Abstract
The present study focused on variation in the fatty acid (FA) composition of the different lipids in black, red and green rice brans. Total lipids were fractionated using TLC into nine subfractions. The lipids comprised mainly triacylglycerols (TAG: 78.0 - 81.6 wt%), free FA (FFA: 5.6 - 8.8 wt%), and phospholipids (PL: 6.3 - 7.0 wt%). The PL components included phosphatidyl choline (52.6 - 57.2 wt%), phosphatidyl inositol (22.3 - 25.2 wt%) and phosphatidyl ethanolamine (11.4 - 16.4 wt%). Comparison of these cultivars showed no significant differences (P > 0.05) in FA composition of TAG, FFA and PL. FA composition of TAG among the three cultivars was characterized in the rice brans: unsaturated FA were predominantly concentrated at the sn-2 position (97.7 - 98.5 wt%) and saturated FA primarily occupied the sn-1 or sn-3 position (29.8 - 31.8 wt%). Comparison of individual PLs revealed significant differences (P < 0.05) in FA composition. Black and red rice brans were very similar, with green rice bran exhibiting a few differences. The results revealed no significant differences (P > 0.05) in the proportional composition of FA with increased degree of milling.
Introduction
Rice (Oryza sativa L.) is one of the staple cereal crops cultivated in the world and feeds more than half of the world's population (Xue et al., 2008). Colored rices, which are found in Japan, are also classified as Oryza sativa L. Rice is primarily processed and consumed as a whole grain. Moreover, rice grain quality is an important economic trait that influences rice production in many rice-producing areas. Although the fat or oil in rice grain is low (i.e., 2 - 3%) and is concentrated in the germ and bran, it is a key determinant of the processing and cooking quality of rice (Zhou et al., 2002a). For instance, the surface lipid content is thought to be an indication of the degree of milling (Siebenmorgan et al., 2006), an important factor in determining the nutritional value and economic return of the milled rice. The amount of bran remaining on the rice kernel after milling affects rice quality, appearance, and texture, and rice is thus milled to the end-use preferences of different consumers (Perdon, et al., 2001; Saleh and Meullenet, 2007). In addition, rice lipid, which frequently forms complexes with starch granules, was shown to affect starch gelatinization, water availability to starch and rice swelling, and thus influences the eating and cooking qualities of rice (Champagne et al., 1990; Marshall et al., 1990; Tester and Morrison, 1990).
Beside dietary consumption, the unique health benefits of rice fat, which includes many unsaturated fatty acids, has drawn much attention (Jennings and Akoh, 2009). A number of studies have shown that rice bran oil reduces the harmful cholesterol (low-density lipoprotein) without affecting the good cholesterol (high-density lipoprotein) in plasma (Kahlon et al., 1992; Sugano and Tsuji, 1997; Wilson et al., 2007). In addition, rice bran oil, which is rich in tocotrienol (Vitamin E), has anti-cancer and anti-radiation effects (Sugano and Tsuji, 1997). On the other hand, some reports have shown that the hydrolysis and oxidation of rice fat are responsible for rice aging and deterioration of grain flavor during storage, and low-oil rice cultivars are more suitable for storage (Zhou et al., 2002b). Therefore, it is important to determine the genetic basis of rice fat synthesis in order to further develop both high-oil and low-oil rice cultivars to address different market needs.
Many investigations on lipid fractions of rice brans have been published (Przybylski et al., 2009; Jennings and Akoh, 2009). However, to the best of our knowledge, no comparative study between different colored rice bran cultivars has been conducted with respect to the rice milling process. Therefore, the objectives of this study are to compare the lipid components and fatty acid (FA) distribution of several acyl lipids in three colored rice bran cultivars, based on differences in the degree of milling.
Materials and Methods
Materials Commercially available mature rice seeds used in this study were from three different colored Japanese cultivars: black (Okunomurasaki), red (Beniroman) and green (Midorimochi) grown in the same district (Asukamura, Takaichigun, Nara) of Japan during the fall of 2012. Black and red rices were nonglutinous rices, and green rice was a glutinous rice. For analysis, seeds were selected for uniformity based on seed weight: 18.2 - 18.8 mg for black, 20.5 - 20.7 mg for red and 18.6 - 18.9 mg for green. The seeds were hand-selected to eliminate cracked or otherwise damaged seeds. Seeds of each cultivar were packed in polyethylene bags under nitrogen gas and placed in a stainless-steel container at −20°C before further analysis.
Reagents and standards All chemicals and solvents used were of analytical grade (Nacalai Tesque, Kyoto, Japan); however, diethyl ether was further purified to remove peroxides. Thin-layer chromatography (TLC) pre-coated silica gel 60 plates (10 × 20 or 20 × 20 cm, 0.25 mm thickness) were purchased from Merck (Darmstadt, Germany). TLC standards mixture, containing monoacylglycerols (MAG), diacylglycerols (DAG), free fatty acids (FFA), triacylglycerols (TAG), steryl esters (SE) and hydrocarbons (HC), was obtained from Nacalai Tesque. A phospholipid (PL) kit from Serdary Research Laboratory (Mississauga, ON, Canada) was used as the PL standard. Lipase from porcine pancreas was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and used after purification with acetone and then diethyl ether, as described previously (Yoshida et al., 2011). Glyceryl-sn-1,3-myristate-sn-2-oleate (Sigma-Aldrich Co.) was used as the TAG standard for enzymatic hydrolysis. Fatty acid methyl esters (FAME) standards (F & OR mixture #3) were obtained from Altech-Applied Science (State College, PA, USA). The internal standards, pentadecane and methyl pentadecanoate, were purchased from Merck, and then 100 mg of each was dissolved in n-hexane (20 mL), respectively. Boron trifluoride (BF3) in methanol (14%; Wako Pure Chemical Inc., Osaka, Japan) was used to prepare the FAME.
Extraction of lipids Rice bran of each cultivar (500 g) was prepared using a domestic miller (BR-CA25, Zojirushi Ltd., Osaka, Japan). Before extraction, brans obtained from half-milled rice and well-milled rice were defined as HMB and WMB, respectively. Total lipids were extracted from 20 g of bran in 300 mL chloroform/methanol (2 : 1, v/v) with vigorous shaking for 15 min at 0°C three times, following the Folch procedure (Folch et al., 1957). These solvents contained 0.01% butylated hydroxytoluene to inhibit oxidative degradation of lipids during analysis. The filtrates were combined and dried in a rotary vacuum evaporator at 35°C The residue was dissolved in 100 mL chloroform/methanol (2 : 1, v/v). The extracted lipids were weighed to determine the lipid content of the rice bran and then transferred to a 25-mL brownglass volumetric flask with chloroform/methanol (2 : 1, v/v) and kept in the dark under nitrogen at −20°C until analysis was conducted.
Lipid analysis According to the previously outlined procedure (Yoshida et al., 2009), total lipids were fractionated by TLC into nine fractions using a solvent system of n-hexane/diethyl ether/acetic acid (80:20:1, v/v/v). Bands corresponding to HC (front), SE (Rf: 0.96), TAG (Rf: 0.58), unknown (Rf: 0.42), FFA (Rf: 0.32), 1,3-DAG (Rf: 0.23), 1,2-DAG (Rf: 0.18), MAG (Rf: 0.12) and PL (origin) were scraped into separate test-tubes (105 − 16 mm; poly (tetrafluoroethylene) coated screw caps). FAME were prepared from the isolated lipids by heating with silica gel for 30 min at 80°C (Kitts et al., 2004). The n-hexane layer containing the FAME was recovered and dried over anhydrous Na2SO4. Then 2-mL aliquots of the extracts were injected into a gas chromatograph (GC: Shimadzu Model-14B, Kyoto, Japan) equipped with a hydrogen flame ionization detector (FID) at 350°C and a polar capillary column (ULBO HE-SS-10 for FAME, fused silica WCOT [no. PSC5481], cyanopropyl silicone, 30 m × 0.32 mm i.d.; Shinwa Chem. Ind., Ltd., Kyoto, Japan) at a column temperature of 180°C.
Helium was used as the carrier gas at a flow rate of 1.5 mL/min, and the GC was operated under a constant pressure of 180 kPa. Both injection and detector temperatures were set at 250°C. The oven temperature was programmed from an initial temperature of 180°C (held for 2 min), and increased to 200°C at a rate of 2°C/min, and then held isothermally (200°C) for 15 min. The component peaks were identified and compared against that of the standard FAME using an electronic integrator (Shimadzu C-R6A). The detection limit was 0.05 wt% of total FA for each FAME in the FAME mixture, and the results are expressed as wt% of total FAME. The other GC conditions were as previously reported (Yoshida et al., 2009).
Samples of the extracted polar lipids, obtained as described above, were further separated by TLC into several fractions with chloroform/methanol/acetic acid/deionized water (170:30:20:7, by volume) as the mobile phase. Bands corresponding to diphosphatidyl glycerol (DPG), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl choline (PC) and phosphatidyl inositol (PI) and others were scraped into separate test tubes, respectively. Then, methyl pentadecanoate (10 - 25 µg) of a standard solution (5 mg/mL) was added to each tube as the internal standard. FAME were prepared by the same method as described above and determined by GC.
Enzymatic hydrolysis of TAG TAG hydrolysis was carried out as previously reported (Yoshida and Alexander, 1983). After approximately 60% of the TAG was hydrolyzed, 0.5 mL of 6 M HCl and 1 mL ethanol were added to stop the reaction. In the preliminary study, no FA (oleic acid) at the sn-2 position of the standard TAG was transferred to the sn-1 or sn-3 position at 60% hydrolysis for 30 min. The FFA and sn-2 MAG bands were scraped into test tubes and then methylated (Kitts et al., 2004). The procedure was checked by comparing the FA compositions of the original TAG and the TAG remaining after partial hydrolysis.
Statistical analyses Data in this study were expressed as means ± SD for at least three independent experiments. Differences between the means of individual groups were assessed by one-way analysis of variance and Tukey's multiple range test using the SAS statistical software package (SAS, Cary, NC, USA). Differences were considered significant at P < 0.05.
Results and Discussion
The content of rice brans and their lipids in colored rice cultivars The bran contents of the black, red and green cultivars (500 g) were 41.9 g (8.4 wt%), 29.2 g (5.9 wt%) and 9.4 g (3.9 wt%) from the HMB, and 76.7 g (15.3 wt%), 59.2 g (11.8 wt%) and 37.5 g (7.5 wt%) from the WMB, respectively. The bran contents of HMB and WMB differed significantly (P < 0.05) among the three cultivars. On the other hand, the lipid contents obtained from these rice brans were 7.4 g (17.7 wt%), 5.4 g (18.4 wt%) and 4.8 g (24.7 wt%) from the HMB and then 11.0 g (14.4 wt%), 8.9 g (15.1 wt%) and 7.9 g (21.1 wt%) from the WMB, for black, red and green cultivars, respectively. The percentage of lipid contents was significantly (P < 0.05) higher in the HMB than in the WMB, and was in the rank order: green > red > black in both rice brans.
Lipid compositions in the rice brans Profiles of the lipid components were compared among the HMB and WMB for the three cultivars (Table 1). Predominant components were TAG (HMB: 78.4 - 81.2 wt%; WMB: 78.0 - 81.6 wt%), followed by FFA (HMB: 6.7 - 7.6 wt%; WMB: 5.6 - 8.8 wt%) and PL (HMB: 6.3 - 6.8 wt%; WMB: 6.5 - 7.0 wt%). When comparing the nine lipid components of the HMB and WMB among all three cultivars, the percentage of TAG was significantly (P < 0.05) lower in the green cultivar than that in the black or red cultivar, while the percentage of FFA was significantly (P < 0.05) higher in the green cultivar than that in the black cultivar. However, with a few exceptions, no substantial differences (P > 0.05) in the content of the lipid components were observed between the values estimated by a combination analysis of TLC and GC using the internal standard (C15:0). Presumably, minor components, such as FFA, 1,3- or 1,2-DAG, and MAG, may be formed by the partial enzymatic hydrolysis of reserve TAG during the storage of rice seeds (Aboul-Nasr et al., 1997; Okunishi and Ohtsubo, 2008). The lipid components resulting from ‘fat by hydrolysis’ in starch granules were determined, showing the presence of FFA with lysolecithin and lysoglycolipids (Hirayama and Matsuda, 1973).
Table 1.
Lipid components of colored rice brans.
1
| Lipid class |
HMB Cultivar |
WMB Cultivar |
| Black |
Red |
Green |
Black |
Red |
Green |
| Hydrocarbons |
29.7 ± 0.7 |
21.5 ± 0.5 |
19.2 ± 0.4 |
77.3 ± 1.9 |
71.5 ± 1.7 |
39.4 ± 1.0 |
|
(0.4)a |
(0.4)a |
(0.4)a |
(0.7)b |
(0.8)b |
(0.5)a |
| Sterylesters |
74.2 ± 1.8 |
64.6 ± 1.6 |
52.7 ± 1.3 |
154.6 ± 3.8 |
89.4 ± 2.3 |
141.7 ± 3.5 |
|
(1.0)a |
(1.2)b |
(l.l)b |
(1.4)c |
(1.0)a |
(1.8)d |
| Triacylgrycerols |
5999 ± 40 |
4374 ± 39 |
3752 ± 38 |
8968 ± 65 |
7276 ± 48 |
6124 ± 42 |
|
(80.7)b |
(81.2)b |
(78.4)a |
(81.2)b |
(81.6)b |
(78.0)a |
| Unknown |
59.3 ± 1.4 |
26.9 ± 0.7 |
24.0 ± 0.5 |
44.2 ± 1.1 |
71.5 ± 1.8 |
23.6 ± 0.6 |
|
(0.8)c |
(0.5)b |
(0.5)b |
(0.4)a |
(0.8)c |
(0.3)a |
| Free fetty acids |
497 ± 12 |
399 ± 10 |
364 ± 9 |
619 ± 15 |
500 ± 12 |
693 ± 16 |
|
(6.7)b |
(7.4)c |
(7.6)c |
(5.6)a |
(5.6)a |
(8.8)d |
| 1, 3-Diacylgrycerols |
118.7 ± 2.9 |
70.0 ± 1.7 |
110.2 ± 2.8 |
176.7 ± 4.3 |
98.3 ± 2.4 |
78.7 ± 1.9 |
|
(1.6)c |
(1.3)b |
(2.3)d |
(1.5)c |
(l.l)a |
(1.0)a |
| 1,2-Diacylgrycerols |
111.2 ± 2.7 |
64.6 ± 1.6 |
63.9 ± 1.5 |
154.6 ± 3.8 |
134.1 ± 3.4 |
78.7 ± 2.0 |
|
(1.5)c |
(1.2)ab |
(1.3)b |
(1.4)b |
(1.5)c |
(1.0)e |
| Monoacylgrycerols |
66.7 ± 1.6 |
26.9 ± 0.7 |
76.7 ± 1.8 |
88.4 ± 2.2 |
98.3 ± 2.5 |
141.7 ± 3.5 |
|
(0.9)b |
(0.5)a |
(1.6)b |
(0.8)b |
(l.l)c |
(1.8)e |
| Phospholipids |
475 ± 11 |
339 ± 8.5 |
326 ± 8.2 |
773 ± 19 |
581 ± 14 |
535 ± 13 |
|
(6.4)a |
(6.3)a |
(6.8)b |
(7.0)c |
(6.5)a |
(6.8)b |
HMB:half-milled rice bran.
WMB:well-milled rice bran.
*Mean values ± standard error. Each value represents the average of three determinations, and is expressed as mg lipid per 500 g of rice. Values in the same row with different superscripts are signifcantly different between the individual cultivars (P < 0.05). Values in parentheses are relative wt% contents of the individual lipids in total lipids.
To clarify the distribution of individual PL in the rice bran lipids, further separation of the PL fraction several fractions (DPG, PE, PC, PI and others) was carried out on TLC in the presence of authentic standards. Comparisons were made for the content of PE, PC, PI and others among the HMB and WMB of the three cultivars (Table 2). Regardless of cultivar, PC, PI and PE were the principal PL in the rice bran lipids, while DPG and PG were minor components (< 2.3 wt%) and others include phosphatidic acid and lyso-PL. However, significant differences (P < 0.05) were observed between the HMB and WMB when comparing DPG, PE and PG contents of the three cultivars. Generally, the percentage of PI was very similar between the HMB (22.3 - 24.5 wt%) and WMB (23.8 - 25.2 wt%) from the three cultivars.
Table 2.
The phospholipid contents in lipid of colored rice brans.
*
| Phospholipid |
HMB Cultivar |
WMB Cultivar |
| Black |
Red |
Green |
Black |
Red |
Green |
| Diphosphatidyl glycerol |
10.9 ± 0.3 |
7.5 ± 0.2 |
7.2 ± 0.2 |
16.2 ± 0.5 |
11.6 ± 0.3 |
10.7 ± 0.3 |
|
(2.3)b |
(2.2)b |
(2.2)b |
(2.1)a |
(2.0)a |
(2.0)a |
| Phosphatidyl ethanolamine |
68.9 ± 1.7 |
55.6 ± 1.3 |
53.5 ± 1.3 |
88.1 ± 2.3 |
69.7 ± 1.7 |
68.0 ± 1.7 |
|
(14.5)c |
(16.4)d |
(16.4)d |
(11.4)a |
(12.0)b |
(12.7)b |
| Phosphatidyl glycerol |
8.5 ± 0.2 |
6.4 ± 0.2 |
5.5 ± 0.2 |
12.4 ± 0.3 |
8.7 ± 0.2 |
8.6 ± 0.2 |
|
(1.8)b |
(1.9)bc |
(1.7)b |
(1.6)a |
(1.5)a |
(1.6)a |
| Phosphatidyl choline |
255.6 ± 6.2 |
180.3 ± 4.3 |
171.5 ± 4.3 |
437.6 ± 10.7 |
332.4 ± 8.1 |
292.0 ± 7.3 |
|
(53.8)a |
(53.0)a |
(52.6)a |
(56.6)b |
(57.2)c |
(54.5)ab |
| Phosphatidyl inositol |
116.4 ± 2.8 |
75.6 ± 1.7 |
73.7 ± 1.7 |
194.8 ± 4.8 |
138.3 ± 3.5 |
132.9 ± 3.3 |
|
(24.5)b |
(22.3)a |
(22.6)a |
(25.2)c |
(23.8)b |
(24.8)b |
| Others |
14.7 ± 0.4 |
14.2 ± 0.4 |
14.7 ± 0.4 |
24.0 ± 0.6 |
20.3 ± 0.5 |
23.6 ± 0.6 |
|
(3.1)a |
(4.2)c |
(4.5)d |
(3.1)a |
(3.5)b |
(4.4)d |
HMB:half-milled rice bran.
WMB:well-milled rice bran.
*Mean values ± standard error. Each value represents the average of three determinations, and is expressed as mg lipid per 500 g of rice. Values in the same row with different superscripts are signifcantly different between the individual cultivars (P < 0.05). Values in parentheses are relative wt% contents of the individual lipids in PL. “Others” include minor PL components such as phosphatidic acid and lysophospholipid.
FA composition of major lipids in the rice brans FA composition of total lipids, TAG, FFA and PL in the rice bran lipids were compared among the HMB and WMB of the three cultivars (Table 3). The principal FA components were generally palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, the percentage of which varied according to these lipid classes. The distribution of total unsaturated FA, particularly linoleic (18:2n-6) and oleic acids, which accounted for 77.2 - 79.3 wt% (total lipids), 77.9 - 79.9 wt% (TAG), 69.4 - 73.5 wt% (FFA), and patterns were very similar within total lipids, TAG, FFA or PL among the HMB and WMB from all three cultivars. These lipids presented with high amounts of 71.6 - 76.1 wt% (PL), respectively.
Some differences (P < 0.05) in FA composition were noted when comparing the four lipid classes (total lipids, TAG, FFA and PL), as shown in Table 3. With a few exceptions for FFA fraction between the HMB and WMB, the percentage of oleic (18:1) acid was significantly (P < 0.05) higher in the black and red bran lipids than in the green bran lipids for the four lipid classes (total lipids, TAG, and PL). On the other hand, the percentage of linoleic (18:2n-6) acid was significantly (P < 0.05) higher in the green rice bran than in the black or red rice bran for total lipids, TAG and PL fractions. It has been demonstrated that there exists distinct differences between nonglutinous and glutinous types of cereals in lipid content and fatty acid composition (Fujino and Mano, 1972; Taira, 1984; Taira and Lee, 1988). These FA distributions for black and red rice brans (nonglutinous) are very similar to the results observed for rice bran lipids in the cultivars Koshihikari, Haenuki, Akitakomachi, Hitomibore and Sasanishiki reported in a previous paper (Yoshida et al., 2011). The data for FA distribution of minor lipid components (SE, 1,3- or 1,2-DAG, and MAG ) in Table 2, were not included in Table 3 because these lipid components were present in too low concentrations to provide reliable results for their FA distributions. Therefore, their FA distribution will be studied in our laboratory in the future.
Table 3.
The fatty acid distributions in major lipid components of colored rice brans.
*
| Bran |
Lipid class |
Cultivar |
Fatty acid (wt-%) |
Total USFA |
| 16:0 |
18:0 |
18:1 |
18:2 |
18:3 |
Others |
| HMB |
Total |
Black |
19.4 ± 1.0a |
1.6 ± 0.1b |
43.2 ± 1.3c |
33.2 ± 1.1a |
1.4 ± 0.1a |
1.2 ± 0.1a |
78.0a |
| Red |
19.3 ± 1.0a |
2.4 ± 0.1c |
40.1 ± 1.2b |
35.2 ± 1.2a |
1.5 ± 0.1a |
1.5 ± 0.1b |
77.2a |
| Green |
19.3 ± 1.0a |
1.6 ± 0.1b |
36.1 ± 1.2a |
40.5 ± 1.3b |
1.5 ± 0.1a |
1.0 ± 0.1a |
78.3a |
| TAG |
Black |
18.5 ± 0.8a |
1.7 ± 0.1a |
46.1 ± 2.1c |
31.5 ± 1.2a |
1.2 ± 0.1a |
1.0 ± 0.1a |
79.0a |
| Red |
17.1 ± 0.7a |
2.0 ± 0.1b |
43.5 ± 2.0b |
34.9 ± 1.3b |
1.3 ± 0.1a |
1.2 ± 0.1b |
79.9a |
| Green |
19.3 ± 0.8b |
1.6 ± 0.1a |
35.1 ± 1.2a |
41.5 ± 1.5C |
1.5 ± 0.1b |
1.0 ± 0.1a |
78.4a |
| sn-2 position |
Black |
1.7 ± 0.1a |
nd |
48.4 ± 2.2c |
48.7 ± 2.3a |
1.2 ± 0.1a |
nd |
98.3a |
| Red |
1.9 ± 0.1a |
nd |
46.7 ± 2.0c |
50.1 ± 2.2a |
1.3 ± 0.1a |
nd |
98.1a |
| Green |
3.5 ± 0.2b |
nd |
35.9 ± 1.2a |
59.1 ± 2.4c |
1.5 ± 0.1b |
nd |
97.7a |
| sn-1, 3 position |
Black |
26.9 ± 1.0b |
2.6 ± 0.1a |
45.0 ± 2.1c |
21.9 ± 1.0a |
1.2 ± 0.1a |
2.4 ± 0.1b |
68.8a |
| Red |
24.7 ± 1.0a |
3.0 ± 0.2b |
41.9 ± 2.0b |
27.0 ± 1.2c |
1.3 ± 0.1a |
2.1 ± 0.1b |
70.2b |
| Green |
27.5 ± 1.1b |
2.4 ± 0.1a |
39.1 ± 1.3a |
27.8 ± 1.2c |
1.4 ± 0.1a |
1.8 ± 0.1a |
68.2a |
| FFA |
Black |
25.2 ± 1.0a |
2.7 ± 0.1b |
41.5 ± 2.1a |
28.7 ± 1.0c |
1.2 ± 0.1b |
0.7 ± 0.1a |
71.6a |
| Red |
24.1 ± 1.0a |
1.9 ± 0.1a |
44.9 ± 2.2b |
27.8 ± 1.1c |
0.6 ± 0.1a |
0.7 ± 0.1a |
73.5a |
| Green |
26.1 ± 1.1b |
1.8 ± 0.1a |
43.8 ± 2.1b |
25.4 ± 1.3a |
1.6 ± 0.1c |
1.3 ± 0.1b |
70.9a |
| PL |
Black |
22.3 ± 1.0b |
1.3 ± 0.1b |
38.6 ± 1.3c |
34.6 ± 1.3a |
1.5 ± 0.1b |
1.7 ± 0.1a |
74.9a |
| Red |
23.1 ± 1.0b |
1.1 ± 0.1b |
38.9 ± 1.2c |
34.7 ± 1.3a |
1.5 ± 0.1b |
0.7 ± 0.1a |
75.4a |
| Green |
25.4 ± 1.1c |
0.8 ± 0.1a |
29.9 ± 1.2a |
41.8 ± 1.8c |
1.3 ± 0.1a |
0.8 ± 0.1a |
73.2a |
| WMB |
Total |
Black |
18.4 ± 0.8a |
1.6 ± 0.1b |
43.3 ± 1.7c |
34.2 ± 1.2a |
1.3 ± 0.1a |
1.2 ± 0.1a |
78.8a |
| Red |
18.3 ± 0.8a |
1.4 ± 0.1a |
42.5 ± 2.0c |
35.3 ± 1.2a |
1.2 ± 0.1a |
1.3 ± 0.1a |
79.3a |
| Green |
19.3 ± 1.0a |
1.6 ± 0.1b |
36.1 ± 1.2a |
40.5 ± 2.0b |
1.5 ± 0.1a |
1.0 ± 0.1a |
78.3a |
| TAG |
Black |
18.5 ± 0.8a |
1.7 ± 0.1a |
46.1 ± 2.0c |
31.5 ± 1.2a |
1.2 ± 0.1a |
1.0 ± 0.1a |
79.0a |
| Red |
17.1 ± 0.8a |
2.0 ± 0.1b |
43.5 ± 2.0b |
34.9 ± 1.2b |
1.3 ± 0.1a |
1.2 ± 0.1b |
79.9a |
| Green |
19.5 ± 1.2b |
1.6 ± 0.1a |
37.0 ± 1.3a |
39.3 ± 1.4c |
1.4 ± 0.1b |
1.2 ± 0.1b |
77.9a |
| sn-2 position |
Black |
2.0 ± 0.1a |
nd |
47.3 ± 2.0c |
49.4 ± 2.1a |
1.3 ± 0.1a |
nd |
98.0a |
| Red |
1.9 ± 0.1a |
nd |
41.6 ± 2.0a |
54.9 ± 2.0b |
1.6 ± 0.1b |
nd |
98.5a |
| Green |
2.3 ± 0.1b |
nd |
37.5 ± 1.5a |
58.6 ± 2.2c |
1.6 ± 0.1b |
nd |
97.7a |
| sn-1, 3 disposition |
Black |
26.8 ± 1.0b |
2.4 ± 0.1a |
41.2 ± 1.4b |
26.6 ± 1.1c |
1.3 ± 0.1a |
1.7 ± 0.1a |
69.3a |
| Red |
24.7 ± 1.0a |
2.8 ± 0.1b |
44.7 ± 1.8c |
24.9 ± 1.0b |
1.2 ± 0.1a |
1.7 ± 0.1a |
70.9b |
| Green |
27.8 ± 1.2b |
2.4 ± 0.1a |
36.9 ± 1.2a |
29.8 ± 1.2c |
1.3 ± 0.1a |
1.8 ± 0.1a |
68.2a |
| FFA |
Black |
24.7 ± 1.0a |
2.2 ± 0.1b |
42.4 ± 1.5b |
26.7 ± 1.2b |
1.8 ± 0.1c |
2.2 ± 0.1c |
69.4a |
| Red |
25.5 ± 1.0a |
1.6 ± 0.1a |
43.6 ± 1.5b |
26.8 ± 1.2b |
1.1 ± 0.1a |
1.4 ± 0.1a |
71.8a |
| Green |
26.2 ± 1.0b |
2.1 ± 0.1b |
40.9 ± 1.3a |
28.6 ± 1.2c |
1.0 ± 0.1a |
1.2 ± 0.1a |
70.8a |
| PL |
Black |
21.8 ± 1.0a |
1.3 ± 0.1b |
40.7 ± 1.3d |
33.7 ± 1.2a |
1.2 ± 0.1a |
1.3 ± 0.1b |
76.1b |
| Red |
23.3 ± 1.1b |
1.3 ± 0.1b |
39.2 ± 1.3c |
34.0 ± 1.3a |
1.2 ± 0.1a |
1.0 ± 0.1b |
74.7a |
| Green |
25.4 ± 1.1c |
1.3 ± 0.1b |
32.1 ± 1.2b |
38.2 ± 1.4b |
1.3 ± 0.1a |
1.7 ± 0.1c |
71.6a |
HMB : half milled rice bran.
WMB : well milled rice bran.
USFA : Unsaturated fatty acids.
nd: not detectable.
*Mean values ± standard error. Each value represents the average of three determinations, and is expressed as relative wt% contents of the individual fatty acids. Values in the same column with different superscripts are signifcantly different between the individual cultivars (P < 0.05). “Others” include minor fatty acid such as C14 : 0, C20 : 0 and C22 : 0.
The characteristics of component and positional distribution of FA in the TAG were compared (Table 3). Linoleic (18:2n-6) acid was predominantly (48.7 - 59.1 wt%) concentrated in the sn-2 TAG molecules, while saturated FA such as palmitic (16:0) and stearic (18:0) acids were primarily sn-1 or sn-3 TAG molecules. With a few exceptions, however, oleic acid was almost evenly distributed in the sn-1, 2 or 3 molecules, corroborating results of previous researchers (Reske et al., 1997). No significant differences (P < 0.05) occurred in the FA distributions among the HMB and WMB from all three cultivars. Taken together, the regiospecific distribution profiles of FA in the TAG were very similar to the results obtained from seed lipids of soybeans and corn (Arcos et al., 2000).
FA compositions of major PL in the rice brans Table 4 shows typical FA distributions of PE, PC and PI in the HMB and WMB of the three cultivars. The major FA in the three PLs were commonly palmitic (16:0), oleic and linoleic (18:2n-6) acids. These FA distributions in PE and PC were very similar among the three cultivars: palmitic (18.3 - 23.9 wt%), oleic (32.6 - 48.8 wt%) and linoleic (27.0 - 41.6 wt%) acids. These PLs contained a high percentage of total unsaturated FA, particularly linoleic (18:2n-6) and oleic acids, which accounted for 73.4 - 79.4 wt% (PE) and 75.6 - 80.6 wt% (PC), respectively. Some differences (P < 0.05) in FA composition were noted among the three PL classes, as shown in Table 4. When comparing FA compositions in the three PLs in the HMB and WMB of these rice bran lipids, the content of oleic acid was significantly (P < 0.05) higher in PC (38.7 - 48.8 wt%) than in PE (32.6 - 37.6 wt%), while the content of linoleic acid (18:2n-6) was significantly (P < 0.05) higher in PE (39.7 - 41.6 wt%) than in PC (27.0 - 35.7 wt%). However, the content of palmitic acid (16:0) was very similar between PE (18.3 - 23.9 wt%) and PC (17.8 - 22.5 wt%), respectively. However, PI was unique in that it had higher saturated FA (41.6 - 44.6 wt%) content than other PLs, although their distributions were very similar among the HMB and WMB from the three cultivars. Particularly, the content of palmitic (16:0) acid was significantly (P < 0.05) higher in PI (39.5 - 41.9 wt%) than in PE (18.3 - 23.9 wt%) or PC (17.8 - 22.5 wt%). Generally, no significant differences (P > 0.05) in the three PLs were observed between the HMB and WMB, while significant differences (P < 0.05) were observed among black or red and green rice brans. The data for FA distributions of minor lipid components, such as DPG, phosphatidic acid, PG and lyso-PL, were not included in Table 4 because the samples were too small to obtain reliable results for these individual PLs. Therefore, these will be considered in future work.
Table 4.
The fatty acid distributions in major phospholipids of colored rice brans.
*
| Bran |
Phospholipid |
Cultivar |
Fatty acid (wt-%) |
Total USFA |
| 16:0 |
18:0 |
18:1 |
18:2 |
18:3 |
Others |
| HMB |
Phosphatidyl ethanolamine |
Black |
18.3 ± 0.5a |
1.6 ± 0.1b |
37.6 ± 0.9c |
40.5 ± 1.0a |
1.1 ± 0.1a |
0.9 ± 0.1b |
79.4c |
| Red |
20.6 ± 0.6a |
1.1 ± 0.1a |
35.6 ± 0.9b |
41.2 ± 1.0a |
0.9 ± 0.1a |
0.6 ± 0.1a |
77.9b |
| Green |
22.9 ± 0.7b |
0.9 ± 0.1a |
32.6 ± 0.7a |
41.6 ± 1.0a |
1.0 ± 0.1a |
1.0 ± 0.1b |
74.3a |
| Phosphatidyl choline |
Black |
21.7 ± 0.5b |
1.3 ± 0.1a |
47.8 ± 1.2c |
28.0 ± 0.7a |
0.5 ± 0.1a |
0.7 ± 0.1a |
76.5a |
| Red |
21.8 ± 0.5b |
1.2 ± 0.1a |
42.3 ± 1.1b |
32.7 ± 0.8b |
1.4 ± 0.1c |
0.6 ± 0.1a |
76.6a |
| Green |
22.5 ± 0.6b |
1.2 ± 0.1a |
39.5 ± 1.0c |
34.8 ± 0.9c |
1.0 ± 0.1b |
1.0 ± 0.1b |
75.6a |
| Phosphatidyl inositol |
Black |
41.9 ± 1.1a |
2.2 ± 0.1b |
29.2 ± 0.7c |
25.5 ± 0.6a |
0.6 ± 0.1a |
0.6 ± 0.1a |
55.4a |
| Red |
41.5 ± 1.1a |
1.9 ± 0.1a |
26.4 ± 0.7b |
28.7 ± 0.7b |
1.0 ± 0.1b |
0.5 ± 0.1a |
56.2a |
| Green |
39.5 ± 1.1a |
1.7 ± 0.1a |
24.2 ± 0.6a |
33.0 ± 0.8c |
1.0 ± 0.1b |
0.6 ± 0.1a |
58.4b |
| WMB |
Phosphatidyl ethanolamine |
Black |
19.3 ± 0.4a |
1.6 ± 0.1b |
36.3 ± 1.2b |
40.8 ± 0.8a |
1.1 ± 0.1a |
0.9 ± 0.1b |
78.4b |
| Red |
20.5 ± 0.5a |
1.1 ± 0.1a |
37.2 ± 0.9c |
39.7 ± 1.0a |
0.9 ± 0.1a |
0.6 ± 0.1a |
77.9b |
| Green |
23.9 ± 0.8b |
0.9 ± 0.1a |
32.6 ± 0.6a |
40.6 ± 1.1a |
1.0 ± 0.1a |
1.0 ± 0.1b |
73.4a |
| Phosphatidyl choline |
Black |
21.7 ± 0.5b |
1.3 ± 0.1a |
48.8 ± 1.2c |
27.0 ± 0.7a |
0.5 ± 0.1a |
0.7 ± 0.1a |
76.5a |
| Red |
17.8 ± 0.4a |
1.2 ± 0.1a |
43.3 ± 1.2b |
35.7 ± 0.9c |
1.4 ± 0.1c |
0.6 ± 0.1a |
80.6b |
| Green |
22.3 ± 0.6b |
1.2 ± 0.1a |
38.7 ± 1.2a |
34.8 ± 0.8c |
2.0 ± 0.1d |
1.0 ± 0.1b |
75.6a |
| Phosphatidyl inositol |
Black |
41.9 ± 1.0a |
2.2 ± 0.1b |
29.2 ± 0.7c |
25.5 ± 0.6a |
0.6 ± 0.1a |
0.6 ± 0.1a |
55.6a |
| Red |
40.5 ± 1.1a |
1.9 ± 0.1a |
27.4 ± 0.7b |
28.7 ± 0.7b |
1.0 ± 0.1b |
0.5 ± 0.1a |
56.2a |
| Green |
39.5 ± 1.0a |
1.7 ± 0.1a |
24.2 ± 0.6a |
33.0 ± 0.8c |
1.0 ± 0.1b |
0.6 ± 0.1a |
58.4b |
HMB : half-milled rice bran.
WMB : well-milled rice bran.
USFA : Unsaturated fatty acids.
*Mean values ± standard error. Each value is expressed as relative wt% contents of the individual FA in each lipid class. Values in the same column with different superscripts are signifcantly different between the individual cultivars (P < 0.05). “Others” include minor FA such as C14 : 0, C20 : 0 and C22:0
Conclusions
The major lipid components in the three different Japanese colored rice bran cultivars were TAG, FFA and PL, while HC, SE, 1,2- and 1,3-DAG were present in minor proportions. The PL components included PC, PI and PE. In general, with a few exceptions, no significant differences (P > 0.05) were observed in the FA distribution patterns among all three cultivars. FA distribution of TAG in the HMB and WMB of all three cultivars was characterized as: unsaturated FA predominantly concentrated at the sn-2 position and saturated FA primary occupying the sn-1 or sn-3 position in these lipids. The distribution patterns in the different acyl lipids and their FA profiles in rice bran lipids were very similar to each other among the three cultivars. Currently, consumer awareness of health food products is increasing and food scientists have been searching for interesting sources of healthful natural components. The results showed that rice bran extracts contain large amounts of nutraceuticals with proven positive health effects (Ha et al., 2006).
Acknowledgements We thank Prof. Bruce Holub of the Department of Human Health and Nutritional Sciences, University of Guelph, Canada, for reviewing and commenting on this manuscript. Financial support for part of this study was provided by a Grant-in-Aid for Special Assistance for Working Expenses of the Private University and Cooperative Research Center of Life Science (‘Academic Frontier’ Project, 2010-2013).
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