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Chemical and biological evaluation of soya-bean flakes autoclaved for different durations
Animal Feed Science and Technology, 31 (1990) 247-259
247
Elsevier Science Publishers B.V., Amsterdam
Chemical and biological evaluation of soya-bean flakes autoclaved for different durations F.H. Kratzer, Sally Bersch, P. Vohra and R.A. Ernst Department of Avian Sciences, University of California, Davis, CA 95616 (U.S.A.) (Received 22 November 1989; accepted for publication 7 May 1990)
ABSTRACT Kratzer, F.H., Bersch, S., Vohra, P. and Ernst, R.A., 1990. Chemical and biological evaluation of soyabean flakes autoclaved for different durations. Anita. Feed Sci. Technol., 31: 247-259. Solvent-extracted soya-bean flakes were autoclaved for 0-180 min at 121 ° C and evaluated by three chick feeding trials and several tests in vitro. Improvement in growth from short periods of heating was related to reduced trypsin inhibitor or urease activity which were highly correlated (r= 0.96 ). After extraction with 0.2% KOH, protein solubility determined by the Kjeldahl nitrogen method was highly correlated ( r = 0.99 ) with that determined by the Coomassie Blue dye-binding method. Protein solubility decreased as the heating duration was increased. A high correlation was also found between Orange G binding and protein solubility (r=0.97); and between Coomassie Blue dye-binding and Orange G binding ( r = 0.98 ). Beyond the initial improvement in growth caused by the destruction of growth inhibitors, the degree of overheating measured by protein solubility, Orange G binding, or formol titration agreed well with decreased growth of chicks. Pancreas weight of < 3.3 g kg-1 body weight is a relatively sensitive test of properly processed soya-bean meal. The effect of overheating soya-bean meal in reducing the growth of chickens was greater in a diet containing 1.2% lysine than when lysine was increased to 1.4%. Coomassie Blue dye-binding and either trypsin inhibitor or urease activity are rapid and convenient methods for evaluating soya-bean meal.
INTRODUCTION
Osborne and Mendel (1917 ) observed that cooked, but not raw, ground soya-beans supported normal growth of rats. Raw soya-bean meal contains antinutrients such as antitrypsin, which depresses growth (Ham et al., 1945; Borcher et al., 1948) and enlarges the pancreas (Chernick et al., 1948; Lepkovsky et al., 1959 ) in chicks, and hemagglutinins or lectins which contribute to growth depression in rats (Liener, 1953; Pallansch and Liener, 1953 ), and chicks (Coates et al., 1970). These are inactivated by heat processing of raw soya-bean meal. Availability of lysine is reduced if soya-bean meal is overheated (Block et al., 1946; Clandinin et al., 1947). During overheating, the epsilon amino group oflysine undergoes a browning or Maillard reaction and lysine availability is reduced (Mauron, 1981 ). 0377-8401/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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F.H. KRATZERETAL.
In addition to trypsin inhibitor, raw soya-bean meal contains another enzyme, urease, which is inactivated by heat processing of the meal. Urease is easily determined by the method of Caskey and Knapp (1944). A residual urease activity to give a change in value of pH of 0.02 is an indicator of properly processed soya-bean meal (Bird et al., 1947 ). The urease test is worthless for detecting overheated meal. The value of the urease test has been questioned by McNaughton et al. ( 1981 ) and Dale et al. (1986), as the gain in body weight of chickens fed on soya-bean samples with zero urease activity was not significantly different from those fed on samples with a residual urease activity giving a change in pH of 0.02. Attempts have been made to find better methods for evaluating processing conditions. These include determination of antitrypsin activity (Kakade et al., 1974) as an indicator ofunderheating, and Orange G dye-binding (Moran et al., 1963), formalin titration (Almquist and Maurer, 1953), and protein solubility in 0.2% KOH (Araba and Dale, 1990) to detect overheating and protein damage. Not all these parameters have been determined on the same samples. A number of reports are available on the optimum heat-processing conditions for soya-bean flakes using laboratory sized autoclaves, with the object of studying relationships between urease activity, antitrypsin activity of the samples, and the growth of chickens fed on these samples (McNaughton and Reece, 1980; McNaughton et al., 1981; Waldroup et al., 1985; Araba and Dale, 1990). Veltmann et al. (1986) compared some commercially available undercooked and overcooked soya-bean meals. A number of these studies used low dietary protein in their feeding experiments. We have extended the studies to compare urease activity, antitrypsin activity, Orange G dye-binding, protein solubility in 0.2% KOH by Kjeldahl and dye-binding methods, formalin titration value, and feeding value for chickens of solvent-extracted soya-bean flakes autoclaved for different times under laboratory conditions in three experiments. MATERIALS AND METHODS
Autoclaving of soya-beanflakes Two lots of solvent-extracted raw flakes were obtained from Central Soya Company, Fort Wayne, IN. In Experiment 1, solvent-extracted raw soya-bean flakes (Lot 1 ) were autoclaved at 121 °C ( 1.055 kg cm -2) for times varying from 3 to 120 min. The autoclave was manually operated and the time was measured when the chamber pressure reached 1.055 kg cm -2. For Experiments 2 and 3, a different autoclave with automatic controls (Castle steam sterilizer, M / C 3423) was used. It was programmed to purge the chamber of air before timing the treatment period. For that reason, the heat treatment in
EVALUATION OF OVERHEATED SOYA-BEAN MEAL
249
Experiments 2 and 3 was somewhat more severe than for an equivalent period of time in Experiment 1. CHEMICAL EVALUATION
Protein solubility The method suggested by Araba and Dale (1990) was used to determine protein solubility in 0.2% KOH. The soluble protein in the supernatant solution was determined by the Kjeldahl method (Association of Official Analytical Chemists, 1975 ). A dye-binding method for soluble protein (Kratzer et al., 1990) measured the capacity of the protein in the KOH filtrate to bind with Coomassie Blue G dye. Lysozyme was used as a reference standard, and the values are expressed as reference protein equivalent (RPE).
Dye-binding Essentially, the method described by Moran et al. ( 1963 ) was used for Crocin Orange G dye-binding. The difference in the intensity of the color of the dye solution before and after reacting with the soya-bean sample indicates the amount of dye bound.
Urease activity The modified method of Caskey and Knapp (1944) as described in American Oil Chemist's Society (1974) was used for the urease determination. It measures the change in pH on incubating the soya-bean meal sample with a buffered urea solution.
Formaldehyde titration The procedure suggested by Almquist and Maurer (1953 ) utilizing titration with base in the presence of formaldehyde was used for evaluating heat damage to soya-bean preparations.
Trypsin inhibitor activity Trypsin inhibitor activity was determined by a modification of the method of Kakade et al. (1974). The soya-bean meal samples were shaken with 0.01 N NaOH for an hour before filtering, and aliquots of the filtrate tested for activity in inhibiting the hydrolysis ofbenzoyl-DL-arginine-p-nitroanilide hydrochloride, a synthetic substrate. The inhibitor activity is expressed in terms of trypsin inhibitor units (TIU).
250
F.H. KRATZERET AL.
BIOLOGICAL EVALUATION
Three feeding experiments were conducted using 2-day-old unsexed broiler (Hubbard) chicks purchased from A & M Hatchery, Santa Rosa, CA. The birds were banded, weighed, and distributed into groups containing 5 chicks each in Experiment 1, and 6 each in Experiments 2 and 3. Each group had approximately the same initial weight. The groups were housed in electrically heated battery cages with the cage temperatures maintained at 35 °C for the first week, 32.2°C for the second, and 29.4°C for the third. Two groups of birds, distributed randomly in the battery cages were fed on each of the experimental diets. Feed and tap water were available at all times. The birds were weighed individually twice weekly. The cumulative feed intake was also measured at that time. At the end of the experiments (Experiment 1,22 days; Experiments 2 and 3, 21 days), birds were killed by cervical dislocation if pancreas weights were to be determined.
Composition of the diets The diets contained coarsely ground raw soya-bean flakes (termed meal), or meal prepared from flakes autoclaved in the manually operated autoclave in Experiment 1, and the automatically controlled autoclave in Experiments 2 and 3. The composition of the diets used in Experiments 1-3 is given in Table 1. The diet in Experiment 1 was calculated to contain about 23% crude protein and 1.4% lysine from a combination of maize and soya-bean meal only. The requirement for lysine for broilers according to the National Research Council (NRC, 1984) is 1.2%. In Experiment 2, calculated lysine level was reduced to 1.2% by incorporating some maize gluten meal into the diets. In Experiment 3, birds were fed on the diets of Experiment 2 and similar diets which were supplemented with lysine to raise its content to 1.4%. The data were checked for statistically significant differences by analysis of variance (ANOVA). The significantly different means were ranked using Duncan's method (Snedecor and Cochran, 1980). RESULTS
Experiment 1 As the autoclaving time increased from 0 to 120 min, the color of the raw soya-bean flakes turned progressively darker. Protein solubility in 0.2% KOH, as determined by the Kjeldahl method, decreased from about 83% for the raw sample to about 32% for the sample heated for 60 min (Table 2). Values for Coomassie Blue dye-binding followed the same trend as Kjeldahl nitrogen values. There is some crossover for the samples autoclaved for 6 and 12 min,
EVALUATIONOF OVERHEATEDSOYA-BEANMEAL
251
TABLE 1 Composition of the diets for Experiments 1-3 Ingredient (g kg- t )
Experiment 1 Lysine 1.4%
Experiment 2 Lysine 1.2%
Maize, ground Soya-bean meal (48% CP) Maize gluten meal (62% CP) Soya-bean oil CaHPO4.2H20 CaCO3 Vitamin pre-mix 4 NaC1, iodized DL-Methionine MnSO4" HaO ZnO Cellulose 5 Lysine
510.7 400.01 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 -
540.0 320.71 50.0 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 -
Calculated composition (%) ME (MJ kg- ~) Crude protein Lysine Methionine Methionine + cystine Calcium Available P
12.93 23.77 1.39 0.60 0.97 1.06 0.59
13.30 23.28 1.19 0.64 1.01 1.05 0.59
Experiment 3 Lysine 1.2%
Lysine 1.4%
537.5
537.5 320.73 50.0 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 2.5
320.73 50.0 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 2.5 13.30 23.26 1.19 0.64 1.01 1.05 0.59
13.30 23.26 1.41 0.64 1.01 1.05 0.59
~Diets contained soya-bean meal autoclaved for 0, 12, 24, or 60 min at 121 °C ( 1.055 kg cm-2). 2Diets contained soya-bean meal autoclaved for 0, 15, 30, 45, 60, 120 or 180 min at 121 ° C ( 1.055 kg cm
- 2 ).
3Diets contained soya-bean meal autoclaved for 0, 7, 15, 21, 30, 45, or 60 min at 121 °C ( 1.055 kg cm-2). 4Vitamin pre-mix supplied the following (in mg kg-t diet): biotin, 0.5; butylated hydroxy toluene, 1000; calcium pantothenate, 30; choline chloride, 1000; folic acid, 5; menadione, 10; niacin, 120; pyridoxine HCI, 10; riboflavin, 10; thiamin HCI, 10; vitamin B 12, 0.01; vitamin A, 10 000 IU; vitamin D3, 4500; vitamin E, 88. 5Solka floc, Brown C., Berlin, NH. CP, crude protein.
possibly owing to inaccuracy in the manual recording of time, after a pressure of 1.055 kg cm -2 was reached in the autoclave. Urease activity was decreased to 32% of the original value after autoclaving for 3 min and to zero after 24 min. Trypsin inhibitor activity (TI) was reduced to about 61 and 37% after autoclaving for 3 and 12 min, respectively, and was still present at a low level in the sample autoclaved for 24 min. Urease is more sensitive to inactivation by heating than TI. Orange G dye-binding index followed the same trend as protein solubility. No significant differences in gain in body weight were observed in chicks
78.0 74,7 75.9
65,2
31.7
13.8
3 6 12
24
60
120
10.7
28.6
64.4
73.6 73.2 72.4
73.1
Coomassie (mg RPE z per 100 mg protein )
0
0
0
0,65 0.50 0.60
2.00
Urease activity (pH change)
0
260
2520
23500 22700 14200
38600
Trypsin inhibitor (units g-~ )
37.0
42.6
47.4
49.0 48.8 49.3
49.5
Orange G binding (mg g- l soya-bean meal)
tThe diet was calculated to contain 1.4% lysine. 2RPE = reference protein equivalent. Significant differences are indicated by different superscripts in a column. Duplicate groups of 5 chicks were used.
83.2
Kjeldahl (%)
Protein solubility (0.2% KOH )
0
Autoclaving time (rain)
Effect of autoclaving soya-bean meal for different times on its nutritional value ~ (Experiment 1 )
TABLE 2
(+_0.71)
Mean 25.5
23.8 ~b ( +_ 1.30) 27.0 ab (+1.23) 28.4 b ( + 1.23)
22.6 a (_+1.23)
Gain in body wt. +_SE (g day- 1)
1,50 (_+0.04)
1.50
1.38
1.56
1.56
Feed/gain ratio
> t-
m
N
bo
EVALUATIONOF OVERHEATEDSOYA-BEANMEAL
253
fed on diets containing soya-bean meal autoclaved for 12, 24, or 60 min at 121 °C. However, chicks on soya-bean meal autoclaved for 60 min had a significantly ( P < 0 . 0 5 ) better weight gain than those fed on raw meal (Table 2 ). The growth data indicate that autoclaving of soya-bean flakes for about 24 min results in optimal growth of chickens, and the growth was not further improved by autoclaving for 60 min, as long as the diets were formulated to contain 1.4% lysine. Significant differences were not observed in the amount of feed needed per unit gain in weight, although there was a trend for improved feed utilization with autoclaving of soya-bean flakes.
Experiment 2 Protein solubility in 0.2% KOH decreased from about 91% in raw samples to about 54% in samples autoclaved for 15 min in the Castle steam sterilizer (Table 3 ), which was a much greater reduction than in the manually operated autoclave (Fig. 1 ), suggesting that purging steam tended to preheat the flakes before the timer became operational. Protein solubility as determined by the Coomassie Blue method followed the same trend as determination by the Kjeldahl method. Urease activity, as well as TI activity, had practically disappeared after autoclaving for more than 15 min. Values for formalin titration as well as Orange G binding index also decreased with an increase in autoclaving time. These reduced indices suggest a reduction in lysine availability (Almquist and Maurer, 1952; Hurrell and Carpenter et al., 1979). An optimum gain in body weight of chicks was observed when they were fed on TABLE 3
Effect of autoclaving soya-bean flakes for different times on some chemically determined indices (Experiment 2)
Autoclaving Protein solubility in time 0.2% KOH (minutes)
Urease activity Trypsin Formalin Orange G binding (pH change) inhibitor (ml 0.05 N (mg g-~ soya(units g - l ) NaOH per 2 g) bean meal)
Kjeldahl Coomassie (%)
(mg RPE l
per 100 mg protein ) 0 15 30 45 60 120 180
90.9 54.1 44.6 30.0 28.9 15.7 12.6
67.3 44.7 32.9 27.4 22.9 11.9 7.7
1.95 0.10 0.10 0.07 0.05 0 0
RPE = reference protein equivalent.
39000 480 0 0 0 0 0
8.8 7.1 6.6 6.0 5.5 5.3 5.2
47.8 40.2 38.9 35.3 34.4 33.5 29.1
254
F.H. KRATZER ET AL.
75 ]O 0 70 ~ 65 0 60' ~ ~
LJ eL r~
55. 50. 45.
"~
40,
o E o
35 30.
0--0 Experiment1 A - - Z ~ Experiment2 [--]--I--I Experiment3
\ \~
A
O
45
60
2O 15
6
I
0
15
30
75
90
~
A
105 120 135 150 165 180
Time, minutes outocloved
Fig. 1, Effect o f duration o f autoclaving soya-bean flakes on Coomassie Blue dye-binding activity. RPE, reference protein equivalent.
TABLE 4 Effect of autoclaving soya-bean flakes for different times on their nutritive value ~ ( Experiment 2 ) Autoclaving time (min) Stock diet o 15 30 45 60 120 180 Mean (_+SE)
Gain in body wt. _+SE (g d a y - ~) 31.2 g (+0.85) 19.6 c (+0.89) 27.4 ef (_+0.82) 25.6 fg (+0.82) 24.2 ae (+_0.82) 21.5 c~ (_+0.85) 8.6 b (_+0.82) 3.5 a (_+0.82) 20.1 (0.99)
Feed/gain ratio
Pancreas weight (g kg- ~body wt, )
1.61
3. I a
1.68
6.2 c
1.39
3.5 a
1.51
3.3 a
1.44
3.5 a
1.53
3.7 ab
1.94
4.5 b
2.83
3.9 "b
1.74 (0.12)
'The diet calculated to contain 1.2% lysine was given for 21 days. Statistically significant differences in a column are denoted by different superscripts. Duplicate groups of 6 birds were used.
EVALUATIONOF OVERHEATEDSOYA-BEANMEAL
255
diets containing soya-bean meal autoclaved for about 30 min, and the diet was calculated to contain 1.2% lysine (Table 4). The protein quality significantly deteriorated if the meal was autoclaved for more than 30 min. Pancreas weights, a good indicator of TI activity (Lepkovsky et al., 1959), suggest the highest activity in raw soya-bean meal. Autoclaving of meal for 15 min reduced the assayed TIU as well as pancreas weights. Heating for 60 min or more again tended to increase pancreas weights, possibly because of reduced body weight. A depression in growth of chicks becomes conspicuous with overheated soya-bean meal at a dietary lysine level of 1.2%. It is possible that in Experiment 1, the effect of a reduction in availability of lysine owing to overheating of soya-bean meal was masked by the excess lysine ( 1.4%) in the diet.
Experiment 3 Protein solubility decreased from about 95% for the raw sample to about 65 and 34% after autoclaving for 15 min and 60 min, respectively (Fig. 1; Table 5 ). The Coomassie index also suggested a decrease in protein solubility with increased heating time, and followed the same trends as in Experiments 1 and 2. The TI activity and urease activity were also not detected after 15 min autoclaving. Orange G binding index also decreased with increased duration of autoclaving. The confirmation of the observation that growth depression of chicks owing to overheating of soya-bean meal can be overcome by increasing the dietary lysine by 0.2% was attempted in Experiment 3 (Table 6 ). Raw, and each TABLE 5 Effect of autoclaving soya-bean flakes for different times on some chemically determined indices (Experiment 3 ) Autoclaving time ( m i n )
0 7 15 21 30 45 60
Solubility in 0.2% KOH Kjeldahl (%)
Coomassie (mg RPE l per 100 mg protein)
94.6 68.9 64.6 47.7 47.9 35.8 34.1
67.6 50.2 45.6 33.7 25.7 19.2 17.2
l RPE = reference protein equivalent.
Trypsin inhibitor (units g - l )
Urease activity (pH change)
Orange G binding (rag g - l soya-bean meal)
36 740 700 180 0 0 0 0
1.30 0 0 0 0 0 0
40.8 34.1 32.0 30.0 28.0 26.6 23.2
256
F.H. KRATZER ET AL.
TABLE 6
Effect of autoclaving duration and supplemental lysine on growth of chickens over 21 days, feed intake/gain ratio, and pancreas weights (Experiment 3 ) Autoclaving time (min)
Stock diet 0 7 15 21 30 45 60
Mean (-+SE)
Body weight gain + SE (g day- l )
Feed/gain ratio
Lysine 1.2%
Lysine 1.2%
29.9 be ( _+0.90) 24.0 d (_+0.94) 31.2 ab (_+0.90) 28.7 c (_+0.90) 29.6 be (_+0.90) 29.6 bc (_+0.90) 26.2 'j (-+0.90) 21.3 e (_+0.90) 27.6 (0.45)
Lysine 1.4%
(g kg- l body wt. )
Lysine 1.4%
1.65 25.9 d (_+0.94) 33.5 a (_+0.94) 33.9 a (-+0.90) 31.7 ab (_+0.90) 32.6 a (_+0.90) 32.9 a (_+0.90) 30.0 be (_+0.94) 31.6 (0.43)
Pancreas weight
Lysine 1.2%
Lysine 1.4%
2.2
1.67
1.65
4.6 b
6.4 a
1.37
1.35
2.3 d
3.2 c
1.45
1.36
3.2 c
3.1 ¢
1.38
1.38
3.3 c
3.1 ~
1.44
1.38
1.48
1.41
1.59
1.38 1.51 (0.05)
1.41 (0.04)
~Statistically significant ( P < 0.05 ) differences are denoted in the experiment by different superscripts after the values. Duplicate groups of 6 birds each were used.
of the heat-processed soya-bean meal samples were given in isocaloric and isonitrogenous diets, but containing either 1.2 or 1.4% lysine. Gain in body weight of chicks was significantly ( P < 0.05 ) lower on the diet containing raw soya-bean meal than with diets containing soya-bean meal autoclaved at 121 °C for 7, 15, 21 or 30 min. Growth was not improved by addition of lysine to diets containing raw soya-bean meal, a fact already well established. When the diet contained 1.2% lysine, optimum gain in body weight of chicks was observed after autoclaving for 7 min when some residual trypsin inhibitor activity was still present. The gain was not significantly improved by additional lysine for this meal. A significant ( P < 0 . 0 5 ) growth depression was observed for chicks fed on soya-bean meal autoclaved for 15 min when lysine was calculated to be 1.2%. But this sample was significantly improved by an addition of 0.2% lysine. The resultant gain in body weight was not significantly different from that of chicks fed on the diet containing soya-bean autoclaved for 7 min. A decrease in lysine availability caused by overheating of soya-bean samples by autoclaving up to 45 min was overcome
EVALUATION OF OVERHEATED SOYA-BEAN MEAL
2 57
by an additional 0.2% dietary lysine. This implies that the deterioration in nutritive value of overheated soya-bean meal is mainly due to a reduction in lysine availability caused by Maillard reaction. Feed/gain ratios were not significantly different in this experiment. Pancreas enlargement was significantly exaggerated by additional lysine for chicks fed on raw soya-bean meal. Significantly ( P < 0.05 ) smaller pancreas weight was observed for birds fed on soya-bean meal autoclaved for 7 min only. The lysine supplement increased pancreas weight for unexplained reasons. Further, autoclaving at 7 min or longer overcame the trypsin inhibitor as evidenced by pancreas weights. DISCUSSION
The improvement in growth and reduction in pancreas weight by autoclaving confirm the presence of antinutrients in the raw soya-bean flakes (Ham et al., 1945; Chernick et al., 1948; Liener, 1953; Coates et al., 1970). The reduction in growth with prolonged autoclaving, especially at marginal lysine levels, confirms the reduction in lysine availability (Block et al., 1946; Clandinin et al., 1947) that is probably due to the Maillard reaction (Mauron, 1981). If the three experiments are compared, high TI values are only observed in soya-bean meal samples with Coomassie Blue values above 60 mg RPE per 100 mg protein. The data on gain in body weights for the three experiments when compared with Coomassie Blue values for protein solubility confirm that soya-bean meals with Coomassie Blue values above 65 mg RPE per 100 mg protein gave lower growth rates because the heat treatment had not been TABLE 7
Correlations between various chemically determined indices for the expression Y = a + b X X
Y
Experiment
a
Coomassie Coomassie Coomassie Orange G Orange G
Orange G Orange G Orange G
1 2 3 1 3 1 2 3 2 2 1 2 3
36.0 28.3 19.4 - 197 -56.0 2.4 0.35 13.6 4.47 - 93.0 4247 - 1011 - 147
Coomassie
Coomassie Coomassie Coomassie Formalin Urease Urease Urease
N Solubility N Solubility N Solubility
N Solubility N Solubility Formalin N Solubility TIU TIU TIU
b
r 0.179 0.286 0.304
5.57 8.67 1.03 1.30 1.15 0.062 20.8 19 160 19 004 28 149
0.98 0.98 0.98 0.98 0.97 0.99 0.99 0.98 0.98 0.99 0.92 0.99 0.99
258
F.H. KRATZER ET AL.
sufficient to destroy the growth inhibitors. The Coomassie Blue value for good quality meal should be between 35 and 50 if the lysine in a broiler diet is 1.2%, and between 25 and 50 if the lysine is increased to 1.4%. Relationships between the various chemically determined indices were calculated by linear regression (Table 7 ). Correlation coefficients were always very high ( r = 0 . 9 ) . This suggests that any of the above indices can be used for evaluation of soya-bean meal quality. Protein solubility can be measured either by the Kjeldahl m e t h o d or by the Coomassie Blue index. The data for the TIU and gain in body weight for the three experiments suggest that activity should be under 5000 T I U g - ~. Soya-bean meal has not been overheated if the Coomassie Blue index is above 35. However, if the meal has a zero urease or trypsin inhibitor activity, and Coomassie Blue index is below 35 but above 25, then raising the lysine in the diet to 1.4% for broilers should overcome the effect of overheating o f soya-bean meal. The methods for the determination of TI or urease activity and Coomassie Blue dye-binding are rapid and the combination gives a good indication of underheating and overheating of soya-bean meals, ACKNOWLEDGMENTS We are grateful to Central Soya Company, Fort Wayne, IN for the solvent extracted soya-bean flakes, and to the Pacific Egg and Poultry Association of Modesto, CA for support o f this study. REFERENCES Almquist, H.J. and Maurer, S., 1953. A method for study of the effectsof heat on the nutritive value of soybean oil meal. Poultry Sci., 32: 549-550. American Oil Chemists Society, 1974. A.O.C.S. Tentative method Ba 9-58, Officialand Tentative Methods of the American Oil Chemists Society. E.W. Link (Editor), Champaign, IL. Araba, M. and Dale, N.M., 1990. Evaluation of protein solubility as an indicator of overprocessing of soybean meal. Poult. Sci., 69: 76-83. Association of Official Analytical Chemists, 1975. Official Methods of Analysis. W. Horwitz (Editor), A.O.A.C.,Washington,DC, pp. 11-16 and 130. Bild, H.R., Boucher, R.V., Caskey, Jr., C.D., Hayward, J.W. and Hunter, J.E., 1947. Urease activity and other chemical criteria as indicators of inadequate heating of soybean oil meal. J. Assoc. Off. Agric. Chem., 30: 354-364. Block, R.J., Cannon, P.R., Wissler, R.W., Steffe,Jr., C.H., Straube, R.L. and Woolridge,R.L., 1946. The effect of baking and toasting on the nutritional value of proteins. Arch. Biochem., 10: 295-301. Borchcr, R., Ackerson, C.W. and Musshel, F.W., 1948. Trypsin inhibitor. IV. Effect of various heating periods on the growth promoting value of soybean oil meal for chicks. Poult. Sci., 27: 601-604. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Caskey, Jr., C.D. and Knapp, F., 1944. Methods for determining inadequately heated soybean meal. Ind. Eng. Chem. (Anal. Edn.), 16: 640-641.
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Chernick, S.S., Lepkovsky, S. and Chaikoff, I.L., 1948. A dietary factor regulating the enzyme content of the pancreas: Changes induced in size and proteolytic activity of the chick pancreas by the ingestion of raw soybean meal. Am. J. Physiol., 155: 33-41. Clandinin, D.R., Cravens, W.W., Elvehjem., C.A. and Halpin, J.G., 1947. Deficiencies in overheated soybean meal. Poult. Sci., 26:150-156. Coates, M.E., Hewitt, D. and Golob, P., 1970. A comparison of effects of raw and heated soyabean meal in the diets of the germ-free and conventional chicks. Br. J. Nutr., 24:213-225. Dale, N., Charles, O.W. and Duke, S., 1986. Reliability of urease activity as an indicator of overprocessing of soybean meal. Poult. Sci., 65 (Suppl. 1 ): 164. Ham, W.E., Sandstedt, R.M. and Mussehl, F.E., 1945. The proteolytic inhibiting substance in the extract from unheated soybean meal and its effect upon growth in chickens. J. Biol. Chem., 161: 635-642. Hurrell, R.F., Lerman, P. and Carpenter, K.J., 1979. Reactive lysine in foodstuffs as measured by a rapid dye-binding procedure. J. Food Sci., 44:1221-1227 and 1231. Kakade, M.L., Rackis, J.J., McGhee, J.E. and Puski, G., 1974. Determination of trypsin inhibitor activity of soy products: A collaborative analysis of an improved procedure. Cereal Chem., 51: 376-382. Kratzer, F.H., Bersch, S. and Vohra, P., 1990. Evaluation of heat-damage to protein by Coomassie Blue G dye-binding. J. Food Sci., 55: 805-807. Lepkovsky, S., Bingham, E. and Pencharz, R., 1959. The fate of the proteolytic enzymes from the pancreatic juice of chicks fed raw and heated soybeans. Poult. Sci., 38:1289-1295. Liener, I.E., 1953. Soyin, a toxic protein from the soybean. 1. Inhibition of rat growth. J. Nutr., 49: 527-539. Mauron, J., 1981. The Maillard reaction in foods: A critical review from the nutritional standpoint. Prog. Food Nutr. Sci., 5: 5-35. McNaughton, J.L. and Reece, F.N., 1980. Effect of moisture content and cooking time on soybean meal urease index, trypsin inhibitor content, and broiler growth. Poult. Sci., 59: 23002306. McNaughton, J.L., Reece, F.N. and Deaton, J.W., 1981. Relationship between color, trypsin inhibitor contents, and urease index of soybean meal and effects on broiler performance. Poult. Sci., 60: 393-400. Moran, Jr., E.T., Jensen, L.S. and McGinnis, J., 1963. Dye binding by soybean and fish meal as an index of quality. J. Nutr., 79: 239-244. N.R.C. (National Research Council), 1984. Nutrient Requirements of Poultry. National Academy Press, Washington, DC. Osborne, T.B. and Mendel, L.B., 1917. The use of soybeans as food. J. Biol. Chem., 32: 369387. Pallansch, M.J. and Liener, I.E., 1953. Soyin, a toxic protein from the soybean. II. Physical characterization. Arch. Biochem. Biophys., 45: 366-374. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods. The Iowa State University Press, Ames, IA, 7th edn., pp. 215-237. Veltman, Jr., J.R., Hansen, B.C., Tanksley, Jr., T.D., Knabe, D. and Linton, S.S., 1986. Comparison of the nutritive value of different heat-treated commercial soybean meals: Utilization by chicks in practical type rations. Poult. Sci., 65:1561-1570. Waldroup, P.W., Ramsey, B.E., Hellwig, H.M. and Smith, N.K., 1985. Optimum processing for soybean meal used in broiler diets. Poult. Sci., 64:2314-2320.
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Elsevier Science Publishers B.V., Amsterdam
Chemical and biological evaluation of soya-bean flakes autoclaved for different durations F.H. Kratzer, Sally Bersch, P. Vohra and R.A. Ernst Department of Avian Sciences, University of California, Davis, CA 95616 (U.S.A.) (Received 22 November 1989; accepted for publication 7 May 1990)
ABSTRACT Kratzer, F.H., Bersch, S., Vohra, P. and Ernst, R.A., 1990. Chemical and biological evaluation of soyabean flakes autoclaved for different durations. Anita. Feed Sci. Technol., 31: 247-259. Solvent-extracted soya-bean flakes were autoclaved for 0-180 min at 121 ° C and evaluated by three chick feeding trials and several tests in vitro. Improvement in growth from short periods of heating was related to reduced trypsin inhibitor or urease activity which were highly correlated (r= 0.96 ). After extraction with 0.2% KOH, protein solubility determined by the Kjeldahl nitrogen method was highly correlated ( r = 0.99 ) with that determined by the Coomassie Blue dye-binding method. Protein solubility decreased as the heating duration was increased. A high correlation was also found between Orange G binding and protein solubility (r=0.97); and between Coomassie Blue dye-binding and Orange G binding ( r = 0.98 ). Beyond the initial improvement in growth caused by the destruction of growth inhibitors, the degree of overheating measured by protein solubility, Orange G binding, or formol titration agreed well with decreased growth of chicks. Pancreas weight of < 3.3 g kg-1 body weight is a relatively sensitive test of properly processed soya-bean meal. The effect of overheating soya-bean meal in reducing the growth of chickens was greater in a diet containing 1.2% lysine than when lysine was increased to 1.4%. Coomassie Blue dye-binding and either trypsin inhibitor or urease activity are rapid and convenient methods for evaluating soya-bean meal.
INTRODUCTION
Osborne and Mendel (1917 ) observed that cooked, but not raw, ground soya-beans supported normal growth of rats. Raw soya-bean meal contains antinutrients such as antitrypsin, which depresses growth (Ham et al., 1945; Borcher et al., 1948) and enlarges the pancreas (Chernick et al., 1948; Lepkovsky et al., 1959 ) in chicks, and hemagglutinins or lectins which contribute to growth depression in rats (Liener, 1953; Pallansch and Liener, 1953 ), and chicks (Coates et al., 1970). These are inactivated by heat processing of raw soya-bean meal. Availability of lysine is reduced if soya-bean meal is overheated (Block et al., 1946; Clandinin et al., 1947). During overheating, the epsilon amino group oflysine undergoes a browning or Maillard reaction and lysine availability is reduced (Mauron, 1981 ). 0377-8401/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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F.H. KRATZERETAL.
In addition to trypsin inhibitor, raw soya-bean meal contains another enzyme, urease, which is inactivated by heat processing of the meal. Urease is easily determined by the method of Caskey and Knapp (1944). A residual urease activity to give a change in value of pH of 0.02 is an indicator of properly processed soya-bean meal (Bird et al., 1947 ). The urease test is worthless for detecting overheated meal. The value of the urease test has been questioned by McNaughton et al. ( 1981 ) and Dale et al. (1986), as the gain in body weight of chickens fed on soya-bean samples with zero urease activity was not significantly different from those fed on samples with a residual urease activity giving a change in pH of 0.02. Attempts have been made to find better methods for evaluating processing conditions. These include determination of antitrypsin activity (Kakade et al., 1974) as an indicator ofunderheating, and Orange G dye-binding (Moran et al., 1963), formalin titration (Almquist and Maurer, 1953), and protein solubility in 0.2% KOH (Araba and Dale, 1990) to detect overheating and protein damage. Not all these parameters have been determined on the same samples. A number of reports are available on the optimum heat-processing conditions for soya-bean flakes using laboratory sized autoclaves, with the object of studying relationships between urease activity, antitrypsin activity of the samples, and the growth of chickens fed on these samples (McNaughton and Reece, 1980; McNaughton et al., 1981; Waldroup et al., 1985; Araba and Dale, 1990). Veltmann et al. (1986) compared some commercially available undercooked and overcooked soya-bean meals. A number of these studies used low dietary protein in their feeding experiments. We have extended the studies to compare urease activity, antitrypsin activity, Orange G dye-binding, protein solubility in 0.2% KOH by Kjeldahl and dye-binding methods, formalin titration value, and feeding value for chickens of solvent-extracted soya-bean flakes autoclaved for different times under laboratory conditions in three experiments. MATERIALS AND METHODS
Autoclaving of soya-beanflakes Two lots of solvent-extracted raw flakes were obtained from Central Soya Company, Fort Wayne, IN. In Experiment 1, solvent-extracted raw soya-bean flakes (Lot 1 ) were autoclaved at 121 °C ( 1.055 kg cm -2) for times varying from 3 to 120 min. The autoclave was manually operated and the time was measured when the chamber pressure reached 1.055 kg cm -2. For Experiments 2 and 3, a different autoclave with automatic controls (Castle steam sterilizer, M / C 3423) was used. It was programmed to purge the chamber of air before timing the treatment period. For that reason, the heat treatment in
EVALUATION OF OVERHEATED SOYA-BEAN MEAL
249
Experiments 2 and 3 was somewhat more severe than for an equivalent period of time in Experiment 1. CHEMICAL EVALUATION
Protein solubility The method suggested by Araba and Dale (1990) was used to determine protein solubility in 0.2% KOH. The soluble protein in the supernatant solution was determined by the Kjeldahl method (Association of Official Analytical Chemists, 1975 ). A dye-binding method for soluble protein (Kratzer et al., 1990) measured the capacity of the protein in the KOH filtrate to bind with Coomassie Blue G dye. Lysozyme was used as a reference standard, and the values are expressed as reference protein equivalent (RPE).
Dye-binding Essentially, the method described by Moran et al. ( 1963 ) was used for Crocin Orange G dye-binding. The difference in the intensity of the color of the dye solution before and after reacting with the soya-bean sample indicates the amount of dye bound.
Urease activity The modified method of Caskey and Knapp (1944) as described in American Oil Chemist's Society (1974) was used for the urease determination. It measures the change in pH on incubating the soya-bean meal sample with a buffered urea solution.
Formaldehyde titration The procedure suggested by Almquist and Maurer (1953 ) utilizing titration with base in the presence of formaldehyde was used for evaluating heat damage to soya-bean preparations.
Trypsin inhibitor activity Trypsin inhibitor activity was determined by a modification of the method of Kakade et al. (1974). The soya-bean meal samples were shaken with 0.01 N NaOH for an hour before filtering, and aliquots of the filtrate tested for activity in inhibiting the hydrolysis ofbenzoyl-DL-arginine-p-nitroanilide hydrochloride, a synthetic substrate. The inhibitor activity is expressed in terms of trypsin inhibitor units (TIU).
250
F.H. KRATZERET AL.
BIOLOGICAL EVALUATION
Three feeding experiments were conducted using 2-day-old unsexed broiler (Hubbard) chicks purchased from A & M Hatchery, Santa Rosa, CA. The birds were banded, weighed, and distributed into groups containing 5 chicks each in Experiment 1, and 6 each in Experiments 2 and 3. Each group had approximately the same initial weight. The groups were housed in electrically heated battery cages with the cage temperatures maintained at 35 °C for the first week, 32.2°C for the second, and 29.4°C for the third. Two groups of birds, distributed randomly in the battery cages were fed on each of the experimental diets. Feed and tap water were available at all times. The birds were weighed individually twice weekly. The cumulative feed intake was also measured at that time. At the end of the experiments (Experiment 1,22 days; Experiments 2 and 3, 21 days), birds were killed by cervical dislocation if pancreas weights were to be determined.
Composition of the diets The diets contained coarsely ground raw soya-bean flakes (termed meal), or meal prepared from flakes autoclaved in the manually operated autoclave in Experiment 1, and the automatically controlled autoclave in Experiments 2 and 3. The composition of the diets used in Experiments 1-3 is given in Table 1. The diet in Experiment 1 was calculated to contain about 23% crude protein and 1.4% lysine from a combination of maize and soya-bean meal only. The requirement for lysine for broilers according to the National Research Council (NRC, 1984) is 1.2%. In Experiment 2, calculated lysine level was reduced to 1.2% by incorporating some maize gluten meal into the diets. In Experiment 3, birds were fed on the diets of Experiment 2 and similar diets which were supplemented with lysine to raise its content to 1.4%. The data were checked for statistically significant differences by analysis of variance (ANOVA). The significantly different means were ranked using Duncan's method (Snedecor and Cochran, 1980). RESULTS
Experiment 1 As the autoclaving time increased from 0 to 120 min, the color of the raw soya-bean flakes turned progressively darker. Protein solubility in 0.2% KOH, as determined by the Kjeldahl method, decreased from about 83% for the raw sample to about 32% for the sample heated for 60 min (Table 2). Values for Coomassie Blue dye-binding followed the same trend as Kjeldahl nitrogen values. There is some crossover for the samples autoclaved for 6 and 12 min,
EVALUATIONOF OVERHEATEDSOYA-BEANMEAL
251
TABLE 1 Composition of the diets for Experiments 1-3 Ingredient (g kg- t )
Experiment 1 Lysine 1.4%
Experiment 2 Lysine 1.2%
Maize, ground Soya-bean meal (48% CP) Maize gluten meal (62% CP) Soya-bean oil CaHPO4.2H20 CaCO3 Vitamin pre-mix 4 NaC1, iodized DL-Methionine MnSO4" HaO ZnO Cellulose 5 Lysine
510.7 400.01 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 -
540.0 320.71 50.0 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 -
Calculated composition (%) ME (MJ kg- ~) Crude protein Lysine Methionine Methionine + cystine Calcium Available P
12.93 23.77 1.39 0.60 0.97 1.06 0.59
13.30 23.28 1.19 0.64 1.01 1.05 0.59
Experiment 3 Lysine 1.2%
Lysine 1.4%
537.5
537.5 320.73 50.0 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 2.5
320.73 50.0 40.0 22.0 10.0 10.0 5.0 2.0 0.2 0.1 2.5 13.30 23.26 1.19 0.64 1.01 1.05 0.59
13.30 23.26 1.41 0.64 1.01 1.05 0.59
~Diets contained soya-bean meal autoclaved for 0, 12, 24, or 60 min at 121 °C ( 1.055 kg cm-2). 2Diets contained soya-bean meal autoclaved for 0, 15, 30, 45, 60, 120 or 180 min at 121 ° C ( 1.055 kg cm
- 2 ).
3Diets contained soya-bean meal autoclaved for 0, 7, 15, 21, 30, 45, or 60 min at 121 °C ( 1.055 kg cm-2). 4Vitamin pre-mix supplied the following (in mg kg-t diet): biotin, 0.5; butylated hydroxy toluene, 1000; calcium pantothenate, 30; choline chloride, 1000; folic acid, 5; menadione, 10; niacin, 120; pyridoxine HCI, 10; riboflavin, 10; thiamin HCI, 10; vitamin B 12, 0.01; vitamin A, 10 000 IU; vitamin D3, 4500; vitamin E, 88. 5Solka floc, Brown C., Berlin, NH. CP, crude protein.
possibly owing to inaccuracy in the manual recording of time, after a pressure of 1.055 kg cm -2 was reached in the autoclave. Urease activity was decreased to 32% of the original value after autoclaving for 3 min and to zero after 24 min. Trypsin inhibitor activity (TI) was reduced to about 61 and 37% after autoclaving for 3 and 12 min, respectively, and was still present at a low level in the sample autoclaved for 24 min. Urease is more sensitive to inactivation by heating than TI. Orange G dye-binding index followed the same trend as protein solubility. No significant differences in gain in body weight were observed in chicks
78.0 74,7 75.9
65,2
31.7
13.8
3 6 12
24
60
120
10.7
28.6
64.4
73.6 73.2 72.4
73.1
Coomassie (mg RPE z per 100 mg protein )
0
0
0
0,65 0.50 0.60
2.00
Urease activity (pH change)
0
260
2520
23500 22700 14200
38600
Trypsin inhibitor (units g-~ )
37.0
42.6
47.4
49.0 48.8 49.3
49.5
Orange G binding (mg g- l soya-bean meal)
tThe diet was calculated to contain 1.4% lysine. 2RPE = reference protein equivalent. Significant differences are indicated by different superscripts in a column. Duplicate groups of 5 chicks were used.
83.2
Kjeldahl (%)
Protein solubility (0.2% KOH )
0
Autoclaving time (rain)
Effect of autoclaving soya-bean meal for different times on its nutritional value ~ (Experiment 1 )
TABLE 2
(+_0.71)
Mean 25.5
23.8 ~b ( +_ 1.30) 27.0 ab (+1.23) 28.4 b ( + 1.23)
22.6 a (_+1.23)
Gain in body wt. +_SE (g day- 1)
1,50 (_+0.04)
1.50
1.38
1.56
1.56
Feed/gain ratio
> t-
m
N
bo
EVALUATIONOF OVERHEATEDSOYA-BEANMEAL
253
fed on diets containing soya-bean meal autoclaved for 12, 24, or 60 min at 121 °C. However, chicks on soya-bean meal autoclaved for 60 min had a significantly ( P < 0 . 0 5 ) better weight gain than those fed on raw meal (Table 2 ). The growth data indicate that autoclaving of soya-bean flakes for about 24 min results in optimal growth of chickens, and the growth was not further improved by autoclaving for 60 min, as long as the diets were formulated to contain 1.4% lysine. Significant differences were not observed in the amount of feed needed per unit gain in weight, although there was a trend for improved feed utilization with autoclaving of soya-bean flakes.
Experiment 2 Protein solubility in 0.2% KOH decreased from about 91% in raw samples to about 54% in samples autoclaved for 15 min in the Castle steam sterilizer (Table 3 ), which was a much greater reduction than in the manually operated autoclave (Fig. 1 ), suggesting that purging steam tended to preheat the flakes before the timer became operational. Protein solubility as determined by the Coomassie Blue method followed the same trend as determination by the Kjeldahl method. Urease activity, as well as TI activity, had practically disappeared after autoclaving for more than 15 min. Values for formalin titration as well as Orange G binding index also decreased with an increase in autoclaving time. These reduced indices suggest a reduction in lysine availability (Almquist and Maurer, 1952; Hurrell and Carpenter et al., 1979). An optimum gain in body weight of chicks was observed when they were fed on TABLE 3
Effect of autoclaving soya-bean flakes for different times on some chemically determined indices (Experiment 2)
Autoclaving Protein solubility in time 0.2% KOH (minutes)
Urease activity Trypsin Formalin Orange G binding (pH change) inhibitor (ml 0.05 N (mg g-~ soya(units g - l ) NaOH per 2 g) bean meal)
Kjeldahl Coomassie (%)
(mg RPE l
per 100 mg protein ) 0 15 30 45 60 120 180
90.9 54.1 44.6 30.0 28.9 15.7 12.6
67.3 44.7 32.9 27.4 22.9 11.9 7.7
1.95 0.10 0.10 0.07 0.05 0 0
RPE = reference protein equivalent.
39000 480 0 0 0 0 0
8.8 7.1 6.6 6.0 5.5 5.3 5.2
47.8 40.2 38.9 35.3 34.4 33.5 29.1
254
F.H. KRATZER ET AL.
75 ]O 0 70 ~ 65 0 60' ~ ~
LJ eL r~
55. 50. 45.
"~
40,
o E o
35 30.
0--0 Experiment1 A - - Z ~ Experiment2 [--]--I--I Experiment3
\ \~
A
O
45
60
2O 15
6
I
0
15
30
75
90
~
A
105 120 135 150 165 180
Time, minutes outocloved
Fig. 1, Effect o f duration o f autoclaving soya-bean flakes on Coomassie Blue dye-binding activity. RPE, reference protein equivalent.
TABLE 4 Effect of autoclaving soya-bean flakes for different times on their nutritive value ~ ( Experiment 2 ) Autoclaving time (min) Stock diet o 15 30 45 60 120 180 Mean (_+SE)
Gain in body wt. _+SE (g d a y - ~) 31.2 g (+0.85) 19.6 c (+0.89) 27.4 ef (_+0.82) 25.6 fg (+0.82) 24.2 ae (+_0.82) 21.5 c~ (_+0.85) 8.6 b (_+0.82) 3.5 a (_+0.82) 20.1 (0.99)
Feed/gain ratio
Pancreas weight (g kg- ~body wt, )
1.61
3. I a
1.68
6.2 c
1.39
3.5 a
1.51
3.3 a
1.44
3.5 a
1.53
3.7 ab
1.94
4.5 b
2.83
3.9 "b
1.74 (0.12)
'The diet calculated to contain 1.2% lysine was given for 21 days. Statistically significant differences in a column are denoted by different superscripts. Duplicate groups of 6 birds were used.
EVALUATIONOF OVERHEATEDSOYA-BEANMEAL
255
diets containing soya-bean meal autoclaved for about 30 min, and the diet was calculated to contain 1.2% lysine (Table 4). The protein quality significantly deteriorated if the meal was autoclaved for more than 30 min. Pancreas weights, a good indicator of TI activity (Lepkovsky et al., 1959), suggest the highest activity in raw soya-bean meal. Autoclaving of meal for 15 min reduced the assayed TIU as well as pancreas weights. Heating for 60 min or more again tended to increase pancreas weights, possibly because of reduced body weight. A depression in growth of chicks becomes conspicuous with overheated soya-bean meal at a dietary lysine level of 1.2%. It is possible that in Experiment 1, the effect of a reduction in availability of lysine owing to overheating of soya-bean meal was masked by the excess lysine ( 1.4%) in the diet.
Experiment 3 Protein solubility decreased from about 95% for the raw sample to about 65 and 34% after autoclaving for 15 min and 60 min, respectively (Fig. 1; Table 5 ). The Coomassie index also suggested a decrease in protein solubility with increased heating time, and followed the same trends as in Experiments 1 and 2. The TI activity and urease activity were also not detected after 15 min autoclaving. Orange G binding index also decreased with increased duration of autoclaving. The confirmation of the observation that growth depression of chicks owing to overheating of soya-bean meal can be overcome by increasing the dietary lysine by 0.2% was attempted in Experiment 3 (Table 6 ). Raw, and each TABLE 5 Effect of autoclaving soya-bean flakes for different times on some chemically determined indices (Experiment 3 ) Autoclaving time ( m i n )
0 7 15 21 30 45 60
Solubility in 0.2% KOH Kjeldahl (%)
Coomassie (mg RPE l per 100 mg protein)
94.6 68.9 64.6 47.7 47.9 35.8 34.1
67.6 50.2 45.6 33.7 25.7 19.2 17.2
l RPE = reference protein equivalent.
Trypsin inhibitor (units g - l )
Urease activity (pH change)
Orange G binding (rag g - l soya-bean meal)
36 740 700 180 0 0 0 0
1.30 0 0 0 0 0 0
40.8 34.1 32.0 30.0 28.0 26.6 23.2
256
F.H. KRATZER ET AL.
TABLE 6
Effect of autoclaving duration and supplemental lysine on growth of chickens over 21 days, feed intake/gain ratio, and pancreas weights (Experiment 3 ) Autoclaving time (min)
Stock diet 0 7 15 21 30 45 60
Mean (-+SE)
Body weight gain + SE (g day- l )
Feed/gain ratio
Lysine 1.2%
Lysine 1.2%
29.9 be ( _+0.90) 24.0 d (_+0.94) 31.2 ab (_+0.90) 28.7 c (_+0.90) 29.6 be (_+0.90) 29.6 bc (_+0.90) 26.2 'j (-+0.90) 21.3 e (_+0.90) 27.6 (0.45)
Lysine 1.4%
(g kg- l body wt. )
Lysine 1.4%
1.65 25.9 d (_+0.94) 33.5 a (_+0.94) 33.9 a (-+0.90) 31.7 ab (_+0.90) 32.6 a (_+0.90) 32.9 a (_+0.90) 30.0 be (_+0.94) 31.6 (0.43)
Pancreas weight
Lysine 1.2%
Lysine 1.4%
2.2
1.67
1.65
4.6 b
6.4 a
1.37
1.35
2.3 d
3.2 c
1.45
1.36
3.2 c
3.1 ¢
1.38
1.38
3.3 c
3.1 ~
1.44
1.38
1.48
1.41
1.59
1.38 1.51 (0.05)
1.41 (0.04)
~Statistically significant ( P < 0.05 ) differences are denoted in the experiment by different superscripts after the values. Duplicate groups of 6 birds each were used.
of the heat-processed soya-bean meal samples were given in isocaloric and isonitrogenous diets, but containing either 1.2 or 1.4% lysine. Gain in body weight of chicks was significantly ( P < 0.05 ) lower on the diet containing raw soya-bean meal than with diets containing soya-bean meal autoclaved at 121 °C for 7, 15, 21 or 30 min. Growth was not improved by addition of lysine to diets containing raw soya-bean meal, a fact already well established. When the diet contained 1.2% lysine, optimum gain in body weight of chicks was observed after autoclaving for 7 min when some residual trypsin inhibitor activity was still present. The gain was not significantly improved by additional lysine for this meal. A significant ( P < 0 . 0 5 ) growth depression was observed for chicks fed on soya-bean meal autoclaved for 15 min when lysine was calculated to be 1.2%. But this sample was significantly improved by an addition of 0.2% lysine. The resultant gain in body weight was not significantly different from that of chicks fed on the diet containing soya-bean autoclaved for 7 min. A decrease in lysine availability caused by overheating of soya-bean samples by autoclaving up to 45 min was overcome
EVALUATION OF OVERHEATED SOYA-BEAN MEAL
2 57
by an additional 0.2% dietary lysine. This implies that the deterioration in nutritive value of overheated soya-bean meal is mainly due to a reduction in lysine availability caused by Maillard reaction. Feed/gain ratios were not significantly different in this experiment. Pancreas enlargement was significantly exaggerated by additional lysine for chicks fed on raw soya-bean meal. Significantly ( P < 0.05 ) smaller pancreas weight was observed for birds fed on soya-bean meal autoclaved for 7 min only. The lysine supplement increased pancreas weight for unexplained reasons. Further, autoclaving at 7 min or longer overcame the trypsin inhibitor as evidenced by pancreas weights. DISCUSSION
The improvement in growth and reduction in pancreas weight by autoclaving confirm the presence of antinutrients in the raw soya-bean flakes (Ham et al., 1945; Chernick et al., 1948; Liener, 1953; Coates et al., 1970). The reduction in growth with prolonged autoclaving, especially at marginal lysine levels, confirms the reduction in lysine availability (Block et al., 1946; Clandinin et al., 1947) that is probably due to the Maillard reaction (Mauron, 1981). If the three experiments are compared, high TI values are only observed in soya-bean meal samples with Coomassie Blue values above 60 mg RPE per 100 mg protein. The data on gain in body weights for the three experiments when compared with Coomassie Blue values for protein solubility confirm that soya-bean meals with Coomassie Blue values above 65 mg RPE per 100 mg protein gave lower growth rates because the heat treatment had not been TABLE 7
Correlations between various chemically determined indices for the expression Y = a + b X X
Y
Experiment
a
Coomassie Coomassie Coomassie Orange G Orange G
Orange G Orange G Orange G
1 2 3 1 3 1 2 3 2 2 1 2 3
36.0 28.3 19.4 - 197 -56.0 2.4 0.35 13.6 4.47 - 93.0 4247 - 1011 - 147
Coomassie
Coomassie Coomassie Coomassie Formalin Urease Urease Urease
N Solubility N Solubility N Solubility
N Solubility N Solubility Formalin N Solubility TIU TIU TIU
b
r 0.179 0.286 0.304
5.57 8.67 1.03 1.30 1.15 0.062 20.8 19 160 19 004 28 149
0.98 0.98 0.98 0.98 0.97 0.99 0.99 0.98 0.98 0.99 0.92 0.99 0.99
258
F.H. KRATZER ET AL.
sufficient to destroy the growth inhibitors. The Coomassie Blue value for good quality meal should be between 35 and 50 if the lysine in a broiler diet is 1.2%, and between 25 and 50 if the lysine is increased to 1.4%. Relationships between the various chemically determined indices were calculated by linear regression (Table 7 ). Correlation coefficients were always very high ( r = 0 . 9 ) . This suggests that any of the above indices can be used for evaluation of soya-bean meal quality. Protein solubility can be measured either by the Kjeldahl m e t h o d or by the Coomassie Blue index. The data for the TIU and gain in body weight for the three experiments suggest that activity should be under 5000 T I U g - ~. Soya-bean meal has not been overheated if the Coomassie Blue index is above 35. However, if the meal has a zero urease or trypsin inhibitor activity, and Coomassie Blue index is below 35 but above 25, then raising the lysine in the diet to 1.4% for broilers should overcome the effect of overheating o f soya-bean meal. The methods for the determination of TI or urease activity and Coomassie Blue dye-binding are rapid and the combination gives a good indication of underheating and overheating of soya-bean meals, ACKNOWLEDGMENTS We are grateful to Central Soya Company, Fort Wayne, IN for the solvent extracted soya-bean flakes, and to the Pacific Egg and Poultry Association of Modesto, CA for support o f this study. REFERENCES Almquist, H.J. and Maurer, S., 1953. A method for study of the effectsof heat on the nutritive value of soybean oil meal. Poultry Sci., 32: 549-550. American Oil Chemists Society, 1974. A.O.C.S. Tentative method Ba 9-58, Officialand Tentative Methods of the American Oil Chemists Society. E.W. Link (Editor), Champaign, IL. Araba, M. and Dale, N.M., 1990. Evaluation of protein solubility as an indicator of overprocessing of soybean meal. Poult. Sci., 69: 76-83. Association of Official Analytical Chemists, 1975. Official Methods of Analysis. W. Horwitz (Editor), A.O.A.C.,Washington,DC, pp. 11-16 and 130. Bild, H.R., Boucher, R.V., Caskey, Jr., C.D., Hayward, J.W. and Hunter, J.E., 1947. Urease activity and other chemical criteria as indicators of inadequate heating of soybean oil meal. J. Assoc. Off. Agric. Chem., 30: 354-364. Block, R.J., Cannon, P.R., Wissler, R.W., Steffe,Jr., C.H., Straube, R.L. and Woolridge,R.L., 1946. The effect of baking and toasting on the nutritional value of proteins. Arch. Biochem., 10: 295-301. Borchcr, R., Ackerson, C.W. and Musshel, F.W., 1948. Trypsin inhibitor. IV. Effect of various heating periods on the growth promoting value of soybean oil meal for chicks. Poult. Sci., 27: 601-604. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Caskey, Jr., C.D. and Knapp, F., 1944. Methods for determining inadequately heated soybean meal. Ind. Eng. Chem. (Anal. Edn.), 16: 640-641.
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