The Black Gap: Understanding the Potential Roles of Black Fungal-Derived Enzymes in Malting and Brewing Quality: A Review

Abstract

The infestation of brewing grains with filamentous fungi can have wide-ranging effects, including poor processability during malting and brewing, diminished storage quality, and potential threats to food safety and human health. Darkly pigmented fungi, also known as dematiaceous fungi, that spoil cereal grains during ripening and storage comprise a rich source of extracellular enzymes, including various cellulolytic enzymes and other polysaccharide-degrading enzymes, along with proteolytic enzymes, that can modify the physicochemical properties of cereal grains, contribute to substrate hydrolysis during germination, and may have a negative influence on malting and brewing properties. This review article addresses the potential impact of dark-pigmented fungi on malting and brewing quality beyond food safety. It summarizes the current knowledge on secreted fungal hydrolytic enzymes involved in barley grain degradation and discusses their potential impact in terms of malting and brewing quality, focusing on dematiaceous fungi and those causing black symptomatology on the grain. Overall, this review highlights the necessity for further research into the impact of dark-pigmented fungi on malting and brewing quality.

Keywords:

• Barley
• enzymes
• fungi
• infected brewing cereals
• malting process

Introduction

Barley (Hordeum vulgare L.) is the primary cereal used in the production of malt and beer. As a natural product, barley grains are naturally colonized with diverse indigenous microflora, including numerous bacterial species, both gram negative and positive; wild yeasts’ and filamentous fungi. Infestation of brewing grains with filamentous fungi has repeatedly been linked to a significant quality-reducing aspect, affecting the processability for malting and brewing proposes, storage quality, food safety, and human health risks.

In recent years the plant-pathogenic fungal genus Fusarium has garnered the most public attention and has been well studied in this respect,^[1–3] though less is known in comparison about the impact of “black fungal” contamination of brewing malt quality.

“Black fungi” describes a group of fungi with darkly pigmented cell walls, often appearing black or dark brown. While Fusarium infestation refers to species belonging to a single genus, the term black fungus is applied to several genera of fungi with different risks of damage, grouped in the form family of Dematiaceae within the Ascomycota division. Their common characteristic is dark-pigmented hyphae and conidiophores due to synthesizing melanin pigments. The most abundant seed-borne black fungi include species of Alternaria, Cladosporium, Drechslera (Helminthosporium, anamorph: Pyrenophora), Epicoccum, Chaetomium, and Stachybotrys.^[4–7] Infestation by members of Aspergillus, Rhizopus, or Penicillium can also cause grains to become discolored, resulting in a black appearance when heavily contaminated. Despite their distinct taxonomic groups from “true” dematiaceous fungi, they are often grouped as “black fungi” in brewing literature due to their common dark symptomatology.

Members of the black fungi group are globally spread.^[8,9] Because dark filamentous fungi and their spores are ubiquitously distributed in a variety of habitats, including soil, air, water, and plants, a certain amount of contamination is inevitably present on cereals. They either infect the cereal grains during ripening in the field or barley and malt during storage and processing.^[10] Between cropping cycles, the fungi survive in infested seeds, or as mycelium or spores in infected plant debris left on or in the soil. The spores are blown long distances by the wind or splashed locally by rain onto plant surfaces, where infection occurs. Temperature and moisture play a crucial role in the growth and sporulation of black fungi. Thus, unfavorable weather conditions like rainy summers, weather-related harvest delays, or improper storage conditions with high humidity can significantly increase the risk of contamination.

To prepare barley grains for brewing, a sequence of chemical and structural modifications is carried out during the malting process. The key objective is to stimulate grain breakdown by its own enzymes. Malting constitutes three main steps: steeping, germinating, and kilning. It starts with steeping; once the required hydration level of around 45% is reached, the embryo starts its metabolic activities. The process of germination is characterized by the development of the embryo; while forming roots and shoots, an extensive array of hydrolytic enzymes are mobilized. The degradation processes occurring here modify the starchy endosperm in consistency by breaking down the cell walls and the protein matrix. Finally, metabolic activity is stopped by drying the grains under the gradual temperature increase to 85 °C during kilning. Color and a range of flavor components are formed during this step.^[11] High-quality malt for brewing is described by analytical attributes, primarily divided into the three hydrolytic processes in the barley kernel during the malting process known as cytolytic, amylolytic, and proteolytic malt modification.^[12]

The malting environment can be considered a complex ecosystem, including not only the germinating grain, but also the microbial communities colonizing the grain.^[13] During steeping, increased water intake initiates the germination of the grain and also promotes fungal growth and activation of dormant spores.^[14] The prevailing warm and humid conditions during barley grain germination and during withering, the early stage of kilning (50 °C for 16 h), provide optimal growth conditions for filamentous fungi, while higher temperatures during kilning (above 60 °C) do not further enhance fungal growth.^[15] Black-pigmented fungi include plant pathogenic and saprophytic species that produce an extensive array of extracellular enzymes that catalyze various chemical reactions with the potential to modify the composition and structure of cereal grains. This can adversely affect malting and brewing properties. The mycelium of filamentous fungi was observed on and under the glume of the grain and within the fruit husk; the aleurone layer and endosperm are usually free of hyphae.^[16,17] Secreted fungal hydrolytic enzymes enter the endosperm and significantly alter specific quality characteristics.^[18] The hydrolytic potential depends on fungal genus and species, some showing more effect on cytolysis, others more on proteolysis or amylolysis. Together with the enzymatic system of the germinating grain, this may lead to an over-modification of the endosperm.

Regarding malting barley, black fungi have not yet been sufficiently pursued from a brewing technology point of view. Much research has been done in the agricultural sector and biotechnology field due to their utilization in industries. But currently, the brewing literature has not yet considered that black fungi and their enzymes may affect malt quality and cause damage similar to that of a Fusarium infestation, despite their significant presence in the microbial colonization of grains.^[14] This review article summarizes the current knowledge on secreted fungal hydrolytic enzymes and their transcriptional regulation involved in barley grain degradation, based on the three main degradation processes: cytolysis, amylolysis, and proteolysis, focusing on dematiaceous fungi and those causing black symptomatology on the grain. Their potential for excessive malt modification is transferred to the malting and brewing process, and the impact in terms of quality is discussed. Understanding the black fungal secretome and how it affects processing and quality could potentially be used to uncover targets for combating fungal enzymes and minimizing their negative impact.

Discussion according to degradation processes

The physiological and biochemical modifications of the barley grain during the malting process are classified into cytolysis (degradation of cell wall polysaccharides), amylolysis (degradation of starch), and proteolysis (degradation of proteins), relating to the main components that are hydrolyzed.^[11] The following summarizes and discusses the potential effects of fungal hydrolases based on these three degradation processes.

1. Cell wall degrading enzymes (CWDE): effect on cytolysis

Cytolysis in germinating barley describes the enzymatic breakdown of structural constituents of cell-wall polysaccharides, mainly barley β-glucan and arabinoxylan.^[12] During the malting process, cytolytic enzymes initiate the degradation of cell-wall polysaccharides of the aleurone layer and endosperm, increasing the permeability and allowing the released enzymes to access the starchy endosperm. This also enhances the efficiency of other degradation processes on the protein matrix and starch granules during mashing. The essential enzymes derived from the barley acting here are glucanases and pentosanases, such as xylanases and arabinosidases.^[19]

Non-starch polysaccharides from barley grain

The main components of the barley grain are the embryo, the aleurone layer, the endosperm, and the husk. Non-starch polysaccharides comprise 22.6–41.1% dry weight^[20] of the mature barley grain, determined by variety, year, climate, and growing environment, and can be divided into three groups: cellulose, hemicellulose, and gums. Barley non-starch polysaccharides are characterized by β-glycosidically linked glucose units.

Cellulose is primarily located in the barley husk, acts as a scaffold, and is composed of β-1,4-linked D-glucose units, which form linear polymeric chains; here, the disaccharide cellobiose is the basic component. During the malting and brewing processes, cellulose remains unmodified. In lautering, the husks play an essential role by forming a filter layer for separating the wort including the soluble malt components from the solids and the less soluble components, such as proteins, polyphenols, lipids, and metal ions at the end of mashing.^[11,21] In addition to cellulose, the husks contain a minor amount of arabinoxylan, a hemicellulose consisting of a linear xylan backbone of β-1,4-linked xylose residues with arabinose substitution and ferulic acid residues, most of which remain (insoluble) in the brewer’s spent grain, locked in a complex matrix.^[22]

Hemicellulose and gums are structural substances of the cell walls of the endosperm and the aleurone. The endosperm cell walls are built up primarily of β-1,3;1,4-glucan (70%), usually referred to as β-glucan, and to a minor extent β-1,4-arabinoxylan (20%).^[23] The surrounding aleurone layer encases the endosperm; the aleurone cell walls are thick and have an inverse ratio of 26% β-glucans and 71% arabinoxylans.^[23] In the aleurone, the majority of the hydrolytic enzymes develop during germination.

During malting, the arabinoxylan structure is degraded in conjunction with β-glucan to allow the efficient breakdown of the major components of the endosperm.^[24] Degradation of β-glucan requires the synergistic interaction of a cellulolytic enzyme complex consisting of endo-1,3-β-glucanase (EC 3.2.1.39), endo-1,4-β-glucanase (EC 3.2.1.4), endo-1,3;1,4-β-glucanase (EC 3.2.1.73), exo-β-glucanase (EC 3.2.1.91) and exo-β-glucosidase (EC 3.2.1.74).^[25,26] Arabinoxylans are degraded by endo-xylanase (EC 3.2.1.8), exo-xylanase (EC 3.2.1.37), arabinofuranosidase (EC 3.2.1.55), β-xylosidase (EC 3.2.1.37), and feruloyl-esterase (EC 3.1.1.73).^[11,25]

Physiological role of fungal CWDE

Phytopathogenic and saprophytic fungi are known to produce a wide range of cell-wall degrading enzymes,^[27] which are thought to play important roles in pathogenicity functions, including penetration, degradation, triggering of plant defense, and symptom expression.^[28] During host invasion, the plant cell wall, primarily made up of cellulose, hemicellulose, and pectin, is the first line of defense against the successful penetration and spread of pathogenic fungi. Secreted hydrolytic enzymes allow them to depolymerize the main structural components of the cell wall and actively penetrate the interior of the host tissue, feeding on the nutrients released there,^[27,29,30] whereas opportunistic fungi usually enter plants through lesions. The expression of genes encoding cell-wall degrading enzymes is regulated by various environmental factors.^[31] Besides their pathogenic activity, CWDE are also suspected to be involved in the synthesis and remodeling of the cell wall during growth and expansion,^[32–34] as glucans are a major component (50-60%) of the fungal cell-wall structure, along with chitin (10-20%) and glycosylated proteins (20-30%).^[35] Carbohydrate-degrading enzymes may play a role in the ability of antagonistic fungi to attack their target fungi due to hydrolysis of the host cell wall.

Fungal CWDE involved in the breakdown of plant cell walls

The black fungal genus Alternaria contains a diversity of saprophytic and pathogenic species and is, along with Fusarium, one of the most prevalent fungi found on barley,^[14] occurring both in the field during growth and postharvest storage. Numerous research studies attested to the cellulolytic and xylanolytic potential of Alternaria spp.^[36–38] A. citri was reported to have both high endo-1,4-β-glucanase and exo-1,4-β-glucanase activity, while A. tenuissima showed only exo-1,4-β-glucanase activity.^[39] A. alternata secretes β-glucosidase,^[40] endo-1,4-β-glucanase and exo-1,4-β-glucanase.^[39] Endo-1,4-β-glucanase randomly cleaves internal β-1,4-glycosidic bonds and breaks down high molecular weight β-glucan polymers into fragments of different molecular masses, which can be further degraded by exo-1,4-β-glucanase and β-glucosidase to cellobiose, laminaribiose, and glucose molecules. High β-glucosidase activity was confirmed for A. alternata cultivars isolated from cereals, namely wheat, and rye.^[41] The enzyme is considered to function during the autolysis of the fungal cell by hydrolyzing laminarin, a 1,3-β-glucan similar to the fungal cell wall; purified β-glucosidase showed best activities at pH 5 and a temperature of 50 °C while being stable in a wide pH (3-8) and temperature range (0 to 60 °C).^[42] The expression of glucanase genes in A. alternata is also related to the pathogenic capability of the host.^[43] Transcriptome analysis of gene expression profiles in the tangerine pathotype of A. alternata revealed that genes encoding CWDEs are required for virulence and are regulated by the transcription factor Ste12, similar to Fusarium graminearum.^[29]

Aspergillus species express a wide range of cellulolytic enzymes and are frequently used to produce industrial enzymes. They are a serious threat to the quality and safety of stored cereals due to the tolerance of low water activity and the production of mycotoxins. Various strains of Aspergillus are able to use both β-glucan and xylan as a single carbon source.^[18] A. niger secretes endo-glucanases, β-glucosidase, endo-xylanases, arabinofuranosidase, feruloyl, and xyloacetyl esterase.^[44] These endo-glucanases and xylanases cleave high-molecular-weight β-glucan and arabinoxylan, respectively, to medium-molecular-weight dextrins. Arabino­furanosidase hydrolyzes the α-1,3-linkage in arabinoxylan, releasing arabinose, while feruloyl- and xyloacetyl-esterases hydrolyze the ester bonds between ferulic acid and arabinose or acetyl and xylose, respectively.^[44] A. niger provides an β-glucosidase performing best between 40–65 °C and a pH of 4.0–5.5, closely matching mashing conditions.^[45] According to the research conducted by Izidoro and Knob, the optimal substrate for xylanase production by A. niger is brewer's grains^[46]; this finding is consistent with the observations made by Noots et al., who discovered xylanase activity in Aspergillus strains during the fermentation of sterile, ungerminated barley, but no β-glucanase activity.^[18]

Penicillium roquefortii and P. verrucosum, which are also responsible for stored grain spoilage, both express endoxylanase. P. funiculosum additionally produces arabinofuranosidase and xylobiase.^[47] Xylobiase, also known as β-xylosidase (EC. 3.2.1.37), hydrolyzes short xylo-oligomers into single xylose molecules. P. digitatum, isolated from corn kernels, is active in cellulose production.^[48]

The Cladosporium genus is one of the most common environmental fungi, including saprobic and dematiaceous species. The fungus C. cladosporioides presents high activities of xylanase and cellulose.^[48–51] Furthermore, high exo-β-1,4- glucanase and moderate endo-β-1,4- glucanase activity was reported.^[52] The characterization of a purified extracellular β-glucosidase revealed optimal activity at 50 °C and a pH of 4.5 using PNPG (p-nitrophenyl-ß-D-glucosidase) as substrate; further competitive inhibition by glucose was observed.^[53]

The black fungus Chaetomium globosum has been detected in barley raw material^[6] and produces varying amounts of β-glucosidase, endo-glucosidase, exo-glucanase, and cellulase, depending on the substrate.^[52,54] Also, Drechslera teres und D. graminea, exclusively found as barley saprophytes, produce endo-β-1,4-xylanase,^[55] contributing to the breakdown of barley arabinoxylan.

Stachybotrys spp. are ubiquitously distributed, preferentially growing as saprobionts on moist, cellulosic, and decaying material such as hay and straw. Stachybotrys chartarum (atra) showed over 200% higher cellulolytic activity in liquid cellulose medium in in-vitro experiments compared to the control.^[56] The action of endo- and exo-1,4-β-glucanase has been identified^[39]; further, a β-glucosidase was verified.^[57] Picart et al. showed that the ß-glucanase Ce12A from Stachybotrys atra exhibited lichenase-like or atypical endoglucanase characteristics. The enzyme showed high activity for β-1,3-1,4-mixed glucans, with a maximum activity for barley β-glucans, whereas it was less active for carboxymethylcellulose (CMC). The hydrolysis of barley β-glucan and lichenan resulted in 3-O-b-cellotriosyl-D-glucose and 3-O-b-cellobiosyl-D-glucose and smaller amounts of glucose and cellobiose.^[58] As a result, the high molecular weight β-glucan is degraded, and the cell walls of the starchy endosperm cells become porous.

In a recent study, species within the genus Rhizopus were detected on barley and barley malt.^[14] Rhizopus spp. occur naturally in soil and air and are also particularly common in postharvest storage situations. Celestino et al. investigated the influence of a β-glucanase, produced by Rhizopus microsporus var. microspores on the brewing process; the isolated enzyme was able to hydrolyze 1,3-1,4-β-glucan and reduced, compared to commercial glucanases, the viscosity and the filtration time of brewing mash.^[59] In other studies, Noots et al. found that β-glucanase and xylanase production by Rhizopus sp. during malting conditions resulted in cell wall modification similar to those observed during the malting of germinating barley. Cell-wall degradation by the fungus alone could be confirmed. Furthermore, a clear correlation between the activity of fungal endo-β-glucanase and an increase in malt friability, which indicates cell-wall degradation analytically, was found.^[18,60]

Effects on the resulting malt and beer quality

Sufficient cell-wall hydrolysis is one of the most important criteria due to its impact on malt, wort, and beer quality. Cytolysis should meanwhile occur mainly in the malt house, where enzymes that are either activated or synthesized during germination release barley polysaccharides from the cell walls and partially degrade them.

The barley husk and the underlying pericarp and seed coat protect the caryopsis from biological and environmental stresses. These tissues consist largely of cellulose and arabinoxylan; minor components further include lignin, lipids, and phenolic compounds.^[11] The secretion of hydrolytic enzymes toward β-1,4-glucosidic and β-1,4-xylosidic linkages is thought to allow plant-pathogenic fungi to penetrate these layers. This may be particularly crucial during the late stages of infection or in filamentous fungi that grow saprophytically on the outer surface of cereal husks.^[3]

Several studies report that cytolytic enzyme potential increases significantly by a combined effect when the grain is infected with filamentous fungi.^[61] Investigating the surface-associated proteome of barley grains, Sultan et al. found a strongly increased xylanolytic activity compared to endogenous xylanase activity. Enzymes affiliated with fungi included β-1,3-glucosidase, exo-β-1,3-glucanase, and β-1,4-xylanase; this group of enzymes is thought to be mainly required for host invasion.^[62] Noots et al. examined the relationship between the modifications of barley cell walls by fungal enzymes during malting using irradiated barley kernels deprived of their physical ability to germinate. Histochemical analysis revealed fungal penetration into the barley endosperm, where fungal enzymes were shown to be released, and the cell wall β-glucan was modified in the absence of barley enzymes.^[18] Complementarily, another study demonstrated that thermostable β-glucanase and xylanase activity was lower after the treatment of barley grains with antifungal agents during the malting process. The resulting congress mash was less filterable and exhibited higher viscosity.^[63]

In the past, high viscosity levels that caused difficulties during lautering and beer filtration were primarily attributed to high molecular weight β-glucan. However, recent studies have shown that arabinoxylan has a significant impact.^[64] The brewing process’s viscosity and filtration performance are largely influenced by the concentration, composition, and final molar masses of non-starch polysaccharides, including arabinoxylan and β-glucan, and these factors are strongly affected by cytolytic degradation processes. Research indicates that the barley β-glucanase and xylanase activity significantly influence the filter cake’s permeability; moreover, a feruloyl esterase, as also produced by A. niger, releases non-crosslinked LMW arabinoxylans of less than 10 kDa, which enhances the wort's flow rate.^[65,66]

An enhanced filterability and faster lautering time have been previously associated with fungal-infested malts due to increased exogenous enzymatic hydrolysis and a lower β-glucan concentration.^[67] Schwarz et al. found strongly increased β-glucanase activities in rejected malts highly contaminated with A. alternata and, to a lesser extent, Epicoccum sp.^[3] Malts infected with Fusarium spp. exhibited elevated xylanase activity and the existence of a heat-resistant microbial β-glucanase. Despite the considerable reduction in β-glucan levels, the wort’s filtration rate was poor, probably due to increased soluble arabinoxylans degraded by fungal xylanases but not impeded in tending to form gels under oxidative conditions.^[2]

Overall, enzymes derived from black fungi can be advantageous in enhancing filtration performance during the brewing process, despite the possibility of gel formation if excessive arabinoxylan degradation occurs. In addition, improved cell-wall degradation can lead to a higher yield of malt extract. Since starch granules and storage proteins are enclosed in the endosperm cell walls, an intensified cytolysis makes the starch more accessible to water and enables better swelling during wort preparation. Consequently, enzymes can more easily break down the starch, leading to a higher yield and more efficient use of raw materials. This could be beneficial for higher utilization, especially in the case of brewing systems using coarse milling.

An intensified cytolytic modification by additional fungal enzymes may have a negative impact on the sensory attributes of beer, such as mouthfeel and palate fullness, because β-glucan and viscosity are known to contribute positively to these.^[68,69] However, currently there is a dearth of studies demonstrating a relationship between mold-derived cytolytic enzymes and the sensory perceptions of beer. Nevertheless, in the brewing industry, gum-degrading enzymes obtained from fungi are commonly used to facilitate separation processes and lower costs without causing any noticeable sensory issues.

Also, a relationship between the activity of fungal arabinoxylanases (xylanases) and glucanases and the occurrence of premature yeast flocculation (PYF) during brewery fermentation has been suggested. Enzymatic degradation of the barley husk arabinoxylan produces acidic high-molecular-weight polysaccharides that have been proposed to cross-link with lectins from the yeast surface, leading to flocculation and precipitation of yeast cells.^[70,71] In a recent study, a positive correlation between xylanase secretion from the black fungus A. tenuissima and the occurrence of PYF has been proven.^[72] Settling out of yeast cells from the fermenting wort results in an incomplete utilization of fermentable sugars and a reduced conversion of undesired volatiles (e.g., diacetyl or acetaldehyde).

2. Amylolytic enzymes: effect on amylolysis

Amylolysis signifies the enzymatic hydrolysis of starch macromolecules into fermentable sugars for the brewing yeast and determines the final extract yield and economy of wort production. Malting is of essential importance since it results in synthesizing and releasing of hydrolytic enzymes, which are necessary for starch degradation into soluble sugars during the subsequent mashing process.^[73]

Starch polysaccharides from barley grain

Starch is composed of two types of macromolecules: amylose and amylopectin. Amylose is a linear polymer of α-1,4-linked D-glucose units, while amylopectin is a highly branched α-1,4-linked D-glucan with varying amounts of α-1,6-linked side chains. Both amylose and amylopectin are present in starch granules within the endosperm, encapsulated in a protein matrix, along with endosperm cell walls containing β-glucan, arabinoxylan, and pectin.^[74] Enzymatic hydrolysis is provided by amylolytic-acting enzymes, a group of hydrolases that specifically cleave the α-glycosidic linkage in starch. The results of hydrolysis are smaller molecules such as maltose, dextrins, and glucose molecules.

In storage tissues such as seeds, starch is hydrolyzed to meet the energy requirements of the growing seedling during germination. Only limited starch breakdown takes place during malting; starch is mainly degraded in the mashing process by the barley hydrolytic endo- and exoenzymes: α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1), β-amylase (1,4-α-D-glucan maltohydrolase, EC 3.2.1.2), to a lesser extent by α-glucosidase (1-4-α-D-glucan glucohydrolase, EC 3.2.1.3) and limited dextrinase (amylopectin1,6 glucosidase, EC 3.2.1.142).

Physiological role of fungal amylolytic enzymes

Secretory hydrolytic enzymes play a major role in filamentous fungi, particularly in nutrient mobilization. As heterotrophic organisms, fungi derive nitrogen and carbon requirements from external organic sources. Complex polysaccharides cannot enter the cell without preliminary hydrolyzation. Given their osmotrophic type of nutrition, they rely on the secretion of extracellular enzymes that convert high-molecular-weight polymers into soluble nutrients that are then transported across the plasma membrane of hyphae. Starch, the major storage component in plants, is an easily accessible energy source. The expression of genes encoding fungal amylolytic enzymes is induced in response to environmental signals: the presence of nutrients, such as starch and its degradation products.^[75,76] Conidia germination is also triggered by appropriate nutrients in the presence of water and air, followed by isotropic growth and the formation of a germ tube.^[77] During conidial outgrowth, secreted amylases ensure the survival of the fungus at the beginning of its new life cycle.^[78,79]

Fungal amylolytic enzymes

Various studies focused on isolated filamentous fungi and their ability to produce extracellular amylolytic enzymes have demonstrated that Alternaria spp. are capable of secreting starch-hydrolyzing enzymes.^[80–82] A. alternata and A. tenuissima are both proven to have high α-amylase activity.^[83] α-amylases randomly cleave internal α-1-4-glycosidic bonds in amylose or amylopectin molecules by an endo-mechanism, generating dextrins and maltose. Lateef et al. purified and characterized an α-amylase from A. alternata, which exhibits thermostable properties (60–90 °C).^[84] This relates to performance during mashing since barley α-amylase acts optimally at temperatures above 65 °C.^[85] The authors also suspect the presence of other amylolytic enzymes, including an α-glucosidase.^[84] A. alternata was recently reported to secrete a glucoamylase with high stability at pH 5. Glucoamylases, further known as γ-amylases, are enzymes that hydrolyze 1-4-glycosidic or 1-6-glycosidic linkages from non-reducing ends of oligo and polysaccharides chains and are capable of complete starch hydrolysis, releasing glucose as the final product.^[86]

According to Abe et al., the most potent amylase-producing fungal isolates obtained from maize grains were those of E. nigrum and C. cladosporioides.^[48] Both were identified as common contaminants of malting barley in our previous research.^[14] Cladosporium resinae, another natural component of the soil microflora,^[87] was shown to produce in addition to α-amylase, an exopullulanase, and two glucoamylases, contributing to starch hydrolysis.^[88–91]

Aspergillus and Rhizopus, two fungal genera causing postharvest damage, are used in industrial applications to produce high yields of saccharolytic enzymes, primarily for enzymatic starch liquefaction in the food and fermentation industries. Aspergillus section Flavi includes domesticated species, such as the yellow koji mold A. oryzae, and harmful representatives, such as A. flavus and A. parasiticus, notorious for contaminating crops. Genetically, A. oryzae is closely related to A. flavus and A. parasiticus; however, due to its widespread use in fermented Japanese foods, its enzyme secretion has been more widely researched. A. oryzae produces vast amounts of amylolytic and proteolytic enzymes. Its α-amylase, known as Taka-amylase A (EC 3.2.1.1), is encoded by three genes amyA (amy1), amyB (amy2), and amyC (amy3)^[92,93] and performs optimally between pH 5 and 6 and a temperature range of 40-55 °C.^[94] In addition, A. oryzae secretes an α-glucosidase and two glucoamylases, GlaA and GlaB, with a pH optimum between 4.0 and 4.5 and a temperature of 56 °C, respectively 65 °C.^[95] Thus, its enzyme repertoire operates in the range of starch degradation occurring during mashing. Amy1, the α-amylase gene of A.flavus, which is nearly identical to amy3 of A. oryzae, is further thought to play a role in the induction of aflatoxin biosynthesis in infected maize kernels. Mycotoxin production alters the host defense system and may help the fungus invade host tissues as an additional virulence factor. The authors hypothesized that, after embryo colonization, the action of A. flavus extracellular α-amylase results in starch degradation products that initiate aflatoxin biosynthesis.^[96,97] Thus, increased enzymatic breakdown elevates the threat to food safety, too.

A. niger provides two types of extracellular α-amylases, an acidic type, being stable at pH 3.2–3.5, and a neutral α-amylase,^[98–101] as well as glucoamylases of varying molecular weights.^[102,103] Both A. niger and A. terreus are reported to secrete thermostable α-amylases with an optimum at 40-60 °C and pH of 4.5-5.5, corresponding to a mash environment.^[104]

As recently reviewed by Higuchi,^[105] Aspergillus amylases can be secreted into the medium or bound to the cell surface.^[106] Secretion can occur in two distinct pathways: from the hyphal tips and septum-directed secretion.^[107–109] Especially in the early phase of conidial germination, favored in the malting process by the ideal conditions of high moisture and moderate temperatures, the secreted hydrolytic enzymes may supply nutrients and help the germ tube penetrate the substrate. Zhu et al.^[110] identified Taka-amylase A (TAA) as the major extracellular protein during the formation of germ tubes. Since no catabolite repression by the presence of glucose in the medium was detected, it was suggested that the regulation of TAA during germination differs from that during the mycelial growth phase. This allows us to conclude that the malting process is a crucial stage, as the prevailing conditions facilitate the germination of fungal spores and the consequent synthesis of enzymes responsible for endosperm degradation.

Effects on the resulting malt and beer quality

Starch undergoes structural modifications during the processing steps of malting and mashing. The main objective of starch hydrolysis is the efficient conversion of starch into fermentable sugars for brewing yeast.

During malting, starch granules are at their raw state (natively ungelatinized starch), composed of concentric alternating crystalline and amorphous layers, a condition in which they are difficult to access enzymatically.^[111] Only by “unfolding” the compact starch granule structure in the first stage of mashing is complete enzymatic hydrolysis possible. Once the gelatinization temperature is reached, starch swells irreversibly, and the crystalline structure of the starch granules is dissolved.

During malting, especially steeping, germination, and the early stage of kilning, during which low temperatures but high moisture prevail, filamentous fungi find favorable growth conditions and sufficient time to break down the starch. However, ungelatinized starch is difficult to access at this processing stage, because susceptibility largely depends on starch molecular fine structures.^[112] Hydrolysis of native starch proceeds by erosion from the surface toward the center of the granule, starting with adsorption of the enzymes onto the granule surface, followed by surface pitting (exo-erosion) and enlarging these cavities/pores until the formation of internal channels occurs (endo-erosion).^[113] Regarding barley enzymes, only α-amylase and α-glucosidase have been shown to attack native starch granules at sub-gelatinization temperature.^[114,115] Several in-vitro studies have reported a similar effect of fungal α-amylases and α-glucosidases capable of degrading raw starch: glucoamylase from Cladosporium gossypiicola causes exo- and endo-corrosion patterns in different starch types, depending on their origin^[116]; Rhizomucor sp. α-amylase was shown to diffuse rapidly into the central part of the granules, where it acts in an inside-out manner^[117]; Rhizopus niveus glucoamylase attacks native barley starch granules^[118]; Aspergillus flavus S2-OY secrets a thermostable α-amylase that hydrolyzes gelatinized and ungelatinized raw starches^[119]; and Penicillium sp. X-1, isolated from decayed raw corn, produces a raw-starch-digesting glucoamylase, although the enzyme does not adsorb onto the granule surfaces.^[120]

When starch is gelatinized and would now accessible, the reaction conditions during mashing are very limited by a pH value maintained around 5.5. Unlike barley enzymes, fungal amylases exhibit activity in wide pH and temperature ranges and are more efficient than malt enzymes in the degradation of starch.^[88] In the mash tun, fungal α-amylase increases the level of fermentable carbohydrates. Since the diastase complex of malt has differing optimum temperatures—barley β-amylase, an exo-amylase that cleaves maltose units from the non-reducing end of both amylose and amylopectin starches, works best between 60–65 °C, while α-amylase, a starch debranching endo-enzyme that is responsible for liquefying the starch, has optimal activity in the range of 65–75 °C^[85]—both enzymes can work simultaneously at the same temperature, but not as effectively as in the respective optimum. Accordingly, the ratio of fermented and non-fermented sugars is affected. Additional fungal α-amylases operating in the optimum of β-amylase, such as from A. oryzae, provide the possibility of synergism, allowing complete starch conversion during mashing to attain the expected final attenuation. In addition, the viscosity decreases rapidly, and lower temperatures might be used during mashing. Added fungal α-glucosidase supports the intrinsic α-amylase during the hydrolysis of amylose, amylopectin, and dextrins to glucose, increasing the content of fermentable carbohydrates and lowering the content of dextrins.^[121] Recent findings indicate that the gelatinization behavior of barley starch depends on the particle size distribution; the gelatinization temperature of small starch granules is higher than that of large starch granules.^[122] Consequently, lots of small starch granules may reduce mashing efficiency.^[123] At this point, fungal enzymes could be beneficial for starch conversion.

The filamentous fungi discussed in this review mainly secrete α-amylases and α-glucosidases, which act synergistically in the degradation of starch molecules, with α-amylase working as a step-maker promoting further degradation by exo-enzymes like α-glucosidase. Sun and Henson^[114] demonstrated a 10.7-fold higher effect on hydrolysis when these two enzymes were combined. Additional degradative amylolytic enzymes yield more fermentable sugars and, thus, higher extracts,^[124] which leads to improved fermentation. In addition to an increased ethanol content, this also promotes the formation of fermentation by-products: beer produced with fungal-infected malt contained higher levels of esters, fusel alcohols, fatty acids, ketones, and dimethylsulfide, as well as Strecker aldehydes and Maillard products.^[67] The latter are known precursor structures of sensory active aging indicators.^[125,126]

The composition of the wort sugar profile is also decisive for fermentation efficiency. Standard brewery wort consists of about 90% carbohydrates, composed of sucrose, fructose, glucose, maltose and maltotriose, and dextrins. External fungal enzymes might cause a shift in these ratios, given that infected malts showed higher levels of α-amylase dextrinization units (DU).^[127] Wort dextrins with different molecular sizes and chain lengths are further said to positively impact the sensory attributes of palate fullness^[128] and mouthfeel.^[69] However, to the best of our knowledge, there are currently no available data supporting a link between fungal enzymes and palate fullness, making it a promising topic for future research.

Regarding the sensory impressions, an increased starch breakdown by fungal amylolytic enzymes is also reported to negatively affect foam stability through decreased wort viscosity.^[124] In contrast, Oliveira et al.^[67] found improved foam stability in beers brewed from infected malt. They suspect the higher concentration of small peptides and amino acids, accompanied by enhanced proteolysis, to positively influence the foam structure. These findings highlight the considerable complexity of the effects of secreted fungal enzymes. It is essential to note that the effects are closely interrelated and that the impairment cannot be limited exclusively to one modification process. In summary, additional amylolytic enzymes by black filamentous fungi may contribute to increased starch degradation. However, the effects on sensory quality parameters must be adequately explored.

3. Proteolytic enzymes: effect on proteolysis

Proteolysis describes the enzymatic breakdown of barley proteins into small peptides and amino acids. Efficient proteolysis is essential to provide a source of assimilable free amino nitrogen (FAN) for the yeast metabolism during fermentation, but also for greater availability of starch to amylases and to break down turbidity components.^[129,130] During the malting process, proteolytic enzymes degrade storage proteins and finish their work in the brewhouse during mashing.

Barley proteins

The optimal average protein content of brewing barley constitutes about 9.5–11.5% of the grain’s dry weight. In the mature grain, proteins are present in multiple structures of the barley kernel. About 40% of the protein is located in the endosperm, where it acts as a reinforcer of the cell walls and protein matrix between starch granules; about 35% functions as non-storage proteins in the embryo; and about 20% is localized in the aleurone layer^[131] as structural components of the aleurone cell walls and as storage proteins in the subaleurone, where it serves as food for the developing embryo.

The barley protein fraction consists mainly of high-molecular-weight proteins, which can be classified according to their solubility as albumins (water-soluble), globulins (salt-soluble), hordeins/prolamines (alcohol-soluble), and glutelins (alkali-soluble).^[132] Hordeins and glutelins are the major barley storage proteins of the subaleurone, whereas albumins and globulins, primarily found in the aleurone and embryo, comprise mainly enzymes and structural components.^[133]

During malting, the storage proteins are broken down by proteases, which are formed and secreted in the endosperm. Depending on the site of the attack, they are classified as either exoproteases or endoproteases—exopeptidases acting on the end of the polypeptide chain and endopeptidases cleave within the middle of the protein chain. Thus, barley proteins are first solubilized by endoproteases and further degraded by exopeptidases.^[134] Catalytic peptidases are further subdivided into aspartic, cysteine, serine, glutamic, threonine, and metalloproteases, referring to the type and structure of the catalytic active center of the enzyme molecule.^[135]

Physiological role of fungal proteases

Proteases are present in all living organisms, including eukaryotes, eubacteria, archaea, and viruses. Fungal proteolytic enzymes are involved in many physiological and pathological processes, having both regulatory and housekeeping functions, e.g., degradation of regulatory proteins, signal transduction, intracellular protein turnover, removal of signal peptides, and conversion of predecessor proteins into their mature structures.^[136,137] Secreted proteases are responsible for nutrient mobilizing because hydrolysis of exogenous proteins supplies the cell with nitrogen sources and ensures the growth and survival of both saprophytic and pathogenic species. They also play an important role in morphogenesis, aerial hyphae formation, conidial discharge, and the spore coat disassembly at dormancy breaking. Furthermore, fungal proteases are considered important factors during pathogenesis, including adhesion to the host tissue, facilitating the penetration of the plant cell wall by degradation of the fibrous glycoproteins,^[138] and interacting with the host plant. In addition, many fungal allergens possess proteolytic activity, boosting the pathogenesis of respiratory allergies.

Fungal proteolytic enzymes

Serine proteinases are predominant among the multiple fungal proteases, although enzymes belonging to other mechanistic classes are also reported.^[139] Serine proteases (EC 3.4.21) are endopeptidases with a serine residue at the active site. These enzymes can be further categorized based on their substrate specificity as either trypsin-like or subtilisin-like. Which serine protease type is secreted may reflect the physiological difference between saprotrophic and phytopathogenic fungi. The latter is more likely to produce trypsin-like serine proteases, suggesting that these may be a determining factor in the virulence of fungal pathogenicity.^[140,141]

Species within the genus Alternaria have been found to produce extracellular serine proteases belonging to the subtilisin- and trypsin-like families.^[138–140] The pH optima of secreted peptidases of A. alternata have been demonstrated in several studies, showing best activities in the range of pH 7–8,^[140,142] whereas Moore et al.^[143] found the optimum rather in a more acidic pH range of 4–7.5. The complete inactivation could be achieved with PMSF and Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK), indicating that it is more likely a trypsin-like protease.^[140] A. solani secretes subtilisin- and trypsin-like proteases with the highest enzymatic activity at neutral and slightly alkaline pH values.^[139,144] However, the ratio of the two exoproteinases is determined by the prevailing environmental conditions, particularly the composition of the growth medium, primarily the available carbon and nitrogen source, temperature, pH, and the origin of the isolate.^[139] In a further study, A. solani protease was also found to belong to the metalloprotease type because the isolated enzyme was inhibited by EDTA but not by PMSF. Maximum enzyme activity was observed in a pH range of 7–10 and a temperature range of 45–50 °C.^[144]

Genomes of Alternaria spp. comprise multiple genes encoding proteases, of which only a few have been studied in detail. Based on gene knockout experiments, Fu et al.^[145] have examined 15 subtilisin-like serine proteases in the tangerine pathotype of A. alternata for their physiological function, revealing that only one of them, namely AaPrb1, is required for pathogenesis. Furthermore, an interaction between AaPrb1 and AaPep4, encoding fungal proteinase A, which is essential for the degradation of autophagosomes, was found.

Serine proteases are also major allergens of prevalent airborne fungi. The proteolytic activity of Epicoccum nigrum (E. purpurascens) has been researched mainly in the context of respiratory allergic disorders. The allergenic glycoprotein Epi p 1, a secretory serine protease, is suggested to mediate host-fungal interactions,^[146] facilitate penetration into the lung epithelium, and promote airway inflammation.^[147,148] According to Bisht et al., the purified enzyme had an optimum of pH 6.5, tested at 37 °C using azoalbumin as substrate; inhibition of its biological function was observed with PMSF, Phosphoramidon, Leupeptin, and Bestatin.^[147] The major allergens Cla c 9 of Cladosporium cladosporioides^[149] and Cla h 9 of C. herbarum^[150] were identified as vacuolar serine proteases; both showed high sequence homology to each other, as well as to other vacuolar serine proteases from Penicillium (Pen ch 18, Pen o 18, Pen c 18) and Aspergillus species (Asp f 18, Asp f 13, Asp fl 13).

Limited studies are available on extracellular proteases of Penicillium spp. The MEROPS peptidase database (http://merops.sanger.ac.uk; Rawlings and Barrett, 1999) has recorded the following entries for the relevant Penicillium representatives: P. chrysogenum: 142 counts of known and putative peptidases, and 45 homologs; P. citrinum: 4 and P. roqueforti, three known and putative peptidases. Extracellular proteome analysis of P. chrysogenum by peptide mass fingerprinting and tandem MS revealed serine carboxypeptidases, secreted serine protease, aspartyl proteases, and one aminopeptidase-like protein.^[151] To our knowledge, however, the respective enzymes have not yet been further characterized.

Genomes of fungi from the Aspergillus genus comprise many known and putative proteases. Accordingly, species of Aspergillus are a major focus of attention for various industrial and pharmaceutical applications. The MEROPS peptidase database (http://merops.sanger.ac.uk; Rawlings and Barrett, 1999) has recorded the following entries for the relevant representatives: A. flavus: 173 known and putative proteases; A. fumigatus: 252; A. niger: 209; A. oryzae: 335; A. parasiticus: 2; A. terreus: and 146; A. versicolor: 1. As recently reviewed by Shamraychuk et al.,^[152] Aspergillus spp. secrete mainly serine proteases.

The Aspergillus niger-derived prolyl endoprotease (AN-PEP) was previously demonstrated to degrade barley hordeins during malting.^[153] AN-PEP showed proteolytic activity between pH 2 and 8, with peak activity at pH 4–5.^[154] Applying AN-PEP on germinating grains resulted in a 46% reduction in hordein fraction, while the treatment did not have a negative impact on malt quality characteristics.^[153] Likewise, the hydrolytic potential of A. oryzae improves the production of gluten-free beer: after Sorghum malting with 1% (w/w) A. oryzae, the extract content of the wort was increased by 27% more fermentable carbohydrates and 24% more FAN.^[155]

The level of peptidase secretion depends on the type of substrate available. Ishida et al.^[156] demonstrated that extracellular nitrogen conditions influence splicing patterns of the A. oryzae serine-type carboxypeptidase gene. In a subsequent study, Maeda et al.^[157] examined the expression of three extracellular dipeptidyl peptidase genes (DppB, DppE, and DppF) from A. oryzae. Since the transcription of DppF, unlike DppB and DppE, was not induced by protein substrates in the culture medium, it was suggested by the authors that DppF might act as a sensor for the presence of substrate in the environment by its resulting dipeptides acting as inducers for the other two proteolytic enzymes.

In addition to their role in nutrient acquisition, extracellular proteases are also reported to be virulence factors of plant pathogenic fungi. A. flavus extracellular alkaline protease (ALP) was observed in infected maize embryo tissue along with increased protein degradation. The fungus is suspected to spread further from the embryo and the aleurone layer into the endosperm. A reduction of ALP by PMSF significantly reduced the level of aflatoxin accumulation in vitro, indicating an essential role for ALP in the infection of maize kernels and subsequent aflatoxin formation.^[158]

Besides Aspergillus spp., members of the genus Rhizopus are used for the industrial production of fermented foods. Due to its highly active proteolytic enzymes and, at the same time, low amylase activity, Rhizopus spp. is used for the production of tempeh, a traditional Indonesian fermented food of soybeans. Fungal enzymes break down the proteins of the bean and thus increase its nutritional value. Today, there are also tempeh variations made from cereal grains, such as barley.^[159] Rhizopus spp. was found to have intracellular, extracellular, and cell wall–bound proteases, among them acidic and alkaline proteases.^[160,161] Aspartic proteases (E.C.3.4.23) are considered the most important proteases secreted by Rhizopus spp. and are highly active and stable in acidic environments. Kumar et al. purified and characterized an extracellular aspartate protease from R. oryzae, which acts optimally at pH 5.5 and is stable within pH 5.5–7.5. The best activity was observed at 60 °C, while in the presence of pepstatin, the proteolytic activity was inhibited by 73% and 97%, respectively.^[162] A secretory alkaline serine protease from R. oryzae was produced using 7% wheat bran as a carbon source in the fermentation medium. The enzyme is stable in the pH range from 3 to 11, with an optimum pH of 8, and further showed thermostable properties, with the highest activity at 60 °C.^[163]

Effects on the resulting malt and beer quality

About one-third of the barley protein content ends up in the finished beer, having a major influence on the resulting quality, including the beer’s body and foam retention. The proteins’ type, quantity, and size distribution are particularly important in this context.^[164] Fungal proteases catalyze further digestion of barley proteins in malt and wort, exceeding the effect of proteases naturally derived from the barley.^[165] This must be considered in terms of malting and brewing quality. Since the fungal proteases referred to above are mainly endoenzymes, it can be hypothesized that they reduce the high-molar-weight protein fraction and thus contribute to increased grain proteolysis.

The predominant fungal serine proteases are characterized by a broad pH and temperature activity range.^[166] Regarding the starchy endosperm, an acidic pH (5.0–5.2) is maintained during germination,^[167] providing optimal conditions for barley proteolytic enzymes to mobilize storage compounds; additionally, this aids in the dissociation of endogenous inhibitors through which the physiological function of proteases is regulated.^[168,169] During the subsequent mashing process, the mash pH value ranges optimally from pH 5.4 to 5.6; nowadays, most industrial mashing processes start at 62 °C.^[11] Considering that the environmental conditions during malting and mashing are similar to those required by external fungal proteases described above, it is reasonable to assume that they remain active throughout these processes. With regard to the field fungus Fusarium, this is underlined by numerous studies.^[2,67,127] Nonetheless, the evidence on black-pigmented fungi is currently scarce. Thus, a transfer between knowledge of secreted black fungal enzymes, the effect of Fusarium infestation, and an intensified proteolysis are drawn upon in some points in this article.

As previously shown for Fusarium sp., contaminated barley malt had higher FAN levels in comparison to uninfected malt.^[67] Wort produced from infected malt is darker in color^{[2,67,127,170]} and contains a higher amount of soluble nitrogen and FAN.^[2,67,155] Both differences can be attributed to the higher proteolytic activity due to additional fungal proteases since the degree of protein modification primarily determines the amount of FAN in the wort. The release of amino acids and reducing sugars, which are precursors of the Maillard reaction, leads to a darker color of the wort caused by the breakdown of protein and starch.^[127] Fungal endopeptidases split in the middle of the chain, resulting in low molecular weight (LMW) groups. Barley infected with F. culmorum showed 30% more soluble protein due to increased solubility in the albumin and glutelin fractions.^[171] Various studies also demonstrate nutritional changes from fermentation with Rhizopus spp., as evidenced by increased crude and soluble protein.^[160] Solid-state fermentation of whole-grain barley by Rhizopus oligosporus led to a significant increase in protein content from 10.25% to 16.85% compared with unfermented barley, showing degradation of macromolecules into LMW.^[172] Similar findings were made in studies of Wang et al.^[173]: during a co-fermentation of de-husked barley by R. oryzae, the amino acid nitrogen, soluble protein, and < 10 kDa peptides accumulated. While a dark color is undesirable for some beer types, the higher nitrogen content can improve yeast metabolism through increased bioavailability of nutrients^[13] and, thus enhance the fermentation process. Beer produced with infected barley malt had a higher amount of soluble amino acids. It exhibited a distinct amino acid profile with significantly elevated levels of threonine and histidine and reduced amounts of glutamine, aspartic acid, glutamic acid, lysine, asparagine, tryptophan, and alanine, compared to the control beer.^[67] A higher availability of amino acids and the way they are utilized by the yeast play a central role in beer flavor formation and can improve sensory quality (e.g., formation of higher alcohols or flavor contribution). However, with intensive fermentation, increased beer pH was also observed in conjunction with mold-contaminated raw materials.^[165]

Even though it might seem that high FAN levels are mainly positive, an excessive breakdown of nitrogenous substances leads to off-flavors, such as diacetyl.^[160] The precursor to diacetyl (2,3-butanedione), α-acetolactate, is a key intermediate in the valine biosynthetic pathway during fermentation.^[174] At high FAN levels, yeast synthesizes valine while consuming more preferred amino acids, resulting in higher formation of α-acetolactate.^[175] Further, foam stability, a key consumer-sensitive parameter of the final beer, is also impaired by an increased proteolysis, which can attack medium-chain polypeptides that stabilize the bubbles in the beer’s head.^[176] The major barley albumin protein Z, along with lipid transfer protein 1 (LTP1), a putative stress protein located in the aleuron layer from the endosperm, play a particular role regarding beer foam stability. Further, Okato et al.^[177] identified barley dimeric alpha-amylase inhibitor-1 (BDAI-1) as a foam-promoting protein. Degradation of foam-promoting proteins, whether by microorganisms or by an intensified modification, causes a reduction of foam stability and, thus, a loss of quality.^[178,179]

It is also suspected that an intensified proteolysis of the malt negatively affects the fullness of the body of the beer, as richness and mouthfeel are made up of HMW polypeptides.^[176] A higher level of malt modification has been shown to decrease palate fullness.^[180] Kato et al. identified 10-20 kDa HMW polypeptide fraction as compounds responsible for an improved mouthfeel, such as the softness and smoothness of beer. This protein fraction contained several foam-stabilizing proteins, like BDAI-1 and LTP1, which are thought to minimize undesired hydrophobic flavor compounds. A subsequent extensive enzymatic degradation of malt proteins negatively affected the taste profile.^[181]

Another problem closely associated with fungal contamination in brewing is gushing, the spontaneous and intensively over-foaming of carbonated beverages. Gushing can occur in two fundamentally different ways: induced by the presence of hydrophobins—small surface-active proteins synthesized by filamentous fungi (primary gushing)^{[2,11,27,182]} or by technical factors such as particles or surface roughness (secondary gushing). Several studies confirm an increased gushing potential of beer caused by hydrophobins produced by Fusarium spp.^[2,11,183]; in this context, F. graminearum, F. culmorum, and F. tricinctum were identified as the main causing agents of primary gushing.^[184] Other fungal species of the genera Aspergillus, Rhizopus, Penicillium, and Nigrospora have also been associated with the occurrence of gushing,^[185] e.g., Penicillium oxalicum infections of grapes contribute to the gushing in sparkling wine.^[186] As reviewed by Shokribousjein et al.,^[178] besides synthesizing hydrophobins, proteolytic degradation of LTP1 and other barley proteins contributes to the gushing factor by leading to a shift in protein composition. This, in turn, affects foam stability, gas solubility, and the formation of microbubbles.^[187]

An infestation of raw material with molds may not only result in the degradation of LTP1, as previously described, but it may also lead to an increase in LTP1. A study conducted by Geißinger et al.^[1] on Fusarium sp. found a change in the albumin composition of barley and barley malt, demonstrating an increased expression of LTP1, likely due to its protective role in the host-pathogen interaction. This is consistent with other studies reporting higher expression levels of LTP1 at high pathogen pressures during humid weather conditions.^[178,188] This shows that a holistic comprehension of the impact of black fungal-derived enzymes in malting and brewing quality requires consideration of not only the fungus’s perspective but also the plant’s or germinating grain’s reaction to it. On the other hand, many fungal external proteases are pathogenic effectors that suppress host immunity by disrupting plant defense mechanisms, including pathogenesis-related (PR) proteins like antifungal proteases, β-1,3-glucanases, or chitinases.^[189] In this context, Fusarium spp. metalloproteases are reported to cleave host-derived enzymes.^[189–191] How this plant-pathogen interaction affects other malting-related enzymes should not be underestimated in understanding the impact of filamentous molds on malting and brewing quality.

Conclusion

This review provides a first-time organized summary of current knowledge on the hydrolytic enzymes secreted by dark-pigmented filamentous fungi and regards them from an entirely new aspect of their impact on malting and brewing quality. It demonstrates the significant contribution these fungi can have on the solubilization processes that occur in the grain kernel during malting and mashing, positively contributing to processability and fermentation to a certain extent, but also possibly negatively impacting important sensory perceptions of the final product. The question of whether these external fungal enzymes have a too strong or only a moderate impact on the overall enzyme repertoire is an important area for future research. Many enzymes produced by black fungi, apart from those of interest to industry (Aspergillus spp. and Penicillium spp.), are still waiting to be fully explored and characterized. Therefore, there is still much to be learned about the complex interactions between fungal enzymes and the malting and brewing process. It should also be noted that heavily contaminated grains with filamentous fungi are usually rejected by maltsters and brewers. Nevertheless, this review serves as motivation for further in-depth research on the impact of black-fungal contamination on malting and brewing quality beyond mycotoxins. A better understanding of the complex interactions between fungi and malt, particularly concerning plant-pathogen interactions, can help develop effective quality control measures and ensure the production of high-quality products. Finally, the insights gained from this review may be applicable to other cereal processing industries, demonstrating the potential for broader impacts beyond the field of malting and brewing.

Author contributions

MB reviewed the literature and drafted the manuscript. BS, MG, and TB edited the manuscript. All authors have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Disclosure statement

The authors report that there are no competing interests to declare.

Additional information

Funding

This work was supported by the IGF Project under Grant AiF 19766 N of the FEI, supported via AiF within the program for promoting the Industrial Collective Research (IGF) of the Federal Ministry of Economic Affairs and Climate Action (BMWK), based on a resolution of the German Parliament. This study was supported by the Forschungskreis der Ernährungsindustrie e.V. (FEI), Wissens­chaftsförderung der Deutschen Brauwirtschaft e.V. (WiFö), and Wissenschaftliche Station für Brauerei in München e.V. AiF Projekt;