Evaluation of in vitro digestibility of Aspergillus oryzae fungal biomass grown on organic residue derived‑VFAs as a promising ruminant feed supplement

Background As demand for high quality animal feed continues to raise,it becomes increasingly important to mini-mize the environmental impact of feed production.An appealing sustainable approach to provide feed fractions is to use organic residues from agro-food industry.In this regard,volatile fatty acids(VFAs)such as acetic,propionic and butyric acids,derived from bioconversion of organic residues can be used as precursors for production of micro-bial protein with ruminant feed inclusion potential.This study aims to investigate the in vitro digestibility of the Asper-gillus oryzae edible fungal biomass cultivated on VFAs-derived from anaerobic digestion of residues.The produced fungal protein biomass,along with hay clover silage and rapeseed meal were subjected to various in vitro assays using two-stage Tilley and Terry(TT),gas,and bag methods to evaluate and compare its digestibility for application in ruminant feed.Results The produced fungal biomass contained a higher crude protein(CP)(41%–49%)and rather similar neutral detergent fiber(NDF)(41%–56%)compared to rapeseed meal.The rumen in vitro dry matter digestibility(IVDMD)of the fungal biomass in the TT method ranged from 82%to 88%(statistically similar to that of the gas method(72%to 85%)).The IVDMD of fungal biomass were up to 26%and 40%greater than that of hay clover silage and rapeseed meal,respectively.The type of substrate and bag method had pronounced effect on the fermentation products(ammonium-N(NH4+-N),total gas and VFAs).Fungal biomass digestion resulted in the highest release of NH4+-N(340–540 mg/L)and the ratio of acetate to propionate ratio(3.5)among subjected substrates.Conclusion The results indicate that gas method can be used as a reliable predictor for IVDMD as well as fermenta-tion products.Furthermore,the high IVDMD and fermentation product observed for Aspergillus oryzae fungal biomass digestion,suggest that the supplementation of fungal biomass will contribute to improving the rumen digestion by providing necessary nitrogen and energy to the ruminant and microbiota.

Isolation and Identification of Glyphosate-degraded Strain Aspergillus oryzae sp.A-F02 and Its Degradation Characteristics

[Objective] The research aimed to isolate the glyphosate-degraded strain and study its degradation characteristics.[Method] A glyphosate-degraded fungal strain A-F02 was isolated from sludge in an aeration tank of a glyphosate manufacture.The fungal strain A-F02 was identified according to morphological characteristics and internal transcribed spacer（ITS）region of nuclear ribosomal DNA sequence analysis.The glyphosate-biodegraded characteristics of strain A-F02 and the influencing factors were studied.[Result] The fungal strain A-F02 was identified as Aspergillus oryzae sp..The glyphosate-biodegraded rate was 86.82% in the mineral salt medium with 1 000 mg/L of glyphosate as the sole source of carbon,after being incubated at 30 ℃ and 150 rpm for 7 d.The biodegradation rates and biomass of the A-F02 were the highest under the culture conditions with glucose（0.5%,w/v）,pH 7.5,30 ℃ and glyphosate（1 500 mg/L）.[Conclusion] The research provided the experimental basis for glyphosate-biodegraded enzyme purification.

Optimization of Solid-State Fermentation with Lactobacillus brevis and Aspergillus oryzae for Trypsin Inhibitor Degradation in Soybean Meal

The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.

Enzymatic Modification of Poly (ethylene terephthalate) Fiber with Lipase from Aspergillus oryzae

Lipase preparation from Aspergillus oryzae could act on ester bonds on the surface of poly （ethylene terephthalate） fibers and a possible hydrolytic product mono （2-hydroxyethyl） terephthalate was released. After the iipase modification, there were more carboxyi groups on the treated poly （ethylene terephthalate） fabric surface that resulted in binding with more cationic dyes. Increased hydrophilicity and antistatic ability of poly （ethylene terephthalate） samples were found based on moisture regain, water contact angle and static half decay time.

Immobilization of Aspergillus Oryzae Mycelium Pellets and Its Application in the Resolution of D, L-Alanine

Aspergillus oryzae 3042 mycelium pellets were immobilized by crosslinking method with reagents of gelatin and formaldehyde. An orthogonal design table was used to determine the optimal immobilization conditions. The L-aminoacylase activity of immobilized mycelium pellets under optimal conditions was assayed. The results show that the L-aminoacylase activity was 1350 U·g^-1 and the activity retention rate was 83%. The properties of the immobilized preparation were studied. Compared with free pellets, the appropriate pH of reaction system changed from 7.0 to 8.0, temperature changed from 52℃ to 63℃, and the ranges of the optimum reaction conditions were all improved. The effect of Co^2＋ on immobilized mycelium pellets was investigated and the favorable concentration was determined. When the immobilized preparation was used for the resolution of D, L-alanine in a packed bed reactor with 10 mm in diameter, 200 mm in height, the operational stability was increased, and the half-life period was 53 d. Thermal stability analysis shows that the immobilized pellets were more stable than the free pellets.

Purification,characterization and antiproliferative activity of L-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity

Objective:To explore the anti-proliferative activity of purified L-asparaginase from Aspergillus oryzae CCT 3940(A.oryzae).Methods:L-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system.The purified enzyme was characterized and used for the antiproliferative assay against nine tumor cell lines and one non-tumor cell line.Results:The free glutaminase L-asparaginase was purified 28.6 fold.L-asparaginase showed high stability under physiological condition,remaining stable in the p H range7.0–8.0 after 1 h incubation at temperature range 30–45℃.The Km and Vmax values of purified L-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL,respectively.The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied.Also,the enzyme from A.oryzae CCT 3940 could inhibit tumor growth of leukemia cell line(K562) with a total growth inhibition value of(3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied.Conclusions:The sensitivity of the cells lines to purified L-asparaginase from A.oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial L-asparaginase from Escherichia coli.The L-asparaginase from A.oryzae CCT 3940 has a high potential for pharmaceutical exploitation in the treatment of leukemia.

IMMOBILIZATION OF AMINOACYLASE FROM ASPERGILLUS ORYZAE ON CHLOROMETHYLATED CROSS-LINKED POLYSTYRENES

A number of chlorumethylated polystyrenes were synthesized and tried to immobilize aminoacylase from Aspergillus oryzae and many factors which affected immobilized enzyme activity were studied in detail. The results indicated that the immobilized enzyme on support(IAR-1) possessed high enzymatic activity and high stability.

Water Retention Value： A Study Model-based by Asperglillus awamori and Aspergillus oryzae Embrace Three Models of Solid Substrate

The goal of this study was to evaluate the water retention value （WRV） of a test solid substrate and a fungal cell in solid state fermentation （SSF）. WRV is the ratio of the weight of water retained after centrifugation under specific conditions by a wet sample to the oven dry weight of the same sample. SSF refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment. Many factors are involved in a successful SSF process. In addition to biological parameters, the SSF process is also dependent on physical factors such as WRV. A centrifugal technique has been modified and applied to the evaluation of WRV. Wheat bran, soybean hulls and rapeseed meal were used as model substrate. Aspergillus awamori and Aspergillus oryzae were used as model microorganism. Results revealed that the ability of wheat bran to retain water in the solid substrate is 56% higher than that of soybean hulls and rapeseed meal. In the term of fungal cell, the ability of A. oryzae to retain water in the cells was higher （73% higher） than that ofA. awamori. In addition, through oven method moisture content loss from A. awamori is 46% higher than that from A. oryzae during drying process. Nevertheless, it can be seen that A. oryzae is able to retain water content about 5 times higher than A. awamori. Through this results, we found that WRV varies depending on solid substrates and microorganisms. This initial information can be beneficial in the SSF process to be carried out.

Aspergillus oryzae reduces IgE binding ability of allergenic egg white proteins

Egg white proteins are one of the major allergens. The objective of this study was to investigate the effect of Aspergillus oryzae cultivation on IgE binding ability of egg white proteins. Effect of A. oryzae on egg white proteins was determined using ninhydrin method,SDS-PAGE, ELISA, fluorescence FITC labeling, MALDITOF-MS and LC-MS/MS analysis. Adding mycelium of A. oryzae ATCC 1011 and 16868 substantially reduced the IgE binding ability of acidified egg white after 24 h incubation. The binding capacity of egg white proteins to IgE in plasma from four egg allergy patients was almost completely lost after incubation with mycelium of ATCC16868. Results from SDS-PAGE, free amino acid analysis,MALDI-TOF-MS and LC-MS/MS indicated that there was no substantial protein degradation during incubation.Therefore, the reduction of IgE binding ability of egg white proteins during A. oryzae treatment was probably due to a loss of ～1700 Da mass including a fragment of the ovomucoid N terminus.

Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae

The chemical diversity of terpenoids is typically established by terpene synthase-catalyzed cyclization and diversified by post-tailoring modifications.Fungal bifunctional terpene synthase(BFTS)associated P450 enzymes have shown significant catalytic potentials through the development of various new terpenoids with different biological activities.This study discovered the BFTS and its related gene cluster from the plant endophytic fungus Didymosphaeria variabile 17020.Heterologous expression of the BFTS in Saccharomyces cerevisiae resulted in the characterization of a major product diterpene variediene(1),along with two new minor products neovariediene and neoflexibilene.Further heterologous expression of the BFTS and one cytochrome P450 enzyme VndE(CYP6138B1)in Aspergillus oryzae NSAR1 led to the identification of seven norditerpenoids(19 carbons)with a structurally unique 5/5 bicyclic ring system.Interestingly,in vivo experiments suggested that the cyclized terpene variediene(1)was modified by VndE along with the endogenous enzymes from the host cell A.oryzae through serial chemical conversions,followed by multi-site hydroxylation via A.oryzae endogenous enzymes.Our work revealed that the two-enzymes biosynthetic system and host cell machinery could produce structurally unique terpenoids.

Conversion of fish processing wastewater into fish feed ingredients through submerged cultivation of Aspergillus oryzae

Fish processing towards production of fillet gives rise to wastewater streams that are ultimately directed to biogas production and/or wastewater treatment.However,these wastewater streams are rich in minerals,fat,and proteins that can be converted to protein-rich feed ingredients through submerged cultivation of edible filamentous fungi.In this study,the origin of wastewater stream,initial pH,cultivation time,and extent of washing during sieving,were found to influence the amount of recovered material from the wastewater streams and its protein content,following cultivation with Aspergillus oryzae.Through culti-vation of the filamentous fungus in sludge,330 kg of material per ton of COD were recovered by sieving,corresponding to 121 kg protein per ton of COD,while through its cultivation in salt brine,210 kg of material were recovered per ton of COD,corresponding to 128 kg protein per ton of COD.Removal ranges of 12-43%,39-92%,and 32-66%for COD,total solids,and nitrogen,respectively,were obtained after A.oryzae growth and harvesting in the wastewater streams.Therefore,the present study shows the versatility that the integration of fungal cultivation provides to fish processing industries,and should be complemented by economic,environmental,and feeding studies,in order to reveal the most promising valorization strategy.

Two-step Enzymatic Synthesis of Guanine Arabinoside

The chemical synthesis of Guanine arabinoside （ara-G） is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside （ara-DA） was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L^-1 uracil arabinoside as arabinose donor, 10 mmol·L^- 1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L^-1 phosphate buffer, and reacted at 60℃ for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.

Improvement of Feeding Value of Rapeseed Meal by Mixed Solid State Fermentation

[Objective] To optimize solid state fermentation conditions of rapeseed meal and thus to reduce glucosinolates and neutral detergent fibers by mixed cultures of Aspergillus oryzae and Tnchoderrna viride. [ Method ] The optimal fermentation conditions were determined by single factor test and orthogonal design. [ Result J The optimum fermentation conditions are as following: inoculum weight ratio (Aspergillus oryzae vs Trichoderma vinde), 1:1 ; inoculum size, 30% ; water content, 40% ; fermentation time, 96 h; and fermentation temperature, 30℃. Under these conditions, glucosinolates were reduced by 90.71% and neutral detergent fibers were degraded by 20.65%. [ Condusion] In laboratory, solid state fermentation with Aspergillus oryzae and Trichoderrna viride can be used to produce high-quality rapeseed meal.

Multi-enzymes Production Studies in Single Tray Solid State Fermentation with Opened and Closed System

The robustness ofA. awamori and A. oryzae as enzyme producers is exploited in fungal fermentation on agricultural solid waste. High-level production of extracellular glucoamylase, protease, cellulase and xylanase has been achieved. Three different types of ＇waste＇ solids （wheat bran, soybean hulls and rapeseed meal） have been used in studies of solid state fermentation （SSF）. The enzymes could be produced in significant levels by continuously supplying oxygen （02） through the tray system known as ＂closed＂ and ＂opened＂ tray systems. A perforated tray system was developed in this study that permits direct access to 02. Testing the tray system with different perforated mesh aperture sizes in this study did not yield different results in growth performance of A. awamori and A. oryzae. A. awamori and A. oryzae can be very versatile in producing various enzymes with different substrates with different starch, protein, hemiceilulose and cellulose contents. These studies indicate that A. awamori is more suitable for the efficient production of multiple enzymes in the closed system including xylanase and cellulase, while the production of glucoamylase and protease is superior in the opened system. A. oryzae is more suitable for the efficient production of protease and cellulase in the closed system, while the production of protease is more favourable the opened system. A. awamori efficiently consumed starch in wheat bran medium and produced very high glucoamylase activity, and after that, the fungus efficiently produced other enzymes to degrade other complex nutrients such as protein, hemicellulose and cellulose. Meanwhile, A. oryzae efficiently consumed protein in rapeseed meal and produced very high protease activity. The ability of both filamentous fungi, to convert biomass through SSF bioconversion will have a great impact on food and agro-industry in every aspect of life from food and medicine to fuel.