Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

Purification and characterization of lipoxygenase from Pleurotus ostreatus

Lipoxygenase was purified homogeneously from cups of Pleurotus ostreatus by Sephacryl S-400 HR gel filtration, Dyematrex Green A affinity, and DEAE-Toyopearl 650M ion-exchange chromatographies. The molecular weight of the enzyme was estimated to be 67,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 66,000 by gel filtration; the isoelectric point was pH 5.1. The optimum pH and temperature of the enzymatic activity were 8.0 and 25 degrees C, respectively. The enzyme contained non-heme iron, and a thiol group seemed to be involved in its activity. The K(m), V(max), and k(cat) values of the enzyme for linoleic acid were 0.13 mM, 23.4 micromol.min(-1).mg(-1), and 25.7 s(-1), respectively. The enzyme showed high specificity toward linoleic acid. When linoleic acid was incubated with the enzyme, 13-hydroperoxy-9Z,11E-octadecadienoic acid was found to be the main oxidative product.     

Characterization and histochemical localization of nonspecific esterase from ascocarps of desert truffle (Terfezia claveryi Chatin)

An esterase activity from Terfezia claveryi Chatin ascocarps, a mycorrhizal hypogeous fungus, is described for the first time. The enzyme was partially purified using phase partitioning in Triton X-114 (TX-114), achieving a reduction of 87% in the triglyceride content and the removal of 63% of phenols. The enzyme showed maximum activity toward short-chain p-nitrophenyl esters, and no interfacial activation was observed, indicating that the enzyme responsible for this activity is an esterase and not a lipase. This esterase presented its maximum activity at pH 7.4 and 60 degrees C. The values obtained for Km at pH 7.4 were 0.3 mM for p-nitrophenyl butyrate and 0.6 mM for p-nitrophenyl acetate with catalytic efficiencies (Vmax/Km) of 0.23 and 0.32, respectively. T. claveryi esterase was inhibited by phenylboric acid, indicating that serine residues were involved in the enzyme activity. This activity was localized only in the hypothecium and was absent from the peridium and gleba.     

Characterization of a GH3 family β-glucosidase from Dictyoglomus turgidum and its application to the hydrolysis of isoflavone glycosides in spent coffee grounds

A recombinant β-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum β-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 °C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-β-D-glucopyranoside (with a K(m) of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-β-D-glucopyranoside, pNP-β-D-xylopyranoside, pNP-β-D-fucopyranoside, and pNP-β-D-galactopyranoside. However, no activity was observed for oNP-β-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-L-arabinopyranoside, and pNP-α-L-rhamnopyranoside. The hydrolytic activity of the β-glucosidase for coffee isoflavones followed the order genistin (with a K(m) of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 μg/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0.6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.     

Relationship Between Hydrolytic Rancidity,Oil Concentration,and Esterase Activity in Rice Bran

Hydrolytic rancidity restricts the utilization of rice bran, reducing its potential value. In the present study, three groups of eight rice cultivars each displaying different levels of oil concentration (high, medium, and low) were cultivated in 1999 and 2000 under field conditions and evaluated for oil content, hydrolytic rancidity, and esterase activity in the bran fraction. Genotype effects were statistically significant for all measured traits (P < 0.05), whereas environment (year) was nonsignificant. Hydrolytic rancidity was strongly correlated with esterase activity (r = 0.89***), but not with oil concentration (r = ‐0.01). A wide variation was found for both hydrolytic rancidity and esterase activity, which ranged from 6.8 to 56.0 mg of C8:0/g of bran (CV = 49.1%) and from 4.3 to 22.8 mg of C8:0/g of bran (CV = 34.3%), respectively. Red bran displayed the lowest values for both hydrolytic rancidity (mean = 10.2 mg of C8:0/g of bran) and esterase activity (mean = 5.4 mg of C8:0/g of bran). Apparently, the low values for hydrolytic rancidity were related to the inhibition effect of bran tannins on lipase activity. In conclusion, cultivar variation was detected for both hydrolytic rancidity and esterase activity in the studied genotypes, esterase activity being the principal factor explaining the variation found for the former trait. Therefore, it may be possible to create new cultivars with increased stability against hydrolytic rancidity by selecting for lower esterase activity.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Microwave-assisted extraction of bound phenolic acids in bran and flour fractions from sorghum and maize cultivars varying in hardness

To release bound phenolic acids, a microwave-assisted extraction procedure was applied to bran and flour fractions obtained from eight sorghum and eight maize cultivars varying in hardness. The procedure was followed by HPLC analysis, and the identities of phenolic acids were confirmed by MS/MS spectra. The extraction of sorghum and maize bound phenolic acids was done for 90 s in 2 M NaOH to release ferulic acid and p-coumaric acid from bran and flour. Two diferulic acids, 8-O-4'- and 8-5'-benzofuran form, were identified and quantitated in sorghum bran, and only the former was found in maize bran. The contents of ferulic acid and diferulic acids in sorghum bran were 416-827 and 25-179 μg/g, respectively, compared to 2193-4779 and 271-819 μg/g in maize. Phenolic acid levels of sorghum were similar between hard and soft cultivars, whereas those of maize differed significantly (p < 0.05) except for ferulic acid in flour. Sorghum phenolic acids were not correlated with grain hardness as measured using a tangential abrasive decortication device. Maize ferulic acid (r = -0.601, p < 0.01), p-coumaric acid (r = -0.668, p < 0.01), and 8-O-4'-diferulic acid (r = -0.629, p < 0.01) were significantly correlated with hardness.     

Characterization of codon-optimized recombinant candida rugosa lipase 5 (LIP5)

Recombinant Candida rugosa lipase 5 (LIP5) has been functionally expressed along with other isoforms in our laboratory. However, the characterization and codon optimization of LIP5 have not been done. In this work, we characterized, codon-optimized and compared LIP5 with commercial lipase. LIP5 activity on hydrolysis of p-nitrophenyl (p-NP) butyrate was optimal at 55 °C as compared with 37 °C of the commercial lipase. Several assays were also performed to determine the substrate specificity of LIP5. p-NP butyrate (C(4)), butyryl-CoA (C(4)), cholesteryl laurate (C(12)), and N-carbobenzoxy-l-tyrosine-p-nitrophenyl ester (l-NBTNPE) were found as preferred substrates of LIP5. Interestingly, LIP5 specificity on hydrolysis of amino acid-derivative substrates was shown to be the highest among any lipase isoforms, but it had very weak preference on hydrolyzing triacylglycerol substrates. LIP5 also displays a pH-dependent maximum activity of a lipase but an esterase substrate preference in general. The characterization of LIP5 along with that of LIP1-LIP4 previously identified shows that each lipase isoform has a distinct substrate preference and catalytic activity.     

Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga)

Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.     

Optimization of acid hydrolysis conditions for feruloylated oligosaccharides from rice bran through response surface methodolgy

Response surface methodology (RSM) was employed to optimize the hydrolysis conditions with trifluoroacetic acid (TFA) to obtain the maximum amount of feruloylated oligosaccharides from rice bran. The TFA concentration and hydrolysis time effects on feruloylated oligosaccharides recovery are studied. The optimum hydrolysis conditions for maximizing feruloylated oligosaccharides recovery were 193 mM TFA concentration and 1.36 h of hydrolysis time. Under these conditions the corresponding acyl ferulic group quantity was 78.63 microg in 1 mL of hydrolysate. The model was experimentally verified with a satisfactory coefficient of R (2) (= 0.96). The quantity of acyl ferulic group in the feruloylated oligosaccharides, purified using Amberlite XAD-4, was 916.12 microg/g of rice bran under the optimum hydrolysis conditions. The proposed method accounted for 54.08% of the total acyl ferulic group in rice bran. The results suggest that the proposed conditions were useful in maximizing recovery of feruloylated oligosaccharides from rice bran.     

Antioxidants and antioxidant activity of several pigmented rice brans

This study investigated the antioxidant content and activity of phenolic acids, anthocyanins, α-tocopherol and γ-oryzanol in pigmented rice (black and red rice) brans. After methanolic extraction, the DPPH free radical scavenging activity and antioxidant activity were measured. The pigmented rice bran extract had a greater reducing power than a normal rice bran extract from a long grain white rice. All bran extracts were highly effective in inhibiting linoleic acid peroxidation (60-85%). High-performance liquid chromatography (HPLC) analysis of antioxidants in rice bran found that γ-oryzanol (39-63%) and phenolic acids (33-43%) were the major antioxidants in all bran samples, and black rice bran also contained anthocyanins 18-26%. HPLC analysis of anthocyanins showed that pigmented bran was rich in cyanidin-3-glucoside (58-95%). Ferulic acid was the dominant phenolic acid in the rice bran samples. Black rice bran contained gallic, hydroxybenzoic, and protocatechuic acids in higher contents than red rice bran and normal rice bran. Furthermore, the addition of 5% black rice bran to wheat flour used for making bread produced a marked increase in the free radical scavenging and antioxidant activity compared to a control bread.     

Prevention of hydrolytic rancidity in rice bran during storage.

The effect of microwave heating, packaging, and storage temperature on the production of free fatty acids (FFA) in rice bran was examined. Freshly milled raw rice bran was adjusted to 21% moisture content and heated in a microwave oven at 850 W for 3 min. Raw and microwave-heated rice bran were packed in zipper-top bags or vacuum-sealed bags and stored at 4-5 or 25 degrees C for 16 weeks. FFA content of bran was measured at 4-week intervals. Total FFA increased rapidly over the 16-week period from the initial value of 2.5% in raw bran stored at 25 degrees C to 54.9% in vacuum bags and 48.1% in zipper-top bags. However, total FFA of raw bran stored at 4-5 degrees C increased at a slower rate from an initial value of 2. 5 to 25.4% in vacuum bags and 19.5% in zipper-top bags. After 16 weeks of storage, total FFA of microwave-heated bran stored at 25 degrees C increased from 2.8 to 6.9 and 5.2%, respectively, for samples stored in vacuum bags and zipper-top bags. Total FFA of microwave-heated samples stored at 4-5 degrees C did not change significantly with storage time. Results showed that hydrolytic rancidity of rice bran can be prevented by microwave heating and that the recommended storage condition for microwaved rice bran is 4-5 degrees C in zipper-top bags.     

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.     

Enzymatic synthesis and properties of highly branched rice starch amylose and amylopectin cluster

We enzymatically modified rice starch to produce highly branched amylopectin and amylose and analyzed the resulting structural changes. To prepare the highly branched amylopectin cluster (HBAPC), we first treated waxy rice starch with Thermus scotoductus alpha-glucanotransferase (TSalphaGT), followed by treatment with Bacillus stearothermophilus maltogenic amylase (BSMA). Highly branched amylose (HBA) was prepared by incubating amylose with Bacillus subtilis 168 branching enzyme (BBE) and subsequently treating it with BSMA. The molecular weight of TSalphaGT-treated waxy rice starch was reduced from 8.9 x 10(8) to 1.2 x 10(5) Da, indicating that the alpha-1,4 glucosidic linkage of the segment between amylopectin clusters was hydrolyzed. Analysis of the amylopectin cluster side chains revealed that a rearrangement in the side-chain length distribution occurred. Furthermore, HBAPC and HBA were found to contain significant numbers of branched maltooligosaccharide side chains. In short, amylopectin molecules of waxy rice starch were hydrolyzed into amylopectin clusters by TSalphaGT in the enzymatic modification process, and then further branched by transglycosylation using BSMA. HBAPC and HBA showed higher water solubility and stability against retrogradation than amylopectin clusters or branched amylose. The hydrolysis rates of HBAPC and HBA by glucoamylase and alpha-amylase greatly decreased. The k cat/ K m value of glucoamylase acting on the amylopectin cluster was 45.94 s(-1)(mg/mL)(-1) and that for glucoamylase acting on HBAPC was 11.10 s(-1)(mg/mL)(-1), indicating that HBAPC was 4-fold less susceptible to glucoamylase. The k cat/ K m value for HBA was 15.90 s(-1)(mg/mL)(-1), or about three times less than that for branched amylose. The k cat/ K m values of porcine pancreatic alpha-amylase for HBAPC and HBA were 496 and 588 s(-1)(mg/mL)(-1), respectively, indicating that HBA and HBAPC are less susceptible to hydrolysis by glucoamylase and alpha-amylase. HBAPC and HBA show potential as novel glucan polymers with low digestibility and high water solubility.     

Action of tyrosinase on ortho-substituted phenols: possible influence on browning and melanogenesis

The action of tyrosinase on ortho-substituted monophenols (thymol, carvacrol, guaiacol, butylated hydroxyanisole, eugenol, and isoeugenol) was studied. These monophenols inhibit melanogenesis because they act as alternative substrates to L-tyrosine and L-Dopa in the monophenolase and diphenolase activities, respectively, despite the steric hindrance on the part of the substituent in ortho position with respect to the hydroxyl group. We kinetically characterize the action of tyrosinase on these substrates and assess its possible effect on browning and melanognesis. In general, these compounds are poor substrates of the enzyme, with high Michaelis constant values, K(m), and low catalytic constant values, k(cat), so that the catalytic efficiency k(cat)/K(m) is low: thymol, 161 ± 4 M(-1) s(-1); carvacrol, 95 ± 7 M(-1) s(-1); guaiacol, 1160 ± 101 M(-1) s(-1).     

Tyrosinase inhibitor from black rice bran

The inhibitor of tyrosinase activity in black rice bran was investigated. The methanol extract from black rice bran was re-extracted with hexane, chloroform, ethyl acetate, or water. The ethyl acetate extract had the most potent inhibition against tyrosinase activity by 80.5% at a concentration of 0.4 mg/mL. Inhibitory compound in the ethyl acetate fraction was isolated by silica gel column chromatography, and identified as protocatechuic acid methyl ester (compound 1) by GC, GC-MS, IR, and 1H and 13C NMR spectroscopy. Compound 1 inhibited 75.4% of tyrosinase activity at a concentration of 0.50 micromol/mL. ID(50) (50% inhibition dose) value of compound 1 was 0.28 micromol/mL. To study the structure-activity relationship, protocatechuic acid (2), vanillic acid (3), vanillic acid methyl ester (4), isovanillic acid (5), isovanillic acid methyl ester (6), veratric acid (7), and veratric acid methyl ester (8) were also assayed.     

Suppressive effect of rice bran incorporation in paddy soil on germination of Monochoria vaginalis and its relationship with electric conductivity

Laboratory experiments were conducted to investigate (1) the effects of the addition of rice (Oryza sativa. L.) bran to paddy soil on the germination of Monochoria vaginalis (Burm. f.) Kunth, and (2) the relationship between the electric conductivity (EC) of the soil solution and germination. Soil samples were collected at 4 sites in Japan. After flooded soils with rice bran had been incubated for 7?d at 30°C, the soil solution was collected using a porous cup and the EC of the soil solution was measured. The amounts of rice bran added to the soil were 0%, 0.3%, 0.6% and 0.9% (weight(w)/w). In the soil solution obtained, seeds of M. vaginalis were incubated for 3?d at 30°C, and the germination percentage was then analyzed. The addition of rice bran suppressed germination, and the degree of suppression increased with increasing content of rice bran. Although the same amount of rice bran was applied to each soil, the degree of growth suppression by rice bran as well as the EC of the soil solution differed among the soils. In each soil, there was a positive correlation between the amount of rice bran and EC, and the degree of growth suppression significantly increased with an increase in EC. When EC was higher than 150?mS?m^?1, seeds of M. vaginalis hardly germinated. There was no significant correlation between the oxidation-reduction potential (Eh) of soil and seed germination, suggesting that EC is a more reliable and convenient indicator than Eh for evaluating the relationship between the addition of organic material and seed germination. In conclusion, the addition of rice bran to soil increases the EC of the soil solution, and EC is one of the factors that suppress the germination of M. vaginalis. The suppressive effect of rice bran on germination is different among soils. This fact is attributed to the difference in EC due to the addition of rice bran. Thus, it is expected that EC can be used as an indicator for determining how much rice bran to add.     

Preparation and functional properties of rice bran protein isolate

Rice bran protein isolate (RBPI) containing approximately 92.0% protein was prepared from unstabilized and defatted rice bran using phytase and xylanase. The yield of RBPI increased from 34% to 74.6% through the use of the enzymatic treatment. Nitrogen solubilities of RBPI were 53, 8, 62, 78, 82, and 80% at pHs 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0, respectively. Differential scanning calorimetry showed that RBPI had denaturation temperature of 83.4 degrees C with low endotherm (0.96 J/g of protein). RBPI had similar foaming properties in comparison to egg white. But emulsifying properties of RBPI were significantly lower than those of bovine serum albumin. The result of amino acid analysis showed that RBPI had a similar profile of essential amino acid requirements for 2-5-year-old children in comparison to that of casein and soy protein isolate.     

Effects of microwave heat, packaging, and storage temperature on fatty acid and proximate compositions in rice bran

The effect of microwave heat, packaging methods, and storage temperatures on proximate and fatty acid compositions of rice bran during 16 weeks of storage was examined. Freshly milled raw rice bran was adjusted to 21% moisture content and microwave heated for 3 min. Raw and microwave-heated brans were packed in zipper-top bags and/or vacuum-sealed bags and stored at 4-5 and/or 25 degrees C for 16 weeks. The moisture content decreased significantly from an initial 8.4 to 6.4% in microwave-heated samples regardless of packaging methods and storage temperatures. Protein, fat, linoleic, and linolenic contents did not change significantly in all raw and microwave-heated samples during 16 weeks of storage. The microwave-heated rice bran packed in zipper-top bags can be stored at 4-5 degrees C for up to 16 weeks without adverse effect on proximate and fatty acid composition quality under the conditions employed in this study.     

Prevention of oxidative rancidity in rice bran during storage.

The effect of microwave heat on lipoxygenase (LOX) activity in rice bran under various storage conditions was examined. Raw rice bran from the long-grain variety Lemont was adjusted to 21% moisture content and heated in a microwave oven at 850 W for 3 min. Raw and microwave-heated rice bran samples were packed in zipper-top bags or vacuum packs and stored at room temperature (25 degrees C) or in the refrigerator (4-5 degrees C) for 16 weeks. Samples were analyzed for LOX activity at 4-week intervals. LOX activity did not significantly change from its initial value at week 0 for zipper-top and vacuum-packed samples while stored at 4-5 degrees C for 12 weeks, but decreased at week 16. Vacuum packing did not show a significant impact on LOX activity during 16 weeks of storage. Microwave-heated samples stored in the refrigerator did not show significant change in LOX activity for up to 12 weeks but showed a significant (p < 0. 05) decrease at 16 weeks. Results showed that oxidative rancidity of rice bran could be prevented by microwave heating the samples, packing in zipper-top bags, and storing at 4-5 degrees C for up to 16 weeks.