ Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5–10 µg kg^?1), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this ‘masked’ DON in final beers exceeded ‘free’ DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).