Upgrading the Bioactive Potential of Hazelnut Oil Cake by Aspergillus oryzae under Solid-State Fermentation.

Hazelnut oil cake (HOC) has the potential to be bioactive component source. Therefore, HOC was processed with a solid-state fermentation (SSF) by Aspergillus oryzae with two steps optimization: Plackett-Burman and Box-Behnken design. The variables were the initial moisture content (X1: 30-50%), incubation temperature (X2: 26-37 °C), and time (X3: 3-5 days), and the response was total peptide content (TPC). The fermented HOC (FHOC) was darker with higher protein, oil, and ash but lower carbohydrate content than HOC. The FHOC had 6.1% more essential amino acid and benzaldehyde comprised 48.8% of determined volatile compounds. Fermentation provided 14 times higher TPC (462.37 mg tryptone/g) and higher phenolic content as 3.5, 48, and 7 times in aqueous, methanolic, and 80% aqueous methanolic extract in FHOC, respectively. FHOC showed higher antioxidant as ABTS+ (75.61 µmol Trolox/g), DPPH (14.09 µmol Trolox/g), and OH (265 mg ascorbic acid/g) radical scavenging, and α-glucosidase inhibition, whereas HOC had more angiotensin converting enzyme inhibition. HOC showed better water absorption while FHOC had better oil absorption activity. Both cakes had similar foaming and emulsifying activity; however, FHOC produced more stable foams and emulsions. SSF at lab-scale yielded more bioactive component with better functionality in FHOC.

Solid-state cultivation of multiple industrial strains of koji mold on different Thai unpolished rice cultivars: biotransformation of phenolic compounds and their effects on antioxidant activity.

Colored rice is abundant in polyphenols, and koji molds have potential for biotransformation. This study aimed to produce Thai-colored rice koji to study its polyphenolic biotransformation. Four industrial koji mold strains: Aspergillus oryzae 6001, A. oryzae 6020, A. sojae 7009, and A. luchuensis 8035, were cultivated on unpolished Thai-colored rice (Riceberry and Sangyod), unpolished Thai white rice (RD43), and polished Japanese white rice (Koshihikari). We discovered that koji molds grew on all the rice varieties. Methanol extracts of all rice kojis exhibited an approximately 2-fold or greater increase in total phenolic content and DPPH antioxidant activity compared to those of steamed rice. Moreover, quercetin, quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, ferulic acid, caffeic acid, protocatechuic acid, vanillic acid, (+)-catechin, and (-)-epicatechin content increased in Riceberry and Sangyod koji samples. Consequently, Aspergillus solid-state cultivation on unpolished Thai-colored rice exhibited higher functionalization than the cultivation of unpolished Thai white rice and polished Japanese white rice.

Changing the polyphenol composition and enhancing the enzyme activity of sorghum grain by solid-state fermentation with different microbial strains.

BACKGROUND: Solid-state fermentation (SSF) has been widely used in the processing of sorghum grain (SG) because it can produce products with improved sensory characteristics. To clarify the influence of different microbial strains on the SSF of SG, especially on the polyphenols content and composition, Lactiplantibacillus plantarum, Saccharomyces cerevisiae, Rhizopus oryzae, Aspergillus oryzae, and Neurospora sitophila were used separately and together for SSF of SG. Furthermore, the relationship between the dynamic changes in polyphenols and enzyme activity closely related to the metabolism of polyphenols has also been measured and analyzed. Microstructural changes observed after SSF provide a visual representation of the SSF on the SG. RESULTS: After SSF, tannin content (TC) and free phenolic content (FPC) were decreased by 56.36% and 23.48%, respectively. Polyphenol oxidase, ß-glucosidase and cellulase activities were increased 5.25, 3.27, and 45.57 times, respectively. TC and FPC were negatively correlated with cellulase activity. A positive correlation between FPC and xylanase activity after 30 h SSF became negative after 48 h SSF. The SG surface was fragmented and porous, reducing the blocking effect of cortex. CONCLUSION: Cellulase played a crucial role in promoting the degradation of tannin (antinutrient) and phenolic compounds. Xylanase continued to release flavonoids while microbial metabolism consumed them with the extension of SSF time. SSF is an effective way to improve the bioactivity and processing characteristics of SG. © 2024 Society of Chemical Industry.

Monaspin B, a Novel Cyclohexyl-furan from Cocultivation of Monascus purpureus and Aspergillus oryzae, Exhibits Potent Antileukemic Activity.

Natural products are a rich resource for the discovery of innovative drugs. Microbial cocultivation enables discovery of novel natural products through tandem enzymatic catalysis between different fungi. In this study, Monascus purpureus, as a food fermentation strain capable of producing abundant natural products, was chosen as an example of a cocultivation pair strain. Cocultivation screening revealed that M. purpureus and Aspergillus oryzae led to the production of two novel cyclohexyl-furans, Monaspins A and B. Optimization of the cocultivation mode and media enhanced the production of Monaspins A and B to 1.2 and 0.8 mg/L, respectively. Monaspins A and B were structurally elucidated by HR-ESI-MS and NMR. Furthermore, Monaspin B displayed potent antiproliferative activity against the leukemic HL-60 cell line by inducing apoptosis, with a half-maximal inhibitory concentration (IC50) of 160 nM. Moreover, in a mouse leukemia model, Monaspin B exhibited a promising in vivo antileukemic effect by reducing white blood cell, lymphocyte, and neutrophil counts. Collectively, these results indicate that Monaspin B is a promising candidate agent for leukemia therapy.

Chicken viscera meal as substrate for the simultaneous production of antioxidant compounds and proteases by Aspergillus oryzae.

The use of chicken waste can contribute to the development of new processes and obtaining molecules with high added value. An experimental design was applied to evaluate the effect of moisture, temperature, and inoculum size on the production of antioxidant peptides and proteases by A. oryzae IOC3999 through solid-state fermentation (SSF) of chicken viscera meal. As a result, the process conditions strongly influenced protease production and antioxidant activity of the fermented products. A global analysis of the results indicated that the most adequate conditions for SSF were (assay 9): 40% initial moisture, 30 °C as the incubation temperature, 5.05 × 106 spores/g as the inoculum size, and 48-h fermentation as the fermentation time. Under this condition, the antioxidant activities for the ABTS- and DPPH-radicals inhibition and ferric reducing antioxidant power (FRAP) methods were 376.16, 153.29, and 300.47 (µmol TE/g), respectively, and the protease production reached 428.22 U/g. Ultrafiltration of the crude extract obtained under optimized fermentation conditions was performed, and the fraction containing peptides with molecular mass lower than 3 kDa showed the highest antioxidant activity. The proteases were biochemically characterized and showed maximal activity at pH values ranging from 5.0 to 6.0 and a temperature of 50 °C. The thermodynamic parameters indicated that the process of thermal protease inactivation is not spontaneous (ΔG*d > 88.78 kJ/mol), increasing with temperature (ΔH*d 27.01-26.88 kJ/mol), and with reduced disorder in the system (ΔS*d < - 197.74 kJ/mol) probably caused by agglomeration of partially denatured enzymes.

Eliciting transcriptomic and antioxidant defensive responses against Rhizoctonia root rot of sorghum using the endophyte Aspergillus oryzae YRA3.

Environmental pollution due to the improper use of the chemical fungicides represents a vital ecological problem, which affects human and animal health, as well as the microbial biodiversity and abundance in the soil. In this study, an endophytic fungus Aspergillus oryzae YRA3, isolated from the wild plant Atractylis carduus (Forssk.) C.Chr, was tested for its biocontrol activity against Rhizoctonia root rot of sorghum. The antagonistic potential of A. oryzae YRA3 was tested against Rhizoctonia solani in vitro. A full inhibition in the growth of R. solani was recorded indicating a strong antagonistic potential for this endophyte. To investigate the chemical composition of its metabolites, GC/MS analysis was used and thirty-two compounds in its culture filtrate were identified. Among these metabolites, some compounds with an antifungal background were detected including palmitic acid, 2-heptanone, and 2,3-butanediol. To these antifungal metabolites the antagonistic activity of A. oryzae YRA3 can be attributed. In the greenhouse experiment, treating of the infected sorghum plants with A. oryzae YRA3 significantly reduced severity of the Rhizoctonia root rot by 73.4%. An upregulation of the defensive genes (JERF3), (POD) and (CHI II) was recorded in sorghum roots when were inoculated with A. oryzae YRA3. In addition, an increment in the activity of peroxidase and polyphenol oxidase, as well as the total phenolic content in the sorghum roots was also recorded. Furthermore, the results obtained from the greenhouse experiment revealed a growth-promoting effect for inoculating the sorghum plants with A. oryzae YRA3. It can be concluded that A. oryzae YRA3 can be a probable biological agent to control this disease in sorghum. However, its evaluation under field conditions is highly needed in the future studies.

In vitro gas production kinetics are influenced by grain processing, flake density, starch retrogradation, and Aspergillus oryzae fermentation extract containing α-amylase activity.

Grain processing such as particle size, flake density, or starch retrogradation can influence ruminal degradability characteristics; however, it is unclear how exogenous α-amylase supplementation interacts with different processed grains. Four experiments were conducted to compare the effects of Aspergillus oryzae fermentation extract (Amaize; Alltech Biotechnology Inc., Nicholasville, KY) supplementation on in vitro gas production kinetics of grain substrates with different processing methods that are common in the feedlot industry. In experiment 1, corn processing (dry-rolled, high-moisture, steam-flaked) and Amaize supplementation (0 or 15 U α-amylase activity/100 mL) were evaluated in a 3 × 2 factorial arrangement of treatments. The rate of gas production for dry-rolled corn was higher (P < 0.001) with Amaize supplementation. In experiment 2, flake density (296, 322, 348, 373, and 399 g/L) and starch retrogradation (storage in heat-sealed foil bags for 3 d at 23 or 55°C) were evaluated in a 5 × 2 factorial arrangement of treatments. There was a flake density × starch retrogradation interaction (P < 0.01) for the rate of gas production because the decrease in the rate of gas production with starch retrogradation was greater at lighter flake densities compared with heavier flake densities. In experiment 3, Amaize supplementation was evaluated across flake densities of nonretrograded steam-flaked corn (stored at 23°C) used in experiment 2. There was a flake density × Amaize interaction (P < 0.01) for the rate of gas production where Amaize supplementation resulted in a lower rate of gas production at lighter flake densities (296, 322, and 348 g/L) but a higher rate of gas production at heavier flake densities (373 and 399 g/L). In experiment 4, Amaize supplementation was evaluated across flake densities of retrograded steam-flaked corn (stored at 55°C) used in experiment 2. Gas production was lower after 24 h with Amaize supplementation for retrograded flakes produced to a density of 322 and 399 g/L while Amaize supplementation did not influence gas production at 24 h at other flake densities. There was a flake density × Amaize interaction for the rate of gas production because Amaize supplementation resulted in a faster (P < 0.01) rate of gas production for all flake densities except retrograded flakes produced to a density of 296 g/L. Enzymatic starch availability was positively correlated with the rate of gas production. These data demonstrate that supplementation of 15 U/100 mL of Amaize resulted in greater rates of gas production for dry-rolled corn, corn steam-flaked to heavier densities, and retrograded steam-flaked corn.

Grain processing has been used for decades to improve digestibility of finishing cattle diets, leading to improved growth and feed efficiency. Grain processing methods that result in changes in particle size, flake density, or starch retrogradation have all been shown to affect the degradability characteristics of nutrients in the rumen. Supplementation of feed additives containing exogenous enzyme activity could have the potential to improve digestibility, growth performance, and feed efficiency of livestock. However, it is unknown how supplementation of exogenous α-amylase activity influences degradability characteristics of different processed grains. The objectives of this study were to compare the effects of Aspergillus oryzae fermentation extract supplementation on in vitro gas production kinetics of grain substrates with different processing methods that are common in the feedlot industry. Enzymatic starch availability of steam-flaked corn, but not dry-rolled or high-moisture corn, was reflective of the rate of in vitro gas production. Increasing flake density and increasing starch retrogradation decreased the rate of in vitro gas production. Supplementation of A. oryzae fermentation extract resulted in increased rates of gas production for dry-rolled corn, corn steam-flaked at heavier densities, and retrograded steam-flaked corn.

Potential of Aspergillus oryzae as a biosynthetic platform for indigoidine, a non-ribosomal peptide pigment with antioxidant activity.

The growing demand for natural pigments in the industrial sector is a significant driving force in the development of production processes. The production of natural blue pigments, which have wide industrial applications, using microbial systems has been gaining significant attention. In this study, we used Aspergillus oryzae as a platform cell factory to produce the blue pigment indigoidine (InK), by genetic manipulation of its non-ribosomal peptide synthetase system to overexpress the indigoidine synthetase gene (AoinK). Phenotypic analysis showed that InK production from the engineered strain was growth associated, owing to the constitutive control of gene expression. Furthermore, the initial pH, temperature, and glutamine and MgSO4 concentrations were key factors affecting InK production by the engineered strain. The pigment secretion was enhanced by addition of 1% Tween 80 solution to the culture medium. The maximum titer of total InK was 1409.22 ± 95.33 mg/L, and the maximum productivity was 265.09 ± 14.74 mg/L·d. Moreover, the recombinant InK produced by the engineered strain exhibited antioxidant activity. These results indicate that A. oryzae has the potential to be used as a fungal platform for overproduction of extracellular non-ribosomal peptide pigments.

Antiproliferative Illudalane Sesquiterpenes from the Marine Sediment Ascomycete Aspergillus oryzae.

The new asperorlactone (1), along with the known illudalane sesquiterpene echinolactone D (2), two known pyrones, 4-(hydroxymethyl)-5-hydroxy-2H-pyran-2-one (3) and its acetate 4, and 4-hydroxybenzaldehyde (5), were isolated from a culture of Aspergillus oryzae, collected from Red Sea marine sediments. The structure of asperorlactone (1) was elucidated by HR-ESIMS, 1D, and 2D NMR, and a comparison between experimental and DFT calculated electronic circular dichroism (ECD) spectra. This is the first report of illudalane sesquiterpenoids from Aspergillus fungi and, more in general, from ascomycetes. Asperorlactone (1) exhibited antiproliferative activity against human lung, liver, and breast carcinoma cell lines, with IC50 values < 100 µM. All the isolated compounds were also evaluated for their toxicity using the zebrafish embryo model.

Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Analysis of phenanthrene degradation by Ascomycota fungi isolated from contaminated soil from Reynosa, Mexico.

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds generated mainly by anthropogenic sources. They are considered toxic to mammals, since they have carcinogenic, mutagenic and genotoxic properties, among others. Although mycoremediation is an efficient, economical and eco-friendly technique for degrading PAHs, the fungal degradation potential of the phylum Ascomycota has not been widely studied. In this work, we evaluated different fungal strains from the polluted soil of 'La Escondida' lagoon in Reynosa, Mexico to know their potential to degrade phenanthrene (PHE). Forty-three soil isolates with the capacity to grow in the presence of PHE (0·1% w/v) were obtained. The fungi Aspergillus oryzae MF13 and Aspergillus flavipes QCS12 had the best potential to degrade PHE. Both fungi germinated and grew at PHE concentrations of up to 5000 mg l-1 and degraded 235 mg l-1 of PHE in 28 days, with and without an additional carbon source. These characteristics indicate that A. oryzae MF13 and A. flavipes QCS12 could be promising organisms for the remediation of sites contaminated with PAHs and detoxification of recalcitrant xenobiotics.

Soybean (Glycine max) hull valorization through the extraction of polyphenols by green alternative methods.

Soybean is one of the greatest crops in the world, with about 348.7 million tons being produced in 2018. Soybean hull is a by-product produced during the processing of soybean to obtain flour and oil. Though not being actually exploited, it is a source of polyphenols with antioxidant activity. Here, the extraction of polyphenols from soybean hull was performed by means of an alkaline hydrolysis treatment, which was optimized by the response surface methodology. At the optimal region, a total phenolic content of 0.72 g gallic acid equivalents per 100 g of soybean hull was obtained with an antioxidant activity of 2.17 mmoles of Trolox equivalents. Polyphenols responsible for the antioxidant activities were identified by LC-MS, including phenolic acids, anthocyanins, stilbenes, and the two main isoflavones of soybean, daidzein and genistein, in their non-glycosylated form. Other alternative extraction methods based on Aspergillus oryzae fermentation and α-amylase hydrolysis are also proposed.

Proximate composition, polyphenols, and antioxidant activity of solid state fermented peanut press cake.

The current research was led to assess the influence of solid-state fermentation (SSF) with Aspergillus oryzae (MTCC 3107) on polyphenols, antioxidant activities, and proximate composition from peanut press cake of variety HNG-10. Total phenolic, flavonoid, and tannin contents were calculated for polyphenols quantification whereas DPPH, ABTS, FRAP, and metal chelating assay were performed for antioxidant activity. Quantification of polyphenols was confirmed by High Performance Liquid Chromatography technique. Maximum value of total phenolic, flavonoid, and tannin content was found to be 25.55 µM/g GAE, 101.17 µM/g QE, and 245.33 µg/g TAE, respectively. The highest inhibition of free radicals scavenging was noticed on the 5th day of fermentation after that decreased gradually with the increase of fermentation time. Significant increase in fat, i.e. 7.05-12.80% and protein content i.e. 44.05-49.60% was observed. Significant difference in proximate composition of fermented and non-fermented press cake concluded that the progressive role of fermentation improved or transformed physico-chemical properties of substrates.

Fungal Hydrophobin RolA Enhanced PETase Hydrolysis of Polyethylene Terephthalate.

Polyethylene terephthalate (PET) becomes one of the most well-known polyesters and is widely used as packaging material. Recently, polyethylene terephthalate hydrolase (PETase) has emerged as a potential biocatalyst demonstrating the ability to degrade polyethylene terephthalate (PET). We showed that the rate of PETase hydrolysis could be significantly increased in the presence of hydrophobin RolA. Hydrophobins represent a class of small fungal protein that has a high surface-active substance and can spontaneously self-assemble at hydrophilic-hydrophobic interfaces. In this work, a class I hydrophobin named RolA was extracted from the mycelium pellet collected from a fermentation culture of Aspergillus oryzae. The SDS-PAGE analysis of the isolated RolA showed the presence of 11 kDa polypeptide. Recombinant PETase from Ideonella sakaiensis was also successfully expressed in Escherichia coli as a soluble protein with molecular weight approximately 30 kDa. The hydrophobin RolA could enhance the PET hydrolysis in the presence of the recombinant PETase. The hydrolysis of PET bottle by RolA-PETase achieved the highest weight loss of 26% in 4 days. It is speculated that the wetting effect of RolA acts on PET surface converts PET to become hydrophilic that leads PETase easier to contact and attack the surface. Graphical Abstract.

Production of GABA-enriched idli with ACE inhibitory and antioxidant properties using Aspergillus oryzae: the antihypertensive effects in spontaneously hypertensive rats.

The present study aimed to develop a fermented food (idli) with enhanced γ-aminobutyric acid (GABA) and angiotensin I-converting enzyme (ACE) inhibitory properties using a GABA-producing fungus. Aspergillus oryzae NSK fermented idli batter and GABA was maximized (451.7 mg kg-1) in 120 h. The ACE inhibitory, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging and nitric oxide radical scavenging activities increased to 41.8%, 1.9 and 0.6 µmol trolox equivalent antioxidant capacity (TEAC) per gram in 120 h, respectively. In contrast, phytic acid and trypsin inhibitor activities decreased to 3.01 g kg-1 and 30.8 mg kg-1, respectively. The systolic blood pressure of spontaneously hypertensive rats in the fermented idli diet group was lower than those fed a plain idli diet. Lipid peroxidation in the plain idli diet group was significantly higher, whereas superoxide dismutase and glutathione reductase activities were significantly lower. The expression of genes ET-1, HSP70, NF-κB and iNOS in the aorta of SHRs that received GABA-containing diets was down-regulated between 2.2 and 3.8 fold. The production of GABA-enriched foods can be a promising approach to lower the blood pressure of spontaneously hypertensive rats.

Amazake made from sake cake and rice koji suppresses sebum content in differentiated hamster sebocytes and improves skin properties in humans.

Amazake is a traditional Japanese health drink. Here, we examined the effects of amazake on skin in cells and humans. Treatment with sake cake or rice koji suppressed intracellular lipid accumulation in differentiated hamster sebocytes, likely through the reduced expression of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA. In double-blind, placebo-controlled trial, seventeen Japanese women ingested either amazake or placebo for 4 weeks. Ingestion of the amazake decreased the sebum content compared to the placebo. The questionnaires showed improvements in "face color," "dark circles under the eyes," "glossy hair," and "waking up well", only in the amazake. In accordance with the questionnaires, additional analysis revealed the change in the L* values under the eyes was statistically increased in the amazake compared to the placebo. These results indicate that amazake may decrease sebum content in cells and humans and increase the L* values under the eyes, with some additional beneficial effects in humans.

Solar activation of fungus coated in photothermal cloth.

Bioorthogonal reactions based on manipulating the physicochemical and biological behavior of natural cells have gained tremendous attention for meeting the demands for multifunctional microorganisms without decreasing cell viability. Described herein is a novel bioorthogonal method for microorganism (Aspergillus oryzae) modification which coats the microorganism with a photothermal conversion cloth for staying bioactive in cold environments. Two steps, including ferric ions primarily binding to the microorganism cell surface, followed by in situ polymerization of pyrrole, are adopted to actualize highly efficient polypyrrole modification on the microorganism surfaces. The production of α-amylase by Aspergillus oryzae and α-amylase catalytic ability are two representative indexes of cold adaptation as confirmed by a starch decomposition test. This strategy for coating microorganisms with photothermal cloth is biocompatible and cost-effective, and can achieve non-contact modulation, which also offers great promise for generating living cell-polymer hybrid structures based on other microorganism systems for low-temperature environmental adaptation.

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis.

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.

Pyridoxal-5'-phosphate-dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi.

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli.

This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, ß-cyclodextrin neutral red desiccated coconut agar (ß-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and ß-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77-71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R2 = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health.