Antioxidant activity of tocopherols, tocotrienols, and gamma-oryzanol components from rice bran against cholesterol oxidation accelerated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride

The antioxidant activities of vitamin E (alpha-tocopherol, alpha-tocotrienol, gamma-tocopherol, and gamma-tocotrienol) and gamma-oryzanol components (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate) purified from rice bran were investigated in a cholesterol oxidation system accelerated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride. All components exhibited significant antioxidant activity in the inhibition of cholesterol oxidation. The highest antioxidant activity was found for 24-methylenecycloartanyl ferulate, and all three gamma-oryzanol components had activities higher than that of any of the four vitamin E components. Because the quantity of gamma-oryzanol is up to 10 times higher than that of vitamin E in rice bran, gamma-oryzanol may be a more important antioxidant of rice bran in the reduction of cholesterol oxidation than vitamin E, which has been considered to be the major antioxidant in rice bran. The antioxidant function of these components against cholesterol oxidation may contribute to the potential hypocholesterolemic property of rice bran.     

Purification and identification of components of gamma-oryzanol in rice bran Oil.

High-purity gamma-oryzanol was obtained from crude rice bran oil using a normal-phase preparative scale HPLC. A reverse-phase HPLC method was used for separating the individual components of gamma-oryzanol present in rice bran oil. Ten fractions were isolated and collected using the reverse-phase HPLC method, and their structures were identified. Identification was accomplished using GC/MS with an electron impact mass spectrum after components were transformed into trimethylsilyl ether derivatives. The 10 components of gamma-oryzanol were identified as Delta(7)-stigmastenyl ferulate, stigmasteryl ferulate, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, Delta(7)-campestenyl ferulate, campesteryl ferulate, Delta(7)-sitostenyl ferulate, sitosteryl ferulate, compestanyl ferulate, and sitostanyl ferulate. Three of these, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate, were major components of gamma-oryzanol.     

Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS

Ferulic acid esters of triterpene alcohols and sterols in rice bran oil have been extensively studied and reported to possess important pharmacological actions. Inconsistent results on the numbers and structures of ferulates have been reported, primarily because of the analytical procedures employed. Conventional methods for analysis of phytosterol content in oil are carried out by characterization of trimethylsilylated derivatives (TMS) using GC-EI-MS after saponification of oils or individual compound isolated from oils. This study developed an LC-MS/MS method for the direct analysis of triterpene alcohol and sterol esters in rice bran oil. In addition to verifying the results of previous research, nine new relatively polar triterpene alcohol and sterol esters were characterized by their retention behaviors in LC and ESI-MS data from both negative- and positive-ion mode. This is the first evidence for the presence of hydroxylated ferulate esters and caffeate esters as part of gamma-oryzanol in rice bran. The method enables rapid and direct on-line characterization of triterpene alcohol and sterol esters in oils. LC-MS/MS equipped with reverse-phase LC and ESI-MS should be well-suited for identification and quantification of the polar metabolites of phytosterols in biological fluids after consumption of rice bran oil or other oils.     

Tocopherol and Tocotrienol Content in Commercial Wheat Mill Streams

The content of tocopherols and tocotrienols, collectively known as vitamin E (tocols), was determined in fractions of roller‐milled wheat grains. The results showed that vitamin E components are present in all major flour fractions of wheat, but that the vitamin E content and composition differed significantly between fractions. The total content of vitamin E, calculated as alpha‐tocopherol equivalents, changed from 16.1 mg α‐TE/g in wheat grain to 12.2 mg α‐TE/g in roller‐milled wheat flour. The germ fraction had the highest content of tocopherols, and the content of α‐tocopherol (195.2 μg/g) was 16 times higher (on average) than in any other fraction. The content of tocotrienols was distributed more uniform in the wheat grain with the highest content in the bran fractions, and the content of β‐tocotrienol was higher than the content of α‐tocopherol in all milling fractions except the wheat germ. The content of β‐tocotrienol was 24.1 μg/g in wheat grain, 25.3–31.0 μg/g in the bran fractions, and 14.3–21.9 μg/g in the fractions of endosperm. Overall, germ and fine bran fractions represent good sources of vitamin E and might be used in breadmaking.     

Two new Monascus metabolites with strong blue fluorescence isolated from red yeast rice

Red yeast rice obtained as cultures of Monascus AS3.4444 on rice was extracted and analyzed by high-performance liquid chromatography (HPLC). Two new Monascus metabolites with similar fluorescence spectra (lambda ex = 396 nm, lambda em = 460 nm) and UV absorption spectra (lambda max = 386 nm) were detected. They were isolated by rechromatography on a silica gel column and semipreparative HPLC, and two strong blue fluorescent compounds were obtained. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), intensive ESI-MS, and nuclear magnetic resonance spectroscopy ( (1)H NMR, (13)C NMR, COSY, and HMBC) studies. High-resolution mass spectrometry indicated the molecular formulas C 21H 24O 5 and C 23H 28O 5. The two new compounds, named monasfluore A and monasfluore B, respectively, contain a alkyl side chain, gamma-lactone, and propenyl group, whereas the more lipophilic compound, monasfluore B, is a higher homologue of monasfluore A, with the more lipophilic octanoyl instead of the hexanoyl side chain.     

Phytochemicals and antioxidant activity of milled fractions of different wheat varieties

The health-promoting effects of whole-grain consumption have been attributed in part to their unique phytochemical contents and profiles that complement those found in fruits and vegetables. Wheat is an important component of the human diet; however, little is known about the phytochemical profiles and total antioxidant activities of milled fractions of different wheat varieties. The objectives of this study were to investigate the distribution of phytochemicals (total phenolics, flavonoids, ferulic acid, and carotenoids) and to determine hydrophilic and lipophilic antioxidant activity in milled fractions (endosperm and bran/germ) of three different wheat varieties, two of which were grown in two environments. Grain samples of each of the wheat varieties were milled into endosperm and bran/germ fractions. Each fraction was extracted and analyzed for total phenolics, ferulic acid, flavonoids, carotenoid contents, and hydrophilic and lipophilic antioxidant activities. Total phenolic content of bran/germ fractions (2867-3120 micromol of gallic acid equiv/100 g) was 15-18-fold higher (p < 0.01) than that of respective endosperm fractions. Ferulic acid content ranged from 1005 to 1130 micromol/100 g in bran/germ fractions and from 15 to 21 micromol/100 g in the endosperm fractions. The bran/germ fraction flavonoid content was 740-940 micromol of catechin equiv/100 g. On average, bran/germ fractions of wheat had 4-fold more lutein, 12-fold more zeaxanthin, and 2-fold more beta-cryptoxanthin than the endosperm fractions. Hydrophilic antioxidant activity of bran/germ samples (7.1-16.4 micromol of vitamin C equiv/g) was 13-27-fold higher than that of the respective endosperm samples. Similarly, lipophilic antioxidant activity was 28-89-fold higher in the bran/germ fractions (1785-4669 nmol of vitamin E equiv/g). Hydrophilic antioxidant activity contribution to the total antioxidant activity (hydrophilic + lipophilic) was >80%. In whole-wheat flour, the bran/germ fraction contributed 83% of the total phenolic content, 79% of the total flavonoid content, 51% of the total lutein, 78% of the total zeaxanthin, 42% of the total beta-cryptoxanthin, 85% of the total hydrophilic antioxidant activity, and 94% of the total lipophilic antioxidant activity. Our results showed that different milled fractions of wheat have different profiles of both hydrophilic and lipophilic phytochemicals. These findings provide information necessary for evaluating contributions to good health and disease prevention from whole-wheat consumption.     

In vitro digestion characteristics of unprocessed and processed whole grains and their components

Chemical composition and in vitro digestion properties of select whole grains, before and after processing, and their components were measured. Substrates included barley, corn, oat, rice, and wheat. In addition to whole grain flours, processed substrates also were tested as were corn bran, oat bran, wheat bran, and wheat germ. Processing of most substrates resulted in higher dry matter and digestible starch and lower resistant starch concentrations. Dietary fiber fractions varied among substrates with processing. Digestion profiles for most substrates correlated well with their chemical composition. Corn bran and rice substrates were the least fermentable. Extrusion rendered barley, corn, and wheat more hydrolytically digestible and barley and oat more fermentatively digestible. Except for corn bran, all components had greater or equal fermentability compared with their native whole grains. Understanding digestion characteristics of whole grains and their components will allow for more accurate utilization of these ingredients in food systems.     

Quantitation of tocotrienol and tocopherol in various rice brans

Rice bran is abundant in bioactive compounds including tocotrienol (T3, unsaturated vitamin E). T3 has been reported about its potential functionalities (i.e., antiangiogenic effect), so much attention has been paid on usability of rice bran T3. Hence, we developed a rapid screening method for T3-rich rice bran by one-step equilibrium direct solvent extraction followed by normal phase high-performance liquid chromatography (HPLC). The method gave high-extraction rate of rice bran T3 and tocopherol (above 90%), and the determination of vitamin E by HPLC was completed within 15 min. Using the method, an average of total T3 content in 109 kinds of rice bran samples was 830 mug/g dry wt. Kouchi-Akamai, Joushuu, and Wataribune were found as the T3-rich rice bran varieties (1350-1430 microg T3/g dry wt). According to T3 ratio against total vitamin E (wt %), the average ratio was 61%. Hirayama, Moritawase, and Kaneko were found as the varieties having the highest T3 ratio (80-86%). Since T3 content in Koshihikari rice bran (the leading variety in Japan) was a little above the average, we cross-fertilized Koshihikari with T3-rich varieties and found that T3 content or ratio in F1 was improved compared with Koshihikari. The varieties found rich in T3 could be used for nutraceutical purpose.     

Simultaneous identification of soyasaponins and isoflavones and quantification of soyasaponin Bb in soy products,using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for simultaneous identification of soyasaponins and soy isoflavones in soy products, based on liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Soy-based nutraceutical products were analyzed by LC/ESI-MS with detection of protonated and sodiated molecular ions, as well as characteristic fragment ions for these compounds. Soy isoflavones were characterized by a strong protonated molecular ion in addition to corresponding [aglycone + H](+) ions. Monitoring the soyasaponin-specific protonated aglycone and dehydrated aglycone ions throughout the chromatogram provided a unique fingerprint for soyasaponin content in the samples. This mass spectrometric fingerprint also allowed immediate classification of the soyasaponin analytes as group A or B soyasaponins, based on the unique masses of aglycone ions observed for each class. Quantification of soyasaponin B(b) in soy-derived materials, based on the use of a purified soyasaponin B(b) standard and a glycyrrhizin internal standard, has been accomplished.     

Stability of Vitamin E in Wheat Flour and Whole Wheat Flour During Storage

The stability of vitamin E during 297 days of storage of wheat flour and whole wheat flour ground on a stone mill or a roller mill, respectively, were studied. One day after milling, the total content of vitamin E, expressed in vitamin E equivalents (α‐TE), was 18.7 α‐TE and 10.8 α‐TE for stone‐milled and roller‐milled wheat flour, respectively. The difference in total vitamin E content was primarily due to the absence of the germ and bran fractions in the roller‐milled flour. The total loss of vitamin E during storage was 24% for stone‐milled wheat flour but 50% for roller‐milled wheat flour. These results indicate that vitamin E, which is present in high amounts in wheat germ, functions as an antioxidant in the stone‐milled wheat flour. Hexanal formation showed that lipid oxidation in roller‐milled flour occurred just after milling, whereas the formation of hexanal in the germ fraction displayed a lack period of 22 days, confirming that vitamin E functions as an effective antioxidant in the wheat germ. Results showed no significant difference in total loss of vitamin E for stone‐milled and roller‐milled whole wheat flour. Total loss after 297 days of storage for both milling methods was ≈32%.     

Cholesterol Response and Foam Cell Formation in Hamsters Fed Rice Bran,Oat Bran,and Cellulose + Soy Protein Diets With or Without Added Vitamin E

Four-week-old male golden Syrian hamsters were fed diets containing cellulose (control, CC), cellulose + soy protein (CS), CS + vitamin E, (CSE), rice bran (RB), RB + vitamin E (RBE), oat bran (OB), and OB + vitamin E (OBE) for six weeks (n = 10/treatment). Diets contained (by weight) 10% total dietary fiber, 3% N, 20% fat, 0.5% cholesterol, and some diets had an additional 0.1% vitamin E. After six weeks, RB and OB diets resulted in significantly higher weight gain than the CC diet. Plasma low-density lipoprotein cholesterol (LDL-C) values and the LDL-C/high-density lipoprotein cholesterol ratio in hamsters fed CSE, RBE, OB, and OBE diets were significantly lower than in those fed CC diet. There were no significant differences in total plasma cholesterol values among the hamsters fed any of the diets. Liver cholesterol in animals fed OB and OBE diets was significantly lower than in all other groups. Foam cell areas in the inner bend of the aortic arch in animals fed all treatment diets were significantly reduced when compared with that in animals fed CC diet. The level of additional dietary vitamin E did not result in further significant reductions in foam cell area. The results of this study suggest that diets containing rice bran, oat bran, or soy protein significantly reduced the development of atherosclerosis in hypercholesterolemic hamsters.     

Quantification of tocopherols, tocotrienols, and γ-oryzanol contents and their distribution in some commercial rice varieties in Taiwan

The eight vitamin E isomers [α-, β-, γ-, and δ-tocopherols (T) and α-, β-, γ-, and δ-tocotrienols (T3)] and γ-oryzanol are known to possess diverse biological activities. This study examined the contents of these compounds and their distribution in 16 commercial rice varieties in Taiwan. Results showed that the order of vitamin E, total T, total T3, and γ-oryzanol contents was rice bran > brown rice > rice husk > polished rice. γ-T3 was the highest vitamin E isomer present in all rice samples, while β-T, β-T3, δ-T, and δ-T3 were present in trace amounts. The Japonica varieties contained a higher total T, total T3, and γ-oryzanol than the Indica varieties. They also have a higher level of α-T and α-T3 but a lower level of γ-T and γ-T3 than the Indica varieties. However, no obvious difference in total T, total T3, and γ-oryzanol content was noted between black- and red-colored rice varieties.     

Content of gamma-oryzanol and composition of steryl ferulates in brown rice (Oryza sativa L.) of European origin

The content of gamma-oryzanol and the composition of steryl ferulates were determined in brown rice of European origin using on-line coupled liquid chromatography-gas chromatography (LC-GC). Analysis of 30 brown rice samples of various cultivars, grown at different sites and in different seasons, revealed the gamma-oryzanol content to range from 26 to 63 mg/100 g. Cycloartenyl ferulate and 24-methylenecycloartanyl ferulate were the major components of gamma-oryzanol followed by campesteryl ferulate, campestanyl ferulate, and beta-sitosteryl ferulate. The proportions of individual steryl ferulates exhibited enormous variability. However, irrespectively of the great variations observed for single steryl ferulates, the proportions of the sum of 4,4'-dimethylsteryl ferulates (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate) and the sum of 4-desmethylsteryl ferulates (campesteryl ferulate, campestanyl ferulate, and beta-sitosteryl ferulate) were rather constant. The significant natural variability observed for gamma-oryzanol content and composition of steryl ferulates were shown to be influenced by environmental conditions but not by the degree of maturity of rice grains.     

Influence of growth temperature on the amounts of tocopherols, tocotrienols, and gamma-oryzanol in brown rice

Brown rice is a valuable source of lipid-soluble antioxidants including ferulated phytosterols (i.e., gamma-oryzanol), tocopherols, and tocotrienols. To evaluate the impact of temperature on the accumulation of these compounds, seeds from six different rice lines grown to maturity in replicate greenhouses in Gainesville, FL, were analyzed. The lines represented Oryza sativa indica, O. sativa japonica, and Oryza glaberrima of different origins. Temperatures were maintained near ambient at one end of each greenhouse and at approximately 4.5 degrees C above ambient at the other end. gamma-Oryzanols, tocopherols, and tocotrienols were extracted from whole seed (i.e., brown rice) and analyzed by HPLC. Tocotrienols and tocopherols varied widely between lines but changed only slightly with respect to temperature. In general, the proportions of alpha-tocotrienol and/or alpha-tocopherol increased at elevated temperature, whereas gamma-tocopherol and gamma-tocotrienol decreased. Six gamma-oryzanol peaks, identified on the basis of absorbance maxima at 330 nm and HPLC-mass spectrometry, were quantified. The most abundant component was 24-methylenecycloartanyl ferulate, present at 40-62% of total. Its levels increased 35-57% at elevated temperature in five of six lines, accounting for most of the change in total gamma-oryzanol. The results suggest that the physiological action of individual ferulated phytosterols should be investigated because their relative proportions in gamma-oryzanol can change.     

Antioxidant activity of steryl ferulate extracts from rye and wheat bran

Antioxidant activity of steryl ferulates from other sources than rice have not yet been studied much, despite the fact that rice steryl ferulates (gamma-oryzanol) have been shown to possess good antioxidant activity. In this study, steryl ferulate extracts from wheat or rye bran were studied for their capability to inhibit hydroperoxide formation in bulk methyl linoleate and methyl linoleate emulsion. Further, their activity to scavenge DPPH radicals was analyzed. The activities were compared to synthetic steryl ferulates, rice steryl ferulates, ferulic acid, and alpha-tocopherol. Nonrice cereal extracts of steryl ferulates exhibited good antioxidant activity, especially in the bulk lipid system. The radical scavenging activity was similar to that of nonesterified ferulic acid, indicating that the ferulic acid moiety is responsible for the antioxidant properties. This study illustrates a new aspect to the health-promoting properties of rye and wheat.     

亚临界丙烷萃取米糠油及其微量活性组分动力学模型

为了揭示亚临界流体萃取米糠油及其微量活性组分的动力学规律,该文以亚临界丙烷为萃取介质,研究了米糠油、γ-谷维素及α-生育酚等活性组分在不同萃取温度和时间的萃取率,并基于 Baümler 模型对目标物的萃取率进行模型拟合,建立相应的动力学模型并验证。结果表明,亚临界萃取过程中目标物的相对萃取率随时间增长递增,且在初始阶段（洗涤过程）的增长速率快于第二阶段（扩散过程）的增长速率;同时,升高萃取温度可同时提高目标产物的扩散系数和相对萃取率;基于Baümler模型可较好拟合亚临界丙烷萃取米糠油、γ-谷维素和α-生育酚的动力学过程（R2>0.95）,所得动力学模型具有较好的预测准确性。由Arrhenius方程可计算出米糠油、α-生育酚和γ-谷维素的亚临界萃取反应活化能,其中米糠油的活化能最低（5.23 kJ/mol）,α-生育酚次之（7.05 kJ/mol）,γ-谷维素最高（9.11 kJ/mol）,表明γ-谷维素最难萃取且对温度依赖程度最高;根据拟合所得动力学模型,对米糠油提取率进行预测,结果表明,该模型对米糠油萃取率的预测准确率达95.8%,而对γ-谷维素和α-生育酚萃取率的预测准确度稍低,分别达94.7%和94.4%,对三者均具有很好的预测准确性。总之,基于 Baümler 模型可以较好地建立米糠油及其有益伴随物的亚临界萃取动力学模型,从而较为准确的预测米糠油及其微量活性组分的萃取率,对米糠油的亚临界萃取工艺具有一定的理论指导意义。     

初级芳香胺的高分辨质谱裂解规律研究

为探究初级芳香胺类化合物的质谱裂解规律,本研究采用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法,在电喷雾正离子模式下采集数据,根据一、二级质谱离子的精确质荷比推导14种初级芳香胺类化合物可能的裂解途径。结果表明,由于分子结构中存在氨基基团,因此初级芳香胺类化合物极易被质子化,更易形成[M+H]⁺。断裂过程主要发生NH₃中性碎片的丢失,产生[M+H-NH₃]⁺特征碎片离子,在此基础上,氯代苯胺类化合物发生碳正离子重排后,丢失氯原子形成[·M+H-NH₃-Cl]⁺碎片离子。甲氧基取代苯胺类化合物可丢失CH₃O基团产生碎片离子[M+H-NH₃-CH₃O]⁺,或发生碳正离子转移重排至苯甲基,进一步丢失CH₃和CH₃O基团。甲基或甲氧基取代的联苯胺类化合物还会发生CN键断裂形成[M+H-NH]⁺,继而丢失NH₃中性碎片形成[M+H-NH-NH₃]⁺。偶氮苯类化合物中高键能的偶氮键不易断裂,碎片离子主要通过两侧的CN键断裂形成。本研究提出的最佳电离方式和质谱裂解规律为初级芳香胺类化合物的快速鉴定提供了重要依据。     

Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer

We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using fluorescein (FL) as the fluorescent probe. The current ORAC(FL) assay is limited in hydrophilic antioxidant due to the aqueous environment of the assay. Lipophilic antioxidants mainly include the vitamin E family and carotenoids, which play a critical role in biological defense systems. In this paper, we expanded the current ORAC(FL) assay to lipophilic antioxidants. Randomly methylated beta-cyclodextrin (RMCD) was introduced as the water solubility enhancer for lipophilic antioxidants. Seven percent RMCD (w/v) in a 50% acetone-H(2)O mixture was found to sufficiently solubilize vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). This newly developed ORAC assay (abbbreviated ORAC(FL-LIPO)) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORAC(FL-LIPO) assay is reliable and robust. For the first time, by using 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid as a standard (1.0), the ORAC values of alpha-tocopherol, (+)-gamma-tocopherol, (+)-delta-tocopherol, alpha-tocopherol acetate, tocotrienols, 2,6-di-tert-butyl-4-methylphenol, and gamma-oryzanol were determined to be 0.5 +/- 0.02, 0.74 +/- 0.03, 1.36 +/- 0.14, 0.00, 0.91 +/- 0.04, 0.16 +/- 0.01, and 3.00 +/- 0.26, respectively. The structural information of oxidized alpha-tocopherol obtained by liquid chromatography/mass spectrometry reveals that the mechanism for the reaction between the vitamin E and the peroxyl radical follows the hydrogen atom transfer mechanism, which is in agreement with the notion that vitamin E is the chain-breaking antioxidant.     

MALDI-TOF MS analysis of food flavonol glycosides

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique that is having a great impact on food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify flavonol glycosides in food samples. 2',4',6'-Trihydroxyacetophenone was chosen as the best matrix because it worked for crude sample extracts and ionized flavonol glycosides in both positive and negative MALDI-TOF MS modes. In the positive mode, multiple ion forms were observed for flavonol glycosides, including [M + H](+), [M + Na](+), [M + K](+), and [M - H + Na + K](+), with further fragmentation through loss of glycosidic residues. The negative mode for all flavonol glycosides resulted in [M - H](-) ion formation without detectable fragmentation. The multiple ions in the positive mode gave more information on individual flavonol glycoside structures than the negative mode. Flavonol glycosides showed similar intensities or responses in the positive mode, while kaempferol glycosides exhibited much less response than quercetin glycosides in negative mode.     

Use of microscopy to assess bran removal patterns in milled rice

During rice milling, the bran and germ are successively removed from the caryopsis (kernel). Because bran and germ contain large quantities of lipid, the amount of lipid remaining on the kernel surface may be used as a method for the assessment of milling quality. Bulk samples of rice pureline varieties and an experimental hybrid were milled for 0, 10, 20, 30, and 40 s. Scanning electron microscopy (SEM) revealed that brown rice kernels had large contours of linear protuberances and depressions running lengthwise along the kernel surface. The protuberances were abraded successively during milling, but varying amounts of material remained in the depressions. Light microscopy combined with the lipid-specific probes Nile Blue A or Sudan Black B demonstrated that the material in the depressions observed with SEM was lipid. Sections of whole, milled rice kernels, prepared using a modified sectioning technique and stained with Nile Blue A, showed that portions of the embryo remain after milling and that lipid is located on or near the surface of the kernel. Differences in quantity and distribution of residual lipid as milling duration increased were documented photographically to indicate the extent to which the bran and embryo components were removed during milling. This paper provides proof of concept that residual lipid is a robust measure of the degree of milling.