The Effect of Phytin on the Phosphorus Requirement of the Chick1

T h e Effect of Phytin on the Phosphorus Requirement of the Chick1 M. B. GILLIS, L. C. N O R M S AND G. F. HEUSER

Agricultural Experiment Station and School of Nutrition, Cornell University, Ithaca, New York (Received for publication September 11, 1948)

HE phosphorus supplied in poultry rations by cereals, cereal by-products, soybeans, and other plant materials is largely in the form of phytic acid and its salts, collectively termed phytin. The utilization of this organically bound form of phosphorus is a question of considerable practical importance since phosphorus is one of the elements most likely to be deficient in unsupplemented rations. In some instances phytin is relied upon to supply a large portion of the total phosphorus requirement of chickens, although the weight of evidence indicates that it is less readily utilized than inorganic phosphates. Lowe, Steenbock, and Krieger (1939) supplemented a low phosphorus basal chick diet with phytin isolated from wheat bran and obtained no increase in calcification. These results were obtained, however, in the absence of supplementary vitamin D or in the presence of suboptimum amounts of this factor. Heuser, Norris, McGinnis, and Scott (1943) presented evidence that growth and bone calcification were sub-normal in chicks receiving adequate levels of vitamin D when the phosphorus supply was principally phytin from natural feed ingredients. Later McGinnis, Norris, and Heuser (1944) showed that, although high levels of vitamin D increased the utilization "of 1

This work was supported in part by a grant from the International Minerals and Chemical Corporation, Chicago, Illinois. 283

cereal phosphorus by the chick, 320 units of vitamin D per 100 grams of feed did not result in normal bone development on a diet containing phosphorus supplied mostly by cereal products. Singsen and Mitchell (1944) suggested that phytin phosphorus is efficiently utilized by chicks when the ration contains a sufficient amount of the phytin splitting enzyme, phytase. This theory appears to be untenable, however, in view of the results of McGinnis (1944) and Spitzer, et al. (1945,1948) who have shown that phytin utilization is not affected by the presence or absence of phytase in the diet, and that this enzyme is naturally present in the digestive tract of animals. Singsen, Matterson, and Scott (1947) have reported that in the case of turkey poults the phytin phosphorus in cereals is not efficiently utilized although vitamin D increases utilization. Vitamin D 3 was found to be more effective in this respect than fish liver oil. No quantitative study has been made to determine the extent to which phytin may be relied upon to meet the chick's phosphorus requirement nor how much inorganic phosphorus is required in a ration containing substantial amounts of phytin. In the absence of any such data, the Poultry Subcommittee of the National Research Council's Committee on Animal Nutrition (1946) has established an allowance of 0.6 percent total phosphorus for chicks with the stipulation that one-third of this amount, or 0.2 percent, must be

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T

284

M. B. GILLIS, L. C. NORRIS AND G. F. HEUSER

EXPERIMENTAL

Day-old White Leghorn male chicks were divided into lots of 15 each at the beginning of the experiments. They were placed in electrically heated battery brooders in rooms from which all sources of ultraviolet irradiation were excluded. The experimental diets to be described were supplied ad libitum during the experimental periods which lasted for four weeks. At the end of this time, the final individual weights were taken and ten representative chicks from each lot were sacrificed for bone ash determinations. The left tibiae were cleaned and ashed according to the standard procedure of the Association of Official Agricultural Chemists. In lots with less than ten survivors all chicks were used for bone ash determinations. Experiment 1 In this experiment the low-phosphorus, purified-type basal diet shown in Table 1 was used. When supplemented with adequate amounts of available calcium and phosphorus, it is nutritionally adequate to support growth which is generally considered optimum at the present time. Before the addition of calcium and phosphorus

TABLE 1.—Low phosphorus basal diet Ingredients Cornstarch Blood fibrin Gelatin Cellophane Soybean oil Wilson's liver "L" Vitamins* Salts* Ca and P supplements

Percent 56.0 + 25.0 5.0 3.0 3.0 1.0 0.5 1.5 5.0 ±

* Gillis, Norris, and Heuser (1948).

supplements it contains approximately 0.05 percent of each of these elements. The design of the first experiment is given in Table 2. All lots received a total of 0.6 percent phosphorus and 1.0 percent calcium to correspond to the recommendations of the National Research Council's Poultry Subcommittee. The form in which the phosphorus was supplied, however, varied from all inorganic to nearly all phytin phosphorus. The phytin in this experiment was supplied by a relatively pure calcium phytate 2 and the inorganic phosphorus by c.p. dicalcium phosphate. The constant level of calcium was maintained by additions of calcium carbonate. The levels of vitamin D 3 shown in Table 2 were fed in connection with the various combinations of the two sources of phosphorus. The vitamin D 3 was supplied in pure form in a corn oil solution.3 The results obtained with respect to both growth and bone ash are also given in Table 2. Excellent growth and calcification were obtained in the lots receiving all inorganic phosphorus in the presence of 10 or more units of vitamin D 3 . In the case of chicks receiving 0.4 percent inorganic phosphorus and 0.2 percent phytin phosphorus, about 40 units of vitamin D 3 were 2

Kindly supplied by the Corn Products Refining Co., New York, N. Y. 8 Courtesy of Winthrop Chemical Co., Rensselaer, N. Y.

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from inorganic sources. Results obtained in this laboratory (Gillis, Norris, and Heuser, 1948, and unpublished data) indicate that this recommendation may be too low with respect to the non-phytin phosphorus requirement. Accordingly, further work has been conducted to determine as precisely as possible the non-phytin phosphorus requirement when both phytin and inorganic phosphorus are present in the diet and the extent to which phytin may be relied upon to meet the chick's requirement for phosphorus. The results of this work are reported in this paper.

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EFFECT OF PHYTIN ON PHOSPHORUS REQUIREMENT TABLE 2.—Effect of replacing inorganic phosphorus with isolated phytin on growth and calcification in chicks Inorganic P : Phytin P :

• 2%

Wt. gm.

Bone ash %

Wt. gm.

Bone ash

0 10 20 40 80 160 320

265 320 325 319

37.8 45.9 46.9 48.2

261 296 295 307 313

34.3 40.4 42.0 46.1 45.1

%

Wt. gm.

Bone ash %

158

30.6

—

—

211 229 246 268

29.7 31.6 32.3 37.8

Wt. gm.

Bone ash

% All dead

— — —

132 139 143

— —. —

27.5 (2)* 27.3 (6) 30.1(11)

Figures in parentheses indicate number of survivors in lots of 15 chicks.

required for growth and calcification. With this and higher levels of vitamin D 3 the results on this combination of the two phosphorus sources were quite satisfactory though slightly below those obtained with all inorganic phosphorus. When phytin supplied two-thirds of the phosphorus in the diet, both growth and bone ash values were greatly depressed. By the end of the fourth week practically all of the- chicks receiving this treatment had developed symptoms of rickets regardless of the level of vitamin D 3 fed. It should be noted that the treatment of the lot in this experiment which received 0.4 percent phytin phosphorus, 0.2 percent inorganic phosphorus, and 40 units of vitamin D 3 meets current recommendations with respect to total calcium and phosphorus, inorganic phosphorus and vitamin D. Growth and calcification in this lot, however, were decidedly unsatisfactory, all chicks developing rickets within the 4 weeks' experimental period. The chicks receiving 0.05 percent inorganic and 0.55 percent phytin phosphorus developed severe rickets within a period of two weeks. High mortality was encountered in all lots receiving this treatment, deaths due to phosphorus deficiency beginning during the second week. Survival was benefited to some extent by the

higher levels of vitamin D3, but the chicks were in such poor condition that none would have survived had the experiment been continued for a short while longer. The results obtained with the two higher levels of phytin in this experiment indicate that increasing the proportion of phytin in the diet emphasizes the need for vitamin D, but that this vitamin does not replace the requirement for non-phytin phosphorus. Experiment 2 In the first experiment relatively pure phytin was supplied in the experimental diets by means of an isolated calcium phytate. In a second experiment of similar design phytin was supplied by means of ordinary feed ingredients naturally high in this form of phosphorus. This was done in order to determine if the results obtained in Experiment 1 were comparable to those which might be expected on a more practical type of diet containing natural sources of phytin. The three rations shown in Table 3 were used in this experiment. Ration 1 is a purified type diet containing wheat bran soaked and washed to remove phytin and included in this ration in order to raise the fiber content to a level comparable with that in rations 2 and 3. The latter two ra-

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Units Vitamin D 3 /100 gm. diet

;

.05% .55%

• 2% • 4%

.4%

•6% • 0%

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M. B. GILLIS, L. C. NORRIS AND G. F. HE-USER

TABLE 3.—Rations used in studying natural sources of phytin Ingredients

Ration No. 1

40.4

—

20.0

— —

30.0 4.0 0.5 1.4 1.0 1.05 1.65

Ration No. 3

—

40.63 20.0

—

15.0 15.0 4.0 0.5 1.4 1.0 .2 2.27

Phytin P

.00%

.20%

.35%

Non-phytin P

.60%

.40%

.25%

* Gillis, Norris, and Heuser (1948). tions contain natural ingredients to supply phytin. These ingredients were analyzed for phytin and total phosphorus, and the combinations of wheat bran, corn meal and soybean meal used were based on the results of the analyses. The total calcium and phosphorus received by all lots was again maintained at levels of 1.0 percent and 0.6 percent respectively. The energy, protein, and fiber contents of the different rations were approximately the same. The experimental design and the results obtained with respect to growth and bone ash values are shown in Table 4. Growth was excellent in the case of the chicks receiving either all inorganic phosphorus or the combination of 0.4 percent inorganic and 0.2 percent phytin phosphorus. Bone ash values were very slightly, but consistently, better in the lots receiving all inorganic phosphorus. In the case of the lots receiving 0.35 percent phytin and 0.25 percent non-phytin phosphorus growth was depressed and bone ash values were definitely inferior. Many of the chicks in these lots developed symptoms of rickets during the 4 weeks' experi-

TABLE 4.—Effect of natural phytin in feedstuffs on chick growth and calcification Non-phytin P: Phytin P :

• 6%

•4%

•25%

•0%

•2%

.35%

Vitamin D 3 Wt. Bone Wt. Bone added per gm. ash gm. ash 100 gm. % % diet 0 20 40 80 160

296 327 325

46.9 47.7 47.0

Wt. Bone gm. ash

%

330

46.2

282

33.6

330 318

46.6 45.5

290 281 279

36.3 39.0 40.9

rus. The reaction of the chicks in the second experiment may have been conditioned by greater body stores of vitamin D since this experiment was conducted later in the hatching season. In this experiment it was also desired to study the effect of supplementing the ration of natural ingredients high in phytin with a level of inorganic phosphorus sufficient to increase the total non-phytin phosphorus to 0.4 percent. Accordingly one lot of chicks received ration 3 supplemented with dicalcium phosphate sufficient to supply 0.15 percent phosphorus, bringing the total non-phytin phosphorus in the diet to 0.4 percent. The results pre-

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Corn starch 40.0 Yellow corn meal —. Raw wheat bran — Washed wheat bran 20.0 Soybean meal — Blood fibrin 30.0 Soybean oil 4.0 Vitamins* 0.5 . Minerals* 1.4 Wilson's liver " L " 1.0 CaHP0 4 (c.p.) 2.38 CaC0 3 .72

Ration No. 2

mental period affording definite evidence that this level of non-phytin phosphorus was inadequate for optimum growth or calcification. The response of chicks to vitamin D in this experiment was different from that obtained in the first experiment. Vitamin D apparently had no effect on growth in any case and affected bone formation only in the case of the chicks receiving the higher level of phytin; whereas in the first experiment both growth and bone ash values were improved in all chicks receiving vitamin D, the response increasing with increasing levels of phytin phospho-

EFFECT OF PHYTIN ON PHOSPHORUS REQUIREMENT TABLE 5.—Effect of supplementing high-phytin diet with inorganic phosphorus

No. 3, Practical Type* None No. 3, Practical Type .15% inorganic P

gm. 281 322

percent 39.0 47.3

sented in Table 5 show that both growth and calcification were significantly improved and that diets of this type are primarily deficient in available phosphorus. It is significant that this ration, comprised largely of practical ingredients, when supplemented with inorganic phosphorus, produced as good growth and calcification in chicks as the purified diets containing all inorganic phosphorus. DISCUSSION The results obtained in these experiments make it increasingly clear that the chemical analysis of a ration for total phosphorus is not a reliable test of its adequacy with respect to this element. The chemical form in which the phosphorus is present must also be taken into consideration. The situation in this respect is somewhat analogous to the determination of crude protein, once considered a sufficient measure of nutritional adequacy but now known to be inadequate because it does not reflect the content of essential amino acids. It seems evident from the data obtained in these experiments that phytin phosphorus is not efficiently utilized by the young chick. This was true of both the isolated material and the phytin present in natural ingredients. In view of the difference in response to vitamin D 3 obtained in the first and second experiment it is impossible to say whether or not the natural sources of phytin were more available than

the isolated calcium phytate. A casual examination of the data indicate that the natural phytin was a slightly more effective source of phosphorus, but this may have been due to the greater body stores of vitamin D as already suggested. The chick can undoubtedly utilize phytin phosphorus to a limited extent in the presence of a substantial amount of the more available inorganic phosphorus. In the presence of not more than 0.05 percent of inorganic phosphorus, however, phytin is extremely unavailable and cannot be relied upon to furnish even the minimum amount of phosphorus required to maintain life. When an adequate level of available inorganic phosphorus was progressively replaced by higher levels of phytin, it resulted in these experiments, in a rapid decline in growth and calcification and a marked increase in the vitamin D requirement. When the diet, containing a total of 0.6 percent phosphorus, included at least 0.4 percent of an available form of inorganic phosphorus, an adequate level of vitamin D 3 resulted in good calcification in all cases. At the levels of non-phytin phosphorus studied below 0.4 percent, however, higher levels of vitamin D 3 did not overcome the phosphorus deficiency. In the case of the isolated calcium phytate, 0.4 percent of inorganic phosphorus was borderline for the chick, and in the case of phytin from natural ingredients, 0.25 percent inorganic phosphorus was definitely too low. Levels of inorganic phosphorus between 0.4 percent and 0.25 percent were not studied in connection with natural phytin, but in view of the inadequacy of the lower level and natural variation in individual chick requirements, 0.4 percent would appear to be a reasonable minimum. This minimum of 0.4 percent inorganic phosphorus is suggested on the basis of results obtained with a chemically pure cal-

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* 0.35% Phytin P; 0.25% Non-phytin P.

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1. The total phosphorus in chick rations as measured by ordinary chemical means is not a reliable index of the nutritional adequacy of this element. 2. For optimum early growth and bone formation the chick requires approximately 0.4 percent of a readily available form of inorganic or non-phytin phosphorus when the diet contains at least 0.6 percent of this element. 3. Replacement of an adequate level of available inorganic phosphorus by progressively higher levels of phytin results in a rapid decline in growth and calcification and a marked increase in the vitamin D

requirement. However, vitamin D does not overcome the adverse effect of supplying too little non-phytin phosphorus. 4. Natural diets high in phytin phosphorus are much more effectively supplemented by increasing the inorganic phosphorus to 0.4 percent than by raising the vitamin D content. REFERENCES

Gillis, M. B., L. C. Norris, and G. F. Heuser, 1948. The utilization by the chick of phosphorus from different sources. J. Nutrition 35:195-207. Heuser, G. F., L. C. Norris, J. McGinnis, and M. L. Scott, 1943. Further evidence of the need for supplementing soybean meal chick rations with phosphorus. Poultry Sci. 22:269-270. Krieger, C. H., R. Bunkfeldt, and H. Steenbock, 1940. Cereals and rickets. X. The availability of phytic acid phosphorus. J. Nutrition 20:7-14. Lowe, J. T., H. Steenbock, and C. H. Krieger, 1939. Cereals and rickets. IX. The availability of phytin-P to the chick. Poultry Sci. 18:40-M. McGinnis, J., 1944. Studies on the utilization by the chick of phosphorus supplied entirely from plant sources. Thesis, Cornell University, Ithaca, New York. McGinnis, J., L. C. Norris, and G. F. Heuser, 1944. Poor utilization of phosphorus in cereals and legumes by chicks for bone development. Poultry Sci. 23:157-159. Poultry Subcommittee, Committee on Animal Nutrition, National Research Council, 1946. Recommended nutrient allowances for poultry, revised. Singsen, E. P., L. D. Matterson, and H. M. Scott, 1947. Phosphorus in poultry nutrition. III. The relationship between the source of vitamin D and the utilization of cereal phosphorus by the poult. J. Nutrition 33:13-26. Singsen, E. P., and H. H. Mitchell, 1944. Soybean meal chick rations need no inorganic phosphorus supplements. Poultry Sci. 23:152-153. • Spitzer, R. R., G. Maruyama, L. Michaud, and P. H. Phillips, 1948. The role of vitamin D in the utilization of phytin phosphorus. J. Nutrition 35: 185-193. Spitzer, R. R. and P. H. Phillips, 1945. Enzymatic relationships in the utilization of soybean oil meal phosphorus by the rat. J. Nutrition 30:183-192.

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cium phosphate which is practically all available to the chick. A number of phosphorus supplements used in poultry rations are not so completely available. It would seem advisable to increase the inorganic phosphorus allowance above this level where a supplement with an appreciably lower availability is used. The chick exhibits a marked difference from the rat and several other mammalian species in its inability to utilize phytin phosphorus with any degree of efficiency even in the presence of adequate vitamin D. A number of workers (Krieger et al. 1940, Spitzer et al. 1945, 1948, and others) have reported that the rat makes effective use of phytic acid and phytin phosphorus when supplied adequate vitamin D, although this form is less efficient than inorganic phosphorus. The reason for this species difference is by no means clear, but undoubtedly derives from the digestive or absorptive peculiarities of birds. Apparently it is not related to the presence of the enzyme phytase in the feed or digestive tract, McGinnis (1944), Spitzer et al. (1948). CONCLUSIONS