Solid-state Bioprocessing Research Articles

Peptidase with Keratinolytic Activity Secreted by Aspergillus terreus During Solid-State Fermentation

The aim of this study was to evaluate peptidase production by Aspergillus terreus in solid-state bioprocess and evaluate its parameters. The best conditions were 5.0 g of wheat bran as substrate, incubation temperature 30°C, inoculum 2.0x105spores/g and 75% saline volume, with production reaching 677 U/mL (5400 U/g culture medium) after 72 h of fermentation. Biochemical characterization of the crude enzymatic extract showed the optimum pH and temperature of 6.5 and 55°C, respectively. The stability at different temperatures and pH values showed that the extract could endure different pH. The evaluation of the ions influence and inhibitors proved that the enzyme required an ion for better activity, which was corroborated with the inhibition of EDTA and PMSF, characterizing serine and/or metallo peptidase. The extract was also tested for specific activities and showed promising results for keratinolytic and collagenolytic activities (0.252 and 0.165 OD/mL, respectively).

Effects of fermentation on antioxidant properties and phytochemical composition of soy germ

Traditional soy-fermented foods, such as miso, douche, natto, and tempeh have been widely used as a dietary supplement in Asian countries, and numerous reports on their phenolics and antioxidant activities have been published. Soy germ contains 10-fold higher phenolics than whole soybean, hence using soy germ as fermentation substrate will be more efficient than whole soybean. Soy germ fermented with Aspergillus niger M46 resulted in a high-efficiency bio-transformation of phenolics and flavonoids to their metabolites, and a diverse secondary metabolic product was also found to response oxidation stress of fungal colonisation. Its antioxidant activity against hydroxyl radicals and superoxide radicals (IC50 = 0.8 and 6.15 µg mL(-1) , respectively) was about 205-fold and 47-fold higher than those of unfermented soy germ (IC50 = 164.0 and 290.48 µg mL(-1) ), respectively. These results were similar to those observed for Trolox, and more active than those of BHT and hesperidin. The β-glucosidase and α-amylase produced during fermentation were mainly responsible for mobilisation of the phenolics. Our results demonstrate that fermented soy germ has the potential to be a good dietary supplement for prevention of oxidative stress-related diseases, and the solid-state bioprocessing strategy could be an innovative approach to enhance the antioxidant activity of soy germ.

Multivariate Analysis of the Stability of Spray-Dried Eupenicillium javanicum Peptidases

Peptidases occupy a central position in the enzyme market because of their importance in many areas, such as for physiological processes, foods, and detergents, as well as in the pharmaceutical, leather, and biotechnology industries. Microbial production is among the major sources of peptidases because it presents many advantages when compared with other methods. In this study, the metallopeptidases produced by the fungus Eupenicillium javanicum under a solid-state fermentation bioprocess were spray-dried. The enzymatic extract was dried using drying adjuvants, and optimal conditions for preserving enzymatic activity were studied following a Box-Behnken experimental design. The spray process factors studied were the air-drying temperature, enzyme feed flow rate, and the proportion of enzyme/additive. The responses analyzed were the dry extract yield and enzymatic activity after spray drying. Additionally, the stability of the dry extracts was assessed during 180 days at 4°C and 25°C. The results revealed extract yields of up to 66.12% and good enzymatic activity for intermediate values of temperature and adjuvant proportions. Furthermore, the dried enzymatic extracts showed potential for future commercial applications because of their stability at 25°C for 180 days.

Bioprocessing of wheat and paddy straw for their nutritional up-gradation

Solid-state bioprocessing of agricultural residues seems to be an emerging and effective method for the production of high quality animal feed. Seven strains of white-rot fungi were selected to degrade wheat and paddy straw (PS) under solid-state conditions. Degradation of different components, i.e., hemicellulose, cellulose and lignin was evaluated along with nutritional parameters including; in vitro digestibility, crude protein, amino acids, total phenolic contents (TPC) etc. Effect of nitrogen-rich supplements on degradation of lignocellulosics was evaluated using two best selected fungal strains (Phlebia brevispora and Phlebia floridensis). The best selected conditions were used to upscale the process up to 200 g batches of wheat and PS. Lignin was selectively degraded up to 30 % with a limited loss of 11-12 % in total organic matter. Finally, the degraded agro-residues demonstrated 50-62 % enhancement in their digestibility. Two-threefold enhancement in other nutritional quality (amino acids, TPCs and antioxidant activity) fortifies the process. Thus the method is quite helpful to design an effective solid-state fermentation system to improve the nutritive quality of agricultural residues by simultaneous production of lignocellulolytic enzyme production and antioxidants.

Bioprocessing of Agricultural Wastes as Optimised Carbon Source and Optimisation of Growth Conditions for Xylanase Production by Aspergillus brasiliensis in Agitated Solid State Fermentation (Ssf)

Objective: Xylanase has been involved in many industrial applications especially in the pulp and paper, baking, detergent as well as food and beverage industries. This enzyme is produced by various microorganisms, mainly from fungal species. Therefore, in this study, Aspergillus brasiliensis ATCC 16404 was investigated for the optimum xylanase production under agitated solid state fermentation (SsF) using different agricultural wastes as alternative inexpensive carbon source. Methods: A. brasiliensis was cultured in 10 g agricultural waste with addition of 2% yeast extract as the nitrogen source up to 120 h of SsF to elucidate xylanase production. Effect of various agricultural wastes such as wheat bran, rice bran, soybean hulls, barley husk, maize and palm kernel cake (PKC) on xylanase activity were enumerated in a stepwise manner, where one parameter was investigated at a time approach to obtain the optimum carbon source for the maximum xylanase production by A. brasiliensis. Thereafter, growth temperatures from 25 to 45°C, initial medium pH from 4 to 10 and agitation speeds of 50 to 200 rpm were also elucidated to determine the optimum growth conditions for the maximum production of xylanase by A. brasiliensis. Results: Based on our results, the highest xylanase activity of 7.30 ± 1.93 U/mL was obtained using wheat bran as the prime carbon source with the initial medium pH 6.5 at 30°C at the agitation speed of 150 rpm. Conclusion: Thus, in our study, the maximum xylanase production by A. brasiliensis using solid state bioprocessing of wheat bran as the carbon source was achieved with the addition of 2% yeast extract as the nitrogen source under the optimum growth conditions of 30°C with medium pH 6.5 at 150 rpm.

Optimization of metalloprotease production by Eupenicillium javanicum in both solid state and submerged bioprocesses

Proteases are enzymes that have great importance in industries and can be produced through bioprocess by microorganisms. Changes in parameters can influence the production rate of these enzymes. The current study evaluated the influence of these parameters on the production of proteases by the fungus Eupenicillium javanicum under both solid state bioprocess (SSB) and submerged bioprocess (SmB), and determined the partial biochemical characterization of the crude enzymatic extract. Our studies have indicated that the best conditions for the production of proteases under SSB were a 10% albumin, 90% wheat bran incubated at 30°C. Under SmB the optimal conditions were medium supplemented with 0.25% casein, medium pH of 5.0 and incubation at 30°C. The biochemical characterization showed that the enzyme is a metalloprotease that presented collagenolytic activity, with optimal pH and temperature of 5.5 and 60°C, respectively. Key words: Metalloprotease, Eupenicillium javanicum, bioprocesses, collagenolytic activity, biochemical characterization

Production of enzymes from Lichtheimia ramosa using Brazilian savannah fruit wastes as substrate on solid state bioprocesses

Background: Enzyme production by solid state bioprocess (SSB) using residues as substrate for microorganisms is an alternative for costs reduction and to avoid their disposal into environment. The aim of this work was to evaluate the physiology of the fungus Lichtheimia ramosa in terms of microbial growth and production of amylases, β-glucosidases, carboxymethylcellulase (CMCase), and xylanases, via SSB, utilizing wastes of the Brazilian savannah fruits bocaiuva ( Acrocomia aculeata ), guavira ( Campomanesia pubescens ) and pequi ( Caryocar brasiliense ) as substrate at different temperatures (25, 30, and 35°C) during 168 hrs. Results: Samples were taken every 24 hrs, which resulted in 8-points kinetic experiments to determine microbiological and enzymatic contents. The best substrate for β-glucosidase activity was pequi waste after 48 hrs at 30°C (0.061 U/mL). For amylase activity, bocaiuva presented itself as the best substrate after 96 hrs at 30°C (0.925 U/mL). CMCase activity was higher in guavira waste after 96 hrs at 35°C (0.787 U/mL). However, the activity was more expressive for xylanase in substrate composed of bocaiuva residue after 144 hrs at 35°C (1.802 U/mL). Conclusions: It was concluded that best growth condition for L. ramosa is at 35°C for all substrates and that xylanase is the enzyme with more potential in SSB, considering the studied Brazilian savannah fruit wastes.

Physiology of Lichtheimia ramosa obtained by solid-state bioprocess using fruit wastes as substrate

Due to the amount of nutrients available in the agroindustrial wastes, these can be converted into high added-value products by the action of microorganisms in solid-state bioprocesses. The aim of this work was to evaluate the growth physiology and lipase production of the fungus Lichtheimia ramosa using the following Brazilian savannah fruit wastes as substrates: bocaiuva (Acrocomia aculeata), pequi (Caryocar brasiliense), guavira (Campomanesia pubescens), araticum (Annona crassiflora) and seriguela (Spondias purpurea). These residues were triturated, homogenized, adjusted to pH 5.0 and 60% moisture, sterilized and packaged in plastic tray-type bioreactors before inoculation with 10% (w/v) of L. ramosa pre-culture medium. The cultivations were conducted in a bacteriological incubator at 30°C for 40days. Samples were taken every 5days and fungi and bacteria contents, proximate composition and lipase activity were evaluated. The maximum fungal counting was observed between 25 and 35days. L. ramosa reached the stationary phase next to 40days in all substrates. Mesophilic and psicrophilic aerobic bacteria were not detected. Protein enrichment was obtained for all media, being superior in seriguela residues (391.66%), followed by pequi (160.04%), araticum (143.31%), guavira (102.42%), and bocaiuva (67.88%). Lipase production was observed in all cultivated media, except in pequi residues that showed decreasing lipase activity. The higher production was observed in guavira (1.12U/g) followed by araticum (0.58U/g), seriguela (0.41U/g) and bocaiuva (0.21U/g) waste substrates. It was concluded that the studied fruit wastes have been successfully utilized as substrates for protein enrichment and lipase production with L. ramosa.

Novel catalytic and effluent decolorization functionalities of sol-gel immobilized Pleurotus ostreatus IBL-02 manganese peroxidase produced from bio-processing of wheat straw

Solid state bio-processing of wheat straw was carried out through an indigenous fungal strain Pleurotus ostreatus IBL-02 under pre-optimized fermentation conditions. The maximum activity, 692±12 U/mL, of the industrially important manganese peroxidase (MnP) enzyme was recorded after five days of still culture incubation. The crude MnP was 2.1-fold purified with a specific activity of 860 U/mg after purification on a Sephadex-G-100 gel column. On native and SDS-PAGE electrophoresis gels, the purified MnP fraction was a single homogenous band of 45 kDa. An active fraction of MnP was immobilized using hydrophobic sol-gel entrapment comprising tetramethoxysilane (T) and propyltrimethoxysilane (P) at different T:P molar ratios. Characterization revealed that after 24 h incubation at varying pH and temperatures, the MnP fraction immobilized at a T:P ratio of 1:2 in the sol-gel retained 82% and 75% of its original activity at pH 4 and 70°C, respectively. The optimally active fraction at a 1:2 T:P ratio was tested against MnSO 4 as a substrate to determine the kinetic catalytic constants K M and V max . To explore the industrial applicability of P. ostreatus IBL-02 MnP, both the free and immobilized MnP were used for the decolorization of four different textile industrial effluents. A maximum of 100% decolorization was achieved for the different textile effluents within the shortest time period. A lower K M , higher V max , hyper-activation, and enhanced acidic and thermal resistance up to 70 °C were the novel catalytic features of the sol-gel immobilized MnP, suggesting that it may be a potential candidate for biotechnological applications particularly for textile bioremediation purposes. Agro-industrial based lignocellulosic waste material i.e. wheat straw was used as a fermentation feed stuff for MnP production. A promising and eco-friendly approach was adopted to immobilize the purified MnP into the sol-gel matrix network. To explore the textile industrial applicability, the immobilized MnP was tested against real dye containing textile effluents.

Evaluation of Antimicrobial, Antioxidant Activities, and Nutritional Values of Fermented Foxtail Millet Extracts by Lactobacillus paracasei Fn032

Solid-state bioprocessing of foxtail millet by Lactobacillus paracasei Fn032 is a biotechnological strategy to produce fermented foxtail millet meal with more beneficial components. The objective of this study was to evaluate the antioxidant, antimicrobial properties and nutritional values of water extracts from fermented foxtail millet flour and its bran with and without protease. Fermented foxtail millet flour with added protease extract showed higher scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals as well as reducing power than fermented foxtail millet flour and fermented foxtail millet bran extracts. Both extracts, fermented foxtail millet flour and fermented foxtail millet flour with protease, showed significant (P < 0.05) effectiveness inhibition abilities on microbial growth when compared with fermented foxtail millet bran extracts. Amino acid profile revealed that fermented foxtail millet flour with protease, with relatively strongest antioxidant, antimicrobial activity, also had the highest total hydrophobic amino acids content (51.39%) and hydrophobic index (8.47 Kj/mol amino acid residue). Moreover, fermented foxtail millet flour with protease revealed the highest protein content, predicted protein efficiency ratio, and protein digestibility. Molecular weight of the whole extracts varied from 180–5000 Da. Based on the results obtained, fermented foxtail millet flour extracts were relatively effective in the antioxidant, antimicrobial properties assayed and might be potential biological values for application in food products.

Decolorization applicability of sol–gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum

BackgroundAn eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents.ResultsA large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol–gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity parameters were below the permissible limits.ConclusionsIn conclusion, G. lucidum MnP was immobilized by sol–gel matrix entrapment with an objective to enhance its practical efficiencies. The MnP was successfully entrapped into a sol- gel matrix of TMOS and PTMS with an overall immobilization efficiency of 93.7%. The sol- gel entrapped MnP seems to have prospective capabilities which can be useful for industrial purposes, especially for bioremediation of industrial effluents.

Isolation, identification and characterization of a novel high level β-glucosidase-producing Lichtheimia ramosa strain

The aim of this work was to prospect for microorganisms in the local environment, selecting those with higher production of cellulolytic and hemicellulolytic enzymes. A total of 36 microorganisms were isolated in a sugarcane-based medium. Colonies were replated in medium containing carboxymethylcellulose as the main carbon source, where all grew, confirming their potential for plant biomass degradation. The enzyme production was evaluated by cultivating the isolated microorganisms in solid-state bioprocess using two different carbon sources, sugarcane bagasse and wheat bran. Among the microorganisms obtained, isolate 11, identified as Lichtheimia ramosa, presented a high level β-glucosidase production of 15.58 U/mL, as well as 2.41 U/mL of xylanase and 2.13 U/mL of CMCase when cultivated on wheat bran. Changes in temperature and cultivation time favored a higher production of β-glucosidase: 17.26 U/mL in wheat bran-based medium at 35 °C for 120 h.

Comparative analysis of solid-state bioprocessing and enzymatic treatment of finger millet for mobilization of bound phenolics

The present work investigates the probable bioprocessing technique to mobilize the bound phenolics naturally found in finger millet cell wall for enriching it with dietary antioxidants. Comparative study was performed between the exogenous enzymatic treatment and solid-state fermentation of grain (SSF) with a food grade organism Rhizopus oryzae. SSF results indicated that at the 6th day of incubation, total phenolic content (18.64 mg gallic acid equivalent/gds) and antioxidant property (DPPH radical scavenging activity of 39.03 %, metal chelating ability of 54 % and better reducing power) of finger millet were drastically enhanced when fermented with GRAS filamentous fungi. During the enzymatic bioprocessing, most of the phenolics released during the hydrolysis, leached out into the liquid portion rather than retaining them within the millet grain, resulting in overall loss of dietary antioxidant. The present study establishes the most effective strategy to enrich the finger millet with phenolic antioxidants.

The Industrial Applicability of Purified Cellulase Complex Indigenously Produced by Trichoderma viride Through Solid-State Bio-processing of Agro-Industrial and Municipal Paper Wastes

An indigenous strain of Trichoderma viride produced high titers of cellulase complex in solid-state bio-processing of agro-industrial orange peel waste, which was used as the growth-supporting substrate. When the conditions of the SSF medium containing 15 g orange peel (50% w/w moisture) inoculated with 5 mL of inoculum were optimal, the maximum productions of endoglucanase (655 ± 5.5 U/mL), exoglucanase (412 ± 4.3 U/mL), and β-glucosidase (515 ± 3.7 U/mL) were recorded after 4 days of incubation at pH 5 and 35 °C. The enzyme with maximum activity (endoglucanase) was purified by ammonium sulfate fractionation and Sephadex G-100 column gel filtration chromatographic technique. Endoglucanase was 5.5-fold purified with specific activity of 498 U/mg in comparison to the crude enzyme. The enzyme was shown to have a molecular weight of 58 kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE). The shelf life profile revealed that the enzyme could be stored at room temperature (30 °C) for up to 45 days without losing much of its activity.

Determining the basic composition and total phenolic compounds of Pleurotus sajor-caju cultivated in three different substrates by solid state bioprocess

Oyster mushrooms (Pleurotus sajor-caju) were grown in grape bagasse, apple pomace or bean straw. Samples were taken for each one of the substrates and analyzed for moisture, ash, carbohydrate, crude fat, crude fiber, crude protein content and total phenolic compounds. The substrate affected all the parameter content and mainly the total phenolic compounds. In general, the nutritional aspects of mushrooms produced using grape bagasse tended to be better. Changes in the total phenolic compound content of the fruit could be explained by the biosorption capacity from substrate. It was possible to confirm that composition depends on cultivation substrate.

Phenolic content, antioxidant activity and antiamylolytic activity of extracts obtained from bioprocessed pineapple and guava wastes

The relationship between phenolic content, antioxidant and antiamylolytic activities was investigated in Rhizopus oligosporus-mediated solid-state bioprocessing of pineapple and guava residues. Two different treatments of fruit residues mixed with soy flour (5g of soy flour and 5g of fruit residue; 1g of soy flour and 9g of fruit residue) were inoculated and incubated at 22oC. Phenol rich extracts (non-concentrated and concentrated 10-fold by boiling) from day 2 and 10 were tested for their ability to inhibit alpha-amylase. In order to correlate the enzymatic inhibition with phenolic-linked antioxidant activity of the extracts, samples were evaluated for total phenolic content and free radical scavenging. Alpha-amylase inhibition was observed for all extracts, concentrated or not, with values ranging from 10.91% to 100% (complete inhibition). A direct correlation between the total phenolic content or the free radical scavenging activity and alpha-amylase inhibition was not found, demonstrating that such an inhibition could be caused by the presence of specific compounds, phenolic or not, rather than by the actual concentration of overall fruit phenolics.

Kinetic characterization of purified laccase produced from Trametes versicolor IBL-04 in solid state bio-processing of corncobs

A locally isolated white rot fungal strain Trametes versicolor IBL-04 produced high laccase activities in solid state bio-processing of corn cobs. Addition of glucose and yeast extract (C: N ratio; 25:1) enhanced laccase synthesis. Addition of Tween-80 and CuSO4 enhanced laccase production to 1012 U/mL under optimized process conditions. Laccase was further purified to 2.89-fold (specific activity of 840 U/mg) by ammonium sulfate fractional precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The purified laccase had a relative molecular mass of 63 kDa as detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Best enzyme activity was at pH 5 and 40oC. Using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate, the enzyme showed maximum activity (Vmax) of 780 U/mL with a corresponding Michaelis constant (Km) value of 73µM. Among the different activators/inhibitors, Cu2+, Mn2+, and Fe2+ stimulated laccase activity, whereas EDTA and cystein inhibited the enzyme. The higher Vmax and lower Km for T. versicolor IBL-04laccase as compared to most of the reported laccases suggests its potential for industrial applications.

Characterization of Exo 1, 4-&amp;lt;i&amp;gt;β&amp;lt;/i&amp;gt; glucanase produced from &amp;lt;i&amp;gt;Tricoderma Viridi&amp;lt;/i&amp;gt; MBL through solid-state bio-processing of orange peel waste

Agro-industrial residues are primarily composed of complex polysaccharides that strengthen the microbial growth for the production of industrially important enzymes like cellulases. In the present study we aimed to characterize the Exo 1, 4-β glucanase that was indigenously produced from Trichoderma viride MBL. T. viride MBL was cultured in the Solid-State medium of orange peel (50% w/w moisture) under optimized fermentation conditions and maximum activity of 412 ± 12 U/mL was recorded after 4th day of incubation at pH 5.5 and 30℃. Exo 1, 4-β glucanase was 4.17-fold purified with specific activity of 642 U/mg in comparison to the crude extract. To confirm its purity and molecular weight, sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS- PAGE) was performed. The enzyme was shown to have a molecular weight of 60 kDa with an optimum pH and temperature of 5 and 50℃, respectively. Lineweaver-Burk reciprocal plot revealed that the kinetic constants Km and Vmax of purified Exo 1, 4-β glucanase were 76 μM and 240 U/mL.

Purification and characterization of laccase produced by Schyzophylum commune IBL-06 in solid state culture of banana stalks

Shyzophyllum commune IBL-06 produced 367 IU/mL of laccase in solid state bioprocess of banana stalk under optimum physical and nutritional parameters. The optimum SSF conditions were рH 4.5; temperature, 35°C; inoculum size, 3mL (106-108 mL-1); moisture content, 60%; C:N ratio in the medium, 15:1 (glucose and ammonium nitrate as additional carbon and nitrogen sources, respectively), ABTS (1mM), 1 mL, and CuSO4 (1mM), 1mL. Laccase was purified 3.95-fold with specific activity of 158 U/mg by ammonium sulfate precipitation, followed by gel filtration chromatography using Sephadex G-100 column. Molecular weight of the laccase was 63 KDa on SDS-PAGE. Purified laccase had an optimum рH of 6.0 and was stable in the рH range from 6 to 7. The optimum temperature was 40 °C, and it displayed considerable stability within the range 30 to 35 °C with 24 h incubation. Km and Vmax were 0.025 mM and 80 mM/min, respectively, using ABTS as a substrate. Copper sulfate increased the activity of purified laccase when used at low concentration, but silver nitrate exerted the strongest inhibition.

Purification and Properties of a Novel Phenolic Antioxidant from Radix astragali Fermented by Aspergillus oryzae M29

The Chinese herb Radix astragalus (RA) has been widely used as a dietary supplement in Asia, and there are numerous reports on its bioactivities. However, there are no reports to date regarding the use of Aspergillus spp. in the culture medium of the RA plant for the production of phenolic antioxidants. In this study, utilizing the fungus Aspergillus to ferment the native RA has successfully resulted in a significant increase in the phenolic contents of RA, and the fermented RA also revealed much better antioxidant activity toward 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, hydroxyl radical, superoxide radical and peroxyl radical than those of unfermented RA. Among these phenolics, a potent novel antioxidant was isolated and identified as 3,4-di(4'-hydroxyphenyl) isobutyric acid with a molecular weight of 272, by ESI-MS (electrospray ionization mass), ¹H NMR (nuclear magnetic resonance), ¹³C NMR, DEPT (distortionless enhancement by polarization transfer)-NMR, HMQC (heteronuclear multiple quantum coherence), and HMBC (heteronuclear multiple bond correlation) spectra. These data demonstrated that the solid-state bioprocessing strategy could be an innovative approach to enhance the antioxidant activity of RA.