Nutrients, composition of tocotrienols, tocopherols, and γ-oryzanol, and antioxidant activity in brown rice before and after biotransformation

Nutrientes, composición de tocotrienoles, tocoferoles y γ-oryzanol, y actividad antioxidante del arroz integral antes y después de la biotransformación

Abstract

Composition of nutraceuticals and nutrients of two varieties of rough rice subjected to biotransformation by germination for 120 h, drying and dehusking, compared to respective control brown rice. An increase in the content of free sugars and soluble fiber, and decrease in insoluble fiber and fat was observed in nutrient composition. Different forms (δ, γ, & α) of tocotrienols and tocopherols were present in both control and biotransformed brown rice. γ-tocotrienol was the major form and its content increased and the total tocopherols, which was low in content, decreased. The γ-oryzanol content and its fractions, namely, cycloartenyl ferulate and 24-methylene cycloartenyl ferulate, did not show any significant alterations. However, the total antioxidant activity decreased. Scanning electron microscopic examination showed enlargement and distinct appearance of starch granules in the biotransformed rice. Biotransformed brown rice was also found to be superior to polished rice in nutritional and nutraceutical qualities.

La composición de nutracéuticos y nutrientes de dos variedades de arroz con cascarilla sujetos a biotransformación por germinación durante 120 horas, secado y descascarillado fue comparada a la de arroz integral control. Se observó un incremento en el contenido de azúcares libres y fibra soluble y una disminución de fibra insoluble y grasa en su composición. Diferentes formas (δ,γy α) de tocotrienoles y tocoferoles se encontraron en el arroz integral control y en el biotransformado. Tocotrienol gamma fue la forma más abundante y su contenido se incrementó, y el total de tocoferoles, que era bajo, se redujo. El contenido de γ-oryzanol y sus fracciones, a saber ferulato cicloartenilo y ferulato de 24-metilén cicloartenilo, no mostró ninguna alteración significativa. Sin embargo, la actividad antioxidante total se redujo. La examinación con microscopio electrónico de barrido mostró un mayor tamaño y apariencia distintiva de los gránulos de fécula del arroz biotransformado. El arroz integral biotransformado también resultó ser superior al arroz pulido en cualidades nutricionales y nutracéuticas.

Keywords:

• biotransformation
• rice
• nutrients
• nutraceuticals
• tocotrienol
• γ-oryzanol

Palabras clave:

• biotransformado
• arroz
• nutrientes
• nutracéuticos
• tocotrienol
• γ-oryzanol

Introduction

Rice is always harvested in the form of paddy (rough rice) and it is milled in suitable rice milling machinery to remove husk and the bran to prepare ready-to-cook rice. The rough rice on dehusking yields brown rice, which contain proteins, fiber, vitamins, minerals, and a number of biofunctional components like tocotrienols, tocopherols, γ-oryzanol, polyphenols, etc., in significant levels. Vitamin E components are well-known natural antioxidants which are known for a number of health benefits. Recent reports suggest fairly uniform antioxidant activity for different tocotrienol and tocopherol components (Xu, Hua, & Godber, 2001). Reports on the reduction in the plasma cholesterol level and cardiovascular protection (Qureshi, Bradlow, Salser, & Brace, 1997) as well as anti-carcinogenic activity of tocotrienols also indicate its health benefits. γ-Oryzanol is a potent antioxidant (Xu et al., 2001) and more effective than vitamin E in reducing oxidative stress-induced degenerative diseases. It reduces plasma cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride and prevents atherosclerosis (Rukmini & Raghuram, 1991) through decreased cholesterol absorption, increased fecal excretion of cholesterol and bile acids, and decreased hepatic biosynthesis of cholesterol. It has tumor inhibitory activity and it is known to prevent gastric ulcer and relives muscle pain through its anti-inflammatory effect (Akihisa et al., 2000). It can also give protection against UV ray exposure induced skin damages. Rice contains numerous phenolic compounds including ferulic and coumaric acids in low amount, and these polyphenolic compounds are potent antioxidants (Itani, Tatemoto, Okamoto, Fujii, & Muto, 2002).

In spite of these nutritional benefits, brown rice is rarely used for food preparation, mainly because of its poor cooking quality, higher chewiness, and poor cooked grain texture. In view of these, the brown rice is scored or polished to remove bran. Normally removal of 5% bran content in brown rice is enough to improve the cooking quality of rice. But very often the rice is polished to 8–12% degree. This results in 65–70% loss of vital nutrients present in germ and bran layers (Champgne, Wood, Juliano, & Bechtel, 2004).

Rice contributes greatly to energy and also protein requirements of more than 50% of the world population and constant efforts are being made to raise the overall nutritional status of rice and rice-based foods through genetic improvement, technological up gradation, fortification, supplementation, and fermentation. Controlled germination or malting of cereals is known to improve their overall nutritional qualities, but reports about application of this technology to rice are scanty (Chavan & Kadam, 1989).

Germination is a natural method to transform the bio molecules in their structural, functional, and nutritional properties. Germination or in vivo biotransformation is reported to enhance the bioavailability of many nutrients and decrease the level of antinutrients. These aspects have been exhaustively reviewed by Chavan and Kadam (1989). Many of the biochemical changes occur mainly due to the activation of hydrolytic enzymes and resultant partial decomposition of macromolecules into oligosaccharides and aminoacids (Yang, Basu, & Ooraikul, 2001). Germinated brown rice is also reported to contain biofunctional components like γ-aminobutyric acid (GABA) (Oh & Oh, 2004), and ferulic acid. Improvement in textural qualities of cereals and their properties will also take place as a result of germination.

Except a few products from germinated brown rice (Ohtsubo, Suzuki, Yasui, & Kasumi, 2005) most of the reports on germinated rice for food products confine to soaking the brown rice and incipient germination. However, a few reports on differently processed, viz., soaked, gas treated, and extruded germinated rice products and their biofunctonal components are available (Ohtsubo et al., 2005). Since rice is mostly consumed in the grain form or after making into flour, information on the nutritional and biofunctional components of germinated brown rice would be useful to the consumers, dieticians, and processors. In view of that, the composition of nutraceuticals and nutrients like γ-oryzanol, tocotrienols, tocopherols, fatty acids, and fiber, proximate content and the total antioxidant activity of the rice prepared by controlled germination of rice were investigated.

Materials and methods

Materials

Two popular varieties of paddy (rough rice) viz., IR 64 (coarse type) and BPT (fine type) were obtained from National Seeds Corporation, Mysore, India. Paddy kept at room temperature for 6 months after harvest were cleaned to free from foreign material, damaged kernels and chaff and used for the studies. All chemicals and solvents used were of analytical grade. Tocopherols and Tocovid capsule, as source of tocotrienols, were from Sigma, USA and Hovid Bhd, Malaysia, respectively.

Germination

Paddy samples (1 kg batch) were washed and soaked in excess water for 16 h, spread on wet jute sheet, and covered with another moist jute sheet and allowed to germinate for 5 days (120 h). During germination, water was sprayed at regular intervals to keep the sprout moist and the sprouts were mixed carefully to facilitate aeration and also to prevent netting of the rootlets. The sprouts were dried in air oven at 50 °C to about 12% moisture content and the rootlets were separated mechanically using a de-rooting machine (Westrup machine, KAMAS, Sweden). The de-rooted paddy was dehusked in rubber roll sheller (Satake Corporation, Japan) and the head rice obtained was used for the studies. Native paddy dehusked in rubber roll sheller was used as control brown rice. A portion of the brown rice was milled to 7% degree of polish in Mcgill miller No.3 with a load of 1 pound and used as a highly polished raw rice sample. The sprouted and dehusked rice, native brown and milled rice samples were stored in air tight container at −4 °C till analysis. The samples were thawed and pulverized into fine (−60 mesh) powder in a hand operated coffee grinder immediately prior to analysis.

Scanning electron microscopy

Cross-section of grain samples prepared by gentle fracturing at 1/4th from germ side, were fixed on metal stub using double-sided sellotape and coated with gold (∼100  °A) in the KSE 24 M high vacuum evaporator. Then the topographical features of the samples were viewed by a scanning electron microscope (LEO 435VP, LEO Electron Microscopy Ltd., Cambridge, England), and the selected regions of the samples depicting distinct morphological features were photographed.

Proximate composition

Moisture, protein, ash, and ether extractives were determined as per AOAC (1999), where as dietary fiber was determined by enzymatic-gravimetric method of Asp, Johansson, Hallmer, and Silijestrom (1983). Total carbohydrates are calculated by the difference, i.e., by substracting from 100 the sum of the values (per 100g) for moisture, protein, fat and ash. The free sugars extracted with 70% ethanol were estimated by phenol–sulfuric acid method of Dubois, Gilles, Hamilton, Rebers, and Smith (1956). The activity of the amylases extracted from samples with 0.05 M sodium phosphate buffer (pH 6.0) was assayed according to Bernfeld (1955). One unit of the amylase activity was defined as the amount of enzyme that catalyzes the liberation of reducing sugars equivalent to 1 μmol of maltose/min under the assay conditions.

Fatty acids

The lipid contents of the samples extracted with chloroform–methanol solvent were saponified using alcoholic KOH and methylated with 14% borontrifluoride. The methylated lipids were extracted in hexane and their fatty acid composition was determined in GC (Shimadzu GC 15A; Recorder – Shimadzu CR7A; Detector-FID; Column – 15% DEGS AW/DMCS packed in 3 m SS column; ageing temperature 210 °C and nitrogen flow 40 ml/min) (Sakina & Gopalkrishna, 2004).

Extraction of sample for assessment of total antioxidant activity and characterization of vitamin E and γ–oryzanol

The flour samples (0.1 g) were extracted with methanol (1 ml) for 1 h with occasional stirring, centrifuged at 3000 rpm and the supernatant was filtered and stored at –20 °C till used (Chen & Bergman, 2005).

Estimation of total antioxidant activity

Total antioxidant activities of different samples were quantified using phosphomolybdenum reagent (Pilar, Manvel, & Mignel, 1999). Results were calculated and expressed as α-tocopherol equivalents using the molar extinction coefficient of α-tocopherol.

Vitamin E characterization and quantification

Characterization of vitamin E (tocopherols and tocotrienols) and quantification was carried out by reverse phase HPLC (Shimadzu CBM-10A system with RF10AXL fluorescent detector and LC10AT pump). The chromatograms were recorded and processed by LC-10A class software. The standards and extracts were separated chromatographically on Merck Hibar C18 column (4.6 mm × 250 mm, 5 μm) using a gradient solvent system consisting of mixture of acetonitrile, methanol, isopropanol, and aqueous acetic acid [45:40:5:10] in pump A and mixture of acetonitrile, methanol, isopropanol [25:70:5] in pump B at a flow rate of 1 ml/min. The fluorescence detector was set at excitation and emission wavelengths of 298 and 328 nm, respectively (Chen & Bergman, 2005). Comparison of the retention times with those of standards permitted the identification of different vitamin E components. Standards of both tocopherol and tocotrienol exhibited a linear response in the range as follows; α 3–45, γ 3–55, and δ 0.4–5 ng.

γ-Oryzanol content and characterization

The γ-oryzanol in the samples was extracted with hexane and the concentration was determined spectrophotometrically by measuring the absorbance at 314 nm followed by calculation using the specific extinction coefficient (358.9) according to the procedure of Seetharamaiah and Prabhakar (1986).

To know the γ-oryzanol composition, the methanolic extracts were fractionated chromatographically on Shimadzu Shim-Pack Prep-ODS(H) column (4.6 mm × 250 mm, 5 μm) using a modified procedure of Rogers et al. (1993) as reported by Chen and Bergman (2005). Gradient solvent system consisted of mixture of acetonitrile, methanol, isopropanol, and aqueous acetic acid [45:40:5:10] in pump A and mixture of acetonitrile, methanol, isopropanol [25:70:5] in pump B set at 1 ml/min. Major fractions were identified according to chromatogram pattern reported by Xu and Godber (1999) and Chen and Bergman (2005). Absorbance spectra at 200–400 nm of major peaks observed in UV-VIS diode array detector (SPD-M10AVP). Peaks were confirmed for the characteristic spectrum of γ-oryzanol which is having λ max in the range 310–330 nm. Further the percentage composition of four major γ-oryzanol components were calculated from the area of the peaks in the chromatogram.

Statistical analysis

The values in the tables are mean ± SD (standard deviation) of three independent determinations and were subjected to Student t-test to study the level of significance at p < 0.05 (Snedecor & Cochran, 1994).

Results and discussion

Proximate composition

Proximate composition of raw brown (RBR) as well as polished rice (PR) and biotransformed brown rice (BTR) are shown in Supplementary Table 1. Protein content in RBR of IR 64 and BPT was 85 and 76 g/kg and that of their BTR counterparts contained 80 and 73 g/kg, respectively. A slight decrease in the protein content may be due to the hydrolytic action of proteases, during germination and its utilization for the development of vegetative growth. It has been reported that hydrolysis of prolamin occurs during germination leading to increase in the contents of some of the essential amino acids and also the γ-amino butyric acid (GABA), a neurotransmitter, (Saikusa, Horino, & Mori, 1994). Total fat contents in brown rice of IR 64 and BPT was 36 and 30 g/kg, respectively, and it decreased by 55% on germination of both the varieties. This may be due to lipolysis and utilization of fat as a source of energy during the initial stage of germination of grains. It may be noted that rice embryo is a good source of fat and the embryo happens to be the first tissue of seeds that takes part actively during germination. Accordingly, the fat content invariably decreases on germination. Total ash, which is an indicator of mineral content, in rice was 13 and 15 g/kg in RBRs of IR 64 and BPT and its content did not show any significant changes in BTR.

A several fold increase in the amylase activity was observed in both IR 64 and BPT after biotransformation, as the increase was from 0.52 to 44.9 U/g in IR 64 and from 0.8 to 46.3 U/g in BPT. Increase in amylase activity during germination and consequent increase in the low molecular weight sugar contents due to starch digestion has been reported in other cereals also (Malleshi & Desikachar, 1982). Free sugars content in BTR increased from 13 to 42 g/kg in IR 64 and from 17 to 51 g/kg in BPT. The activity of amylases increase when the rice malt slurry is heated and this leads to complete hydrolysis of the starch and resultant decrease in water holding capacity of the starch, or in other terms decrease in the dietary bulk of starchy foods (Malleshi & Desikachar, 1982). This is highly desirable in development of number of specialty foods such as infant, weaning, and enteral foods from BTR.

Scanning electron microscopy

Scanning electron photomicrographs (Supplementary Figure 1) of the inner endosperm of the rice kernels show that, in the raw brown rice, the compound starch granules are in compact form surrounded by proteinaceous matter, but in BTR the endosperm granules are less compact with slightly large size due to partial digestion of cell walls. This could be due to partial digestion of the smaller granules besides loss of cells facilitating the swelling of bigger granules by providing ample space for the bigger granules to expand in situ. Normally during germination of cereals proteases and cell wall digesting material digest the matrix covering starch granules and facilitate amylase to easily access the starch granule. Individual as well as compound starch granules were also clearly visible after germination.

Nutrients, nutraceuticals, and antioxidant activity

The content of some nutrients and nutraceuticals in the native, milled and biotransformed IR 64 and BPT rice is given in Supplementary Table 2. Dietary fiber, considered as a phytochemical with nutraceutical properties and its content in RBR of IR 64 and BPT were 46.0 and 40.5 g/kg and its content decreased to 44.5 and 37.6 g/kg, respectively in biotransformed rice. However, the decrease from 41.5 to 36.8 g/kg in IR 64 and 37 to 31.7 g/kg in BPT was mainly in the form of insoluble fiber. On the contrary, soluble fiber contents in RBR increased from 4.5 to 7.7 g/kg in IR 64 and 3.5 to 5.9 g/kg in BPT. These changes may be due to the hydrolytic action of enzymes on the non-starchy polysaccharides, the main dietary fiber constituents. There are conflicting reports about the changes in the dietary fiber content on malting of cereals. Increase in insoluble fiber and decrease in soluble fiber content during germination was reported by Ohtsubo et al. (2005), which may be due to the inclusion of the rootlets of sprouts during analysis. Decrease in dietary fiber in our experiment may be due to the use of de-vegetated material, and similar observation was made by Rao and Muralikrishna (2004) also. Biotransformed brown rice from both the varieties was found to be superior with respect to the content of soluble fiber. Since, fully polished rice is devoid of bran layers, the content of insoluble dietary fiber in HPR of IR 64 and BPT was 10.3 and 9.0 g/kg, respectively and soluble fiber was below the level of detection. Presence of both soluble and insoluble fiber in biotransformed brown rice provides nutritional advantages over polished rice. Reports on physiologic responses of soluble and insoluble dietary fiber components and their health benefits (Plaami 1997) and the role of fiber in pre-germinated brown rice, in lowering the blood glucose (Seki et al., 2005) have been published.

Gas chromatographic analysis of fatty acids showed that major fatty acids present in the lipids of both raw and germinated rice of IR 64 and BPT were palmitic, oleic, and linoleic acids, forming 21, 40, and 36%, respectively. The remaining 3% consisted of myristic, stearic, and linolenic acids. There was no noticeable change in the fatty acid composition in BTR compared to RBR. More or less similar results were obtained for BPT rice also. In the fully polished rice also there was no difference in fatty acid composition compared to the respective BTRs. The higher proportion of unsaturated fatty acids in BTRs makes them nutritionally superior even though the fat content is very low.

Characterization of vitamin E by reverse phase HPLC showed the presence of δ, γ, and α tocotrienols and also the tocopherols in both RBR and BTR of theboth varieties of rice (Supplementary Figure 2). Generally the β form of tocotrienols and tocopherols incereals is negligible (Ha et al., 2006) and even the minute quantity present elutes along with γ form in the reversed phase HPLC. Among the constituents of vitamin E, major components in both rice varieties were γ-tocotrienol, α-tocopherol, and γ-tocopherol. Content of total vitamin E in RBR of IR 64 and BPT were 29.4 mg/kg and 26.5 mg/kg, respectively. Biotransformation did not cause any significant change in the vitamin E content in IR 64 (28.4 mg/kg), however its content increased considerably (35.3 mg/kg) in BPT. Further analysis of the vitamin E components showed an increase in γ-tocotrienol from 20.2 to 23.1 mg/kg in IR 64 and from18.4 to 28.0 mg/kg in BPT and consequent increase in total tocotrienols in both the rice varieties. But γ-tocopherols decreased from 4.97 to 0.69 mg/kg in IR 64 and 4.7 to 0.75 mg/kg in BPT. Even though significant change was not observed in α-tocopherol in IR 64, its increase was substantial in BPT. Decrease in γ-tocopherol resulted in consequent reduction in the content of total tocopherols. However these changes did not affect the content of total vitamin E, since its contribution was only around 25%.

There are no reports on changes in vitamin E components in rice on germination unlike in pulses where the major vitamin E component happens to be tocopherols. Increase in α-tocopherol content during germination and decrease in the content of γ-tocopherol in lupin seeds have been reported by Frias, Miranda, Doblado, and Concepcion (2005). Reports suggest that α-tocopherol is formed by the transformation of γ-tocopherol (Shintani & Dean, 1998) and which could explain the decrease in γ-tocopherol observed in both rice varieties in our investigation. Increase in α-tocopherol in the germinated rice was observed in BPT, but no significant change was observed in the case of IR 64. This difference may be due to the increased metabolic activity in BPT rice on germination as evidenced by the higher percentage increase in γ-tocotrienols.

Increase in the major vitamin E component in rice namely γ-tocotrienol and also retention or increase in total vitamin E contents in the biotransformed rice indicate that germination is a promising method for enhancement of nutraceutical properties of rice. These components if taken as supplements are reported to have a number of beneficial effects (Qureshi et al., 1997).

The content of γ-oryzanol in brown rice of IR 64 and BPT were 280 and 295 mg/kg, respectively and germination did not alter its content. Content of γ-oryzanol in BTR of rice varieties was ∼5 times higher than that in the polished rice in both the varieties. Since γ-oryzanol is a mixture of ferulic acid esters of triterpene alcohols and sterols, further to know whether there is any change in γ-oryzanol composition on biotransformation, characterization of γ-oryzanol using HPLC was carried out in IR 64 variety only. It showed (Supplementary Figure 3) the presence of major fractions like cycloartenyl ferulate (21%), 24-methylene cycloartenyl ferulate (40%), campesteryl ferulate (26%), and sitosteryl ferulate (13%) in control brown rice and no significant changes were observed in their values in biotransformed rice. γ-Oryzanol fractions are reported to have high antioxidant activity with highest activity for 24 methylene cycloartenyl ferulate (Xu et al., 2001), and anti-inflammatory properties with highest activity for cycloartenyl ferulate and for 24 methylene cycloartenyl ferulate (Akihisa et al., 2000). Since the contents of these components are not affected during the biotransformation, such rice would be more biopotent.

Total antioxidant activity of methanolic extracts of the BTR was 25% lower compared to RBR (8.2 and 8.1 M α-tocopherol equivalent/kg) in IR 64 as well as BPT. Total antioxidant activity is contributed by vitamins, γ-oryzanol, phytate, phenolics, etc. (Kikuzaki, Hisamoto, Hirose, Akiyama, & Taniguchi, 2002). Rao and Muralikrishna (2006) also reported the presence of feruloyl arabinoxylans and its antioxidant potential in germinated rice. Even though the presence of these phenolic compounds is very low they contribute in the total antioxidant activity and other health beneficial effects. Decrease in the total antioxidant activity in BTR may be due to the leaching of soluble polyphenols. However, ∼75% of the activity is retained in spite of various processing steps. BTR of both varieties are also superior to polished rice in having 120% more total antioxidant activity.

Conclusions

Germination or biotransformation of IR 64 and BPT variety of paddy brought changes in nutritional and phytochemical nutraceutical characteristics. In the biotransformed brown rice, increase in free sugars due to amylase activity as well as increase in health beneficial soluble fiber was observed among nutrients. Increase in the content of γ-tocotrienol and total tocotrienol, and complete retention of γ-oryzanol content and its fractions were observed among nutraceuticals. However, decrease in tocopherols, which is a minor component, and moderate loss in total antioxidant activity were observed in biotransformed rice. In spite of various processing steps, there is improvement and maximal retention of major nutraceuticals and bioactivity in biotransformed brown rice, and it is nutritionally and nutraceutically superior to conventional highly polished rice.

Supplementary material

Supplementary Table 1. Proximate composition of native and biotransformed brown, and raw polished IR 64 and BPT rice.

Tabla adicional 1. Composición aproximada de arroz integral autóctono, biotransformado y pulido, IR 64 y BPT.

	IR 64			BPT
	Raw brown	Biotransformed brown	Raw polished	Raw brown	Biotransformed brown	Raw polished
Moisture (g/kg)	112 + 2.5^a	78 + 6.1^b	113 + 4.4^a	125 + 4.7^a	66 + 6^b	124 + 4.2^a
Protein (g/kg)	85 + 3.6^a	80 + 5.0^a	82 + 4.2^a	76 + 3.2^a	73 + 5.5^a	71 + 3.1^a
Fat (g/kg)	36 + 3.1^a	17 + 2.6^b	6.0 + 0.6^c	30 + 3.2^a	15.7 + 2.5^b	5.7 + 1.5^c
Ash (g/kg)	13.0 + 1.4^a	12.5 + 1.5^a	9.0 + 0.5^b	15 + 1.5^a	15.9 + 1.7^a	11.0 + 1.7^b
Carbohydrate (g/kg)	750 + 1.3^a	810 + 2.0^b	790 + 3.2^c	750 + 8.3^a	830 + 11.6^b	790 + 3.6^c
Free sugar (g/kg)	13 + 1.0^a	42 + 3.1^b	3.0 + 0.3^c	17 + 1.3^a	51 + 4.7 ^b	4.1 + 0.3^c
Values are mean ± SD of three independent observations. Values with different superscripts (a, b, c) in a row were significantly different at p < 0.05 on independent testing of these two varieties.
Los Valores son promedio ± desvinción estándar de tes observaciones independientes. Valores con letras distintas (a, b, c) en una fila fueron significativamente diferentes a p < 0,05 en pruebas independientes de estas dos variedades.

Supplementary Table 2. Some nutritional and nutraceutical components of raw and biotransformed brown, and polished IR 64 and BPT rice.

Tabla adicional 2. Algunos componentes nutricionales y nutracéuticos de arroz integral, biotransformado y pulido, IR64 y BPT.

	IR 64			BPT
	Raw brown	Biotransformed brown	Raw polished	Raw brown	Biotransformed brown	Raw polished
Insoluble fibre (g/kg)	41.5+1.3^a	36.8+2.4^b	10.3+1.1^c	37.0+2.0^a	31.7+2.5^b	9.0+1.0^c
Soluble fibre (g/kg)	4.5+0.5^a	7.7+0.4^b	ND	3.5+0.2^a	5.9+0.2^b	ND
Total fibre (g/kg)	46.0+0.9^a	44.5+2.6^a	10.3+1.1^b	40.5+2.2^a	37.6+2.7^a	9.0+1.0^b
Palmitic acid (%)	21.0+1.7^a	19.0+1.0^a	20.0+1.8	22.0+2.2^a	22.0+0.6^a	23.0+1.0
Oleic acid (%)	40.0+1.5^a	39.0+1.1^a	41.0+2.1	41.0+2.1^a	39.0+2.1^a	38.0+3.5
Linoleic acid (%)	36.0+0.6^a	38.0+2.4^a	36.0+2.9^a	35.0+1.7^a	36.0+1.7^a	36.0+2.5^a
γ-Tocotrienol (mg/kg)	20.2+1.0^a	23.1+0.7^b	6.4+0.2^c	18.4+0.8^a	28.0+0.9^b	6.8+0.3^c
Total tocotrienols (mg/kg)	21.8+0.6^a	25.5+1.3^b	6.7+0.2^c	19.3+0.8^a	29.8+1.0^b	7.0+0.3^c
γ-Tocopherol (mg/kg)	4.97+0.8^a	0.69+0.2^b	0.71+0.04^b	4.7+1.0^a	0.75+0.1^b	0.1+.0.02^c
α-Tocopherol (mg/kg)	2.54+0.5^a	2.20+0.2^a	0.39+0.006^b	2.5+0.2^a	4.7+0.02^b	0.86+0.04^c
Total tocopherols (mg/kg)	7.61+0.5^a	2.90+0.0.2^b	1.10+0.1^c	7.20+1.1^a	5.5+0.3^b	0.97+0.06 ^c
Total vitamin E (mg/kg)	29.4+0.8^a	28.4+1.5^a	7.8+0.2^b	26.5+1.8^a	35.3+1.3^b	7.97+0.3^c
Oryzanol (mg/kg)	280+20.6^a	296+18.6^a	54.0+4.5^b	295+19.1^a	297+21.1^a	66+5.7^b
Total antioxidant activity (M α-Toco pherol Eq:/kg)	8.2+0.62^a	6.4+0.2^b	2.9+0.4^c	8.1+0.5^a	6.5.0+0.66^b	3.3+0.25^c
Values are mean ± SD of three independent observations. Values with different superscripts (a, b, c) in a row significantly different at p < 0.05 for IR 64 and BPT variety separately.
Los valores son promedio ± desviación estándar de tres observaciones independientes. Valores con letras distintas (a, b, c) en una fila fueron significativamente diferentes a p < 0,05 para IR 64 y por separado.

Supplementary Figure 1. Scanning electron photomicrographs (×500) of the cross section of IR 64 rice showing the endospermal changes in (A) raw brown (B) bitotransformed and (C) raw polished.

Figura adicional 1. Fotomicrogramas (×500) de escaneado electrónico de la sección transversal de arroz IR 64 mostrando los cambios en el endospermo en (A) arroz integral, (B) biotransformado y (C) arroz pulido.

Supplementary Figure 2. HPLC Chromatograms of vitamin E in (A) IR 64 raw brown rice and (B) biotransformed brown rice (a, b, c = δ, γ, α tocotrienols; d, e, f = δ, γ, α tocopherols).

Figura adicional 2. Cromatogramas HPLC de vitamina E en (A) arroz integral IR 64 y (B) arroz integral biotransformado (a, b, c = δ, γ, α tocotrienoles; d, e, f = δ, γ, α tocoferoles).

Supplementary Figure 3. HPLC chromatogram of γ-oryzanol fractions in IR64 raw brown rice (CAF = cycloartenyl ferulate, MCAF = 24-methylene cycloartenyl ferulate, CF = campesteryl ferulate, SF = sitosteryl ferulate).

Figura adicional 3. Cromatograma HPLC de fracciones de γ-oryzanol en arroz integral IR 64 (CAF = ferulato cicloartenilo, MCAF = ferulato 24-metilén cicloartenilo, CF = campesteril ferulato, SF = sitosteril ferulato).