**Chemistry Department, International Rice Research Institute, Los Banos, Laguna, Philippines Received August 11, 1977

Transcription

1 Agric. Biol. Chem., 42 (3), 565 `570, 1978 Protein Quality of a High Protein Milled Rice in Rats õ Kiku MURATA,* Tadao KITAGAWA and Bienvenido 0. JULIANO** Food and Nutrition Laboratory, Faculty of the Science of Living, Osaka City University, Sugimoto-cho, Sumiyoshi-ku, Osaka, Japan **Chemistry Department, International Rice Research Institute, Los Banos, Laguna, Philippines Received August 11, 1977 The use of partially destarched rice flour with the same amino acid pattern as raw rice protein, and prepared by ƒ -amylolysis of cooked rice, permitted the comparison of the protein quality of a high (11 %)-protein milled rice (IR ) with egg and Japanese milled rice (6.7 protein) in rats at identical dietary protein levels. Net protein utilizztion values by carcass nitrogen analysis relative to egg were 55 to 56 % for IR rice and 56 % for the Japanese rice. Relative nutritive value of 1R rice protein using 0, 4, 8, 12 and 15% dietary protein levels was 44 to 48 % of egg as compared to 46 to 51 % for the Japanese rice. As part of the International Rice Research Institute (IRRI) breeding program to improve the protein content of rice,' biological testing of protein quality in rats and man is essential. Although the protein quality of rices differing in protein content has been determined in rats by protein efficiency ratio (PER)," nitrogen growth index," net protein utilization (NPU)3) and relative nutritive value (RNV),4) low protein samples cannot be run above dietary protein levels of 5 to 7 %. The slope ratio technique employed by Hegsted and Juliano4) utilizes rice levels of 0, 28, 56 and 84 %, which represent different protein levels for different rice samples. The NPU procedure of Eggum and Juliano3) requires correction for low protein rice since each rat is fed 10 g dry matter and 150 mg N per day. Recent studies by Murata et al.5,6) demon strated the feasibility of preparing partially destarched rice flour (concentrated rice protein) by ƒ -amylase treatment of cooked milled rice. This technique was applied to a high-protein rice IR and the preparation was utilized to compare its protein quality relative to egg õ This work was presented in part at the 30th Meeting of the Food and Nutrition Society of Japan held in Sendai, May, * Present address: Teikoku Women's University, Thodacho, Moriguchi City, Osaka, Japan. protein and to a Japanese rice with average protein content by NPU and RNV methods. MATERIALS AND METHODS Materials. IR rough rice was obtained from the IRRI 1974 dry-season crop. It was dehulled and milled in a Satake type SB-2B one-pass rice husker - pearler and stored at 4 C prior to air shipment to Osaka. Japanese milled rice grown in Fukui Prefecture in 1973 and fresh eggs were obtained in Osaka. Egg powder was prepared by mincing, lyophilizing and powdering boiled eggs after removing the shell. The egg powder was defatted three times with hexane and stored in a vacuum desiccator, Commercial, spraydried whole egg powder was purchased from Taiyo Food Co. and also defatted with hexane. Milled rice was ground into a fine flour and part of it was used for the preparation of partially destarched rice flour as previously described.l.l> Bacterial a- amylase (230,000 U/g, protease-free, Ueda Chem. Ind. Ltd., Osaka) was employed to destarch cooked rice flour. Net protein utilization. Young male rats of Sprague-Dawley strain were used; six rats per diet. Mean body weight was 59 g for experiment I and 57 g for experiment 11. A protein-free diet and diets con taining 10% protein were fed ad libitum to the rats for 10 days (Table I). The composition of the mineral and vitamin mixtures was according to Harper.') In experi ment I, the NPU of IR rice, commercial egg powder (61.1 % protein), and lyophilized egg (71-73% protein) were compared by nitrogen balance and carcass

2 566 K. MURATA, T, KITAGAWA and B. 0, JULIANO TABLE I. COMPOSITION OF DIETS FOR NET PROTEIN UTILIZATION AND RELATIVE NUTRITIVE VALUE EXPERIMENTSa) a) In g/100 g diet. Chocola A (0.05 ml) was added to supply 1500 I. U. vitamin A and 150I. U. vitamin D per 100 g diet.b ) Protein-free diet contains 88.9 g starch/100 g diet for net protein utilization and 89.9 g starch/100 g diet for relative nutritive value experiments. Oil and salt mixture in the egg diets were reduced cor responding to the fat and mineral contribution of the egg powder preparations.c) After Harper.7) d) Choline-Cl 0.5 g/ml of 50% ethanol. nitrogen methods. In experiment II, NPU was obtained by the carcass nitrogen method to compare IR rice, Japanese rice, and commercial egg powder. Urine and feces of four rats in each group were collected for 48 hr between the 7th and 10th day of feeding, and analyzed for nitrogen by semimicro Kjeldahl method.',') Procedure for analysis of carcass nitrogen was as described previously.') Relative nutritive value. A group of six young male rats of the Sprague-Dawley strain was assigned per diet. IR milled rice and egg were fed at die tary protein level of 1, 2, 4, 8, 12 and 15 %. Rice with destarched rice flour or starch constituted 89.9% of the diet as previously described6) (Table I). Initial mean body weight was 51 g. A protein-free diet was included and a group was killed at the beginning of the experiment. The rice and the egg diets were fed to the rats ad libitum for 21 days. Food intake and body weight were measured every 1 or 2 days during the experimental period. Temperature was maintained at C. At the end of the feeding period, all rats were killed, the content of the gut discarded and the whole carcasses stored at 20 C until analyzed. Frozen carcasses were chopped, placed in tared beakers and dried at 80 C and then at C to obtain the body water content. Increase in water content was calculated by subtracting the initial mean body water of the rats killed at the start of the experi ment. Hexane was added to the dried carcasses in the beaker to extract the fat. Extraction was repeated three times. The defatted, dry carcasses were ground into a powder in a Waring blendor and their nitrogen content was determined in triplicate by semimicro Kjeldahl method.6) The data for 0, 4, 8, 12 and 15% dietary protein levels were used to calculate RNV for rice but only the data for 0, 4 and 8 % protein diets were included for egg because the growth of rats receiving 12 and 15% egg protein was not proportionate to those receiving 4 and 8 % protein. Earlier results also showed maximum growth of the rats at 10% egg protein level.') The slopes of the regression lines between nitrogen intake and a) body weight gain, b) body water gain, and c) body nitrogen increase were calculated as previously described.8,i) RNV was calculated as the ratio of the slope of the rice group to the corresponding slope of the egg group multiplied by 100. Other analyses. Electron microscopy of concen trated rice protein was as described previously.") Albumin was extracted with water and globulin with 0.5 M NaCl and assayed as previously reported.") Electrophoresis on polyacrylamide disc gels was run by standard procedure.11) Amino acid analysis of IR milled rice and concentrated rice protein was determined at IRRI in a Beckman Spinco Model 120C analyzer employing the 2 hr run with PA-28 and PA-35 resinṣ11) RESULTS AND DISCUSSION Properties of destarched rice flour The IR milled rice with I I % protein gave a destarched rice flour with 36 to 41 protein. Earlier preparations from 7 % protein Japanese rice only had 27 to 34% protein.') Transmission electron microscopy

3 a Protein Quality of a High Protein Milled Rice in Rats 567 TABLE 11. CONTENT OF SELECTED AMINO ACIDS IN IR MILLED RICE AND ) Ing/16gN. DESTARCHED RICE FLOURa) of destarched rice flour showed that the protein bodies were intact after cooking, ƒ -alnylolysis and freeze-drying." In contrast to milled rice protein, with 2.6% albumin and 3.3% globulin, the destarched flour protein had only 0.7% albumin and 0.2% globulin. Disc gel electrophoresis showed many albumin and globulin bands for IR milled rice."' But in destarched flour, albumin was left in the stacking gel and in the case of globulin, no bands were noted in the electrophoregram. Amino acid analysis showed almost identical aminograms for the milled rice and the destarched flour protein (Table II). Thus, the destarched flour had almost the same amino acid score (56%) as the milled rice protein (58 %) based on 5.5 g lysine/ 16 g N as 100 %." Net protein utilization NPU obtained for IR rice using nitrogen balance was about 63 % of commercial egg powder (Table III). By carcass nitrogen analysis, IR milled rice protein had NPU of 55 to 56 % of commercial egg protein, which was similar to its amino acid score (Table II) and to the NPU obtained for Japanese rice of 56%. Lyophilized egg showed somewhat higher NPU than commercial egg. Using a constant energy intake and 9 % protein diet (dry basis), other samples of IR milled rice had NPU of 64.2 to 71.0%,',"' and egg had NPU of %14) by nitrogen balance technique in growing rats. In a nitrogen balance study with Filipino children," the NPU of IR milled rice with 11.0%. protein was 60 % after correction for reported endogenous nitrogen losses." Nishizawa et al."' reported NPU by nitrogen balance in growing rats of concentrated rice protein from a Japanese rice as 76.4% for milled rice control and 74.0% for high-protein, ureasprayed, milled rice as compared to 84.3% for casein. Relative nutritive value The regression equations including nitrogenfree diet for lyophilized egg and IR milled rice based on gain in body weight, nitrogen and water showed that high-protein nilled rice had RNV of 44 to 48 % relative to lyophilized egg (Table IV). The intercepts were not identical for rice and egg proteins and were higher than obtained for the proteinfree diet as reported also by Hegsted and Juliano.4) Hegsted and Juliano4) reported TABLE III. NET PROTEIN UTILIZATION Or MILLED RICE AND EGG IN GROWING RATSa) a) Dietary protein level of 10%. b) Mean±standard deviation. Numbers in parenthesis are based on commercial egg powder as 100.

4 568 K. MURATA, T. KITAGAWA and B. 0. JULIANO TABLE IV. ESTIMATES OF RELATIVE NUTRITIVE VALUE IN GROWING RATS OF IR RICE AS COMPARED TO EGGa) a) Based on six rats per diet and 0, 4 and 8 % protein diet for egg and 0, 4, 8, 12 and 15 % protein diet for IR milled rice.b) Correlation coefficient significant at l % level. TABLE V. ESTIMATES OF RELATIVE NUTRITIVE VALUE IN GROWING RATS OF IR RICE AS COMPARED TO EGGa) a) Based on 6 rats per diet and 4 and 8 % protein diet for egg and 4, 8, 12 and 15 % protein diet for IR milled rice. b) Correlation coefficient significant at I % level. RNV for other samples of IR milled rices of 51 % (12.3% protein sample) and 53 -(11.8 % protein sample) based on lactalbumin. These data could be compared directly with our data since defatted egg has RNY of 99 % of laetalbumin.8) Hegsted et al.9) also found identical results of RNV for a high (19 %) protein rice flour of 44 to 45 % based on gain of body water, nitrogen, and weight in rats. The corresponding RNV obtained for Japanese rice with 6.7 % protein was 51 % of commercial egg protein based on body weight gain data, 46 % based on body nitrogen gain and 51 % based on body water gains' Thus the RNV of 44 to 48 % for IR rice with 11.0 % protein (Table IV) were not much lower than the RNV for the Japanese rice. Rela tive utilizable protein (RNV X protein content) was higher for IR ( %) than for Japanese rice ( %). These results,are consistent with our earlier studies that the additional protein of high protein rice increased the utilizable protein of the rice.', 3,4,13,15) When the data for the nitrogen-free diet are excluded in the calculation of RNV,18) lower slopes were obtained particularly for egg (Table V). Intercepts were also higher for egg than for IR rice. In addition RNV of IR rice relative to egg was consistently higher than when a nitrogen-free diet was included in the calculation of RNV (Table IV). By contrast, no change or slight decrease was noted for RNV of the Japanese rice when the nitrogen-free diet was excluded." Corresponding RNV for Japanese rice of 44 to 51 % are lower than RNV of 52 to 57 % for IR rice which contrasts with the RNV including the nitrogen-free diet. A sample of IR milled rice with 10% protein had a RNV in growing rats of 76 % of casein using 2, 5 and 8 % protein diets."' As casein has an RNV of 75 %,8) its corresponding RNV is

5 Protein Quality of a High Protein Milled Rice in Rats 569 TABLE VI. ESTIMATES OF RELATIVE NUTRITIVE VALUE IN GROWING RATS OF IR RICE AS COMPARED TO EGG AT Low PROTEIN LEVELa) a) Based on 6 rats per diet and 0, 1 and 2% protein in the diet. b> Correlation coefficient significant at I % level. 57% of lactalbumin. Higher RNV relative to lyophilized egg were obtained for IR milled rice protein when only the low-protein diets (0, 1 and 2 %) were considered in the regression line (Table VI) as compared to when the protein range 0, 4, 8, 12 and 15 % were considered (Table IV). Exclusion of the nitrogen-free diet in the cal culation of RNV at low dietary protein levels of 1 and 2 % resulted in RNV of IR relative to egg of 89 % by body weight gain, 69 % by body nitrogen gain, and 77 % by body water gain. At the low protein levels, exclu sion of the nitrogen-free diet resulted in a decrease of RNV of IR rice relative to egg. Using dietary protein levels of 0, 1, 2, 3, 4 and 5%, Bressani et al," showed that the re lative quality based on weight gain for four milled rices with 6.0 to 15.2% protein (cor rected for differences in Kjeldahl factors of 6.25 for casein and 5.95 for rice) ranged from 77.0 to 54.5 % based on a value of 75 % for casein, since casein has an RNV of 75%'.` Again when only dietary protein levels giving net weight increase were used to calculate the regression equation, RNV for the four rites dropped to 45 to 40%, These results reflect the same trend we now report for 1 to 2 dietary protein and 4 to 15 % dietary protein data in Table VI and Table IV, respectively. However, the increases in RNV by omitting the nitrogen-free diet data (Table V) from the response curve (Table IV) are not consistent with these observations.6,8) The contributing factor was that although the slopes for the dose response of IR decreased, those of egg decreased even more (Table V) Lesser changes in the slope of egg had been noted previously."' Our use of the same dietary protein range for all rices via the addition of destarched rice flour reduced the dependence of RNV on protein level in the diet and resulted in more comparable and accurate data among rices differing in protein. For the same protein range of milled rice (6 to 11 %) for which we obtained 48 to 51 % and 51 to 57 % RNV based on body weight gain (with and without the nitrogen-free diet, respectively), Hegsted and Juliano4) obtained a wider RNV range (68 to 78 %) using dietary rice levels of 0, 28, 56 and 84%, regardless of protein content of rice. Bressani et al.2) based on carcass nitrogen gain, found the RNV of milled rice ( % protein) would be 48 to 67% of lactalbumin (and egg protein) based on an RNV of 75 % for casein. The above studies using a-amylase destarch ed cooked rice showed that the decrease in protein quality as a result of an increase in protein content is considerably reduced when the rat assays are done at the standard dietary protein level, which is 10 % for NPU and from 0 to 8 or 15 % for RNV. Differences in protein quality were much higher when low or nonidentical dietary protein levels were used to assay rites differing in protein content. The relative utilizable protein of high protein rice was higher and proportional to protein content of the grain. Factors contributing to the similarity of protein quality among rice

6 570 K. MURATA, T. KITAGAWA and B. O. JULIANO varieties are the high digestibility in rats of protein of all raw milled rices3 13) and the very slight change observed in the content of the first limiting amino acid lysine in the protein with an increase in protein content in rice.'-" Our analysis showed the amino acid score of protein of the Japanese milled rice to be 59 % as against 58% for IR rice protein. Acknowledgements. The work at IRRI was supported in part by National Institutes of Health (U.S.A.) contract NOi-AM The work at Osaka City University was supported in part by the Essential Amino Acid Research Committee, Japan. Y. Asada and K. Sasaki assisted in the rat feeding trials. Transmission electron microscopy was by N. Harris, Univ. of Durham, England. Electrophoresis and extraction studies on albumin and globulin were made by G. B. Cagampang. The u-amylase was a gift of Ueda Chem. Ind. Co., Ltd. REFERENCES 1) B. 0. Juliano and H. M. Beachell, "CIMMYT/ Purdue Univ. High-quality Protein Maize," Dowden, Hutchinson and Ross, Inc., Stroudsburg, Pa., 1974, p ) R. Bressani, L. G. Elias and B. O. Juliano, J. Agric. Food Chem., 19, 1028 (1971). 3) B. 0. Eggum and B. 0. Juliano, J. Sei. Food Agric., 24, 921 (1973). 4) D. M. Hegsted and B. O. Juliano, J. Nutr., 104, 772 (1974). 5) K. Murata, Y. Tanaka and Y. Kawaguchi, Nutr. Ren. Int (1973). 6) K. Murata, M. Nishikaze and M. Tanaka, J. Nutr. Sci. Vitaminol., 23, 125 (1977). 7) A. E. Harper, J. Nutr., 68, 408 (1958). 8) D. M. Hegsted, "Protein-enriched cereal foods for world needs," ed. by M. Milner, Am. Asso. Cereal Chemists, St. Paul, Minn., 1969, p ) D. M. Hegsted, R. Neff and J. Worcester, J. Agric. Food Chem., 16, 190 (1968). 10) N. Harris and B. O. Juliano, Ann. Bot., 41, 1 (1977). 11) G. B. Cagampang, A. A. Perdon and B. O. Juliano, Phytochemistry., 15, 1425 (1976). 12) International Rice Research Institute, Annual report for IRRI, Los Baiios, Philippines, 1977, p ) B. O. Eggum and B. O. Juliano, J. Sci. Food Agric. 26, 425 (1975). 14) B. O. Eggum, "A Study of Certain Factors Influencing Protein Utilization in Rats and Pigs," Agric. Res. Lab. Publ., No. 406, 1973, p ) B. V. Roxas, C. Ll. Intengan and B. O. Juliano, Nutr. Rep. Int., 11, 393 (1975). 16) P.-C. Huang and T.-C. Tung, "Protein Requirement of Infants of about One Year of Age," Paper Presented at 9th Int. Nutr. Congr., Mexico, ) N. Nishizawa, I. Kitahara, T. Noguchi, S. Hareyama and K. Hongo, Agric. Biol. Chem., 41, 477 (1977). 18) Protein-Calorie Advisory Group of the United Nations. Protein Methods for Cereal Breeders as Related to Human Nutritional Requirements. PAG Guideline No. 16, PAG Bull., 5 (2),22 (1975). 19) International Rice Research Institute. Annual Report for IRRI, Los Banos, Philippines, 1976, p. 11.