J. Sci. Food Agric. 1979, 30, 475481
Nutrient Content and Distribution in Milling Fractions of Rice Grain Adoracion P. Resurreccion, Bienvenido 0. Juliano and Yonemi Tanakaa Chemistry Department, International Rice Research Institute, Los Barios, Laguna, Philippines (Manuscript received 6 September 1978)
Nutrient content and distribution in a low-protein (7.5 %) rice and a high-protein (10.8 %) rice were studied by analysing successive abrasive milling fractions of brown rice. Non-starch constituents decreased from the surface to the centre of the grain in both rices, except that the highest protein fraction in high-protein rice was the subaleurone layer. Starch and amylose contents of starch increased progressively from the surface to the centre of the grain and were lower in high-protein grain. Although this low-protein rice had lower total ash content than the high-protein rice, other samples of the same low-protein rice had comparable ash content to the highprotein rice. Glutelin accounted for 87-93% of milled rice protein. Protein and protein bodies of the sub-aleurone layer and inner endosperm have similar aminograms, and electrophoretic patterns using analytical and SDS-polyacrylamide disc gels. 1. Introduction Nutrient distribution in brown rice has been studied in detail by analysis of its successive abrasive milling f r a c t i ~ n s . l -High-protein ~ rices have been shown to have a more even distribution of protein, but the outer endosperm has the highest protein f r a c t i ~ n . ~The # s outer layers of milled rice are more rich in non-starch constituents, including B vitamins, than the core of the endo~perm.l-~> Although the brown rices contained similar B-vitamin contents, high-protein milled rice had higher contents of thiamin, riboflavin and phytin P than low-protein rice,g but similar content of ash and total fibre.10 As part of our study of the nutritive value of high-protein rices, we examined the nutrient content and distribution in a low-protein rice (IR32) and a high-protein rice (IR480-5-9). Dietary fibre analysis by the neutral detergent method was also undertaken in consideration of recent interest in the importance of fibre in health and mineral absorption in cereal-based diets.ll Particular interest was placed on the protein of the sub-aleurone layers, since these cell layers contain crystalline protein bodies that are absent in the inner endosperm.l2>13 2. Experimental
Samples of rough rice (IR480-5-9 and IR32) were obtained from the 1975-76 crop of the International Rice Research Institute farm. They were dehulled and milled in Satake dehullers. These have been used for study of protein quality of milled ri~e.149~5 Abrasive milling was done with a Satake grain-testing mill TM-05 to obtain successive milling layers. Samples were ground by passing them through a Udy cyclone mill with 60-mesh screen prior to analysis. Samples were analysed in dublicate for moisture content from loss in weight after 1 h at 13O0C,l6 for protein by micro-Kjeldahl N multiplied by the factor 5.95,lG for crude fat by extraction with refluxing petroleum ether in Soxhlet extractors, for crude fibre16and for dietary fibre by the neutral a Present address: Fukuoka Agricultural Senior High School, Oosano Dazaifu-machi, Chikusi-gun, Fukuoka, Japan.
0022-5142/79/0500-0475 $02.00 0 1979 Society of Chemical Industry 415
A. P. Resurreccion et al.
416
detergent method,16 and for crude ash by dry ashing 1 h at 325°C and then 16 h at 490°C. K, Mg, Fe and Zn were determined on the ash by atomic absorption spectroscopy and P by colorimetry in 0.43 % ammonium molybdate and 1 % ascorbic acid in 1.08~-HC1.Thiamin contents of brown and milled rice were determined by the thiochrome method, using Taka-diastase, thiochrome decalso, and a Coleman Model 12C electronic photofluorometer and calculated as pg of thiamin (not thiamin hydrochloride) per gram of sample.lG Riboflavin was determined by the fluorometric method.16Phytin P was analysed by extraction with 3 irichloroacetic acid (TCA), precipitation with magnesium nitrate and calcium acetate, and ashing of the precipitate for P determination.17 Protein fractions of sub-aleurone and inner endosperm were extracted, precipitated by TCA, and analysed for Kjeldahl N as previously described.18 Protein extract was analysed by analytical disc gel electrophoresis, and SDS-/I-mercaptoethanol extract was subjected to SDS-polyacrylamide gel electrophoresis.~g~ 20 Protein bodies were prepared by destarching raw, gelatinised, and cooked sub-aleurone and inner endosperm with crystalline bovine pancreatic a-amylase (Sigma Chem. C O ) . Milled ~ ~ rice and protein bodies were hydrolysed under N2 in ~N-HCIin a sealed tube at 110°C for 23 h and subjected to amino acid analysis in a Spinco Model 120C analyser with Spinco PA-35 and AA-15 or Hamilton H70 resins.'s, 19 3. Results and discussion 3.1. Gross composition Low-protein and high-protein brown rices had similar content of crude fibre, crude fat and Zny Table 1. Nutrient content in milling fractions of a low-protein and a high-protein rice grainu
Endosperm
Nutrient
Sample
Moisture (%) Protein ( % N x 5.95) Starch ( P/,anhydroglucose) Amylose (%) Crude fibre (%) Dietary fibre (%) Crude fat (%) Crude ash
(x)
Total P (%) Total K (%) Total Mg (%) Total Fe (parts 10 Total Zn (parts Phytin P (%)
G,
Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein
Brown rice (0-100) 10.4 10.7 7.5 10.8 69.2 66.3 27.6 22.6 0.7 0.7 2.2 1.8 3.0 2.7 0.81 1.58 0.16 0.38 0.20 0.28 0.08 0.16 12 16 28 30 0.10 0.28
Milled rice (90-100) 11.6 11.1
6.8 10.5 79.7 74.8 28.9 23.4 0.1 0.1 0.6 0.5 0.5 0.4 0.3
0.8 0.06 0.16 0.10 0.12 0.02 0.05 4 5 17 24 0.02 0.07
-
Bran (0-6) 9.3 10.6 14.2 14.0 14.6 11.7 6.1 1.6 8.8 6.3 25.8 18.5 20.7 20.9 5.8 11.9 1.00 2.60 1.12 2.09 0.60 1.33 105 89 124 103 0.84 2.28
Polish (6-12) 11.1 10.7 13.1 18.8 42.9 35.6 15.6 11.2 2.4 2.1 7.0 4.7 12.8 13.7 3.5 6.7 0.67 1.61 0.68 1.18 0.41 0.76 53 44 57 65 0.60 1.58
1R32 (1976 dry season) and lR480-5-9 (1975 wet season), respectively.
Subaleurone (12-20) 11.9 11.4 12.3 20.4 66.1 57.1 25.8 19.0 0.1 0.2 0.3 0.6 3.0 2.9 1 .o I .8 0.21 0.35 0.21 0.24 0.10 0.14 17 16 22 47 0.21 0.24
Middle (20-30) 12.1 10.3 10.2 16.0 75.5 67.1 28.6 23.1 0.1 0.1 0.4 0.3 0.3 0.3 0.4 0.6 0.06 0.09 0.08 0.06 0.01 0.02 4 6 14 28 0.010 0.025
Inner (30-100) 11.5 9.4 5.4 7.4 76.9 74.8 30.6 25.8 0.1 0.1 0.2 0.4 0.2 0. I 0.2 0.3 0.05 0.05 0.07 0.04 0.004 0.003 3 2 14 20 0.005 0.003
Nutrient content and distribution in rice grain
477
but the high-protein rice had higher crude ash (P, K, Mg and Fe) but lower starch and dietary fibre than the low-protein rice (Table 1). Differences in amylose content was unrelated to protein per se since IR32 is high in amylose (> 25 %) and IR480-5-9 is intermediate in amylose (20-25 %). Among 38 samples of brown rice with 6.7-11.5% protein, crude fat content was relatively constant at 2.1-3.2 %.22 Processing of both low-protein and high-protein brown rices to milled rices resulted in a decrease in all nutrient levels except starch and amylose (Table 1). Loss of protein from milling was greater for low-protein rice than for high-protein rice (Table 2). This is because the bran of low-protein rice was found to have a higher percentage of the total protein and protein content decreased progressively with each subsequent inner layer. By contrast, the sub-aleurone layer is the highest protein content fraction for the high-protein rice. Similar differences in protein distribution is reported between these two types of rice by Mitsuda and Murakami,* and Houston.3 Starch and amylose content increased progressively with the degree of milling in both samples (Tables 1 and 2) as it is present mainly in the endosperm.12 Although crude fibre and dietary fibre levels were similar in the two brown rice samples, the bran of low-protein rice tended to be higher in fibre. By contrast, reported 9.2-21.1 % crude fibre and crude fat distribution was similar in both samples. Mod et 28.744.7 % neutral detergent fibre in rice bran, 0.3 % crude fibre and 2 . 4 2 . 9% neutral detergent fibre in milled rice, and 1 . 1 % crude fibre and 3.8-6.0% neutral detergent fibre in brown rice. Ash content was nearly twice as high in the high-protein brown rice as in the low-protein rice (Table l), but its distribution was similar in both samples (Table 2). All milling fractions of the highTable 2. Nutrient distribution in milling fractions of a low-protein and a high-protein rice grain0 Endosperm
Nutrient Protein ( % N x 5.95) Starch (%,anhydroglucose) Amylose (%) Crude fibre (%) Dietary fibre (%) Crude fat (%) Crude ash (%) Total P (%) Total K
(7;)
Total Mg (%)
Total Fe (parts 10-6) Total Zn (parts Phytin P (%)
Sample
Bran (0-6)
Polish (6-12)
Sub-aleurone ( 1 2-20)
Middle (20-30)
Inner (30-100)
Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein
11.5 8.1 1.2 1.0 1.3 0.4 69.5 63 .O 71.3 63.4 51.3 52.1 42.5 46.1 38.0 48.0 37.0 50.3 49.7 56.7 47.2 47.4 31.2 20.2 46.8 53.6
10.6 10.9 3.1 3.2 3.4 3.0 19.0 21 .o 19.3 16.1 31.8 34.1 25.7 26.3 25.4 29.8 22.4 28.4 34.0 32.4 23.9 23.5 14.4 12.7 33.4 37.1
13.3 15.7 7.5 6.9 1.5 6.7
13.7 15.4 10.8 10.1 10.3 10.2 1.3 1.7 1.8 1.7 1.2 1.3 4.9 3.9 3.8 2.8 4.4 2.4 1.4 1.4 3.0 5.3 5.9 9.1 0.9 1.0
50.9 49.9 76.8 78.8 77.5 79.7 9.2 11.6 6.5 16.0 5.8 2.9 17.1 13.7 22.2 10.8 27.0 11.2 3.9 1.5 15.7 12.4 41.1 45.7 3.2 0.8
1 .o
2.7 1.1 2.8 9.9 9.6 9.8 9.4 10.6 8.6 9.2 1.7 11.0 8.0 10.2 11.4 7.4 12.3 15.6 7.5
"Percentage of total nutrient content of the five milling fractions in Table 1. IR32 (1976 dry season) IR480-5-9 (1975 wet season), respectively.
A. P.Resurreccion er al.
418
protein samples had higher ash content. Bran, polish and the sub-aleurone layer had higher levels of P, K, and Mg in high-protein rice than in low-protein rice, and the milled rice also reflected this difference. By contrast, there was little difference in Fe levels between the two rices, but the lowprotein rice had higher Fe levels in bran and polish. In the case of Zn, total content in brown rice was similar, but more of it was in the endosperm in high-protein rice. The results are consistent with the reported distribution and highest concentration of P, K, Mg and Fe either in the aleurone layer or the pericarp.1324925 At least 80% of brown rice P and 40% of milled rice P and 90% of rice bran P is phytin P.26 Phytin is mainly present in the form of salts of potassium and magnesium as globoid inclusions in rice aleurone protein parti~les.2~ Phytate P was 74% of brown rice P in high-protein rice and 60% in low-protein rice. It comprised only 44 % of milled rice P in high-protein rice and 25 % in lowprotein rice. In the bran, phytate P contributed 88% of total P in high-protein rice and 84% in low-protein rice. Phytin in rice bran has been reported to be a pepsin inhibitor.27 The protein of cooked high-protein milled rice had 60.0% apparent digestibility as compared to 66.2 % apparent digestibility for low-protein milled rice in Filipino ~hi1dren.l~ However, the true protein digestibility of raw milled rice in rats was higher in the high-protein rice (100.4%) than in the low-protein rice (98.1 %).14 The rices had phytin P content of 0.07 and 0.02%, respectively and their crude and dietary fibre contents were similar (Table 1). Probably, the difference in phytate content of these milled rices is not involved in the difference in apparent digestibility of their protein in children. Other brown rice samples of the low-protein rice (IR32) had higher ash content (1.20-1.53 %) than the sample in Table 1 , indicating that the low-protein brown rice sample used in the detailed study was unusually low in ash content. The composition of another sample of IR32 rice with 7.7 % protein was 1.34% crude ash, 65.9%starch, 0.7 % crude fibre, 1.7% neutral detergent fibre, 3.0% crude fat, 0.24% total P, 0.25 % total K, 0.14% total Mg, 18 parts 10-6 total Fe, and 19 total Zn. Thus, the main nutrient affected by protein change was starch. parts Similar decreases in thiamin, riboflavin, phytin P and other non-starch constituents were also noted in milling other samples of low-protein (IR8)and high-protein (IR480-5-9) rices (Table 3). In this pair o f samples, the high-protein rice also had a higher crude ash content. Four samples of IR8 and eight other samples of IR480-5-9 showed variable content of milled rice protein (7.5-12.4%), ash (0.324.77%),total P (0.084.16 %), phytate P (0.024.07%),and Zn (10-22 parts Degree of milling is an additional variable affecting composition in milled rice. Crude fibre (0.6-1.5%), crude fat ( 0 . 3 4 6 % ) and thiamin levels (0.24.8 pg g-1) were also not Table 3. Nutrient content of brown and milled rices of two samples ditrering in protein content Low-protein ricea
High-protein rice0
Nutrient
Brown rice
Milled rice
Brown rice
Milled rice
Moisture (%) Protein (%N x 5.95) Crude fat (%) Crude fibre (%) Dietary fibre (%) Crude ash (%) Total P (%) Phytin P (%) Total Zn (parts Thiamin (pg g-1) Riboflavin (pg g-1)
10.1 8.5 2.3 1.1 1.8 1.36 0.30 0.21 20 3.97 0.48
9.9 7.6 0.3 0.6 1.3 0.36 0.08 0.03 10 0.20 0.19
11.2 11.6 3.1 2.2 2.7 1.74 0.30 0.25 25 3.61 0.50
10.7 10.8 0.5 1.1 1.5 0.55 0.13 0.06 16 0.82 0.23
a
IR8, 1974 dry season crop. 113480-5-9, 1974 dry season crop.
Nutrient content and distribution in rice grain
419
necessarily higher in higher-protein milled rices. Our data are also consistent with the reported uneven distribution within milled rice of protein, fat, ash, amylose, starch, Mg, P, K, phytate and vitarnin~.b-~ 3.2. Protein
Amino acid composition showed lower lysine content for protein in flour and destarched flour from sub-aleurone of IR480-5-9 rice than from inner endosperm, but no difference was noted for IR32 rice (Table 4). Results with destarched flour from raw and gelatinised milled rice and with cooked milled rices were similar.21Normand et al.4 and Kennedy and Schelstraete28also found no appreciable difference in aminogram of various overmilling fractions of milled rice. A study of the corresponding albumin, globulin, prolamin and glutelin contents of the subaleurone and inner endosperm of high-protein and low-protein rices showed that the difference in protein content between the four samples was mainly in glutelin content (Table 4). Glutelin made up 87-93 % of protein of these milling fractions. The low lysine content of protein in the sub-aleurone layer of IR480-5-9 rice may be due to its higher prolamin content than the three other samples. Table 4. Aminogram and protein fractions of sub-aleurone and inner endosperm (core) of high-protein and lowprotein milled rices and destarched rices prepared from them by a-amylolysis of cooked rice (g per 16.8 g N) Sub-aleurone layer (12-20 %) High-protein rice= Amino acid LYs His NH3 Arg ASP Thr Ser Glu Pro CYs GlY Ala Val Met Ile Leu Tyr Phe Protein
Flour
Low-protein rice*
Destarched flour
Flour
( %)"
Prolamin
Destarched flour
Flour
Destarched flour
3.63 2.62 2.57 8.82 10.5 3.62 4.84 22.8 5.15 2.46 4.76 6.06 6.36 2.16 4.49 9.12 5.24 5.98
3.50 2.49 2.20 9.06 9.92 3.68 5.25 22.8 5.09 3.46 4.68 6.26 6.38 3.00 4.49 9.46 5.96 6.10
4.17 2.58 2.55 9.13 10.2 3.61 4.87 20.8 5.57 2.47 4.73 6.18 6.13 2.30 4.27 8.92 4.74 5.90
3.88 2.41 2.26 9.11 9.92 3.52 4.92 21.2 5.08 3.20 4.90 6.38 6.53 3.14 4.52 9.58 6.23 5.87
3.60 2.50 2.78 8.26 11.3 3.66 4.80 20.6 5.40 2.29 5.08 6.42 6.47 2.30 4.59 9.12 3.58 5.91
3.54 2.46 2.40 9.04 10.2 3.64 5.16 22.4 5.15 3.28 4.58 6.06 6.44 3.61 4.51 9.16 6.22 6.05
21.7
85.6
12.6
79.0
7.34
80.2
5.20
84.0
0.4(2)
0.5 (4)
0.04 (1)
0.1 (2)
1.2 (6)
1 .O (8)
0 . 3 (4)
0 . 5 (10)
1.o (4)
0 . 2 (2)
0.2 (3)
0.1 (2)
19.1 (88)
10.9 (87)
6.8 (93)
4.5 (87)
( %)"
Glutelin
Flour
Low-protein rice0
3.22 2.37 2.46 8.88 9.97 3.46 5.10 23.0 5.06 3.33 4.46 5.99 6.59 2.88 4.54 9.53 6.68 6.18
(%)" Globulin
Destarched flour
High-protein riced
3.33 2.63 2.74 9.05 9.90 3.51 4.99 22.50 5.30 2.26 4.32 5.94 5.97 2.72 4.28 9.12 6.25 6.05
( %)"
Albumin
Inner endosperm (70-100 %)
( %)" ~
IR480-5-9, 1975 wet season crop. 1R32, 1976 dry season crop. c Based on milled rice at 12% moisture.
480
A. P. Resurreccion et a/.
Albumin content of total protein was higher in the sub-aleurone layer than in the inner endosperm, in agreement with Houston et a/.29The proportion of globulin in the protein of sub-aleurone was higher than that in the inner endosperm protein only in the high-protein rice. Based on these results, the crystalline protein bodies and the small spherical protein bodies present only in the sub-aleurone layer'3 together probably have the same aminogram as the common large spherical protein bodies. Analytical disc gel electrophoresis of albumins and globulins from the sub-aleurone and inner endosperm of low-protein and high-protein rices indicated little difference in the electrophoregram of albumins in the four samples. Albumins consisted of two major bands and at least three minor ones as reported earlier.20Two bands that were minor in sub-aleurone albumins were more intense in the albumins from the inner endosperm of low-protein rice. By contrast, globulins from the inner endosperm showed more minor bands than globulins from the sub-aleurone in both rices. Both exhibited two major globulin bands as reported earlier.20 SDS-polyacrylamide disc gel electrophoregrams of the total protein extract of the sub-aleurone and inner endosperm of both rices showed mainly the three major subunit fractions of glutelin with molecular wt 38 000, 25 000, and 16 OOO;19 this confirms the fact that glutelin is the predominant protein in these samples (Table 4).
4. Conclusions Nutrient analysis of milling fractions of low-protein rice and a high-protein rice showed that all non-starch constituents decreased from surface to centre of the grain. However, the milling fraction with the highest protein was the sub-aleurone layer in the high-protein sample; bran had the highest protein level in the low-protein sample. The difference in milled-rice protein was mainly in the glutelin content. The only other major nutrient in which low-protein and high-protein rice differed was starch content. Protein of the sub-aleurone layer and inner endosperm had similar aminograms and electrophoregrams of protein subunits in both rices, but protein of subaleurone layer was richer in albumin.
Acknowledgement The work was supported in part by contract N01-AM-7-0726 from the National Institutes of Health (USA). References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13.
14. 15. 16. 17. 18.
19. 20. 21. 22. 23.
Barber, S . In Rice Chemisiry and Technology (Houston, D. F., Ed.), Am. Assoc. Cereal Chemists, Inc., St Paul, Minn., 1972, p. 215. Houston, D. F.; Mohammad, A.; Wasserman, T.; Kester, E. B. Cereal Chem. 1964, 41, 514. Houston, D. F. Rice.. 1967, 70 (8). 12. Normand, F. L.; Soignet, D. M.; Hogan, J. T.; Deobald, H. J. Rice J . 1966, 69 (9). 13. Kennedy, B. M.; Schelstraete, M.; del Rosario, A. R. Cereal Cliem. 1974, 51, 435. Kennedy, B. M.; Schelstraete, M. Cereal Chern. 1975, 52, 173. Kennedy, B. M.; Schelstraete, M.; Tamai, K. Cereal Cltem. 1975, 52, 182. Mitsuda, H.; Murakami, K. Physiol. Plants 1969, 8, 1. International Rice Research Institute Annual repurf f o r 1971 Los Baiios, Philippines, 1972, p. 7. International Rice Research Institute Annual reportfor 1972 Los Bafios, Philippines, 1973, p. 9. Ismail-Beigi, F.; Reinhold, J. G.; Faraji, B.; Abadi, P. J . Nufr. 1977, 107, 510. Harris, N . ; Juliano, B. 0. Ann. B o f . 1977, 41, 1. Bechtel. D. B.; Pomeranz, Y.Ant. J . But. 1978, 65, 684. Eggum, B. 0.; Resurreccion, A. P.; Juliano, B. 0. Nurr. Rep. I n f . 1977, 16, 649. Roxas, B. V.; Intengan, C. L.: Juliano, 9.0. J . Nutr. 1979, 109, 843. Am. Assoc. Cereal Chemists, Inc. Cereal Lnboraiory Methuds 1962, 7th edn. Bourdet, A.; Feillet, P. Cereal Chum. 1967, 44, 457. Tecson, E. M. S . ; Esmama, B. V.; Lontok, L. P.; Juliano, B. 0. Cereal Cl7rm. 1971, 48, 168. Villareal, R . M.; Juliano, B. 0. Phytocheniistry 1978, 17, 177. Perdon, A. A.; Juliano, B. 0. Phytocheniistry 1978, 17, 351. Resurreccion, A. P.; Juliano, B. 0 . ; Eggum, B. 0. Nufr. Rep. Int. 1978, 18, 170. International Rice Research Institute Annual repurf for 1975 Los Baiios, Philippines, 1976, p. 111. Mod, R. R.; Conkerton, E. J.; Ory, R. L.; Normand, F. L. J. Agric. Food Chem. 1978, 26, 1031.
Nutrient content and distribution in rice grain
481
24. Tanaka, K.; Yoshida, T.; Asada, K.; Kasai, 2. Arch. Biochem. Biophys. 1973, 155, 136. 25. Tanaka, K.; Yoshida, T.; Kasai, Z. Soil Sci. Plant Nutr. 1974, 20, 87. 26. McCall, E. P.; Jurgens, J. F.; Hoffpaiur, C. L.; Pons, Jr, W. A.; Stark, Jr, S. M.; Cucullu, A . F.; Heinzelman, D. C.; Arino, V. 0.; Murray, M. D. J. Agric. Food Chern. 1953, 1, 988. 21. Kanaya, K.; Yasumoto, K . ; Mitsuda, H. Eiyo To Sehokuryo 1976, 29, 341. 28. Kennedy, B. M.; Schelstraete, M. Cereal Chem. 1974, 51, 448. 29. Houston, D. F.; Iwasaki, T.; Mohammad, A . ; Chen, L. J. Agric. Food Chem. 1968,16, 720.
32
Nutrient Content and Distribution in Milling Fractions of Rice Grain Adoracion P. Resurreccion, Bienvenido 0. Juliano and Yonemi Tanakaa Chemistry Department, International Rice Research Institute, Los Barios, Laguna, Philippines (Manuscript received 6 September 1978)
Nutrient content and distribution in a low-protein (7.5 %) rice and a high-protein (10.8 %) rice were studied by analysing successive abrasive milling fractions of brown rice. Non-starch constituents decreased from the surface to the centre of the grain in both rices, except that the highest protein fraction in high-protein rice was the subaleurone layer. Starch and amylose contents of starch increased progressively from the surface to the centre of the grain and were lower in high-protein grain. Although this low-protein rice had lower total ash content than the high-protein rice, other samples of the same low-protein rice had comparable ash content to the highprotein rice. Glutelin accounted for 87-93% of milled rice protein. Protein and protein bodies of the sub-aleurone layer and inner endosperm have similar aminograms, and electrophoretic patterns using analytical and SDS-polyacrylamide disc gels. 1. Introduction Nutrient distribution in brown rice has been studied in detail by analysis of its successive abrasive milling f r a c t i ~ n s . l -High-protein ~ rices have been shown to have a more even distribution of protein, but the outer endosperm has the highest protein f r a c t i ~ n . ~The # s outer layers of milled rice are more rich in non-starch constituents, including B vitamins, than the core of the endo~perm.l-~> Although the brown rices contained similar B-vitamin contents, high-protein milled rice had higher contents of thiamin, riboflavin and phytin P than low-protein rice,g but similar content of ash and total fibre.10 As part of our study of the nutritive value of high-protein rices, we examined the nutrient content and distribution in a low-protein rice (IR32) and a high-protein rice (IR480-5-9). Dietary fibre analysis by the neutral detergent method was also undertaken in consideration of recent interest in the importance of fibre in health and mineral absorption in cereal-based diets.ll Particular interest was placed on the protein of the sub-aleurone layers, since these cell layers contain crystalline protein bodies that are absent in the inner endosperm.l2>13 2. Experimental
Samples of rough rice (IR480-5-9 and IR32) were obtained from the 1975-76 crop of the International Rice Research Institute farm. They were dehulled and milled in Satake dehullers. These have been used for study of protein quality of milled ri~e.149~5 Abrasive milling was done with a Satake grain-testing mill TM-05 to obtain successive milling layers. Samples were ground by passing them through a Udy cyclone mill with 60-mesh screen prior to analysis. Samples were analysed in dublicate for moisture content from loss in weight after 1 h at 13O0C,l6 for protein by micro-Kjeldahl N multiplied by the factor 5.95,lG for crude fat by extraction with refluxing petroleum ether in Soxhlet extractors, for crude fibre16and for dietary fibre by the neutral a Present address: Fukuoka Agricultural Senior High School, Oosano Dazaifu-machi, Chikusi-gun, Fukuoka, Japan.
0022-5142/79/0500-0475 $02.00 0 1979 Society of Chemical Industry 415
A. P. Resurreccion et al.
416
detergent method,16 and for crude ash by dry ashing 1 h at 325°C and then 16 h at 490°C. K, Mg, Fe and Zn were determined on the ash by atomic absorption spectroscopy and P by colorimetry in 0.43 % ammonium molybdate and 1 % ascorbic acid in 1.08~-HC1.Thiamin contents of brown and milled rice were determined by the thiochrome method, using Taka-diastase, thiochrome decalso, and a Coleman Model 12C electronic photofluorometer and calculated as pg of thiamin (not thiamin hydrochloride) per gram of sample.lG Riboflavin was determined by the fluorometric method.16Phytin P was analysed by extraction with 3 irichloroacetic acid (TCA), precipitation with magnesium nitrate and calcium acetate, and ashing of the precipitate for P determination.17 Protein fractions of sub-aleurone and inner endosperm were extracted, precipitated by TCA, and analysed for Kjeldahl N as previously described.18 Protein extract was analysed by analytical disc gel electrophoresis, and SDS-/I-mercaptoethanol extract was subjected to SDS-polyacrylamide gel electrophoresis.~g~ 20 Protein bodies were prepared by destarching raw, gelatinised, and cooked sub-aleurone and inner endosperm with crystalline bovine pancreatic a-amylase (Sigma Chem. C O ) . Milled ~ ~ rice and protein bodies were hydrolysed under N2 in ~N-HCIin a sealed tube at 110°C for 23 h and subjected to amino acid analysis in a Spinco Model 120C analyser with Spinco PA-35 and AA-15 or Hamilton H70 resins.'s, 19 3. Results and discussion 3.1. Gross composition Low-protein and high-protein brown rices had similar content of crude fibre, crude fat and Zny Table 1. Nutrient content in milling fractions of a low-protein and a high-protein rice grainu
Endosperm
Nutrient
Sample
Moisture (%) Protein ( % N x 5.95) Starch ( P/,anhydroglucose) Amylose (%) Crude fibre (%) Dietary fibre (%) Crude fat (%) Crude ash
(x)
Total P (%) Total K (%) Total Mg (%) Total Fe (parts 10 Total Zn (parts Phytin P (%)
G,
Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein
Brown rice (0-100) 10.4 10.7 7.5 10.8 69.2 66.3 27.6 22.6 0.7 0.7 2.2 1.8 3.0 2.7 0.81 1.58 0.16 0.38 0.20 0.28 0.08 0.16 12 16 28 30 0.10 0.28
Milled rice (90-100) 11.6 11.1
6.8 10.5 79.7 74.8 28.9 23.4 0.1 0.1 0.6 0.5 0.5 0.4 0.3
0.8 0.06 0.16 0.10 0.12 0.02 0.05 4 5 17 24 0.02 0.07
-
Bran (0-6) 9.3 10.6 14.2 14.0 14.6 11.7 6.1 1.6 8.8 6.3 25.8 18.5 20.7 20.9 5.8 11.9 1.00 2.60 1.12 2.09 0.60 1.33 105 89 124 103 0.84 2.28
Polish (6-12) 11.1 10.7 13.1 18.8 42.9 35.6 15.6 11.2 2.4 2.1 7.0 4.7 12.8 13.7 3.5 6.7 0.67 1.61 0.68 1.18 0.41 0.76 53 44 57 65 0.60 1.58
1R32 (1976 dry season) and lR480-5-9 (1975 wet season), respectively.
Subaleurone (12-20) 11.9 11.4 12.3 20.4 66.1 57.1 25.8 19.0 0.1 0.2 0.3 0.6 3.0 2.9 1 .o I .8 0.21 0.35 0.21 0.24 0.10 0.14 17 16 22 47 0.21 0.24
Middle (20-30) 12.1 10.3 10.2 16.0 75.5 67.1 28.6 23.1 0.1 0.1 0.4 0.3 0.3 0.3 0.4 0.6 0.06 0.09 0.08 0.06 0.01 0.02 4 6 14 28 0.010 0.025
Inner (30-100) 11.5 9.4 5.4 7.4 76.9 74.8 30.6 25.8 0.1 0.1 0.2 0.4 0.2 0. I 0.2 0.3 0.05 0.05 0.07 0.04 0.004 0.003 3 2 14 20 0.005 0.003
Nutrient content and distribution in rice grain
477
but the high-protein rice had higher crude ash (P, K, Mg and Fe) but lower starch and dietary fibre than the low-protein rice (Table 1). Differences in amylose content was unrelated to protein per se since IR32 is high in amylose (> 25 %) and IR480-5-9 is intermediate in amylose (20-25 %). Among 38 samples of brown rice with 6.7-11.5% protein, crude fat content was relatively constant at 2.1-3.2 %.22 Processing of both low-protein and high-protein brown rices to milled rices resulted in a decrease in all nutrient levels except starch and amylose (Table 1). Loss of protein from milling was greater for low-protein rice than for high-protein rice (Table 2). This is because the bran of low-protein rice was found to have a higher percentage of the total protein and protein content decreased progressively with each subsequent inner layer. By contrast, the sub-aleurone layer is the highest protein content fraction for the high-protein rice. Similar differences in protein distribution is reported between these two types of rice by Mitsuda and Murakami,* and Houston.3 Starch and amylose content increased progressively with the degree of milling in both samples (Tables 1 and 2) as it is present mainly in the endosperm.12 Although crude fibre and dietary fibre levels were similar in the two brown rice samples, the bran of low-protein rice tended to be higher in fibre. By contrast, reported 9.2-21.1 % crude fibre and crude fat distribution was similar in both samples. Mod et 28.744.7 % neutral detergent fibre in rice bran, 0.3 % crude fibre and 2 . 4 2 . 9% neutral detergent fibre in milled rice, and 1 . 1 % crude fibre and 3.8-6.0% neutral detergent fibre in brown rice. Ash content was nearly twice as high in the high-protein brown rice as in the low-protein rice (Table l), but its distribution was similar in both samples (Table 2). All milling fractions of the highTable 2. Nutrient distribution in milling fractions of a low-protein and a high-protein rice grain0 Endosperm
Nutrient Protein ( % N x 5.95) Starch (%,anhydroglucose) Amylose (%) Crude fibre (%) Dietary fibre (%) Crude fat (%) Crude ash (%) Total P (%) Total K
(7;)
Total Mg (%)
Total Fe (parts 10-6) Total Zn (parts Phytin P (%)
Sample
Bran (0-6)
Polish (6-12)
Sub-aleurone ( 1 2-20)
Middle (20-30)
Inner (30-100)
Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein Low protein High protein
11.5 8.1 1.2 1.0 1.3 0.4 69.5 63 .O 71.3 63.4 51.3 52.1 42.5 46.1 38.0 48.0 37.0 50.3 49.7 56.7 47.2 47.4 31.2 20.2 46.8 53.6
10.6 10.9 3.1 3.2 3.4 3.0 19.0 21 .o 19.3 16.1 31.8 34.1 25.7 26.3 25.4 29.8 22.4 28.4 34.0 32.4 23.9 23.5 14.4 12.7 33.4 37.1
13.3 15.7 7.5 6.9 1.5 6.7
13.7 15.4 10.8 10.1 10.3 10.2 1.3 1.7 1.8 1.7 1.2 1.3 4.9 3.9 3.8 2.8 4.4 2.4 1.4 1.4 3.0 5.3 5.9 9.1 0.9 1.0
50.9 49.9 76.8 78.8 77.5 79.7 9.2 11.6 6.5 16.0 5.8 2.9 17.1 13.7 22.2 10.8 27.0 11.2 3.9 1.5 15.7 12.4 41.1 45.7 3.2 0.8
1 .o
2.7 1.1 2.8 9.9 9.6 9.8 9.4 10.6 8.6 9.2 1.7 11.0 8.0 10.2 11.4 7.4 12.3 15.6 7.5
"Percentage of total nutrient content of the five milling fractions in Table 1. IR32 (1976 dry season) IR480-5-9 (1975 wet season), respectively.
A. P.Resurreccion er al.
418
protein samples had higher ash content. Bran, polish and the sub-aleurone layer had higher levels of P, K, and Mg in high-protein rice than in low-protein rice, and the milled rice also reflected this difference. By contrast, there was little difference in Fe levels between the two rices, but the lowprotein rice had higher Fe levels in bran and polish. In the case of Zn, total content in brown rice was similar, but more of it was in the endosperm in high-protein rice. The results are consistent with the reported distribution and highest concentration of P, K, Mg and Fe either in the aleurone layer or the pericarp.1324925 At least 80% of brown rice P and 40% of milled rice P and 90% of rice bran P is phytin P.26 Phytin is mainly present in the form of salts of potassium and magnesium as globoid inclusions in rice aleurone protein parti~les.2~ Phytate P was 74% of brown rice P in high-protein rice and 60% in low-protein rice. It comprised only 44 % of milled rice P in high-protein rice and 25 % in lowprotein rice. In the bran, phytate P contributed 88% of total P in high-protein rice and 84% in low-protein rice. Phytin in rice bran has been reported to be a pepsin inhibitor.27 The protein of cooked high-protein milled rice had 60.0% apparent digestibility as compared to 66.2 % apparent digestibility for low-protein milled rice in Filipino ~hi1dren.l~ However, the true protein digestibility of raw milled rice in rats was higher in the high-protein rice (100.4%) than in the low-protein rice (98.1 %).14 The rices had phytin P content of 0.07 and 0.02%, respectively and their crude and dietary fibre contents were similar (Table 1). Probably, the difference in phytate content of these milled rices is not involved in the difference in apparent digestibility of their protein in children. Other brown rice samples of the low-protein rice (IR32) had higher ash content (1.20-1.53 %) than the sample in Table 1 , indicating that the low-protein brown rice sample used in the detailed study was unusually low in ash content. The composition of another sample of IR32 rice with 7.7 % protein was 1.34% crude ash, 65.9%starch, 0.7 % crude fibre, 1.7% neutral detergent fibre, 3.0% crude fat, 0.24% total P, 0.25 % total K, 0.14% total Mg, 18 parts 10-6 total Fe, and 19 total Zn. Thus, the main nutrient affected by protein change was starch. parts Similar decreases in thiamin, riboflavin, phytin P and other non-starch constituents were also noted in milling other samples of low-protein (IR8)and high-protein (IR480-5-9) rices (Table 3). In this pair o f samples, the high-protein rice also had a higher crude ash content. Four samples of IR8 and eight other samples of IR480-5-9 showed variable content of milled rice protein (7.5-12.4%), ash (0.324.77%),total P (0.084.16 %), phytate P (0.024.07%),and Zn (10-22 parts Degree of milling is an additional variable affecting composition in milled rice. Crude fibre (0.6-1.5%), crude fat ( 0 . 3 4 6 % ) and thiamin levels (0.24.8 pg g-1) were also not Table 3. Nutrient content of brown and milled rices of two samples ditrering in protein content Low-protein ricea
High-protein rice0
Nutrient
Brown rice
Milled rice
Brown rice
Milled rice
Moisture (%) Protein (%N x 5.95) Crude fat (%) Crude fibre (%) Dietary fibre (%) Crude ash (%) Total P (%) Phytin P (%) Total Zn (parts Thiamin (pg g-1) Riboflavin (pg g-1)
10.1 8.5 2.3 1.1 1.8 1.36 0.30 0.21 20 3.97 0.48
9.9 7.6 0.3 0.6 1.3 0.36 0.08 0.03 10 0.20 0.19
11.2 11.6 3.1 2.2 2.7 1.74 0.30 0.25 25 3.61 0.50
10.7 10.8 0.5 1.1 1.5 0.55 0.13 0.06 16 0.82 0.23
a
IR8, 1974 dry season crop. 113480-5-9, 1974 dry season crop.
Nutrient content and distribution in rice grain
419
necessarily higher in higher-protein milled rices. Our data are also consistent with the reported uneven distribution within milled rice of protein, fat, ash, amylose, starch, Mg, P, K, phytate and vitarnin~.b-~ 3.2. Protein
Amino acid composition showed lower lysine content for protein in flour and destarched flour from sub-aleurone of IR480-5-9 rice than from inner endosperm, but no difference was noted for IR32 rice (Table 4). Results with destarched flour from raw and gelatinised milled rice and with cooked milled rices were similar.21Normand et al.4 and Kennedy and Schelstraete28also found no appreciable difference in aminogram of various overmilling fractions of milled rice. A study of the corresponding albumin, globulin, prolamin and glutelin contents of the subaleurone and inner endosperm of high-protein and low-protein rices showed that the difference in protein content between the four samples was mainly in glutelin content (Table 4). Glutelin made up 87-93 % of protein of these milling fractions. The low lysine content of protein in the sub-aleurone layer of IR480-5-9 rice may be due to its higher prolamin content than the three other samples. Table 4. Aminogram and protein fractions of sub-aleurone and inner endosperm (core) of high-protein and lowprotein milled rices and destarched rices prepared from them by a-amylolysis of cooked rice (g per 16.8 g N) Sub-aleurone layer (12-20 %) High-protein rice= Amino acid LYs His NH3 Arg ASP Thr Ser Glu Pro CYs GlY Ala Val Met Ile Leu Tyr Phe Protein
Flour
Low-protein rice*
Destarched flour
Flour
( %)"
Prolamin
Destarched flour
Flour
Destarched flour
3.63 2.62 2.57 8.82 10.5 3.62 4.84 22.8 5.15 2.46 4.76 6.06 6.36 2.16 4.49 9.12 5.24 5.98
3.50 2.49 2.20 9.06 9.92 3.68 5.25 22.8 5.09 3.46 4.68 6.26 6.38 3.00 4.49 9.46 5.96 6.10
4.17 2.58 2.55 9.13 10.2 3.61 4.87 20.8 5.57 2.47 4.73 6.18 6.13 2.30 4.27 8.92 4.74 5.90
3.88 2.41 2.26 9.11 9.92 3.52 4.92 21.2 5.08 3.20 4.90 6.38 6.53 3.14 4.52 9.58 6.23 5.87
3.60 2.50 2.78 8.26 11.3 3.66 4.80 20.6 5.40 2.29 5.08 6.42 6.47 2.30 4.59 9.12 3.58 5.91
3.54 2.46 2.40 9.04 10.2 3.64 5.16 22.4 5.15 3.28 4.58 6.06 6.44 3.61 4.51 9.16 6.22 6.05
21.7
85.6
12.6
79.0
7.34
80.2
5.20
84.0
0.4(2)
0.5 (4)
0.04 (1)
0.1 (2)
1.2 (6)
1 .O (8)
0 . 3 (4)
0 . 5 (10)
1.o (4)
0 . 2 (2)
0.2 (3)
0.1 (2)
19.1 (88)
10.9 (87)
6.8 (93)
4.5 (87)
( %)"
Glutelin
Flour
Low-protein rice0
3.22 2.37 2.46 8.88 9.97 3.46 5.10 23.0 5.06 3.33 4.46 5.99 6.59 2.88 4.54 9.53 6.68 6.18
(%)" Globulin
Destarched flour
High-protein riced
3.33 2.63 2.74 9.05 9.90 3.51 4.99 22.50 5.30 2.26 4.32 5.94 5.97 2.72 4.28 9.12 6.25 6.05
( %)"
Albumin
Inner endosperm (70-100 %)
( %)" ~
IR480-5-9, 1975 wet season crop. 1R32, 1976 dry season crop. c Based on milled rice at 12% moisture.
480
A. P. Resurreccion et a/.
Albumin content of total protein was higher in the sub-aleurone layer than in the inner endosperm, in agreement with Houston et a/.29The proportion of globulin in the protein of sub-aleurone was higher than that in the inner endosperm protein only in the high-protein rice. Based on these results, the crystalline protein bodies and the small spherical protein bodies present only in the sub-aleurone layer'3 together probably have the same aminogram as the common large spherical protein bodies. Analytical disc gel electrophoresis of albumins and globulins from the sub-aleurone and inner endosperm of low-protein and high-protein rices indicated little difference in the electrophoregram of albumins in the four samples. Albumins consisted of two major bands and at least three minor ones as reported earlier.20Two bands that were minor in sub-aleurone albumins were more intense in the albumins from the inner endosperm of low-protein rice. By contrast, globulins from the inner endosperm showed more minor bands than globulins from the sub-aleurone in both rices. Both exhibited two major globulin bands as reported earlier.20 SDS-polyacrylamide disc gel electrophoregrams of the total protein extract of the sub-aleurone and inner endosperm of both rices showed mainly the three major subunit fractions of glutelin with molecular wt 38 000, 25 000, and 16 OOO;19 this confirms the fact that glutelin is the predominant protein in these samples (Table 4).
4. Conclusions Nutrient analysis of milling fractions of low-protein rice and a high-protein rice showed that all non-starch constituents decreased from surface to centre of the grain. However, the milling fraction with the highest protein was the sub-aleurone layer in the high-protein sample; bran had the highest protein level in the low-protein sample. The difference in milled-rice protein was mainly in the glutelin content. The only other major nutrient in which low-protein and high-protein rice differed was starch content. Protein of the sub-aleurone layer and inner endosperm had similar aminograms and electrophoregrams of protein subunits in both rices, but protein of subaleurone layer was richer in albumin.
Acknowledgement The work was supported in part by contract N01-AM-7-0726 from the National Institutes of Health (USA). References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13.
14. 15. 16. 17. 18.
19. 20. 21. 22. 23.
Barber, S . In Rice Chemisiry and Technology (Houston, D. F., Ed.), Am. Assoc. Cereal Chemists, Inc., St Paul, Minn., 1972, p. 215. Houston, D. F.; Mohammad, A.; Wasserman, T.; Kester, E. B. Cereal Chem. 1964, 41, 514. Houston, D. F. Rice.. 1967, 70 (8). 12. Normand, F. L.; Soignet, D. M.; Hogan, J. T.; Deobald, H. J. Rice J . 1966, 69 (9). 13. Kennedy, B. M.; Schelstraete, M.; del Rosario, A. R. Cereal Cliem. 1974, 51, 435. Kennedy, B. M.; Schelstraete, M. Cereal Chern. 1975, 52, 173. Kennedy, B. M.; Schelstraete, M.; Tamai, K. Cereal Cltem. 1975, 52, 182. Mitsuda, H.; Murakami, K. Physiol. Plants 1969, 8, 1. International Rice Research Institute Annual repurf f o r 1971 Los Baiios, Philippines, 1972, p. 7. International Rice Research Institute Annual reportfor 1972 Los Bafios, Philippines, 1973, p. 9. Ismail-Beigi, F.; Reinhold, J. G.; Faraji, B.; Abadi, P. J . Nufr. 1977, 107, 510. Harris, N . ; Juliano, B. 0. Ann. B o f . 1977, 41, 1. Bechtel. D. B.; Pomeranz, Y.Ant. J . But. 1978, 65, 684. Eggum, B. 0.; Resurreccion, A. P.; Juliano, B. 0. Nurr. Rep. I n f . 1977, 16, 649. Roxas, B. V.; Intengan, C. L.: Juliano, 9.0. J . Nutr. 1979, 109, 843. Am. Assoc. Cereal Chemists, Inc. Cereal Lnboraiory Methuds 1962, 7th edn. Bourdet, A.; Feillet, P. Cereal Chum. 1967, 44, 457. Tecson, E. M. S . ; Esmama, B. V.; Lontok, L. P.; Juliano, B. 0. Cereal Cl7rm. 1971, 48, 168. Villareal, R . M.; Juliano, B. 0. Phytocheniistry 1978, 17, 177. Perdon, A. A.; Juliano, B. 0. Phytocheniistry 1978, 17, 351. Resurreccion, A. P.; Juliano, B. 0 . ; Eggum, B. 0. Nufr. Rep. Int. 1978, 18, 170. International Rice Research Institute Annual repurf for 1975 Los Baiios, Philippines, 1976, p. 111. Mod, R. R.; Conkerton, E. J.; Ory, R. L.; Normand, F. L. J. Agric. Food Chem. 1978, 26, 1031.
Nutrient content and distribution in rice grain
481
24. Tanaka, K.; Yoshida, T.; Asada, K.; Kasai, 2. Arch. Biochem. Biophys. 1973, 155, 136. 25. Tanaka, K.; Yoshida, T.; Kasai, Z. Soil Sci. Plant Nutr. 1974, 20, 87. 26. McCall, E. P.; Jurgens, J. F.; Hoffpaiur, C. L.; Pons, Jr, W. A.; Stark, Jr, S. M.; Cucullu, A . F.; Heinzelman, D. C.; Arino, V. 0.; Murray, M. D. J. Agric. Food Chern. 1953, 1, 988. 21. Kanaya, K.; Yasumoto, K . ; Mitsuda, H. Eiyo To Sehokuryo 1976, 29, 341. 28. Kennedy, B. M.; Schelstraete, M. Cereal Chem. 1974, 51, 448. 29. Houston, D. F.; Iwasaki, T.; Mohammad, A . ; Chen, L. J. Agric. Food Chem. 1968,16, 720.
32
