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Apparent Biological Value of Pelleted and Autoclaved Torula Yeast Measured in Various Ways1
Apparent Biological Value of Pelleted and Autoclaved Torula Yeast Measured in Various Ways1 C. GlTLER, 2 J . S. FlNLAYSON, 3 C. A . BAUMANN AND M . L . SUNDE Departments of Poultry Husbandry and Biochemistry, College of Agriculture, University of Wisconsin, Madison 6, Wisconsin (Received for publication March 20, 1958)
ORULA yeast appears to be a good source of protein for chicks fed practical type diets. Klose and Fevold (1945) reported that 80% of the fish meal in a cereal ration could be replaced by torula without decrease in the rate of gain, methionine being the limiting amino acid in the yeast. Ringrose (1949) and Vedvik (1952) showed that 50-60% of the soybean oil meal in a corn-soy diet could be effectively replaced by torula. On purified diets, however, chicks grow very poorly when fed torula yeast as the sole source of protein (Vedvik, 1952; Gitler, 1956). The present studies deal with attempts to improve the protein quality of torula yeast by pelleting, autoclaving, or extracting with alkali. The products were tested in chicks by ordinary growth studies, and measurements were also made in rats by a spaced feeding technique. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison, Wisconsin. This work was supported in part by grants from the Sulphite Pulp Manufacturer's Research League, Appleton, Wisconsin, and by the Research Committee of the Graduate School, with funds from the Wisconsin Alumni Research Foundation. 1 Preliminary reports of these data were presented at the 45th Annual Meeting of the Poultry Science Association, North Carolina State College, Raleigh, North Carolina 1956. 2 Present Address—Instituto Nacional de Nutricion, Dr. Jimenez 261, Mexico D.F., Mexico. 3 Public Health Service Research Fellow of the National Cancer Institute, Radiophysics Institute, Karolinska Sjukhuset, Stockholm 60, Sweden.
METHODS
Chick experiments. In all series day old chicks, New Hampshire male X Single Comb White Leghorn female cross or Arbor Acres White Plymouth Rocks, were weighed individually, randomized according to weights into groups, housed in electrically heated, wire floored batteries, and given feed and water ad libitum. Weights were recorded at weekly intervals. The experimental period was 4 weeks in all cases. Rat experiments. Holtzman strain male rats 21 days of age were housed in individual wire-bottom cages and were given water ad libitum. Rats were trained and selected for spaced-feeding experiments by the method of Finlayson and Baumann (1956). The torula yeast* was commercial feed grade (Torulopsis utilis) that had been grown aerobically on spent sulphite liquor. The crude protein content was found to be 46% (NX6.25). Von Soden and Dirr (1942) and Dirr and Decker (1944) reported 20 to 36% of yeast nitrogen to be supplied by non-protein compounds, and hence the alpha-amino nitrogen in our samples was also determined;! 83% of the total nitrogen proved * Obtained through the courtesy of Dr. P. L. Pavcek from the Lake States Yeast Corp., Inc., Rhinelander, Wisconsin. t Hydrolysed with 6N HC1 for 1 hour, concentrated in vacuo followed by determination of the a-amino nitrogen by the nitrous acid method of Van Slyke and Kirk (1933).
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BIOLOGICAL V A L U E OF T O R U L A Y E A S T S
RESULTS AND DISCUSSION
Chick experiments. In experiment A torula yeast was fed as the sole source of protein or with various levels of soybean oil meal in a practical cereal diet containing methionine (Table 1). The total protein in the diet was 20.7% of which 7.6% was supplied by the cereal proteins and 13.1% from various combinations of soybean oil meal and torula yeast At most levels of administration growth of the chicks fed yeast was comparable to the control group (32% soybean oil meal) except where very high levels of yeast were fed (Table 2, experiment A). When more than 20% of the diet was supplied by tort Pabst Laboratories and Jos. Schlitz Brewing Co., Milwaukee, Wisconsin.
TABLE 1.—Basal diet Ingredients per kg. of ration gms. 430 320
Ground yellow corn Soybean oil meal Wheat bran Wheat middlings Alfalfa meal Fish solubles
so so 50 30
Bone meal Granite grit Oyster shell Salt (iodized) Feeding oil (1,500 A—300 D) DL-Methionine MnS0 4
gms. 30 10 20 S 5 O.S 0.22 mgs. 3.2 10.0 5.0 14.0 8.0
Riboflavin Niacin Ca Pantothenate Penicillin Vitamin B12 (micrograms)
ula, the finely powdered product became impacted in the beaks of the chicks resulting in decreased food consumption and weight gains. Pelleting of the torula allowed complete substitution of the soybean oil meal by the yeast without significantly diminishing the weight gains. However, the improvement in growth when the torula was pelleted appeared in some groups to be greater than could be exTABLE 2.—The effect on chick growth of substituting torula yeast (A) and pelleted torula yeast (B) for soybean oil meal in a practical ration Experiment B
A Group 1
Soybean Torula oil meal yeast
Average weights 2 wks.
1 2 3 4 5 6 7 8 9
%
%
12 8 4 0
20 24 28 32
32 28 24 20 16
0 4 8 12 16
gms. 147 143 148 148 148f 151t 161 144 149 138
4 wks.
2 wks.
4 wks.
gms. gms. gms. 336+ 9« 128 3 4 4 + 1 1 * 333+ 6 123 3 3 2 ± 5 317+ 7 136 360+ 13 327 ± 8 135 351± 8 323+ 9f 134 355± 8 318+ 8f 330±12 126 335+ 9 290+ 8 136 345 ± 8 304+ 9 130 3 2 0 + 6 259+13 131 329 ± 8
1 25 New Hampshire XSingle Comb White Leghorn chicks were used. * Standard Errors. t Duplicate groups.
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to be alpha-amino nitrogen, and the "true protein" (alpha-amino nitrogen X 6.25) value of the yeast was 39.2%. In some experiments the torula yeast was pelleted in a commercial pelleting machine to 5/32 of an inch. A-K torula yeast* consisted of torula yeast that had been subjected to mild alkaline treatment to extract the nucleic acids. The alkali-soluble protein was precipitated by adjustment to the isoelectric point. The press-cake together with the alkali-soluble protein were dried and ground and this constituted the material fed. According to the manufacturer it contained 5 1 % of crude protein, 3 to 4% of chloride and 0.8 to 1.1% of sulphate. Brewers' yeast% (Saccharomyces cerevisiae) was obtained as a by-product from the manufacture of beer. It contained 5 1 % of crude protein (NX6.25) and 42.3% of protein (alpha-amino NX6.25). Autoclaved torula was torula yeast mixed with an equal volume of distilled water and autoclaved at 15 lbs. pressure and 121°C. for 30 minutes, oven dried at 121°C. and ground to its original fineness.
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C . GlTLER, J . S. FlNLAYSON, C . A . BAUMANN AND M . L . SUNDE
were used per group.
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the untreated yeast. The results with autoclaved torula resembled those with the untreated yeast rather than the pelleted yeast. Allred el al. (1957a,b) reported an improvement in the growth rate of chicks and poults fed a practical diet that was pelleted over that on the untreated diet. This improvement survived grinding to the original fineness but the effect of pelleting was not reproduced by autoclaving the materials tested. The present results parallel those of Allred el al.; once the torula was pelleted, its protein value for the chick was about equal to that of soybean oil meal. Allred et al. (1957b) reported that this effect was largely due to All rations contained 16% of protein the effect on the grain portion of the diet. (alpha amino NX6.25), the assumption In these studies the protein was also afbeing made that all the nitrogen in fish fected by pelleting. meal was protein nitrogen. Torula, pelJThe low growth obtained when the proleted torula and autoclaved torula were tein source was torula supplemented with added at levels equivalent in N to 25, 50, methionine (Table 3 groups 5, 9,13) sugand 75% of the fish meal. The pelleted gested that other amino acids were limittorula was ground prior to incorporation ing. In the next series of experiments the into the diets to a texture approximately proteins of torula, A-K torula and brewequal to the original yeast. Methionine ers' yeast were supplemented with variwas added in all groups to raise the level ous amino acids. The diet was the same to 0.7% of the diet. The Ca and P levels as that employed in experiment C except were adjusted to 1.5 and 0.8% respec- that the methionine was omitted from the tively by adding the required levels of TABLE 3.—The value of torula, pelleted torula and CaC0 3 and Ca 2 (HP0 4 ) 2 . autoclaved torula as substitutes for fishmeal in a purified diet (Expt. C) When torula was substituted for fish meal, 25% replacement did not affect the Average weights* Amt. rate of growth, but substitution of 50, 75, Additions to basal added1 2 wks. 4 wks. and 100% decreased the growth rate by gm. gm. % 18, 29, and 48%, of the control level re1. None 143 289 2. Torula Yeast 25 159 280 spectively. Replacement of up to 50% of 3. Torula Yeast 50 140 237 4. Torula Yeast 75 121 205 the fish meal by pelleted torula resulted in 5. Torula Yeast 100 107 151 6. Pelleted Torula Yeast2 25 170 302 growth comparable to that in the control 7. Pelleted Torula Yeast 50 144 273 8. Pelleted Torula Yeast 75 129 240 group, but substitution by 75 or 100% of 9. Pelleted Torula Yeast 100 114 189 10. Autoclaved Torula Yeast 25 150 294 the fish meal resulted in a reduction in 11. Autoclaved Torula Yeast 50 140 207 12. Autoclaved Torula Yeast 75 109 189 growth by 17 and 35% respectively. At 13. Autoclaved Torula Yeast 100 106 169 all levels, chicks fed the pelleted and then 1 Percent of the dietary fish meal replaced. The pelleted yeast was ground previous to incorporation ground torula yeast grew faster than the into2 the diet. All diets contained 16% protein (Amino NX6.25). * Ten New Hampshire X Single Comb White Leghorn chicks chicks receiving comparable amounts of plained by changes in the texture of the diet (Table 2, groups 3, 4 and 5). The possibility that pelleting improved the protein quality of torula yeast was tested in Experiment C with a semi-synthetic diet containing in g. per 100 grams; menhaden fish meal (60% protein) 26, salts V (Briggs el al., 1943) 6, soybean oil 5, choline chloride 0.2, vitamin A and D oil (1,500 I. U. of A and 300 I. C. U. of D per g) 0.5 and dextrin to 100 grams; the vitamins in mg. per 100 grams were riboflavin 0.9, thiamine-HCl 0.6, niacin 5, calcium pantothenate 2, pyridoxine-HCl 0.8, menadione 0.02, biotin 0.03, folic acid 0.2, vitamin B J2 0.002 and a tocopherol acetate 0.3.
BIOLOGICAL VALUE OF TORULA YEASTS
TABLE 4.—The effect of amino acid supplementation on the protein quality of torula, A-K torula and brewers' yeast Group
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 8 4
Source of protein
Torula yeast 1-M -M+H +M +A hM+A+G -M + A + G + T A-K Torula yeast +M +M+A +M +A +G +M +A + G + T Brewers' yeast +M +M+A +M +A +G + M +A + G + T Casein-Gelatin -f-M Fish meal + M
Average weights at 4 weeks DM
E1'*
fi,j
G2.3
gm. 80 174
gm. 93 162 152 170 167 154 79 217 225 231 260 108 172 176 180
gm.
gm.
— — — •— — — 108
— 184
— 158
212 222 78 190 240 267 267 88 180 196
—
194 256
— — — — —
224
238
— 261
240
—
—
—
— •
• —
—
205 194 218
— 198 232 204 209 — 158 222 204 209 — 266
Diets contained 16% total protein (NX6.25). Diets contained 16% total protein (Amino N X6.25). New Hampshire XS.C.W. Leghorn Chicks (10 per group). Arbor Acres White Rocks (10 per group) M = 0 . 3 % DL—Methionine A=0.5% Arginine G=0.25% Glycine T = 0 . 2 % Tryptophan H = 0 . 5 % Histidine
was obtained only when A-K torula supplied the protein (Table 4, line 11 vs. 17). Rat Experiments. The basal diet for both spaced and ad libitum feeding contained alcohol-extracted casein 12, Wesson salts 4, corn oil 5, glucose monohydrate (Cerelose) to 100, and complete vitamins including 50 mg. of a-tocopherol per kg. of diet (Finlayson and Baumann, 1956). When yeast was to be tested as the sole source of protein, the casein was omitted. Additions to the ration were made at the expense of the carbohydrate. The yeasts were added at levels of crude protein ranging from 10 to 30%, and growth on these diets was compared with that on comparable levels of added casein. Since torula yeast contained 46% of crude protein and brewers' yeast, 51%, the desired level of 30% of protein in the diet was furnished by 65 or 59% of the respective yeasts in the diets. Increasing the yeast supplement by 17% (viz., to the level of 35% protein to correct for the non-protein N present) caused no con-
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basal mixture and the fish meal was replaced by yeast, or a combination of casein and gelatin and the required level of dextrin. In experiments D, E and F the yeasts were incorporated into the diets on the basis of their crude protein content (NX6.25) while in experiment G the "true protein" content was used. The amino acid composition of the diets was calculated according to values for torula and A-K torula obtained from Dr. A. J. Wiley of the Sulfite Researcher's Mfg. League (Gitler, 1956) and those of Block and Boiling (1945) for brewers' yeast. These calculations showed that by direct comparison to the recommended levels (Bird, 1954), corrected to 16% protein, only methionine and perhaps arginine were limiting. However, estimation of the amino acid content of torula by the method of Flodin (1953) using the methionine content as unity, showed a deficiency in torula yeast of about 0.5% arginine and 0.25% glycine from the optimal ratios. These levels were therefore chosen for supplementation and in addition the possibility of tryptophan being destroyed during the preparation of the A-K torula warranted the testing of the supplemental value of this amino acid. Additions were made at the expense of the whole diet. The chicks grew poorly on the diets in which the unsupplemented yeasts supplied the protein (Table 4 groups 1, 7, and 12). The addition of methionine to the yeasts doubled the rate of growth (groups 2, 8 and 13). In addition a response was obtained to added arginine especially in the trial in which the yeasts were incorporated on the basis of their "true protein" content (Experiment G groups 2 vs. 4, 8 vs. 9). The response to glycine and tryptophan, while apparent with A-K torula was in general too variable to be significant. It should be noted that growth comparable to that on gelatin and casein
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C. GlTLER, J. S. FlNLAYSON, C. A. BAUMANN AND M . L. SUNDE
TABLE 5.—Growth1 of spaced-fed rats on 12% casein plus yeast Additional protein3 Supplement Series
Casein
Torula Yeast
H I
J
K H I
k H I
0% gm.
10% gm.
20% gm.
30% gm.
35% gm.
14±3 66+4 15±2 33±2 53±4 72±3 23±3 47±1 77±1 31±8 42 + 8 53±3
22±6 6 ± 5 4+7 23±3 30 + 4
Autoclaved Torula
J
K
38+5 48±5 46±4 45 + 2 48±5 56±3 56 + 4
Brewers' Yeast
I K
50±6 39±2 31±6
Autoclaved Brewers'
I K
55 + 2 57+3
1 Grams gain at 2 weeks+standard error for 5 rats. 2 Added to the 12% casein basal diet at the expense of carbohydrate.
was fed either as a supplement to casein or as the sole source of protein, and under both spaced and ad libitum feeding conditions. When yeast was the sole source of protein, autoclaving failed to improve growth whether the feeding was spaced (Table 6, series L) or ad libitum (Table 6, series M). Moreover when yeast was supplemented to casein in diets fed ad libitum, no effect of autoclaving was seen, regardless of the protein level (Table 6, series M and N). However, in spacedfeeding experiments, 10% of torula protein was as good a supplement to 12% of casein as was an additional 10% of casein itself (Table 6, series M). In all series, changes in feed efficiency closely paralleled those in weight gain. Axtmayer (1946) reported an improvement in the protein quality of torula yeast due to autoclaving, whereas Goyco and Asenjo (1948), who autoclaved for a longer period, reported no such change. However, when the latter workers followed Axtmayer's method of autoclaving, they observed a slightly higher rate of growth in rats fed the heated yeast, though the difference was not statistically significant. The autoclaving procedure used in the present studies was similar to that of Axtmayer. From our work it appeared that the measurable effect of heat treatment is dependent on the feeding conditions; i.e. improvement due to autoclaving was observed only when the yeast was space-fed at high levels. It was hoped when the studies with rats were undertaken, that- biological values of the various yeasts could be reported. Substituting weight gains of rats fed 30% protein supplements (Table 5) into the biological value formula of Finlayson and Baumann (1956) yielded the following figures: intact torula yeast 2, autoclaved torula 39, brewers' yeast 24, autoclaved brewers' 52. These values for the intact
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sistent change in the growth rate (Table 5, series H and I). When spaced-fed at the level of 30% of crude protein, untreated torula yeast was definitely inferior to casein (Table 5, series H). Moreover, extraction with ether or with dilute alkali (A-K torula) did not alter its nutritive quality. On the other hand, when torula was autoclaved, its nutritive value appeared to be increased considerably (Table 5, series H). In subsequent series the spaced-feeding of commercial torula yeast at protein levels above 20% resulted in a depression of growth, whereas the growth of rats fed autoclaved torula was remarkably constant at all levels. Autoclaving torula improved growth significantly in 5 of 7 trials, autoclaving brewers' yeast, in 2 of 2 (Table 5). Improvement due to autoclaving occurred in all cases in which the yeast was supplemented at a protein level above 20%, but only 1 of 3 cases at 20% or less. At these lower levels torula was as good a supplement to 12% of casein as was additional casein itself (Table 5, protein levels 10 and 20). Next, untreated or autoclaved yeast
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BIOLOGICAL VALUE OF TORULA YEASTS TABLE 6.—Responses of rats to untreated and autoclaved yeast at various protein levels Untreated Gain, 1 gms.
'0
spaced
F.E. 2
Autoclaved Gain, 1 gms.
22 Torula 22 Torula+.125% Methionine
32 + 5
22±2
45 + 9
40 + 2
F.E. !
spaced
12
30 Casein 30 Torula 30 Brewers'
77 + 1 30+4 31 + 6
.44 .25 .27
85±4 56 + 4 57±3
.45 .37 .39
ad lib.
12
10 Casein 10 Torula 10 Brewers'
70 + 8 81+4 80 + 3
.50 .50 .51
72 + 2 70 + 6 71 + 5
.54 .48 .49
ad lib.
12
30 Casein 30 Torula 30 Brewers'
64 + 5 43 + 4 60 + 2
.65 .50 .65
45 + 3 57 + 4
.56 .65
30 Casein 30 Torula 30 Brewers'
79 + 3 23 + 1 46 + 6
.68 .32 .54
24+2 43 + 4
.35 .53
M
ad lib. 1 2
Mean 2 week gain + standard error for 5 rats. Feed efficiency: grams gained per gram food eaten.
yeasts are considerably lower than those reported by other workers (Goyco and Asenjo, 1949). The formula, therefore, which was satisfactory when applied to purified proteins, apparently does not yield absolute biological values for materials such as yeast, although the results probably have comparative value. Autoclaving might have improved the digestibility of the yeast in rats under conditions of spaced-feeding, in which differences in absorption would be accentuated by the presence of high levels of yeast. This, however, would not explain the beneficial effect of pelleting in the chick experiments. The results of the experiments with chicks would indicate the possible use of torula yeast in practical diets to meet a part of the total dietary protein. Its high content of readily available lysine (Tsien et ak, 1957) makes it especially suited to supplementation of diets high in cereal proteins. SUMMARY
1. The apparent biological value of
pelleted torula yeast for the chick was found to be equivalent to that of soybean oil meal when fed in a practical cereal diet. When the original yeast was used, growth of chicks was not affected by substitution of 60% of soybean oil meal, but a 75 to 100% replacement reduced the weight gain by 15 and 23% respectively. Food consumption decreased due to impaction in the beaks of the chicks fed the high levels of the original intact yeast. 2. The improvement in growth observed after pelleting of torula as compared to the intact yeast appeared due primarily to changes in texture. Experiments with semi-synthetic diets suggested that the process of pelleting may have improved the nutritional value of the yeast protein. Autoclaving of the yeast failed to result in a comparable improvement in the apparent biological value of the torula for chicks. 3. Growth on torula, alkali-extracted torula, or brewers' yeast was improved by supplements of methionine and arginine. Other amino acids studied gave no con-
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C. GITLER, J. S. FINLAYSON, C. A. BAUMANN AND M. L. SUNDE
REFERENCES Allred, J. B., L. S. Jensen and J. McGinnis, 1957a. Factors affecting the response of chicks and poults to feed pelleting. Poultry Sci. 36: 517-523. Allred, J. B., R. E. Fry, L. S. Jensen and J. McGinnis, 1957b. Studies with chicks on improvement in nutritive value of feed ingredients by pelleting. Poultry Sci. 36: 1284-1289. Axtmayer, J. H., 1946. Cited in Goyco, J. A., and C. F. Asenjo, 1947. The net protein value of food yeast. J. Nutrition, 33: 593-600. Bird, H. R., 1954. Nutrition requirements for poultry. National Research Council, Publication 301. Block, R. J., and D. Boiling, 1945. The Amino Acid Composition of Proteins and Foods. Charles C Thomas, Springfield, Illinois publisher. Briggs, G. M., Jr., T. O. Luckey, C. A. Elvehjem and E. B. Hart, 1943. Studies on two chemically unidentified water soluble vitamins necessary for the chick. J. Biol. Chem. 148: 163-172.
Dirr, K., and P. Decker, 1944. Uber den Wert der Wuchshefe fur die menschliche Ernahrung. V. Uber den Reineiweissgehalt der Hefe. Biochem. Z. 316: 245-248. Finlayson, J. S., and C. A. Baumann, 1956. Responses of rats to urea and related substances. The use of a spaced-feeding technique. J. Nutrition, 59: 211-221. Flodin, N . W., 1953. Amino acids and proteins. Their place in human nutrition problems. J. Agr. Food Chem. 1: 222-235. Gitler, C , 1956. Master's degree thesis, University of Wisconsin, Madison. Goyco, J. A., and C. F . Asenjo, 1948. Studies on edible yeasts Puerto Rico J. Publ. Health Trop. Med. 23:471-^97. Goyco, J. A., and C. F. Asenjo, 1949. Net protein and growth-promoting values of three different types of yeast prepared under identical conditions. J. Nutrition, 38: 517-526. Klose, A. A., and H. L. Fevold, 1945. Nutritional value of yeast protein for the rat and the chick. J. Nutrition, 29: 421-130. Ringrose, R. C , 1949. Nutritive properties of torula yeast for poultry. Poultry Sci. 28: 75-83. Soden, O. von, and K. Dirr, 1942. Uber den Wert der Wuchshefen fur die menschliche Ernahrung III. Verdaulichkeit in vitro von verschieidenen Hefen in Vergleich zu anderen Eiweisstragern der menschlichen Ernahrung. Biochem Z. 312: 252262. Tsien, W. S., E. L. Johnson and I. E. Liener, 1957. The availability of lysine from torula yeast. Arch. Biochem. Biophys. 71:414-422. Van Slyke, D. D., and E. Kirk, 1933. Comparison of gasometric, colorimetric and titrimetric determinations of amino nitrogen in blood and urine. J. Biol. Chem. 102: 651-682. Vedvik, J. R., 1952. Master's Thesis, University of Wisconsin, Madison.
BOOK REVIEWS (Continued from page 1313) become extremely complex. . . . I t should be helpful, therefore, to take stock from time to time and to sum up descriptively these guides to information and literature. . . . I t is hoped that the present work will be considered a step in this direction." The reviewer is perfectly willing to admit that the subject is most complex, that decisions must be made in many debatable or borderline areas, and that such a compilation must be kept within reason-
able limits. Nevertheless he is not certain that the present volume has accomplished its purpose, and he is certain that there are serious omissions. For example, the list of textbooks in poultry husbandry given on page 89 can, by no stretch of the imagination, be considered complete or comprehensive. One would have thought that, somewhere in such a book, space could have been found for a list of major scientific journals publishing manuscripts in
(Continued on page 1324)
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sistent growth response. 4. In rats trained to eat for 2 hours per day, autoclaving improved the growth promoting qualities of both torula and brewers' yeasts when supplemented at high levels to a diet containing 12% of casein. Autoclaving caused no improvement in growth on torula yeast fed as the sole source of protein under ad libitum conditions, or when supplemented to casein at low levels (10% protein or less). Under these latter conditions, torula yeast appeared to be as good a supplement to 12% casein as was brewers' yeast or additional casein, regardless of the method of feeding.
ORULA yeast appears to be a good source of protein for chicks fed practical type diets. Klose and Fevold (1945) reported that 80% of the fish meal in a cereal ration could be replaced by torula without decrease in the rate of gain, methionine being the limiting amino acid in the yeast. Ringrose (1949) and Vedvik (1952) showed that 50-60% of the soybean oil meal in a corn-soy diet could be effectively replaced by torula. On purified diets, however, chicks grow very poorly when fed torula yeast as the sole source of protein (Vedvik, 1952; Gitler, 1956). The present studies deal with attempts to improve the protein quality of torula yeast by pelleting, autoclaving, or extracting with alkali. The products were tested in chicks by ordinary growth studies, and measurements were also made in rats by a spaced feeding technique. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison, Wisconsin. This work was supported in part by grants from the Sulphite Pulp Manufacturer's Research League, Appleton, Wisconsin, and by the Research Committee of the Graduate School, with funds from the Wisconsin Alumni Research Foundation. 1 Preliminary reports of these data were presented at the 45th Annual Meeting of the Poultry Science Association, North Carolina State College, Raleigh, North Carolina 1956. 2 Present Address—Instituto Nacional de Nutricion, Dr. Jimenez 261, Mexico D.F., Mexico. 3 Public Health Service Research Fellow of the National Cancer Institute, Radiophysics Institute, Karolinska Sjukhuset, Stockholm 60, Sweden.
METHODS
Chick experiments. In all series day old chicks, New Hampshire male X Single Comb White Leghorn female cross or Arbor Acres White Plymouth Rocks, were weighed individually, randomized according to weights into groups, housed in electrically heated, wire floored batteries, and given feed and water ad libitum. Weights were recorded at weekly intervals. The experimental period was 4 weeks in all cases. Rat experiments. Holtzman strain male rats 21 days of age were housed in individual wire-bottom cages and were given water ad libitum. Rats were trained and selected for spaced-feeding experiments by the method of Finlayson and Baumann (1956). The torula yeast* was commercial feed grade (Torulopsis utilis) that had been grown aerobically on spent sulphite liquor. The crude protein content was found to be 46% (NX6.25). Von Soden and Dirr (1942) and Dirr and Decker (1944) reported 20 to 36% of yeast nitrogen to be supplied by non-protein compounds, and hence the alpha-amino nitrogen in our samples was also determined;! 83% of the total nitrogen proved * Obtained through the courtesy of Dr. P. L. Pavcek from the Lake States Yeast Corp., Inc., Rhinelander, Wisconsin. t Hydrolysed with 6N HC1 for 1 hour, concentrated in vacuo followed by determination of the a-amino nitrogen by the nitrous acid method of Van Slyke and Kirk (1933).
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1315
BIOLOGICAL V A L U E OF T O R U L A Y E A S T S
RESULTS AND DISCUSSION
Chick experiments. In experiment A torula yeast was fed as the sole source of protein or with various levels of soybean oil meal in a practical cereal diet containing methionine (Table 1). The total protein in the diet was 20.7% of which 7.6% was supplied by the cereal proteins and 13.1% from various combinations of soybean oil meal and torula yeast At most levels of administration growth of the chicks fed yeast was comparable to the control group (32% soybean oil meal) except where very high levels of yeast were fed (Table 2, experiment A). When more than 20% of the diet was supplied by tort Pabst Laboratories and Jos. Schlitz Brewing Co., Milwaukee, Wisconsin.
TABLE 1.—Basal diet Ingredients per kg. of ration gms. 430 320
Ground yellow corn Soybean oil meal Wheat bran Wheat middlings Alfalfa meal Fish solubles
so so 50 30
Bone meal Granite grit Oyster shell Salt (iodized) Feeding oil (1,500 A—300 D) DL-Methionine MnS0 4
gms. 30 10 20 S 5 O.S 0.22 mgs. 3.2 10.0 5.0 14.0 8.0
Riboflavin Niacin Ca Pantothenate Penicillin Vitamin B12 (micrograms)
ula, the finely powdered product became impacted in the beaks of the chicks resulting in decreased food consumption and weight gains. Pelleting of the torula allowed complete substitution of the soybean oil meal by the yeast without significantly diminishing the weight gains. However, the improvement in growth when the torula was pelleted appeared in some groups to be greater than could be exTABLE 2.—The effect on chick growth of substituting torula yeast (A) and pelleted torula yeast (B) for soybean oil meal in a practical ration Experiment B
A Group 1
Soybean Torula oil meal yeast
Average weights 2 wks.
1 2 3 4 5 6 7 8 9
%
%
12 8 4 0
20 24 28 32
32 28 24 20 16
0 4 8 12 16
gms. 147 143 148 148 148f 151t 161 144 149 138
4 wks.
2 wks.
4 wks.
gms. gms. gms. 336+ 9« 128 3 4 4 + 1 1 * 333+ 6 123 3 3 2 ± 5 317+ 7 136 360+ 13 327 ± 8 135 351± 8 323+ 9f 134 355± 8 318+ 8f 330±12 126 335+ 9 290+ 8 136 345 ± 8 304+ 9 130 3 2 0 + 6 259+13 131 329 ± 8
1 25 New Hampshire XSingle Comb White Leghorn chicks were used. * Standard Errors. t Duplicate groups.
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to be alpha-amino nitrogen, and the "true protein" (alpha-amino nitrogen X 6.25) value of the yeast was 39.2%. In some experiments the torula yeast was pelleted in a commercial pelleting machine to 5/32 of an inch. A-K torula yeast* consisted of torula yeast that had been subjected to mild alkaline treatment to extract the nucleic acids. The alkali-soluble protein was precipitated by adjustment to the isoelectric point. The press-cake together with the alkali-soluble protein were dried and ground and this constituted the material fed. According to the manufacturer it contained 5 1 % of crude protein, 3 to 4% of chloride and 0.8 to 1.1% of sulphate. Brewers' yeast% (Saccharomyces cerevisiae) was obtained as a by-product from the manufacture of beer. It contained 5 1 % of crude protein (NX6.25) and 42.3% of protein (alpha-amino NX6.25). Autoclaved torula was torula yeast mixed with an equal volume of distilled water and autoclaved at 15 lbs. pressure and 121°C. for 30 minutes, oven dried at 121°C. and ground to its original fineness.
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C . GlTLER, J . S. FlNLAYSON, C . A . BAUMANN AND M . L . SUNDE
were used per group.
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the untreated yeast. The results with autoclaved torula resembled those with the untreated yeast rather than the pelleted yeast. Allred el al. (1957a,b) reported an improvement in the growth rate of chicks and poults fed a practical diet that was pelleted over that on the untreated diet. This improvement survived grinding to the original fineness but the effect of pelleting was not reproduced by autoclaving the materials tested. The present results parallel those of Allred el al.; once the torula was pelleted, its protein value for the chick was about equal to that of soybean oil meal. Allred et al. (1957b) reported that this effect was largely due to All rations contained 16% of protein the effect on the grain portion of the diet. (alpha amino NX6.25), the assumption In these studies the protein was also afbeing made that all the nitrogen in fish fected by pelleting. meal was protein nitrogen. Torula, pelJThe low growth obtained when the proleted torula and autoclaved torula were tein source was torula supplemented with added at levels equivalent in N to 25, 50, methionine (Table 3 groups 5, 9,13) sugand 75% of the fish meal. The pelleted gested that other amino acids were limittorula was ground prior to incorporation ing. In the next series of experiments the into the diets to a texture approximately proteins of torula, A-K torula and brewequal to the original yeast. Methionine ers' yeast were supplemented with variwas added in all groups to raise the level ous amino acids. The diet was the same to 0.7% of the diet. The Ca and P levels as that employed in experiment C except were adjusted to 1.5 and 0.8% respec- that the methionine was omitted from the tively by adding the required levels of TABLE 3.—The value of torula, pelleted torula and CaC0 3 and Ca 2 (HP0 4 ) 2 . autoclaved torula as substitutes for fishmeal in a purified diet (Expt. C) When torula was substituted for fish meal, 25% replacement did not affect the Average weights* Amt. rate of growth, but substitution of 50, 75, Additions to basal added1 2 wks. 4 wks. and 100% decreased the growth rate by gm. gm. % 18, 29, and 48%, of the control level re1. None 143 289 2. Torula Yeast 25 159 280 spectively. Replacement of up to 50% of 3. Torula Yeast 50 140 237 4. Torula Yeast 75 121 205 the fish meal by pelleted torula resulted in 5. Torula Yeast 100 107 151 6. Pelleted Torula Yeast2 25 170 302 growth comparable to that in the control 7. Pelleted Torula Yeast 50 144 273 8. Pelleted Torula Yeast 75 129 240 group, but substitution by 75 or 100% of 9. Pelleted Torula Yeast 100 114 189 10. Autoclaved Torula Yeast 25 150 294 the fish meal resulted in a reduction in 11. Autoclaved Torula Yeast 50 140 207 12. Autoclaved Torula Yeast 75 109 189 growth by 17 and 35% respectively. At 13. Autoclaved Torula Yeast 100 106 169 all levels, chicks fed the pelleted and then 1 Percent of the dietary fish meal replaced. The pelleted yeast was ground previous to incorporation ground torula yeast grew faster than the into2 the diet. All diets contained 16% protein (Amino NX6.25). * Ten New Hampshire X Single Comb White Leghorn chicks chicks receiving comparable amounts of plained by changes in the texture of the diet (Table 2, groups 3, 4 and 5). The possibility that pelleting improved the protein quality of torula yeast was tested in Experiment C with a semi-synthetic diet containing in g. per 100 grams; menhaden fish meal (60% protein) 26, salts V (Briggs el al., 1943) 6, soybean oil 5, choline chloride 0.2, vitamin A and D oil (1,500 I. U. of A and 300 I. C. U. of D per g) 0.5 and dextrin to 100 grams; the vitamins in mg. per 100 grams were riboflavin 0.9, thiamine-HCl 0.6, niacin 5, calcium pantothenate 2, pyridoxine-HCl 0.8, menadione 0.02, biotin 0.03, folic acid 0.2, vitamin B J2 0.002 and a tocopherol acetate 0.3.
BIOLOGICAL VALUE OF TORULA YEASTS
TABLE 4.—The effect of amino acid supplementation on the protein quality of torula, A-K torula and brewers' yeast Group
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 8 4
Source of protein
Torula yeast 1-M -M+H +M +A hM+A+G -M + A + G + T A-K Torula yeast +M +M+A +M +A +G +M +A + G + T Brewers' yeast +M +M+A +M +A +G + M +A + G + T Casein-Gelatin -f-M Fish meal + M
Average weights at 4 weeks DM
E1'*
fi,j
G2.3
gm. 80 174
gm. 93 162 152 170 167 154 79 217 225 231 260 108 172 176 180
gm.
gm.
— — — •— — — 108
— 184
— 158
212 222 78 190 240 267 267 88 180 196
—
194 256
— — — — —
224
238
— 261
240
—
—
—
— •
• —
—
205 194 218
— 198 232 204 209 — 158 222 204 209 — 266
Diets contained 16% total protein (NX6.25). Diets contained 16% total protein (Amino N X6.25). New Hampshire XS.C.W. Leghorn Chicks (10 per group). Arbor Acres White Rocks (10 per group) M = 0 . 3 % DL—Methionine A=0.5% Arginine G=0.25% Glycine T = 0 . 2 % Tryptophan H = 0 . 5 % Histidine
was obtained only when A-K torula supplied the protein (Table 4, line 11 vs. 17). Rat Experiments. The basal diet for both spaced and ad libitum feeding contained alcohol-extracted casein 12, Wesson salts 4, corn oil 5, glucose monohydrate (Cerelose) to 100, and complete vitamins including 50 mg. of a-tocopherol per kg. of diet (Finlayson and Baumann, 1956). When yeast was to be tested as the sole source of protein, the casein was omitted. Additions to the ration were made at the expense of the carbohydrate. The yeasts were added at levels of crude protein ranging from 10 to 30%, and growth on these diets was compared with that on comparable levels of added casein. Since torula yeast contained 46% of crude protein and brewers' yeast, 51%, the desired level of 30% of protein in the diet was furnished by 65 or 59% of the respective yeasts in the diets. Increasing the yeast supplement by 17% (viz., to the level of 35% protein to correct for the non-protein N present) caused no con-
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basal mixture and the fish meal was replaced by yeast, or a combination of casein and gelatin and the required level of dextrin. In experiments D, E and F the yeasts were incorporated into the diets on the basis of their crude protein content (NX6.25) while in experiment G the "true protein" content was used. The amino acid composition of the diets was calculated according to values for torula and A-K torula obtained from Dr. A. J. Wiley of the Sulfite Researcher's Mfg. League (Gitler, 1956) and those of Block and Boiling (1945) for brewers' yeast. These calculations showed that by direct comparison to the recommended levels (Bird, 1954), corrected to 16% protein, only methionine and perhaps arginine were limiting. However, estimation of the amino acid content of torula by the method of Flodin (1953) using the methionine content as unity, showed a deficiency in torula yeast of about 0.5% arginine and 0.25% glycine from the optimal ratios. These levels were therefore chosen for supplementation and in addition the possibility of tryptophan being destroyed during the preparation of the A-K torula warranted the testing of the supplemental value of this amino acid. Additions were made at the expense of the whole diet. The chicks grew poorly on the diets in which the unsupplemented yeasts supplied the protein (Table 4 groups 1, 7, and 12). The addition of methionine to the yeasts doubled the rate of growth (groups 2, 8 and 13). In addition a response was obtained to added arginine especially in the trial in which the yeasts were incorporated on the basis of their "true protein" content (Experiment G groups 2 vs. 4, 8 vs. 9). The response to glycine and tryptophan, while apparent with A-K torula was in general too variable to be significant. It should be noted that growth comparable to that on gelatin and casein
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C. GlTLER, J. S. FlNLAYSON, C. A. BAUMANN AND M . L. SUNDE
TABLE 5.—Growth1 of spaced-fed rats on 12% casein plus yeast Additional protein3 Supplement Series
Casein
Torula Yeast
H I
J
K H I
k H I
0% gm.
10% gm.
20% gm.
30% gm.
35% gm.
14±3 66+4 15±2 33±2 53±4 72±3 23±3 47±1 77±1 31±8 42 + 8 53±3
22±6 6 ± 5 4+7 23±3 30 + 4
Autoclaved Torula
J
K
38+5 48±5 46±4 45 + 2 48±5 56±3 56 + 4
Brewers' Yeast
I K
50±6 39±2 31±6
Autoclaved Brewers'
I K
55 + 2 57+3
1 Grams gain at 2 weeks+standard error for 5 rats. 2 Added to the 12% casein basal diet at the expense of carbohydrate.
was fed either as a supplement to casein or as the sole source of protein, and under both spaced and ad libitum feeding conditions. When yeast was the sole source of protein, autoclaving failed to improve growth whether the feeding was spaced (Table 6, series L) or ad libitum (Table 6, series M). Moreover when yeast was supplemented to casein in diets fed ad libitum, no effect of autoclaving was seen, regardless of the protein level (Table 6, series M and N). However, in spacedfeeding experiments, 10% of torula protein was as good a supplement to 12% of casein as was an additional 10% of casein itself (Table 6, series M). In all series, changes in feed efficiency closely paralleled those in weight gain. Axtmayer (1946) reported an improvement in the protein quality of torula yeast due to autoclaving, whereas Goyco and Asenjo (1948), who autoclaved for a longer period, reported no such change. However, when the latter workers followed Axtmayer's method of autoclaving, they observed a slightly higher rate of growth in rats fed the heated yeast, though the difference was not statistically significant. The autoclaving procedure used in the present studies was similar to that of Axtmayer. From our work it appeared that the measurable effect of heat treatment is dependent on the feeding conditions; i.e. improvement due to autoclaving was observed only when the yeast was space-fed at high levels. It was hoped when the studies with rats were undertaken, that- biological values of the various yeasts could be reported. Substituting weight gains of rats fed 30% protein supplements (Table 5) into the biological value formula of Finlayson and Baumann (1956) yielded the following figures: intact torula yeast 2, autoclaved torula 39, brewers' yeast 24, autoclaved brewers' 52. These values for the intact
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sistent change in the growth rate (Table 5, series H and I). When spaced-fed at the level of 30% of crude protein, untreated torula yeast was definitely inferior to casein (Table 5, series H). Moreover, extraction with ether or with dilute alkali (A-K torula) did not alter its nutritive quality. On the other hand, when torula was autoclaved, its nutritive value appeared to be increased considerably (Table 5, series H). In subsequent series the spaced-feeding of commercial torula yeast at protein levels above 20% resulted in a depression of growth, whereas the growth of rats fed autoclaved torula was remarkably constant at all levels. Autoclaving torula improved growth significantly in 5 of 7 trials, autoclaving brewers' yeast, in 2 of 2 (Table 5). Improvement due to autoclaving occurred in all cases in which the yeast was supplemented at a protein level above 20%, but only 1 of 3 cases at 20% or less. At these lower levels torula was as good a supplement to 12% of casein as was additional casein itself (Table 5, protein levels 10 and 20). Next, untreated or autoclaved yeast
1319
BIOLOGICAL VALUE OF TORULA YEASTS TABLE 6.—Responses of rats to untreated and autoclaved yeast at various protein levels Untreated Gain, 1 gms.
'0
spaced
F.E. 2
Autoclaved Gain, 1 gms.
22 Torula 22 Torula+.125% Methionine
32 + 5
22±2
45 + 9
40 + 2
F.E. !
spaced
12
30 Casein 30 Torula 30 Brewers'
77 + 1 30+4 31 + 6
.44 .25 .27
85±4 56 + 4 57±3
.45 .37 .39
ad lib.
12
10 Casein 10 Torula 10 Brewers'
70 + 8 81+4 80 + 3
.50 .50 .51
72 + 2 70 + 6 71 + 5
.54 .48 .49
ad lib.
12
30 Casein 30 Torula 30 Brewers'
64 + 5 43 + 4 60 + 2
.65 .50 .65
45 + 3 57 + 4
.56 .65
30 Casein 30 Torula 30 Brewers'
79 + 3 23 + 1 46 + 6
.68 .32 .54
24+2 43 + 4
.35 .53
M
ad lib. 1 2
Mean 2 week gain + standard error for 5 rats. Feed efficiency: grams gained per gram food eaten.
yeasts are considerably lower than those reported by other workers (Goyco and Asenjo, 1949). The formula, therefore, which was satisfactory when applied to purified proteins, apparently does not yield absolute biological values for materials such as yeast, although the results probably have comparative value. Autoclaving might have improved the digestibility of the yeast in rats under conditions of spaced-feeding, in which differences in absorption would be accentuated by the presence of high levels of yeast. This, however, would not explain the beneficial effect of pelleting in the chick experiments. The results of the experiments with chicks would indicate the possible use of torula yeast in practical diets to meet a part of the total dietary protein. Its high content of readily available lysine (Tsien et ak, 1957) makes it especially suited to supplementation of diets high in cereal proteins. SUMMARY
1. The apparent biological value of
pelleted torula yeast for the chick was found to be equivalent to that of soybean oil meal when fed in a practical cereal diet. When the original yeast was used, growth of chicks was not affected by substitution of 60% of soybean oil meal, but a 75 to 100% replacement reduced the weight gain by 15 and 23% respectively. Food consumption decreased due to impaction in the beaks of the chicks fed the high levels of the original intact yeast. 2. The improvement in growth observed after pelleting of torula as compared to the intact yeast appeared due primarily to changes in texture. Experiments with semi-synthetic diets suggested that the process of pelleting may have improved the nutritional value of the yeast protein. Autoclaving of the yeast failed to result in a comparable improvement in the apparent biological value of the torula for chicks. 3. Growth on torula, alkali-extracted torula, or brewers' yeast was improved by supplements of methionine and arginine. Other amino acids studied gave no con-
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N
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C. GITLER, J. S. FINLAYSON, C. A. BAUMANN AND M. L. SUNDE
REFERENCES Allred, J. B., L. S. Jensen and J. McGinnis, 1957a. Factors affecting the response of chicks and poults to feed pelleting. Poultry Sci. 36: 517-523. Allred, J. B., R. E. Fry, L. S. Jensen and J. McGinnis, 1957b. Studies with chicks on improvement in nutritive value of feed ingredients by pelleting. Poultry Sci. 36: 1284-1289. Axtmayer, J. H., 1946. Cited in Goyco, J. A., and C. F. Asenjo, 1947. The net protein value of food yeast. J. Nutrition, 33: 593-600. Bird, H. R., 1954. Nutrition requirements for poultry. National Research Council, Publication 301. Block, R. J., and D. Boiling, 1945. The Amino Acid Composition of Proteins and Foods. Charles C Thomas, Springfield, Illinois publisher. Briggs, G. M., Jr., T. O. Luckey, C. A. Elvehjem and E. B. Hart, 1943. Studies on two chemically unidentified water soluble vitamins necessary for the chick. J. Biol. Chem. 148: 163-172.
Dirr, K., and P. Decker, 1944. Uber den Wert der Wuchshefe fur die menschliche Ernahrung. V. Uber den Reineiweissgehalt der Hefe. Biochem. Z. 316: 245-248. Finlayson, J. S., and C. A. Baumann, 1956. Responses of rats to urea and related substances. The use of a spaced-feeding technique. J. Nutrition, 59: 211-221. Flodin, N . W., 1953. Amino acids and proteins. Their place in human nutrition problems. J. Agr. Food Chem. 1: 222-235. Gitler, C , 1956. Master's degree thesis, University of Wisconsin, Madison. Goyco, J. A., and C. F . Asenjo, 1948. Studies on edible yeasts Puerto Rico J. Publ. Health Trop. Med. 23:471-^97. Goyco, J. A., and C. F. Asenjo, 1949. Net protein and growth-promoting values of three different types of yeast prepared under identical conditions. J. Nutrition, 38: 517-526. Klose, A. A., and H. L. Fevold, 1945. Nutritional value of yeast protein for the rat and the chick. J. Nutrition, 29: 421-130. Ringrose, R. C , 1949. Nutritive properties of torula yeast for poultry. Poultry Sci. 28: 75-83. Soden, O. von, and K. Dirr, 1942. Uber den Wert der Wuchshefen fur die menschliche Ernahrung III. Verdaulichkeit in vitro von verschieidenen Hefen in Vergleich zu anderen Eiweisstragern der menschlichen Ernahrung. Biochem Z. 312: 252262. Tsien, W. S., E. L. Johnson and I. E. Liener, 1957. The availability of lysine from torula yeast. Arch. Biochem. Biophys. 71:414-422. Van Slyke, D. D., and E. Kirk, 1933. Comparison of gasometric, colorimetric and titrimetric determinations of amino nitrogen in blood and urine. J. Biol. Chem. 102: 651-682. Vedvik, J. R., 1952. Master's Thesis, University of Wisconsin, Madison.
BOOK REVIEWS (Continued from page 1313) become extremely complex. . . . I t should be helpful, therefore, to take stock from time to time and to sum up descriptively these guides to information and literature. . . . I t is hoped that the present work will be considered a step in this direction." The reviewer is perfectly willing to admit that the subject is most complex, that decisions must be made in many debatable or borderline areas, and that such a compilation must be kept within reason-
able limits. Nevertheless he is not certain that the present volume has accomplished its purpose, and he is certain that there are serious omissions. For example, the list of textbooks in poultry husbandry given on page 89 can, by no stretch of the imagination, be considered complete or comprehensive. One would have thought that, somewhere in such a book, space could have been found for a list of major scientific journals publishing manuscripts in
(Continued on page 1324)
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sistent growth response. 4. In rats trained to eat for 2 hours per day, autoclaving improved the growth promoting qualities of both torula and brewers' yeasts when supplemented at high levels to a diet containing 12% of casein. Autoclaving caused no improvement in growth on torula yeast fed as the sole source of protein under ad libitum conditions, or when supplemented to casein at low levels (10% protein or less). Under these latter conditions, torula yeast appeared to be as good a supplement to 12% casein as was brewers' yeast or additional casein, regardless of the method of feeding.