(introduction...)

Objectives
Experimental details
Conclusions and comments

E. Vargas and R. Bressani
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

To evaluate the protein quality of rice-and bean diets.
To determine the effect of supplementary animal protein and energy density on the quality of rice-andbean diets.
To estimate the protein and amino acid needs of normal adult individuals fed such diets.

Experimental details

1. Subjects Number: ten per experimental run.
Experimental runs: four.
Age: 20 to 31 years.
Sex: male.
Racial origin: Maya Indian and Spanish.

TABLE 1. Ascending Protein Intake Sequence

Protein intake	Calorie intake
level (g/kg/day)	level (g/kg/day)	No. of days	Diet fed
0.6	45 & 50	6	Regular diet
0.0	45 & 50	3	Nitrogen-free diet
0.2	45 & 50	2	Rice and bean*
0.4	45 & 50	2	Rice and bean
0.6	45 & 50	2	Rice and bean

*Rice and bean (60:40 protein distribution) in study I with 45 kcal/kg/day; in study 2 with 50 kcal/kg/days; in study 3 with 10 per cent milk protein substitution with 45 kcal/kg/day and in study 4 with 10 per cent milk protein substitution with 50 kcal/kg/day.

Physiological status: normal.

Nutritional status: acceptable-weight 49.1 to 65.0 kg; height 157 to 172 cm.

Health status: free of chronic infections and free of intestinal parasites.

2. Study Environment Location:
Metabolic Unit of the Division of Food Science, INCAP. Climatic characteristics: temperature 21 to 25 C (day); relative humidity 77 to 85 per cent; altitude 1,470 m above sea level; months of September to December 1979.

3. Physical Activity Normal
(laboratory technicians and institution maintenance crew).

4. Duration of Study
Total time per Study nine days.

Procedure: short-term, multiple-point nitrogen-balance assay.

Protein intake changes: every two days, as indicated in table 1.

TABLE 2. Nitrogen-Free Diet Composition per Assay

Food	1	2	3	4	Calories from each ingredient (kcal)
Instant coffee, g	3	3	3	3	3
White sugar, g	25	25	25	25	100
Apple or pineapple marmalade, g	30	30	30	30	78
Wheat starch bread,1 g	300	300	300	300	801
Margarine, g	80	80	80	80	576
Cornstarch soup²	480	480	480	480	144
Vegetable (Guisquil), g	100	100	100	100	52
Apple (with peel), g	200	200	200	200	116
Artifical fruit-flavoured drink (glasses)	3	3	3	3	228
Cookies (units)³	2	1	1	1	100/unit
Carbonated drinks (units)	1	-	1	-	136
Vitamin/mineral supplement (pills) 4	1	1	1	1	-
Total calories	2,434	2,198	2,334	2,198

1,3 Prepared from wheat starch (Jolly Joan, Ener-G Foods, Inc., P.O. Box 24723, Seattle, Washington 98124, USA).
2 Prepared from cornstarch and margarine, and seasoned with aromatic herbs. Herbs were not consumed
4 UNIT TMR.

5. Diets Table 2 describes the ingredient composition of the basal diet fed (nitrogen-free diet), and table 3 shows the protein content of the rice and beans used in the four studies. Table 3 also describes the vitamin and mineral composition.

The protein and energy intake from rice and beans was calculated every two days. It was given to the subjects in three equal portions, or during lunch or dinner. Large batches of raw rice and beans were purchased to reduce the variability that could be caused by differences in quality. The beans were cooked in large lots by soaking for 14 to 16 hours followed by cooking at 151bs/inch2 (1.05 kg/cm2) for 30 minutes. The material was then stored frozen. Rice was steamed.

TABLE 3. Protein Content of Protein Sources

Food	Moisture	Dry matter	Protein (N x 6.25)
	(%)	(%)	(%)*
Rice	68.5	31.5	2.68
Beans	76.1	23.9	7.38
Basal diet (nitrogen-free diet)	76.4	23.6	0.35

*Fresh basis.

6. Measurements and indicators
Composition of diets: AOAC methods of proximate chemical analysis.

Digestibility: apparent and true.

Nitrogen balance: apparent-does not include other losses.

Indicators of protein quality and utilization: (a) linear regression analysis (y = a + bx) of nitrogen intake (Nl) to nitrogen retention (NR) and of nitrogen absorption (NA) to NR; N i for N R = 0; (b) quadratic regression analysis (y = a + bx + cx²) of Nl to NR and of NA to NR, used primarily to estimate recommended protein intake as obtained by calculating the first derivative dy/dx = b + cx, where x (Nl or NA) is equal to-(b/2c).

Energy: digestibility and metabolizable energy at 0.6 9 protein intake and by measuring energy in food, faeces, and urine.

Other determinations and measurements: none.

Summary of Main Results Protein digestibility (apparent) of the rice and bean diet (60:40) was not affected by a difference in energy intake of 45 to 50 kcal (59.1 vs. 59.6 per cent). It was increased by the 10 per cent milk replacement of the protein in the rice and bean mixture without being affected by energy intake (65.3 and 64.6 per cent). The protein digestibility of milk was 75.6 per cent. Energy digestibility of the diets varied from 93.3 to 94.6 per cent, while metabolizable energy varied from 91.9 to 92.9 per cent.

The linear coefficient of regression between Nl and NR was not affected by energy intake without milk (0.75 and 0.79) or with milk (0.95 and 0.86), but milk supplementation improved it significantly, and was no different from milk alone (0.91). The Nl for NR = 0 followed the same trend. For 45 and 50 kcal/kg/day the values without milk were 95.6 and 92.9 mg/kg/day, and with milk, 78.4 and 80.8 mg/kg/day. The milk reference value used was 86.6 mg/kg/day. The relative coefficients of regression to milk equal to 100 per cent were 82.4, 86.8, 104.4, and 94.5 per cent.

The effect of milk was attributed to an improvement in protein digestibility rather than to an improvement in essential amino acid balances, as judged by amino acid content and by the linear regression coefficients of NA to NR, which were statistically alike (0.99, 1.02, 1.10, and 1.04 for diets, and 0.91 for milk). Using the quadratic regression equations, the protein intakes for maximum nitrogen retention were 0.79, 0.79, 0.69, and 0.74, with diets of better quality giving lower values. Correcting for differences in digestibility, ail values were similar, with an average of 74.8 mg N/kg/ day. This value was interpreted to represent the amount needed for all of the population to be in positive and maximal balance.

Conclusions and comments

1. The short term assay yields data calculated and interpreted in the same way as that from the longterm, conventional method. It is sensitive to differences in quality and reduces problems that may develop in subjects.

2. Rice and bean diets (60:40 protein ratio) are not improved by energy intake; however, replacement of 10 per cent of the protein by milk protein increases quality through an improvement in digestibility.

3. Although present interpretation of results from linear regression analysis provides useful data for estimating protein digestibility, protein quality, and intakes for maintenance, the quadratic regression analysis should be included as part of the whole analysis. This should be tested further.

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Ricardo Bressani, Delia A. Navarrete, Emilio Vargas, and Olivia Gutierrez
Division of Agricultural and Food Sciences, Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objective

The staple foods for large population groups in Latin America are common beans (Phaseolus vulgaris) and maize or rice. The cereal grains are sometimes replaced by tubers (e.g.. plantain! or roots (e.g., cassava or yam). These studies were carried out to evaluate the protein nutritional quality of common beans mixed with starch, a cereal, or plantain, using a short-term, multi-level nitrogen-balance assay.

Experimental details

1. Subjects
Four sets of experiments were carried out. A total of 32 healthy young men volunteered for the study. They were laboratory technicians and maintenance employees. They were Spanish or mixed Spanish-Mayan Indian, from 23 to 35 years old, with weight ranges from 46.5 to 65.0 kg and heights from 157 to 172 cm. They did not have chronic infections or intestinal parasites.

2. Study Environment
The men lived in their homes in Guatemala City and worked at INCAP. All of their meals were eaten in the Division of Food Sciences Metabolic Unit. The daily ambient temperature ranged from 21° to 25° C in some studies and from 27° to 32° C during others. Relative humidity ranged from 72 to 85 per cent. Guatemala City is 1,510 m above sea level.

TABLE 1. Basal Diet Used in Human Metabolic Studies

Food	Amount (g)	kcal
Instant coffee	3	3
Sugar	25	100
Apple jelly	30	78
Toasted bread*	300	801
Margarine	80	576
Soup * *	480	144
Chayote	100	52
Apple	200	116
Artificially flavoured drink, glass	3	228
Starch cookies,*units	2	200
Carbonated beverage, bottle	1	136
Total energy	-	2,434

* Made from wheat starch only
** Made from cornstarch.

3. Physical Activity
All men performed their usual chores.

4. Duration of the Study
After subjects had consumed the standard protein diet for several days, a nineday experiment was carried out. During the first three days the men ate the diet described in table 1, which provided only between 20 and 30 mg N/kg/day. After that they ate the experimental diet in which protein content was changed every two days. The three two day periods of protein intake provided 0.2, 0.4, and 0.6 9 protein/kg/day. The details of this short-term, multiple nitrogen-balance assay have been described elsewhere (Bressani et al., in H.L. Wilcke, D.T. Hopkins, and D.H. Waggle, eds., Soy Protein and Human Nutrition [Academic Press, New York, 1979] ).

5. Experimental Diets
The four experimental diets were as follows:

TABLE 2. Regression Equation between Nitrogen Intake and Nitrogen Retained of Subjects on a Diet of Wheat Starch and Black Beans (Phaseolus vulgaris)

Subject	a 1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
1F-HM	-39.7 +0.32	124.1	0.56
2F-AF	-100.9+0.86	117.3	0.93
3F-GM	-50.6 + 0.47	107.6	0.71
4F-WH	-85.9 + 0.80	107.4	0.83
5F-SY	-35.9 + 0.30	119.7	0.99
6F-AS	-63.1 +0.62	101.8	0.96
Average	-62.6 + 0.55	113.8	0.75

1 NR=a+b(NI).

TABLE 3. Regression Equation between Nitrogen Intake and Nitrogen Retained of Subjects on a Diet of Tortilla and Black Beans (Phaseolus vulgaris)

Subjects	a1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
7T- FS	-89.5 + 0.94	95.2	0 96
8T-VO	-105.9 + 0.89	118.9	0.82
10T-AL	-83.5 + 0.91	91.7	0 96
11T-WS	-88.4 + 0.91	97.1	0 94
12T-EM	-80.6 + 0.95	84.8	0 99
Average	-86.9 + 0.89	97.6	0.89

1. Common beans with wheat starch were the main energy sources (6 men participated).
2. Corn tortillas and black beans. The maize-bean mixture was provided in proportions of 70:30 on a dry-weight basis (6 men participated).
3. Rice and common beans fed in a 60:40 ratio, based on protein content (10 men participated). d. Common beans and cooked, mature plantain, with black beans providing between 30 and 32 per cent of total energy (12 men participated).

1 NR=a+b(NI).

The initial low-nitrogen diet provided between 2,400 and 2,500 kcal/day. Energy intake was adjusted to 45 to 50 kcal/kg/day to meet each individual's energy needs and to allow him to maintain body weight throughout the experiment. A multivitamin and mineral tablet was provided each day. Water intake was maintained at a constant level for this study.

6. Indicators and Measurements
a. The composition of the diets was calculated with AOAC methods of proximate chemical analysis. b. Urine and faeces were collected as 24 hour pools. Faecal markers were used to separate faecal collections. c. The nitrogen contents of diets, urine, and faeces were determined using the macro Kjeldahl technique. Apparent nitrogen balance was calculated by subtracting urinary and faecal nitrogen from dietary nitrogen. Apparent digestibilties were calculated, not including obligatory faecal nitrogen losses,

Summary of main results

Tables 2 to 5 show the individual regression equations of apparent nitrogen balance on nitrogen intake. Table 6 shows the nitrogen intake required to obtain equilibrium and the amounts of foods in the diet that provided such nutrients. Table 7 shows the apparent digestibility of the proteins fed in various experiments.

Conclusions and comments

1. Larger amounts of beans are needed to obtain nitrogen equilibrium when they are eaten with starch or plantains than when they are eaten with cereal grains (see table 7). This is due both to the nitrogen contribution of the cereal grains and to the sulphur amino acids that they provide. The difference in the protein quality of the mixtures is demonstrated by the higher regression coefficients of the bean-andmaize or beanand-rice mixtures.

TABLE 4. Regression Equations between Nitrogen Intake and Nitrogen Retention of Subjects Fed Rice and Black Beans (Phaseolus vulgaris) in a 60:40 Protein Ratio

Subject	a1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
M.R.	- 92.7 + 0.76	121.5	0.92
R.A.	- 60.6 + 0.63	96.9	0.96
L.J.	- 65.9 + 0.81	81.8	0.96
F.M.	- 81.8 + 0.78	105.1	0.97
A.G.	- 76.8 + 0.79	96.9	0.95
C.E.	- 54.8 + 0.79	69.5	0.99
J.L.	-54.5 + 0.55	98.8	0.98
G.P.	- 70.1 +0.70	100.4	0.95
O.B.	- 84.8 + 0.92	92.3	0.96
R.C.	- 86.1 +0.87	98.6	0.92
Average	-71.6 +0.75	95.6	0.87

¹ NR-a+B(NI),

Protein digestibilities were low in the four studies, more so when the bean-and-plantain diet was used. The polyphenolic compounds in beans increase faecal nitrogen output.

Plantain also contains polyphenolic compounds that may add to the faecal nitrogen excretion.

2. More sulphur-containing amino acids are needed in the bean-paste diets eaten by populations who also consume starchy foods such as plantain or cassava. This does not seem to be so important in the diet of populations who consume cereal grains in addition to beans.

TABLE 5. Regression Equation between Nitrogen Intake and Nitrogen Retained of Subjects on a Diet of Plantains and Black Beans (Phaseolus vulgaris)

Subjects	a1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
11P-F.M.	- 121.7 + 1.20	101.4	0.91
12P-O.B.	- 54.0 + 0.47	114.9	0.82
13P-R.A.	- 75.6 + 0.70	108.0	0.99
14P-S.P.	- 66.9 + 0.53	126.2	0.76
15P-R.S.	- 53.4 + 0.34	1 57.0	0.73
16P-O. B.	--69.7 + 0.63	110.6	0.99
17P-H. R.	- 109.8 + 0.92	119.3	0.92
18P-M.R.	- 63.6 +0.29	219.3	0.87
19P-O.H.	- 126.3 + 1.51	83.6	0.85
20P-N. Ro.	- 75.3 + 0.64	117.6	0.96
Average	-81.7 + 0.73	111.9	0.77

¹ NR=a+b(NI).

TABLE 6. Nitrogen Intake for Nitrogen Equilibrium and Amounts of Foods Needed

Diet	Nitrogen intake for nitrogen equilibrium (mg/kg/day)	Beans/day		Other food/day
		dried wt. (g)	cooked wt. (g)	dried wt (g)	cooked wt. (g)
Beans/starch	114	186	638	-	-
Beans/plantain	112	185	636	-	855
Beans/maize	98	82	170	193	495
Beans/rice	95	52	194	281	802

TABLE 7. Apparent Protein Digestibility of Black Beans (Phaseolus vulgaris) Fed with Starchy Foods and Cereal Grains

Diet	Nitrogen intake (mg/kg/day)	Apparent protein digestibility (%)
Beans/starch	115.6 ± 0.9	60.0 ± 2.2
Beans/plantain	117.4 ± 0.6	52.5 ± 4.0
Beans/maize	127.6 ± 0.5	61.0 ± 9.0
Beans/rice	102.5 ± 1.1	59.1 ± 7.4