Martin Rühl

Food and Feed Enzymes

Humans have benefited from the unique catalytic properties of enzymes, in particular for food production, for thousands of years. Prominent examples include the production of fermented alcoholic beverages, such as beer and wine, as well as bakery and dairy products. The chapter reviews the historic background of the development of modern enzyme technology and provides an overview of the industrial food and feed enzymes currently available on the world market. The chapter highlights enzyme applications for the improvement of resource efficiency, the biopreservation of food, and the treatment of food intolerances. Further topics address the improvement of food safety and food quality.

Characterization of novel insect associated peptidases for hydrolysis of food proteins

Insects are able to feed on a broad spectrum of nutritional sources, due to a variable enzymatic system which can be endogenic or provided by associated microorganisms. This enzymatic system may be employed for the hydrolysis of industrial relevant proteins. Several grain pests were screened for their ability to hydrolyze storage proteins from wheat and rice as well as casein. Zymograms identified hydrolytic activities of the lesser grain borer Rhizopertha dominica against gluten and rice protein. Besides, R. dominica showed the highest prolyl-specific peptidase activity among all tested insects. Enzyme extracts of R. dominica were purified via anion exchange chromatography using a fast protein liquid chromatography system. Two of the purified peptidase fractions were able to hydrolyze peptides from wheat and barley relevant for celiac disease showing a proline preferential cleaving pattern.

Prolyl-specific peptidases for applications in food protein hydrolysis

Various food proteins including, e.g. gluten, collagen and casein are rich in l-proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

Analysis of the volatilome of Calocybe gambosa

The volatilomes of fresh and dried fruiting bodies of Calocybe gambosa were analyzed and compared in this study for the first time. Aroma compounds were extracted by means of liquid/liquid extraction, purified by solvent assisted flavor evaporation (SAFE), and identified by gas chromatography-mass spectrometry and olfactometry. An aroma extract dilution analysis (AEDA) was performed to identify the key flavor compounds, and the main volatiles were quantified by GC flame ionization detection. The key odor compound of fresh fruiting bodies of C. gambosa was (E)-non-2-enal, which, together with (E)-non-2-en-1-ol, was responsible for the characteristic flour- and cucumber-like odor. The aroma profile of commercially available dried fruiting bodies of C. gambosa showed a different pattern. In the dried fruiting bodies, odor compounds like 3-methylbutanoic acid were dominating, and (E)-non-2-enal was not detectable. Thus, the aroma quality of commercially available dried fruiting bodies of C. gambosa differs significantly from that of freshly collected specimens.

Dikaryotic fruiting body development in a single dikaryon of Agrocybe aegerita and the spectrum of monokaryotic fruiting types in its monokaryotic progeny

Using monokaryotic offspring from several dikaryotic parental strains, the phenomenon of monokaryotic fruiting has been previously analysed in the commercially cultivated high-quality edible mushroom Agrocybe aegerita, revealing a variety of monokaryotic fruiting types. Here, we report a single dikaryotic A. aegerita strain, A. aegerita AAE-3, and 40 monokaryons derived from it, which exhibit a wide spectrum of monokaryotic fruiting types, including a rare, previously unknown type. Advantageously, the selected parental strain A. aegerita AAE-3 completes its life cycle within three weeks by the formation of dikaryotic fruiting bodies of typical agaric morphology on malt extract agar plates. In order to morphologically compare normal dikaryotic fruiting to monokaryotic fruiting, histology was performed from all dikaryotic fruiting body development stages and all fruiting types of monokaryotic origin. No clamp connections or dikaryotic hyphae were observed within the plectenchyma of monokaryotic fruiting stages. Among the monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons, we also characterised the rare ‘stipe type’ here described as ‘elongated initials type’ as no differentiation into a future cap and stipe was seen. The two mating-compatible monokaryotic strains representing the extremes of the fruiting type spectrum observed, A. aegerita AAE-3-13 (‘mycelium type’) and A. aegerita AAE-3-32 (‘abortive + true homokaryotic fruiting fruiter type, AHF + THF fruiter type’), were also found to readily produce oidia (arthrospores). In order to obtain a set of mating-compatible monokaryons covering the whole observed spectrum of monokaryotic fruiting, the two monokaryons A. aegerita AAE-3-40 (‘initials type’) and A. aegerita AAE-3-37 (‘elongated initials type’) have been selected for their mating compatibility with A. aegerita AAE-3-32 and A. aegerita AAE-3-13, respectively. Together with the parental dikaryotic strain A. aegerita AAE-3, this set of standard monokaryons could prove useful for studies exploring the factors regulating monokaryotic fruiting in comparison to dikaryotic mushroom formation.

Comprehensive analysis of the volatilome of Scytinostroma portentosum

Fruiting bodies of the corticoid fungus Scytinostroma portentosum, known as mothball crust, have been sampled from a dead branch of sallow. Volatile organic compounds of the samples were extracted by means of liquid/liquid extraction and purified by solvent assisted flavour evaporation. In addition, solid-phase microextraction was applied on one fruiting body sample. The odour active compounds were identified by gas chromatography-mass spectrometry and olfactometry on two columns of different polarity and described by sensory detection. Furthermore, an aroma extract dilution analysis was performed to identify the main flavour compounds. The main odour compounds of S. portentosum with an FD factors ≥16 were 3-chloroindole responsible for the typical mothball odour, the mushroom odours oct-1-en-3-ol and oct-1-en-3-one, methyl p-anisate having an anise-like smell, the bloomy and sweet smelling terpenes linalool and nerolidol, as well as benzylacetone and methyl hexadecanoate. Herewith, a comprehensive aroma active volatilome of S. portentosum is presented.

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes

BackgroundAgrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology.ResultsHere, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery.ConclusionsThe genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.