‘Emerging’ mycotoxins in cereals processing chains: Changes of enniatins during beer and bread making

Enniatins represent an emerging food safety issue because of their extensive incidence, documented in recent decades, in various small grain cereals. This study was concerned with the fate of these Fusarium mycotoxins within malting, brewing, milling and baking, when employed for the processing of contaminated barley and wheat. Besides enniatins A, A1, B and B1, also deoxynivalenol and its conjugated form (deoxynivalenol-3-glucoside) were determined in almost all tested cereal-based samples. Significant decline of enniatins occurred within all technologies, with the largest drop in their concentrations observed in the brewing process. While enniatins were not detectable in final beers, they were almost quantitatively transferred to spent grains, probably because of their limited water solubility. Regarding bread baking, levels of enniatins decreased down to 30% of their concentration in the initial flour used for baking. In this case, degradation at higher temperatures might be assumed.     

MALTING CHARACTERISTICS OF FINGER MILLET,SORGHUM AND BARLEY*

The malting characteristics of the finger millet variety Imele (FI), sorghum varieties Andivo (SA) and Ingumba (SI) and the barley variety Research (BR) were compared in relation to the brewing of traditional African opaque beer as well as conventional lager beer. The investigations include (a) the effect of steeping and germination conditions, (b) the influence of gibberellic acid and kilning temperature on the activity of important brewing enzymes and (c) an appraisal of the brewing potential of the worts obtained. FI, SA and SI malts were considered unsuitable as barley malt extenders for conventional lager beers, but FI and possibly SI malts would be suitable for tropical lager beer manufacture.     

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).     

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).     

THE PRESENCE OF TWO DIMETHYL SULPHIDE PRECURSORS IN MALT,THEIR CONTROL BY MALT KILNING CONDITIONS,AND THEIR EFFECT ON BEER DMS LEVELS

The precursor of dimethyl sulphide (DMS) which is formed during barley germination and is present in green malt differs from that present in malt kilned at high (> ～75°C) temperatures in that it cannot be metabolized to DMS by yeast. It is therefore termed ‘inactive’ precursor to distinguish it from the ‘active’ precursor present in kilned malt, which is metabolized to DMS by yeast. During malt kilning, inactive precursor is changed into the active form, but only at temperatures at which destruction of both precursors is also taking place. Therefore, for any kiln there is an optimum temperature range over which maximum conversion of inactive to active precursor can be combined with minimum destruction of both precursors. The DMS in beers brewed from green malt or malts kilned only at low (< ～70°C) temperatures is almost entirely the result of precursor destruction during wort boiling, final levels being governed not only by the conditions of boiling, but also by the extent of losses during fermentation and subsequent processing. In contrast, the DMS in beers brewed from malts kilned at higher temperatures is mostly formed during fermentation, and since beer DMS levels can be related to wort and beer DMS precursor levels, they can be better predicted and controlled. The method used for the measurement of total DMS precursor levels in malts is described in detail.     

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.     

THE MEASUREMENT OF DIMETHYL SULPHIDE PRECURSOR IN MALTS,ADJUNCTS, WORTS AND BEERS

Dimethyl sulphide in beer originates from a precursor in the malt. A method is described for the measurement of this precursor in brewing raw materials, wort and beer. The precursor levels in various green malts, kilned malts, and adjuncts are given. Excess methionine inhibits precursor uptake by yeast during fermentation. Residual precursor is still present in some commercial bottled beers.     

Micromalting triticale: Optimising processing conditions

Small-scale trials were carried out to optimise the conditions for micromalting triticale grain, using three varieties grown in the UK. Compared with barley, triticale grains required short steeping periods with a short air rest. They malted rapidly, and produced malts with high hot water extracts after 4 or 5 days' germination. Applications of gibberellic acid and potassium bromate during malting significantly altered the qualities of malts. Gibberellic acid increased hot water extracts, soluble nitrogen levels and malting losses. When the additives were used in combination, high hot water extracts were obtained and malting losses were reduced relative to controls, but unexpectedly the level of total soluble nitrogen was elevated and did not significantly differ from that obtained using gibberellic acid alone. Both additives usefully reduced wort viscosities, which were unacceptably high by barley malt brewing standards. Wort separation from small-scale mashes was slow.     

Malting for Brewhouse Performance*

In many instances brewhouse performance cannot be predicted from a finished malt specification. This is particularly so for factors such as lautering, yeast performance, filtration and head character. There are two aspects involved in improving the brewing performance of malt. Firstly, an understanding of the malt characteristics affecting particular aspects of the brewing process and secondly an understanding of how the malting process affects or can be controlled to optimise these characteristics and thus their performance. In this work the relationship between barley variety, steeping pattern, malt quality and brewing performance is investigated. Six different barley varieties were micromalted in a Seeger micromalting unit under four different steeping regimes. Sub-samples were taken at intervals during steeping for enzyme analysis and measurement of water distribution. Following steeping, the samples were germinated and kilned using standard micromalting conditions. Finished malts were analysed by standard EBC methods for routine malt quality parameters including apparent attenuation limit. Worts were tested for total β-glucan content, β-glucan molecular weight distribution, filter plugging potential and carbohydrate levels. A sub set of malt samples were then micro-brewed and tested for β-glucan molecular weight (MW) distribution, beer filterability and foam stability.     

A study on malt modification,used as a tool to reduce levels of beer hordeins

Storage proteins from barley, wheat and rye are toxic to gluten sensitive consumers. These consumers include those suffering from coeliac disease, which account for up to 1% of the global population, and non‐coeliac gluten sensitivity that may affect even greater numbers of the population. Codex Alimentarius has published guidelines and limits of gluten in gluten‐free foods, which are applied in Europe, and similar guidelines apply in the rest of the world. The storage proteins present in barley are hordeins. These proteins are broken down and used by the plant as a source of amino acids during germination and growth of the barley embryo. The objective of this study was to extend the germination stage of the malting process and look at the effect on beer hordeins. Standard MEBAK methods were used to develop an extended malting process and produce three different malts, germinated for 3, 5 or 7 days. The quality of malt was assessed and model beers were produced from each malt to test the effect of modification on levels of beer hordeins. Malt germinated for 7 days produced beer 18 mg/kg hordeins corresponding to a reduction of 44% compared with the beer made from malt germinated for 3 days characterized by a hordein content equal to 32 mg/kg. The malting loss was increased during the 7 days of germination but otherwise all malts were of high quality. The results showed that malting conditions have a significant impact on beer hordeins. Copyright © 2018 The Institute of Brewing & Distilling     

Effects of nitrogen application on malt modification and dimethyl sulfide precursor production in two Japanese barley cultivars

BACKGROUND: Nitrogenous components have a great influence on both malt and beer qualities. Barley storage proteins are degraded during the germination process, in which amino acids and small peptides are released. Some of these compounds relate to dimethyl sulfide precursor production in the malting process. In this study, barley and malt qualities were investigated using two Japanese barley cultivars, Sukai Golden and Mikamo Golden, with several different nitrogen (N) treatments. RESULTS: Nitrogen top‐dressing treatments efficiently increased N and sulfur (S) concentrations in grains. A difference in malt modification was induced by these treatments without any change in protease activity in malts. S‐Methyl methionine (SMM) concentration in malt of Sukai Golden with low‐N treatment was 1.8–2.1 times higher than that with higher‐N treatments. Methionine concentration in malts was not significantly affected by N treatments of both cultivars, while grain S level was not consistent under any treatments. CONCLUSION: Results show that low‐N treatment increases SMM concentration in malts despite major S‐containing amino acids of malts being not highly affected by the difference in nutrient status of grains. Further investigations are necessary into aspects of both metabolic profiles in barley germination and SMM degradation in the kilning process. Copyright © 2008 Society of Chemical Industry     

The effect of germination and kilning on the cyanogenic potential,amylase and alcohol levels of sorghum malts used for burukutu production

Grain sorghum of the red and white varieties was malted by steeping in water for 18 h, germinated over 5 days and kilned at 50 °C. The malts were analysed for amylase activities and cyanogenic potential and used to produce burukutu, an alcoholic beverage. The alcohol content of the burukutu was recovered by distillation and determined by the refractive index method. α‐Amylase activity peaked on malting day 3 and was higher in the white malts. β‐Amylase activity peaked on day 3 in the red malts and on day 4 in the white malts, but was higher in the red malts. Dhurrinase activity was highest on malting day 4, with a higher activity in the red malts. Kilning at 50 °C reduced the activities of these enzymes. The dhurrin content increased during germination and was consistently higher in the white malts, in which there was evidence of dhurrin mobilisation. In the red malts the dhurrin content increased during germination but decreased progressively after kilning; evidence of dhurrin mobilisation was apparent as from malting day 4. Burukutu produced from the red malts gave higher alcohol contents than that from the white malts. Maximum alcohol yields were obtained on malting day 3 in the red malts and on day 5 in the white malts. © 2000 Society of Chemical Industry     

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。     

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.     

Optimization of Malting Conditions for Two Black Rice Varieties,Black Non‐Waxy Rice and Black Waxy Rice (Oryza sativa L. Indica)

Two black rice varieties, “black non‐waxy” and “black waxy”, were investigated as possible raw materials for the production of malt. The malting conditions were optimised using response surface methodology. The three process parameters were steeping, germination time and temperature. Each parameter was tested at three levels: adjustment degrees of steeping were 38, 41, and 44%, germination times were 6, 7, and 8 days, and the temperatures were 20, 25 and 30°C. At the end of the germination process, all samples were kilned at 50°C for 24 h, and shoot/rootlets were removed before a detailed quality assessment was performed. Data analysis was performed using the Design Expert Statistic Program. The optimal conditions found for both rice varieties were as follows: germination time of 8 days at 30°C and 44% grain moisture. Although the extract yield, and a‐amylase and β‐amylase activities of both rice malts were lower than barley malt, the higher activity of limit‐dextrinase enzyme and apparent attenuation limit (AAL), which was higher than 80%, suggests that rice malt has potential for use in brewing.     

Effect of Germination Moisture and Time on Pearl Millet Malt Quality — With Respect to Its Opaque and Lager Beer Brewing Potential

The effect of germination moisture and time on pearl millet malt quality was investigated. Two pearl millet varieties SDMV 89004 and 91018 were germinated at 25°C under three different watering regimes for 5 days. As with sorghum malting, diastatic power, beta‐amylase activity, free α‐amino nitrogen (FAN), hot water extract and malting loss all increased with level of watering. However, pearl millet malt had a much higher level of beta‐amylase and higher FAN than sorghum malt and a similar level of extract. Malting losses were similar or lower than with sorghum. Thus, it appears that pearl millet malt has perhaps even better potential than sorghum malt in lager beer brewing, at least as a barley malt extender, especially in areas where these grains are cultivated and barley cannot be economically cultivated. Also, its increased use in commercial opaque beer brewing, where sorghum malt is currently used, could be beneficial.     

Utilization of the Linear Mode of MALDI‐TOF Mass Spectrometry in the Study of Glycation During the Malting Process

The process of glycation during the malting process was monitored by the linear mode of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF MS). Water‐soluble proteins were investigated and two hulled barley varieties, Jersey and Tolar, were compared to the hulless line KM 1910. The crude extracts of the proteins obtained from the grain, the malt, and aliquots collected every 24 h during the malting process, were mixed with the matrix (2,6‐dihydroxyacetophenone) and analyzed by mass spectrometry. The protein composition of the barley changed during the malting process. The protein patterns did not differ significantly between the three varieties of the barley grains. However, significant differences between the malts were evident. Results showed the influence of the malting process on the glycation of certain water‐soluble barley proteins, nonspecific lipid transfer protein 1 (LTP1) and protein Z, of which the glycated forms survived the brewing process. These major barley proteins are very important for the formation and stability of beer foam and glycation may prevent their precipitation. Analysis results indicated that slight glycation of the proteins had occurred on the second day of malting. The linear mode of MALDI‐TOF mass spectrometry was used as a fast and simple method for monitoring the patterns of low‐molecular weight barley proteins with regard to barley variety discrimination. This procedure also enables the selection of barley varieties suitable for the malting industry.     

Influence of barley variety, timing of nitrogen fertilisation and sunn pest infestation on malting and brewing

BACKGROUND: This paper presents a multivariate approach to investigate the influence of barley variety, timing of nitrogen fertilisation and sunn pest infestation on malting and brewing. Four spring and two winter barley varieties were grown in one location in southern Europe. Moreover, one of the spring varieties was infested with sunn pest, in order to study the effects of this pest on malting quality, and subjected to different nitrogen fertilisation timing regimes. The samples were micromalted, mashed, brewed and analysed. RESULTS: The data showed that even though the two winter barleys seemed to be the best regarding their physical appearance (sieving fraction I + II > 82%), this superiority was not confirmed in the malt samples, which showed low values of Hartong extract (27.1%) and high values of pH (6.07–6.11) and β‐glucan content (12.5–13.2 g kg^?1), resulting in low‐quality beers. The barley sample subjected to postponed fertilisation had a total nitrogen content (19.5 g kg^?1 dry matter) exceeding the specification for malting barley and gave a beer with a low content of free amino nitrogen (47 mg L^?1) and high values of viscosity (1.99 cP) and β‐glucan content (533 mg L^?1). The beer obtained from the barley sample subjected to pest attack had good quality parameters. CONCLUSION: All spring barleys gave well‐modified malts and consequently beers of higher quality than the winter barleys. Moreover, postponed fertilisation was negatively related to the quality of the final beer, and sunn pest infestation did not induce important economic losses in the beer production chain. Copyright © 2010 Society of Chemical Industry     

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     

Influence of the malting parameters on the haze formation of beer after filtration

Malting changes the chemical and enzymatical composition of barley. During malting, enzymes are synthesized, cell walls (pentosans, proteins, etc.) degraded and starch becomes available for enzymatic attack. The progress of germination defines the final beer quality and processability in several aspects: mouthfeel, foam and haze formation (different proteins), processability (viscosity caused by certain substances, like β-glucan), fermentation progress (FAN, sugar content), etc. The objective of this research was to study the influence of different modified malt on turbidity in beer after filtration. This was achieved by analyzing selected malts at different germination stages and afterward studying their influence on the final beer composition, focusing on protein content and composition. Protein fractions were analyzed using a Lab-on-a-Chip technique, which separates the proteins—based on their molecular weight—by capillary electrophoresis. This analysis was supported by the use of two-dimensional gel electrophoresis (2D-PAGE). Additionally, common malt and beer analyses and turbidity and filterability measurements were performed. The protein composition could be followed from malt to beer with both the Lab-on-a-Chip technique and 2D-PAGE. No differences in protein composition could be seen in the final protein composition of the beer. However, it could be observed, with Lab-on-a-Chip technique, that high amounts of a protein fraction with a size of 25–28 kDa caused increased turbidity in the beer.