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Comparative Protein Digestibility in Growing Rats of Cooked Rice and Protein Properties of Indica and Japonica Milled Rices
Journal of Cereal Science 33 (2001) 183–191 doi:10.1006/jcrs.2000.0355, available online at http://www.idealibrary.com on
Comparative Protein Digestibility in Growing Rats of Cooked Rice and Protein Properties of Indica and Japonica Milled Rices Sigurd Boisen∗, Joy Bartolome A. Duldulao†, Evelyn Mae T. Mendoza‡ and Bienvenido O. Juliano§ ∗ Danish Institute of Animal Science, Department of Nutrition, Research Centre Foulum, DK-8830 Tjele, Denmark; † Philippine Rice Research Institute, Maligaya, 3119 Mun˜oz, Nueva Ecija, Philippines; ‡ University of the Philippines Los Ban˜os, Biochemistry Laboratory, Institute of Plant Breeding, 4031 College, Laguna, Philippines; § Philippine Rice Research, Institute Los Ban˜os, 4031 College, Laguna, Philippines. Received 1 June 1999
ABSTRACT Eight indica and japonica milled rices with low amylose content and low starch gelatinisation temperature were analysed for cooked rice energy and N balance in growing rats and for protein properties. Digestible energy values were similar. Japonica rices Koshihikari and Sasanishiki had higher true digestibility (TD) and net protein utilisation in rats than indica rices IR24 and PR2338315. Similar results were obtained from in vitro proteolysis. High TD of cooked rices was not significantly correlated with low prolamin content in raw rice and with low waxy gene product (protein bound to starch granule) but was significantly correlated with low cysteine content in protein and with low denatured prolamin content in cooked rice. In two pairs of cooked waxy milled rices, which are devoid of waxy gene product, japonica rice still tended to show higher rat TD and in vitro protein digestibility than indica rice, consistent with lower cysteine content in japonica protein. 2001 Academic Press
Keywords: cooked-rice protein digestibility, indica vs. japonica, protein properties, waxy gene product, cysteine content, protein content, glutelin content, prolamin content.
INTRODUCTION The storage protein bodies (PB) in the rice endosperm are the prolamin-rich spherical PB I and the glutelin-rich segmented crystalline PB II1. True protein digestibility (TD) of milled rice in growing rats have been shown2 to be reduced from 100% to 85–90% by cooking the raw rice in boiling water. The poorly digestible fraction was shown
Corresponding author: B. Juliano. Tel: (63-49) 536-3633; Fax: (63-49) 536-3515; E-mail: [email protected]. S. Boisen and J. B. A. Duldulao are the senior authors. 0733–5210/01/030183+09 $35.00/0
to be the core proteins of the prolamin-rich PB I3,4. But indica and japonica rices are reported to have similar (85–90%) TDs3,5 in man. Collaborative N balance studies in growing rats with the late Dr Bjørn O. Eggum of the Danish Institute of Animal Science (DIAS) involved 12 indica cooked rices (TD 83·3–97·4%) up to 19922,4,6. Higher TD (94·6–99·8%) were obtained with more recent studies on seven japonica cooked rices9,10, but there was a tendency for TD to decrease with increase in amylose content (AC, r=−0·76, p<0·05). The TD of the combined 19 cooked milled rices correlated both with protein content (r=−0·83, p<0·01) and AC (r=−0·68, p<0·01). 2001 Academic Press
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The waxy gene product (granule-bound starch synthase) of japonica and indica nonwaxy rice differ only by 16 individual nucleotides11. Indica rice starch has more than twice as much 60-kD subunit waxy gene product12,13 as japonica rice starch, reflecting lower specific activity of the granule-bound starch synthase14. The starch synthase is relatively stable and extractable only after starch gelatinisation. Interestingly, high-AC indica rice in Taiwan has 2% higher protein content than low-AC japonica rice15. Dr K. Tanaka of Kyoto Prefectural University has reported (unpubl., 1985) on the higher in vitro pepsin digestibility of protein in raw rice in three japonica (78–80%) than in three indica rices (68, 69 and 78%). Protein contents were 7·4–8·8% for japonica and 7·5–10·4% for indica rices. The protein of japonica rice has been reported to be 20% prolamin, but indica rice protein has 30% prolamin16. The prolamin and glutelin fractions of milled rice protein increase with an increase in grain protein content17. Because of these considerations, a collaborative study was undertaken on cooked milled rice at similar low AC to determine; (1) whether or not japonica rice protein has greater TD in growing rats than indica rice protein; (2) whether the difference is related to the increase in prolamin content with an increase in protein content of milled rice; and (3) whether this difference is in any way related to the greater amount of waxy gene product (granule-bound starch; synthase) in indica rice.
MATERIALS AND METHODS Materials Brown rice samples of low AC and low starch gelatinisation temperature (GT) were obtained from the 1996 crop from various sources: two popular Japanese japonica rices, Koshihikari and Sasanishiki from Masao Yokoo, National Agricultural Research Center, Tsukuba, Japan; four Philippine rices: IR24, Sinandomeng, PR2338315, and PRJ 3 from University of the Philippines Los Ban˜os (UPLB)-PhilRice Los Ban˜os and PhilRice Maligaya; Thai variety Khao Dawk Mali 105 from Ngamchuen Kongseree, Pathumthani Rice Research Center, Thanyaburi, Pathumthani, Thailand; and U.S. long-grain variety Toro 2 from Anna M. McClung, ARS-USDA Rice Research,
Beaumont, TX, USA. All had low AC (15–20%) and low GT (alkali spreading value 6–7). Malagkit Sungsong and IR29 low GT waxy rices were obtained early in 1999 from PhilRice Maligaya. Taikeng Glu. 5 and Taichung Sen Glu. 1 low-GT waxy milled rices were obtained from Sheu-chih Sheng, Taichung District Agricultural Improvement Station, Tatsuen Hsiang, Changhua, Taiwan.
Methods Chemical studies on protein of these cooked and raw milled rices were undertaken at the Institute of Plant Breeding by J. B. A. Duldulao as his M. S. thesis at UPLB. Indica-japonica classification was checked by the seedling isozyme method18 at the Isozyme Laboratory of D. S. Brar at the International Rice Research Institute, and on waxy gene product content of the rice starch13. Rice flour (40 mg) was purified by five protein extractions with buffer A (55 m Tris HCl pH 6·8 with 2% (w/v) SDS, 5% (w/v) -mercaptoethanol and 10% (v/v) glycerol). The resulting starch granules were boiled in 10 volumes of buffer A for 10 min. After cooling, 10 volumes of buffer were added and the slurry was centrifuged. A 20-L supernate was used for SDS-PAGE. Crude protein content was determined by micro-Kjeldahl method using the factor 6·25 or by Lowry method. Prolamin, albumin-globulin and glutelin contents were determined on raw and cooked milled rice flour by sequential extraction of 400 mg flour for 2 h each with 8 mL of 60% 1-PrOH, 0·7 NaCl (pH 7·5), and 3% (w/w) SDS and 0·5% dithiothreitol (pH 8)19. Rough and brown rices that pass the chemical studies at PhilRice were milled after at least 4 months after harvest (aged), washed and boiled in 1·3 times its weight in water, freeze-dried and airshipped to DIAS. At DIAS, the eight samples were run together for amino acid analysis10, digestible energy (DE), true digestibility (TD), biological value (BV), and net protein utilisation (NPU) in five growing rats each10 and their in vitro protein (EDN) and dry matter enzymic digestibility (EDDM) values at the ileal level20 and in vitro organic matter enzymic digestibility (EDOM) at the faecal level21. Standard deviation (S.D.) was 0·2 g/16 g N for lysine, 0·1 g/16 g N for cysteine and methionine10, 2% for in vitro EDN20, and 5% for in vitro EDDM20 and EDOM21. Amino acid
Rice protein digestibility and properties
Table I
Classification of eight milled rices in terms of indica-japonica and starch properties (PhilRice Los Ban˜os)
Propertya
Glaszmann’s group Waxy gene product Waxy gene protein (% dry basis) Apparent amylose (% dry basis) Alkali spreading value a
185
Koshihikari Sasanishiki
Toro 2
Sinandomeng PRJ 3
Khao PR23383-15 IR24 Dawk Mali 105
VI Wxb 0·07
VI Wxb 0·06
0 Wxb 0·11
I Wxb 0·08
I Wxb 0·06
I Wxb 0·08
I Wxa 0·13
I Wxa 0·20
15·2
15·5
14·0
13·0
11·8
11·1
14·8
13·0
7·0
7·0
6·0
5·7
7·0
6·3
4·1
7·0
Glaszmann’s18 group: 1 (indica), group VI ( japonica); waxy gene product: Wxa (indica), Wxb ( japonica).
score was calculated based on the FAO/WHO/ UNU amino acid pattern for the preschool child with lysine content of 5·8% as 100%22. Amino acid score X TD was also calculated23. Part of the raw milled rice was tested for alkali spreading value24 as index of starch GT and for AC by iodine colorimetry25. Protein extracts and fractions and waxy gene product were dispersed in buffer A in a boiling water bath for 2 min and subjected to SDS-10% gel PAGE26 with protein standards lysozyme (14·3 kD), trypsinogen (24 kD), pepsin (34·7kD), ovalbumin (45 kD), and bovine serum albumin (66 kD). Polyacrylamide gels used were 0·75 mm thick, 8 cm long and 7 mm wide. Gels were stained with Coomassie blue and protein bands quantified by QuantiScan software (Biosoft). In vitro protein TD was also determined at UPLB on raw and cooked milled rice flours20. Residual proteins after pepsin and pancreatin digestion were extracted with cold buffer A (residual proteins I) and with boiling buffer A (residual proteins II) and the extracts also subjected to SDS-PAGE. S.D. were calculated and some data were subjected to Duncan’s multiple range test27. RESULTS AND DISCUSSION Classification into indica and japonic types The eight rices were all verified to have low AC and low GT (Table I). An exception was the mixture of high-GT grains (5 of 12) in PR2338315. One out of 12 grains had high GT in Sinandomeng. The rices had their expected Glaszmann18 grouping of I for indica and VI for japonica, except for Toro 2 which could not be classified (group 0). Khao Dawk Mali 105 was in
group I, although an earlier sample gave group VI28. Classification by waxy gene product in the endosperm using purified rice starch showed high waxy gene product (indica, Wxa) only for PR23383-15 and IR24 and Wxb ( japonica) for the other six samples, including Khao Dawk Mali 105 and Toro 2, in conformity with previous results28. The presence or absence of the 3 subunit of glutelin28 could not be resolved from the SDSPAGE and could not be used for indica-japonica classification28.
Aminogram and balance in rats The freeze-dried cooked milled rices had 91·3– 93·2% dry matter or 6·8–8·7% moisture (Table II). Protein content range was 6·19–10·87% and was lower in japonica than in indica rices. Ash content was 2·87–5·09%. Energy content was 17·5–18·4 J/g dry matter. The amino acid values were used without correction for N recovery since the N recovery was already high at 88·9–93·4% (Table II). Lysine was the first limiting amino acid (3·1–3·4 g/16 g N) of rice protein corresponding to an amino acid score of 53–59%. Koshihikari and Sasanishiki protein had the highest lysine content and amino acid score. IR24, PR23383-15 and PRJ 3 proteins had the lowest lysine value. Other essential amino acids were present in amounts closer to the reference pattern. However, Koshihikari and Sasanishiki had lower cysteine content of protein than the other rices and had lower tyrosine content than IR24 and PR23383-15. Koshihikari protein has the highest phenylalanine, arginine and leucine levels. Digestible energy (DE) in growing rats was sim-
186
Table II
S. Boisen et al.
Properties, aminogram, rat balance data and in vitro digestibility of japonica and indica cooked milled rices (Danish Institute of Animal Science)
Property
Dry matter (%) Protein (% N×6·25) Crude ash (%) Gross energy ( J/g dry matter) Lys (g/16 g N) Met (g/16 g N) Cys (g/16 g N) Thr (g/16 g N) Ile (g/16 g N) Leu (g/16 g N) Val (g/16 g N) Phe (g/16 g N) Tyr (g/16 g N) Arg (g/16 g N) His (g/16 g N) Ala (g/16 g N) Asp (g/16 g N) Glu (g/16 g N) Gly (6/16 g N) Pro (g/16 g N) Ser (g/16 g N) N recovery (%) Amino acid scorea (%) Rat balance data Digestible energy (% of intake)±SD True digestibility (% of N intake)±SD Biological value (% of absorbed N)±SD NPU (% of N intake) ±SD Amino acid score X TD (%) EDNb (% at ileal level) EDDMb (% at ileal level) EDOMb (% at faecal level)
Koshihikari Sasanishiki
Toro 2
Sinandomeng
PRJ 3
Khao Dawk Mali 105
PR23383-15 IR24
93·2 6·19 2·98 17·5
92·7 6·47 2·87 17·8
91·8 8·38 2·98 18·2
91·3 7·82 3·20 18·3
92·6 9·41 5·09 18·4
92·3 10·87 3·09 18·3
92·0 8·63 4·51 18·3
91·8 8·97 3·44 18·2
3·4 2·4 2·0 3·3 4·4 8·1 6·1 5·3 3·7 8·3 2·5 5·5 8·9 17·3 4·5 4·7 5·3 93·9 59
3·4 2·4 2·0 3·2 4·2 7·7 5·9 4·9 3·5 7·9 2·4 5·2 8·5 16·5 4·3 4·4 5·0 89·8 59
3·2 2·7 2·1 3·3 4·1 7·4 5·8 4·9 3·8 7·9 2·5 5·1 8·5 16·4 4·2 4·4 4·9 89·0 55
3·2 3·1 2·3 3·4 4·1 7·4 5·8 5·0 3·7 7·4 2·3 5·2 8·1 16·7 4·1 4·4 4·7 88·4 55
3·1 2·7 2·2 3·3 4·3 7·9 5·9 5·1 4·2 7·9 2·4 5·3 8·2 17·6 4·1 4·7 5·0 91·5 53
3·2 2·3 2·1 3·2 4·3 7·8 6·0 5·1 4·3 8·0 2·4 5·2 8·4 17·3 4·1 4·5 5·0 91·7 55
3·1 2·7 2·3 3·3 4·1 7·6 5·9 5·1 4·1 7·6 2·4 5·2 8·1 17·0 4·1 4·6 4·9 89·9 53
3·1 2·7 2·2 3·3 4·2 7·7 5·9 5·1 4·0 7·5 2·3 5·3 8·1 17·2 4·1 4·4 4·9 89·6 53
97·4 ±0·5 98·8 ±2·5 77·3 ±3·1 76·3 ±3·9
97·5 ±0·4 98·6 ±2·0 81·5 ±2·0 80·3 ±1·7
97·4 ±0·4 97·2 ±1·7 70·6 ±2·5 68·2 ±1·4
97·2 ±0·6 94·6 ±3·1 75·8 ±3·1 71·6 ±1·6
96·9 ±0·7 93·2 ±3·3 73·9 ±3·1 68·9 ±4·8
97·2 ±0·7 94·1 ±3·1 72·5 ±2·1 68·2 ±1·7
96·9 ±0·2 92·5 ±1·1 74·1 ±3·4 68·5 ±2·9
97·0 ±0·3 92·6 ±1·8 71·7 ±2·5 66·5 ±3·4
58 90·0
58 88·5
54 87·1
52 82·3
50 82·6
52 85·0
52 83·3
49 81·5
82·8
77·4
84·5
80·1
78·3
79·4
71·7
76·7
98·3
98·3
98·1
98·0
97·6
97·5
97·2
97·6
a
Based on 5·8% lysine in FAO/WHO/UNU22 pattern as 100%. EDN means enzyme digestible N (protein) at ileal level; EDDM means enzyme digestible dry matter at ileal level; EDOM is enzyme digestible organic matter at faecal level.
b
ilar at 96·9–97·5% of intake (Table II). TD was 92·5–98·8% and highest for Koshihikari, Sasanishiki and Toro 2, as in EDN values. PR2338315 and IR24 had lower TD than japonica rices. Koshihikari and Sasanishiki had the lowest cyst-
eine content in protein. Amino acid digestibility in cooked milled has been shown to be lowest for cysteine2, suggesting disulphide bond formation. Biological value was 70·6–81·5% and was highest for Sasanishiki and lowest for Toro 2 and IR24.
Rice protein digestibility and properties
NPU was 66·5–80·3% and highest for Sasanishiki and lowest for IR24. Rat TD correlated positively with NPU and lysine level in protein and negatively with cysteine and tyrosine levels in protein (Table IV). Amino acid score corrected for TD was highest for Koshihikari and Sasanishiki and lowest for IR24 and PRJ 3 (Table II). Toro 2 had intermediate value.
187
In vitro EDN at the ileal level was 81·5–90·0% and was highest for the two japonica samples and Toro 2 and lowest for IR24. In vitro EDDM at the ileal level was 71·7–84·5%, highest for Toro 2 and lowest for PR23383-15, and did not follow TD in rats (Table II). EDOM at the faecal level was high at 97·2–98·3% and was similar to DE values in rats. In vitro digestible protein (DP) of raw milled rice was 81·1–89·2% (Table III). It was also highest for Koshihikari and Sasanishiki, followed by Toro 2. By contrast, TD of raw rice protein in growing rats was 100%2. Corresponding DP values for cooked milled rice were lower at 77·0–82·6%. IR24 had the lowest value in both sets, whereas japonica rices, Koshihikari and Sasanishiki, and Toro 2 had the highest values. Cooked rice DP values were less than the EDN values reported in Table II, probably due to differences in the batches of proteases used. Undigested proteins in raw rice were soluble in cold (residual proteins I) and boiling buffer A (residual proteins II), but was only extracted in boiling buffer A from protease-treated cooked rice (Table III). Protease-treated cooked rice had higher residual protein than the corresponding raw rice. Rat TD correlated positively with the three in vitro assays, EDN of cooked rice and DP of raw and cooked rice (Table IV). EDN correlated significantly with DP of raw and cooked rice and NPU and with lysine content of protein and negatively with cysteine content in protein and behaved closer to in vitro DP of raw rice than to DP of cooked rice. In vitro DP of cooked rice correlated with DP of raw rice and negatively with cysteine content of protein. In vitro DP of raw rice correlated with NPU and lysine content of protein and negatively with cysteine content of protein.
values were low at 0·25–0·40% (4–6% of protein). Low prolamin values of 0·23–0·44% (3–7% of protein) were obtained using 60% 1-PrOH solvent without -mercaptoethanol. Glutelin values were high (4·80–6·87% of milled rice; 87–91% of protein) but included the 10–16 kD prolamin subunits, as verified by SDS-PAGE. The addition of -mercaptoethanol to 60% 1-PrOH should have extracted the 10–16 kD prolamin subunits. Rat TD did not correlate significantly with albumin-globulin and prolamin contents, but correlated negatively with glutelin content (Table IV). Correcting for the prolamin contamination in glutelin, the corrected prolamin values of raw rice were 0·72–1·22% (11–17% of protein) and corrected glutelin values were 3·90–6·09% (72–83% of protein). Cooking, as expected, reduced protein extractability to 64–81% (mean 71%) due to heat denaturation (Table III). ‘Albumin-globulin’ values were 0·12–0·26% (2–5% of protein), ‘prolamin’ content was 0·05–0·09% and ‘glutelin’ content was 4·16–5·63%. The denatured prolamin (10–16 kD) values in cooked rice flour, which was extracted only by boiling and not by cold buffer A, was 0·13–0·34% of cooked rice and was lower in Koshihikari, Sasanishiki, Sinandomeng and Toro 2 than in PR23383-15, IR24, PRJ 3 and Khao Dawk Mali 105 (Table III). Correlation coefficient with rat TD was significant (Table IV). The denatured prolamin probably represents the high cysteine prolamin fraction29 that became denatured on cooking due to disulphide bonding to cystine. Which explains why TD correlated with cysteine content of protein (Table IV). Protein content of cooked rice in Table III was lower than that in Table II, except for Koshihikari. However, the protein values from the two laboratories were correlated (r=0·96, p<0·01). Rat TD correlated positively with lysine content in the protein and negatively with protein content, glutelin content and with cysteine and tyrosine levels in protein (Table IV). Interestingly DIAS protein content correlated with lysine (r=−0·75, p<0·05) and tyrosine (r=0·91, p<0·01) in protein and with glutelin content (r=0·85, p<0·01) and may explain the significant correlation of TD with these properties.
Prolamin and other protein fractions
Waxy gene product
Total protein extraction from raw rice was 94– 106% (mean 100%)(Table III). Albumin-globulin
Rat TD did not correlate significantly negatively with waxy gene product content of milled rice
In vitro digestibility
188
Table III
S. Boisen et al.
Properties of raw and cooked milled rices (Institute of Plant Breeding, UP Los Ban˜os-PhilRice Los Ban˜os)
Property of milled rice Raw milled rice Protein (% N×62·5) ±SD In vitro digestible protein (%) ±SD Res. proteins I (%) Res. proteins II (%) Albumin-globulina,b (%) Prolamina,b (%) Glutelina (%) ±SD Percent N extraction Waxy gene product (%) Total After proteolysis Cooked milled rice Protein (% N×6·25) ±SD In vitro digestible protein (%) ±SD Res. protein II (%) “Albumin-globulin”a,b (%) “Prolamin”a,b (%) “Glutelin”b (%) ±SD Percent N extraction Denatured “prolamin” (%) Waxy gene product (%) Total After proteolysis
Koshihikari
Sasanishiki
Toro 2
Sinandomeng PRJ 3
Khao PR23383-15 Dawk Mali 105
IR24
5·35 ±0·06 89·2 ±0·2 2·5 9·5 0·34c 0·30d 4·80f ±0·02 102
5·46 ±0·25 88·6 ±0·0 1·6 8·6 0·25f 0·27e 5·18e ±0·00 104
6·44 ±0·42 87·2 ±0·4 1·5 12·7 0·34c 0·23f 5·54a ±0·01 95
5·83 ±0·06 81·4 ±0·0 3·6 9·9 0·27e 0·23f 5·68c ±0·02 106
7·38 ±0·16 82·2 ±1·2 1·6 6·1 0·38b 0·32c 6·77a ±0·11 101
7·74 ±0·16 82·8 ±0·4 1·8 4·7 0·27e 0·44a 6·87a ±0·07 98
7·91 ±0·07 82·7 ±1·0 1·0 6·0 0·31d 0·34b 6·49b ±0·05 102
6·64 ±0·05 81·1 ±0·4 1·6 8·0 0·40a 0·44a 5·39d ±0·05 94
0·07 0·09
0·06 0·06
0·11 0·10
0·08 0·08
0·06 0·06
0·08 0·08
0·13 0·11
0·20 0·08
6·10 ±0·02 80·0 ±0·4 20·5 0·26a 0·08b 4·56e ±0·01 80
6·21 ±0·25 81·1 ±0·3 20·5 0·22b 0·07c 4·16f ±0·00 72
7·79 ±0·13 82·6 ±0·1 12·7 0·19d 0·07c 5·63a ±0·02 76
6·91 ±0·02 78·0 ±0·3 13·1 0·21c 0·06d 4·20f ±0·00 65
7·92 ±0·27 77·9 ±1·3 16·5 0·12g 0·09a 4·85c ±0·07 64
8·35 ±0·06 79·1 ±0·7 17·5 0·15f 0·06d 5·63a ±0·03 70
7·51 ±0·09 77·6 ±0·6 14·3 0·16e 0·05e 4·75d ±0·06 66
7·17 ±0·36 77·0 ±0·8 16·3 0·19d 0·07c 5·25b ±0·03 77
0·17
0·15
0·13
0·17
0·34
0·32
0·31
0·27
0·07 0·00
0·06 0·00
0·11 0·00
0·08 0·00
0·06 0·00
0·08 0·00
0·13 0·00
0·20 0·00
a
SD<0·01%. Protein values in the same line followed by the same letter are not significantly different at p=0·05 by Duncan’s27 multiple range test. c No residual protein I was extracted. b
(Table IV). SDS-PAGE of residual protein of in vitro protease-digested milled rice showed mainly the 13±3 kD prolamin band in both raw and cooked rice, but only the raw rice had some waxy gene product at MW 60 kD. By contrast, TD of raw rice protein in rats is 100%2. The enzymes used probably did not have adequate amylase activity to hydrolyze the raw starch protecting the waxy gene product in vitro. However, the decrease in waxy gene product in IR24 raw rice from 0·20% to 0·08% due to proteolysis (Table III) is difficult
to explain. The gelatinized starch in cooked rice facilitated the in vitro hydrolysis of waxy gene product. The positive but not significant correlation between AC and rat TD (Table IV) was because of the higher AC content of Koshihikari and Sasanishiki (Table I). This contrasts with the earlier negative value on 19 cooked milled rices differing in AC and GT2,6–10. Protein content and AC were also correlated (r=−0·87, p<0·01). The poor starch digestibility of IR36 based
Rice protein digestibility and properties
Table IV
Simple correlation coefficients between digestibility values and other grain properties of eight milled rices
Property of milled rice
Simple correlation coeeficients with
True digestibility (TD) in rats (% of cooked rice protein intake) Enzyme digestible nitrogen (EDN) (% of cooked rice protein) Digestible protein of cooked rice (DPcooked)(% of protein) Digestible protein of raw rice (DPraw)(% of protein) Enzyme digestible dry matter (% of cooked rice dry matter) Biological value (% of absorbed N) Net protein utilisation (% of cooked rice ingested N) Protein content (DIAS) (% in cooked rice) Protein content (LB) (% in cooked) Protein content (LB) (% in raw) Lysine in protein (g/16 g N) Cysteine in protein (g/16 g N) Tyrosine in protein (g/16 g N) Albumin-globulin (% in raw rice) Prolamin (% in raw rice) Glutelin (% in raw rice) Denatured prolamin (% in cooked rice) Amylose content (% in raw rice) Waxy gene product (% in rice) ∗
189
Significant at p=0·05.
∗∗
TD
EDN
DPcooked
DPraw
1·00
0·93∗∗
0·83∗
0·95∗∗
1·00
0·81∗
0·98∗
1·00
0·83∗ 1·00
0·63
0·51
0·66
0·48
0·58 0·80∗
0·50 0·72∗
0·18 0·44
0·52 0·74∗
−0·75∗
−0·62
−0·39
−0·71∗
−0·64 −0·76∗ 0·93∗∗ −0·83∗ −0·78∗ −0·34 −0·57 −0·71∗ −0·82∗
−0·51 −0·57 0·89∗∗ −0·88∗∗ −0·56 −0·31 −0·37 −0·55 −0·61
−0·17 −0·39 0·62 −0·72∗ −0·52 −0·30 −0·55 −0·41 −0·72∗
−0·57 −0·64 0·86∗∗ −0·85∗∗ −0·65 −0·23 −0·47 −0·63 −0·68
0·61 −0·52
0·61 −0·49
0·41 −0·40
0·70 −0·43
Highly significant at p=0·01.
amylose extender mutant even in raw form8 is probably due to the presence, together with the polyhedral granules, of irregularly shaped ae granules29, which waxy gene product is not readily digested probably because of a different granule structure and higher GT. By contrast its polyhedral granules are readily digested with their waxy gene product. Waxy milled rices Verification of the above results was undertaken with two pairs of japonica and indica waxy cooked milled rices, which are devoid of the waxy gene product11,12. Glaszmann18 method verified the classification for the Philippine rices (Table V), but the Taiwanese rices were received as milled rice and could not be tested. However, Taikeng Glu. 5 is a short grain typical of japonica rice and Taichung Sen Glu. 1 is a long grain typical of indica rice. The indica rice had higher protein
content, particularly for the Taiwanese pair (Table V). The waxy samples had similar digestible energy values (Table V). The indica samples tended to have lower TD but only the difference for the Taikeng-Taichung pair was significant. Differences in BV and NPU were not significant. The EDN and EDDM values tended to be lower also for indica rices but the differences were also significant only for the Taiwanese pair. Amino acid score X TD was also lowest for Taichung Sen Glu. 1. Lysine content of protein and amino acid score were lower in Taichung Sen Glu. 1 than in the others. Indica rice protein had higher cysteine and methionine contents than japonica rice protein. The tyrosine trend in the eight low-AC rices (Table IV) was not shown by the waxy rices. The lysine difference probably reflected the difference in protein content31, but protein content showed a negative correlation with cysteine in protein and a
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Table V
Properties, rat balance data, aminogram and in vitro digestibility of two pairs of japonica and indica cooked waxy milled rices
Property
Philippine Malagkit Sungsong
Glaszmann’s18 group Amylose content (% dry basis) Gross energy ( J/g dry matter) Rat balance data Digestible energy (% ±SD) True digestibility (% ±SD) Biological value (% ±SD) NPU (% ±SD) EDNa (% at ileal level) EDDMb (% at ileal level) Protein (% N×6·25 dry basis) Albumin-globulin (% of raw) Prolamin (% of raw rice) Glutelin (% of raw rice) Lys (g/16 g N) Met (g/16 g N) Cys (g/16 g N) Thr (g/16 g N) Ile (g/16 g N) Leu (g/16 g N) Val (g/16 g N) Phe (g/16 g N) Tyr (g/16 g N) Arg (g/16 g N) His (g/16 g N) Ala (g/16 g N) Asp (g/16 g N) Glu (g/16 g N) Gly (g/16 g N) Pro (g/16 g N) Ser (g/16 g N) N recovery (%) Amino acid scoreb (%) Amino acid scoreb×TD (%)
Taiwanese IR29 I
Taikeng Glu. 5
Taichung Sen Glu. 1
VI 1·7 17·3
2·0 17·2
(VI) 1·3 17·4
(I) 1·8 17·5
98·0±0·4 97·9±1·2 77·2±2·2 75·5±2·1 85·1 80·5 9·19 0·3 0·5 8·4 3·2 2·4 2·1 3·3 4·3 8·0 5·9 5·0 4·0 7·9 2·4 5·3 8·5 17·4 4·2 4·3 5·2 90·7 54 53
96·9±0·7 93·8±3·0 76·5±1·7 71·7±2·1 81·7 74·1 9·58 0·3 0·6 8·7 3·2 2·7 2·3 3·3 4·4 7·9 5·8 4·9 3·9 7·3 2·3 5·3 8·1 17·0 4·1 4·4 5·0 89·3 56 53
97·2±0·6 97·2±1·9 73·7±2·9 71·6±3·0 87·3 80·4 9·88 0·4 0·8 8·7 3·2 2·4 2·1 3·2 4·3 7·8 5·8 4·8 4·0 8·0 2·4 5·2 8·4 16·9 4·3 4·4 5·1 89·0 55 53
97·0±0·5 93·1±0·9 71·4±3·3 66·5±3·7 77·0 71·1 10·75 0·2 0·6 9·9 3·0 2·6 2·3 3·3 4·3 7·7 5·8 4·9 4·1 7·4 2·3 5·2 8·0 17·0 4·0 4·3 5·0 90·4 52 48
a
EDN means enzyme digestible protein (N) at ileal level; EDDM means enzyme digestible dry matter at ileal level. b Based on 5·8% lysine in FAO/WHO/UNU22 pattern as 100%.
positive but not significant correlation with tyrosine in protein31. The prolamin content was the highest for Taikeng Glu. 5. Denatured prolamin in cooked rice could not be estimated because of the greater solubility of gelatinised waxy starch in buffer A. Thus this study of eight low-AC and four waxy low-GT cooked milled rices verified the higher rat TD and in vitro protein digestibility of japonica rice over indica rice. The difference was not related to lower prolamin content and lower waxy gene product level of starch in japonica, but was related to lower cysteine level in protein and lower level of denatured prolamin in the cooked rice. The
higher level of cysteine in indica protein probably contributes to this greater prolamin denaturation. The higher protein content in indica rice was verified. Acknowledgements We would like to thank the suppliers of the rice samples, the technical assistance of L.T. Roferos, the Biochemistry and Analytical Services Laboratory, Institute of Plant Breeding, UPLB and the IRRI Isozyme Laboratory and editing by Tess Rola. J.B.A.D. acknowledges the scholarship extended by PhilRice for his M.S. programme at UPLB.
Rice protein digestibility and properties
REFERENCES 1. Tanaka, K., Sugimoto, T., Ogawa, M. and Kasai, Z. Isolation and characterization of two types of protein bodies in the rice endosperm. Agricultural and Biological Chemistry 44 (1980) 1633–1639. 2. Eggum, B.O., Resurreccion, A.P. and Juliano, B.O. Effect of cooking on nutritional value of milled rice in rats. Nutrition Reports International 16 (1977) 649–655. 3. Tanaka, Y., Resurreccion, A.P., Juliano, B.O. and Bechtel, D.B. Properties of whole and undigested fraction of protein bodies of milled rice. Agricultural and Biological Chemistry 42 (1978) 2015–2023. 4. Resurreccion, A.P., Li, X., Okita, T.W. and Juliano, B.O. Characterization of poorly digested protein of cooked rice protein bodies. Cereal Chemistry 70 (1993) 101–104. 5. Tanaka, Y., Hayashida, S. and Hongo, M. Quantitative relation between feces protein particles and rice protein bodies. Nippon Nogei Kagaku Kaishi 49 (1975) 425–429. 6. Eggum, B.O., Juliano, B.O., Ibabao, M.G.B., Perez, C.M. and Carangal, V.R. Protein and energy utilization in boiled rice-legume diets and boiled cereals in growing rats. Plant Foods for Human Nutrition 37 (1987) 237–245. 7. Eggum, B.O., Juliano, B.O., Perez, C.M. and Acedo, E.F. The resistant starch, undigestible energy and undigestible protein contents of raw and cooked milled rice. Journal of Cereal Science 18 (1993) 159–170. 8. Eggum, B.O., Juliano, B.O., Perez, C.M. and Khush, G.S. Starch, energy, and protein utilization by rats in milled rice of IR36-based amylose extender mutant. Cereal Chemistry 70 (1993) 275–279. 9. Eggum, B.O., Satoh, H. and Juliano, B.O. Protein quality evaluation of cooked rice for protein mutants in growing rats. Cereal Chemistry 71 (1994) 199–201. 10. Eggum, B.O. and Juliano, B.O. Properties and protein quality in growing rats of a low-glutelin content rice mutant. Cereal Chemistry 74 (1997) 200–201. 11. Wang, Z.Y., Zheng, F.Q., Shen, G.Z., Gao, J.P., Snustad, D.P., Li, M.G., Zhang, J.L. and Hong, M.M. The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene. Plant Journal 7 (1995) 613–622. 12. Villareal, C.P. and Juliano, B.O. Comparative levels of waxy gene product of endosperm starch granules of different rice ecotypes. Starch/Sta¨rke 41 (1989) 369–371. 13. Villareal, C.P. and Juliano, B.O. Waxy gene factor and residual protein of rice starch granules. Starch/Sta¨rke 38 (1986) 118–119. 14. Villareal, C.P. and Juliano, B.O. Comparative starch synthase activity of indica and japonica developing grain. Starch/Sta¨rke 45 (1993) 114–115. 15. Song, S., Hsu, A.N. and Hong, M.C. The grading system and quality evaluation of major releasing rice varieties in Taiwan. In ‘Rice Grain Quality, Proceedings of a Symposium, Changhua, Taiwan, 7–9 Apr. 1988’, (S.Song and M.C.Hong, eds.), Taichung District Agricultural Station, Changhua, Taiwan, China (1988) pp. 327–340. 16. Hibino, T., Kidzu, T., Masumura, T., Ohtsubo, K.,
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Tanaka, K., Kawabata, K. and Fujii, S. Amino acid composition of rice prolamin polypeptides. Agricultural and Biological Chemistry 53 (1989) 313–318. Cagampang, G.B., Cruz, L.J., Espiritu, S.G., Santiago, R.G. and Juliano, B.O. Studies on the extraction and composition of rice proteins. Cereal Chemistry 43 (1966) 145–155. Glaszmann, J.C. Isozymes and classification of Asian rice varieties. Theoretical and Applied Genetics 74 (1987) 21–30. Huebner, F.R., Bietz, J.A., Webb, B.D. and Juliano, B.O. Rice cultivar identification by high-performance liquid chromatography of endosperm proteins. Cereal Chemistry 67 (1990) 129–135. Boisen, S. and Fernandez, J.A. Prediction of the apparent ileal digestibility of protein and amino acids in feedstuffs and feed mixtures for pigs by in vitro analyses. Animal Feed Science and Technology 51 (1995) 29–43. Beames, R.M., Helm, J.H., Eggum, B.O., Boisen, S., Bach Knudsen, K.E. and Swift, M.L. A comparison of methods for measuring the nutritive value for pigs of a range of hulled and hulless barley cultivars. Animal Feed Science and Technology 62 (1996) 189–201. WHO. Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series 724. World Health Organization, Geneva (1985). 206 pp. FAO. Protein quality evaluation. Report of Joint FAO/ WHO Expert Consultation. FAO Food and Nutrition Paper 51. Food and Agriculture Organization, Rome (1991). 66 pp. Little, R.R., Hilder, G.B. and Dawson, E.H. Differential effect of dilute alkali on 25 varieties of milled white rice. Cereal Chemistry 35 (1958) 111–126. Juliano, B.O., Perez, C.M., Blakeney, A.B., Castillo, T.D., Kongseree, N., Laignelet, B., Lapis, E.T., Murty, V.V.S., Paule, C.M. and Webb, B.D. International cooperative testing on the amylose content of milled rice. Starch/Sta¨rke 33 (1981) 157–162. See, Y.P. and Jackowski, G. Estimating molecular weights of polypeptides by SDS gel electrophoresis. In ‘Protein Structure’ (T.E. Creighton, ed.). IRL Press, Oxford, UK (1989) pp 1–19. Duncan, D.B. Multiple range and multiple F tests. Biometrics 11 (1955) 1–42. Resurreccion, A.P., Villareal, C.P., Parco, A., Second, G. and Juliano, B.O. Classification of cultivated rice into indica and japonica types by isozyme, RFLP and two milled-rice methods. Theoretical and Applied Genetics 89 (1994) 14–18. Kim, W.T. and Okita, T.W. Structure, expression and heterogeneity of the rice seed prolamins. Plant Physiology 88 (1988) 649–655. Kaushik, R.P. and Khush, G.S. Endosperm mutants in rice: gene expression in japonica and indica backgrounds. Cereal Chemistry 68 (1991) 487–491. Hegsted, D.M. and Juliano, B.O. Difficulties in assessing the nutritional quality of rice protein. Journal of Nutrition 104 (1974) 772–781.
Comparative Protein Digestibility in Growing Rats of Cooked Rice and Protein Properties of Indica and Japonica Milled Rices Sigurd Boisen∗, Joy Bartolome A. Duldulao†, Evelyn Mae T. Mendoza‡ and Bienvenido O. Juliano§ ∗ Danish Institute of Animal Science, Department of Nutrition, Research Centre Foulum, DK-8830 Tjele, Denmark; † Philippine Rice Research Institute, Maligaya, 3119 Mun˜oz, Nueva Ecija, Philippines; ‡ University of the Philippines Los Ban˜os, Biochemistry Laboratory, Institute of Plant Breeding, 4031 College, Laguna, Philippines; § Philippine Rice Research, Institute Los Ban˜os, 4031 College, Laguna, Philippines. Received 1 June 1999
ABSTRACT Eight indica and japonica milled rices with low amylose content and low starch gelatinisation temperature were analysed for cooked rice energy and N balance in growing rats and for protein properties. Digestible energy values were similar. Japonica rices Koshihikari and Sasanishiki had higher true digestibility (TD) and net protein utilisation in rats than indica rices IR24 and PR2338315. Similar results were obtained from in vitro proteolysis. High TD of cooked rices was not significantly correlated with low prolamin content in raw rice and with low waxy gene product (protein bound to starch granule) but was significantly correlated with low cysteine content in protein and with low denatured prolamin content in cooked rice. In two pairs of cooked waxy milled rices, which are devoid of waxy gene product, japonica rice still tended to show higher rat TD and in vitro protein digestibility than indica rice, consistent with lower cysteine content in japonica protein. 2001 Academic Press
Keywords: cooked-rice protein digestibility, indica vs. japonica, protein properties, waxy gene product, cysteine content, protein content, glutelin content, prolamin content.
INTRODUCTION The storage protein bodies (PB) in the rice endosperm are the prolamin-rich spherical PB I and the glutelin-rich segmented crystalline PB II1. True protein digestibility (TD) of milled rice in growing rats have been shown2 to be reduced from 100% to 85–90% by cooking the raw rice in boiling water. The poorly digestible fraction was shown
Corresponding author: B. Juliano. Tel: (63-49) 536-3633; Fax: (63-49) 536-3515; E-mail: [email protected]. S. Boisen and J. B. A. Duldulao are the senior authors. 0733–5210/01/030183+09 $35.00/0
to be the core proteins of the prolamin-rich PB I3,4. But indica and japonica rices are reported to have similar (85–90%) TDs3,5 in man. Collaborative N balance studies in growing rats with the late Dr Bjørn O. Eggum of the Danish Institute of Animal Science (DIAS) involved 12 indica cooked rices (TD 83·3–97·4%) up to 19922,4,6. Higher TD (94·6–99·8%) were obtained with more recent studies on seven japonica cooked rices9,10, but there was a tendency for TD to decrease with increase in amylose content (AC, r=−0·76, p<0·05). The TD of the combined 19 cooked milled rices correlated both with protein content (r=−0·83, p<0·01) and AC (r=−0·68, p<0·01). 2001 Academic Press
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The waxy gene product (granule-bound starch synthase) of japonica and indica nonwaxy rice differ only by 16 individual nucleotides11. Indica rice starch has more than twice as much 60-kD subunit waxy gene product12,13 as japonica rice starch, reflecting lower specific activity of the granule-bound starch synthase14. The starch synthase is relatively stable and extractable only after starch gelatinisation. Interestingly, high-AC indica rice in Taiwan has 2% higher protein content than low-AC japonica rice15. Dr K. Tanaka of Kyoto Prefectural University has reported (unpubl., 1985) on the higher in vitro pepsin digestibility of protein in raw rice in three japonica (78–80%) than in three indica rices (68, 69 and 78%). Protein contents were 7·4–8·8% for japonica and 7·5–10·4% for indica rices. The protein of japonica rice has been reported to be 20% prolamin, but indica rice protein has 30% prolamin16. The prolamin and glutelin fractions of milled rice protein increase with an increase in grain protein content17. Because of these considerations, a collaborative study was undertaken on cooked milled rice at similar low AC to determine; (1) whether or not japonica rice protein has greater TD in growing rats than indica rice protein; (2) whether the difference is related to the increase in prolamin content with an increase in protein content of milled rice; and (3) whether this difference is in any way related to the greater amount of waxy gene product (granule-bound starch; synthase) in indica rice.
MATERIALS AND METHODS Materials Brown rice samples of low AC and low starch gelatinisation temperature (GT) were obtained from the 1996 crop from various sources: two popular Japanese japonica rices, Koshihikari and Sasanishiki from Masao Yokoo, National Agricultural Research Center, Tsukuba, Japan; four Philippine rices: IR24, Sinandomeng, PR2338315, and PRJ 3 from University of the Philippines Los Ban˜os (UPLB)-PhilRice Los Ban˜os and PhilRice Maligaya; Thai variety Khao Dawk Mali 105 from Ngamchuen Kongseree, Pathumthani Rice Research Center, Thanyaburi, Pathumthani, Thailand; and U.S. long-grain variety Toro 2 from Anna M. McClung, ARS-USDA Rice Research,
Beaumont, TX, USA. All had low AC (15–20%) and low GT (alkali spreading value 6–7). Malagkit Sungsong and IR29 low GT waxy rices were obtained early in 1999 from PhilRice Maligaya. Taikeng Glu. 5 and Taichung Sen Glu. 1 low-GT waxy milled rices were obtained from Sheu-chih Sheng, Taichung District Agricultural Improvement Station, Tatsuen Hsiang, Changhua, Taiwan.
Methods Chemical studies on protein of these cooked and raw milled rices were undertaken at the Institute of Plant Breeding by J. B. A. Duldulao as his M. S. thesis at UPLB. Indica-japonica classification was checked by the seedling isozyme method18 at the Isozyme Laboratory of D. S. Brar at the International Rice Research Institute, and on waxy gene product content of the rice starch13. Rice flour (40 mg) was purified by five protein extractions with buffer A (55 m Tris HCl pH 6·8 with 2% (w/v) SDS, 5% (w/v) -mercaptoethanol and 10% (v/v) glycerol). The resulting starch granules were boiled in 10 volumes of buffer A for 10 min. After cooling, 10 volumes of buffer were added and the slurry was centrifuged. A 20-L supernate was used for SDS-PAGE. Crude protein content was determined by micro-Kjeldahl method using the factor 6·25 or by Lowry method. Prolamin, albumin-globulin and glutelin contents were determined on raw and cooked milled rice flour by sequential extraction of 400 mg flour for 2 h each with 8 mL of 60% 1-PrOH, 0·7 NaCl (pH 7·5), and 3% (w/w) SDS and 0·5% dithiothreitol (pH 8)19. Rough and brown rices that pass the chemical studies at PhilRice were milled after at least 4 months after harvest (aged), washed and boiled in 1·3 times its weight in water, freeze-dried and airshipped to DIAS. At DIAS, the eight samples were run together for amino acid analysis10, digestible energy (DE), true digestibility (TD), biological value (BV), and net protein utilisation (NPU) in five growing rats each10 and their in vitro protein (EDN) and dry matter enzymic digestibility (EDDM) values at the ileal level20 and in vitro organic matter enzymic digestibility (EDOM) at the faecal level21. Standard deviation (S.D.) was 0·2 g/16 g N for lysine, 0·1 g/16 g N for cysteine and methionine10, 2% for in vitro EDN20, and 5% for in vitro EDDM20 and EDOM21. Amino acid
Rice protein digestibility and properties
Table I
Classification of eight milled rices in terms of indica-japonica and starch properties (PhilRice Los Ban˜os)
Propertya
Glaszmann’s group Waxy gene product Waxy gene protein (% dry basis) Apparent amylose (% dry basis) Alkali spreading value a
185
Koshihikari Sasanishiki
Toro 2
Sinandomeng PRJ 3
Khao PR23383-15 IR24 Dawk Mali 105
VI Wxb 0·07
VI Wxb 0·06
0 Wxb 0·11
I Wxb 0·08
I Wxb 0·06
I Wxb 0·08
I Wxa 0·13
I Wxa 0·20
15·2
15·5
14·0
13·0
11·8
11·1
14·8
13·0
7·0
7·0
6·0
5·7
7·0
6·3
4·1
7·0
Glaszmann’s18 group: 1 (indica), group VI ( japonica); waxy gene product: Wxa (indica), Wxb ( japonica).
score was calculated based on the FAO/WHO/ UNU amino acid pattern for the preschool child with lysine content of 5·8% as 100%22. Amino acid score X TD was also calculated23. Part of the raw milled rice was tested for alkali spreading value24 as index of starch GT and for AC by iodine colorimetry25. Protein extracts and fractions and waxy gene product were dispersed in buffer A in a boiling water bath for 2 min and subjected to SDS-10% gel PAGE26 with protein standards lysozyme (14·3 kD), trypsinogen (24 kD), pepsin (34·7kD), ovalbumin (45 kD), and bovine serum albumin (66 kD). Polyacrylamide gels used were 0·75 mm thick, 8 cm long and 7 mm wide. Gels were stained with Coomassie blue and protein bands quantified by QuantiScan software (Biosoft). In vitro protein TD was also determined at UPLB on raw and cooked milled rice flours20. Residual proteins after pepsin and pancreatin digestion were extracted with cold buffer A (residual proteins I) and with boiling buffer A (residual proteins II) and the extracts also subjected to SDS-PAGE. S.D. were calculated and some data were subjected to Duncan’s multiple range test27. RESULTS AND DISCUSSION Classification into indica and japonic types The eight rices were all verified to have low AC and low GT (Table I). An exception was the mixture of high-GT grains (5 of 12) in PR2338315. One out of 12 grains had high GT in Sinandomeng. The rices had their expected Glaszmann18 grouping of I for indica and VI for japonica, except for Toro 2 which could not be classified (group 0). Khao Dawk Mali 105 was in
group I, although an earlier sample gave group VI28. Classification by waxy gene product in the endosperm using purified rice starch showed high waxy gene product (indica, Wxa) only for PR23383-15 and IR24 and Wxb ( japonica) for the other six samples, including Khao Dawk Mali 105 and Toro 2, in conformity with previous results28. The presence or absence of the 3 subunit of glutelin28 could not be resolved from the SDSPAGE and could not be used for indica-japonica classification28.
Aminogram and balance in rats The freeze-dried cooked milled rices had 91·3– 93·2% dry matter or 6·8–8·7% moisture (Table II). Protein content range was 6·19–10·87% and was lower in japonica than in indica rices. Ash content was 2·87–5·09%. Energy content was 17·5–18·4 J/g dry matter. The amino acid values were used without correction for N recovery since the N recovery was already high at 88·9–93·4% (Table II). Lysine was the first limiting amino acid (3·1–3·4 g/16 g N) of rice protein corresponding to an amino acid score of 53–59%. Koshihikari and Sasanishiki protein had the highest lysine content and amino acid score. IR24, PR23383-15 and PRJ 3 proteins had the lowest lysine value. Other essential amino acids were present in amounts closer to the reference pattern. However, Koshihikari and Sasanishiki had lower cysteine content of protein than the other rices and had lower tyrosine content than IR24 and PR23383-15. Koshihikari protein has the highest phenylalanine, arginine and leucine levels. Digestible energy (DE) in growing rats was sim-
186
Table II
S. Boisen et al.
Properties, aminogram, rat balance data and in vitro digestibility of japonica and indica cooked milled rices (Danish Institute of Animal Science)
Property
Dry matter (%) Protein (% N×6·25) Crude ash (%) Gross energy ( J/g dry matter) Lys (g/16 g N) Met (g/16 g N) Cys (g/16 g N) Thr (g/16 g N) Ile (g/16 g N) Leu (g/16 g N) Val (g/16 g N) Phe (g/16 g N) Tyr (g/16 g N) Arg (g/16 g N) His (g/16 g N) Ala (g/16 g N) Asp (g/16 g N) Glu (g/16 g N) Gly (6/16 g N) Pro (g/16 g N) Ser (g/16 g N) N recovery (%) Amino acid scorea (%) Rat balance data Digestible energy (% of intake)±SD True digestibility (% of N intake)±SD Biological value (% of absorbed N)±SD NPU (% of N intake) ±SD Amino acid score X TD (%) EDNb (% at ileal level) EDDMb (% at ileal level) EDOMb (% at faecal level)
Koshihikari Sasanishiki
Toro 2
Sinandomeng
PRJ 3
Khao Dawk Mali 105
PR23383-15 IR24
93·2 6·19 2·98 17·5
92·7 6·47 2·87 17·8
91·8 8·38 2·98 18·2
91·3 7·82 3·20 18·3
92·6 9·41 5·09 18·4
92·3 10·87 3·09 18·3
92·0 8·63 4·51 18·3
91·8 8·97 3·44 18·2
3·4 2·4 2·0 3·3 4·4 8·1 6·1 5·3 3·7 8·3 2·5 5·5 8·9 17·3 4·5 4·7 5·3 93·9 59
3·4 2·4 2·0 3·2 4·2 7·7 5·9 4·9 3·5 7·9 2·4 5·2 8·5 16·5 4·3 4·4 5·0 89·8 59
3·2 2·7 2·1 3·3 4·1 7·4 5·8 4·9 3·8 7·9 2·5 5·1 8·5 16·4 4·2 4·4 4·9 89·0 55
3·2 3·1 2·3 3·4 4·1 7·4 5·8 5·0 3·7 7·4 2·3 5·2 8·1 16·7 4·1 4·4 4·7 88·4 55
3·1 2·7 2·2 3·3 4·3 7·9 5·9 5·1 4·2 7·9 2·4 5·3 8·2 17·6 4·1 4·7 5·0 91·5 53
3·2 2·3 2·1 3·2 4·3 7·8 6·0 5·1 4·3 8·0 2·4 5·2 8·4 17·3 4·1 4·5 5·0 91·7 55
3·1 2·7 2·3 3·3 4·1 7·6 5·9 5·1 4·1 7·6 2·4 5·2 8·1 17·0 4·1 4·6 4·9 89·9 53
3·1 2·7 2·2 3·3 4·2 7·7 5·9 5·1 4·0 7·5 2·3 5·3 8·1 17·2 4·1 4·4 4·9 89·6 53
97·4 ±0·5 98·8 ±2·5 77·3 ±3·1 76·3 ±3·9
97·5 ±0·4 98·6 ±2·0 81·5 ±2·0 80·3 ±1·7
97·4 ±0·4 97·2 ±1·7 70·6 ±2·5 68·2 ±1·4
97·2 ±0·6 94·6 ±3·1 75·8 ±3·1 71·6 ±1·6
96·9 ±0·7 93·2 ±3·3 73·9 ±3·1 68·9 ±4·8
97·2 ±0·7 94·1 ±3·1 72·5 ±2·1 68·2 ±1·7
96·9 ±0·2 92·5 ±1·1 74·1 ±3·4 68·5 ±2·9
97·0 ±0·3 92·6 ±1·8 71·7 ±2·5 66·5 ±3·4
58 90·0
58 88·5
54 87·1
52 82·3
50 82·6
52 85·0
52 83·3
49 81·5
82·8
77·4
84·5
80·1
78·3
79·4
71·7
76·7
98·3
98·3
98·1
98·0
97·6
97·5
97·2
97·6
a
Based on 5·8% lysine in FAO/WHO/UNU22 pattern as 100%. EDN means enzyme digestible N (protein) at ileal level; EDDM means enzyme digestible dry matter at ileal level; EDOM is enzyme digestible organic matter at faecal level.
b
ilar at 96·9–97·5% of intake (Table II). TD was 92·5–98·8% and highest for Koshihikari, Sasanishiki and Toro 2, as in EDN values. PR2338315 and IR24 had lower TD than japonica rices. Koshihikari and Sasanishiki had the lowest cyst-
eine content in protein. Amino acid digestibility in cooked milled has been shown to be lowest for cysteine2, suggesting disulphide bond formation. Biological value was 70·6–81·5% and was highest for Sasanishiki and lowest for Toro 2 and IR24.
Rice protein digestibility and properties
NPU was 66·5–80·3% and highest for Sasanishiki and lowest for IR24. Rat TD correlated positively with NPU and lysine level in protein and negatively with cysteine and tyrosine levels in protein (Table IV). Amino acid score corrected for TD was highest for Koshihikari and Sasanishiki and lowest for IR24 and PRJ 3 (Table II). Toro 2 had intermediate value.
187
In vitro EDN at the ileal level was 81·5–90·0% and was highest for the two japonica samples and Toro 2 and lowest for IR24. In vitro EDDM at the ileal level was 71·7–84·5%, highest for Toro 2 and lowest for PR23383-15, and did not follow TD in rats (Table II). EDOM at the faecal level was high at 97·2–98·3% and was similar to DE values in rats. In vitro digestible protein (DP) of raw milled rice was 81·1–89·2% (Table III). It was also highest for Koshihikari and Sasanishiki, followed by Toro 2. By contrast, TD of raw rice protein in growing rats was 100%2. Corresponding DP values for cooked milled rice were lower at 77·0–82·6%. IR24 had the lowest value in both sets, whereas japonica rices, Koshihikari and Sasanishiki, and Toro 2 had the highest values. Cooked rice DP values were less than the EDN values reported in Table II, probably due to differences in the batches of proteases used. Undigested proteins in raw rice were soluble in cold (residual proteins I) and boiling buffer A (residual proteins II), but was only extracted in boiling buffer A from protease-treated cooked rice (Table III). Protease-treated cooked rice had higher residual protein than the corresponding raw rice. Rat TD correlated positively with the three in vitro assays, EDN of cooked rice and DP of raw and cooked rice (Table IV). EDN correlated significantly with DP of raw and cooked rice and NPU and with lysine content of protein and negatively with cysteine content in protein and behaved closer to in vitro DP of raw rice than to DP of cooked rice. In vitro DP of cooked rice correlated with DP of raw rice and negatively with cysteine content of protein. In vitro DP of raw rice correlated with NPU and lysine content of protein and negatively with cysteine content of protein.
values were low at 0·25–0·40% (4–6% of protein). Low prolamin values of 0·23–0·44% (3–7% of protein) were obtained using 60% 1-PrOH solvent without -mercaptoethanol. Glutelin values were high (4·80–6·87% of milled rice; 87–91% of protein) but included the 10–16 kD prolamin subunits, as verified by SDS-PAGE. The addition of -mercaptoethanol to 60% 1-PrOH should have extracted the 10–16 kD prolamin subunits. Rat TD did not correlate significantly with albumin-globulin and prolamin contents, but correlated negatively with glutelin content (Table IV). Correcting for the prolamin contamination in glutelin, the corrected prolamin values of raw rice were 0·72–1·22% (11–17% of protein) and corrected glutelin values were 3·90–6·09% (72–83% of protein). Cooking, as expected, reduced protein extractability to 64–81% (mean 71%) due to heat denaturation (Table III). ‘Albumin-globulin’ values were 0·12–0·26% (2–5% of protein), ‘prolamin’ content was 0·05–0·09% and ‘glutelin’ content was 4·16–5·63%. The denatured prolamin (10–16 kD) values in cooked rice flour, which was extracted only by boiling and not by cold buffer A, was 0·13–0·34% of cooked rice and was lower in Koshihikari, Sasanishiki, Sinandomeng and Toro 2 than in PR23383-15, IR24, PRJ 3 and Khao Dawk Mali 105 (Table III). Correlation coefficient with rat TD was significant (Table IV). The denatured prolamin probably represents the high cysteine prolamin fraction29 that became denatured on cooking due to disulphide bonding to cystine. Which explains why TD correlated with cysteine content of protein (Table IV). Protein content of cooked rice in Table III was lower than that in Table II, except for Koshihikari. However, the protein values from the two laboratories were correlated (r=0·96, p<0·01). Rat TD correlated positively with lysine content in the protein and negatively with protein content, glutelin content and with cysteine and tyrosine levels in protein (Table IV). Interestingly DIAS protein content correlated with lysine (r=−0·75, p<0·05) and tyrosine (r=0·91, p<0·01) in protein and with glutelin content (r=0·85, p<0·01) and may explain the significant correlation of TD with these properties.
Prolamin and other protein fractions
Waxy gene product
Total protein extraction from raw rice was 94– 106% (mean 100%)(Table III). Albumin-globulin
Rat TD did not correlate significantly negatively with waxy gene product content of milled rice
In vitro digestibility
188
Table III
S. Boisen et al.
Properties of raw and cooked milled rices (Institute of Plant Breeding, UP Los Ban˜os-PhilRice Los Ban˜os)
Property of milled rice Raw milled rice Protein (% N×62·5) ±SD In vitro digestible protein (%) ±SD Res. proteins I (%) Res. proteins II (%) Albumin-globulina,b (%) Prolamina,b (%) Glutelina (%) ±SD Percent N extraction Waxy gene product (%) Total After proteolysis Cooked milled rice Protein (% N×6·25) ±SD In vitro digestible protein (%) ±SD Res. protein II (%) “Albumin-globulin”a,b (%) “Prolamin”a,b (%) “Glutelin”b (%) ±SD Percent N extraction Denatured “prolamin” (%) Waxy gene product (%) Total After proteolysis
Koshihikari
Sasanishiki
Toro 2
Sinandomeng PRJ 3
Khao PR23383-15 Dawk Mali 105
IR24
5·35 ±0·06 89·2 ±0·2 2·5 9·5 0·34c 0·30d 4·80f ±0·02 102
5·46 ±0·25 88·6 ±0·0 1·6 8·6 0·25f 0·27e 5·18e ±0·00 104
6·44 ±0·42 87·2 ±0·4 1·5 12·7 0·34c 0·23f 5·54a ±0·01 95
5·83 ±0·06 81·4 ±0·0 3·6 9·9 0·27e 0·23f 5·68c ±0·02 106
7·38 ±0·16 82·2 ±1·2 1·6 6·1 0·38b 0·32c 6·77a ±0·11 101
7·74 ±0·16 82·8 ±0·4 1·8 4·7 0·27e 0·44a 6·87a ±0·07 98
7·91 ±0·07 82·7 ±1·0 1·0 6·0 0·31d 0·34b 6·49b ±0·05 102
6·64 ±0·05 81·1 ±0·4 1·6 8·0 0·40a 0·44a 5·39d ±0·05 94
0·07 0·09
0·06 0·06
0·11 0·10
0·08 0·08
0·06 0·06
0·08 0·08
0·13 0·11
0·20 0·08
6·10 ±0·02 80·0 ±0·4 20·5 0·26a 0·08b 4·56e ±0·01 80
6·21 ±0·25 81·1 ±0·3 20·5 0·22b 0·07c 4·16f ±0·00 72
7·79 ±0·13 82·6 ±0·1 12·7 0·19d 0·07c 5·63a ±0·02 76
6·91 ±0·02 78·0 ±0·3 13·1 0·21c 0·06d 4·20f ±0·00 65
7·92 ±0·27 77·9 ±1·3 16·5 0·12g 0·09a 4·85c ±0·07 64
8·35 ±0·06 79·1 ±0·7 17·5 0·15f 0·06d 5·63a ±0·03 70
7·51 ±0·09 77·6 ±0·6 14·3 0·16e 0·05e 4·75d ±0·06 66
7·17 ±0·36 77·0 ±0·8 16·3 0·19d 0·07c 5·25b ±0·03 77
0·17
0·15
0·13
0·17
0·34
0·32
0·31
0·27
0·07 0·00
0·06 0·00
0·11 0·00
0·08 0·00
0·06 0·00
0·08 0·00
0·13 0·00
0·20 0·00
a
SD<0·01%. Protein values in the same line followed by the same letter are not significantly different at p=0·05 by Duncan’s27 multiple range test. c No residual protein I was extracted. b
(Table IV). SDS-PAGE of residual protein of in vitro protease-digested milled rice showed mainly the 13±3 kD prolamin band in both raw and cooked rice, but only the raw rice had some waxy gene product at MW 60 kD. By contrast, TD of raw rice protein in rats is 100%2. The enzymes used probably did not have adequate amylase activity to hydrolyze the raw starch protecting the waxy gene product in vitro. However, the decrease in waxy gene product in IR24 raw rice from 0·20% to 0·08% due to proteolysis (Table III) is difficult
to explain. The gelatinized starch in cooked rice facilitated the in vitro hydrolysis of waxy gene product. The positive but not significant correlation between AC and rat TD (Table IV) was because of the higher AC content of Koshihikari and Sasanishiki (Table I). This contrasts with the earlier negative value on 19 cooked milled rices differing in AC and GT2,6–10. Protein content and AC were also correlated (r=−0·87, p<0·01). The poor starch digestibility of IR36 based
Rice protein digestibility and properties
Table IV
Simple correlation coefficients between digestibility values and other grain properties of eight milled rices
Property of milled rice
Simple correlation coeeficients with
True digestibility (TD) in rats (% of cooked rice protein intake) Enzyme digestible nitrogen (EDN) (% of cooked rice protein) Digestible protein of cooked rice (DPcooked)(% of protein) Digestible protein of raw rice (DPraw)(% of protein) Enzyme digestible dry matter (% of cooked rice dry matter) Biological value (% of absorbed N) Net protein utilisation (% of cooked rice ingested N) Protein content (DIAS) (% in cooked rice) Protein content (LB) (% in cooked) Protein content (LB) (% in raw) Lysine in protein (g/16 g N) Cysteine in protein (g/16 g N) Tyrosine in protein (g/16 g N) Albumin-globulin (% in raw rice) Prolamin (% in raw rice) Glutelin (% in raw rice) Denatured prolamin (% in cooked rice) Amylose content (% in raw rice) Waxy gene product (% in rice) ∗
189
Significant at p=0·05.
∗∗
TD
EDN
DPcooked
DPraw
1·00
0·93∗∗
0·83∗
0·95∗∗
1·00
0·81∗
0·98∗
1·00
0·83∗ 1·00
0·63
0·51
0·66
0·48
0·58 0·80∗
0·50 0·72∗
0·18 0·44
0·52 0·74∗
−0·75∗
−0·62
−0·39
−0·71∗
−0·64 −0·76∗ 0·93∗∗ −0·83∗ −0·78∗ −0·34 −0·57 −0·71∗ −0·82∗
−0·51 −0·57 0·89∗∗ −0·88∗∗ −0·56 −0·31 −0·37 −0·55 −0·61
−0·17 −0·39 0·62 −0·72∗ −0·52 −0·30 −0·55 −0·41 −0·72∗
−0·57 −0·64 0·86∗∗ −0·85∗∗ −0·65 −0·23 −0·47 −0·63 −0·68
0·61 −0·52
0·61 −0·49
0·41 −0·40
0·70 −0·43
Highly significant at p=0·01.
amylose extender mutant even in raw form8 is probably due to the presence, together with the polyhedral granules, of irregularly shaped ae granules29, which waxy gene product is not readily digested probably because of a different granule structure and higher GT. By contrast its polyhedral granules are readily digested with their waxy gene product. Waxy milled rices Verification of the above results was undertaken with two pairs of japonica and indica waxy cooked milled rices, which are devoid of the waxy gene product11,12. Glaszmann18 method verified the classification for the Philippine rices (Table V), but the Taiwanese rices were received as milled rice and could not be tested. However, Taikeng Glu. 5 is a short grain typical of japonica rice and Taichung Sen Glu. 1 is a long grain typical of indica rice. The indica rice had higher protein
content, particularly for the Taiwanese pair (Table V). The waxy samples had similar digestible energy values (Table V). The indica samples tended to have lower TD but only the difference for the Taikeng-Taichung pair was significant. Differences in BV and NPU were not significant. The EDN and EDDM values tended to be lower also for indica rices but the differences were also significant only for the Taiwanese pair. Amino acid score X TD was also lowest for Taichung Sen Glu. 1. Lysine content of protein and amino acid score were lower in Taichung Sen Glu. 1 than in the others. Indica rice protein had higher cysteine and methionine contents than japonica rice protein. The tyrosine trend in the eight low-AC rices (Table IV) was not shown by the waxy rices. The lysine difference probably reflected the difference in protein content31, but protein content showed a negative correlation with cysteine in protein and a
190
S. Boisen et al.
Table V
Properties, rat balance data, aminogram and in vitro digestibility of two pairs of japonica and indica cooked waxy milled rices
Property
Philippine Malagkit Sungsong
Glaszmann’s18 group Amylose content (% dry basis) Gross energy ( J/g dry matter) Rat balance data Digestible energy (% ±SD) True digestibility (% ±SD) Biological value (% ±SD) NPU (% ±SD) EDNa (% at ileal level) EDDMb (% at ileal level) Protein (% N×6·25 dry basis) Albumin-globulin (% of raw) Prolamin (% of raw rice) Glutelin (% of raw rice) Lys (g/16 g N) Met (g/16 g N) Cys (g/16 g N) Thr (g/16 g N) Ile (g/16 g N) Leu (g/16 g N) Val (g/16 g N) Phe (g/16 g N) Tyr (g/16 g N) Arg (g/16 g N) His (g/16 g N) Ala (g/16 g N) Asp (g/16 g N) Glu (g/16 g N) Gly (g/16 g N) Pro (g/16 g N) Ser (g/16 g N) N recovery (%) Amino acid scoreb (%) Amino acid scoreb×TD (%)
Taiwanese IR29 I
Taikeng Glu. 5
Taichung Sen Glu. 1
VI 1·7 17·3
2·0 17·2
(VI) 1·3 17·4
(I) 1·8 17·5
98·0±0·4 97·9±1·2 77·2±2·2 75·5±2·1 85·1 80·5 9·19 0·3 0·5 8·4 3·2 2·4 2·1 3·3 4·3 8·0 5·9 5·0 4·0 7·9 2·4 5·3 8·5 17·4 4·2 4·3 5·2 90·7 54 53
96·9±0·7 93·8±3·0 76·5±1·7 71·7±2·1 81·7 74·1 9·58 0·3 0·6 8·7 3·2 2·7 2·3 3·3 4·4 7·9 5·8 4·9 3·9 7·3 2·3 5·3 8·1 17·0 4·1 4·4 5·0 89·3 56 53
97·2±0·6 97·2±1·9 73·7±2·9 71·6±3·0 87·3 80·4 9·88 0·4 0·8 8·7 3·2 2·4 2·1 3·2 4·3 7·8 5·8 4·8 4·0 8·0 2·4 5·2 8·4 16·9 4·3 4·4 5·1 89·0 55 53
97·0±0·5 93·1±0·9 71·4±3·3 66·5±3·7 77·0 71·1 10·75 0·2 0·6 9·9 3·0 2·6 2·3 3·3 4·3 7·7 5·8 4·9 4·1 7·4 2·3 5·2 8·0 17·0 4·0 4·3 5·0 90·4 52 48
a
EDN means enzyme digestible protein (N) at ileal level; EDDM means enzyme digestible dry matter at ileal level. b Based on 5·8% lysine in FAO/WHO/UNU22 pattern as 100%.
positive but not significant correlation with tyrosine in protein31. The prolamin content was the highest for Taikeng Glu. 5. Denatured prolamin in cooked rice could not be estimated because of the greater solubility of gelatinised waxy starch in buffer A. Thus this study of eight low-AC and four waxy low-GT cooked milled rices verified the higher rat TD and in vitro protein digestibility of japonica rice over indica rice. The difference was not related to lower prolamin content and lower waxy gene product level of starch in japonica, but was related to lower cysteine level in protein and lower level of denatured prolamin in the cooked rice. The
higher level of cysteine in indica protein probably contributes to this greater prolamin denaturation. The higher protein content in indica rice was verified. Acknowledgements We would like to thank the suppliers of the rice samples, the technical assistance of L.T. Roferos, the Biochemistry and Analytical Services Laboratory, Institute of Plant Breeding, UPLB and the IRRI Isozyme Laboratory and editing by Tess Rola. J.B.A.D. acknowledges the scholarship extended by PhilRice for his M.S. programme at UPLB.
Rice protein digestibility and properties
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