Proteomics and Metabolomics Reveal that an Abundant α-Glucosidase Drives Sorghum Fermentability for Beer Brewing.

Sorghum (Sorghum bicolor), a grass native to Africa, is a popular alternative to barley for brewing beer. The importance of sorghum to beer brewing is increasing because it is a naturally gluten-free cereal, and climate change is expected to cause a reduction in the production of barley over the coming decades. However, there are challenges associated with the use of sorghum instead of barley in beer brewing. Here, we used proteomics and metabolomics to gain insights into the sorghum brewing process to advise processes for efficient beer production from sorghum. We found that during malting, sorghum synthesizes the amylases and proteases necessary for brewing. Proteomics revealed that mashing with sorghum malt required higher temperatures than barley malt for efficient protein solubilization. Both α- and ß-amylase were considerably less abundant in sorghum wort than in barley wort, correlating with lower maltose concentrations in sorghum wort. However, metabolomics revealed higher glucose concentrations in sorghum wort than in barley wort, consistent with the presence of an abundant α-glucosidase detected by proteomics in sorghum malt. Our results indicate that sorghum can be a viable grain for industrial fermented beverage production, but that its use requires careful process optimization for efficient production of fermentable wort and high-quality beer.

Mashing performance as a function of malt particle size in beer production.

Significant innovations have occurred over the past 50 years in the malting and brewing industries, focused on optimization of the beer mashing, boiling and fermentation processes. One of the challenges faced in beer brewing has been in the malting process to obtain the desired malt and wort quality to produce high-quality beer products. The hydrolytic enzymes produced during grain germination are mostly entrapped inside the cellular matrices of the grain. The intra-grain diffusion of enzymes for in-situ hydrolysis, as well as diffusion of enzymes to wort, depends upon the malt size and malt size fractions obtained after milling. This review investigates the relationship between varying barley grain particle size distribution and the efficiency of the malting and mashing processes. Recommended ideal particle size of barley grain before and after milling are proposed based on the review of existing literature. Each brewing batch of grains with a proportion of >80% plump grains (>2.5 mm in size) is suggested to be the optimal size before milling, whereas the optimum grain particle size after milling ranged between 0.25 and 0.5 mm. The current review will summarize the theoretical aspects for malt milling and the particle size characteristics for optimizing the brewing process.

Posttranslational Modifications Drive Protein Stability to Control the Dynamic Beer Brewing Proteome.

Mashing is a key step in beer brewing in which starch and proteins are solubilized from malted barley in a hot water extraction and digested to oligomaltose and free amino nitrogen. We used SWATH-MS to measure the abundance and site-specific modifications of proteins throughout a small-scale pale ale mash. Proteins extracted from the malt at low temperatures early in the mash decreased precipitously in abundance at higher temperatures late in the mash due to temperature/time-induced unfolding and aggregation. We validated these observations using experimental manipulation of time and temperature parameters in a microscale pale ale mash. Correlation analysis of temperature/time-dependent abundance showed that sequence and structure were the main features that controlled protein abundance profiles. Partial proteolysis by barley proteases was common early in the mash. The resulting proteolytically clipped proteins were particularly sensitive and were preferentially lost at high temperatures late in the mash, while intact proteins remained soluble. The beer brewing proteome is therefore driven by the interplay between protein solubilization and proteolysis, which are in turn determined by barley variety, growth conditions, and brewing process parameters.

Brewing with malted barley or raw barley: what makes the difference in the processes?

Malted barley is the main source for fermentable sugars used by yeasts in the traditional brewing of beers but its use has been increasingly substituted by unmalted barley and other raw grain adjuncts in recent years. The incorporation of raw grains is mainly economically driven, with the added advantage of improved sustainability, by reducing reliance on the malting process and its associated cost. The use of raw grains however, especially in high proportion, requires modifications to the brewing process to accommodate the lack of malt enzymes and the differences in structural and chemical composition between malted and raw grains. This review describes the traditional malting and brewing processes for the production of full malt beer, compares the modifications to these processes, namely milling and mashing, when raw barley or other grains are used in the production of wort-a solution of fermentable extracts metabolized by yeast and converted into beer, and discusses the activity of endogenous malt enzymes and the use of commercial brewing enzyme cocktails which enable high adjunct brewing.

The chemical profiling of fatty acids during the brewing process.

BACKGROUND: Although fatty acids have a beneficial effect on yeast growth during fermentation, their effect on foam and sensory stability of beer is negative. In general, long-chain fatty acids originate from raw materials, whereas short-chain acids are produced by yeast during fermentation. If the concentration of short-chain fatty acids, especially isovaleric and butyric acid, overreaches a sensory threshold, then an unpleasant aroma, such as cheesy or sweaty feet, can be formed in beer. RESULTS: The distribution of fatty acids, from the preparation of sweet wort to the final beer, was studied using chemometric evaluation. Differences were observed between the decoction and infusion system using four barley varieties. Attention was paid to the behavior of short-chain fatty acids, namely isovaleric acid. The concentration of isovaleric acid in commercial beers brewed in infusion and decoction systems was approximately 1.4 and 1.0 mg L-1 , respectively. The same trend was observed in experimental samples (1.3 and 0.5 mg L-1 , respectively). This phenomenon was confirmed experimentally; based on the results, this possibly explains why, during the fermentation, isovaleric acid is coupled with the redox state of yeast cell, which is given by the wort composition (i.e. by the mashing process). CONCLUSION: The formation of isovaleric acid is not only caused by microbiology infection or by oxidized hops, but also is influenced by the mashing process. © 2018 Society of Chemical Industry.

Investigation of changes in rheological properties during processing of fermented cereal beverages.

Many indigenous fermented foods and beverages consumed throughout the world are produced at home or in crafts enterprises. The production of fermented beverages on a large commercial or industrial scale requires clearly established technical and technological requirements. This study shows a novel way to investigate the optimal process parameters of the Kyrgyz traditional fermented beverage Bozo using rotational rheological parameters. Five significant process parameters were investigated like cooking of millet porridge, the mashing temperature, the mashing time under conditions of mixing and viscosity changes of the end product during storage. According to the gelatinization temperature of millet porridge, cooking parameters were recommended at T = 79 °C and t = 30 min. The optimum mashing temperature of millet porridge was determined to be 65 °C and mashing time under stirring conditions of millet porridge was found to be 10 min. The viscosity of the beverage Bozo was investigated after 7, 14 and 21 days of storage at 5, 10, 20, and 30 °C. The effective viscosity of Bozo was calculated using the Casson model, which increased from 39.67 to 51.25 Pa·s after 21 days of storage. The effect of temperature on effective viscosity of Bozo and the activation energy was calculated using an Arrhenius-type equation. The parameters obtained make it possible to provide food manufacturers useful information for boiling, mashing and storage parameters after fermentation as well as quality control of Bozo.

Direct determination of amino acids in brewery worts produced by different processes by capillary zone electrophoresis.

The direct and simultaneous determination of cysteine, histidine, phenylalanine, lysine, tryptophan and arginine in brewery worts by capillary zone electrophoresis (CZE) was applied to evaluate the effects of temperature control and protease supplementation during mashing on the changes of these amino acids (AAs) wort composition. A cation exchange resin was used for AAs extraction from wort samples prior to CZE determination. The separation was achieved using a 50 mmol/L phosphate buffer at pH 12.5, containing 0.4 mmol/L cetyltrimethylammonium bromide (CTAB), as background electrolyte (BGE) solution; -20 kV; 20°C and hydrodynamic injection time of 15 s, at 50 mbar. Recovery evaluation using worts led to values between 83.1 and 96.2%, demonstrating the method feasibility, which was successfully applied in the quantification of AAs in wort samples. This study showed that temperature control and addition of exogenous proteases in the mashing may increase the AAs concentration in wort, improving the final product quality (beer). The present method is a good alternative for monitoring specific AAs in worts and their determination can allow the brewing process optimization.

Characterization of the molar mass distribution of macromolecules in beer for different mashing processes using asymmetric flow field-flow fractionation (AF4) coupled with multiple detectors.

The macromolecular composition of beer is largely determined by the brewing and the mashing process. It is known that the physico-chemical properties of proteinaceous and polysaccharide molecules are closely related to the mechanism of foam stability. Three types of "American pale ale" style beer were prepared using different mashing protocols. The foam stability of the beers was assessed using the Derek Rudin standard method. Asymmetric flow field-flow fractionation (AF4) in combination with ultraviolet (UV), multiangle light scattering (MALS) and differential refractive index (dRI) detectors was used to separate the macromolecules present in the beers and the molar mass (M) and molar mass distributions (MD) were determined. Macromolecular components were identified by enzymatic treatments with ß-glucanase and proteinase K. The MD of ß-glucan ranged from 106 to 108 g/mol. In addition, correlation between the beer's composition and foam stability was investigated (increased concentration of protein and ß-glucan was associated with increased foam stability).

Construction of a highly thermostable 1,3-1,4-ß-glucanase by combinational mutagenesis and its potential application in the brewing industry.

OBJECTIVES: To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-ß-glucanase by combinational mutagenesis and to test its effect in congress mashing. RESULTS: A mutant ß-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg-1 and 189 s-1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml-1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively. CONCLUSIONS: The mutant ß-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

Modeling and Optimization of Triticale Wort Production Using an Artificial Neural Network and a Genetic Algorithm.

Triticale grain, a wheat-rye hybrid, has been reported to comply very well with the requirements for modern brewing adjuncts. In this study, two triticale varieties, in both unmalted and malted forms, were investigated at various ratios in the grist, applying different mashing regimes and concentrations of the commercial enzyme Shearzyme® 500 L with the aim of evaluating their impact on wort production. In order to capture the complex relationships between the input (triticale ratio, enzyme ratio, mashing regime, and triticale variety) and output variables (wort extract content, wort viscosity, and free amino nitrogen (FAN) content in wort), the study aimed to implement the use of artificial neural networks (ANNs) to model the mashing process. Also, a genetic algorithm (GA) was integrated to minimize a specified multi-objective function, optimizing the mashing process represented by the ANN model. Among the solutions on the Pareto front, one notable set of solutions was found with objective function values of 0.0949, 0.0131, and 1.6812 for the three conflicting objectives, respectively. These values represent a trade-off that optimally balances the different aspects of the optimization problem. The optimized input variables had values of 23%, 9%, 1, and 3 for the respective input variables of triticale ratio, enzyme ratio, mashing regime, and triticale variety. The results derived from the ANN model, applying the GA-optimized input values, were 8.65% w/w for wort extract content, 1.52 mPa·s for wort viscosity, and 148.32 mg/L for FAN content in wort. Comparatively, the results conducted from the real laboratory mashing were 8.63% w/w for wort extract content, 1.51 mPa·s for wort viscosity, and 148.88 mg/L for FAN content in wort applying same input values. The presented data from the optimization process using the GA and the subsequent experimental verification on the real mashing process have demonstrated the practical applicability of the proposed approach which confirms the potential to enhance the quality and efficiency of triticale wort production.

The Impact of 10 Unmalted Alternative Adjuncts on Wort Characteristics.

Consumers are more than ever in search of novel and exciting beer choices, and brewers are, therefore, continuously experimenting to adapt their product portfolio. One interesting way to naturally incorporate novel flavors and tastes is by using alternative adjuncts, but this is not always an easy and straightforward process. In this study, a 40% unmalted alternative adjunct (einkorn, emmer, spelt, khorasan, quinoa, amaranth, buckwheat, sorghum, teff, and tritordeum) or reference (barley malt, unmalted barley, and unmalted wheat) was added to 60% barley malt, after which three different laboratory mashing processes (Congress mash, Congress mash with pre-gelatinization of the adjunct, and Evans mash) were performed, and their behavior during mashing and the resulting wort characteristics were investigated in detail. Overall, the extraction process of all 10 unmalted alternative adjuncts was not complete for all three laboratory mashing processes, whereby Congress mashing resulted in the highest extract and fastest filtration, whereas Evans mashing resulted in the lowest extract and slowest filtration. Pre-gelatinization of the unmalted was generally only beneficial for adjuncts with high onset starch gelatinization temperatures. This process also inactivated endogenous enzymes in the unmalted adjuncts, which had an adverse effect on the mashing process.

Modification mechanism of protein in rice adjuncts upon extrusion and its effects on nitrogen conversion during mashing.

The traditional production of wort with adjunct-introduced was achieved by double mashing procedure, which hindered the utilization of proteins in adjunct and led to a deficiency of nitrogen in wort. In this study, the modification mechanism of the extrusion pretreatment on the structure characterization of rice flour protein was investigated. The decoction mashing procedure was performed to enhance the nitrogen conversion of the extruded rice adjunct. Decreased solubility along with disrupted secondary and tertiary structures of rice protein were observed after extrusion. As a result, the total nitrogen, free amino nitrogen, and free amino acids content of wort with extruded rice adjunct-introduced were improved by 23.28 %, 34.67 %, and 7.33 %, respectively, which could be verified by the electrophoretic patterns of the wort protein. The application of extrusion as a pretreatment of adjuncts can promote the protein availability of adjuncts in the decoction mashing stage.

Successful Treatment of Microsporidial Keratoconjunctivitis (MKC) With a Combination of Topical Voriconazole 1% and Gatifloxacin 0.5%: A Large Case Series of 29 Patients.

OBJECTIVE: This study aims to report the successful treatment of microsporidial keratoconjunctivitis (MKC) with the combination of topical drops of voriconazole (1%) and gatifloxacin (0.5%) in all 29 patients. Demography, clinical profile, and previous treatment history were also analyzed. METHODS: A retrospective, non-comparative case series of all Gram stain-proven MKC from September 2021 to October 2022 was included in this study. Patients were given antimicrobials such as topical drops of voriconazole 1%, gatifloxacin 0.5%, or a combination of both in 29 patients based on the treatment response. Topical steroids were added to 31 patients for corneal haziness. RESULTS: A total of 33 patients were found to be positive for microsporidiosis confirmed by Gram staining. Twenty-four (72.7%) were men and nine (27.3%) were women. The mean age was 34.45±12. The presenting symptoms were mainly redness in 30 patients (90.9%), followed by watering in 13 (39.4%), foreign body sensation in 10 (30.3%), etc. Among the 23 patients (69%), a history of risk factors was identified, with 17 patients (51.5%) specifically reporting dust exposure as a major cause. MKCs were successfully treated with antimicrobials such as voriconazole 1% in three patients, gatifloxacin 0.5% in one patient, and a combination of both in 29 patients. Topical steroids were added to 31 patients for corneal haziness. At the last follow-up, a visually insignificant nummular corneal scar was noted in six patients. No drop in vision was noted in any of these patients at the end of the follow-up. No cases progressed to stromal keratitis and no surgical intervention was required in any cases. CONCLUSIONS: We successfully treated all 29 cases with a combination of voriconazole and gatifloxacin without requiring any surgical intervention or encountering stromal complications. This successful treatment in all 29 cases offers valuable insights into the potential of this drug combination, possibly attributable to its additive action or broad-spectrum coverage across various species.

Effects of the mashing process and macromolecule contents on the filterability of barley malt.

The filterability of barley malt is a critical quality parameter in beer brewing. The effects of two mashing processes (processes A and B) on the filterability of the three barley malts and their macromolecule contents were investigated. Filtration volume increased by 4%, 9%, and 13% for the Baudin, Ganpi, and Gangpi malts, respectively, and the final filtration volume of Gangpi was still poorer than that of Baudin. A downward mashing process (process C) was applied to measure the ß-glucan, arabinoxylans (AX), the polymeric arabinoxylan (PAX), and high molecular weight nitrogen (HMWN) content. The ß-glucan degradation rate of well-modified malt during malting was higher than that of poorly modified malt, whereas the PAX and HMWN solubilization rates during malting were lower in well-modified malt than in poorly modified malt. The filterability of poorly modified malt did not effectively improve with an initial mashing phase at 37℃. ß-Glucan degradation and PAX and HMWN solubilization during malting were critical for ensuring malt quality. Thus, the goal of predicting the filterability of malt was achieved by applying a downward mashing process. PRACTICAL APPLICATION: Quality of wort and beer as well as production efficiency is affected by the malt quality. The filterability of barley malt can affect the production efficiency and quality of wort. The change in the macromolecule contents during malting is important to ensure the production of high-quality malt. The results of this study can provide a good method for the detection of malt filtration performance, and it also may contribute to the purpose of prediction the changes in barley malt and the resulting barley malt filterability.

A kinetic study on the thermal inactivation of barley malt α-amylase and ß-amylase during the mashing process.

To obtain an efficient conversion of starch into fermentable sugars and dextrins during the brewing process, mashing time-temperature profiles need to promote starch gelatinisation and enzyme activity while avoiding thermal inactivation of the amylases. This study focused on the second part of this balance by investigating the thermal stability of α-amylase and ß-amylase of Planet barley malt throughout mashing. Thermal inactivation in wort was modelled for both enzymes resulting in the estimation of thermal inactivation kinetic parameters such as rate constant of thermal inactivation kT (the rate of thermal inactivation of an enzyme at a constant temperature), activation energy for thermal inactivation Ea, decimal reduction time DT (the time needed to inactivate 90% of the enzyme activity at a given temperature) and the z-value. First-order inactivation was observed for α-amylase. For ß-amylase, fractional conversion inactivation occurred with a residual fraction of 13% of the ß-amylase activity that remained after prolonged heating at 72.5 °C. The ß-amylase protein population hence seems to consist of thermolabile and thermostable isoforms. The kinetic parameters for thermal inactivation of the enzymes were used to predict their residual activities throughout a laboratory-scale mashing process. The predicted residual activities met the experimentally determined residual enzyme activities closely, except for ß-amylase at temperatures higher than 72.5 °C. The results obtained in this work allow designing new mashing processes or tailoring existing processes towards variability in the input material, barley malt, without the need for trial-and-error experiments.

Proteolytic activities and profiles as useful traits to select barley cultivars for beer production.

Barley malting depends on hydrolytic enzymes that degrade storage macromolecules. Identifying barley cultivars with proteolytic activity that guarantees appropriate foaming, flavor, and aroma in the beer is of great importance. In this work, the proteolytic activity and profiles of brewing malt from Mexican barley cultivars were analyzed. Data showed that Cys- (at 50°C) and Ser-proteases (at 70°C) are the major contributors to proteolytic activity during mashing. Essential amino acids, necessary for fermentation and production of good flavor and aroma in beer, were detected at the end of mashing. According to our results, Mexican cultivar HV2005-19 exhibits similar proteolytic activities as those from cultivar Metcalfe, which is one of the most utilized for the brewing industry. Moreover, we propose Cys- and Ser-proteases as biochemical markers during mashing at 50 and 70°C, respectively, to select barley cultivars for beer production. PRACTICAL APPLICATIONS: Proteolytic activity, which depends on activation and de novo synthesis of proteases in the aleurone layer of barley seeds, is crucial in beer production. Identifying new barley varieties that have optimal proteolytic activities is of great interest for genetic improvement programs. In this study, we propose the variety HV2005-19 as a genotype with Cys- and Ser-proteases activity similar to that from Metcalfe, which is a top variety in the brewing industry.

Starch hydrolysis during mashing: A study of the activity and thermal inactivation kinetics of barley malt α-amylase and ß-amylase.

Hydrolysis of starch is key in several industrial processes, including brewing. Here, the activity and inactivation kinetics of amylases throughout barley malt mashing are investigated, as a prerequisite for rational optimisation of this process. Varietal differences were observed in the activity of α- and ß-amylases as a function of temperature for six barley and malt varieties. These differences were not reflected in the resulting wort composition after mashing, using three isothermal phases of 30 min at 45 °C, 62 °C and 72 °C with intermediate heating by 1 °C/min. Thermal inactivation kinetics parameters determined for α- and ß-amylases of an industrially relevant malt variety in a diluted system showed that enzymes were inactivated at lower temperatures than expected. The obtained kinetic parameters could predict α-amylase, but not ß-amylase inactivation in real mashing conditions, suggesting that ß-amylase stability is enhanced during mashing by components present or formed in the mash.

Gelatinization or Pasting? The Impact of Different Temperature Levels on the Saccharification Efficiency of Barley Malt Starch.

Efficient enzymatic hydrolysis of cereal starches requires a proper hydrothermal pre-treatment. For malted barley, however, the exact initial temperature is presently unknown. Therefore, samples were micro-mashed according to accurately determined gelatinization and pasting temperatures. The impact on starch morphology, mash viscometry and sugar yields was recorded in the presence and absence of an amylase inhibitor to differentiate between morphological and enzymatic effects. Mashing at gelatinization onset temperatures (54.5-57.1 °C) led to negligible morphological and viscometric changes, whereas mashing at pasting onset temperatures (57.5-59.8 °C) induced significant starch granule swelling and degradation resulting in increased sugar yields (61.7% of upper reference limit). Complete hydrolysis of A-type and partial hydrolysis of B-type granules was achieved within only 10 min of mashing at higher temperatures (61.4-64.5 °C), resulting in a sugar yield of 97.5% as compared to the reference laboratory method mashing procedure (65 °C for 60 min). The results indicate that the beginning of starch pasting was correctly identified and point out the potential of an adapted process temperature control.

Characteristic changes in malt, wort, and beer produced from different Nigerian rice varieties as influenced by varying malting conditions.

Gluten-free beer could be produced with rice, although the latter would primarily serve as adjunct in combination with barley malt in today's brewing. However, the recent growing realisation of the potential and applications of rice malt for brewing an all-rice malt beer through varying malting conditions cannot be overlooked. In this study, therefore, the characteristic changes in malt, wort, and beer from different Nigerian rice varieties (FARO 44, FARO 57, NERICA 7) as influenced by varying malting conditions (steeping duration (18, 24 and 30 h), germination periods (2, 3 and 4 days) and kilning temperatures (50 and 55 °C)), were investigated. Rice (grain) samples were examined by thousand kernel weight (TKW), germinative energy (GE), germinative capacity (GC), and degree of steeping (DoS). To ensure that rice wort/beer with unique beer style and enhanced attributes, comparable to barley wort/beer is produced, malting conditions that produced rice malts with peak diastatic power (DP), cold water extract (CWE), and hot water extract (HWE) were selected. Peak DP, CWE and HWE were obtained at FARO 44 (18 h steeping, 3 days germination, 55 °C kilning (S18G3K55°)), FARO 57 (30 h steeping, 2 days germination, 50 °C kilning (S30G2K50°)) and NERICA 7 (24 h steeping, 3 days germination, 55 °C kilning (S24G3K55°)). Selected malts were further tested for moisture content, total nitrogen, malt yield and malting loss and subsequently progressed to wort and beer production. Wort's pH, total soluble nitrogen (TSN), brix, kolbach index (KI), free amino nitrogen (FAN), dextrose equivalent (DE), original extract (OE) and sugar profile were determined, as well as beer's pH, colour, apparent extract (AE), alcohol by volume (%ABV), turbidity and sensory attributes. Rice grain varied significantly (p < 0.05) in TKW, GE, GC and DoS across varieties. Despite wort's pH, TSN, DE, OE as well as beer pH, colour, AE and turbidity resembling (p > 0.05) across varieties, wort's brix, KI, FAN, sugar profile as well as beer's %ABV, differed significantly (p < 0.05). Sensory attributes of appearance, colour, mouthfeel, and overall acceptability in beer differed noticeably (p < 0.05), except for aroma and taste (p > 0.05). Overall, the rice beer, though very slightly hazy, represented a pale yellow light lager, which is indicative of its peculiar beer style. Besides increased DP and enhanced hydrolysis, varying malting conditions of current study could serve as a pathway of reducing the cost of exogenous (commercial) enzymes or barley malt imports, together with decreasing barley's dependency for brewing in the tropics.

Fate and Behavior of Field-Applied Pesticides during Malting and Mashing Processes.

The present work aimed to study the fate of field-applied pesticides during malting and mashing processes. Twenty-four field-collected barley samples were subject to micromalting followed by lab-scale mashing to investigate the carryover of residual pesticides from barley to malt and then from malt to sweet wort. The citrate-buffered QuEChERS sample preparation method was adapted for simultaneous determination of 57 pesticide residues in grain, malt, spent grains, and sweet wort samples using ultra-performance liquid chromatography coupled with tandem mass spectroscopy (UPLC-MS/MS). Residues of four fungicides (fenpropimorph, pyraclostrobin, tebuconazole, and trifloxystrobin) and two insecticides (chlorpyrifos and pirimiphos-methyl), frequently found in the barley samples, were investigated in detail in this study. The carryover percentages of these pesticides to malt, against the concentration of residues in barley grain, ranged from 22% for pirimiphos-methyl up to 78% for fenpropimorph. The results confirm a general rule that residues of pesticides with log P values >2 remain on the malt, but it was found that their transfer potential is more related to its individual physical-chemical properties but does not much correlate to their log P values. In the second part of the study, a noticeable carryover from malt to sweet wort was observed for pyraclostrobin, fenpropimorph, and tebuconazole residues, and these values ranged from 2 to 15%. Moreover, the analysis of pesticide residues in spent grain after mashing revealed that the spent grain samples contain on average once as much pyraclostrobin and tebuconazole residues as the original malt. It was concluded that (1) pyraclostrobin and tebuconazole residues could be incorporated into or associated with macromolecules in barley grain to form "hidden" (bound) forms, and (2) the parent compounds are subsequently released from their hidden forms during mashing.