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Autoclaved Egg White as a Protein Source for Chick Diets Low in Zinc1
Autoclaved Egg White as a Protein Source for Chick Diets Low in Zinc1 JAMES M. HEMPE and J. E. SAVAGE Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211 (Received for publication July 21, 1989)
1990 Poultry Science 69:959-965
inhibitors adversely affect pancreatic function and cause both pancreatic hypertrophy and a Spray-dried egg white contains approxi- reduced activity of proteolytic enzymes in mately 80% protein and less than 1 mg of zinc mice (Kirschenmann and Schneeman, 1982). per kg. Based on its amino acid composition, The purpose of the present experiments was egg white would appear to be an excellent to develop a low-zinc diet based on egg white source of protein for use in low-zinc diets for chicks. However, egg white also contains for the chick that would be nutritionally avidin, a heat-labile protein that complexes adequate when supplemented with zinc. The with and decreases the absorption of biotin experimental design included an assessment of the effects of avidin and of egg-white protein(Eakin et al, 1941; Gyorgy et al., 1941). Spray-dried egg white was a satisfactory ase inhibitors on the chick as well as the source of protein in low-zinc diets for rats development of procedures to eliminate such when the diets were supplemented with extra effects. biotin in order to overcome the adverse effects of avidin (Luecke et al., 1968; Forbes et al., MATERIALS AND METHODS 1979). Supplemental biotin improved growth Sexed, broiler-strain chicks (Hubbard by and reduced the incidence of perosis in chicks fed egg-white diets (Jukes and Bird, 1942). Hubbard cross) were obtained from a commerBut a diet for chicks based on egg white cial hatchery on the day they were hatched. that is nutritionally adequate except for zinc The chicks were divided into equal weight has not been demonstrated. One reason may be groups of 10 each. The groups were housed in that raw egg white also contains proteinase stainless-steel, electrically-heated battery inhibitors that deactivate trypsin and chymo- brooders; the rooms were temperature-contrypsin in vitro (Rhodes et al., 1960). These trolled (24 C) with continuous light. Feed and distilled water were supplied for ad libitum consumption in stainless-steel or glass containers for the duration of each experiment. Body 'Contribution from the Missouri Agricultural Experiweights were recorded weekly. ment Station, Journal Series Number 10344. INTRODUCTION
959
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ABSTRACT Spray-dried egg white was evaluated either as the sole source or the primary source of protein in purified, low-zinc diets for chicks. The effects of unheated and autoclaved egg white were compared for chicks fed diets supplemented with graded amounts of biotin. Growth rate and pancreas weight were used as the criteria of nutritional adequacy. The data indicated that extra biotin did not completely overcome the adverse effects of unheated egg white. Chicks fed unheated egg white and excess biotin had decreased body weights and showed signs of pancreatic hypertrophy, probably due to proteinase inhibitors in the egg white. Autoclaving rehydrated and spray-dried egg white for 30 rain at 121C, but not at 80 C, markedly reduced the biotin-binding and proteinase-inhibitor activities of unheated egg white. Extracting autoclaved egg white with water did not improve the growth rate or reduce the amount of zinc in the egg white. Chicks fed diets of autoclaved egg white and gelatin with low concentrations of zinc showed symptoms of severe zinc deficiency. Chicks fed diets with adequate concentrations of zinc had body weights similar to those fed casein-gelatin control diets. When graded amounts of zinc were added to the diet, the growth rate of the chicks and the tissue concentration of zinc reflected zinc intake. These data demonstrated that rehydrated, spray-dried egg white autoclaved for 30 min at 121 C is an excellent source of protein for use in low-zinc diets for chicks. (Key words: zinc, purified diet, egg white, avidin, trypsin inhibitor)
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HEMPE AND SAVAGE TABLE 1. Composition of the diets
Ingredient
Egg white, basal
Casein-gelatin, control
(%) 32.4
51.9 6.0 5.5 1.0 2.0 1.0 .2
24.4 5.0 .9 .3 54.3 4.8 5.5 .3 1.0 2.0 1.0 .3 .2
Autoclaved or unheated. Composition (grams per kilogram of premix): CaHP04-2H20, 581.6; Ca3(P04)2, 116.9; CaC0 3 , 58.523; K2C(>31-1/2H20,168.7; MgS0 4 ,58.4; Ferric citrate, 9.4; MnS0 4 H 2 0, 6.0; KK>3, .23; CuS0 4 , .24; and Na 2 Se0 3 -5H 2 0, .0065. Supplied (grams per kilogram of diet): Ca, 11.3; P, 7.0, and K, 4.4. Supplied (milligrams per kilogram of diet): Mg, 649; Fe, 86; Mn, 107; I, 7.5; Cu, 5.3; and Se, .11. 3 Solka Floe, James River Corporation, Berlin, NH. ''in glucose carrier, supplied (per kilogram of diet): retinyl palmitate, 20,000 IU; cholecalciferoL 2,000 ICU; and DLtocopheryl acetate, 20 IU. Supplied (milligrams per kilogram of diet): menadione sodium bisulfite, 2; ethoxyquin, 125; thiamine HCl, 10; riboflavin, 10; pyndoxine HCl, 15; D-calcium pantothenate, 30; niacin, 75; foiacin, 2; and cyanocobalamin, .03. Reagent-grade zinc carbonate in a glucose carrier, supplied 50 mg of zinc per kilogram of diet ^D-biotin in a glucose carrier; supplied .3 mg of biotin per kg of diet
Diets The ingredient compositions for the eggwhite, basal diet and the casein-based control diet are shown in Table 1. Without added zinc, the diet based on egg white was analyzed to contain .9 mg of zinc per kg of diet. This formulation was followed in all five experiments with minor adjustments in the ratios of egg white, glucose, and soybean oil. When gelatin was added to the basal diet, the amounts of egg white, glucose, and soybean oil were adjusted in order to keep the dietary amounts of crude protein (26%) and of energy (3,400 kcal per kg of diet) approximately the same. The chicks were fed the casein diet as a positive control in Experiments 1, 2, and 3. Biotin was omitted from the vitamin premix in Experiment 1,2, and 4 to study the effects of the autoclaving temperature on avidin activity.
2
Milton G. Waldbaum Co., Wakefield, NE. Amsco model 2053, American Sterilizer Co., Erie, PA.
3
Supplements of D-biotin were added to those diets to supply .3,1.5, or 3.0 mg of biotin per kg of diet, representing 2, 10, and 20 times the biotin requirement (National Research Council, 1984). Pilot studies indicated that feather growth was abnormal in chicks fed diets containing unheated, spray-dried egg white. This prompted a study of the value of gelatin as an amino acid supplement. The effects were also determined of autoclaving dry egg white versus rehydrated egg white and of extracting autoclaved egg white with deionized, distilled water. Stainless-steel or plastic equipment was used for all processing procedures involving egg white. Autoclaved, rehydrated egg white was prepared by mixing 10 kg of spray-dried egg white2 with 12 L of deionized, distilled water. The mixture was poured into flat pans to a depth of approximately 5 cm and was autoclaved in a laboratory sterilizer3 at either 80 C or 121 C. After autoclaving, the hardened cake was cut into strips approximately 5 cm wide and then ground in a food-chopper.
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Spray-dried egg white Casein Gelatin L-arginine (free-base) DL-methionine Glucose monohydrate Soybean oil Mineral premix2 Sodium chloride Cellulose3 Vitamin premix4 Zinc premix5 Biotin premix6 Choline chloride
LOW-ZINC CHICK DIET
Experimental Design
diets containing: 1) rehydrated egg white autoclaved at 121 C, which was either extracted or not extracted with deionized, distilled water, and 2) the same diet witii and without a 5% gelatin supplement. The fifth group was fed a diet for which the sole source of protein was spray-dried egg white autoclaved at 121 C in the dry form. Experiment 4. Duplicate groups of 10 male chicks were assigned to 14 dietary treatments. In twelve of me treatments, the growm rates and pancreas weights were compared for chicks fed diets containing: 1) unheated egg white, rehydrated egg white autoclaved at 80 C, or rehydrated egg white autoclaved at 121 C; 2) the same diets with a supplement of .3 mg or of 1.5 mg of biotin per kg of diet; and 3) the same diets witii and without a 5% gelatin supplement. Two additional groups were fed diets containing unheated egg white and 3.0 mg of biotin per kg of diet, eiuier with or without a supplement of 5% gelatin. Final body weights were recorded at 21 days of age. The chicks used to determine the pancreas response were reweighed on Day 26, when the pancreases were removed and weighed. Experiments. Groups of 10 male chicks were assigned to nine dietary treatments. All diets contained 5% gelatin and rehydrated egg white autoclaved at 121 C. These diets were similar to the one described in Table 1, except that die zinc premix contained graded concentrations of zinc (6, 8, 10,12, 14, 16, 18, 20, or 50 mg per kg), supplied as zinc carbonate. All diets were supplemented with .3 mg of biotin per kg of diet.
In addition to body weight, comparisons were made of pancreas weight and pancreas weight as a percentage of body weight in Experiments 1,2, and 4. These data were used as indicators of pancreatic hypertrophy to assess the effects of egg-white proteinase inhibitors on pancreatic function. To avoid confounding the effects of the proteinase inhibitors with those of avidin, pancreas data were collected only from chicks fed egg-white diets supplemented with 1.5 or 3.0 mg of biotin per kg of diet. Final body weights were determined at either 21 or 28 days of age. Bioassays at 3 wk and at 4 wk were observed to be statistically similar. Experiment 1. Duplicate groups of 10 male chicks were assigned to eight dietary treatments. The chicks were fed diets containing: 1) unheated egg white or rehydrated egg white autoclaved at 121 C; 2) the same diet with a Analytical Procedures supplement of .3 or 1.5 mg of biotin per kg of Samples of the diet, tibia, and pancreas were diet; 3) the same diet with and without a dried overnight in a vacuum oven at 60 C. The supplement of 5% gelatin. Experiment 2. Duplicate groups of 10 female samples were weighed and were dry-ashed for chicks were assigned to five dietary treatments. 18 h at 500 C. All ash residues were heated with In four of the treatments, comparisons were nitric acid on a hot plate and then were reashed at made of the growth rates and the pancreas the same temperature. Following the second weights for chicks fed diets containing 1.5 mg of ashing, the residues were dissolved in dilute biotin per kg of diet and: 1) with rehydrated egg HCl; the zinc concentration was determined by white autoclaved at 80 C or at 121 C; 2) with and flame atomic-absorption spectrophotometry. without a 5% gelatin supplement. The fifth For plasma zinc analyses, whole blood was group was fed a diet containing rehydrated egg collected by cardiac puncture following an white autoclaved at 80 C and supplemented with overnight fast. The blood was placed in heparinized tubes and was centrifuged at 2,000 x g for .3 mg of biotin per kg of diet. Experiment3. Groups of 10 male chicks were 10 min. One mL of plasma was diluted with 4 assigned to five dietary treatments. Four treat- mL of 1% HCl in deionized, distilled water, and ments compared the growth rates of chicks fed then analyzed as above.
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Water-extracted egg white was prepared by soaking the ground, autoclaved egg white in deionized, distilled water for 10 min and then siphoning die supernate. This procedure was repeated three times. Because extraction removed most of the sodium chloride, diets containing water-extracted egg white were supplemented with .3% of sodium chloride. Following grinding (or grinding and extraction), me autoclaved egg white was dried for 24 h in a forced-air oven at 60 C. Then, die egg white was ground in a hammer mill and was stored at room temperature. A similar procedure was used to make spray-dried egg white, autoclaved in the dry form except that the egg white was not rehydrated prior to autoclaving.
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HEMPE AND SAVAGE
TABLE 2. Effect of autociaving procedure, dietary biotin concentration, and gelatin supplementation on the growth rate of chicks fed egg-white diets1 Autociaving procedure2 Protein source
Temperature (Q
Rehydrated Extracted
Gelatin (%)
Added biotin
Experiment 1 Spray-dried egg white 121
Yes
(c\ \ti)
1.5 .3 1.5 .3 .3
730"b 696 bc 572 d 285 f
(16) (18) (11) (9)
751" 743*b 676° 359" 692 bc
(18) (18) (16) ( 8) (20)
Casein Experiment 3 Spray-dried egg white 121 121 121 Casein
Yes Yes
Yes Yes
1.5 1.5 .3 .3
761 b 772"b 319°
(14) (17) (8)
794ab
814*
(12) (13)
803"
(12)
Yes Yes No
1.5 1.5 1.5 .3
928 b 943b 204°
998"b 1,009"
( 9) (10)
o-7-yab
(9)
1.5 .3 1.5 .3 3.0 1.5 .3
618" 523 b 414 d 242 ef 428 cd 275 e 21 l f
625" 625" 601" 257** 516 b 473 bc 261 ef
(15) (15) (13) (14) (19) (20) (13)
Yes No No
Experiment 4 Spray-dried egg white 121
Yes
No
80
Yes
No
No
(9) (8) (6)
(15) (14) (12) (12) (20) (18) (18)
Within an experiment, values with no common superscripts are significantly different (P<.05). Mean body weights at 28 days (Experiments 1 to 3) or at 21 days of age (Experiment 4). Male chicks were used in Experiments 1,3, and 4; female chicks were used in Experiment 2. Ten chicks (Experiment 3) or 20 chicks (Experiments 1,2, and 4) were assigned to each dietary treatment group. The numbers in parentheses represent the number of chicks remaining at the end of the experiment 2 When rehydrated, egg white was mixed at a ratio of 1:1.2 with deionized, distilled water before autociaving. The autociaving time was 30 rain. Some of the autoclaved egg white was extracted with deionized, distilled water.
Statistical Analyses Two-way (Experiments 1, 2, and 4) or oneway (Experiments 3 and 5) analyses of variances were performed using the general linear models procedure of the Statistical Analysis System (SAS Institute, 1982). Differences between the means for dietary treatments concerning body weights, pancreas weights, and zinc concentrations in tissue were tested by Fisher's Least Significant Differences (LSD) method (Snedecor and Cochran, 1980). RESULTS AND DISCUSSION
Classic symptoms of severe biotin deficiency were observed in the chicks fed diets
containing unheated egg white and 2 times the biotin requirement (.3 mg per kg). These symptoms, collectively described as "egg white injury" or as "egg white syndrome" (Ringrose et al., 1931; Eakin et al., 1941; Gyorgy et al., 1941; Jukes and Bird, 1942) included reduced growth (Table 2), increased mortality, perosis, and dermatitis. The growth rate was significantly increased and the deficiency symptoms were reduced in chicks fed the unheated egg white plus 1.5 or 3.0 mg of biotin per kg of diet. Excess biotin did not enhance the growth rate for chicks fed rehydrated egg white autoclaved at 121 C (Table 2). A slight growth depression was observed in only one of the groups fed diets containing .3 mg of biotin per
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Yes
No Casein Experiment 2 Spray-dried egg white 121 80
5
0
(mg/kg)
LOW-ZINC CHICK DIET
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TABLE 3. Effect of autoclaving on pancreas size in chicks fed diets based on egg white1
Experiment 1 2 4
Autoclaved egg white
Autoclaving temperature
Added biotin
Pancreas weight
Pancreas weight in proportion to body weight
Yes No Yes Yes Yes Yes No
(Q 121 ... 121 80 121 80 ...
(mgperkg) 1.5 1.5 1.5 1.5 1.5 1.5 3.0 L5
(g) 2.00* 1.99* 1.90* 1.91* 1.78b 2.08b 252* 2.22b
(%) .266 b .339* .239* .249* .202c .262b .324* .355*^
kg of diet (Experiment 4). In contrast, the growtii of chicks fed diets containing rehydrated egg white autoclaved at 80 C was markedly affected by the concentration of dietary biotin. These data indicated that avidin in raw, spray-dried egg white was inactivated when the egg white was rehydrated and was autoclaved for 30 min at 121 C, but not at 80 C. Despite the beneficial effect of the added biotin in the diets with unheated egg white, the growth rate of chicks fed those diets was less than that of those fed diets containing egg white autoclaved at 121 C (Table 2). In all likelihood, this reduced growth rate cannot be attributed to avidin, since the diets supplemented with 3.0 mg of biotin per kg of diet (20 times the requirement) contained enough biotin to exceed the predicted binding capacity of the avidin in the egg white. Raw egg white also contains other heatlabile, antinutritional proteins mat may adversely affect chick growth. Proteinase inhibitors in egg white reduce in vitro proteolysis by trypsin and chymotrypsin (Rhodes et al., 1960). Kirschenmann and Schneeman (1982) observed pancreatic hypertrophy and an increased synthesis of pancreatic enzymes in mice fed unheated egg white. In the present experiment, the pancreas weights of chicks fed the unheated egg white were equal to or significantly greater than those for the chicks fed diets containing egg white autoclaved at 121 C (Table 3), despite the marked difference in body weights (Table 2). When expressed as a percentage of body weight, the pancreas weights of die chicks fed
the diets with unheated egg white were significantly greater than those of the chicks fed autoclaved egg white. The greater relative size of the pancreas in these chicks suggests that the proteinase inhibitors in the diets with unheated egg white induced pancreatic hypertrophy and were responsible for the depressed growth. Although autoclaving the rehydrated, spraydried egg white at 121 C apparently eliminated both avidin and proteinase-inhibitor activity, autoclaving the egg white at 80 C produced less conclusive results. The growth data (Table 2) show that avidin was not inactivated at 80 C eimer in Experiment 2 or Experiment 4. The pancreas weights (Table 3), however, suggest that the proteinase inhibitors were inactivated in Experiment 2, but only partially so in Experiment 4. The inconsistency of these results may be the result of the fact that 80 C is the transition temperature for the heatdenaturation of the trypsin inhibitor (Lineweaver and Murray, 1947). Because denaturation temperature is affected by both pH and by heating time, the slight differences in egg-white autoclaving conditions between experiments may have altered the degree of proteinase inhibitor inactivation at 80 C. In an effort to simplify the procedures for processing egg white, the effects were compared of autoclaving spray-dried egg white at 121 C in the dry versus the rehydrated form. The growth of the chicks fed diets containing egg white autoclaved in the dry form was severely depressed, compared to that of chicks fed the autoclaved, rehydrated egg white (Experiment 3, Table 2). The extent of the
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*~cWithin an experiment, values in each column with no common superscripts are significantly different (P<.05). Mean pancreas weights at 28 days (Experiments 1 and 2) or at 26 days of age (Experiment 4).
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HEMPE AND SAVAGE
TABLE 4. Effect of graded levels of zinc from zinc carbonate on the growth and zinc concentrations in tissue of chicks fed diets based on autoclaved egg white1 Added zinc (mg per kg)
(g) 240 s 313 b 422 c 589 d 619 d 624 d 612 d 643 d 622 d
Zinc concentration Plasma (g per mL) .51 a .61 ab .63 ab JS1* .87°* 1.05d 1.42e 1.67f 1.75f
Tibia
Pancreas —
a
33.9 41.3 a 56.4 b 76.6° 118.6d 138.3e 168.2f 209.08 259.211
i„\
55.5" 59.2 a 62.4 ab 55.8 a 62.0 ab 66.8 ab 85.6b 120.0° 161.4d
a-b
Values within a column with no common superscripts are significantly different (P<05). Means of 10 male chicks per treatment Body weights were recorded and samples were collected at 21 days of age. The concentrations of zinc in (he tibia and pancreas are expressed on a dry-weight basis, and plasma zinc is based on volume. 1
growth depression was similar to that observed in chicks fed the diets with unheated egg white, suggesting that autoclaving spray-dried egg white in the dry form did not appreciably inactivate the intrinsic, antinutritional proteins. Parsons and Kelly (1933) reported that dried eggs took markedly more heating time to "detoxify" than rehydrated eggs. Kelly and Scott (1968) showed that autoclaving dry egg white at 121 C for more than 5 min markedly reduced chick growth. Because of its high sodium content, spraydried egg white has been reported as unsuitable for chick or turkey diets (Dewar and Siller, 1971). However, in the present experiment, reducing the content of sodium chloride in such diets by extracting the autoclaved egg white with deionized, distilled water did not affect chick growth (Experiment 3, Table 2). The amounts of sodium ions and of chloride ions in autoclaved egg white were markedly reduced by water extraction (from 1.4 to .27% and from 1.23 to .02%, respectively). In contrast, the zinc concentration of the egg white was unaffected. These data indicated that the concentration of sodium chloride in diets based on autoclaved egg white (approximately .4%) was not toxic; also, that water extraction was not beneficial. The combined growth data from Experiments 1 through 4 indicated that the growth of chicks fed egg white autoclaved at 121 C was significantly improved by supplementation with 5% gelatin (P<.05). The amino acid composition of the egg-white diet may have
been improved by the use of arginine, or glycine, or both, supplied by the gelatin. The growth of the chicks fed diets containing 5% gelatin and autoclaved egg white was equal to or greater than that of the chicks fed the casein-gelatin control diet (Table 2). The growth-promoting effect of gelatin was greatest when it was added to diets containing unheated, rather than autoclaved, egg white. Replacing part of the egg white with 5% gelatin reduced the concentrations of avidin and proteinase inhibitors in the diet by 20%, which may partly explain the greater response to gelatin using unheated egg white. The growth rate of the chicks fed a diet with autoclaved egg white similar to that described in Table 1 (except that it included 5% gelatin and .3 mg of biotin per kg of diet) was more than 30% greater than that previously reported for chicks fed egg white diets supplemented with adequate zinc (Davies and Motzok, 1971; Lease and Johnston, 1980; Dewar and Downie, 1984). The basal diets described by those investigators contained 1.3, 6, and 7 mg of zinc per kg of diet, respectively, more than the .9 mg of zinc per kg in the basal diets based on egg white used in the present experiments. Table 4 lists the body weights as well as the zinc concentrations in the plasma, tibia, and pancreas for chicks fed the diets containing rehydrated egg white autoclaved at 121 C, 5% gelatin, and graded concentrations of added zinc. In pilot studies, poor survival was observed with chicks fed less than 6 mg of supplemental
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6 8 10 12 14 16 18 20 50
Body weight
LOW-ZINC CHICK DIET
REFERENCES Cotterill, O. J., and J. L. Glauert, 1979. Nutrient values for shell, liquid/frozen, and dehydrated eggs derived by linear regression analysis and conversion factors. Poultry Sci. 58:131-134. Davies, M. I., and I. Motzok, 1971. Zinc deficiency in the chick: effect on tissue alkaline phosphatases. Comp.
Biochem. Physiol. 40B: 129-137. Dewar, W. A., and J. N. Downie, 1984. The zinc requirement of broiler chicks and turkey poults fed on purified diets. Br. J. Nutr. 51:467-477. Dewar, W. A., and W. G. Siller, 1971. Sodium toxicity resulting from feeding hen egg albumen powder to turkey poults. Br. Poult. Sci. 12:535-543. Eakin, R. E., E. E. SnelL and R. J. Williams, 1941. The concentration and assay of avidin, the injury-producing protein in raw egg white. J. Biol. Cbem. 140: 535-543. Forbes, R. M, D. E. Weingartner, H. M. Parker, R. R. Bell, and J. W. Erdman, Jr., 1979. Bioavailability to rats of zinc, magnesium and calcium in casein-, egg- and soy protein-containing diets. J. Nutr. 109:1652-1660. Gyorgy, P., C. S. Rose, R. E. Eakin, E. E. SneU, and R. J. Williams, 1941. Egg-white injury as the result of nonabsorption or inactivation of biotin. Science 93: 477-478. Jukes, T. H., and F. H. Bird, 1942. Prevention of perosis by biotin. Proc. Soc. Exp. Biol. Med. 49:231-232. Kelly, M., and H. M Scott, 1968. Autoclaving time in relation to the nutritional quality of dried egg white. Poultry Sci. 47:850-352. Kirschenmann, S. G., and B. O. Schneeman, 1982. Pancreatic enzymes, bile acids and cholesterol levels in mice fed raw or heated egg albumen. J. Food Sci. 47: 714-715, 719. Lease, J. G., and R. K. Johnston, 1980. A comparison of raw and cooked dried egg white as a source of protein for the chick. Poultry Sci. 59:1800-1806. Lineweaver, H., and C. W. Murray, 1947. Identification of the trypsin inhibitor of egg white with ovomucoid. J. Biol. Chem. 171:565-581. Luecke, R. W., R. E. Olman, and B. V. Baltzer, 1968. Zinc deficiency in the rat: effect on serum and intestinal alkaline phosphatase activities. J. Nutr. 94:344-350. National Research Council, 1984. Nutrient requirements of poultry. 8th ed. Natl. Acad. Sci., Washington, DC. Parsons, H. T., and E. Kelly, 1933. The character of the dermatitis-producing factor in dietary egg white as shown by certain chemical treatments. J. Biol. Chem. 100:645-652. Rhodes, M. B., N. Bennett, and R. E. Feeney, 1960. The trypsin and chymotrypsin inhibitors from avian egg whites. J. Biol. Chem. 235:1686-1693. Ringrose, A. T., L. C. Norris, and G. F. Heuser, 1931. The occurrence of a pellagra-like syndrome in chicks. Poultry Sci. 10:166-177. SAS institute, 1982. SAS® User's Guide: Statistics. SAS Inst. Inc., Cary, NC. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. 7th ed. Iowa State University Press, Ames, IA.
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zinc per kg of diet. The data indicated that the zinc requirement for growth in chicks fed the diet based on autoclaved egg white was approximately 14 mg per kg. Unlike growth, the plasma zinc concentration after fasting did not exhibit a plateau response until the dietary zinc concentration exceeded 20 mg per kg. The zinc concentration in the tibia was significantly different at nearly every dietary concentration of zinc and did not reach a clearly defined plateau within the range of dietary zinc used in the current experiment. However, the zinc concentration in the pancreas was unaffected at low concentrations of dietary zinc, but increased markedly after the amount required for growth was surpassed. These data demonstrate that commercially available, spray-dried egg white is not a suitable source of protein for semipurified chick diets. The detrimental effects observed when using spray-dried egg white as the major protein source can be eliminated by first rehydrating and then autoclaving the egg white for 30 min at 121 C. In the present study, the growth rate and zinc concentrations in the tissue of chicks fed diets containing autoclaved egg white, 5% supplemental gelatin, and graded concentrations of zinc clearly indicated that egg white prepared by this procedure is an excellent source of protein for use in purified, low-zinc diets. Since spray-dried egg white is also relatively or exceptionally low in calcium, phosphorus, copper, manganese, iron, lipids, and fat-soluble vitamins (Cotterill and Glauert, 1979), autoclaved egg white may also be useful in studies involving mose nutrients.
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1990 Poultry Science 69:959-965
inhibitors adversely affect pancreatic function and cause both pancreatic hypertrophy and a Spray-dried egg white contains approxi- reduced activity of proteolytic enzymes in mately 80% protein and less than 1 mg of zinc mice (Kirschenmann and Schneeman, 1982). per kg. Based on its amino acid composition, The purpose of the present experiments was egg white would appear to be an excellent to develop a low-zinc diet based on egg white source of protein for use in low-zinc diets for chicks. However, egg white also contains for the chick that would be nutritionally avidin, a heat-labile protein that complexes adequate when supplemented with zinc. The with and decreases the absorption of biotin experimental design included an assessment of the effects of avidin and of egg-white protein(Eakin et al, 1941; Gyorgy et al., 1941). Spray-dried egg white was a satisfactory ase inhibitors on the chick as well as the source of protein in low-zinc diets for rats development of procedures to eliminate such when the diets were supplemented with extra effects. biotin in order to overcome the adverse effects of avidin (Luecke et al., 1968; Forbes et al., MATERIALS AND METHODS 1979). Supplemental biotin improved growth Sexed, broiler-strain chicks (Hubbard by and reduced the incidence of perosis in chicks fed egg-white diets (Jukes and Bird, 1942). Hubbard cross) were obtained from a commerBut a diet for chicks based on egg white cial hatchery on the day they were hatched. that is nutritionally adequate except for zinc The chicks were divided into equal weight has not been demonstrated. One reason may be groups of 10 each. The groups were housed in that raw egg white also contains proteinase stainless-steel, electrically-heated battery inhibitors that deactivate trypsin and chymo- brooders; the rooms were temperature-contrypsin in vitro (Rhodes et al., 1960). These trolled (24 C) with continuous light. Feed and distilled water were supplied for ad libitum consumption in stainless-steel or glass containers for the duration of each experiment. Body 'Contribution from the Missouri Agricultural Experiweights were recorded weekly. ment Station, Journal Series Number 10344. INTRODUCTION
959
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ABSTRACT Spray-dried egg white was evaluated either as the sole source or the primary source of protein in purified, low-zinc diets for chicks. The effects of unheated and autoclaved egg white were compared for chicks fed diets supplemented with graded amounts of biotin. Growth rate and pancreas weight were used as the criteria of nutritional adequacy. The data indicated that extra biotin did not completely overcome the adverse effects of unheated egg white. Chicks fed unheated egg white and excess biotin had decreased body weights and showed signs of pancreatic hypertrophy, probably due to proteinase inhibitors in the egg white. Autoclaving rehydrated and spray-dried egg white for 30 rain at 121C, but not at 80 C, markedly reduced the biotin-binding and proteinase-inhibitor activities of unheated egg white. Extracting autoclaved egg white with water did not improve the growth rate or reduce the amount of zinc in the egg white. Chicks fed diets of autoclaved egg white and gelatin with low concentrations of zinc showed symptoms of severe zinc deficiency. Chicks fed diets with adequate concentrations of zinc had body weights similar to those fed casein-gelatin control diets. When graded amounts of zinc were added to the diet, the growth rate of the chicks and the tissue concentration of zinc reflected zinc intake. These data demonstrated that rehydrated, spray-dried egg white autoclaved for 30 min at 121 C is an excellent source of protein for use in low-zinc diets for chicks. (Key words: zinc, purified diet, egg white, avidin, trypsin inhibitor)
960
HEMPE AND SAVAGE TABLE 1. Composition of the diets
Ingredient
Egg white, basal
Casein-gelatin, control
(%) 32.4
51.9 6.0 5.5 1.0 2.0 1.0 .2
24.4 5.0 .9 .3 54.3 4.8 5.5 .3 1.0 2.0 1.0 .3 .2
Autoclaved or unheated. Composition (grams per kilogram of premix): CaHP04-2H20, 581.6; Ca3(P04)2, 116.9; CaC0 3 , 58.523; K2C(>31-1/2H20,168.7; MgS0 4 ,58.4; Ferric citrate, 9.4; MnS0 4 H 2 0, 6.0; KK>3, .23; CuS0 4 , .24; and Na 2 Se0 3 -5H 2 0, .0065. Supplied (grams per kilogram of diet): Ca, 11.3; P, 7.0, and K, 4.4. Supplied (milligrams per kilogram of diet): Mg, 649; Fe, 86; Mn, 107; I, 7.5; Cu, 5.3; and Se, .11. 3 Solka Floe, James River Corporation, Berlin, NH. ''in glucose carrier, supplied (per kilogram of diet): retinyl palmitate, 20,000 IU; cholecalciferoL 2,000 ICU; and DLtocopheryl acetate, 20 IU. Supplied (milligrams per kilogram of diet): menadione sodium bisulfite, 2; ethoxyquin, 125; thiamine HCl, 10; riboflavin, 10; pyndoxine HCl, 15; D-calcium pantothenate, 30; niacin, 75; foiacin, 2; and cyanocobalamin, .03. Reagent-grade zinc carbonate in a glucose carrier, supplied 50 mg of zinc per kilogram of diet ^D-biotin in a glucose carrier; supplied .3 mg of biotin per kg of diet
Diets The ingredient compositions for the eggwhite, basal diet and the casein-based control diet are shown in Table 1. Without added zinc, the diet based on egg white was analyzed to contain .9 mg of zinc per kg of diet. This formulation was followed in all five experiments with minor adjustments in the ratios of egg white, glucose, and soybean oil. When gelatin was added to the basal diet, the amounts of egg white, glucose, and soybean oil were adjusted in order to keep the dietary amounts of crude protein (26%) and of energy (3,400 kcal per kg of diet) approximately the same. The chicks were fed the casein diet as a positive control in Experiments 1, 2, and 3. Biotin was omitted from the vitamin premix in Experiment 1,2, and 4 to study the effects of the autoclaving temperature on avidin activity.
2
Milton G. Waldbaum Co., Wakefield, NE. Amsco model 2053, American Sterilizer Co., Erie, PA.
3
Supplements of D-biotin were added to those diets to supply .3,1.5, or 3.0 mg of biotin per kg of diet, representing 2, 10, and 20 times the biotin requirement (National Research Council, 1984). Pilot studies indicated that feather growth was abnormal in chicks fed diets containing unheated, spray-dried egg white. This prompted a study of the value of gelatin as an amino acid supplement. The effects were also determined of autoclaving dry egg white versus rehydrated egg white and of extracting autoclaved egg white with deionized, distilled water. Stainless-steel or plastic equipment was used for all processing procedures involving egg white. Autoclaved, rehydrated egg white was prepared by mixing 10 kg of spray-dried egg white2 with 12 L of deionized, distilled water. The mixture was poured into flat pans to a depth of approximately 5 cm and was autoclaved in a laboratory sterilizer3 at either 80 C or 121 C. After autoclaving, the hardened cake was cut into strips approximately 5 cm wide and then ground in a food-chopper.
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Spray-dried egg white Casein Gelatin L-arginine (free-base) DL-methionine Glucose monohydrate Soybean oil Mineral premix2 Sodium chloride Cellulose3 Vitamin premix4 Zinc premix5 Biotin premix6 Choline chloride
LOW-ZINC CHICK DIET
Experimental Design
diets containing: 1) rehydrated egg white autoclaved at 121 C, which was either extracted or not extracted with deionized, distilled water, and 2) the same diet witii and without a 5% gelatin supplement. The fifth group was fed a diet for which the sole source of protein was spray-dried egg white autoclaved at 121 C in the dry form. Experiment 4. Duplicate groups of 10 male chicks were assigned to 14 dietary treatments. In twelve of me treatments, the growm rates and pancreas weights were compared for chicks fed diets containing: 1) unheated egg white, rehydrated egg white autoclaved at 80 C, or rehydrated egg white autoclaved at 121 C; 2) the same diets with a supplement of .3 mg or of 1.5 mg of biotin per kg of diet; and 3) the same diets witii and without a 5% gelatin supplement. Two additional groups were fed diets containing unheated egg white and 3.0 mg of biotin per kg of diet, eiuier with or without a supplement of 5% gelatin. Final body weights were recorded at 21 days of age. The chicks used to determine the pancreas response were reweighed on Day 26, when the pancreases were removed and weighed. Experiments. Groups of 10 male chicks were assigned to nine dietary treatments. All diets contained 5% gelatin and rehydrated egg white autoclaved at 121 C. These diets were similar to the one described in Table 1, except that die zinc premix contained graded concentrations of zinc (6, 8, 10,12, 14, 16, 18, 20, or 50 mg per kg), supplied as zinc carbonate. All diets were supplemented with .3 mg of biotin per kg of diet.
In addition to body weight, comparisons were made of pancreas weight and pancreas weight as a percentage of body weight in Experiments 1,2, and 4. These data were used as indicators of pancreatic hypertrophy to assess the effects of egg-white proteinase inhibitors on pancreatic function. To avoid confounding the effects of the proteinase inhibitors with those of avidin, pancreas data were collected only from chicks fed egg-white diets supplemented with 1.5 or 3.0 mg of biotin per kg of diet. Final body weights were determined at either 21 or 28 days of age. Bioassays at 3 wk and at 4 wk were observed to be statistically similar. Experiment 1. Duplicate groups of 10 male chicks were assigned to eight dietary treatments. The chicks were fed diets containing: 1) unheated egg white or rehydrated egg white autoclaved at 121 C; 2) the same diet with a Analytical Procedures supplement of .3 or 1.5 mg of biotin per kg of Samples of the diet, tibia, and pancreas were diet; 3) the same diet with and without a dried overnight in a vacuum oven at 60 C. The supplement of 5% gelatin. Experiment 2. Duplicate groups of 10 female samples were weighed and were dry-ashed for chicks were assigned to five dietary treatments. 18 h at 500 C. All ash residues were heated with In four of the treatments, comparisons were nitric acid on a hot plate and then were reashed at made of the growth rates and the pancreas the same temperature. Following the second weights for chicks fed diets containing 1.5 mg of ashing, the residues were dissolved in dilute biotin per kg of diet and: 1) with rehydrated egg HCl; the zinc concentration was determined by white autoclaved at 80 C or at 121 C; 2) with and flame atomic-absorption spectrophotometry. without a 5% gelatin supplement. The fifth For plasma zinc analyses, whole blood was group was fed a diet containing rehydrated egg collected by cardiac puncture following an white autoclaved at 80 C and supplemented with overnight fast. The blood was placed in heparinized tubes and was centrifuged at 2,000 x g for .3 mg of biotin per kg of diet. Experiment3. Groups of 10 male chicks were 10 min. One mL of plasma was diluted with 4 assigned to five dietary treatments. Four treat- mL of 1% HCl in deionized, distilled water, and ments compared the growth rates of chicks fed then analyzed as above.
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Water-extracted egg white was prepared by soaking the ground, autoclaved egg white in deionized, distilled water for 10 min and then siphoning die supernate. This procedure was repeated three times. Because extraction removed most of the sodium chloride, diets containing water-extracted egg white were supplemented with .3% of sodium chloride. Following grinding (or grinding and extraction), me autoclaved egg white was dried for 24 h in a forced-air oven at 60 C. Then, die egg white was ground in a hammer mill and was stored at room temperature. A similar procedure was used to make spray-dried egg white, autoclaved in the dry form except that the egg white was not rehydrated prior to autoclaving.
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TABLE 2. Effect of autociaving procedure, dietary biotin concentration, and gelatin supplementation on the growth rate of chicks fed egg-white diets1 Autociaving procedure2 Protein source
Temperature (Q
Rehydrated Extracted
Gelatin (%)
Added biotin
Experiment 1 Spray-dried egg white 121
Yes
(c\ \ti)
1.5 .3 1.5 .3 .3
730"b 696 bc 572 d 285 f
(16) (18) (11) (9)
751" 743*b 676° 359" 692 bc
(18) (18) (16) ( 8) (20)
Casein Experiment 3 Spray-dried egg white 121 121 121 Casein
Yes Yes
Yes Yes
1.5 1.5 .3 .3
761 b 772"b 319°
(14) (17) (8)
794ab
814*
(12) (13)
803"
(12)
Yes Yes No
1.5 1.5 1.5 .3
928 b 943b 204°
998"b 1,009"
( 9) (10)
o-7-yab
(9)
1.5 .3 1.5 .3 3.0 1.5 .3
618" 523 b 414 d 242 ef 428 cd 275 e 21 l f
625" 625" 601" 257** 516 b 473 bc 261 ef
(15) (15) (13) (14) (19) (20) (13)
Yes No No
Experiment 4 Spray-dried egg white 121
Yes
No
80
Yes
No
No
(9) (8) (6)
(15) (14) (12) (12) (20) (18) (18)
Within an experiment, values with no common superscripts are significantly different (P<.05). Mean body weights at 28 days (Experiments 1 to 3) or at 21 days of age (Experiment 4). Male chicks were used in Experiments 1,3, and 4; female chicks were used in Experiment 2. Ten chicks (Experiment 3) or 20 chicks (Experiments 1,2, and 4) were assigned to each dietary treatment group. The numbers in parentheses represent the number of chicks remaining at the end of the experiment 2 When rehydrated, egg white was mixed at a ratio of 1:1.2 with deionized, distilled water before autociaving. The autociaving time was 30 rain. Some of the autoclaved egg white was extracted with deionized, distilled water.
Statistical Analyses Two-way (Experiments 1, 2, and 4) or oneway (Experiments 3 and 5) analyses of variances were performed using the general linear models procedure of the Statistical Analysis System (SAS Institute, 1982). Differences between the means for dietary treatments concerning body weights, pancreas weights, and zinc concentrations in tissue were tested by Fisher's Least Significant Differences (LSD) method (Snedecor and Cochran, 1980). RESULTS AND DISCUSSION
Classic symptoms of severe biotin deficiency were observed in the chicks fed diets
containing unheated egg white and 2 times the biotin requirement (.3 mg per kg). These symptoms, collectively described as "egg white injury" or as "egg white syndrome" (Ringrose et al., 1931; Eakin et al., 1941; Gyorgy et al., 1941; Jukes and Bird, 1942) included reduced growth (Table 2), increased mortality, perosis, and dermatitis. The growth rate was significantly increased and the deficiency symptoms were reduced in chicks fed the unheated egg white plus 1.5 or 3.0 mg of biotin per kg of diet. Excess biotin did not enhance the growth rate for chicks fed rehydrated egg white autoclaved at 121 C (Table 2). A slight growth depression was observed in only one of the groups fed diets containing .3 mg of biotin per
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Yes
No Casein Experiment 2 Spray-dried egg white 121 80
5
0
(mg/kg)
LOW-ZINC CHICK DIET
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TABLE 3. Effect of autoclaving on pancreas size in chicks fed diets based on egg white1
Experiment 1 2 4
Autoclaved egg white
Autoclaving temperature
Added biotin
Pancreas weight
Pancreas weight in proportion to body weight
Yes No Yes Yes Yes Yes No
(Q 121 ... 121 80 121 80 ...
(mgperkg) 1.5 1.5 1.5 1.5 1.5 1.5 3.0 L5
(g) 2.00* 1.99* 1.90* 1.91* 1.78b 2.08b 252* 2.22b
(%) .266 b .339* .239* .249* .202c .262b .324* .355*^
kg of diet (Experiment 4). In contrast, the growtii of chicks fed diets containing rehydrated egg white autoclaved at 80 C was markedly affected by the concentration of dietary biotin. These data indicated that avidin in raw, spray-dried egg white was inactivated when the egg white was rehydrated and was autoclaved for 30 min at 121 C, but not at 80 C. Despite the beneficial effect of the added biotin in the diets with unheated egg white, the growth rate of chicks fed those diets was less than that of those fed diets containing egg white autoclaved at 121 C (Table 2). In all likelihood, this reduced growth rate cannot be attributed to avidin, since the diets supplemented with 3.0 mg of biotin per kg of diet (20 times the requirement) contained enough biotin to exceed the predicted binding capacity of the avidin in the egg white. Raw egg white also contains other heatlabile, antinutritional proteins mat may adversely affect chick growth. Proteinase inhibitors in egg white reduce in vitro proteolysis by trypsin and chymotrypsin (Rhodes et al., 1960). Kirschenmann and Schneeman (1982) observed pancreatic hypertrophy and an increased synthesis of pancreatic enzymes in mice fed unheated egg white. In the present experiment, the pancreas weights of chicks fed the unheated egg white were equal to or significantly greater than those for the chicks fed diets containing egg white autoclaved at 121 C (Table 3), despite the marked difference in body weights (Table 2). When expressed as a percentage of body weight, the pancreas weights of die chicks fed
the diets with unheated egg white were significantly greater than those of the chicks fed autoclaved egg white. The greater relative size of the pancreas in these chicks suggests that the proteinase inhibitors in the diets with unheated egg white induced pancreatic hypertrophy and were responsible for the depressed growth. Although autoclaving the rehydrated, spraydried egg white at 121 C apparently eliminated both avidin and proteinase-inhibitor activity, autoclaving the egg white at 80 C produced less conclusive results. The growth data (Table 2) show that avidin was not inactivated at 80 C eimer in Experiment 2 or Experiment 4. The pancreas weights (Table 3), however, suggest that the proteinase inhibitors were inactivated in Experiment 2, but only partially so in Experiment 4. The inconsistency of these results may be the result of the fact that 80 C is the transition temperature for the heatdenaturation of the trypsin inhibitor (Lineweaver and Murray, 1947). Because denaturation temperature is affected by both pH and by heating time, the slight differences in egg-white autoclaving conditions between experiments may have altered the degree of proteinase inhibitor inactivation at 80 C. In an effort to simplify the procedures for processing egg white, the effects were compared of autoclaving spray-dried egg white at 121 C in the dry versus the rehydrated form. The growth of the chicks fed diets containing egg white autoclaved in the dry form was severely depressed, compared to that of chicks fed the autoclaved, rehydrated egg white (Experiment 3, Table 2). The extent of the
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*~cWithin an experiment, values in each column with no common superscripts are significantly different (P<.05). Mean pancreas weights at 28 days (Experiments 1 and 2) or at 26 days of age (Experiment 4).
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HEMPE AND SAVAGE
TABLE 4. Effect of graded levels of zinc from zinc carbonate on the growth and zinc concentrations in tissue of chicks fed diets based on autoclaved egg white1 Added zinc (mg per kg)
(g) 240 s 313 b 422 c 589 d 619 d 624 d 612 d 643 d 622 d
Zinc concentration Plasma (g per mL) .51 a .61 ab .63 ab JS1* .87°* 1.05d 1.42e 1.67f 1.75f
Tibia
Pancreas —
a
33.9 41.3 a 56.4 b 76.6° 118.6d 138.3e 168.2f 209.08 259.211
i„\
55.5" 59.2 a 62.4 ab 55.8 a 62.0 ab 66.8 ab 85.6b 120.0° 161.4d
a-b
Values within a column with no common superscripts are significantly different (P<05). Means of 10 male chicks per treatment Body weights were recorded and samples were collected at 21 days of age. The concentrations of zinc in (he tibia and pancreas are expressed on a dry-weight basis, and plasma zinc is based on volume. 1
growth depression was similar to that observed in chicks fed the diets with unheated egg white, suggesting that autoclaving spray-dried egg white in the dry form did not appreciably inactivate the intrinsic, antinutritional proteins. Parsons and Kelly (1933) reported that dried eggs took markedly more heating time to "detoxify" than rehydrated eggs. Kelly and Scott (1968) showed that autoclaving dry egg white at 121 C for more than 5 min markedly reduced chick growth. Because of its high sodium content, spraydried egg white has been reported as unsuitable for chick or turkey diets (Dewar and Siller, 1971). However, in the present experiment, reducing the content of sodium chloride in such diets by extracting the autoclaved egg white with deionized, distilled water did not affect chick growth (Experiment 3, Table 2). The amounts of sodium ions and of chloride ions in autoclaved egg white were markedly reduced by water extraction (from 1.4 to .27% and from 1.23 to .02%, respectively). In contrast, the zinc concentration of the egg white was unaffected. These data indicated that the concentration of sodium chloride in diets based on autoclaved egg white (approximately .4%) was not toxic; also, that water extraction was not beneficial. The combined growth data from Experiments 1 through 4 indicated that the growth of chicks fed egg white autoclaved at 121 C was significantly improved by supplementation with 5% gelatin (P<.05). The amino acid composition of the egg-white diet may have
been improved by the use of arginine, or glycine, or both, supplied by the gelatin. The growth of the chicks fed diets containing 5% gelatin and autoclaved egg white was equal to or greater than that of the chicks fed the casein-gelatin control diet (Table 2). The growth-promoting effect of gelatin was greatest when it was added to diets containing unheated, rather than autoclaved, egg white. Replacing part of the egg white with 5% gelatin reduced the concentrations of avidin and proteinase inhibitors in the diet by 20%, which may partly explain the greater response to gelatin using unheated egg white. The growth rate of the chicks fed a diet with autoclaved egg white similar to that described in Table 1 (except that it included 5% gelatin and .3 mg of biotin per kg of diet) was more than 30% greater than that previously reported for chicks fed egg white diets supplemented with adequate zinc (Davies and Motzok, 1971; Lease and Johnston, 1980; Dewar and Downie, 1984). The basal diets described by those investigators contained 1.3, 6, and 7 mg of zinc per kg of diet, respectively, more than the .9 mg of zinc per kg in the basal diets based on egg white used in the present experiments. Table 4 lists the body weights as well as the zinc concentrations in the plasma, tibia, and pancreas for chicks fed the diets containing rehydrated egg white autoclaved at 121 C, 5% gelatin, and graded concentrations of added zinc. In pilot studies, poor survival was observed with chicks fed less than 6 mg of supplemental
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6 8 10 12 14 16 18 20 50
Body weight
LOW-ZINC CHICK DIET
REFERENCES Cotterill, O. J., and J. L. Glauert, 1979. Nutrient values for shell, liquid/frozen, and dehydrated eggs derived by linear regression analysis and conversion factors. Poultry Sci. 58:131-134. Davies, M. I., and I. Motzok, 1971. Zinc deficiency in the chick: effect on tissue alkaline phosphatases. Comp.
Biochem. Physiol. 40B: 129-137. Dewar, W. A., and J. N. Downie, 1984. The zinc requirement of broiler chicks and turkey poults fed on purified diets. Br. J. Nutr. 51:467-477. Dewar, W. A., and W. G. Siller, 1971. Sodium toxicity resulting from feeding hen egg albumen powder to turkey poults. Br. Poult. Sci. 12:535-543. Eakin, R. E., E. E. SnelL and R. J. Williams, 1941. The concentration and assay of avidin, the injury-producing protein in raw egg white. J. Biol. Cbem. 140: 535-543. Forbes, R. M, D. E. Weingartner, H. M. Parker, R. R. Bell, and J. W. Erdman, Jr., 1979. Bioavailability to rats of zinc, magnesium and calcium in casein-, egg- and soy protein-containing diets. J. Nutr. 109:1652-1660. Gyorgy, P., C. S. Rose, R. E. Eakin, E. E. SneU, and R. J. Williams, 1941. Egg-white injury as the result of nonabsorption or inactivation of biotin. Science 93: 477-478. Jukes, T. H., and F. H. Bird, 1942. Prevention of perosis by biotin. Proc. Soc. Exp. Biol. Med. 49:231-232. Kelly, M., and H. M Scott, 1968. Autoclaving time in relation to the nutritional quality of dried egg white. Poultry Sci. 47:850-352. Kirschenmann, S. G., and B. O. Schneeman, 1982. Pancreatic enzymes, bile acids and cholesterol levels in mice fed raw or heated egg albumen. J. Food Sci. 47: 714-715, 719. Lease, J. G., and R. K. Johnston, 1980. A comparison of raw and cooked dried egg white as a source of protein for the chick. Poultry Sci. 59:1800-1806. Lineweaver, H., and C. W. Murray, 1947. Identification of the trypsin inhibitor of egg white with ovomucoid. J. Biol. Chem. 171:565-581. Luecke, R. W., R. E. Olman, and B. V. Baltzer, 1968. Zinc deficiency in the rat: effect on serum and intestinal alkaline phosphatase activities. J. Nutr. 94:344-350. National Research Council, 1984. Nutrient requirements of poultry. 8th ed. Natl. Acad. Sci., Washington, DC. Parsons, H. T., and E. Kelly, 1933. The character of the dermatitis-producing factor in dietary egg white as shown by certain chemical treatments. J. Biol. Chem. 100:645-652. Rhodes, M. B., N. Bennett, and R. E. Feeney, 1960. The trypsin and chymotrypsin inhibitors from avian egg whites. J. Biol. Chem. 235:1686-1693. Ringrose, A. T., L. C. Norris, and G. F. Heuser, 1931. The occurrence of a pellagra-like syndrome in chicks. Poultry Sci. 10:166-177. SAS institute, 1982. SAS® User's Guide: Statistics. SAS Inst. Inc., Cary, NC. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. 7th ed. Iowa State University Press, Ames, IA.
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zinc per kg of diet. The data indicated that the zinc requirement for growth in chicks fed the diet based on autoclaved egg white was approximately 14 mg per kg. Unlike growth, the plasma zinc concentration after fasting did not exhibit a plateau response until the dietary zinc concentration exceeded 20 mg per kg. The zinc concentration in the tibia was significantly different at nearly every dietary concentration of zinc and did not reach a clearly defined plateau within the range of dietary zinc used in the current experiment. However, the zinc concentration in the pancreas was unaffected at low concentrations of dietary zinc, but increased markedly after the amount required for growth was surpassed. These data demonstrate that commercially available, spray-dried egg white is not a suitable source of protein for semipurified chick diets. The detrimental effects observed when using spray-dried egg white as the major protein source can be eliminated by first rehydrating and then autoclaving the egg white for 30 min at 121 C. In the present study, the growth rate and zinc concentrations in the tissue of chicks fed diets containing autoclaved egg white, 5% supplemental gelatin, and graded concentrations of zinc clearly indicated that egg white prepared by this procedure is an excellent source of protein for use in purified, low-zinc diets. Since spray-dried egg white is also relatively or exceptionally low in calcium, phosphorus, copper, manganese, iron, lipids, and fat-soluble vitamins (Cotterill and Glauert, 1979), autoclaved egg white may also be useful in studies involving mose nutrients.
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