Thermostable Α-amylase Research Articles

Non-target Effects of Hyperthermostable α-Amylase Transgenic Nicotiana tabacum in the Laboratory and the Field.

Thermostable α-amylases are important enzymes used in many industrial processes. The expression of recombinant Pyrococcus furiosus α-amylase (PFA) in Nicotiana tabacum has led to the accumulation of high levels of recombinant protein in transgenic plants. The initial steps to registering the transgenic tobacco at a commercial production scale and growing it in the field requires a risk assessment of potential non-target effects. The objective of this study was to assess the effect of feeding on transgenic tobacco with 2 indigenous insect species commonly associated with wild and commercial tobacco involving plants grown and evaluated under laboratory and field conditions. The highest levels of PFA ranged from 1.3 to 2.7 g/kg leaf fresh weight produced in the field-grown cultivars Con Havana and Little Crittenden, respectively. These two cultivars also had the highest nicotine (ranging from 4.6 to 10.9 mg/g), but there was little to no negative effect for either tobacco hornworm Manduca sexta L. or aphid Myzus nicotianae (Blackman). Both laboratory and field trials determined no short term (5 days) decrease in the survival or fecundity of the tobacco aphid after feeding on PFA transgenic tobacco compared to non-transgenic plants. In the field, tobacco hornworm larvae showed no differences in survival, final larval weights or development time to adult stage between transgenic lines of four cultivars and their corresponding wild type controls. Laboratory studies confirmed the field trial results indicating the low risk association of PFA expressed in tobacco leaves with tobacco hornworms and aphids that would feed on the transgenic plants.

Low molecular weight alkaline thermostable α-amylase from Geobacillus sp. nov.

Industrial demands for enzymes that are stable in a broad range of conditions are increasing. Such enzymes, one of which is α-amylase, could be produced by extremophiles. This study reports a thermostable α-amylase produced by a newly isolated Geobacillus sp. nov. from a geothermal area. The phylogenetic analysis of the 16S rRNA gene showed that the isolate formed a separate branch with 95% homology to Geobacillus sp. After precipitation using ammonium sulphate followed by ion-exchange chromatography, the enzyme produced a specific activity of 25.1 (U/mg) with a purity of 6.5-fold of the crude extract. The molecular weight of the enzyme was approximately 12.2 kDa. The optimum activity was observed at 75 °C and pH 8. The activity increased in the presence of Ba2+ and Fe2+ but decreased in the presence of K+ and Mg2+. Ca2+ and Mn2+ increased the activity slightly. The activity completely diminished with the addition of Cu2+. EDTA and PMSF also sharply reduced enzyme activity. Although the stability was moderate, the low molecular weight could be an important feature for its future applications.

Proteinaceous Residue Removal from Oat β-Glucan Extracts Obtained by Alkaline Water Extraction.

Background: Wet methods of 1-3, 1-4 -β-D-glucan isolation from cereals differ mainly in the type of grain fraction used as raw material, the solid-liquid ratio of β-glucan in raw material vs. solvent used, and the type of aqueous solvent modification (alkali, neutral or acidic). All these factors impact the characterization of the residues finally found in extracts. Oat bran is a rich source of globulin fraction which can be transferred into the extracts, especially when a high pH is employed. Methods: A multi-stage (enzymatic and acidic) purification procedure was performed to remove the residues, especially starch and protein, from β-glucan isolates from oat of different molar mass. Pancreatin, thermostable α-amylase, amyloglucosidase, and papain were used for consecutive residue removal. Three levels of low pH = 4.5, 3.5 and 3.0 were also tested for effective protein precipitation. Results: The starch hydrolysis and liquefaction significantly facilitate the proteinaceous matter removal although papain usage showed an intensive unfavorable impact on β-glucan molar mass. Soluble protein content was significantly decreased after pancreatin and α-amylase treatment, while the significant reduction of amine nitrogen was noted after complete starch hydrolysis and a second acidification step. Conclusions: A complex procedure employing different enzymes is needed to successfully reduce the possibly bioactive residues in isolated oat β-glucan fractions.

Thermostable and mesophilic α-amylase: Effects on wheat starch physicochemical properties and their applications in extruded noodles

We compare modification effects of thermostable and mesophilic α-amylases (i.e. TS-αA and MS-αA) under high-performance extrusion treatment. As confirmed by size exclusion chromatography, a portion of the wheat starch extracted from extruded noodles (ENs) was modified into a water-soluble mixture of oligosaccharides and dextrin. X-Ray diffraction and differential scanning calorimeter investigations demonstrated that the appropriate concentrations of both enzymes promote starch gelatinisation, whilst their excess usage presents negative effects due to the difficulty of overlaying or adhering to starch fragments. From these findings, innovative ENs with a well-developed porous structure was successfully created to enhance the rehydration and palatability of ENs. Compared with the overall TS-αA modification results, the use of MS-αA concentration of 1.6‰ is recommended as the optimum choice for producing ENs. The current study is the first to compare the modification effects of TS-αA and MS-αA during the high-performance extrusion and to illustrate their promising applications in ENs.

Purification, biochemical, and molecular characterization of a novel extracellular thermostable and alkaline α-amylase from Tepidimonas fonticaldi strain HB23.

The present study investigated the purification, biochemical, and molecular characterization of a novel thermostable α-amylase (TfAmy48) from Tepidimonas fonticaldi strain HB23. MALDI-TOF/MS analysis indicated that the purified enzyme is a monomer with a molecular mass of 48,138.10 Da. The results from amino-acid sequence analysis revealed high homology between the 25 NH2-terminal residues of TfAmy48 and those of Gammaproteobacteria α-amylases. The optimum pH and temperature values for α-amylase activity were pH 8 and 80 °C, respectively. Thin-layer chromatography (TLC) analysis showed that the final hydrolyzed products of the enzyme from soluble potato starch were maltopentaose, maltose, and maltotriose, which indicate that TfAmy48 possessed an endo-acting pattern. Compared to Termamyl®300 L, TfAmy48 showed extreme stability and tolerance towards organic solvents and excellent compatibility with some commercial laundry detergents. These proprieties make TfAmy48 enzyme a potential candidate as a cleaning bioadditive in detergent composition. The Tfamy48 gene encoding TfAmy48 was cloned, sequenced, and heterologously-expressed in the extracellular fraction of Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rTfAmy48) were similar to those of native one. The highest sequence identity value (97%) was obtained with PsAmy1 α-amylase from Pseudomonas sp. strain KFCC10818, with only 16 amino-acid (aa) residues of difference.

Evaluation of brewer's spent grain hydrolysate as a substrate for production of thermostable α-amylase by Bacillus stearothermophilus

In the present study, BSG was hydrolysed using cellulolytic enzymes and used as a growth medium supplement for cultivation of the thermophilic bacterium, Bacillus stearothermophilus in the production of α-amylase. A central composite design involving five parameters and four levels viz. starch, peptone, KCl, and MgSO4 along with BSG hydrolysate was used to derive the optimal media composition. The fermentation was conducted using shake flasks for 36 h at a temperature of 50 °C and pH 7.0 at 220 rpm. Optimization trials revealed that maximal amylase production (198.09 U/ml) occurred with a medium composition of starch (0.2% w/v), peptone (0.2% w/v), KCl·4 H2O (0.02% w/v), MgSO4·7 H2O (0.01% w/v) and hydrolysate (0.22% v/v). A 1.3-fold increase in amylase activity was obtained following novel media composition. All the factors considered in the study were found to be significant. The enzyme was purified by three step purification strategy, characterised and tested for anti-biofilm activity.

Identification of α-amylase gene by PCR and activity of thermostable α-amylase from thermophilic Anoxybacillus thermarum isolated from Remboken hot spring in Minahasa, Indonesia

Information of thermophilic bacteria in Indonesian hot springs is limited, especially in the province of North Sulawesi. The study aims to isolate and identify thermophilic bacteria from Remboken hot springs at Minahasa Indonesia, identify the α-amylase gene and its enzyme activity. To identify the thermophilic bacteria, it was conducted the morphological analysis and biochemistry tests. Th microbial species was identified using PCR and sequencing of 16S rRNA followed by BLAST search on the EZBioCloud database, whereas the α-amylase gene was identified by Polymerase chain reaction (PCR). Starch hydrolysis test was used to analyze α-amylase activity qualitatively. It was done using iodine method (Fuwa method). The results showed that thermophilic bacteria isolated belong to the species Ureibacillus suwonensis, Anoxybacillus thermarum, and Anoxybacillus mongoliensis. PCR analysis showed that Anoxybacillus thermarum FRM-RBK02 possesses an α-amylase gene. The strain was able to hydrolyze amylose, indicated by a clear zone around the growth; hence exhibiting α-amylase activity. The the crude amylase of Anoxybacillus thermarum FRM-RBK02 exhibited optimum activity at 80°C and pH 7.0. Overall, Anoxybacillus thermarum FRM-RBK02 has α-amylase gene and could produce thermostable α-amylase with amylolytic capability.

Pilot-scale production of a highly thermostable α-amylase enzyme fromThermotoga petrophilacloned intoE. coliand its application as a desizer in textile industry

In this study, the industrial applications of a highly thermostable α-amylase as a desizer in the textile industry was evaluated. The cloned gene was expressed in different media (ZBM, LB, ZYBM9, and ZB) with IPTG (isopropyl β-d-1-thiogalactopyranoside) used as an inducer. Lactose was also used as an alternate inducer for the T7 promoter system in E. coli. For the large-scale production of the enzyme, different parameters were optimized. The maximum enzyme production was achieved when the volume of medium was 70% of the total volume of fermenter with a 2.0 vvm air supply and 20% dissolved oxygen at a 200 rpm agitation rate. Under all the optimized conditions, the maximum enzyme production was 22.08 U ml−1 min−1 with lactose (200 mM) as an inducer in ZBM medium. The desizing potential of the purified α-amylase enzyme was calculated with different enzyme concentrations (50–300 U ml−1) at different temperatures (50–100 °C), and pHs (4–9) with varying time intervals (30–120 min). The highest desizing activity was found when 150 U ml−1 enzyme units were utilized at 85 °C and at 6.5 pH for 1 h.

The role of thermostable proteinaceous α-amylase inhibitors in slowing starch digestion in pasta

Starch in pasta is slowly digested, which is nutritionally advantageous because it reduces post-prandial glycemia, a risk factor for type 2 diabetes. We examine the role of proteinaceous amylase inhibitors, providing insights into how wheat proteins slow starch digestion. Gluten samples were extracted in the laboratory and also commercially obtained, and incubated in water at 37 °C and 100 °C. The centrifuged supernatant and gluten pellet were incubated with porcine α-amylase, and enzymatic activity measurement revealed that the supernatant from gluten heated to 100 °C retained the capacity to significantly reduce amylolytic activity. Confocal microscopy showed that the gluten pellet had become porous following heat treatment. These results are consistent with an increased concentration of soluble proteins after cooking. Proteins in the soluble fraction and gluten pellet were identified with mass spectrometry proteomics, which screened a limited number of endogenous double-headed α-amylase/trypsin inhibitors (α-AI(s)), found to be enriched in the supernatant after cooking. Endogenous α-AI(s) play a role in slowing starch digestion by interacting with α-amylase as it penetrates the gluten network that entraps the starch, resulting in an immobilization of the enzyme onto the gluten network, so that the digestion of starch entrapped by the gluten network is inhibited.

A Novel Raw Starch Hydrolyzing Thermostable α-Amylase Produced by Newly Isolated Bacillus mojavensis SO-10: Purification, Characterization and Usage in Starch Industries

ABSTRACT The aim of this study is the production, purification, and characterisation of thermostable raw starch hydrolyzing α-amylase produced by Bacillus mojavensis SO-10. The maximum production conditions of α-amylase were found at 36th hour, 35 °C and pH 7.0. We utilized three steps to purify the thermostable α-amylase and as a result, 34-fold and 18% yield were obtained. The molecular weight of purified α-amylase was determined as 73 kD. The Km and Vmax rates were detected as 0.010 mM and 3.38 µmol min−1, respectively. This purified α-amylase exhibited the highest activity at pH 5.0-6.0 and 70 oC and showed stability over a wide variety of pH and temperature at 4.0-8.0, and 40-50 oC, respectively. The thermostable purified α-amylase exhibited stability in the presence of denaturing agents and heavy metal ions. The purified enzyme hydrolyzed the raw starches of corn and wheat grains in the ratio of 36.7% and 39.2% respectively. The end-yields of soluble starch hydrolysis were analyzed by thin-layer chromatography (TLC). In addition, the usage of purified α-amylase in clarification of apple juice and domestic washing detergent industries were evaluated.

Identification of Four Thermophilic Geobacillus Isolates from Hammam Pharaon, Sinai, Egypt

The present study was conducted to identify thermophilic bacteria from local habitats and to test their ability for producing thermostable α-Amylase, cellulase, lipase and protease in order to pave the way for their use in industrial processes. Out of 29 isolates initially obtained mainly from Hammam Pharaon, four potential isolates were selected based on thermo-tolerance ability and enzyme activity. The four isolates were characterized using phenotypic and 16S rRNA sequencing which recognized the isolates as Geobacillus. All four isolates were found to be variable for indole test, methyl red and rod shaped morphology. However, they were found to be variable for citrate utilization, catalase test starch hydrolysis, gelatin hydrolysis, casein hydrolysis, and Tween 80 hydrolysis. These bacteria grow at temperatures ranging from 50 to 90°C and the pH ranged from 6 to 9. This study added a lot to our knowledge about the thermophilic bacteria inhabiting Hammam Pharaon and their characters that would open the door for their use in biotechnological applications..

Scale-up thermostable α-amylase production in lab-scale fermenter using rice husk as an elicitor by Bacillus licheniformis-AZ2 isolated from Qinarje Hot Spring (Ardebil Prov. of Iran)

Background and purpose: Amylases are commercially important enzymes with various biotechnological, clinical and medical applications. This study aimed at scaling up α-amylase production elicited by rice husk in stirred-fermenter using Bacillus lichneniformis-AZ2 isolated from Qinarje Hot Spring. Materials and methods: Effect of temperature, aeration rate and agitation speed on bacterial growth and ɑ-amylase production were investigated under batch fermentation process in a 3-Lit stirred-fermenter. OFAT method was followed to select optimum level of each parameter. Other factors were set upon the results of previous experiments carried out in shake-flask scale. Results: Maximum α-amylase production of 17.66 ± 0.87 U/mL (2.1 folds more than shake-flask cultures) was achieved in stirred-fermenter with optimized agitation speed of 100 rpm and 1 vvm aeration rate at 37oC after 60 h of incubation. This time was shorter than the corresponding fermentation time obtained from shake-flask experiments by half. A comparison of kinetic parameters of fermentation in stirred-fermenter and shake-flask cultures revealed that B. licheniformis-AZ2 was more active to synthesize ɑ-amylase in fermenter. In shaken cultures Qx, Qp, Yp/x, µmax, qp and td, were 0.27 (g/L/h), 228.6 (U/L/h), 13.64 (U/g), 0.055 (h-1), 0.76 (U/g/h) and 12.48 h, whereas in stirred-fermenter the above values were 0.40 (g/L/h), 723.1 (U/L/h), 45.17 (U/g), 0.120 (h-1), 5.42 (U/g/h) and 5.78 h, respectively. Conclusions: SmF in stirred-fermenter is a potential strategy for ɑ-amylase production. Although for commercialization further studies are needed in pilot-scale. Rice husk as a low-cost agro-waste is preferable to use as the carbon and energy sources, which provides a great ɑ-amylase elicitation.

Introduction of novel thermostable α-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related α-amylases under five individual GH13 subfamilies.

Among the thermophilic Bacillaceae family members, α-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus α-amylase genes displayed ≥ 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (≤ 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (≤ 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 α-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli α-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 α-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around α-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around α-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3.

Production, purification and characterization of raw starch hydrolyzing thermostable acidic α-amylase from hot springs, India

Alpha-amylase is an important hydrolytic enzyme used for various industrial processes. In the present study, Geobacillus bacterium (K1C), producing a thermostable α-amylase was isolated from Manikaran hot springs, India. We have purified and characterized the biochemical properties of α-amylase. The optimum temperature and pH for α-amylase activity was 80 °C and pH 6.0 respectively. The far-UV CD spectra of the enzyme indicated the presence of random coil conformation and showed an intermediate phase during temperature-induced unfolding. In the presence of substrate, thermostability of the α-amylase was increased as 50% initial activity was retained at 70 °C for 6 h and at 80 °C for 2 h. Moreover, the enzyme also showed remarkable pH stability as 90% of the initial activity was retained even after 48 h of incubation at pH 5.0, 6.0 and 7.0. Interestingly, amylase activity of the purified enzyme was Ca2+independent, whereas the complete inhibition of activity was observed in the presence of Cu2+, Pb2+, and Hg2+. The purified α-amylase was stable in the presence of detergents, organic solvents and Proteinase K. Furthermore, it exhibited the ability to hydrolyze raw starches (e.g. rice, wheat, corn, potato) efficiently; thus this enzyme has the potential to be used for industrial applications.

Purification and characterization of novel thermostable and Ca-independent α-amylase produced by Bacillus amyloliquefaciens BH072

In the present study, a novel α-amylase produced by Bacillus amyloliquefaciens BH072 was purified and characterized. The molecular weight of purified α-amylase was approximately 68 kDa, determined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and ten amino acid of N-terminal was NSGLNGYLTH. The kinetic parameters Km and Vmax were 4.27 ± 0.21 mg/mL and 987.34 ± 23.34 U/mg, respectively. Purified α-amylase showed maximal activity at pH 7 and 60 °C. Enzyme remained stable in pH range 6.0–7.0 and 50–80 °C. The activity of the α-amylase was Ca2+ independent and stability in the presence of surfactant, oxidizing and bleaching agents. The β-mercaptoethanol and EDTA greatly enhanced and reduced α-amylase activity, respectively. This enzyme has high hydrolysis rate toward corn, wheat and potato starch and hydrolyzes soluble starch to glucose, maltose, maltotriose and maltotetraose, indicating that the α-amylase represents a promising candidate for applications in the food industry.

Refolding of Misfolded Inclusion Bodies of Recombinant α-Amylase: Characterization of Cobalt Activated Thermostable α-Amylase from Geobacillus SBS-4S

Refolding of Misfolded Inclusion Bodies of Recombinant α-Amylase: Characterization of Cobalt Activated Thermostable α-Amylase from Geobacillus SBS-4S

Comparisons of Modern U.S. and Canadian Malting Barley Cultivars with Those from Pre-Prohibition: II. Amylolytic Enzyme Activities and Thermostabilities

ABSTRACTAmylolytic enzyme activities and thermostabilities in North American pre-Prohibition and modern malting barley cultivars were compared throughout Congress mashing. The mean α-amylase activity was significantly higher in modern cultivars throughout mashing than in pre-Prohibition varieties, although the reverse was observed for mean thermostabilities, expressed as percent of initial activity. The mean limit dextrinase activity in modern cultivars was higher early in mashing than in pre-Prohibition varieties, but modern cultivar mean thermostability was less than in pre-Prohibition varieties at the end of mashing. There were no significant differences in population means for β-amylase or α-glucosidase activities during mashing although mean thermostabilities for both enzymes were greater in pre-Prohibition cultivars than in modern cultivars. A comparison of individual genotypes showed the four highest α-amylase activities were in modern cultivars, with Charles having the most thermostable (116%) and Tradition the least thermostable (21%). Among pre-Prohibition cultivars, Oderbrucker (132%) and Manchuria (127%) had the most thermostable α-amylases. Hanna and Manchuria had the highest β-amylase activities at 30 and 45 min of mashing. No cultivar had β-amylase activity beyond 45 min of mashing. At the end of mashing, Silver King, Hannchen and Oderbrucker had the highest α-glucosidase activities and thermostabilities (88, 52, and 53%, respectively). Among modern cultivars, highest α-glucosidase activities and thermostabilities were in Metcalfe (39%), Tradition (47%) and Charles (42%). The highest limit dextrinase activities, followed by their thermostabilities, at the end of mashing were in Manchuria (57%) and Hannchen (60%) followed by Tradition (44%), Metcalfe (26%) and Charles (37%).

Bioextrusion of Broken Rice in the Presence of Divalent Metal Salts: Effects on Starch Microstructure and Phenolics Compounds

Broken rice has become more popular to be sustainably used due its low-cost and potential utilization for preparing purified/modified starches and fermented products. The use of broken rice as substrate of an enzymatic extrusion process modified by divalent metal salts was investigated. Six metal salts with concentrations 0.6, 1.2, and 1.8 mmol/100g (db of starch) were premixed with a thermostable α-amylase and with broken rice before being extruded. Extrudates were characterized using RVA, X-ray, FT–IR, and SEM techniques. RVA results showed that Mg(II), Ca(II), Mn(II), and Zn(II) decreased extrudate viscosity whereas Fe(II) and Cu(II) increased it compared to extrusion performed in absence of salts. DNS test demonstrated that nontransition metals Mg(II) and Ca(II) activated the enzyme and improved the extrusion performance, while transition metals Mn(II), Fe(II), Cu(II), and Zn(II) inhibited the activity of the enzyme. X-ray Diffraction (XRD) showed that all ions contributed to slightly increase the ext...

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses.

Phenolic retention of brown rice after extrusion with mesophilic α-amylase

Abstract Based on the results of phenolic retention of brown rice treated by extrusion with thermostable α-amylase (ETA), this work investigated the phenolic retention of brown rice after extrusion with mesophilic α-amylase (EMA). Similarly, EMA significantly increased phenolic retention of brown rice with the increasing enzyme level, and the phenolic retention was positively correlated with reducing sugars. However, brown rice treated by EMA had higher retention rate of free phenolic content, free antioxidant activity, and total identified free phenolic acids than that treated by ETA at different enzyme levels, although the corresponding reducing sugars produced by EMA were lower than by ETA. These results indicated that the extrusion temperature had greater effect on the phenolic retention than enzymatic hydrolysis during enzymatic extrusion. In addition, brown rice treated by EMA with 0.5% enzyme had a high retention of free flavonoids at 72.4%. Therefore, EMA might be a better processing technology for brown rice to improve its nutrition.