The Nitrogen Content in the Fruiting Body and Mycelium of Pleurotus Ostreatus and Its Utilization as a Medium Component in Thraustochytrid Fermentation.

Following the idea of a circular bioeconomy, the use of side streams as substitutes for cultivation media (components) in bioprocesses would mean an enormous economic and ecological advantage. Costly compounds in conventional media for the production of the triterpene squalene in thraustochytrids are the main carbon source and complex nitrogen sources. Among other side streams examined, extracts from the spent mycelium of the basidiomycete Pleurotus ostreatus were best-suited to acting as alternative nitrogen sources in cultivation media for thraustochytrids. The total nitrogen (3.76 ± 0.01 and 4.24 ± 0.04%, respectively) and protein (16.47 ± 0.06 and 18.57 ± 0.18%, respectively) contents of the fruiting body and mycelium were determined. The fungal cells were hydrolyzed and extracted to generate accessible nitrogen sources. Under preferred conditions, the extracts from the fruiting body and mycelium contained 73.63 ± 1.19 and 89.93 ± 7.54 mM of free amino groups, respectively. Cultivations of Schizochytrium sp. S31 on a medium using a mycelium extract as a complex nitrogen source showed decelerated growth but a similar squalene yield (123.79 ± 14.11 mg/L after 216 h) compared to a conventional medium (111.29 ± 19.96 mg/L, although improvable by additional complex nitrogen source).

Squalene production under oxygen limitation by Schizochytrium sp. S31 in different cultivation systems.

The triterpene squalene is widely used in the food, cosmetics and pharmaceutical industries due to its antioxidant, antistatic and anti-carcinogenic properties. It is usually obtained from the liver of deep sea sharks, which are facing extinction. Alternative production organisms are marine protists from the family Thraustochytriaceae, which produce and store large quantities of various lipids. Squalene accumulation in thraustochytrids is complex, as it is an intermediate in sterol biosynthesis. Its conversion to squalene 2,3-epoxide is the first step in sterol synthesis and is heavily oxygen dependent. Hence, the oxygen supply during cultivation was investigated in our study. In shake flask cultivations, a reduced oxygen supply led to increased squalene and decreased sterol contents and yields. Oxygen-limited conditions were applied to bioreactor scale, where squalene accumulation and growth of Schizochytrium sp. S31 was determined in batch, fed-batch and continuous cultivation. The highest dry matter (32.03 g/L) was obtained during fed-batch cultivation, whereas batch cultivation yielded the highest biomass productivity (0.2 g/L*h-1). Squalene accumulation benefited from keeping the microorganisms in the growth phase. Therefore, the highest squalene content of 39.67 ± 1.34 mg/g was achieved by continuous cultivation (D = 0.025 h-1) and the highest squalene yield of 1131 mg/L during fed-batch cultivation. Volumetric and specific squalene productivity both reached maxima in the continuous cultivation at D = 0.025 h-1 (6.94 ± 0.27 mg/L*h-1 and 1.00 ± 0.03 mg/g*h-1, respectively). Thus, the choice of a suitable cultivation method under oxygen-limiting conditions depends heavily on the process requirements. KEY POINTS: • Measurements of respiratory activity and backscatter light of thraustochytrids • Oxygen limitation increased squalene accumulation in Schizochytrium sp. S31 • Comparison of different cultivation methods under oxygen-limiting conditions.

Comparative Untargeted Metabolic Profiling of Different Parts of Citrus sinensis Fruits via Liquid Chromatography-Mass Spectrometry Coupled with Multivariate Data Analyses to Unravel Authenticity.

Differences between seven authentic samples of Citrus sinensis var. Valencia peel (albedo and flavedo) and juices from Spain and Uruguay, in addition to a concentrate obtained from Brazil, were investigated by untargeted metabolic profiling. Sixty-six metabolites were detected by nano-liquid chromatography coupled to a high-resolution electrospray-ionization quadrupole time-of-flight mass spectrometer (nLC-ESI-qTOF-MS) belonging to phenolic acids, coumarins, flavonoid glycosides, limonoids, terpenes, and fatty acids. Eleven metabolites were detected for the first time in Citrus sinensis and identified as citroside A, sinapic acid pentoside, apigenin-C-hexosyl-O-pentoside, chrysoeriol-C-hexoside, di-hexosyl-diosmetin, perilloside A, gingerol, ionone epoxide hydroxy-sphingenine, xanthomicrol, and coumaryl alcohol-O-hexoside. Some flavonoids were completely absent from the juice, while present most prominently in the Citrus peel, conveying more industrial and economic prospects to the latter. Multivariate data analyses clarified that the differences among orange parts overweighed the geographical source. PCA analysis of ESI-(-)-mode data revealed for hydroxylinoleic acid abundance in flavedo peel from Uruguay the most distant cluster from all others. The PCA analysis of ESI-(+)-mode data provided a clear segregation of the different Citrus sinensis parts primarily due to the large diversity of flavonoids and coumarins among the studied samples.

Impact of Agro-Industrial Side-Streams on Sesquiterpene Production by Submerged Cultured Cerrena unicolor.

The quality and harvest of essential oils depend on a large number of factors, most of which are hard to control in an open-field environment. Therefore, Basidiomycota have gained attention as a source for biotechnologically produced terpenoids. The basidiomycete Cerrena unicolor (Cun) was cultivated in submerged culture, and the production of sesquiterpenoids was analyzed via stir bar sorptive extraction (SBSE), followed by thermo-desorption gas chromatography coupled with mass spectrometry (TDS-GC-MS). Identification of aroma-active sesquiterpenoids was supported by GC, coupled with an olfactory detection port (ODP). Following the ideal of a circular bioeconomy, Cun was submerged (up-scalable) cultivated, and supplemented with a variety of food industrial side-streams. The effects of the different supplementations and of pure fatty acids were evaluated by liquid extraction and analysis of the terpenoids via GC-MS. As sesquiterpenoid production was enhanced by the most by lipid-rich side-streams, a cultivation with 13C-labeled acetate was conducted. Data confirmed that lipid-rich side-streams enhanced the sesquiterpene production through an increased acetyl-CoA pool.

Mycelium vs. Fruiting Bodies of Edible Fungi-A Comparison of Metabolites.

Edible mushrooms are widely appreciated for their appealing flavours, low caloric values and high content of presumably health-protecting metabolites. Their long history of safe use together with the looming worldwide food crisis have revived the idea of generating meat analogues and protein isolates by the controlled fermentation of mycelia of these edible fungi as a dietary option. The occurrence of proteins, polysaccharides, smaller metabolites, metal ions and toxins in mycelia and fruiting bodies is compared among the three most popular species, Agaricus bisporus (button mushroom), Pleurotus ostreatus (oyster mushroom), Lentinus edodes (shiitake) and some closely related species. Large effects of substrate chemistry, strain, developmental stage and ecological interactions result in a wide variation of the concentrations of some metabolites in both mycelial cells and fruiting bodies. This is obviously a result of the high adaptation abilities required to survive in natural habitats. Fungal bioprocesses are decoupled from agricultural production and can be operated anytime, anywhere, and on any scale according to demand. It is concluded that fungal biomass, if produced under food-grade conditions and on an industrial scale, could provide a safe and nutritious meat substitute and protein isolates with a high biological value for future vegan foods.

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides.

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.

Production of an Anise- and Woodruff-like Aroma by Monokaryotic Strains of Pleurotus sapidus Grown on Citrus Side Streams.

The production of natural flavors by means of microorganisms is of great interest for the food and flavor industry, and by-products of the agro-industry are particularly suitable as substrates. In the present study, Citrus side streams were fermented using monokaryotic strains of the fungus Pleurotus sapidus. Some of the cultures exhibited a pleasant smell, reminiscent of woodruff and anise, as well as herbaceous notes. To evaluate the composition of the overall aroma, liquid/liquid extracts of submerged cultures of a selected monokaryon were prepared, and the volatiles were isolated via solvent-assisted flavor evaporation. Aroma extract dilution analyses revealed p-anisaldehyde (sweetish, anisic- and woodruff-like) with a flavor dilution factor of 218 as a character impact compound. The coconut-like, herbaceous, and sweetish smelling acyloin identified as (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone also contributed to the overall aroma and was described as an aroma-active substance with an odor threshold in air of 0.2 ng L-1 to 2.4 ng L-1 for the first time. Supplementation of the culture medium with isotopically substituted l-tyrosine elucidated this phenolic amino acid as precursor of p-anisaldehyde as well as of (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone. Chiral analysis via HPLC revealed an enantiomeric excess of 97% for the isolated product produced by P. sapidus.

Enzymatic hydrolysis of kaempferol 3-O-(2‴-O-sinapoyl-ß-sophoroside), the key bitter compound of rapeseed (Brassica napus L.) protein isolate.

BACKGROUND: The use of rapeseed protein for human nutrition is primarily limited by its strong bitterness, which is why the key bitter compound, kaempferol 3-O-(2‴-O-sinapoyl-ß-sophoroside), is enzymatically degraded. RESULTS: Mass spectrometry analyses of an extract from an untreated rapeseed protein isolate gave three signals for m/z 815 [M-H]. The predominant compound among the three compounds was confirmed as kaempferol-3-O-(2‴-O-sinapoyl-ß-sophoroside). Enzymatic hydrolysis of this key bitter compound was achieved using a sinapyl ester cleaving side activity of a ferulic acid esterase (FAE) from the basidiomycete Schizophyllum commune (ScoFAE). Recombinant ferulic acid esterases from Streptomyces werraensis (SwFAE) and from Pleurotus eryngii (PeFAE) possessed better cleavage activity towards methyl sinapate but did not hydrolyze the sinapyl ester linkage of the bitter kaempferol sophoroside. CONCLUSION: Kaempferol-3-O-(2‴-O-sinapoyl-ß-sophoroside) was successfully degraded by enzymatic treatment with ScoFAE, which may provide a means to move the status of rapeseed protein from feed additive to food ingredient. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus.

The valuable aroma compound piperonal with its vanilla-like olfactory properties is of high interest for the fragrance and flavor industry. A lipoxygenase (LOXPsa 1) of the basidiomycete Pleurotus sapidus was identified to convert piperine, the abundant pungent principle of black pepper (Piper nigrum), to piperonal and a second volatile product, 3,4-methylenedioxycinnamaldehyde, with a vanilla-like odor through an alkene cleavage. The reaction principle was co-oxidation, as proven by its dependence on the presence of linoleic or α-linolenic acid, common substrates of lipoxygenases. Optimization of the reaction conditions (substrate concentrations, reaction temperature and time) led to a 24-fold and 15-fold increase of the piperonal and 3,4-methylenedioxycinnamaldehyde concentration using the recombinant enzyme. Monokaryotic strains showed different concentrations of and ratios between the two reaction products.

Phenylacrylic acids addition to potato and sweet potato showed no impact on acrylamide concentration via oxa-Michael-addition during frying.

Three phenolic acids, p-coumaric, ferulic and caffeic acid as well as cinnamic acid were added to raw potatoes and sweet potatoes before frying. A distinct mitigation of acrylamide was not detected. Fried samples were analysed for postulated adducts of a direct reaction between acrylamide and these phenolic acids using LC-MS. In a model system with pure compounds (phenylacrylic acid and acrylamide) heated on 10% hydrated silica gel one specific adduct (respective m/z for M â€‹+ â€‹H+) was formed in each reaction. MS/MS-data suggested an oxa-Michael formation of 3-amino-3-oxopropyl-phenylacrylates, which was confirmed by de novo syntheses along an SN2 substitution of 3-chloropropanamide. Exemplarily, the structure of the ester was confirmed for p-coumaric acid by NMR-data. Standard addition revealed that 3-amino-(3-oxopropyl-phenyl)-acrylates occurred neither in fried potato nor in sweet potato, while a formation was shown in phenylacrylic acid plus acrylamide supplemented potatoes and sweet potatoes.

Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

Generation of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants.

Traditional smoke flavours bear the risk of containing a multitude of contaminating carcinogenic side-products. Enzymatic decarboxylation of ferulic acid released from agro-industrial side-streams by ferulic acid esterases (FAE) enables the sustainable generation of pure, food grade 4-vinylguaiacol (4-VG), the impact compound of smoke flavour. The first basidiomycetous ferulic acid decarboxylase (FAD) was isolated from Schizophyllum commune (ScoFAD) and heterologously produced by Komagataella phaffii. It showed a molecular mass of 21 kDa, catalytic optima at pH 5.5 and 35°C, and a sequence identity of 63.6% to its next relative, a FAD from the ascomycete Cordyceps farinosa. The ScoFAD exhibited a high affinity to its only known substrate ferulic acid (FA) of 0.16 mmol L-1 and a turnover number of 750 s-1. The resulting catalytic efficiency kcat KM-1 of 4,779 L s-1 mmol-1 exceeded the next best known enzyme by more than a factor of 50. Immobilised on AminoLink Plus Agarose, ScoFAD maintained its activity for several days. The combination with FAEs and agro-industrial side-streams paves the way for a new generation of sustainable, clean, and safe smoke flavours.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Formation of Decatrienones with a Pineapple-like Aroma from 1-13C-Acetate by Cell Cultures of the Birch Polypore, Fomitopsis betulina.

During submerged cultivation, the edible basidiomycete Fomitopsis betulina (previously Piptoporus betulinus) developed a fruity odor, strongly reminding of pineapple. Olfactometric analysis showed that this impression was mainly caused by the two (5E/Z,7E,9)-decatrien-2-ones. At the time of maximum concentration on the 5th day, the (5E/5Z)-ratio was 94:6. Three hypotheses were experimentally examined to shed light onto the genesis of the uncommon volatiles: first, an indirect effect of agro-industrial side-streams, such as cabbage cuttings, supporting good growth; second, an unsaturated odd-numbered fatty acid precursor; and third, a polyketide-like pathway. In the presence of 1-13C- or 2-13C-acetate up to five acetates were incorporated into the molecular ions of the C10-body. Addition of 1-13C-pyruvate or 1-13C-lactate did not confirm an odd-numbered starter of the polyketide chain. None of the methylketones was found in pineapple or any other food before.

(5E/Z,7E,9)-Decatrien-2-ones, Pineapple-like Flavors from Fomitopsis betulina-Structure Elucidation and Sensorial Properties.

During the cultivation of the edible mushroom Fomitopsis betulina on agro-industrial side streams, a pleasant flavor strongly reminiscent of pineapple was perceived. Aroma extract dilution analyses identified two flavor components with a distinct pineapple odor. On the basis of mass spectrometric data, a Wittig reaction of (E)-penta-2,4-dien-1-yltriphosphonium bromide with ethyl levulinate was conducted. The resulting (5E/Z,7E,9)-decatrien-2-ones were identical to the compounds isolated from the fungal culture. Some structurally related methyl ketones were synthesized, confirmed by nuclear magnetic resonance and mass spectrometry, and their odor was characterized. The lowest odor threshold and most characteristic pineapple-like odor was found for (5Z,7E,9)-decatrien-2-one. Global minimum energy calculation of the methyl ketones and the comparison to (1,3E,5Z)-undecatriene, a character impact compound of fresh pineapple, showed that a chain length of at least 10 carbon atoms and a terminal double bond embedded in a "L"-shaped conformation were common to compounds imparting an intense pineapple-like odor. Both (5E/Z,7E,9)-decatrien-2-ones have not been described as natural flavor compounds.

Response of the sesquiterpene synthesis in submerged cultures of the Basidiomycete Tyromyces floriformis to the medium composition.

Tyromyces floriformis, a potent fungal sesquiterpene producer, was grown Cerrena unicolor, as a model organism in submerged culture to search for chemicals affecting sesquiterpene biosynthesis in vitro. Thirty-one sesquiterpenes and sesquiterpenoids were identified in the supernatant, among them the fruity α-ylangene as the main volatile. Additives, such as some polysaccharides or lipids, did not affect the qualitative product spectrum but strongly affected the quantitative synthesis. Rye arabinoxylan and other polysaccharides, such as chitin, starch, and agarose, almost blocked the synthesis of α-ylangene. Single addition of the building blocks of arabinoxylan, arabinose, xylose, or ferulic acid showed no inhibitory effect, whereas 0.05% (w/v) 32-α-l-arabinofuranosyl-xylobiose and larger oligosaccharides resulted in a significant suppression. In contrast, addition of acetyl donors boosted the α-ylangene concentration by 1 order of magnitude up to >40 mg L-1. Both increased as well as decreased α-ylangene concentrations correlated with the intracellular sesquiterpene cyclase activity. Similar experiments using submerged cultured Cerrena unicolor, Postia placenta, and Coprinopsis cinerea showed that the additives affected fungal sesquiterpenoid synthesis differently. Whereas the addition of acetyl donors boosted the synthesis in all biphasic cultures, it was inhibited by polysaccharides in fungi preferably interacting with lignified plants. In contrast, Cerrena unicolor, known for a symbiotic lifestyle with wasps, responded by forming higher concentrations of the possibly insect-attracting sesquiterpenes.

Long-Term Monokaryotic Cultures of Pleurotus ostreatus var. florida Produce High and Stable Laccase Activity Capable to Degrade ß-Carotene.

An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching ß-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles.

High moisture extrusion of lupin protein: influence of extrusion parameters on extruder responses and product properties.

BACKGROUND: High moisture extrusion (HME) of lupin protein concentrate and isolate (50:50) mixture was performed by varying the extrusion parameters, such as barrel temperature (138-180 °C), water feed (40-68%) and screw speed (400-1800 rpm). The effect of extrusion parameters on extruder responses [die pressure, product temperature, torque and specific mechanical energy (SME)] and product properties [colour, cutting force, cooking yield, microstructure and in vitro protein digestibility (IVPD)] was evaluated. RESULTS: The multiple regression analysis of the results revealed that the water feed had a significant negative linear effect on the extruder responses considered, as well as on colour difference and cutting force of extrudates. Screw speed had a positive linear effect on product temperature, SME and cooking yield. Barrel temperature affected extruder responses and product properties to a lesser extent. Scanning electron microscopy showed that denser microstructure and higher number of fibre layers were created by increasing temperature and screw speed along with decreasing water feed. The results of IVPD of selected extrudates showed that the increase in barrel temperature decreased the IVPD, whereas the increase in water feed resulted in higher IVPD. The screw speed had no significant effect on IVPD. CONCLUSION: The study demonstrates that the use of lupin protein is feasible to produce meat analogues with HME which could enhance the possibilities to meet the growing protein demands for human consumption. © 2018 Society of Chemical Industry.

BadGluc, a ß-glucosidase from Bjerkandera adusta with anthocyanase properties.

A glycosidase of the basidiomycete Bjerkandera adusta (BadGluc) was found in screenings to possess a strong decolorizing ability towards malvidin-3-galactoside, an anthocyanin abundant in various berry fruits. The BadGluc was purified from the culture supernatant via FPLC, and the corresponding gene was identified which showed low similarity to other characterized glucosidases. Scanning the primary sequence with PROSITE no active site motif was detected. Eventually, a specific 18 aa consensus pattern was identified manually. The active site motif possessed an undescribed sequence which was only found in a few hypothetical proteins. The corresponding gene was cloned and expressed in Pichia pastoris GS115 yielding activities up to 100 U/L using 4-nitrophenyl-ß-d-glucopyranoside (pNPG) as substrate. The enzyme possessed a good temperature (70% after 1 h at 50°C) and pH stability (70% between pH 2 and 7.5), and preferably catalysed the hydrolysis of delphinidin-3-glucoside and cyanidin-3-glucoside, regardless of the position of the terminal Hexa-His tag. This novel glucosidase worked in aqueous solution as well as on pre-stained fabrics making it the first known candidate anthocyanase for applications in the detergent and food industries.

Enzymatic mitigation of 5-O-chlorogenic acid for an improved digestibility of coffee.

A p-coumaroyl esterase from Rhizoctonia solani was used to decrease 5-O-chlorogenic acid (5-CQA) content in coffee powder. HPLC-UV showed a decline of up to 98% of 5-CQA after the enzyme treatment. Effects on aroma were determined by means of aroma extract dilution analysis. Flavour dilution factors of treated and control extract differed in four volatile compounds only. Effect on aroma and taste was evaluated by sensory tests. No significant differences were perceived, and no off-flavour nor off-taste was noted. As chlorogenic acids are suspected to cause stomach irritating effects in sensitive people, the enzyme treatment offers a technically feasible approach to improve the quality of coffee beverages by reducing 5-CQA concentration without significantly affecting the aroma and taste profile.