Safety evaluation of the food enzyme pectinesterase from the genetically modified Trichoderma reesei strain RF6201.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Trichoderma reesei strain RF6201 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.532 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,880. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Trichoderma reesei strain RF6197.

The food enzyme endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Trichoderma reesei strain RF6197 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.156 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 6,410. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and matches were found with a number of pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Biocatalytic Production of Odor-Active Fatty Aldehydes from Fungal Lipids.

Odor-active fatty aldehydes are important compounds for the flavor and fragrance industry. By a coupled enzymatic reaction using an α-dioxygenase (α-DOX) and an aldehyde dehydrogenase (FALDH), scarcely available aldehydes from the biotransformation of margaroleic acid [17:1(9Z)] were characterized and have shown highly interesting odor profiles, including citrus-like, soapy, herbaceous, and savory notes. In particular, (Z)-8-hexadecenal and (Z)-7-pentadecenal exhibited notable meaty odor characteristics. Submerged cultivation of Mortierella hyalina revealed the accumulation of the above-mentioned, naturally uncommon fatty acid 17:1(9Z). Its production was significantly increased by the modulation of culture conditions, whereas the highest accumulation was observed after 4 days at 24 °C and l-isoleucine supplementation. The lipase-, α-DOX-, and FALDH-mediated biotransformation of M. hyalina lipid extract resulted in a complex aldehyde mixture with a high aldehyde yield of ∼50%. The odor qualities of the formed aldehydes were assessed by means of gas chromatography-olfactometry, and several of the obtained fatty aldehydes have been sensorially described for the first time. To assess the aldehyde mixture's potential as a flavor ingredient, a sensory evaluation was conducted. The obtained product exhibited intense citrus-like, green, and soapy odor impressions.

Formation of a Meat-Like Flavor by Submerged Cultivated Laetiporus montanus.

Basidiomycota are natural sources of aroma compounds. When grown in submerged cultures, the fungus Laetiporus montanus (LMO) forms a spicy and meat-like aroma. It thus represents an interesting candidate for the production of natural savory flavors. To identify the key aroma compounds of LMO grown submerged in malt extract peptone medium, the volatiles were isolated by means of automated solvent assisted flavor evaporation (aSAFE). An aroma extract dilution analysis was performed by means of gas chromatography-olfactometry coupled with a flame ionization detector (GC-FID-O). In the aSAFE extract of LMO, 24 aroma-active compounds were detected. 5-Butyl-2(5H)-furanone (FD 4096), perceived as coconut-like, was determined as the compound with the highest FD factor. (E,E)-2,4-Decadienal, (E,Z)-2,4-decadienal, and sotolon were identified as responsible key compounds for the spicy odor of the submerged cultures. Moreover, supplementation of the cultures of LMO, Laetiporus sulphureus, and Laetiporus persicinus with 13C-labeled thiamine hydrochloride resulted in the formation of 2-methyl-3-(methylthio)furan (MMTF), a compound with a pronounced meaty flavor. The concentrations of MMTF were further increased to 19-27 µg L-1 by additional supplementation of the cultures with ascorbic acid. The results of this study indicate potential for the biotechnological production of a meat-like flavor by Laetiporus species.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Aspergillus oryzae strain AR-183.

The food enzyme endo-polygalacturonase (1→4)-α-d-galacturonan glycanohydrolase EC 3.2.1.15 is produced with the genetically modified Aspergillus oryzae strain AR-183 by AB ENZYMES GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation. Since residual amounts of total organic solids (TOS) are removed by repeated washing or distillation, dietary exposure to the food enzyme TOS from coffee demucilation and from the production of flavouring extracts was considered not necessary. For the remaining three food processes, dietary exposure was estimated to be up to 0.087 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 11,494. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR-962.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus oryzae strain AR-962 by AB Enzymes GmbH. The genetic modifications did not give rise to safety concerns. The food enzyme was free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation. Since residual amounts of total organic solids are removed by repeated washing or distillation, dietary exposure to the food enzyme total organic solids (TOS) from the production of flavouring extracts and coffee demucilation was considered not necessary. For the remaining three food processes, dietary exposure to the food enzyme-TOS was estimated to be up to 0.647 mg TOS/kg bw per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,546. A search for the similarity of the amino acid sequence to those of known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Effect of Ecdysterone on the Hepatic Transcriptome and Lipid Metabolism in Lean and Obese Zucker Rats.

Conflicting reports exist with regard to the effect of ecdysterone, the predominating representative of steroid hormones in insects and plants, on hepatic and plasma lipid concentrations in different rodent models of obesity, fatty liver, and diabetes, indicating that the effect is dependent on the rodent model used. Here, the hypothesis was tested for the first time that ecdysterone causes lipid-lowering effects in genetically obese Zucker rats. To test this hypothesis, two groups of male obese Zucker rats (n = 8) were fed a nutrient-adequate diet supplemented without or with 0.5 g ecdysterone per kg diet. To study further if ecdysterone is capable of alleviating the strong lipid-synthetic activity in the liver of obese Zucker rats, the study included also two groups of male lean Zucker rats (n = 8) which also received either the ecdysterone-supplemented or the non-supplemented diet. While hepatic and plasma concentrations of triglycerides and cholesterol were markedly higher in the obese compared to the lean rats (p < 0.05), hepatic and plasma triglyceride and cholesterol concentrations did not differ between rats of the same genotype fed the diets without or with ecdysterone. In conclusion, the present study clearly shows that ecdysterone supplementation does not exhibit lipid-lowering actions in the liver and plasma of lean and obese Zucker rats.

Biotechnological Production and Sensory Evaluation of ω1-Unsaturated Aldehydes.

Lipid extracts of the fungus Flammulina velutipes were found to contain various scarce fatty acids including dodec-11-enoic acid and di- and tri-unsaturated C16 isomers. A biotechnological approach using a heterologously expressed carboxylic acid reductase was developed to transform the fatty acids into the respective aldehydes, yielding inter alia dodec-11-enal. Supplementation studies gave insights into the fungal biosynthesis of this rarely occurring acid and suggested a terminal desaturation of lauric acid being responsible for its formation. A systematic structure-odor relationship assessment of terminally unsaturated aldehydes (C7-C13) revealed odor thresholds in the range of 0.24-22 µg/L in aqueous solution and 0.039-29 ng/L in air. In both cases, non-8-enal was identified as the most potent compound. All aldehydes exhibited green odor qualities. Short-chained substances were additionally associated with grassy, melon-, and cucumber-like notes, while longer-chained homologs smelled soapy and coriander leaf-like with partly herbaceous nuances. Dodec-11-enal turned out to be of highly pleasant scent without off-notes.

Characterization of the Nutritional Composition of a Biotechnologically Produced Oyster Mushroom and its Physiological Effects in Obese Zucker Rats.

SCOPE: Sustainable protein sources are needed to meet the increasing protein demands of a continuously growing world population. This study is focused on the biotechnological production of a protein rich oyster mushroom (Pleurotus sajor-caju; PSC) by valorization of an agricultural side stream and the evaluation of the physiological effects of PSC in a rat model of metabolic syndrome. METHODS AND RESULTS: PSC is produced via submerged cultivation in a 150 L bioreactor that utilizes isomaltulose molasses as its sole carbon source, and is further analyzed for its nutritional composition. A feeding trial is performed using Zucker rats which are fed a 5% PSC supplemented diet, for 4 weeks. Biochemical analyses reveal a significant reduction of the liver lipid concentrations and liver inflammation in the PSC fed obese rats in comparison to the obese rats from the control group. Hepatic qPCR analyses, differential transcript profiling, and enzyme activity measurements reveal a number of altered pathways that may be responsible for these anti-steatotic and anti-inflammatory effects of the mushroom. CONCLUSION: Bioconversion of a low quality agricultural side stream to an improved protein source is performed by submerged cultured PSC, and the obtained mycelium shows strong anti-steatotic and anti-inflammatory effects.

Supplementation of Sulfur-Containing Amino Acids or Essential Amino Acids Does Not Reverse the Hepatic Lipid-Lowering Effect of a Protein-Rich Insect Meal in Obese Zucker Rats.

The present study tested the hypothesis that the liver lipid-lowering effect of insect meal (IM) is caused by its low methionine concentration. A total of fifty, male obese Zucker rats were randomly assigned to five groups of 10 rats each (casein (C), IM, IM + Met, IM + Cys, and IM + EAA). While group C received a diet with casein, the IM-fed groups received a diet with IM as the protein source. In groups IM + Met, IM + Cys and IM + EAA, the diets were additionally supplemented with methionine, cysteine and essential amino acids (EAA), respectively. Hepatic concentrations of triacylglycerols and cholesterol, and hepatic mRNA levels and activities of lipogenic and cholesterogenic enzymes were markedly lower in the IM-fed groups than in group C (p < 0.05). All of these parameters either did not differ across the IM-fed groups or were only slightly higher in groups IM + Met, IM + Cys and IM+EAA than in the group IM. In conclusion, the results indicate that a difference in the amino acid composition between IM and casein, a low concentration of methionine in IM and a reduced cysteine synthesis secondary to a decreased methionine availability resulting from feeding IM are not causative for the lipid-lowering effect of IM.

Aroma Characterization and Safety Assessment of a Beverage Fermented by Trametes versicolor.

A cereal-based beverage was developed by fermentation of wort with the basidiomycete Trametes versicolor. The beverage possessed a fruity, fresh, and slightly floral aroma. The volatiles of the beverage were isolated by liquid-liquid extraction (LLE) and additionally by headspace solid phase microextraction (HS-SPME). The aroma compounds were analyzed by a gas chromatography system equipped with a tandem mass spectrometer and an olfactory detection port (GC-MS/MS-O) followed by aroma (extract) dilution analysis. Thirty-four different odor impressions were perceived, and 27 corresponding compounds were identified. Fifteen key odorants with flavor dilution (FD) factors ranging from 8 to 128 were quantitated, and their respective odor activity values (OAVs) were calculated. Six key odorants were synthesized de novo by T. versicolor. Furthermore, quantitative changes during the fermentation process were analyzed. To prepare for the market introduction of the beverage, a comprehensive safety assessment was performed.

Heterologous expression of the msp2 gene from Marasmius scorodonius.

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca(2+), and hemin.

Novel peroxidases of Marasmius scorodonius degrade beta-carotene.

Two extracellular enzymes (MsP1 and MsP2) capable of efficient beta-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of approximately 150 and approximately 120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of approximately 23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).