Niger Strain Research Articles

Profiling of bioactive secondary metabolites from Aspergillus niger against a guava wilt pathogen, Fusarium oxysporum f. sp. psidii.

Guava wilt is a devastating soil-borne disease that causes significant losses in guava orchards. Management of the disease is very challenging once established in the field. Therefore, there is a need to explore for an effective, economical, and sustainable management strategies. Aspergillus niger, a bio-control fungus, has been demonstrated effectiveness against various soil-borne pathogens including guava wilt pathogens. It produces a diverse hydrolysing enzymes and secondary metabolites. However, no extensive study has been undertaken to profile the secondary metabolites of A. niger. In this investigation, we assessed eleven A. niger strains (AN-1 to AN-11) against four guava wilt pathogens (Fusarium oxysporum f. sp. psidii, F. falciforme, F. chlamydosporum, and F. verticillioides) using a dual culture assay. All strains demonstrated effective by restricting the mycelial growth of pathogens, among them AN-11 displayed maximum inhibition of 86.33%, followed by the AN-3 (84.27%). The UPLC-QToF-ESIMS analysis was undertaken to explore the secondary metabolites of AN-11 responsible for inhibiting F. oxysporum f. sp. psidii. The crude extracts were obtained from F. oxysporum f. sp. psidii, AN-11 and their interaction using ethyl acetate as a solvent. After evaporating, the crude fractions were analysed using UPLC-QToF-ESIMS with an Acquity UPLC and a SCIEX SelexION Triple QuadTM 5500 System. From the ethyl acetate extract of F. oxysporum f. sp. psidii, approximately 14 metabolites involved in pathogenicity were identified. Similarly, analysis of AN-11 crude extract revealed 25 metabolites, and notably, 41 metabolites were identified during the interaction between AN-11 and F. oxysporum f. sp. psidii, including kotanin, isokotanin A, aurofusarin, kojic acid, pyranonigrin, aurasperone F, hexylitaconic acid, asperazine, bicoumanigrin, chloramphenicol, cephalosporin C, fusarin C, zearalonone, fonsecin B, malformin A, and others. Among these, 21 metabolites were produced only during the interaction and have antimicrobial properties. This study highlights the significant potential of the AN-11 strain in generating a diverse array of non-volatile secondary metabolites with antimicrobial properties. These metabolites could be further extracted and investigated for their efficacy against other soil borne pathogens and potentially developed into formulations for controlling plant diseases.

Aspergillus niger Strain AM1 as an Agent for Biodegradation of Petroleum and Petroleum Products

Aspergillus niger Strain AM1 as an Agent for Biodegradation of Petroleum and Petroleum Products

Synthesis, Spectroscopy and Biological Studies of Copper and Silver Complexes Derived from 2-Substituted Amino Propanoic Acid

Synthesis of copper and silver complexes were prepared from novel Schiff base ligand. These synthesized ligand and their metal complexes were characterized by elemental analysis, FT-IR, 1H NMR, UV-Vis and Mass spectrometry. The coordination of amino nitrogen, phenolic oxygen, and carboxylato oxygen atoms is shown by the infrared spectra.The Schiff bases and its complexes were screened for their in vitro antibacterial and antifungal studies against the bacteria Pseudomonas Aeruginosa (PA, Gram -ve), Bacillus Cirroflagellosus (BC, Gram +ve), Penicillium Notatium (PN) and Aspergillus Niger (AN) strains. The results of the antibacterial investigation show that the copper and silver complexes possess more potent bactericide and fungicide properties than the ligand.

Oxygen Consumption in Filamentous Pellets of Aspergillus niger: Microelectrode Measurements and Modeling.

Filamentous fungi cultivated as biopellets are well established in biotechnology industries. A distinctive feature of filamentous fungi is that hyphal growth and fungal morphology affect product titers and require tailored process conditions. Within the pellet, mass transfer, substrate consumption, and biomass formation are intricately linked to the local hyphal fraction and pellet size. This study combined oxygen concentration measurements with microelectrode profiling and three-dimensional X-ray microtomography measurements of the same fungal pellets for the first time. This allowed for the precise correlation of micromorphological information with local oxygen concentrations of two Aspergillus niger strains (hyperbranching and regular branching). The generated results showed that the identified oxygen-penetrated outer pellet regions exhibited a depth of 90-290 µm, strain-specific, with the active part percentage in the pellet ranging from 18% to 69%, without any difference between strains. Using a 1D continuum diffusion consumption model, the oxygen concentration in the pellets was computed depending on the local hyphal fraction. The best simulation results were achieved by individually estimating the oxygen-related biomass yield coefficient of the consumption term within each examined pellet, with an average estimated value of 1.95 (± 0.72) kg biomass per kg oxygen. The study lays the foundation for understanding oxygen supply in fungal pellets and optimizing processes and pellet morphologies accordingly.

Safety evaluation of the food enzyme glucan-1,4-α-glucosidase from the non-genetically modified Aspergillus niger strain DP-Azh100.

The food enzyme glucan-1,4-α-glucosidase (4-α-d-glucan glucohydrolase; EC 3.2.1.3) is produced with the non-genetically modified Aspergillus niger strain DP-Azh100 by Genencor International B.V. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in four food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the two remaining processes. It was estimated to be up to 1.390 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 at the highest dose tested of 1000 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 719. A search for the homology of the amino acid sequence of the food enzyme to known allergens was made and one match to a respiratory allergen was found. Known sources of food allergens were used in the food enzyme manufacturing process. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low. 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.

Isolation and Molecular Identification of Fungal Contamination on the Eggshells of Lepidochelys olivacea L. Collected from Coastal Waters

Background: One of the causes of the decline in the Olive ridley turtle population is the low egg hatching rate due to contamination by pathogenic fungi. Purpose: This study aims to evaluate the results of the isolation and molecular identification of fungal contamination on the eggshells of olive ridley turtles that failed to hatch from three coastal waters in Bali province. Method: Sampling was done using the purposive sampling technique. Samples of failed hatching eggshells from the coast were collected, as many as 2 eggs/semi-natural nest at TCEC. Samples were cultured on PDA media. Macroscopic and molecular identification methods were carried out to determine the species level of fungal isolates. Results: The results showed that 10 fungal isolates were successfully collected. Isolates KL1 and SG1 had white colony surface characteristics, isolates KL4, SY2 and SY3 showed black colonies, and isolates KL2, KL3, SG2, SG3, SY4 had green colonies. Three similar isolates were sent for further molecular identification. The BLASTn results of the nucleotide sequences of isolates SY2 and SG3 showed a similarity to Aspergillus niger strain PHY105 (91%), while fungal isolate KL1 had a similarity to Fusarium solani strain 87, by 98%. Conclusion: This study provides new information about the pathogenic fungi, Fusarium solani strain 87 and Aspergillus niger strain PHY105, which are suspected to be the cause of the hatching failure of olive ridley turtle eggs.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Transcriptomics identify the triggering of citrate export as the key event caused by manganese deficiency in Aspergillus niger.

Citric acid is produced on industrial scale by batch fermentation of the filamentous fungus Aspergillus niger. High-yield citric acid production requires a low (<5 ppb) manganese(II) ion concentration in the culture broth. However, the requirement for this deficiency has not been investigated on a functional genomics level. Here, we compared the transcriptome of a citric acid hyper-producer A. niger strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. We observed that Mn2+ deficiency triggers an upregulation of the citrate exporter gene cexA and provides functional evidence that this event is responsible for citrate overflow. In addition to the industrial relevance, this is the first study that examined the role of Mn2+ ion deficiency in a heterotrophic eukaryotic cell on a genome-wide scale.

Safety evaluation of the food enzyme carboxypeptidase C from the genetically modified Aspergillus niger strain PEG.

The food enzyme carboxypeptidase C (EC 3.4.16.5) is produced with the genetically modified Aspergillus niger strain PEG by DSM Food Specialties B.V. 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 nine food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.053 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the carboxypeptidase to be used in the toxicological studies, because both strains were derived from the same recipient strain, the location of the inserts was comparable, no partial inserts were present and the production methods were essentially the same. Genotoxicity tests did not raise 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 1850 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 901. A homology search for the amino acid sequence of the food enzyme to known allergens was made and one match with a wheat allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially in wheat-allergic individuals, but the likelihood is low. 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 an extension of use of a food enzyme containing endo-polygalacturonase, pectinesterase, pectin lyase and non-reducing end α-l-arabinofuranosidase activities from the non-genetically modified Aspergillus niger strain PEC.

The food enzyme has four declared activities: endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase (endo-cleaving); EC 3.2.1.15), pectinesterase (pectin pectylhydrolase; EC 3.1.1.11), pectin lyase ((1-4)-6-O-methyl-α-d-galacturonan lyase; EC 4.2.2.10) and non-reducing end α-l-arabinofuranosidase (α-l-arabinofuranoside non-reducing end α-l-arabinofuranosidase; EC 3.2.1.55). It is produced with the non-genetically modified Aspergillus niger strain PEC by DSM Food Specialties B.V. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant has requested to extend its use to include four additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.612 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (204 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 333. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Synthesis and antimicrobial activity of benzoxazine/benzothiazine appended 1,2,3-triazole hybrids

In the present context to rationalize the search for new effective antimicrobial agents against the infections caused by microbes, a series of benzoxazine/benzothiazine appended 1,2,3-triazole hybrids were designed and synthesized via click reaction with ingredients of two or more bioactive moieties through the concept of molecular hybridization. Various spectral techniques such as FTIR, 1H NMR, 13C NMR, 2D NMR and HRMS were utilized for structural elucidation of the synthesized hybrids. The serial dilution method was employed for performing the antimicrobial activity of synthesized triazoles against four bacterial (S. aureus, B. subtilis, E. coli, K. pneumoniae) and two fungal (C. albicans, A. niger) strains. The antibacterial results demonstrated that a couple of hybrids show noteworthy outcomes related to standard medication. Compound 4r was indicated great action against E. coli with MIC values 0.0259 µmol/mL. Based on outcomes of antibacterial activity, molecular docking study was carried out with E. coli DNA gyrase to understand the binding behavior. To check out the drug-likeness, in silico ADME (Absorption, Distribution, Metabolism and Excretion) studies of synthesized hybrids were also analyzed and found that compounds can be developed as potential oral drug candidates.

Role of Copper Ions in Resistance of Modern Polymer Composite Materials to Fungal Damage

Resistance of polymer composite materials to biodamage is one of the pressing problems of our time. Incorporation of Cu2O (I) in the composition of a polymer composite based on the ED-20 epoxy resin increases its biocidal properties. Under conditions of mineral and organic contamination, the area of the samples affected by micromycetes was found to decrease with increasing concentration of dispersed particles in the composite. The affected area of the samples filled with the particles encapsulated in polylactide was 1.5 times smaller than that of the composites filled with non-encapsulated particles. Сopper oxide had a toxic effect on the Aspergillus niger strain dominant on the surface of the samples, causing a decrease in the average radial growth rate on the Czapek-Dox agar medium and in the biomass weight concentration during the growth of micromycetes in a liquid medium compared to the variant without Cu2O.

Fungal Peptidomelanin: A Novel Biopolymer for the Chelation of Heavy Metals.

Melanin is an amorphous, highly heterogeneous polymer found across all kingdoms of life. Although the properties of melanin can greatly vary, most forms are insoluble and strongly absorb light, appearing dark brown to black. Here, we describe a water-soluble form of melanin (peptidomelanin) secreted by the spores of Aspergillus niger (strain: melanoliber) during germination. Peptidomelanin is composed of an insoluble L-DOPA core polymer that is solubilized via short, copolymerized heterogeneous peptide chains forming a "corona" with a mean amino acid length of 2.6 ± 2.3. Based on in vitro experiments, we propose a biochemical copolymerization mechanism involving the hydroxylation of tyrosynylated peptides. Peptidomelanin is capable of chelating heavy metals such as lead, mercury, and uranium (as uranyl) in large quantities. Preliminary data indicates that peptidomelanin may have applications for the remediation of heavy metals in situ, including in agricultural settings.

Comparison of enhanced neural network and response surface models in predicting bio-dissolution of aluminum and vanadium from bauxite residue by isolated Aspergillus niger strains

Background: Recovery of Al and V from red mud, a hazardous residue of the Bayer process, using bioleaching is a nature-based waste management solution that simultaneously improves environment protection and metallurgical recoveries.Methods: For the first time, a novel hybrid multilayer Perceptron (MLP) network enhanced by an Imperialist Competitive Algorithm (ICA), and a response surface developed based on a factorial experiment design methodology (RSM) were employed and compared for, prediction optimization and optimization prediction of Al and V bioleaching by strains of A. Niger microorganisms isolated from pistachio shells and grape skins. The controlling variables were fungi source (A), adoption strategy (B), solid activation (C), solid percent (D), and bioleaching time. Considering the stochastic nature of ICA, a multi-criteria-ranking system based on accuracy and error indices was developed to select the best MLP. Probability value at 95% confidence interval, lack-of-fit, analysis of variance, R2, Adj. R2, and predicted R2 were the judges for the determination of the developed model's statistical significance.Significant Findings: Based on ANOVA, between A, B, C, and D, the effectiveness orders of A > D > C>AC>AD on Al, and A>AD>C>AC>D on V bioleaching were obtained. The superiority of the developed hybrid ICA-MLP model over the developed RSM model in predicting Al and V dissolution was determined by NSE, RSME, MAE, and MEDAE. Parameters optimization and consequently evaluating optimum condition repeatability through three repeating experiments resulted in maximum dissolution recoveries of RSM: 96.5% for Al and 91.2% for V, and ICA-MLP: 97.1% for Al and 90.3% for V. Considering the lower relative errors of repeating validation tests, it can be concluded that, although both models provide reasonable results, but the ICA-MLP methodology is more reliable (relative error <1.8%).

Mitigating the detrimental impacts of low- and high-density polyethylene microplastics using a novel microbial consortium on a soil-plant system: Insights and interactions

The accumulation of polyethylene microplastics (PE-MPs) in soil has raised considerable concerns; however, the effects of their persistence and mitigation on agroecosystems have not been explored. This study aimed to assess the detrimental effects of PE-MPs on a soil-plant system and evaluate their mitigation using a novel microbial consortium (MC). We incorporated low-density polyethylene (LDPE) and high-density polyethylene (HDPE) at two different concentrations, along with a control (0 %, 1 %, and 2 % w/w) into the sandy loam soil for a duration of 135 days. The samples were also treated with a novel MC and incubated for 135 days. The MC comprised three bacterial strains (Ralstonia pickettii (MW290933) strain SHAn2, Pseudomonas putida strain ShA, and Lysinibacillus xylanilyticus XDB9 (T) strain S7–10F), and a fungal strain (Aspergillus niger strain F1–16S). Sunflowers were subsequently cultivated, and physiological growth parameters were measured. The results showed that adding 2 % LDPE significantly decreased soil pH by 1.06 units compared to the control. Moreover, adding 2 % HDPE resulted in a more significant decrease in soil electrical conductivity (EC) relative to LDPE and the control. A dose-dependent increase in dissolved organic carbon (DOC) was observed, with the highest DOC found in 2 % LDPE. The addition of higher dosages of LDPE reduced soil bulk density (BD) more than HDPE. The addition of 2 % HDPE increased the water drop penetration time (WDPT) but decreased the mean weight diameter of soil aggregates (MWD) and water-stable aggregates (WSA) compared to LDPE. The results also revealed that higher levels of LDPE enhanced soil basal respiration (BR) and microbial carbon biomass (MBC). The interaction of MC and higher MP percentages considerably reduced soil pH, EC, BD, and WDPT but significantly increased soil DOC, MWD, WSA, BR, and MBC. Regarding plant growth, incorporating 2 % PE-MPs significantly reduced physiological responses of sunflower: chlorophyll content (Chl; −15.2 %), Fv/Fm ratio (-25.3 %), shoot dry weight (ShD; −31.3 %), root dry weight (RD; −40 %), leaf area (LA; −38.4 %), and stem diameter (StemD; −25 %) compared to the control; however, the addition of novel MC considerably reduced and ameliorated the harmful effects of 2 % PE-MPs on the investigated plant growth responses.

Gene function characterization in Aspergillus niger using a dual resistance marker transformation system mediated by Agrobacterium tumefaciens

Aspergillus niger is a well-known workhorse for the industrial production of enzymes and organic acids. This fungus can also cause postharvest diseases in fruits. Although Agrobacterium tumefaciens-mediated transformation (ATMT) based on antibiotic resistance markers has been effectively exploited for inspecting functions of target genes in wild-type fungi, it still needs to be further improved in A. niger. In the present study, we re-examined the ATMT in the wild-type A. niger strains using the hygromycin resistance marker and introduced the nourseothricin resistance gene as a new selection marker for this fungus. Unexpectedly, our results revealed that the ATMT method using the resistance markers in A. niger led to numerous small colonies as false-positive transformants on transformation plates. Using the top agar overlay technique to restrict false positive colonies, a transformation efficiency of 87 ± 18 true transformants could be achieved for 106 conidia. With two different selection markers, we could perform both the deletion and complementation of a target gene in a single wild-type A. niger strain. Our results also indicated that two key regulatory genes (laeA and veA) of the velvet complex are required for A. niger to infect apple fruits. Notably, we demonstrated for the first time that a laeA homologous gene from the citrus postharvest pathogen Penicillium digitatum was able to restore the acidification ability and pathogenicity of the A. niger ΔlaeA mutant. The dual resistance marker ATMT system from our work represents an improved genetic tool for gene function characterization in A. niger.

Investigation of heat-resistant antifungal agents from Bacillus amyloliquefaciens and Bacillus subtilis for biocontrol of mycotoxigenic fungi

Fungal infections of food and feed with toxigenic strains results in the accumulation of harmful metabolites. In this study, the antifungal activity of two indigenous bacterial strains Bacillus amyloliquefaciens and Bacillus subtilis isolated from camel feed was investigated. The potential of the bacterial cell free supernatant (CFS) to reduce mycotoxins synthesis was evaluated, and the impact of their antifungal metabolites on the fungal hyphae was examined. High antagonistic activity was exhibited by both bacterial strains. The best activity was observed against two mycotoxigenic A. niger strains (Sp31 and Sp33) with inhibition zones of 36.75 ± 0.7 mm and 35.75 ±1.5 mm, by strains B. amyloliquefaciens and B. subtilis, respectively. The effects of these bacterial strains’ CFSs on mycotoxins synthesis were investigated, and both CFSs were found to reduce the mycotoxins synthesis by A. parasiticus, A. niger and Penicillium spp., in a dose-dependent manner. Moreover, the fungal cell wall morphology was significantly altered by the bacterial CFS at a very low concentration of 1 %. The heat stability of the antifungal metabolites was evaluated at various temperatures ranging from −20 °C to 100 °C, and the metabolites were found to be highly stable, retaining their antifungal activity against A. niger. The antifungal activity of B. amyloliquefaciens and B. subtilis was stable at −20 °C for 236 and 34 days of storage, respectively. These findings suggest that both bacterial strains are exceptional candidates in the biocontrol of mycotoxigenic fungi and in the reduction of their toxic metabolites.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus niger strain ASP.

The food enzyme asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. 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. The food enzyme is intended to be used in the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.792 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 at the highest dose tested of 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. 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 3-phytase from the non-genetically modified Aspergillus niger strain PHY93-08.

The food enzyme 3-phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase EC 3.1.3.8) is produced with the non-genetically modified Aspergillus niger strain PHY93-08 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in nine food manufacturing processes. Since residual amounts of food enzyme-total organic solids (TOS) are removed in two of the food manufacturing processes, dietary exposure was calculated only for the remaining seven processes. It was estimated to be up to 0.763 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise safety concerns. 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 2560 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 3355. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. 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.

Purification and biochemical characterization of a novel thermostable endo-polygalacturonase from Aspergillus niger strain HO32 and its suitability for clarification of orange juice

A novel endo-polygalacturonase (PGC-AN64) from Aspergillus niger HO32 was produced (88.22 U/mL) on orange peel by-product (OPP), purified, biochemically characterized, and its utility in clarification of orange juice was elucidated. The enzyme was purified by ammonium sulfate fractionation (80 %) and gel filtration chromatography on Sephacryl® S-200 HR column. Its pH and temperature optima were 6.5 and 70 °C, respectively, and was stable over a pH range of 5–7 and temperature 60–80 °C. The 26-residue NH2-terminal sequence of PGC-AN64 showed high homology with those of Aspergillus pectinases. Metal ions (Na+, Ca2+, Mg2+, Mn2+, and Zn2+) positively affect PGC-AN64 activity, while Ba2+, Cd2+, and Cr3+ negatively affect its original activity. Interestingly, PGC-AN64 exhibited a considerable substrate specificity and catalytic efficiency compared to the commercial enzymes PECLYVE V (P1) and PECLYVE CP (P2). More interestingly and compared to P1 and P2, PGC-AN64 showed that the clarification process had a significant effect on the transmittance percentages with 84.68 ± 2.08, 88.05 ± 1.79, and 93.13 ± 3.58 % for PGC-AN64, P1, and P2, respectively. Furthermore, color parameters after the clarification process increase in the L* and b* values, and led to a decrease in the a* value. The obtained results suggest that PGC-AN64 revealed its potential in orange juice clarification.