The Effect of Fortification of Processed Soya Flour with Dl-methionine Hydroxy Analogue or Dl-methionine on the Digestibility, Biological Value, and Net Protein Utilization of the Proteins as Studied in Children.

The true digestibility coefficient, biological value, and net available protein of diets based on processed soya flour supplemented with dl-methionine hydroxy analogue (MHA) or dl-methionine (at a level of 1.2 g/16 g N) have been determined in children aged 8-9 years. The mean daily intake of protein by the children on the different diets was maintained a t a level of about 1.2 g/kg body weight. Supplementation of soya flour with dl-methionine brought about a marked increase in the biological value and net protein utilization of the proteins. MHA was, however, slightly less effective than dl-methionine in this respect. The biological value and net protein utilization of the different proteins were as follows: soya flour, 63.5 and 53.3; soya flour + NIHA, 71.5 and 61.4; soya flour + methionine, 74.9 and 64.7; and skim milk powder, 82.6 and 72.0. Introduction During recent years, studies have been carried out by several workers on the use of oilseed meals and legumes as supplen~ents to human diets and also for the treatment of protein malnutrition in children (1-3). Legume proteins, in general, are deficient in methionine (4). I t has been shown by certain workers in experiments with animals that fortification of legume proteins with dlmethionine brings about a marked improvement in their nutritive value ( 5 , 6). In an earlier publication from this laboratory, it was reported that fortification of so>-a-bean proteins with dl-methionine hydroxy analogue (MHA) increased the protein efficiency ratio and net protein utilization in albino rats almost to the same extent as that obtained with dl-methionine (7). The present paper describes the results of studies with children. Experimental .Materials Spray-dried skim milk powder of good quality was used. Processed full-fat soya flour was prepared according to Narayana Rao et al. (8). The essential amino acid composition of the proteins of the soya flour, skim milk powder, and the low-protein diet was determined according to the methods used by Krishnamurthy et al. (9). The mean intakes of the essential ainino acids from the different diets were calculated by using the above values. The sample of calcium salt of dl-methionine hydroxy analogue (goy0 purity) used in this study was kindly supplied by Monsanto Chemical Company, Canadian Journal of Biochemistry. Volume 42 (1964) 377 C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y 20 3. 19 6. 16 0. 22 0 on 0 4/ 20 /1 1 Fo r pe rs on al u se o nl y. 378 CANADIAN JOURKAL OF BIOCHEMISTRY. VOL. 42, 19G4 U.S.A. The product was reported by the manufacturers to contain 78.8% pure acid. dl-Methionine (E. IbIerck, U.S.A.) was used as the source of ~nethionine. The required quantities of soya-bean flour were fortified with dl-methionine or MHA a t a level of 1.2 g / l 6 g N by dry mixing in a inechailical miser. Subjects The subjects were eight girls aged 8-9 years and were residents of a boarding home in Mysore city. The ages, heights, and weights of the girls are given in Table I. All of them belonged to the low-income groups of the population and were accusto~lled to consunling diets based on cereals, nlillets, and le, '*~iilles. TABLE I Ages, heights, and weights of the children a t the beginning of the test Age Height \i7eig h t Girl No. (years) (cm ) (kg) Diets The composition of the low-protein diet used in this study is given in Table 11. This diet provided 1459 calories and contained about 2.8 g protein (N X 6.25). The children received the low-protein diet throughout the experiment. In addition, the children received either soya flour (with or without added dl-methionine or MHA) or skiin inilk powder as a source of protein during the first four periods of the experiment. They were fed three tiines a clay, i.e. in the morning, noon, and night. The mineral salts and vitaininized starch were mixed with the tapioca flour and corn starch. Tapioca flour was given in the form of unleavened bread while the corn starch was made into sweet and savory vermicelli-lilte preparations. In addition, the children received a vegetable soup and a sweetened drink containing ascorbic acid. Vitamins A and D were added to the vegetable oil. Full-fat soya flour (41.0 g) was given in three equal doses along with breakfast, lunch, and dinner in the form of sweet pudding. Sltim rnilk powder (56.8 g) was also given in three equal closes (after reconstitution in 6 tiines the weight of water and addition of cane sugar) along with the three meals. The mean daily intake of protein on the soya flour or sltim milk powder diets was maintained a t a level of about 1.2 g/kg body weight. Feeding of Children and Collection of Urine and Faeces The metabolism period consisted of five periods of 10 clays each; period 1, C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y 20 3. 19 6. 16 0. 22 0 on 0 4/ 20 /1 1 Fo r pe rs on al u se o nl y. PARTHASARATHY ET AL.: PROTEIN UTILIZATION TABLE I 1 Mean daily intake (g) of foodstuffs by the children on different diets* t Diets based on Low; protein Skim milk Foodstuffs diet Soya flour1 powder Basal low-protein diet Tapioca flour (washed with dilute alkali) Corn starch Sugar Peanut oil (fortified with vitamins A and D) Salt mixture5 Vitaminized starch11 Supplen~ents Processed full-fat soya flour Skim milk powder *All the diets supplied in addition (g/day): common salt, 8.0; onion, 14.0; talnarinti fruit pulp, 5.0; non-leafy vegetables (knolkhol, brinjals, ladies finger, and radish white), GO.0; condiments (red chillies and mustard) 3.0. T h e protein content ( N X 6.25) of the different diets were as follows: low-protein diet, 2.8 g; soya flour diet, 24.4 g; and skim milk powder diet. 25.8 g. tEach child received 50 mg of ascorbic acid daily in the form of a sweetened drink. $In the 2nd and 3rd period, each child in addition was given 260 mg of dl-rnethionine or methionine hydroxy analogue respectively. BOsborne and Mendel salt mixture. IiProvided the daily requirements of B vitamins as recommended by the (C.S.A.) Xational Research Council, Food and Nutrition Board (1958). soya flour diet; period 2, soya flour + dl-methionine diet; period 3, so>-a flour + MHA diet; period 4, skim milk powder diet; and period 5, low-protein diet. The first 5 days on each diet were treated as a preliminary period for the children to get accustomed to the diet and the collectioil of urine and faeces was confined to the last 5 days in each period. Carmine was used as a nlarlcer for the collection of faeces. The daily excretion of creatinine in urine was determined as a check for the quantitative collection of urine. The dailjexcretion of creatinine in the subjects ranged from 457 ing to 511 mg per day and of creatine 48 to 75 mg per day. In the same subject the daily excretion of creatinine and creatine on different days during the metabolisnl period did not differ by more than 4y0, indicating thereby that the collection of urine was allnost quantitative. Duplicate samples of the different diets consunled daily by each child were collected and dried a t GO-65O C in a cabinet drier. 'The). were powdered and kept in glass-stoppered bottles for analysis. The urine and faeces were preserved according to n h r t h ~ r et al. (10). Total nitrogen in diet, urine, and faeces were determined by the micro-Kjeldhal method. The pattern of diets consuined by the children during the different periods is given in Table 11. The essential amino acid conlposition of the diets is given in Table 111. Data regarding the amino acid intake of children on the different diets, as coinpared with children's amino acid requireinents as reported by Nakagawa ct al. (1114), are given in Table IV. The digestibility coefficient, biological value, net protein utilization (NPU) C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y 20 3. 19 6. 16 0. 22 0 on 0 4/ 20 /1 1 Fo r pe rs on al u se o nl y. 3 80 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 42. 1964 TABLE I11 Essential amino acid content (g/16 g N) of the mixed proteins of different diets Diets based on F A 0 Idea 1 Soya flour reference reference + methionine Skim milk protein protein Xinillo acid Soya flour or MHA powder pattern (15) pattern (16) Arginine Histidine Lysine Leucine Isoleuciile hlethionine Cystine Total sulphur amino acids Phenylalaniile Threonine Tryptophan Valiile Protein score *Methonine or MHA. ?.Amino acids limiting as compared to FA0 pattern. $Amino acids limiting as compared to Ideal reference protein pattern. TABLE IV Mean daily intake (mg/kg) of esseiltial amino acids by the children from the different diets as compared with the amino acid requirements Diets based on Amino Basal Soya flour acid* low-protein + methionine Skim milk requireAmino acid diet Soya flour or MHA powder ments Arginine Histidine Lysine Leucine Isoleucine Methioniile Cystine Total sulphur amino acids Phenylalanine Threonine Tryptophail Valine *Data of Nakagawa el al. (11-14). tMethionine or MHA. and net available protein were calculated according to the following formulae: N intake faecal N Apparent digestibility coefficient = 100 X N intake N intake (faecal N endogenous True digestibility coefficient = 100 X faecal N) N intake C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y 20 3. 19 6. 16 0. 22 0 on 0 4/ 20 /1 1 Fo r pe rs on al u se o nl y. PARTHASARATHY ET AL.: PROTEIN UTILIZATION 381 N intake (faecal N endogenous faecal N) (urinary N endogenous urinary N) Biological value = 100 X N intake (faecal N endogenous faecal N) true digestibility coefficient X biological value NPU(0,) = 100 protein intake X NPU(,,) Net available protein = 100 Statistical Treatment of Data 'The data were analyzed by the analysis of variance method appropriate for randomized block design, considering each subject as a block and differences tested for significance by using a one-sided or two-sided t test, whichever is appropriate. Results Data regarding the daily urinary and faecal endogenous nitrogen on the lowprotein diet are given in Table V. The mean daily balance of nitrogen, digestibilitjcoefficient, biological value, and net protein utilization of the protein in children fed on diets based on soya (with or without added MHA or dl-methionine) or on skiin milk powder is given in Table VI. The net available protein on the different diets is given in Table VII. TABLE V Daily urinary and faecal excretion (g ) of nitrogen by the children on the low-protein diet Girl KO. Urinary Faecal 'I'otal 1 2 3 4 1 6 ; hlean value with its standard error (7 d.f.) Essential Amino Acid Intake and Requirements (Tables 111 and I V) The protein scores of the different diets as compared with FA0 reference protein pattern and Ideal reference protein pattern (16) calculated according to the method of FA0 Conlmittee (15) are as follows: soya flour diet, 76 and 67; soya flour + methionine or MHA diet, 93 and 78; and milk diet, 79 and 68 respect ivelj-. Data regarding the essential amino acid intakes and requirements of the children are given in Table IV. I t is evident that soya-bean protein a t a level of 1.2 g/kg body weight provided the essential amino acid requirements of children as assessed by Nakagawa et al. (11-14) even after allowance is made for the loss of 16% of the protein in digestion. C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y 20 3. 19 6. 16 0. 22 0 on 0 4/ 20 /1 1 Fo r pe rs on al u se o nl y.