- Email: [email protected]
The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meal
Animal Feed Science and Technology, 16 (1986) 151--156
151
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
Short Communication THE EFFECT OF ENZYMATIC TREATMENT ON AMINO ACID CONTENT AND NITROGEN CHARACTERISTICS OF FEATHER MEAL
M.C. P A P A D O P O U L O S
I
Department of Animal Science, Agricultural University,P.O. Box 338, 6700 A H Wageningen (The Netherlands) (Received 14 June 1984; accepted for publication 30 M a y 1986)
ABSTRACT Papadopoulos, M.C., 1986. The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meal. Anita. Feed Sci. Technol., 16: 151-156.
Samples of feather meal were prepared by proteolytic enzyme treatment. Changes in amino acid content, in vitro protein digestibility and solubility of the enzyme-treated feather meals by comparison with samples with no enzyme addition are discussed. It was concluded that, although samples subjected to enzymatic treatment showed some losses in most of amino acids, their higher values for in vitro protein digestibility and solubility are considered to be favourable, unless other modifications to the protein structure affect the nutritive value of feather meal.
INTRODUCTION
Feather, a byproduct from the rendering industry, is almost pure keratin protein, but in its natural state is resistant to digestive enzymes when fed to an animal, because of cystine-disulfide bonds. Thermal treatments, therefore, are needed to make feathers more digestible (Papadopoulos et al., 1985, 1986). Experiences with this product by feeding monogastric species (Papadopoulos, 1985) and ruminants (Aderibigbe and Church, 1983) are, however, quite divergent, a factor which stimulates wavering interest. As requirements for high quality protein increase it will become more and more necessary and desirable to change the properties of several protein sources to meet the animals' protein needs. The functional and nutritional properties of food proteins may be improved by the application of enzymatic modifications (Whitaker, 1977). Previous reports contain some data on enzymatically-treated feathers (Noval and Nickerson, 1959; El'Present address: Wessanen Nederland B.V., P.O. Box 630, 1500 EP Zaandam, The Netherlands.
0377-8401/86/$03.50
© 1986 Elsevier Science Publishers B.V.
152 mayergi and Smith, 1971). However, these works have only been specifically directed to the keratinolytic activity of the microorganisms used. In this study, feather meal was enzyme-treated to various degrees and an a t t e m p t was made to measure the changes in amino acid content and nitrogen characteristics as related to the extent of enzyme-treatment. MATERIALS AND METHODS
Enzymatic treatment Substrate preparation Feather meal substrate was prepared in the following way. Fresh white feathers of broiler chicks were obtained from a local poultry dressing plant, cleaned and subjected to steam processing in a laboratory autoclave at 345 kPa for 60 min and with 60% moisture content. The general procedure employed in the preparation and processing of raw feathers has been described in previous work (Papadopoulos et al., 1986).
Enzyme source Maxatase, a commercial proteolytic enzyme, which is active at alkaline range of pH, was obtained as a sample from a manufacturing company (Gist-Brocades NV, Delft). Its activity was 330 000 Delft Units g-~.
Assay procedure Two hundred g of powdered feather meal, prepared as previously described, was suspended in 100 ml of h o t water ca. 58°(2 and immersed in a waterbath. The pH was maintained with concentrated ammonia {25%) at 8.5 and the required a m o u n t of enzyme added to the aqueous suspension of the feather meal. The mixture was gently agitated at 52°C for 2 h then heated at 870(2 for 5 min to inactivate the enzyme, cooled rapidly and freeze
Determination of reaction time (preliminary trial). In this trial the reaction was allowed to proceed for 5 h and samples were withdrawn each 30 rain. There were two replicates per test sample. A control series w i t h o u t added enzyme was run concurrently with the test series. When hydrolysis was terminated, the suspension was centrifuged at 18000 × g for 60 min. The solids were washed once with distilled water and centrifuged again under the same conditions. The total supernatant liquid was freeze-dried, as shown in Fig. 1. The criterion for assessing the effectiveness of the enzyme's action was the ability to increase the solubility of the feather meal protein. This
153
MIXING
i
H20 AUTOCLAVED FEATHER MEAL
t #
AMMONIA ENZYME
1 I HYDROLYSIS IN STIRRED FLASK
l I ENZYME INACTIVATION
HEATING
!CENTRIFUGAT'OI N
1
SLUDGE
I SOPERNATAN'1 1 I F~EEZE~'ED I 1
Unconverted Protein and other Insoluble Meter/a/
HYDROLYZED PROTEIN
Fig. 1. Diagrammatic representation o f the preparation o f e n z y m a t i c hydrolysis. OO2
.U ~
x\
001
>-
~j u:
0
120
-001
180
240
300
TIME (MIN)
Fig. 2. The relative enzymatic conversion rate ( R E C R ) in relation to time (T), w h e n enzyme (0.4%) was incubated with feather meal at 52°C and p H 8.6 or 6, based on regression analysis. PECR = 3 . 1 9 9 x 10 -~ -- 1 2 . 4 0 2 x 10 -6 x T (at pH 8.5). PECR=1.594× 10-2--12.078× 10 -5 × T ( a t p H 6 . 0 ) .
is defmed as: Enzymatic conversion rate (ECR) =
wt of the supernatant (freeze-dried)
wt of the sludge + supernatant (freezedried) The relative ECR (RECR) is a linear expression (Fig. 2) when the natural--
154
log R E C R is regressed against time (Shipley and Clark, 1972). The time required for the two enzymatic treatments, at p H 8.5 and 6, to reach the same R E C R values is approximately 2 h. Therefore, 2 h of enzymatic hydrolysis was chosen as the optimum reaction time for the enzymatic treatment of feather meals.
Analytical methods The methods used for amino acid analysis, in vitro protein digestibility in pepsin-HC1 solution and nitrogen solubility in 0.02 N NaOH and 6 N HCI, were those described in a previous work (Papadopoulos et al., 1986). RESULTS A N D DISCUSSION
The degree of changes in amino acid content and nitrogen characteristics of enzyme-treated feather meal, is shown in Table I. The amino acid content, with the exception of tyrosine and phenylalanine, were lower in the enzymetreated feather meal by comparison with samples with no enzyme. Within the enzyme-treated feather meals the contents of threonine, cystine, lysine, arginine, tyrosine, phenylalanine, serine, glutamic acid and proline were decreased, while the contents of valine, methionine, isoleucine, aspartic acid, glycine and alanine were increased as a result of increased enzyme concentration {0.20--0.60%}. Leucine and histidine did not show clear trend. The changes which occur in feather meal protein as a result of technological processing can result from different types of reactions. Possible mechanisms for the modifications of amino acid residues have been discussed by several investigators (Hurrell et al., 1976; Papadopoulos et al., 1985, 1986), although there is no satisfactory explanation for the observed increases in tyrosine and phenylalanine of the enzymatically processed samples. The deviations occurring in the amino acid composition in our study could probably be explained by different orders of kinetic parameters for individual nutrients which might occur in thermal processing {Karel, 1979). An interesting feature of this study is that although amino acid content of the test feather meal decreased as a result of added enzyme, in vitro protein digestibility and nitrogen solubility were increased (Table I). Within the enzyme-treated feather meals there were clear increases in digestibility and solubility when enzyme concentrations increased from 0.20 to 0.60%. These findings are the result of the greater extent of denaturation during enzymatic treatment. Protein digestion in vitro by pepsin-HCl test is an accepted means of evaluating digestibility of proteins of animal feeds {Papadopoulos, 1985). Determination of protein solubility in alkali and acid solutions have also been used as measures of the functional properties of foods by food tech-
155
TABLE I Effect of enzymatic treatment feather meal', = Item
o n t h e a m i n o a c i d c o n t e n t s a n d n i t r o g e n c h a r a c t e r i s t i c s in
Feather meal Control 3
E n z y m e a d d i t i o n (%)4 0.20
0.40
0.60
Threonine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Aspartic acid Serine Glutamic acid Proline Glycine Alanine
4.77 c 3.91 c 7.77 c 0.59 c 4.97b 7.51 b 2.30 a 4.21 a 1.95 b 0.67 b 6.53 c 6.56 c 11.61 c 10.93 c 9.17b 7.13 c 4.34 b
4.74 c 3.61 b 7.15 a 0.52 a 4.81a 7 . 4 5 ab 2.78 c 4.66c 1.93 b 0.63 a 6.43 b 5.90 a 10.76 b 10.84 bc 8 . 9 4 ab 6.35 a 3.96 a
4.49 ba 3 . 7 8 bc 7.15 a 0.56 b 4.81a 7.30 a 2.51 b 4.35 b 1 . 9 0 ab 0.64 a 6 . 3 1 ab 5.93 a 1 0 . 5 8 ab 10.35 a 8.78 a 6.33 a 3.98 a
4.59 b 3.52 a 7.32 b 0.54 b 4.86 a 7 . 4 4 ab 2.68 bc 4.57 c 1.92 b 0.63 a 6 . 3 5 ab 5 . 9 8 ab 1 0 . 6 0 ab 1 0 . 5 7 ab 8.9lab 6.47 b 4.08 a
Cp s PDP 6 NSS 7 NSH s
97.32 91.80 74.29 37.20
97.75 94.54 86.19 75.81
97.63 94.98 88.35 76.70
97.19 94.42 89.48 82.08
a a a a
a b b b
a b b b
a b bc c
' Comparison between treatments. 2 Values represent the average of three replicates. 3Feather meal without enzyme addition. 4 w t / w t f e a t h e r m e a l d r y m a t t e r basis. s C r u d e p r o t e i n (N x 6 . 3 5 ) . 6 I n v i t r o p r o t e i n d i g e s t i b i l i t y in p e p s i n - - H C 1 . 7 N i t r o g e n s o l u b i l i t y in 0 . 0 2 N N a O H . 8 N i t r o g e n s o l u b i l i t y in 6 N HC1. a ' b ' C V a l u e s in t h e s a m e r o w w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P < 0 . 0 5 ) .
nologists (Clatterbuek et al., 1980). Digestibility is a major factor affecting the quality of feather meal in animal feeding since feather protein in the natural state is not readily digestible due to cystine-disulfide bonds. Thus, cleavage of these bonds makes the feather protein more soluble and susceptible to digestive proteolytic enzymes. Because in the enzyme-treated feather meal the cystine content is significantly lower and nitrogen characteristics appeared to be significantly increased in comparison with samples treated without enzyme, it is suggested that changes in cystine content are used in the evaluation of the nutritive value of feather meal as a feedstuff
156
in animal feeding. Higher values for in vitro protein digestibility are considered to be favourable, while the losses of amino acids other than cystine, may not be of much nutritional significance unless other structural modifications to the protein affect its nutritive value. The beneficial effect however of enzymatic treatment on feather meal protein quality as determined in the laboratory, should be further investigated in vivo in order to determine the full potential of the method employed as an alternative m e t h o d to conventional processing of feather meal. ACKNOWLEDGEMENTS
The author wishes to express appreciation to Dr. A.R. El Boushy for his interest in this study and A.E. R o o d b e e n for his assistance with the enzymatic preparations.
REFERENCES Aderibigbe, A.O. and Church, D.C., 1983. Feather and hair meals for ruminants. I. Effect of degree of processing on utilization of feather meal. J. Anita. Sci., 56: 1198-1207. Clatterbuck, K.L., Kehrberg, N.L. and Marable, N.L., 1980. Solubility and in vitro digestibility of soy flours, concentrates and isolates. J. Food Sci., 45: 931--935. Elmayergi, H.H. and Smith, R.E., 1971. Influence of growth of Streptomyces fradiae on pepsin-HC1 digestibility and methionine content of feather meal. Can. J. Microb., 17: 1067--1072. Hurrell, R.F., Carpenter, K.J., Sinclair, W.J., Otterburn, M.S. and Asquith, R.S., 1976. Mechanisms of heat damage in proteins. 7. The significance of lysine containing isopeptides and of lanthionine in heated proteins. Br. J. Nutr., 35: 383--395. Karel, M., 1979. Prediction of nutrient losses and optimalization of processing conditions. In: S.R. Tannenbaum (Editor), Nutritional and Safety Aspects of Food Processing. Marcel Dekker, Inc., New York, pp. 233--263. Noval, J.J. and Nickerson, W.J., 1959. Decomposition of native keratin by Streptomyces fradiae. J. Bacteriol., 77: 251--263. Papadopoulos, M.C., 1985. Processed chicken feathers as feedstuff for poultry and swine. A review. Agric. Wastes, 14: 275--290. Papadopoulos, M.C., El Boushy, A.R. and Roodbeen, A.E., 1985. The effect of varying autoclaving conditions and added sodium hydroxide on amino acid content and nitrogen characteristics of feather meal. J. Sci. Food Agric., 36: 1219--1226. Papadopoulos, M.C., El Boushy, A.R., Roodbeen, A.E. and Ketelaars, E.H., 1986. Effects of processing time and moisture content on amino acid composition and nitrogen characteristics of feather meal. Anita. Feed Sci. Technol., 14: 279--290. Shipley, R.A. and Clark, R.E., 1972. Tracer Methods for in vivo Kinetics. Academic Press, NY, 239 pp. Whitaker, J.R., 1977. Enzymatic modifications of proteins applicable to foods. In: R.E. Feeney and J.R. Whitaker (Editors), Food Proteins -- Improvement through Chemical and Enzymatic Modification, ACS, Washington, DC, pp. 95--155.
151
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
Short Communication THE EFFECT OF ENZYMATIC TREATMENT ON AMINO ACID CONTENT AND NITROGEN CHARACTERISTICS OF FEATHER MEAL
M.C. P A P A D O P O U L O S
I
Department of Animal Science, Agricultural University,P.O. Box 338, 6700 A H Wageningen (The Netherlands) (Received 14 June 1984; accepted for publication 30 M a y 1986)
ABSTRACT Papadopoulos, M.C., 1986. The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meal. Anita. Feed Sci. Technol., 16: 151-156.
Samples of feather meal were prepared by proteolytic enzyme treatment. Changes in amino acid content, in vitro protein digestibility and solubility of the enzyme-treated feather meals by comparison with samples with no enzyme addition are discussed. It was concluded that, although samples subjected to enzymatic treatment showed some losses in most of amino acids, their higher values for in vitro protein digestibility and solubility are considered to be favourable, unless other modifications to the protein structure affect the nutritive value of feather meal.
INTRODUCTION
Feather, a byproduct from the rendering industry, is almost pure keratin protein, but in its natural state is resistant to digestive enzymes when fed to an animal, because of cystine-disulfide bonds. Thermal treatments, therefore, are needed to make feathers more digestible (Papadopoulos et al., 1985, 1986). Experiences with this product by feeding monogastric species (Papadopoulos, 1985) and ruminants (Aderibigbe and Church, 1983) are, however, quite divergent, a factor which stimulates wavering interest. As requirements for high quality protein increase it will become more and more necessary and desirable to change the properties of several protein sources to meet the animals' protein needs. The functional and nutritional properties of food proteins may be improved by the application of enzymatic modifications (Whitaker, 1977). Previous reports contain some data on enzymatically-treated feathers (Noval and Nickerson, 1959; El'Present address: Wessanen Nederland B.V., P.O. Box 630, 1500 EP Zaandam, The Netherlands.
0377-8401/86/$03.50
© 1986 Elsevier Science Publishers B.V.
152 mayergi and Smith, 1971). However, these works have only been specifically directed to the keratinolytic activity of the microorganisms used. In this study, feather meal was enzyme-treated to various degrees and an a t t e m p t was made to measure the changes in amino acid content and nitrogen characteristics as related to the extent of enzyme-treatment. MATERIALS AND METHODS
Enzymatic treatment Substrate preparation Feather meal substrate was prepared in the following way. Fresh white feathers of broiler chicks were obtained from a local poultry dressing plant, cleaned and subjected to steam processing in a laboratory autoclave at 345 kPa for 60 min and with 60% moisture content. The general procedure employed in the preparation and processing of raw feathers has been described in previous work (Papadopoulos et al., 1986).
Enzyme source Maxatase, a commercial proteolytic enzyme, which is active at alkaline range of pH, was obtained as a sample from a manufacturing company (Gist-Brocades NV, Delft). Its activity was 330 000 Delft Units g-~.
Assay procedure Two hundred g of powdered feather meal, prepared as previously described, was suspended in 100 ml of h o t water ca. 58°(2 and immersed in a waterbath. The pH was maintained with concentrated ammonia {25%) at 8.5 and the required a m o u n t of enzyme added to the aqueous suspension of the feather meal. The mixture was gently agitated at 52°C for 2 h then heated at 870(2 for 5 min to inactivate the enzyme, cooled rapidly and freeze
Determination of reaction time (preliminary trial). In this trial the reaction was allowed to proceed for 5 h and samples were withdrawn each 30 rain. There were two replicates per test sample. A control series w i t h o u t added enzyme was run concurrently with the test series. When hydrolysis was terminated, the suspension was centrifuged at 18000 × g for 60 min. The solids were washed once with distilled water and centrifuged again under the same conditions. The total supernatant liquid was freeze-dried, as shown in Fig. 1. The criterion for assessing the effectiveness of the enzyme's action was the ability to increase the solubility of the feather meal protein. This
153
MIXING
i
H20 AUTOCLAVED FEATHER MEAL
t #
AMMONIA ENZYME
1 I HYDROLYSIS IN STIRRED FLASK
l I ENZYME INACTIVATION
HEATING
!CENTRIFUGAT'OI N
1
SLUDGE
I SOPERNATAN'1 1 I F~EEZE~'ED I 1
Unconverted Protein and other Insoluble Meter/a/
HYDROLYZED PROTEIN
Fig. 1. Diagrammatic representation o f the preparation o f e n z y m a t i c hydrolysis. OO2
.U ~
x\
001
>-
~j u:
0
120
-001
180
240
300
TIME (MIN)
Fig. 2. The relative enzymatic conversion rate ( R E C R ) in relation to time (T), w h e n enzyme (0.4%) was incubated with feather meal at 52°C and p H 8.6 or 6, based on regression analysis. PECR = 3 . 1 9 9 x 10 -~ -- 1 2 . 4 0 2 x 10 -6 x T (at pH 8.5). PECR=1.594× 10-2--12.078× 10 -5 × T ( a t p H 6 . 0 ) .
is defmed as: Enzymatic conversion rate (ECR) =
wt of the supernatant (freeze-dried)
wt of the sludge + supernatant (freezedried) The relative ECR (RECR) is a linear expression (Fig. 2) when the natural--
154
log R E C R is regressed against time (Shipley and Clark, 1972). The time required for the two enzymatic treatments, at p H 8.5 and 6, to reach the same R E C R values is approximately 2 h. Therefore, 2 h of enzymatic hydrolysis was chosen as the optimum reaction time for the enzymatic treatment of feather meals.
Analytical methods The methods used for amino acid analysis, in vitro protein digestibility in pepsin-HC1 solution and nitrogen solubility in 0.02 N NaOH and 6 N HCI, were those described in a previous work (Papadopoulos et al., 1986). RESULTS A N D DISCUSSION
The degree of changes in amino acid content and nitrogen characteristics of enzyme-treated feather meal, is shown in Table I. The amino acid content, with the exception of tyrosine and phenylalanine, were lower in the enzymetreated feather meal by comparison with samples with no enzyme. Within the enzyme-treated feather meals the contents of threonine, cystine, lysine, arginine, tyrosine, phenylalanine, serine, glutamic acid and proline were decreased, while the contents of valine, methionine, isoleucine, aspartic acid, glycine and alanine were increased as a result of increased enzyme concentration {0.20--0.60%}. Leucine and histidine did not show clear trend. The changes which occur in feather meal protein as a result of technological processing can result from different types of reactions. Possible mechanisms for the modifications of amino acid residues have been discussed by several investigators (Hurrell et al., 1976; Papadopoulos et al., 1985, 1986), although there is no satisfactory explanation for the observed increases in tyrosine and phenylalanine of the enzymatically processed samples. The deviations occurring in the amino acid composition in our study could probably be explained by different orders of kinetic parameters for individual nutrients which might occur in thermal processing {Karel, 1979). An interesting feature of this study is that although amino acid content of the test feather meal decreased as a result of added enzyme, in vitro protein digestibility and nitrogen solubility were increased (Table I). Within the enzyme-treated feather meals there were clear increases in digestibility and solubility when enzyme concentrations increased from 0.20 to 0.60%. These findings are the result of the greater extent of denaturation during enzymatic treatment. Protein digestion in vitro by pepsin-HCl test is an accepted means of evaluating digestibility of proteins of animal feeds {Papadopoulos, 1985). Determination of protein solubility in alkali and acid solutions have also been used as measures of the functional properties of foods by food tech-
155
TABLE I Effect of enzymatic treatment feather meal', = Item
o n t h e a m i n o a c i d c o n t e n t s a n d n i t r o g e n c h a r a c t e r i s t i c s in
Feather meal Control 3
E n z y m e a d d i t i o n (%)4 0.20
0.40
0.60
Threonine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Aspartic acid Serine Glutamic acid Proline Glycine Alanine
4.77 c 3.91 c 7.77 c 0.59 c 4.97b 7.51 b 2.30 a 4.21 a 1.95 b 0.67 b 6.53 c 6.56 c 11.61 c 10.93 c 9.17b 7.13 c 4.34 b
4.74 c 3.61 b 7.15 a 0.52 a 4.81a 7 . 4 5 ab 2.78 c 4.66c 1.93 b 0.63 a 6.43 b 5.90 a 10.76 b 10.84 bc 8 . 9 4 ab 6.35 a 3.96 a
4.49 ba 3 . 7 8 bc 7.15 a 0.56 b 4.81a 7.30 a 2.51 b 4.35 b 1 . 9 0 ab 0.64 a 6 . 3 1 ab 5.93 a 1 0 . 5 8 ab 10.35 a 8.78 a 6.33 a 3.98 a
4.59 b 3.52 a 7.32 b 0.54 b 4.86 a 7 . 4 4 ab 2.68 bc 4.57 c 1.92 b 0.63 a 6 . 3 5 ab 5 . 9 8 ab 1 0 . 6 0 ab 1 0 . 5 7 ab 8.9lab 6.47 b 4.08 a
Cp s PDP 6 NSS 7 NSH s
97.32 91.80 74.29 37.20
97.75 94.54 86.19 75.81
97.63 94.98 88.35 76.70
97.19 94.42 89.48 82.08
a a a a
a b b b
a b b b
a b bc c
' Comparison between treatments. 2 Values represent the average of three replicates. 3Feather meal without enzyme addition. 4 w t / w t f e a t h e r m e a l d r y m a t t e r basis. s C r u d e p r o t e i n (N x 6 . 3 5 ) . 6 I n v i t r o p r o t e i n d i g e s t i b i l i t y in p e p s i n - - H C 1 . 7 N i t r o g e n s o l u b i l i t y in 0 . 0 2 N N a O H . 8 N i t r o g e n s o l u b i l i t y in 6 N HC1. a ' b ' C V a l u e s in t h e s a m e r o w w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y (P < 0 . 0 5 ) .
nologists (Clatterbuek et al., 1980). Digestibility is a major factor affecting the quality of feather meal in animal feeding since feather protein in the natural state is not readily digestible due to cystine-disulfide bonds. Thus, cleavage of these bonds makes the feather protein more soluble and susceptible to digestive proteolytic enzymes. Because in the enzyme-treated feather meal the cystine content is significantly lower and nitrogen characteristics appeared to be significantly increased in comparison with samples treated without enzyme, it is suggested that changes in cystine content are used in the evaluation of the nutritive value of feather meal as a feedstuff
156
in animal feeding. Higher values for in vitro protein digestibility are considered to be favourable, while the losses of amino acids other than cystine, may not be of much nutritional significance unless other structural modifications to the protein affect its nutritive value. The beneficial effect however of enzymatic treatment on feather meal protein quality as determined in the laboratory, should be further investigated in vivo in order to determine the full potential of the method employed as an alternative m e t h o d to conventional processing of feather meal. ACKNOWLEDGEMENTS
The author wishes to express appreciation to Dr. A.R. El Boushy for his interest in this study and A.E. R o o d b e e n for his assistance with the enzymatic preparations.
REFERENCES Aderibigbe, A.O. and Church, D.C., 1983. Feather and hair meals for ruminants. I. Effect of degree of processing on utilization of feather meal. J. Anita. Sci., 56: 1198-1207. Clatterbuck, K.L., Kehrberg, N.L. and Marable, N.L., 1980. Solubility and in vitro digestibility of soy flours, concentrates and isolates. J. Food Sci., 45: 931--935. Elmayergi, H.H. and Smith, R.E., 1971. Influence of growth of Streptomyces fradiae on pepsin-HC1 digestibility and methionine content of feather meal. Can. J. Microb., 17: 1067--1072. Hurrell, R.F., Carpenter, K.J., Sinclair, W.J., Otterburn, M.S. and Asquith, R.S., 1976. Mechanisms of heat damage in proteins. 7. The significance of lysine containing isopeptides and of lanthionine in heated proteins. Br. J. Nutr., 35: 383--395. Karel, M., 1979. Prediction of nutrient losses and optimalization of processing conditions. In: S.R. Tannenbaum (Editor), Nutritional and Safety Aspects of Food Processing. Marcel Dekker, Inc., New York, pp. 233--263. Noval, J.J. and Nickerson, W.J., 1959. Decomposition of native keratin by Streptomyces fradiae. J. Bacteriol., 77: 251--263. Papadopoulos, M.C., 1985. Processed chicken feathers as feedstuff for poultry and swine. A review. Agric. Wastes, 14: 275--290. Papadopoulos, M.C., El Boushy, A.R. and Roodbeen, A.E., 1985. The effect of varying autoclaving conditions and added sodium hydroxide on amino acid content and nitrogen characteristics of feather meal. J. Sci. Food Agric., 36: 1219--1226. Papadopoulos, M.C., El Boushy, A.R., Roodbeen, A.E. and Ketelaars, E.H., 1986. Effects of processing time and moisture content on amino acid composition and nitrogen characteristics of feather meal. Anita. Feed Sci. Technol., 14: 279--290. Shipley, R.A. and Clark, R.E., 1972. Tracer Methods for in vivo Kinetics. Academic Press, NY, 239 pp. Whitaker, J.R., 1977. Enzymatic modifications of proteins applicable to foods. In: R.E. Feeney and J.R. Whitaker (Editors), Food Proteins -- Improvement through Chemical and Enzymatic Modification, ACS, Washington, DC, pp. 95--155.