Assessment of Hydrolyzate Solutions for Nutrition ARTHUR J. MUELLER Department of Nutritional Research, M e a d Johnson and Company, Evansville, Ind.
THE
need of an animal for a particular food substance is frequentlyindicated by its willingness to eat thematerial. S o m a times the amount of food eaten voluntarily may be used in the measurement of ita efficacy, even though appetite per se is unreliable as an exact index of need ($)-for example, the ability of rats to determine the presence of tryptophane in the diet, as indicated by an increase or decrease in food consumption, has often been noted ( I ) . In making solutions of protein hydrolyzates, it became necessary to know whether processing had deleteriously affected their nutritive value. It was believed that if the solution were evaporated on a nitrogen-free diet, certain constituents might be harmfully atrected from a nutritional standpoint. The simplest alternative was to offer the solution to rats as their sole source of nitrogen and determine whether sufficient solution would be ingested for rapid growth. The hydrolyzates incorporated into the basal diet a t levels of 1.2, 1.7, and 2.4% nitrogen are originally made in powder form. Therefore they can be mixed directly with the basal diet. The only hydrolyzate evaporated on the basal diet was one of the two purchased on the open market (Table I). Accordingly, young rats were supplied a basal dry diet containing all growth essentials save protein; and water and the hydrolyzate solution were offered in separate drinking tubes. Among ten rats there would usually be one or two which would not drink the solutions, and did not grow, even though others in the group showed good gains. This unwillingness of an occasional rat to drink the solution makes the average growth figures subject to considerable variation. The more dependable assessment of growth can be made by
incorporating the dry hydrolyzate in the diet. By comparing thia growth with the average growth when a solution of the same material was drunk, an estimate of the usefulness of this procedure can be made. Dry preparations are not always available for assay, and perhaps some information concerning the nutritive value of a solution may be obtained by the procedure. EXPERIMENTAL
Groups of ten rats, 21 to 23 days of age and weighing about 50 grams, equally distributed as to sex and litter, were placed in individual wire-screen cages, and given a diet of the following composition: dextrin, 82; lard, 9; salt mixture (2), 4; cod liver oil, 2; wheat germ oil, 1; brewer's yeast, 2; thiamine, 0.0006; riboflavin, 0.0002%. Water and the hydrolyzate solution were offered by means of two drinking tubes attached to the side of the c y . The volumes consumed daily were noted. he tubes which contained the 10% hydrolyzate solution were cleaned and refilled every other day. To minimize bacterial growth, 0.5% of sodium benzoate was added. This preservative is more effective than 5% ethyl alcohol and has no deleterious effect on the animal, nor does it affect the taste of the solution. The rats were weighed weekly for four weeks. I n order to com are the average growth with the results obtained by the usuap technique the same lots of protein hydrolyzate were incorporated in the basal diet and fed to roups of ten rats each. Three levels of intake were employe3, equivalent approximately to 10, 14, and 20% of the dried hydrolyzate but calculated to supply 1.2, 1.7, or 2.4% of nitrogen, respectively. The average gain in weight for a period of four weeks was determined.
The data are presented in two tables, similarly arranged, and comparison is made between the average growth of the animals receiving the solution and those fed the hydrolyzate in the diet. Growth on the solution was not so consistent Table 1. Average Gain per Rat per Day for Groups of N o t L e n Than 10 Rats Supplied as growth on the material incorpoProtein Hydrolysatee in Solution or in Diet rated in the diet a t a fixed percentage, 10% Nitrogen Level in Diet Average Gain but if only a qualitative comparison is Preparation Solution 1 . 2 % 1.7% 2.4% 10% s o h . 1 . 2 % N 1 . 7 % N 2 . 4 % N made, it is obvious that when the hy&oms par prom of nitrogen inpeatsd drolyzate in the diet promoted growth, 1.31 2.07 2.55 12.8 14.9 12.8 11.8 2.24 13.2 11.4 the same hydrolyzate in solution was also 2.68 16.8 13.5 16.5 effective. 17.3 I n Table I, the assays under prepara16.6 tion 403 show that considerable variation 38 2.49 2.77 8.00 16.4 2.37 14.4 was observed when the solution wtw 2.11 15.6 1.80 13.8 offered to M e r e n t groups of rats. Of the 2.03 15.0 2.00 14.3 six assays, four were reasonably consistent 1.85 2 . 6 0 3 . 0 4 402 2.64 15.0 17.1 19.0 12.7 with each other and two gave results con2.39 11.9 3.24 siderably higher and lower than these four. 14.3 3.09 12.1 When the dry preparation 38 was in3.15 13.4 corporated in the diet and fed to six g r o u p 1.39 2 . 2 5 10,012 1.71 16.4 12.8 15.6 1.25 1.17 2 . 2 4 16.4 12.6 14.5 of rats, five of the assay groups were 10,014 2.21 1.50 2 . 0 8 14.4 13.6 16.4 consistent and only one gave a slightly 1.09 1.52 15.1 12.3 different value. 40 -0.06 0.81 1.44 9.1 8.5 0.85 8.0 This was true also with the fivetriala 415 0.67 0 . 7 3 1.68 2.63 7.9 11.8 15.9 13.6 of preparation 402 incorporated in the 1.18 10.4 diet. The other values in the table likePreparation A - 0.26 - 0 . 1 4 wise indicate that the solution assay is Preparation B 0.33 1.59 7.45 12.9 a much less exact procedure than the Acid-hydrolyaed caaein 0.60 -0.31 -0.50 usual one of incorporating a fixed amount Code numben, in both Tables I and I1 indicate experimental lots of Ami en (a pancreatic hyof the material in the diet. drolysate of casein) made during the development of the product. Because of fifferencea in preparation, aome l o b romoted better growth than others Preparations A and B were wchased solutiona Table I1 presents data on single assays of of casein hydrorycatee which yere fed by tube and'also evaporated on the basal $et. The acid-hyvarious hydrolyzates given by drinking drolyaed casein waa made by boihng with sulfuric acid in the usual way. tube, and assays of the same material incor-
-
639.
640
INDUSTRIAL AND ENGINEERING CHEMISTRY
Table 11. Average Gain per Rat per Day for Groups of 10 Rats Supplied Protein Hydrolyzates in Solution and in Diet Preparation
10,001 10,002 10,003 10,004 10,005 10.006 10,007 10,008 10,009 10,010 10,011 10,013
Nitrogen Level Average Gain in Diet 1.2% 1.7% ’ 10% soln. 1.2%“ 1.7%” Grads per gram of nitrogen Grama gain per day ingested 1.92 2.68 2.40 16.5 17.3 17.6 1.32 11.2 1.56 2.46 16.4 16.3 1.93 14.8 2.08 3.49 17.8 18.8 1.36 1.93 3.24 12.9 16.5 17.9 1.90 2.68 2.54 14.4 17.4 17.3 1.23 1.64 2.83 14.7 17.5 17.5 1.80 2.86 0.86 10.5 15.1 17.3 12.6 1.66 3.04 1.08 17.0 19.1 1.05 10.7 1.65 2.49 17.7 17.3 1.34 11.2 19.1 1.95 2.80 17.3 0.90 12.8 16.1 15.9 1.66 2.75 2.12 17.5 15.1 15.9 1.55 2.46
10% Solution
porated in the diet a t two intake levels. In no case did the average growth of the rats receiving the 10% solution equal the growth when 14% of the hydrolyzate (1.7% nitrogen) was incorporated in the diet. Only in three instances was there close approximation in growth (10,001, 10,005, and 10,013) and this can only be taken t,o indicate that all the rats in these particular groups drank
Vol. 17, No. 10
the offered solution in good amount. I n general the average gain in the solution group approximated that of 10% (1.2QJ, nitrogen) of the hydrolyzate, since the average growth of all the solution groups was 1.51 grams and of the groups fed 1.2% nitrogen was 1.78 grams. However, the individual groups fed 1.2% nitrogen varied from -75% to +209% of the gain on the corresponding solutions, so that the variability in the results precludes a single assay of a solution from being conclusive. CONCLUSION
When solutions of hydrolyzates are offered to rats as the sole source of dietary nitrogen, a rough approximation of their value for growth can be obtained. The accuracy of the procedure cannot be compared with that of the classic methods for the assessment of biological value of proteins. The procedure may be helpful when dry preparations are not available for standard biological assay. LITERATURE CITED
(1) Cox, W.M.,Jr., and Mueller, A. J., Proe. SOC.Expt. Biol. Med., 42, 658-63 (1939). (2) Nutrition Reas., 2, 199 (1944).
Electrogravimetric Determination of Copper in Copper-Base and Tin-Base Alloys By Controlled Potential Electrolysis JAMES J. LINGANE, Mallinckrodt Chemical Laboratory, Harvard University, Cambridge, Mass.
(4) a relatively simple apparatus was described which automatically controls the potential of an electrode at any desired constant value during the entire course of an electrolysis, and thus renders the application of “graded potential” procedures (6) as convenient as the much less selective “constant current” methods. The present paper demonstrates the utility of this apparatus for the direct determination of copper in the presence of tin, antimony, lead, and various other metals, and presents a procedure for determining copper in tin-base and copper-base alloys that requires no preliminary separations. In the procedure described herein the copper is deposited from a slightly acid tartrate solution, which, for the separation of copper from tin, possesses a number of advantages over the hydrochloric acid solution used in the well-known Schoch-Brown method (1, 6, 7 ) ; although Schoch and Brown used tartrate to separate copper from antimony, it has not previously been used to separate copper from tin. From an acidic tartrate solution cupric copper is reduced directly to the metal, whereas reduction from a hydrochloric acid solution is complicated by stepwise reduction through the cuprous state. Stannic tin forms a much more stable complex ion with tartrate ion than with chloride ion; indeed, the stannic tartrate complex is so stable that no reduction wave is observed with the dropping mercury electrode with stannic tin in acidic, neutral, or basic tartrate solutions ( 2 ) . A few centigrams of copper can be determined accurately in the presence of as much as 2 grams of tin by the procedure described below. Furthermore, both antimonous and antimonic antimony form sufficiently stable complex ions with tartrate ion to permit the determination of cJpper in the presence of antimony without difficulty, whereas antimony codeposits more or less completely with copper from a hydrochloric acid solution. With the exception of bismuth, the other metals commonly present in tin-base and copper-base alloys
I
S A previous paper
also form more or less stable tartrate complexes, which fact effectively prevents their interfering with bhe determination of copper. APPARATUS
A cylindrical platinum gauze cathode 5 cm. high and 5 cm. in diameter was used. A platinum gauze cylinder 5 cm. high and 2.5 cm. in diameter was employed as anode, and it was mounted inside and coaxially with the cathode. Efficient stirring was provided by a motor-driven glass stirrer whose shaft passed down through the center of the anode cylinder. The blades of the stirrer were in the form of a large U, whose arms projected well up into the annular space between the electrodes. A saturated calomel electrode was used to control the cathode potential. A 6-mm. tube filled with a 3% agar gel in saturated potassium chloride served as a salt bridge and its tip was placed outside and as close sts possible to the cathode cylinder a t about its middle. An ordinary 250-cc. beaker served as the electrolysis cell. A thick (1.25-cm.) piece of Bakelite plate, with clamps for the electrode leads and holes for the stirrer shaft and salt bridge, was used 89 a cover. The electrical circuit, by means of which the potential of the cathode was controlled automatically to within t0.02 volt at any desired value, has been described in detail (4). This apparatus functioned very satisfactorily without attention during the entire course of an electrolysis. PROCEDURE
A 0.5- to 2-gram sample of the alloy was weighed into a covered 250-cc. beaker and dissolved in a warm mixture of 8 cc. of 12 N hydrochloric acid and 2 cc. of 16 N nitric acid. The nitric acid was added in several small portions as needed. The solution was boiled very gently for a minute or two to remove most of the oxides of nitrogen and chlorine. Then 100 cc. of a solution containing 23 grams (0.10 mole) of reagent quality sodium tartrate dihydrate, 1 gram of urea to remove oxides of nitrogen, and 10 cc. of 5 N sodium hydroxide, were added. The solution was diluted to about 200 cc., treated with 1 to 2 grams of hydroxylamine
