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Aleurone Cells and the Digestibility of Wheat Mill Feeds1
Low
PROTEIN LAYER DIETS
egg weight was also shown when feed conversion was calculated. An average requirement for feed per dozen eggs was 1.88 kg. for the S.C.W.L. and 1.83 for the hybrids. When the data were given in terms of feed per 100 gm. of egg, the S.C.W.L. were less efficient in utilization of total feed or protein by approximately 30%. The basis for a difference in feed intake appeared to be due to body size as the S.C.W.L. were 250 gm. larger than the hybrids at the end of the trial.
REFERENCES Bray, D. J., 1960. Studies with corn-soya laying
diets. 2. Optimum combinations of corn and soybean protein. Poultry Sci. 43: 396-401. Britzman, D. C , and C. W. Carlson, 1965. Limiting amino acids in corn-soybean meal diets for laying hens. Feedstuffs, 37: (43) 20-21. Carlson, C. W., and E. Guenthner, 1969. Response of laying hens fed typical corn-soy diets to supplements of methionine and lysine. Poultry Sci. 48: 137-143. Chavez, R., J. M. Thomas and B. L. Reid, 1966. The utilization of non-protein nitrogen by laying hens. Poultry Sci. 45: 547-553. Johnson, D. Jr., and H. Fisher, 1958. The amino acid requirement of laying hens. 3. Minimal requirement levels of amino acids: techniques and development of diet. Brit. J. Nutr. 12: 276-285. Merck and Co., 1961. Amino Acid Values in Feedstuffs. Rahway, N. J. Steel, G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Inc., New York. Young, R. J., M. L. Griffith, I. D. Desai and M. L. Scott, 1965. The response of laying hens fed low protein diets to glutamic acid and diammonium citrate. Poultry Sci. 34: 1428.
Aleurone Cells and the Digestibility of Wheat Mill Feeds1 R. M. SAUNDERS, H. G. WALKER, JR. AND G. O. KOHLER Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710 (Received for publication April 8, 1969)
P
ELLETING feeds is usually advantageous for several reasons. Where improved growth has been shown, however, workers do not agree on the factor(s) responsible for the improvement. The relative value of pellets versus mash and grain in poultry nutrition has been the subject of a review by Calet (1965) where current speculations to account for the observed differences have been listed. I t appears certain that chemical and/or physical 1 Part of this work was presented at the 54th Annual Meeting of the American Association of Cereal Chemists, Chicago, Illinois, April 1969.
changes must have occurred during the pelleting process to account for the large differences found. Recently Cave et al. (1965a, b) found that wheat milling fractions gave relatively low metabolizable energy values and poor protein utilization when they constituted an important part of chick diets. By steam-pelleting wheat bran, the metabolizable energy in chick diets was increased as much as 30% over unpelleted mash. An increase of 17% was found for steam-pelleted wheat shorts over unpelleted material. The present paper re-
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
ACKNOWLEDGEMENTS
The authors thank Merck & Co., Railway, N.J. for the L-Lysine HC1 used and for a Grant-in-aid that partially supported this work. Our thanks are also given to the Dow Chemical Co., Midland Mich, for the D-L methionine.
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TABLE 1.—Proximate analysis oj the Canadian bran, American bran, and shorts used in chick feeding experiments
Canadian bran American bran Shorts
8.60 9.48 13.57
2.79 2.94 3.15
4.00 4.62 6.45
8.36 8.23 8.45
5.94 5.80 4.74
MATERIALS AND METHODS*
A Canadian hard red spring wheat bran in both mash and steam-pelleted form was obtained from Dr. J.D. Summers of the University of Guelph, Ontario, Canada. An American hard red spring wheat bran was steam pelleted in this laboratory in a Lab model California Pellet Mill. The die was 1-^ inch thick with J-inch diameter holes. The moisture level was 16%. Wheat shorts, supplied by International Milling Company, was from a North Dakota #2 grade hard red spring wheat and was reground three times in a Brabendor Senior Quadrumat Mill. Proximate analysis of the two brans and shorts are shown in Table 1. "Grits" refers to small sand grit from the Monterey, California area, water washed, kiln dried and screened (10-20 mesh). It was included in the diet at 2% level. In the experiments where grit was added to the bran ration, grit had also been included in the commercial ration for a period of 48 hours prior to removal of the commercial feed. Autoclaving was carried out at 15 p.s.i.g. for 20 minutes. * Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.
Amino acids were determined on acid hydrolysates (6 N HC1 for 24 hours in tubes sealed under vacuum) by ion-exchange chromatography with a Phoenix amino acid analyzer (Kohler and Palter, 1967). RESULTS AND DISCUSSION
After feeding bran to chicks, microscopic examination of the contents of the lower intestine and feces showed a number of intact cells from the aleurone layer had not been digested. Empty aleurone cells were easily distinguished from full, undigested cells. The latter had a brownish granular appearance and usually con-
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
ports findings which explain why the biological availability of wheat bran nutrients is increased by the pelleting procedure. A preliminary report of this work has appeared (Saunders et al., 1968).
Bran rations were fed to chicks in the following manner: Eight-week-old New Hampshire chicks in groups of three, previously maintained on a commercial diet, were starved for 24 hours and then allowed access ad libitum to the wetted bran product as the sole feed. After 24 hours the chicks were killed and the lower small intestinal contents (Laws and Moore, 1963) were removed and retained. Fecal samples were collected from the cage. Intestinal contents and fecal droppings were washed by shaking about 1 g. of the material with 50 ml. of water and filtering through fine nylon mesh (0.007inch diameter holes). The solids left in the nylon mesh were thoroughly washed with running water. Microscopic examination and photography were carried out at this stage on a portion of the solids. The remainder was dried over KOH in a desiccator and then analyzed for nitrogen (Kjeldahl). Aleurone cells were counted at a magnification of 210. In one series of experiments fecal droppings only were collected and analyzed for nitrogen content. Eight groups of chicks in groups of three were fed each ration. The fecal sample was a composite sample from the three birds.
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A L E U R O N E DIGESTIBILITY i.
*?F5L '-~>*> „W
tained small globules. T h e granular material could be stained with protein stains and the globules with fat stains. Figure 1 illustrates a section of aleurone containing both full and empty cells. Figure 2 shows the full cells a t higher magnification. T h e globules are clearly evident. Tables 2 and 3 summarize the results of a number of feeding experiments. I n Table 2 the first column gives the percentage of empty aleurone cells (i.e., contents digested) of the total cell count for both feces and intestinal contents after different feeds or treatments. T h e percentages were determined by counting not less than 1500 aleurone cells in each sample. T h e values for intestinal contents are averaged from separate determinations on three individual birds; fecal values are from a composite sample from the same three birds. In all cases the percent of e m p t y cells is greater in the pel-
leted diets than in the mash diets, a difference t h a t was statistically significant at the 9 5 % confidence level. The table also lists the protein content of the same samples of washed intestinal and fecal solids. T h e residual protein level (nonutilized b y the animal) decreased as the number of empty cells increased, and lowest residual protein was always associated with pelleted feeds. When autoclaved wheat bran mash was used as feed, the empty aleurone cell counts and residual protein levels were about the same as those of untreated mash. Table 3 shows a statistical analysis when similar feeds were fed to eight groups of three birds. This series of experiments was carried out about 9 months after the experiments reported under Table 2. T h e American pellets were a different batch. Slight differences in digestibility of the bran are apparent b u t
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
FIG. 1. Aleurone cells (X42) present in feces after feeding steam-pelleted wheat bran to chicks. Dark cells, non-utilized material; white cells, empty (i.e., contents utilized).
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R. M. SAUNDERS, H. G. W A L K E R , J R . AND G. 0 . K O H I . E R
the relative protein digestibilities follow the same trend. Protein in pelleted bran feeds was utilized better than in nonpelleted bran. T h e differences in residual protein were significant at the 9 5 % confidence level. In this experiment, autoclaved mash actually showed a statistically significant decrease in protein utilization when compared to the regular mash. This was probably due to Maillard reactions leading to undigestible protein fragments.
where breakages occur, the contents have been utilized. This was also the case where aleurone appeared to be dented or crinkled. Since autoclaving itself causes no inincrease in aleurone cell utilization, the major factor leading to improved digestibility must be associated with the pelleting step. Steam treatment may help soften cell walls prior to pelleting, and it m a y be t h a t it is the shear and compression forces
Figure 3 shows a magnified section of aleurone tissue isolated from feces from a chick fed steam-pelleted bran. I t is clear t h a t some cell walls were broken as a result of the physical stress of pelleting. None of the walls of the intact cells appear to have been damaged, b u t all the empty cells show evidence of breakage. I t is evident t h a t rupturing the cell walls has given digestive enzymes greater access to cellular contents since, without exception,
TABLE 2.—Number of empty aleurone cells1 and residual protein2 in wheat bran feeds after digestion by chicks
Feed Canadian mash Canadian pellets American mash American pellets American mash, autoclaved 1 2 3
E m p t y aleurone cells, %
Protein content, 3 %
Intestine
Feces
Intestine
Feces
28.8 43.2 15.0 35.6 18.3
32.8 50.2 21.4 40.0 22.6
7.47 6.61 8.41 6.73 8.15
7.12 6.27 8.38 6.38 7.92
Average from three chicks. Pooled sample same three chicks. N X5.7. Protein content of washed intestinal and fecal solids.
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FIG. 2. Aleurone cells (X105) present in feces after feeding steam-pelleted wheat bran to chicks.
ALEURONE DIGESTIBILITY
TABLE 3.—Residual protein in wheal bran and shorts feeds after digestion by chicks
Feed Canadian Canadian American American American American
F
mash pellets mash pellets mash, autoclaved mash+grits
Shorts Shorts milled 3X
n Standard St£? en ' deviation /o 7.75 7.01 8.27 7.41 8.72 7.81
0.13 0.18 0.14 0.29 0.43 0.21
7.13 5.19
0.40 0.26
2
1
NX5.7. Calculated from eight batches of three birds each. 2
cally (P<0.05) significant. Feeding grit with all mash feed to layers was shown to result in an increase in feed utilization (Mcintosh et al., 1962). These workers suspected that the grit raised the energy of the grain by somehow increasing the
FIG. 3. Cell wall breakage in aluerone cells (X105) present in feces after feeding steampelleted wheat bran to chicks.
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of pelleting that actually damage the heavy cell walls. Booth and Moran (1946), experimenting with human subjects, showed that "digestion [utilization] of bran consists essentially of the absorption of the contents of the aleurone cells." Later Rohrlich and Rasmus (1955) clearly showed that digestion of aleurone contents occurred only when the cell walls were ruptured. Recently, Olsen and Slinger (1968) suggested that the aleurone layer of cells may be made more readily available to rats during pelleting of wheat bran. Two further experiments help substantiate the idea that mechanical cell wall rupture improves the digestibility of aleurone cells. When grit was added to a mash ration and fed to chicks, protein digestion increased as shown by fecal analysis (Table 3). This increase was statisti-
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R. M. SAUNDERS, H. G. WALKER, JR. AND G. O. KOHLER
1965), and phosphorus (Summers et ah, 1967) availability in grains. Improvement in the case of fat and nitrogen is probably due to cell wall breakage. However, improvement in availability of phytin phosphorus for growth is probably due to hydrolysis during steaming. To maximize the digestibility of wheat milling fractions, it is apparent that some form of processing must be carried out to disrupt as many aleurone cells as possible. Steam-pelleting is one method to do this but the effect is variable. For example with the American bran the resultant increase in aleurone protein utilization was about 12% and 30% with two different batches of pellets. The Canadian bran showed a gain of 11 to 14%. The effect of temperature and moisture is probably of vital importance during pelleting. During the preparation of protein concentrates from bran and shorts it was shown that the moisture level had a profound effect, probably by affecting the brittleness of the aleurone cell walls (Fellers et ah, 1966). A similar effect would certainly be of importance during pelleting. Further research is required on the steam-pelleting process to optimize the aleurone layer disintegration. Increased nutrient availability resulting from cell-wall rupture is probably of widespread importance. Since it is known that aleurone cell walls have a high cellulose content (McMasters et ah, 1964), in all likelihood, it is more than coincidence that favorable feeding results after pelleting have been connected with the cellulose content of the grain, i.e., the greatest improvement is registered with the "low energy" (high roughage) rations (Calet, 1965). Such an effect is not restricted to grains. For example, feed efficiency and weight gains of ruminants were increased when high cellulose-containing grasses were pelleted (Feed Management, 1968). With chicks on an alfalfa diet, metaboliz-
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availability of the energy elements. The likely explanation is that aleurone cell walls are broken by the grinding action of the grit in the gizzard, leading to increased digestibility. When a wheat shorts ration was reground three times and fed to chicks, protein digestibility increased significantly (Table 3). Apparently the remilling ruptured enough additional cell walls to improve overall digestibility. Occasionally an aleurone cell in the feces contained mainly starch granules visible after iodine staining. Many aleurone cells contained large fat globules which are not normally observed in cells from this layer. They have been observed proviously in aleurone digested by an in vitro procedure (Chick et ah, 1947). We did not see globules in cells with broken walls. Since the aleurone layer probably constitutes 30 to 50% of the bran fraction and a somewhat lower proportion of the shorts fraction, an appreciable portion of the diet was excreted instead of utilized in each case. The aleurone layer is the major wheat bran component containing significant amounts of nutritionally important materials. For example in a hand dissected sample, Hinton et ah (1947, 1953) established that the aleurone layer contained almost all the B-vitamins and over 75% of the protein of bran. With a bran sample isolated by a combination of chemical and physical means, Shetlar et ah (1947) showed that a combined hyaline-aleurone fraction (83% aleurone) contained 90% of the protein, 94% of the fat, and 60% of the ash in whole bran. I t is thus probable that with the increased protein utilization found in these experiments, there is concurrently an increase in utilization of fats, vitamins and minerals. Previous reports note that pelleting has a beneficial effect on nitrogen (Leveille and Fisher, 1959), fat (Sell and Thompson
ALEURONE DIGESTIBILITY
ACKNOWLEDGMENTS
Our thanks are extended to Dr. J. D. Summers for the Canadian bran mash and pellets; to Dr. F. T. Jones for the photomicroscopy; to Mrs. M. Allis for the amino acid analyses; and to Mr. H. M. Wright for other analyses. REFERENCES Booth, R. G., and T. Moran, 1946. Digestibility of high-extraction wheaten flours. Lancet, 251: 119122. Calet, C , 1965. The relative value of pellets versus mash and grain in poultry nutrition. World's Poultry Sci. J. 21: 23-52. Cave, N. A. G., S. J. Slinger, J. D. Summers and G. C. Ashton, 1965a. The nutritional value of wheat milling by-products for the growing chick. I. Availability of energy. Cereal Chem. 42: 523532. Cave, N. A. G., S. J. Slinger, J. D. Summers and G. C. Ashton, 1965b. The nutritional value of wheat milling by-products for the growing chick. II. Evaluation of protein. Cereal Chem. 42: 533538. Chick, H., M. E. M. Cutting, C. J. Martin and E. B. Slack, 1947. Observations on the digestibility and nutritive value of the nitrogenous constituents of wheat bran. Brit. J. Nutr. 1: 161-182. Fellers, D. A., A. D. Shepherd, N. J. Bellard and A. P. Mossman, 1966. Protein concentrates by dry milling of wheat millfeeds. Cereal Chem. 43: 715-725. Feed Management, 1968. Pelleting—the pros and cons. 19(2): 66, 68.
Hinton, J. J. C , 1947. Distribution of vitamin Bi and nitrogen in wheat grain. Proc. Roy. Soc. 134B: 418-429. Hinton, J. J. C , F. G. Peers and B. Shaw, 1953. The B-vitamins in wheat: The unique aleurone layer. Nature, 172: 993-995. Kohler, G. O., and R. Palter, 1967. Studies on methods for amino acid analysis of wheat products. Cereal Chem. 44: 512-520. Laws, B. M., and J. H. Moore, 1963. Some observations on the pancreatic amylase and intestinal maltase of the chick. Can. J. Biochem. Physiol. 41:2107-2121. Leveille, G. A., and H. Fisher, 1959. Amino acid requirements for maintenance in the adult rooster. II. The requirements for glutamic acid, histidine, lysine and arginine. J. Nutr. 69: 289294. Mcintosh, J. I., S. J. Slinger, I. R. Sibbald and G. C. Ashton, 1962. Factors affecting the metabolizable energy content of poultry feeds. 7. The effects of grinding, pelleting and grit feeding on the availability of the energy of wheat, corn, oats and barley. 8. A study on the effects of dietary balance. Poultry Sci. 41: 445-456. McMasters, M. M., M. Bradbury and J. J. C. Hinton, 1964. In Wheat: Chemistry and Technology (edit, by I. Hlynka) (American Association of Cereal Chemists, Inc., St. Paul, Minn.) pp. 55-97. Olsen, E. M., and S. J. Slinger, 1968. Effect of steam pelleting and regrinding on the digestibility of protein in cereal grains, soybean meal and wheat bran by the rat. Can. J. Animal Sci. 48: 35-39. Rohrlich, M., and R. Rasmus, 1955. Untersuchungen liber die verdanlichkeit des eiweiss der aleuronzellen. Die Wissenschaftlich Mullerei, 8: 33-39. Saunders, R. M., H. G. Walker and G. O. Kohler, 1968. The digestibility of steam-pelleted wheat bran. Poultry Sci. 47: 1636-1637. Sell, J. L., and O. J. Thompson, 1965. The effects of ration pelleting and level of fat on the efficiency of nutrient utilisation by the chicken. Brit. Poultry Sci. 6: 345-354. Shetlar, M. R., G. T. Rankin, J. F. Lyman and W. G. France, 1947. Investigation of the proximate chemical composition of the separate bran layers of wheat. Cereal Chem. 24: 111-122. Summers, J. D., S. J. Slinger and G. Cisneros, 1967. Some factors affecting the biological availability of phosphorus in wheat by-products. Cereal Chem. 44: 318-323. Summers, J. D., 1968. The effects of steam pelleting on the metabolizable energy value of dehydrated alfalfa meal. U.S. Dept. Agr. ARS 74-46, pp. 105-110.
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able energies were substantially higher with pelleted alfalfa than with unpelleted alfalfa (Summers, 1968). It is likely that the cell-rupturing effect described here is the most important transformation leading to a higher metabolizable energy. Both steam-pelleting and remilling seem to be effective in increasing cell-wall breakage. Experiments are presently underway to investigate other processing conditions, and to develop methods leading to improved digestibility of wheat milling fractions, forages, and cereals in both foods and feeds.
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PROTEIN LAYER DIETS
egg weight was also shown when feed conversion was calculated. An average requirement for feed per dozen eggs was 1.88 kg. for the S.C.W.L. and 1.83 for the hybrids. When the data were given in terms of feed per 100 gm. of egg, the S.C.W.L. were less efficient in utilization of total feed or protein by approximately 30%. The basis for a difference in feed intake appeared to be due to body size as the S.C.W.L. were 250 gm. larger than the hybrids at the end of the trial.
REFERENCES Bray, D. J., 1960. Studies with corn-soya laying
diets. 2. Optimum combinations of corn and soybean protein. Poultry Sci. 43: 396-401. Britzman, D. C , and C. W. Carlson, 1965. Limiting amino acids in corn-soybean meal diets for laying hens. Feedstuffs, 37: (43) 20-21. Carlson, C. W., and E. Guenthner, 1969. Response of laying hens fed typical corn-soy diets to supplements of methionine and lysine. Poultry Sci. 48: 137-143. Chavez, R., J. M. Thomas and B. L. Reid, 1966. The utilization of non-protein nitrogen by laying hens. Poultry Sci. 45: 547-553. Johnson, D. Jr., and H. Fisher, 1958. The amino acid requirement of laying hens. 3. Minimal requirement levels of amino acids: techniques and development of diet. Brit. J. Nutr. 12: 276-285. Merck and Co., 1961. Amino Acid Values in Feedstuffs. Rahway, N. J. Steel, G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Inc., New York. Young, R. J., M. L. Griffith, I. D. Desai and M. L. Scott, 1965. The response of laying hens fed low protein diets to glutamic acid and diammonium citrate. Poultry Sci. 34: 1428.
Aleurone Cells and the Digestibility of Wheat Mill Feeds1 R. M. SAUNDERS, H. G. WALKER, JR. AND G. O. KOHLER Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710 (Received for publication April 8, 1969)
P
ELLETING feeds is usually advantageous for several reasons. Where improved growth has been shown, however, workers do not agree on the factor(s) responsible for the improvement. The relative value of pellets versus mash and grain in poultry nutrition has been the subject of a review by Calet (1965) where current speculations to account for the observed differences have been listed. I t appears certain that chemical and/or physical 1 Part of this work was presented at the 54th Annual Meeting of the American Association of Cereal Chemists, Chicago, Illinois, April 1969.
changes must have occurred during the pelleting process to account for the large differences found. Recently Cave et al. (1965a, b) found that wheat milling fractions gave relatively low metabolizable energy values and poor protein utilization when they constituted an important part of chick diets. By steam-pelleting wheat bran, the metabolizable energy in chick diets was increased as much as 30% over unpelleted mash. An increase of 17% was found for steam-pelleted wheat shorts over unpelleted material. The present paper re-
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
ACKNOWLEDGEMENTS
The authors thank Merck & Co., Railway, N.J. for the L-Lysine HC1 used and for a Grant-in-aid that partially supported this work. Our thanks are also given to the Dow Chemical Co., Midland Mich, for the D-L methionine.
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R. M. SAUNDERS, H. G. WALKER, JR. AND G. 0. KOHLER
TABLE 1.—Proximate analysis oj the Canadian bran, American bran, and shorts used in chick feeding experiments
Canadian bran American bran Shorts
8.60 9.48 13.57
2.79 2.94 3.15
4.00 4.62 6.45
8.36 8.23 8.45
5.94 5.80 4.74
MATERIALS AND METHODS*
A Canadian hard red spring wheat bran in both mash and steam-pelleted form was obtained from Dr. J.D. Summers of the University of Guelph, Ontario, Canada. An American hard red spring wheat bran was steam pelleted in this laboratory in a Lab model California Pellet Mill. The die was 1-^ inch thick with J-inch diameter holes. The moisture level was 16%. Wheat shorts, supplied by International Milling Company, was from a North Dakota #2 grade hard red spring wheat and was reground three times in a Brabendor Senior Quadrumat Mill. Proximate analysis of the two brans and shorts are shown in Table 1. "Grits" refers to small sand grit from the Monterey, California area, water washed, kiln dried and screened (10-20 mesh). It was included in the diet at 2% level. In the experiments where grit was added to the bran ration, grit had also been included in the commercial ration for a period of 48 hours prior to removal of the commercial feed. Autoclaving was carried out at 15 p.s.i.g. for 20 minutes. * Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.
Amino acids were determined on acid hydrolysates (6 N HC1 for 24 hours in tubes sealed under vacuum) by ion-exchange chromatography with a Phoenix amino acid analyzer (Kohler and Palter, 1967). RESULTS AND DISCUSSION
After feeding bran to chicks, microscopic examination of the contents of the lower intestine and feces showed a number of intact cells from the aleurone layer had not been digested. Empty aleurone cells were easily distinguished from full, undigested cells. The latter had a brownish granular appearance and usually con-
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
ports findings which explain why the biological availability of wheat bran nutrients is increased by the pelleting procedure. A preliminary report of this work has appeared (Saunders et al., 1968).
Bran rations were fed to chicks in the following manner: Eight-week-old New Hampshire chicks in groups of three, previously maintained on a commercial diet, were starved for 24 hours and then allowed access ad libitum to the wetted bran product as the sole feed. After 24 hours the chicks were killed and the lower small intestinal contents (Laws and Moore, 1963) were removed and retained. Fecal samples were collected from the cage. Intestinal contents and fecal droppings were washed by shaking about 1 g. of the material with 50 ml. of water and filtering through fine nylon mesh (0.007inch diameter holes). The solids left in the nylon mesh were thoroughly washed with running water. Microscopic examination and photography were carried out at this stage on a portion of the solids. The remainder was dried over KOH in a desiccator and then analyzed for nitrogen (Kjeldahl). Aleurone cells were counted at a magnification of 210. In one series of experiments fecal droppings only were collected and analyzed for nitrogen content. Eight groups of chicks in groups of three were fed each ration. The fecal sample was a composite sample from the three birds.
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A L E U R O N E DIGESTIBILITY i.
*?F5L '-~>*> „W
tained small globules. T h e granular material could be stained with protein stains and the globules with fat stains. Figure 1 illustrates a section of aleurone containing both full and empty cells. Figure 2 shows the full cells a t higher magnification. T h e globules are clearly evident. Tables 2 and 3 summarize the results of a number of feeding experiments. I n Table 2 the first column gives the percentage of empty aleurone cells (i.e., contents digested) of the total cell count for both feces and intestinal contents after different feeds or treatments. T h e percentages were determined by counting not less than 1500 aleurone cells in each sample. T h e values for intestinal contents are averaged from separate determinations on three individual birds; fecal values are from a composite sample from the same three birds. In all cases the percent of e m p t y cells is greater in the pel-
leted diets than in the mash diets, a difference t h a t was statistically significant at the 9 5 % confidence level. The table also lists the protein content of the same samples of washed intestinal and fecal solids. T h e residual protein level (nonutilized b y the animal) decreased as the number of empty cells increased, and lowest residual protein was always associated with pelleted feeds. When autoclaved wheat bran mash was used as feed, the empty aleurone cell counts and residual protein levels were about the same as those of untreated mash. Table 3 shows a statistical analysis when similar feeds were fed to eight groups of three birds. This series of experiments was carried out about 9 months after the experiments reported under Table 2. T h e American pellets were a different batch. Slight differences in digestibility of the bran are apparent b u t
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
FIG. 1. Aleurone cells (X42) present in feces after feeding steam-pelleted wheat bran to chicks. Dark cells, non-utilized material; white cells, empty (i.e., contents utilized).
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R. M. SAUNDERS, H. G. W A L K E R , J R . AND G. 0 . K O H I . E R
the relative protein digestibilities follow the same trend. Protein in pelleted bran feeds was utilized better than in nonpelleted bran. T h e differences in residual protein were significant at the 9 5 % confidence level. In this experiment, autoclaved mash actually showed a statistically significant decrease in protein utilization when compared to the regular mash. This was probably due to Maillard reactions leading to undigestible protein fragments.
where breakages occur, the contents have been utilized. This was also the case where aleurone appeared to be dented or crinkled. Since autoclaving itself causes no inincrease in aleurone cell utilization, the major factor leading to improved digestibility must be associated with the pelleting step. Steam treatment may help soften cell walls prior to pelleting, and it m a y be t h a t it is the shear and compression forces
Figure 3 shows a magnified section of aleurone tissue isolated from feces from a chick fed steam-pelleted bran. I t is clear t h a t some cell walls were broken as a result of the physical stress of pelleting. None of the walls of the intact cells appear to have been damaged, b u t all the empty cells show evidence of breakage. I t is evident t h a t rupturing the cell walls has given digestive enzymes greater access to cellular contents since, without exception,
TABLE 2.—Number of empty aleurone cells1 and residual protein2 in wheat bran feeds after digestion by chicks
Feed Canadian mash Canadian pellets American mash American pellets American mash, autoclaved 1 2 3
E m p t y aleurone cells, %
Protein content, 3 %
Intestine
Feces
Intestine
Feces
28.8 43.2 15.0 35.6 18.3
32.8 50.2 21.4 40.0 22.6
7.47 6.61 8.41 6.73 8.15
7.12 6.27 8.38 6.38 7.92
Average from three chicks. Pooled sample same three chicks. N X5.7. Protein content of washed intestinal and fecal solids.
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library on April 9, 2015
FIG. 2. Aleurone cells (X105) present in feces after feeding steam-pelleted wheat bran to chicks.
ALEURONE DIGESTIBILITY
TABLE 3.—Residual protein in wheal bran and shorts feeds after digestion by chicks
Feed Canadian Canadian American American American American
F
mash pellets mash pellets mash, autoclaved mash+grits
Shorts Shorts milled 3X
n Standard St£? en ' deviation /o 7.75 7.01 8.27 7.41 8.72 7.81
0.13 0.18 0.14 0.29 0.43 0.21
7.13 5.19
0.40 0.26
2
1
NX5.7. Calculated from eight batches of three birds each. 2
cally (P<0.05) significant. Feeding grit with all mash feed to layers was shown to result in an increase in feed utilization (Mcintosh et al., 1962). These workers suspected that the grit raised the energy of the grain by somehow increasing the
FIG. 3. Cell wall breakage in aluerone cells (X105) present in feces after feeding steampelleted wheat bran to chicks.
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of pelleting that actually damage the heavy cell walls. Booth and Moran (1946), experimenting with human subjects, showed that "digestion [utilization] of bran consists essentially of the absorption of the contents of the aleurone cells." Later Rohrlich and Rasmus (1955) clearly showed that digestion of aleurone contents occurred only when the cell walls were ruptured. Recently, Olsen and Slinger (1968) suggested that the aleurone layer of cells may be made more readily available to rats during pelleting of wheat bran. Two further experiments help substantiate the idea that mechanical cell wall rupture improves the digestibility of aleurone cells. When grit was added to a mash ration and fed to chicks, protein digestion increased as shown by fecal analysis (Table 3). This increase was statisti-
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R. M. SAUNDERS, H. G. WALKER, JR. AND G. O. KOHLER
1965), and phosphorus (Summers et ah, 1967) availability in grains. Improvement in the case of fat and nitrogen is probably due to cell wall breakage. However, improvement in availability of phytin phosphorus for growth is probably due to hydrolysis during steaming. To maximize the digestibility of wheat milling fractions, it is apparent that some form of processing must be carried out to disrupt as many aleurone cells as possible. Steam-pelleting is one method to do this but the effect is variable. For example with the American bran the resultant increase in aleurone protein utilization was about 12% and 30% with two different batches of pellets. The Canadian bran showed a gain of 11 to 14%. The effect of temperature and moisture is probably of vital importance during pelleting. During the preparation of protein concentrates from bran and shorts it was shown that the moisture level had a profound effect, probably by affecting the brittleness of the aleurone cell walls (Fellers et ah, 1966). A similar effect would certainly be of importance during pelleting. Further research is required on the steam-pelleting process to optimize the aleurone layer disintegration. Increased nutrient availability resulting from cell-wall rupture is probably of widespread importance. Since it is known that aleurone cell walls have a high cellulose content (McMasters et ah, 1964), in all likelihood, it is more than coincidence that favorable feeding results after pelleting have been connected with the cellulose content of the grain, i.e., the greatest improvement is registered with the "low energy" (high roughage) rations (Calet, 1965). Such an effect is not restricted to grains. For example, feed efficiency and weight gains of ruminants were increased when high cellulose-containing grasses were pelleted (Feed Management, 1968). With chicks on an alfalfa diet, metaboliz-
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availability of the energy elements. The likely explanation is that aleurone cell walls are broken by the grinding action of the grit in the gizzard, leading to increased digestibility. When a wheat shorts ration was reground three times and fed to chicks, protein digestibility increased significantly (Table 3). Apparently the remilling ruptured enough additional cell walls to improve overall digestibility. Occasionally an aleurone cell in the feces contained mainly starch granules visible after iodine staining. Many aleurone cells contained large fat globules which are not normally observed in cells from this layer. They have been observed proviously in aleurone digested by an in vitro procedure (Chick et ah, 1947). We did not see globules in cells with broken walls. Since the aleurone layer probably constitutes 30 to 50% of the bran fraction and a somewhat lower proportion of the shorts fraction, an appreciable portion of the diet was excreted instead of utilized in each case. The aleurone layer is the major wheat bran component containing significant amounts of nutritionally important materials. For example in a hand dissected sample, Hinton et ah (1947, 1953) established that the aleurone layer contained almost all the B-vitamins and over 75% of the protein of bran. With a bran sample isolated by a combination of chemical and physical means, Shetlar et ah (1947) showed that a combined hyaline-aleurone fraction (83% aleurone) contained 90% of the protein, 94% of the fat, and 60% of the ash in whole bran. I t is thus probable that with the increased protein utilization found in these experiments, there is concurrently an increase in utilization of fats, vitamins and minerals. Previous reports note that pelleting has a beneficial effect on nitrogen (Leveille and Fisher, 1959), fat (Sell and Thompson
ALEURONE DIGESTIBILITY
ACKNOWLEDGMENTS
Our thanks are extended to Dr. J. D. Summers for the Canadian bran mash and pellets; to Dr. F. T. Jones for the photomicroscopy; to Mrs. M. Allis for the amino acid analyses; and to Mr. H. M. Wright for other analyses. REFERENCES Booth, R. G., and T. Moran, 1946. Digestibility of high-extraction wheaten flours. Lancet, 251: 119122. Calet, C , 1965. The relative value of pellets versus mash and grain in poultry nutrition. World's Poultry Sci. J. 21: 23-52. Cave, N. A. G., S. J. Slinger, J. D. Summers and G. C. Ashton, 1965a. The nutritional value of wheat milling by-products for the growing chick. I. Availability of energy. Cereal Chem. 42: 523532. Cave, N. A. G., S. J. Slinger, J. D. Summers and G. C. Ashton, 1965b. The nutritional value of wheat milling by-products for the growing chick. II. Evaluation of protein. Cereal Chem. 42: 533538. Chick, H., M. E. M. Cutting, C. J. Martin and E. B. Slack, 1947. Observations on the digestibility and nutritive value of the nitrogenous constituents of wheat bran. Brit. J. Nutr. 1: 161-182. Fellers, D. A., A. D. Shepherd, N. J. Bellard and A. P. Mossman, 1966. Protein concentrates by dry milling of wheat millfeeds. Cereal Chem. 43: 715-725. Feed Management, 1968. Pelleting—the pros and cons. 19(2): 66, 68.
Hinton, J. J. C , 1947. Distribution of vitamin Bi and nitrogen in wheat grain. Proc. Roy. Soc. 134B: 418-429. Hinton, J. J. C , F. G. Peers and B. Shaw, 1953. The B-vitamins in wheat: The unique aleurone layer. Nature, 172: 993-995. Kohler, G. O., and R. Palter, 1967. Studies on methods for amino acid analysis of wheat products. Cereal Chem. 44: 512-520. Laws, B. M., and J. H. Moore, 1963. Some observations on the pancreatic amylase and intestinal maltase of the chick. Can. J. Biochem. Physiol. 41:2107-2121. Leveille, G. A., and H. Fisher, 1959. Amino acid requirements for maintenance in the adult rooster. II. The requirements for glutamic acid, histidine, lysine and arginine. J. Nutr. 69: 289294. Mcintosh, J. I., S. J. Slinger, I. R. Sibbald and G. C. Ashton, 1962. Factors affecting the metabolizable energy content of poultry feeds. 7. The effects of grinding, pelleting and grit feeding on the availability of the energy of wheat, corn, oats and barley. 8. A study on the effects of dietary balance. Poultry Sci. 41: 445-456. McMasters, M. M., M. Bradbury and J. J. C. Hinton, 1964. In Wheat: Chemistry and Technology (edit, by I. Hlynka) (American Association of Cereal Chemists, Inc., St. Paul, Minn.) pp. 55-97. Olsen, E. M., and S. J. Slinger, 1968. Effect of steam pelleting and regrinding on the digestibility of protein in cereal grains, soybean meal and wheat bran by the rat. Can. J. Animal Sci. 48: 35-39. Rohrlich, M., and R. Rasmus, 1955. Untersuchungen liber die verdanlichkeit des eiweiss der aleuronzellen. Die Wissenschaftlich Mullerei, 8: 33-39. Saunders, R. M., H. G. Walker and G. O. Kohler, 1968. The digestibility of steam-pelleted wheat bran. Poultry Sci. 47: 1636-1637. Sell, J. L., and O. J. Thompson, 1965. The effects of ration pelleting and level of fat on the efficiency of nutrient utilisation by the chicken. Brit. Poultry Sci. 6: 345-354. Shetlar, M. R., G. T. Rankin, J. F. Lyman and W. G. France, 1947. Investigation of the proximate chemical composition of the separate bran layers of wheat. Cereal Chem. 24: 111-122. Summers, J. D., S. J. Slinger and G. Cisneros, 1967. Some factors affecting the biological availability of phosphorus in wheat by-products. Cereal Chem. 44: 318-323. Summers, J. D., 1968. The effects of steam pelleting on the metabolizable energy value of dehydrated alfalfa meal. U.S. Dept. Agr. ARS 74-46, pp. 105-110.
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able energies were substantially higher with pelleted alfalfa than with unpelleted alfalfa (Summers, 1968). It is likely that the cell-rupturing effect described here is the most important transformation leading to a higher metabolizable energy. Both steam-pelleting and remilling seem to be effective in increasing cell-wall breakage. Experiments are presently underway to investigate other processing conditions, and to develop methods leading to improved digestibility of wheat milling fractions, forages, and cereals in both foods and feeds.
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