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Lysine in cassava based diets: I. A note on the effect of heat on digestibility and available lysine
Food Chemistry 15 (1984) 261-268
Lysiine in Cassava Based Diets: I. A Note on the Effect of Heat on Digestibility and Available Lysine S. R. A. Adewusi & O. L. Oke Chemistry Department, University of Ife, Ile-Ife, Nigeria (Received: 22 June, 1984) ABSTRACT Heating Conophor seeds and Amaranthus leaf protein concentrate (LPC) for 24 h at 121 °C reduced the digestibility of their crude protein contents. Loss in available lysine was moderate in LPC alone and cassava-LPC mixtures heated for 24 h at 121 °C (< 45 % loss) but high in Conophor seeds and glucose-LPC mixtures (72 and 80 % loss respectively).
INTRODUCTION The use of leaf protein concentrate (LPC) as a protein supplement and cassava as an energy source in the developing countries has been advocated (Oke, 1975). However, Knipfel et al. (1975) pointed out that heating protein alone or protein-carbohydrate mixtures reduced feed intake, weight gain and digestibility of crude protein in rats. It is well known that during processing of feedstuffs and other foods a lot of heat is either used or generated. When LPC and cassava are processed into feeds and stored in the hot and humid climates of the tropics damage due to the Maillard reaction is possible. The purpose of this study is to investigate heat damage to protein-carbohydrate mixtures during processing. In this paper, we report the effect of heat on Conophor seeds (model for a naturally occurring protein-carbohydrate-oil mixture (Oke & Fafunson, 1975)), LPC-cassava and LPC-glucose mixtures (models for feed proteincarbohydrate mixtures), and LPC alone (model for protein in the absence 261 Food Chemistry 0308-8146/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
262
s. R. A. Adewusi, O. L. Oke
of carbohydrate). The heat effect was assessed by determining in vitro digestibility and available lysine. Several methods were used and results compared.
MATERIALS A N D METHODS LPC was obtained from Amaranthus spp. using IBP pulper and press. Conophor tetracarpidium seeds were purchased locally, dried and milled to a fine powder. Authentic samples for available lysine assay were obtained from Dr K. J. Carpenter (Cambridge University, UK). Cassava flour was prepared in the Teaching and Research Farm, University of Ife. In vitro digestibility was determined by the pepsin-pancreatin procedure (Saunders et al., 1973) and papain digestion (Buchanan & Byers, 1969). Nitrogen in the residue was analysed by the micro-Kjeldahl method. Sugar content of cassava flour was determined by the procedure outlined by Ketiku & Oyenuga (1972). Cassava flour and LPC were mixed together in the ratios 1:1, 1:2 and 2:1 by weight. Glucose and LPC were also mixed in the same ratios. All samples were spread thinly on aluminium foil and heated at 121 °C. Available lysine was determined by the FDNB method of Carpenter (1960) as modified by Booth (1971) and also by the TNBS method of Kakade & Liener (1969).
RESULTS A N D DISCUSSION The LPC used in this experiment contained 67 ~o crude protein (CP), 6.7 and 5.0 g of available lysine by F D N B and TNBS methods, respectively, per 16 gN. Conophor seeds contained 25 ~o CP, 7.1 and 5.6 g of available lysine by FDNB and TNBS methods, respectively, per 16 g N. To demonstrate the reliability of results obtained in our laboratory, we analysed samples obtained from Dr Carpenter's laboratory and compared both sets of results as shown in Table 1. These results indicate that the TNBS assay consistently gave lower values than the F D N B assay. The sugar content of cassava flour presented in Table 2 shows that values for total sugar content and reducing sugars which may react with lysine in Maillard reactions are small but vary from sample to sample.
Lysine in cassava based diets: I
263
TABLE 1 Available Lysine Contents of Some Protein Samples as Determined by FDNB and TNBS Methods and Compared to Literature Values
Sample
Amaranthus LPC Conophor seeds BPA X 841 b Chicken muscle--X902 b X902 heated 8 h at 121 °C b Groundnut meal b Groundnut meal heated 4h at 121 °C Casein
% Crude protein
FDNB TNBS (g available lysine per 16 g N)
Literature value (FDNB)
67.0 25-0 89"0 91.0 87.0 44.2
6"7 7" 1 12"26 8.60 8.10 3.00
5-0 5"6 9.82 6.98 7.20 2.58
ND a ND 12.20 b 8.94 b 7.26 b 3-19 b
41-6 84.0
1.80 7.80
1.44 5.54
1.61 b 8.17 c
ND, not determined. b Samples and FDNB available lysine values were obtained from Dr Carpenter's laboratc,ry, while at University of Cambridge, UK. c Value obtained from Friedman & Finley (1975). Generally glucose was present in the highest concentration ( 0 . 1 8 - 1 . 3 0 g / 1 0 0 g) a n d m a l t o s e t h e l o w e s t . T h e effect o f h e a t o n t h e d i g e s t i b i l i t y o f c r u d e p r o t e i n o f C o n o p h o r s e e d s a n d A m a r a n t h u s L P C s h o w n in F i g s 1 a n d 2 i n d i c a t e s t h a t d i g e s t i b i l i t y o f C o n o p h o r s e e d p r o t e i n w a s r e d u c e d b y a l m o s t 60 % w h e n h e a t e d f o r 24 h a t 121 °C. T h e p a p a i n d i g e s t i o n m e t h o d i n d i c a t e d n o TABLE 2 The Composition and Concentration of Sugars in Cassava Flour Preparations
Constituents
Range (g/lOO g dry sample) ~
Total sugar Reducing sugar Glucose Fructose Maltose Sucrose (by difference)
0.72~4.50 0.30°2'76 0.18-1.30 0.10-0.99 0-0.49 b 0.42 1.74
a Values show range of three determinations from cassava flour prepared at different times of the year. b Maltose was not detected in one preparation.
264
S. R. A. Adewusi, O. L. Oke
I
i00 ,[I
o
o PEPSIN- PANCREATIN
•
~
PAPAIN
DIGESTION
DIGESTION
80>. -- 6 O J
_
40"
o Fig. I.
i
~
HOURS
OF
1'2
J~
HEATING
AT
2'0
2"4
121°C
In vitro digestibility of heated Conophor seeds using pepsin-pancreatin and
papain methods.
c
~ PEPSIN- PANCREAT IN
IOOJ
=~ PAPAIN
DIGESTION
DIGESTION
|
80~ >. I-re
6o-
I-
~40" 20-
0 Fig. 2.
4
~
HOURS
OF
12
16
HEATING
AT
2'o
24
121°C
In vitro digestibility of heated Amaranthus leaf protein concentrate using
pepsin-pancreatin and papain methods.
Lysine in cassava b a s e d diets." 1
265
90-
80-
70~r
=
RATIO
I:I
----
RATIO
1:2
O
R A T I O 2:1
CASSAVA : L P C
60" --~
~
O
,,
:
,.
: '"
50"
uJ
_J ~ 30 LL O ~
m O .J
20'
~" ~o
o
i HOURS
Fig. 3.
~ OF
r2 HEATING
ig AT
~
2;4
121°C
The effect of heat on available lysine loss in cassava-leaf protein concentrate mixtures, using the F D N B method.
measurable digestibility after 24 h. Digestibility ofAmaranthus LPC also decreased to about 0 ~ by both papain and pepsin-pancreatin methods after 24-h heating at 121 °C. The effect of heat on the available lysine content of cassava-LPC and glucose-LPC mixtures, LPC alone and Conophor seeds, shown in Figs 3 to 6, indicated the following: (1) Cassava-LPC mixtures of ratio 1:2 seemed to be most affected, losing 45 ~ of the available lysine when heated for 24 h at 121 °C. (2) About 80 ~ of the available lysine was lost when a glucose-LPC mixture (ratio 1 : 2) was heated for 24 h at 121 °C. This showed that the deleterious effect of the Maillard reaction increased because of the presence of reducing sugars (Knipfel et al., 1975). (3) When LPC was heated alone, about 45 ~ of the available lysine was lost in 24 h.
OF
HEATING AT
,, "
2:1
J21oc
:
:
"
GLUCOSE:LPC
1:2 80-
IO.
20.
IO-
20-
o
o
~ OF
~ HOURS
o
o
HEATING
,~
FDNB
TNBS
AT
,~
2o 121°¢
METHOD
METHOD
2:4 Fig. 5. The effect of heat on available lysine loss in Amaranthus leaf protein concentrate (LPC) alone using the F D N B and TNBS methods.
o
o
30-
30-
50-
40-
..J
=,
40.
50
Fig. 4. The effect of heat on available lysine loss in glucose-leaf protein concentrate mixtures, using the FDNB method.
o
5
~Z 6 0 "
HOURS
,,
RATIO h i
u~ 60-
o
a
70"
~
70.
8O
c
o
•
901
~v
to o~ o~
267
Lysine in cassava based diets." I a
o
T N B S
METt-tC)D
o
o
FDN B
METHOD
/,.~t-
90" BO" 70' 60, SO. 40 30 20 IO
o
~
~
&
8
HOURS
Fig. 6.
~b OF
1'2
HEATING
14 AT
3~
18
26
2~
2:4
121°C
The effectof heat on the available lysineloss of Conophor seedsusing the FDNB and TNBS methods.
(4) The effect of heat on available lysine content of Conophor seeds was similar to that obtained for glucose-LPC mixtures (72 % available lysine was lost in 24 h). (5) In experiments where both F D N B and TNBS methods were used, the TNBS method appeared to be more sensitive but this impression might be due to the low available lysine values often obtained by this method. In conclusion, it has been shown that the effect of heat on digestibility and available lysine increases with time and the presence of reducing sugars. Because cassava has a low level of reducing sugars, it appears that Maillard reactions will be insignificant in cassava based diets. In addition, the F D N B assay is recommended for quantitative estimation of available lysine while the less cumbersome TNBS (available lysine) and papain (in vitro digestibility) methods should be used in screening feeds and feedstuffs for heat damage. ACKNOWLEDGEMENT We ~Lre grateful to Dr O . A . Afolabi for a critical review of the manuscript.
268
S. R. A. Adewusi, O. L. Oke
REFERENCES Booth, V. H. (1971). Problems in the determination of FDN B-available lysine. J. Sci. Fd Agric., 22, 658. Buchanan, R. A. & Byers, M. (1969). Interference by cyanide with the measurement of papain hydrolysis. J. Sci. Fd Agric., 20, 364. Carpenter, K. J. (1960). The estimation of the available lysine in animal protein foods. Biochem. J., 77, 604. Friedman, M. & Finley, J. W. (1975). Vinyl compounds as reagents for available lysine in proteins. In: Protein-nutritional quality in foods and feeds, Part 1 (Friedman, M. (Ed.)). Marcel Dekker Inc., New York, p. 503. Kakade, M. L. & Liener, I. E. (1969). Determination of available lysine in proteins. Analyt. Biochem., 27, 273. Ketiku, A. O. & Oyenuga, V. A. (1972). Changes in the carbohydrate constituents of cassava root tuber during growth. J. Sci. Fd Agric., 23, 1451. Knipfel, J. B., Botting, H. G. & McLaughlan, J. M. (1975). Nutritional quality of several proteins as affected by heating in the presence of carbohydrates. In: Protein-nutritional quality o/foods and feeds, Part 2 (Friedman, M. (Ed.)). Marcel Dekker Inc., New York, p. 375. Oke, O. L. (1975). A case for vegetable proteins in developing countries. Wld Rev. Nutr. Diet., 23, 259. Oke, O. L. & Fafunson, M. A. (1975). Lesser known oil-seeds--the nutritional value of Conophor seeds in vitro and in rats. Nutr. Rep. Intl, 12, 41. Saunders, R. M., Connor, M. A., Booth, A. N., Bickoff, E. M. & Kohler, G. O. (1973). Measurement of digestibility of alfalfa protein concentrates by in vitro and in vivo methods. J. Nutr., 103, 530.
Lysiine in Cassava Based Diets: I. A Note on the Effect of Heat on Digestibility and Available Lysine S. R. A. Adewusi & O. L. Oke Chemistry Department, University of Ife, Ile-Ife, Nigeria (Received: 22 June, 1984) ABSTRACT Heating Conophor seeds and Amaranthus leaf protein concentrate (LPC) for 24 h at 121 °C reduced the digestibility of their crude protein contents. Loss in available lysine was moderate in LPC alone and cassava-LPC mixtures heated for 24 h at 121 °C (< 45 % loss) but high in Conophor seeds and glucose-LPC mixtures (72 and 80 % loss respectively).
INTRODUCTION The use of leaf protein concentrate (LPC) as a protein supplement and cassava as an energy source in the developing countries has been advocated (Oke, 1975). However, Knipfel et al. (1975) pointed out that heating protein alone or protein-carbohydrate mixtures reduced feed intake, weight gain and digestibility of crude protein in rats. It is well known that during processing of feedstuffs and other foods a lot of heat is either used or generated. When LPC and cassava are processed into feeds and stored in the hot and humid climates of the tropics damage due to the Maillard reaction is possible. The purpose of this study is to investigate heat damage to protein-carbohydrate mixtures during processing. In this paper, we report the effect of heat on Conophor seeds (model for a naturally occurring protein-carbohydrate-oil mixture (Oke & Fafunson, 1975)), LPC-cassava and LPC-glucose mixtures (models for feed proteincarbohydrate mixtures), and LPC alone (model for protein in the absence 261 Food Chemistry 0308-8146/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
262
s. R. A. Adewusi, O. L. Oke
of carbohydrate). The heat effect was assessed by determining in vitro digestibility and available lysine. Several methods were used and results compared.
MATERIALS A N D METHODS LPC was obtained from Amaranthus spp. using IBP pulper and press. Conophor tetracarpidium seeds were purchased locally, dried and milled to a fine powder. Authentic samples for available lysine assay were obtained from Dr K. J. Carpenter (Cambridge University, UK). Cassava flour was prepared in the Teaching and Research Farm, University of Ife. In vitro digestibility was determined by the pepsin-pancreatin procedure (Saunders et al., 1973) and papain digestion (Buchanan & Byers, 1969). Nitrogen in the residue was analysed by the micro-Kjeldahl method. Sugar content of cassava flour was determined by the procedure outlined by Ketiku & Oyenuga (1972). Cassava flour and LPC were mixed together in the ratios 1:1, 1:2 and 2:1 by weight. Glucose and LPC were also mixed in the same ratios. All samples were spread thinly on aluminium foil and heated at 121 °C. Available lysine was determined by the FDNB method of Carpenter (1960) as modified by Booth (1971) and also by the TNBS method of Kakade & Liener (1969).
RESULTS A N D DISCUSSION The LPC used in this experiment contained 67 ~o crude protein (CP), 6.7 and 5.0 g of available lysine by F D N B and TNBS methods, respectively, per 16 gN. Conophor seeds contained 25 ~o CP, 7.1 and 5.6 g of available lysine by FDNB and TNBS methods, respectively, per 16 g N. To demonstrate the reliability of results obtained in our laboratory, we analysed samples obtained from Dr Carpenter's laboratory and compared both sets of results as shown in Table 1. These results indicate that the TNBS assay consistently gave lower values than the F D N B assay. The sugar content of cassava flour presented in Table 2 shows that values for total sugar content and reducing sugars which may react with lysine in Maillard reactions are small but vary from sample to sample.
Lysine in cassava based diets: I
263
TABLE 1 Available Lysine Contents of Some Protein Samples as Determined by FDNB and TNBS Methods and Compared to Literature Values
Sample
Amaranthus LPC Conophor seeds BPA X 841 b Chicken muscle--X902 b X902 heated 8 h at 121 °C b Groundnut meal b Groundnut meal heated 4h at 121 °C Casein
% Crude protein
FDNB TNBS (g available lysine per 16 g N)
Literature value (FDNB)
67.0 25-0 89"0 91.0 87.0 44.2
6"7 7" 1 12"26 8.60 8.10 3.00
5-0 5"6 9.82 6.98 7.20 2.58
ND a ND 12.20 b 8.94 b 7.26 b 3-19 b
41-6 84.0
1.80 7.80
1.44 5.54
1.61 b 8.17 c
ND, not determined. b Samples and FDNB available lysine values were obtained from Dr Carpenter's laboratc,ry, while at University of Cambridge, UK. c Value obtained from Friedman & Finley (1975). Generally glucose was present in the highest concentration ( 0 . 1 8 - 1 . 3 0 g / 1 0 0 g) a n d m a l t o s e t h e l o w e s t . T h e effect o f h e a t o n t h e d i g e s t i b i l i t y o f c r u d e p r o t e i n o f C o n o p h o r s e e d s a n d A m a r a n t h u s L P C s h o w n in F i g s 1 a n d 2 i n d i c a t e s t h a t d i g e s t i b i l i t y o f C o n o p h o r s e e d p r o t e i n w a s r e d u c e d b y a l m o s t 60 % w h e n h e a t e d f o r 24 h a t 121 °C. T h e p a p a i n d i g e s t i o n m e t h o d i n d i c a t e d n o TABLE 2 The Composition and Concentration of Sugars in Cassava Flour Preparations
Constituents
Range (g/lOO g dry sample) ~
Total sugar Reducing sugar Glucose Fructose Maltose Sucrose (by difference)
0.72~4.50 0.30°2'76 0.18-1.30 0.10-0.99 0-0.49 b 0.42 1.74
a Values show range of three determinations from cassava flour prepared at different times of the year. b Maltose was not detected in one preparation.
264
S. R. A. Adewusi, O. L. Oke
I
i00 ,[I
o
o PEPSIN- PANCREATIN
•
~
PAPAIN
DIGESTION
DIGESTION
80>. -- 6 O J
_
40"
o Fig. I.
i
~
HOURS
OF
1'2
J~
HEATING
AT
2'0
2"4
121°C
In vitro digestibility of heated Conophor seeds using pepsin-pancreatin and
papain methods.
c
~ PEPSIN- PANCREAT IN
IOOJ
=~ PAPAIN
DIGESTION
DIGESTION
|
80~ >. I-re
6o-
I-
~40" 20-
0 Fig. 2.
4
~
HOURS
OF
12
16
HEATING
AT
2'o
24
121°C
In vitro digestibility of heated Amaranthus leaf protein concentrate using
pepsin-pancreatin and papain methods.
Lysine in cassava b a s e d diets." 1
265
90-
80-
70~r
=
RATIO
I:I
----
RATIO
1:2
O
R A T I O 2:1
CASSAVA : L P C
60" --~
~
O
,,
:
,.
: '"
50"
uJ
_J ~ 30 LL O ~
m O .J
20'
~" ~o
o
i HOURS
Fig. 3.
~ OF
r2 HEATING
ig AT
~
2;4
121°C
The effect of heat on available lysine loss in cassava-leaf protein concentrate mixtures, using the F D N B method.
measurable digestibility after 24 h. Digestibility ofAmaranthus LPC also decreased to about 0 ~ by both papain and pepsin-pancreatin methods after 24-h heating at 121 °C. The effect of heat on the available lysine content of cassava-LPC and glucose-LPC mixtures, LPC alone and Conophor seeds, shown in Figs 3 to 6, indicated the following: (1) Cassava-LPC mixtures of ratio 1:2 seemed to be most affected, losing 45 ~ of the available lysine when heated for 24 h at 121 °C. (2) About 80 ~ of the available lysine was lost when a glucose-LPC mixture (ratio 1 : 2) was heated for 24 h at 121 °C. This showed that the deleterious effect of the Maillard reaction increased because of the presence of reducing sugars (Knipfel et al., 1975). (3) When LPC was heated alone, about 45 ~ of the available lysine was lost in 24 h.
OF
HEATING AT
,, "
2:1
J21oc
:
:
"
GLUCOSE:LPC
1:2 80-
IO.
20.
IO-
20-
o
o
~ OF
~ HOURS
o
o
HEATING
,~
FDNB
TNBS
AT
,~
2o 121°¢
METHOD
METHOD
2:4 Fig. 5. The effect of heat on available lysine loss in Amaranthus leaf protein concentrate (LPC) alone using the F D N B and TNBS methods.
o
o
30-
30-
50-
40-
..J
=,
40.
50
Fig. 4. The effect of heat on available lysine loss in glucose-leaf protein concentrate mixtures, using the FDNB method.
o
5
~Z 6 0 "
HOURS
,,
RATIO h i
u~ 60-
o
a
70"
~
70.
8O
c
o
•
901
~v
to o~ o~
267
Lysine in cassava based diets." I a
o
T N B S
METt-tC)D
o
o
FDN B
METHOD
/,.~t-
90" BO" 70' 60, SO. 40 30 20 IO
o
~
~
&
8
HOURS
Fig. 6.
~b OF
1'2
HEATING
14 AT
3~
18
26
2~
2:4
121°C
The effectof heat on the available lysineloss of Conophor seedsusing the FDNB and TNBS methods.
(4) The effect of heat on available lysine content of Conophor seeds was similar to that obtained for glucose-LPC mixtures (72 % available lysine was lost in 24 h). (5) In experiments where both F D N B and TNBS methods were used, the TNBS method appeared to be more sensitive but this impression might be due to the low available lysine values often obtained by this method. In conclusion, it has been shown that the effect of heat on digestibility and available lysine increases with time and the presence of reducing sugars. Because cassava has a low level of reducing sugars, it appears that Maillard reactions will be insignificant in cassava based diets. In addition, the F D N B assay is recommended for quantitative estimation of available lysine while the less cumbersome TNBS (available lysine) and papain (in vitro digestibility) methods should be used in screening feeds and feedstuffs for heat damage. ACKNOWLEDGEMENT We ~Lre grateful to Dr O . A . Afolabi for a critical review of the manuscript.
268
S. R. A. Adewusi, O. L. Oke
REFERENCES Booth, V. H. (1971). Problems in the determination of FDN B-available lysine. J. Sci. Fd Agric., 22, 658. Buchanan, R. A. & Byers, M. (1969). Interference by cyanide with the measurement of papain hydrolysis. J. Sci. Fd Agric., 20, 364. Carpenter, K. J. (1960). The estimation of the available lysine in animal protein foods. Biochem. J., 77, 604. Friedman, M. & Finley, J. W. (1975). Vinyl compounds as reagents for available lysine in proteins. In: Protein-nutritional quality in foods and feeds, Part 1 (Friedman, M. (Ed.)). Marcel Dekker Inc., New York, p. 503. Kakade, M. L. & Liener, I. E. (1969). Determination of available lysine in proteins. Analyt. Biochem., 27, 273. Ketiku, A. O. & Oyenuga, V. A. (1972). Changes in the carbohydrate constituents of cassava root tuber during growth. J. Sci. Fd Agric., 23, 1451. Knipfel, J. B., Botting, H. G. & McLaughlan, J. M. (1975). Nutritional quality of several proteins as affected by heating in the presence of carbohydrates. In: Protein-nutritional quality o/foods and feeds, Part 2 (Friedman, M. (Ed.)). Marcel Dekker Inc., New York, p. 375. Oke, O. L. (1975). A case for vegetable proteins in developing countries. Wld Rev. Nutr. Diet., 23, 259. Oke, O. L. & Fafunson, M. A. (1975). Lesser known oil-seeds--the nutritional value of Conophor seeds in vitro and in rats. Nutr. Rep. Intl, 12, 41. Saunders, R. M., Connor, M. A., Booth, A. N., Bickoff, E. M. & Kohler, G. O. (1973). Measurement of digestibility of alfalfa protein concentrates by in vitro and in vivo methods. J. Nutr., 103, 530.