Strain Of Aspergillus Oryzae Research Articles

Construction of Aspergillus oryzae strains with organelles labeled with fluorescence and observation of organelle morphology

The organelles in the multi-nucleated filamentous fungus Aspergillus oryzae present polymorphism. To observe the organelle morphology in A. oryzae and provide references for the localization prediction of unknown proteins and the disclosure of biological reaction pathways in A. oryzae, we fused different subcellular localization signals with green fluorescent protein (GFP) to obtain different subcellular localization vectors, which were then transferred into A. oryzae by Agrobacterium tumefaciens-mediated transformation. The A. oryzae reporter strains with fluorescence-labeled nuclei, mitochondria, endoplasmic reticulum, vacuole, lipid droplets, peroxisome, and Golgi apparatus were successfully constructed. Furthermore, staining with small-molecule specific dyes was carried out to validate the co-localization of fluorescence-labeled mitochondria, nuclei, and lipid droplets in the reporter strains, which further confirmed that the reporter strains were successfully constructed. The distribution and morphology of fluorescence-labeled organelles were observed at different growth stages and under different culture conditions. The constructed reporter strains provide basic tools for studying the organelle morphology, localization of unknown target proteins, and subcellular localization in A. oryzae.

Comparative Genome Analysis of Japanese Field-Isolated Aspergillus for Aflatoxin Productivity and Non-Productivity.

Aspergillus flavus produces aflatoxin, a carcinogenic fungal toxin that poses a threat to the agricultural and food industries. There is a concern that the distribution of aflatoxin-producing A. flavus is expanding in Japan due to climate change, and it is necessary to understand what types of strains inhabit. In this study, we sequenced the genomes of four Aspergillus strains isolated from agricultural fields in the Ibaraki prefecture of Japan and identified their genetic variants. Phylogenetic analysis based on single-nucleotide variants revealed that the two aflatoxin-producing strains were closely related to A. flavus NRRL3357, whereas the two non-producing strains were closely related to the RIB40 strain of Aspergillus oryzae, a fungus widely used in the Japanese fermentation industry. A detailed analysis of the variants in the aflatoxin biosynthetic gene cluster showed that the two aflatoxin-producing strains belonged to different morphotype lineages. RT-qPCR results indicated that the expression of aflatoxin biosynthetic genes was consistent with aflatoxin production in the two aflatoxin-producing strains, whereas the two non-producing strains expressed most of the aflatoxin biosynthetic genes, unlike common knowledge in A. oryzae, suggesting that the lack of aflatoxin production was attributed to genes outside of the aflatoxin biosynthetic gene cluster in these strains.

Optimisation of fermentation conditions for the production of gamma-aminobutyric acid (GABA)-rich soy sauce

This study addresses the challenge of enhancing gamma-aminobutyric acid (GABA) content in soy sauce through optimized fermentation condition. Using a multiple starter culture, consisting of Aspergillus oryzae strain NSK, Bacillus cereus strain KBC and Tetragenococcus halophilus strain KBC, the incubation conditions including the percentage of bacterial inoculum (10, 15 and 20 %), pH (3, 5 and 7) and agitation speed (100, 150 and 200 rpm) were optimized through Response Surface Methodology (RSM). Under the optimal conditions (20 % inoculum, pH 7 and stirring at 100 rpm), the multiple starter culture generated 128.69 mg/L of GABA after 7 days and produced 239.08 mg/L of GABA after 4 weeks of fermentation, which is 36 % higher than under non-optimized conditions (153.48 mg/L). Furthermore, sensory analysis revealed high consumer acceptance of the fermented soy sauce than the control (soy sauce without any treatment and additional bacteria) and commercial soy sauce. Consumers indicated that the starter culture offered an improved umami taste and reduced bitter, sour and salty flavours compared to the commercial product. Under optimal fermentation conditions determined by RSM statistical analysis, the multiple starter culture is able to produce high levels of GABA and is more likely to be accepted by consumers. The findings of this research have the potential to impact the food sector by offering a functional soy sauce with added health benefits and also being well-received by consumers.

PSIII-18 Effects of HiPhorius and Ronozyme HiPhos on performance of nursery pigs

Abstract The objective of this study was to evaluate the efficacy of the new phytase HiPhorius (encoded by a 6-phytase gene from Citrobacter braakii and expressed from Aspergillus oryzae; dsm-firmenich) compared with Ronozyme HiPhos (a 6-phytase product produced with a genetically modified strain of the filamentous fungus Aspergillus oryzae; dsm-firmenich) on performance of nursery pigs. A total of 528 weaned pigs [PIC Camborough female × PIC 337 boar; 19 d of age, initial body weight (BW) = 6.5 kg] were blocked by initial BW and gender and allotted to 48 pens which were randomly assigned to 1 of 4 dietary treatments (10 to 12 pigs/pen; 12 pens/treatment), including T1) Ronozyme HiPhos 3,000 FYT/kg, T2) HiPhorius 1,000 FYT/kg, T3) HiPhorius 1,500 FYT/kg, and T4) HiPhorius 2,000 FYT/kg. Pigs were fed a 3-phase feed-budget program, consisting of 1.4 kg/pig in Phase 1, 5.5 kg/pig in Phase 2, and Phase 3 diets fed to d 42 postweaning. Diets were formulated to be isocaloric and contained 1.41, 1.34, and 1.27% SID Lys for phase 1, 2, and 3, respectively. Phytase levels were tested at the same dose in phase 1 and 2 diets, while in phase 3, phytase levels were tested at 2,000 and 1,000 FYT/kg, respectively, for the Ronozyme HiPhos and HiPhorius treatments. During d 0-15 and d 0-22, pigs fed HiPhorius 1000 had reduced (P < 0.05) feed conversion than Ronozyme HiPhos and HiPhorius 2000. No significant treatment effects were observed on average daily gain (ADG) or average daily feed intake (ADFI) during these periods. During d 22-42, pigs fed HiPhorius 2,000 had greater ADG (P = 0.08), final BW (P = 0.09), and ADFI (P = 0.03) than HiPhorius 1,000 and Ronozyme HiPhos; however, there were no treatment effects on feed conversion. Overall (d 0 - 42), feeding HiPhorius 2,000 improved ADG compared with feeding Ronozyme HiPhos, HiPhorius 1,000, and HiPhorius 1,500 (476 vs. 458, 449, and 458 g, respectively; P = 0.07); however, similar ADFI and feed conversion were observed among treatments. Data from this study suggest that feeding HiPhorius at 2,000 FYT/kg provided greater ADG compared with feeding HiPhorius at reduced amounts or Ronozyme HiPhos. While the two sources of phytase had similar responses on nursery ADFI and feed conversion overall, feeding HiPhorius at 1,000 FYT/kg resulted in decreased feed conversion during the first 3 wk post-weaning.

Safety evaluation of the food enzyme carboxypeptidase D from the genetically modified Aspergillus oryzae strain NZYM-MK.

The food enzyme carboxypeptidase D (EC 3.4.16.6) is produced with the genetically modified Aspergillus oryzae strain NZYM-MK by Novozymes A/S. It is free from viable cells of the production organism and its DNA. The genetic modifications do not give rise to safety concerns. The food enzyme is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.908 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 2220 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 2445. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found, one with a food allergen (wheat). The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to wheat, cannot be excluded, but will not exceed that of wheat consumption. 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 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. 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 five food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for uses in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in three food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining four processes. Dietary exposure was up to 0.575 mg TOS/kg body weight (bw) per day in European populations. When combined with the NOAEL reported in the previous opinion (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 1739. Based on the data provided for the previous evaluation 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.

Safety evaluation of the food enzyme leucyl aminopeptidase from the genetically modified Aspergillus oryzae strain NZYM-BU.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the genetically modified Aspergillus oryzae strain NZYM-BU by Novozymes A/S. 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. Dietary exposure to the food enzyme TOS was estimated to be up to 1.508 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 4,928 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 3,268. 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 by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of an extension of use 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. 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 five food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in three food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining four processes. Dietary exposure was up to 0.087 mg TOS/kg body weight (bw) per day in European populations. When combined with the NOAEL reported in the previous opinion (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 11,494. Based on the data provided for the previous evaluation 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.

Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform

BackgroundCordycepin (3′-deoxyadenosine) is an important bioactive compound in medical and healthcare markets. The drawbacks of commercial cordycepin production using Cordyceps spp. include long cultivation periods and low cordycepin yields. To overcome these limitations and meet the increasing market demand, the efficient production of cordycepin by the GRAS-status Aspergillus oryzae strain using a synthetic biology approach was developed in this study.ResultsAn engineered strain of A. oryzae capable of cordycepin production was successfully constructed by overexpressing two metabolic genes (cns1 and cns2) involved in cordycepin biosynthesis under the control of constitutive promoters. Investigation of the flexibility of carbon utilization for cordycepin production by the engineered A. oryzae strain revealed that it was able to utilize C6-, C5-, and C12-sugars as carbon sources, with glucose being the best carbon source for cordycepin production. High cordycepin productivity (564.64 ± 9.59 mg/L/d) was acquired by optimizing the submerged fermentation conditions.ConclusionsThis study demonstrates a powerful production platform for bioactive cordycepin production by A. oryzae using a synthetic biology approach. An efficient and cost-effective fermentation process for cordycepin production using an engineered strain was established, offering a powerful alternative source for further upscaling.

Safety evaluation of the food enzyme leucyl aminopeptidase from non-genetically modified Aspergillus oryzae strain NZYM-EX.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non-genetically modified microorganism Aspergillus oryzae strain NZYM-EX by Novozymes A/S. The food enzyme is free from viable cells of the production organism. It is intended to be used in eight food manufacturing processes: processing of dairy products for the production of (1) flavouring preparations, (2) modified milk proteins; processing of plant- and fungal-derived products for the production of (3) protein hydrolysates, (4) soy sauce; processing of meat and fish products for the production of (5) protein hydrolysates; processing of cereals and other grains for the production of (6) baked products, (7) brewed products; (8) processing of yeast and yeast products. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.577 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 440 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 763. A search for 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 by 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.

Characteristics of Koji Using Liquid Starter for Soy Sauce Production

Soy sauce is a widely consumed seasoning derived from soybeans and wheat. This study explored the application of innovative techniques to enhance the traditional soy sauce preparation process. Fungi were isolated from a commercial koji starter, and the Aspergillus oryzae strain BJ-1 was identified. Additionally, an examination of the methods to optimize the medium composition for liquid starters revealed the impact of varying the medium composition on mycelial growth and enzyme activity. Specifically, compositions containing >10% defatted soybean meal and wheat in a 55:45 ratio resulted in elevated mycelial growth and enzymatic activity, making them promising candidates for koji production. The effect of different inoculation ratios of liquid starter on the characteristics of koji was also investigated, and a 10% inoculum was found to be preferable because of its advantageous characteristics of enzyme activities and pH for soy sauce production. This study contributes to the enhancement of the efficiency and safety of soy sauce production through innovative liquid culture techniques.

Safety and efficacy of a feed additive consisting of endo-1,4-beta-xylanase (produced by Aspergillus oryzaeDSM 33700) (RONOZYME® WX (CT/L)) for all poultry species and all Suidae (DSM nutritional products ltd).

The additive RONOZYME® WX (CT/L) contains endo-1,4-beta-xylanase produced with a genetically modified strain of the filamentous fungus Aspergillus oryzae; the additive is currently authorised for poultry for fattening, weaned piglets, pigs for fattening, lactating sows and laying hens. The applicant has requested to change the production strain, substituting strain A. oryzae DSM 26372 with A. oryzae DSM 33700, and to extend the use of the additive to all poultry species and all Suidae. RONOZYME® WX (CT/L), manufactured with the production strain A. oryzae DSM 33700, did not give rise to safety concerns with regard to the genetic modification of the production strain. No viable cells of the production strain nor its DNA were detected in an intermediate product representative of both final formulations of the additive. RONOZYME® WX (CT/L) was considered safe for all poultry species and all Suidae at the recommended inclusion levels. The use of RONOZYME® WX CT and L manufactured with the production strain A. oryzae DSM 33700 raised no concerns for consumers. RONOZYME® WX L is not an eye irritant; however, no conclusions could be drawn on the potential of RONOZYME® WX CT to be an eye irritant. Both formulations are not irritant to the skin, but due to the lack of data, the FEEDAP Panel was not able to conclude on the potential of both formulations of the additive to be skin sensitisers. Due to the proteinaceous nature of the active substance, the additive is considered a respiratory sensitiser. The additive manufactured by A. oryzae DSM 33700 raises no safety concerns for the environment. The additive has the potential to be efficacious in all poultry species and all Suidae at 100 and 200 FXU/kg complete feed, respectively.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus oryzae strain NZYM-SP.

The food enzyme asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) is produced with the genetically modified Aspergillus oryzae strain NZYM-SP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used to prevent acrylamide formation in food processing. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.101 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 Panelidentified a no observed adverse effect level of 880 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 8,713. 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 Panelconsidered that a risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Exogenous Phytase Improves Growth Performance, Nutrient Retention, Tibia Mineralization, and Breast Meat Quality in Ross-308 Broilers

To evaluate the effect of exogenous phytase (Ronozyme HiPhos-L, produced by the strain of Aspergillus oryzae), 1050 one-day-old Ross-308 broilers with an initial average body weight of 50.29 ± 0.98 g were randomly assigned to one of seven dietary treatments with 10 replicates and 15 chicks/cage. During starter (d 1–7), grower (d 8–21), and finisher (d 21–35) periods, broilers were allowed to feed: positive control (PC), negative control (NC), and NC diet supplemented with 250, 500, 1000, 1500, and 3000 U/kg of phytase. Broilers fed NC diet supplemented with graded levels of phytase (250 U/kg to 3000 U/kg) showed a linear increase (p < 0.05) in body weight gain and feed intake during the starter and the overall trial period with no adverse effect on feed conversion ratio. The nutrient utilization of broilers showed neither an increase nor a decrease with the PC and NC diet until d 35 but when the NC diet was supplemented with a graded dose of phytase, the broilers showed a linear increase (p < 0.05) in dry matter, gross energy, calcium (Ca), ash, and phosphorus (P) utilization and a tendency to increase (p < 0.10) nitrogen retention at the end of the starter phase. Unlike the starter phase, broilers fed the PC diet showed increased (p < 0.05) ash and Ca retention at the end of the grower and finisher phase compared to those fed the NC diet. In addition, the phytase group showed a linear increase (p < 0.05) in ash, Ca, and P utilization at the end of d 21 and 35. Moreover, the inclusion of graded levels of phytase in the NC diet showed a linear increase (<0.0001) in bone (ash, Ca, and P) mineralization in broilers. Also, they showed a linear increase (p < 0.05) in gizzard weight, breast muscle, color of lightness, redness, and pH and a decreased cooking loss and drip loss. Though the standard diets were able to compensate for the P deficiency and promote performance and bone mineralization, the elevated (1000 FTU/kg) levels of phytase in the NC diet showed a higher response. Thus, we suggest that incorporating the exogenous enzyme phytase into the broiler feed in excess of 1000 units (U)/kg would be more beneficial to achieve better productivity.

Antioxidant and Antimicrobial Activity of Cuscuta reflexa ROXB. and Few Members of Family Convolvulaceae

Antioxidant activity of 12 species, of which one was Cuscuta reflexa Roxb. which belongs to the family of Cuscutaceae as well as 11 of Convolvulaceae, collected from district Bhimber, Mirpur and Kotli were measured by using ABTS, DPPH and FRAP assay. The total phenol and flavonoid contents of the whole plant of all the selected species were investigated. Ipomoea eriocarpa showed a comparatively higher amount of total polyphenols (0.98 ± 0.073 mg/gdw). The maximum DPPH value was shown by Ipomoea carnea Jacq (5.6%) and the minimum value was shown by Convolvulus arvensis L. extracts (3.0 %). ABTS was the highest by Convolvulus prostrastus and the lowest by Ipomoea carnea. In FRAP assay, the highest value was 2.75 by Ipomoea hederacia and the lowest value was 0.31 by I. arachnosperma Welw. The antifungal and antibacterial activity of the methanolic extracts of the whole plant of all the selected species was carried out by using both bacterial and fungal strains. Two gram positive bacteria namely Staphylococcus aureus and Bacillus subtilis, two gram negative bacteria namely E. coli and Pseudomonas aeruginosa, two fungal strains of Aspergillus niger and Aspergillus oryzae were used to carry out antimicrobial activity. For the comparison of inhibition zones showed by plant extracts against bacterial and fungal strains. Erythromycin, tetracycline and cefoparazone were used as standard for bacterial strains. Moreover, fungivine and Terbinafine were used as standard antibiotics against fungal strains. Among fungal strains, the highest zone of inhibition was shown by Cuscuta reflexa Roxb. extract against Aspergillus niger, that is 5.55±0.3, while the lowest zone of inhibition, that is, 0.60±0.28mm was shown by Evolvulus alsinoides (L.) extract against Aspergillus oryzae. Plants from the Convolvulaceae family have had frequent traditional uses for different therapeutic purposes. Furthermore, all plants selected for this study showed strong antimicrobial, antifungal, and antioxidant activity. Their further detailed phytochemical studies can help to develop plant-based therapeutic agents

Novel cadmium (II) N2O2‐Schiff base compounds: X‐ray crystal structure, Hirshfeld surface study, thermal behavior, antimicrobial, DNA cleavage power, antioxidant, and cytotoxicity activity

A new N2O2‐tetradentate Schiff base ligand entitled as N,N′‐((ethane‐1,2‐diybis [oxy])bis (ethan‐2,1‐diyl))bis(3‐(4‐methoxyphenyl)prop‐2‐en‐1‐imine was prepared by direct reaction between 3‐(4‐methoxyphenyl)‐2‐propenal and 1,8‐diamino‐3,6‐dioxaoctane. After characterization of the ligand, some novel cadmium coordination compounds formulated as CdLX2 (X: Cl−, Br−, I−, N3−, SCN−and NO3−) were synthesized and characterized by physical and spectral techniques. Typically crystal structure of cadmium nitrate coordination compound was determined suggesting crystallization in monoclinic system (with space group of I2/a) and a coordination geometry of square anti‐prism around the Cd (II) ion. Moreover, CdLBr2 and CdLI2 compounds were prepared in nanostructured dimensions by sonochemical method confirmed by SEM, EDX, and XRD techniques. In continuation, thermal behavior investigation of the compounds including thermal stabilities, thermal decomposition steps, and thermo‐kinetic parameters was performed based on Coats‐Redfern equations from room temperature to 900 °C. Furthermore, all the compounds were subjected to bioassay against two Gram‐positive bacteria of Staphylococcus aureus and Bacillus subtilis, two Gram‐negative bacteria of Pseudomonas aeruginosa and Escherichia coli, and two fungal strains of Aspergillus oryzae and Candida albicans. The results showed that all the synthesized compounds act well as antimicrobial agents. DNA cleavage power of the materials was also evaluated using extracted DNA from E. coli bacterium. The cytotoxic activities of some compounds were investigated against the prostate cancer cell lines (PC3) and human umbilical vein endothelial cells (HUVEC) (as normal cells) by MTT assay and nitric oxide level evaluation method as compared with cis‐platin confirming anti‐cancer effects of them. Finally, the antioxidant properties of the N2O2‐ligand and its cadmium coordination compounds were examined by DPPH and FRAP methods suggesting all the compounds have good antioxidant activity as compared with standard samples.

Screening of lignin-degrading fungi and bioaugmentation on the directional humification of garden waste composting

The effective lignin-degrading strains mesophilic Sordaria tomentoalba (MSDA1) and thermophilic Thermomyces lanuginosus (HDGA2) were screened and used for the directional humification of garden waste (GW) composting. The strains MSDA1 and HDGA2 demonstrated significant Laccase (Lac), Lignin peroxidase (LiP), and Manganese peroxidase (MnP) activities, of which MnP activity was the highest at 746.31 U/L and 901.48 U/L, respectively. The composting BA-1 group was inoculated with the fungi MSDA1, HDGA2, M1 (mesophilic cellulose-degrading strain Aspergillus oryzae), and ZJ3 (thermophilic cellulose-degrading strain Thermoascus aurantiacus) on day 0, while BA-2 group was inoculated with the fungi MSDA1 and M1 on day 0 and the fungi HDGA2 and ZJ3 on day 3, respectively. The attained humic acid (HA) was 122.2 g/kg and 131.81 g/kg in BA-1 and BA-2 groups, respectively, significantly higher than the control group (72.97 g/kg). The organic matter contents of BA-2 (51.28%) were higher than that of CK (30.55%) and BA-1 (22.04%), and HA/fulvic acid (FA) were 1.26 (CK), 2.04 (BA-1) and 3.10 (BA-2), respectively. The results revealed that bioaugmentation could promote the directional humification by providing more FA precursors with the addition of lignocellulosic degradation fungi. Ammonium nitrogen (NH4+−N) could be fixed by FA during the high-temperature period, promoting NH4+−N being assimilated into amino acids and then converted to HA. Bioaugmentation effectively increased the HA and N content of the compost. The sequential addition of mesophilic and thermophilic fungi improved the quality of the directional humification compost.

Safety evaluation of the food enzyme inulinase from the genetically modified Aspergillus oryzae strain MUCL 44346.

The food enzyme inulinase (1-β-d-fructan fructanohydrolase; EC 3.2.1.7) is produced with the genetically modified Aspergillus oryzae strain MUCL 44346 by PURATOS NV. 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 the production of fructo-oligosaccharides (FOS) from inulin extracted from chicory roots. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.01 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 (NOAEL) of 100 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 10,000. 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 tomato allergens. 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 tomato, cannot be excluded. However, the likelihood of allergic reactions is expected not to exceed the likelihood of allergic reactions to tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme 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.

Solid‐state and submerged fermentation effects on functional properties of pea protein‐enriched flour

AbstractBackground and ObjectivesModification of plant proteins is often required to make them suitable substitutes for animal proteins in food applications. The effects of fermentation on the quality and functional properties of pea protein‐enriched flour (PPEF) were evaluated in this study. PPEF was fermented by Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus, Lactobacillus plantarum, and Bacillus subtilis strains using solid‐state and submerged fermentation (SSF and SmF, respectively).FindingsProtein contents significantly increased following fermentation for all samples, while lipid contents significantly decreased in SSF and increased in SmF samples. Surface properties were modified: enhanced zeta potential and decreased surface hydrophobicity were observed. At pH 7, emulsification activities and stabilities increased in A. oryzae and R. oligosporus samples, and emulsion stability increased for all fermented samples. Water and oil holding capacities increased after fermentation. SmF‐PPEF showed generally more significant changes in these properties.ConclusionsFermentation improved the functional properties of PPEF to varying degrees, depending on the mode of fermentation, pH conditions, and fermenting strains.Significance and NoveltyThis research demonstrates that fermentation can be efficient in modifying various functional properties of legume proteins, thus, helping increase their values.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus oryzae strain NZYM-OA.

The food enzyme asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified microorganism Aspergillus oryzae strain NZYM-OA by Novozymes A/S. 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 various food manufacturing processes to prevent acrylamide formation. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.051 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 1,182 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 23,176. 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, under the intended conditions of use, the risk of allergic reactions by 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.