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).     

The study of deoxynivalenol and its masked metabolites fate during the brewing process realised by UPLC–TOFMS method

The co-occurrence of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON-3-Glc) has been recently reported in malt and beer. In this study, the concentration changes were monitored within the brewing process of four beer brands: light, dark tap and two lagers, produced from ground malt mixtures differing in composition, and also mycotoxins content. A simple and rapid method employing DON-dedicated immunoaffinity columns (IAC) for the selective pre-concentration, followed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC–TOFMS) system for the reliable quantification at (ultra)trace levels, was validated for all experimental matrices. The results document the key role of the malt contamination nature. While in the first monitoring period a significant increase of both DON and DON-3-Glc occurred (up to 250% and 450%, respectively), fairly different trends were observed when new malts were used for identical technological processing (in some beers a decrease of DON and only a small increase of DON-3-Glc occurred). Worth noticing, that the outcome of the brewing process was surprisingly reproducible for a particular malt mixture. In the final phase, a small monitoring study comparing Czech and Austrian alcohol-free and conventional beers was carried out.     

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.     

Fundamental study on the influence of Fusarium infection on quality and ultrastructure of barley malt

Barley infection with Fusarium species has been a long standing problem for the malting and brewing industries. In this study, we evaluate the impact of Fusarium culmorum infected raw barley on the final malt quality. Barley grains were infected for 5 days at optimum fungal growth conditions. Grains were fully characterized and compared to standard barley grains. Due to fungal infection, germinative energy of infected barley grains decreased by 45%; its water sensitivity increased dramatically, and grains accumulated 199 μg/kg of deoxynivalenol (DON). Barley grains were subsequently malted for 8 days, fully characterized and compared to standard malt grains. Fungal growth behavior was evaluated during malting using a PCR-based assay and mycotoxins were measured using HPLC. Fungal biomass increased in grains, during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Kernel ultrastructure was evaluated using scanning electron and confocal laser scanning microscopy. Infected malt grains were characterized by extreme structural proteolytic, (hemi)-cellulolytic and starch deterioration with increased friability and fragmentation. Infected grains had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt in comparison to the control. The results of this study revealed that 20% F. culmorum infected barley kernels lead to a significant reduction in malt quality as well as mycotoxin formation.     

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。     

Incidence of trichothecenes and zearalenone in poultry feed mixtures from Slovakia

A total of 50 samples of poultry feed mixtures of Slovakian origin were analyzed for eight toxicologically significant Fusarium mycotoxins, namely zearalenone (ZON), A-trichothecenes: diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2) and B-trichothecenes: deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON) and nivalenol (NIV). The A-trichothecenes and the B-trichothecenes were detected by means of high pressure liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and gas chromatography electron capture detection (GC-ECD), respectively. Reversed phase-high performance liquid chromatography with a fluorescence detector (RP-HPLC-FLD) was used for ZON detection. The most frequent mycotoxin detected was T-2, which was found in 45 samples (90%) in relatively low concentrations ranging from 1 to 130 microg kg(-1) (average 13 microg kg(-1)), followed by ZON that was found in 44 samples (88%) in concentrations ranging from 3 to 86 microg kg(-1) (average 21 microg kg(-1)). HT-2 and DON were detected in 38 (76%) and 28 (56%) samples, respectively, in concentrations of 2 to 173 (average 18 microg kg(-1)) for HT-2 and 64 to 1230 microg kg(-1) sample (average 303 microg kg(-1)) for DON. The acetyl-derivatives of DON were in just four samples, while NIV was not detected in any of the samples investigated. In as many as 22 samples (44%), a combination of four simultaneously co-occurring mycotoxins, i.e. T-2, HT-2, ZON and DON, was revealed. Despite the limited number of samples investigated during this study poultry feed mixtures may represent a risk from a toxicological point of view and should be regarded as a potential source of the Fusarium mycotoxins in Central Europe. This is the first reported study dealing with zearalenone and trichothecene contamination of poultry mixed feeds from Slovakia.     

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.     

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.     

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.     

江苏单二大麦麦芽主要酿造品质的形成

江苏啤酒大麦在国内啤酒行业具有重要的作用, 但其质量也存在一些明显的不足, 影响了其广泛应用于啤酒生产中。本文对江苏啤酒大麦及其麦芽中存在的微生物及其影响、所形成的真菌毒素进行了综述。并针对江苏啤酒大麦麦芽过滤性能的缺陷原因进行了探讨, 为进一步推进江苏啤酒大麦产业链的健康发展提供了思路。     

Influence of barley variety and malting process on lipid content of malt

The lipid content of a beer affects its ability to form a stable head of foam and plays an important role in beer staling. The concentration and the quality of lipids in beer depend on their composition in the raw materials and on the brewing process and they may exert considerable influence on beer quality. This paper presents an investigation of the influence of barley variety and malting process on the lipid content of finished malt. Five barley samples, grown in Italy, representing 4 spring barley and 1 winter barley were used. The samples were micro-malted and analysed. The aim of this research was to verify the influence of different barley varieties on the lipid content of malt and also on the changes in fatty acid (FA) profile during the malting process. Lipid content and FA profile were evaluated. Principal component analysis (PCA) was used to establish relationships between the different samples. An evaluation of the correlation between lipid content of barleys and the quality of the resulting malts was also conducted. The data showed that the total lipid content during the malting process decreased significantly as barley was converted into malt. Different barley varieties present different FA contents and different FA patterns. The correlation between the lipid content of barley and the quality of the resulting malt confirmed the negative influence of lipids.     

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。     

Protein changes during malting and brewing with focus on haze and foam formation: a review

Beer is a complex mixture of over 450 constituents and, in addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides, and lipids. In beer, several different protein groups, originating from barley, barley malt, and yeast, are known to influence beer quality. Some of them play a role in foam formation and mouthfeel, and others are known to form haze and have to be precipitated to guarantee haze stability, since turbidity gives a first visual impression of the quality of beer to the consumer. These proteins are derived from the malt used and are influenced, modified, and aggregated throughout the whole malting and brewing process. During malting, barley storage proteins are partially degraded by proteinases into amino acids and peptides that are critical for obtaining high-quality malt and therefore high-quality wort and beer. During mashing, proteins are solubilized and transferred into the produced wort. Throughout wort boiling proteins are glycated and coagulated being possible to separate those coagulated proteins from the wort as hot trub. In fermentation and maturation process, proteins aggregate as well, because of low pH, and can be separated. The understanding of beer protein also requires knowledge about the barley cultivar characteristics on barley/malt proteins, hordeins, protein Z, and LTP1. This review summarizes the protein composition and functions and the changes of malt proteins in beer during the malting and brewing process. Also methods for protein identification are described.     

NEW ANALYSES IN MALTING AND BREWING*

Recent advances in our knowledge of the plant physiological and biochemical processes surrounding malting and brewing have prompted the development of a new generation of analytical techniques. These techniques are based upon the measurement of biochemical and physical parameters which have lately been shown to directly influence the quality of both malt and beer. Recognition of these parameters, coupled with a deep insight of the physical and chemical changes that can occur during the entire malting and brewing production chain, allow strategic emplacement of new analyses in order to provide a superior specific monitoring of malting and brewing. In the present context we submit several examples of these novel analyses, spanning from barley control to analysis of pasteurising efficiency. Characteristic for all the analyses discussed, is the ease and rapidity with which they can be performed. The following methods are described: (1) Measurement of both pre-harvest sprouting and germination efficiency in barley. (2) Malt modification analysis to measure malting efficiency, time of kilning and malt homogeneity. (3) Rapid β-glucan analysis in malt, wort and beer. (4) Immunological analysis to determine the amount and type of additives in finished beer as well as addition of adjunct. (5) Enzymic and immunological control of beer pasteurisation.     

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.     

Multivariate analysis for feasibility of Korean six‐row barleys for beer brewing

The feasibility of using six‐row barley, which is produced more often than two‐row barley (malting barley) in Korea, for beer brewing was studied. Beer was brewed from one variety of two‐row barley (Jinyang, malting barley) and four varieties of six‐row barley (Jasujungchal and Hinchalssal which are unhulled; Dahyang and Samgwangchal which are hulled). Using principal component analysis of the material properties in malting, mashing and fermentation, and the sensory properties of beer, the barley was categorized into three groups: group 1 (Jinyang and Dahyang), group 2 (Samgwangchal and Hinchalssal) and group 3 (Jasujungchal). Group 1 was distinctive for extract (dry basis), Brix and carbonation; group 2 was characterized by alcohol, foam stability and sour odour; and group 3 was characterized by malt protein and sour taste. The brewing qualities of group 1 were superior to those of the other groups. Among the Korean six‐row barley varieties, Dahyang was found to be the most suitable for beer production. Copyright © 2014 The Institute of Brewing & Distilling     

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.     

PROANTHOCYANIDIN-FREE BARLEY—MALTING AND BREWING

Colloidal haze in beer is due to the precipitation of proteins by proanthocyanidins. Carlsberg Research Center reported in 1977 that the use of barley mutants which block the biosynthesis of catechins and proanthocyanidins in the grain prevents the formation of haze in beer. The results from ten years of malting and brewing research with proanthocyanidin-free barley and malt are reviewed.