Evaluation of Protein Solubility as an Indicator of Overprocessing Soybean Meal

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1 Evaluation of Protein Solubility as an Indicator of Overprocessing Soybean Meal M. ARABA and N. M. DALE 1 Poultry Science Division, University of Georgia, Athens, Georgia 362 (Received for publication December 23, 1988) ABSTRACT Three experiments were conducted to compare urease activity (UA), orange G-binding capacity (OGBC), and protein solubility (PS) in a potassium-hydroxide solution of soybean meal (SBM) as indicators of overprocessing. In two additional experiments, amino-acid supplementation was employed in an attempt to overcome the growth depression associated with overheating SBM. Subsamples of commercially prepared lots of dehulled SBM were heated for varying periods of time in an autoclave at 121 C. In Experiments 1,2, and 3, UA dropped to zero (ph change) after approximately 1 min of cooking. The assay was unable to differentiate between meals that were heated for longer periods. The OGBC of SBM was decreased by autoclaving. However, die narrow range of the orange G values that corresponded to degrees of heat treatment indicated that this test may be of limited value in identifying overprocessed SBM. By contrast, PS values decreased with each increase of autoclaving time, and did not approach zero even with 8 min of heating. Adding.2% lysine either singly or in combination with.2% arginine or.1% methionine, or both, totally overcame the growth depression due to overprocessing. The weight gains and feed conversions of broiler chicks were negatively correlated with autoclaving times in excess of 1 minutes. In all experiments, PS values more closely reflected the impaired chick performance from overheated SBM than UA or OGBC. The PS values below 7% suggested overprocessed SBM. (Key words: soybean meal, overprocessing, protein solubility, urease activity, orange G binding) INTRODUCTION As has long been recognized, soybean meal (SBM) may reduce its nutritive value (Evans and McGinnis, 1946; Block et al., 1946; Clandinin et al., 1947; Fritz et al., 1947; Balloun et al., 1953; Moran et al, 1963; Hill and Olsen, 1967; Warnick and Anderson, 1968). Damage to essential amino acids such as lysine (Warnick and Anderson, 1968) and arginine (Renner et al., 1953) by the excess heating of SBM markedly reduces its protein availability to poultry. The most common assay used to evaluate SBM for overprocessing is the urease-activity test. Urease levels per se are of little interest to poultry nutritionists, but they are used as indicators of the possible presence of toxic factors such as trypsin inhibitors. However, Abraham et al. (1971) noted that the ureaseactivity test does not reflect the intensity of the heat treatment on SBM quality after the urease enzyme has been totally inactivated. Moreover, total destruction of urease in SBM does not necessarily result in an impairment of chick l To whom correspondence should be addressed Poultry Science 69:76-83 growth (McNaughton and Reece, 198; Dale et al., 1986). Soybean meal with urease activity as low as.1 for a ph change did not result in reduced chick performance, compared to SBM with a higher urease value (De Schrijver, 1977). In addition, low urease activity in SBM can also be the result of a lengthy storage of underheated meals (De Schrijver, 1977). From these reports, two basic problems with urease activity as a measure of overprocessing for SBM are apparent. First, low levels of urease activity are not necessarily correlated with impaired chick performance. Second, because urease activity at zero is not always indicative of heat-damaged meal and because there is no negative scale in the urease test, the assay is of no value in determining the relative severity of overprocessing. Other in vitro tests such as Cresol red (Olomucki and Bornstein, 196) and assays of orange G binding (Udy, 1956; Moran et al, 1963) were suggested as means of determining protein quality. The capacity of these compounds to bind proteins increases or decreases, respectively, when the products are subjected to increasing heat treatment. Smith and Circle (1938) demonstrated that an alkali solution can extract up to 95% of the nitrogenous matter of oil-free SBM. Olcott and

2 SOYBEAN OIL MEAL OVERPROCESSING AND PROTEIN SOLUBILITY 77 Fontaine (1941) showed that the solubility of cottonseed protein in a dilute, sodium-chloride solution was decreased by heating. Also, Lyman et al. (1953) determined that a similar relationship exists with cottonseed heating; generally, its protein solubility was associated with impaired chick growth when the chicks were fed cooked cottonseed meals. Sometimes the protein-solubility assay has been applied to SBM by the poultry industry. Rinehart, working with Purina Mills Inc., St. Louis, MO in the late 196's, apparendy was the first to employ protein solubility in potassium hydroxide as a way of evaluating the SBM processing. During the past decade, the assay has gained wide popularity in Brazil and possibly other countries. Unfortunately, since the use of this technique has been virtually all by private companies, an evaluation of its application has yet to appear in the scientific literature. The purpose of the present studies was to evaluate the protein solubility of SBM in a.2% solution of potassium hydroxide as means of identifying overheated SBM with reduced nutritive value. In addition, the effect on chick performance of supplementing the diet with amino acid when the diet contained SBM of varying protein solubility was evaluated by means of trials in vivo. MATERIALS AND METHODS General. Experiments 1, 2, and 3 were conducted in order to compare various chemical tests and their ability to correlate closely with chick performance. In two additional studies, Experiments 4 and 5, amino-acid supplementation was employed in an attempt to overcome the growth depression associated with meals in which the solubility of the protein was low. Five lots of commercially produced, dehulled, solvent-extracted SBM were obtained from a local feed mill. To simulate overheating, subsamples of each lot were spread in shallow (2.54 cm) aluminum pans and were processed in an autoclave at 121 C for varying lengths of time. Meals were emptied from pans after autoclaving and airdried. In Vitro Analyses. Urease activities were determined following the method described by Caskey and Knapp (1944). The dye-binding procedure for lysine availability was that of Moran et al. (1963). Protein solubility in potassium hydroxide was determined as follows: 1.5 g (± 1 g) of a SBM sample, ground in a Udy mill (Udy Corporation, Boulder, CO) so it would pass through a.5- mm screen, mixed with 75 ml of.2% (.36 normal, ph 12.5) potassium hydroxide, then stirred for 2 min on a magnetic stir plate. The mixture was centrifuged at 2,7 rpm for 15 min. The supernatant was decanted, avoiding centrifugate, and filtered through glass wool. About ml were recovered in a 5 ml beaker. Fifteen ml, in duplicate from a single filtrate, were transferred to Kjeldahl tubes giving a.3-g aliquot of the original sample (1.5 g x 15 ml per 75 ml). Twelve-and-a-half ml of concentrated H2SO4, 2 Kjeltab, and 2 ml of 3% H2O2 were added to each tube. Total nitrogen was determined by the Kjeldahl method, and the protein content was calculated. For the original samples, the crude-protein content was also determined. Protein solubility was expressed as a percentage of the total protein soluble in a.2% solution of potassium hydroxide. In Vivo Trials. All experiments used dayold, male broiler chicks obtained from a commercial hatchery. The chicks were allotted to experimental treatments with 5 pens of 8 birds each. The chicks were distributed so that the average body weight per pen was approximately equal. Replicate pens were randomly assigned to each experimental treatment. The birds were maintained in thermostatically controlled, Petersime battery brooders (Petersime Co., Gettysburg, OH) for 3 wk. (Experiment 1 lasted only 18 days.) Feed and water were supplied on an ad libitum basis. Body weight and feed consumption were recorded at 15 and 18 days of age in Experiment 1, and at 2 and 3 wk of age in subsequent trials. Weight gains and feed conversions were determined. Mortality was recorded daily. Each SBM sample was incorporated in a practical basal diet. The same formulation was used in all experiments (Table 1). The diets were designed to be limiting in protein (17.5%) and especially in lysine (.81%); also, to be sensitive to the possible destruction of these nutrients caused by overheating for starting broiler chicks. Using the Statistical Analysis System (SAS Institute, 1985), the data from each experiment were analyzed on a pen basis by a one-way ANOVA. Significant differences between treatment means were estimated by using Duncan's multiple range test (1955).

3 78 ARABA AND DALE TABLE 1. Composition and calculated composition of basal diet, Experiments 1 to 5 Ingredient Ground yellow corn Dehulled soybean meal 1 Corn gluten meal Defluorinated phosphate Poultry fat Ground limestone Sodium chloride Vitamin premix Mineral mix 3 DL-methionine Total Calculated composition ME, kcal/kg CP, % Lysine, % Methionine, % Methionine + cystine, % Percentage , Subjected to different autoclaving times at 121 C. Vitamin premix provides (per kilogram of diet): vitamin A, 5,5 IU; vitamin D 3, 1,1 ICU; vitamin E, 11IU; riboflavin, 4.4 mg; Ca pantothenate, 12 mg; nicotinic acid, 44 mg; choline chloride, 22 mg; vitamin B12, 6.6 ig; menadione, 1.1 mg (as MSBC); folic acid,.55 mg; d-bioun,.11 mg; thiamine, 2.2 mg (as thiamine mononitrate); pyridoxine, 2.2 mg (as pyridoxine HC1); and ethoxyquin, 125 mg per kg of diet. ^race-mineral mix provides (mg/kg of diet): Mn, 6; Zn, 5; Fe, 3; Cu, 5; I, 1.5. In Experiment 1, subsamples of SBM (49.3% protein, determined) were autoclaved for, 5, 1, 2,, and 8 min. All subsamples were analyzed for urease activity, dye-binding capacity, and protein solubility. Chicks were fed diets containing 2% of each SBM sample, according to the design already described. Experiment 2 was identical to Experiment 1, except that autoclaving times of, 4, 8, 12, 16, and 2 min were employed in order to simulate more closely the effect of mild overprocessing. Because autoclaving the meal did not produce a significant growth depression among chicks in Experiment 2, the SBM samples were cooked for a longer time (, 5, 1, 2, and min) in Experiment 3. Experiment 4 was conducted to study the effect of amino-acid supplementation on the performance of chicks fed SBM with reduced protein solubility. Samples of SBM were autoclaved for, 2, and min. Two diets were prepared with meals from each cooking time. One served as a control; the other was supplemented with.2% L-lysine,.2% L- arginine, and.1% DL-methionine. Thus, six dietary treatments were employed. The aminoacid mixture replaced an equivalent amount of corn in the basal rations. As a positive growth response to lysine, arginine and methionine supplementation was noted in Experiment 4. Experiment 5 was conducted to determine which amino acid would be the most limiting under the conditions extant. In addition to two control diets containing SBM that had been autoclaved for to min, the chicks were fed seven diets containing SBM that had been autoclaved for min supplemented with.2% L-lysine,.2% L-arginine, and.1% DL-methionine, individually or in all combinations. RESULTS The deleterious effect of prolonged heating on the nutritive value of SBM was shown repeatedly during the investigations reported here. The degree to which the meal could be heat-damaged was tested by urease activity, dye-binding capacity, and the protein solubility of the meal. Experiment 1. The urease activity of the SBM used in this trial was.3 units of ph change prior to any further processing in the autoclave (Table 2). With 5 min of autoclaving, the urease activity dropped to.2 and was zero for processing times of 1 min or longer. The capacity of the SBM to bind Orange G was decreased by heat treatment. The reduction was only 4.4 points (mg/g of meal) between the nonautoclaved meal and that which had been autoclaved for 8 min. The protein solubility of the meal in a solution of.2% potassium hydroxide before autoclaving was 86%. After subsequent heating, the percentage of solubility dropped steadily, reaching.8% at 8 min of autoclaving time. At 18 days of age, the growth of the chicks decreased as the autoclaving time increased (Table 2). These differences became significant (P<.5) following 1 min of processing, with significant decreases observed from 1 to 2, 2 to, and to 8 min. Feed conversions became poorer as the autoclaving time increased. Experiment 2. The SBM used in this study had an initial urease activity of.2 units of ph change (Table 3). After 4 min of autoclaving, the value dropped to.1. Further heating reduced the value to zero. The dye bound to

4 SOYBEAN OIL MEAL OVERPROCESSING AND PROTEIN SOLUBILITY 79 TABLE 2. Effect of autoclaving of soybean meal on chick performance (1 to IS days) and on protein solubility, urease activity, and orange G-binding capacity, Experiment 1 Treatment (min) Weight gain 45" 445 a 424 a 393 b 316 c 219 d Feed:gain ratio 1.79 c IXl** 1.83^ 1.89 b 2.4 b 2.55 a Protein solubility Urease activity (ph units of change).3.2 a-di Means within each column with no common superscripts are significantly different (P<,5). the meal was diminished from 77.1 mg/g of meal to 71.3 by cooking for 16 min. Meals cooked for 16 and 2 min had identical binding capacities for orange G. By contrast, protein solubility decreased with each increase in autoclaving time from to 16 min. No significant differences (P<.5) in either weight gain for the chicks or feed conversion with any of the test diets were observed (Table 3). No differences were observed in the ratios for feed conversion. Experiment 3. In this trial, the urease activity of the SBM fed to chicks to 3 wk of age was units of ph change before any further heating in the autoclave (Table 4). Since there is no negative scale in the urease test, further heating produced no change in activity. The capacity of the meal to bind orange G decreased from 76.4 mg/g of meal to 74.8, to 71.7, to 7.9, and finally to 7. as the autoclaving time increased from to 5, 1, 2, and min, respectively. The protein solubility of the meal decreased markedly with each increment of heat-treatment time, from 82.3% Orange G bound (mg/g of meal) to 72.6, 66.9, 6.5, and 46.1%, respectively. An increase in autoclaving time from 1 to 2, and from 2 to min caused reductions in weight gain and in feed conversion which were significant (P<.5) when the meal had been autoclaved for min. Experiment 4. Prior to autoclaving, the meal used in this study had a urease activity of.2 units of ph change (Table 5). This value dropped to.1 when the meal was autoclaved for 2 min. Autoclaving for min did not cause any further decrease in the urease activity of the meal. When SBM was autoclaved for, 2, and min, its capacity to bind orange G was 74.8, 71.4, and 71.8 mg/g of meal, respectively. Autoclaving decreased protein solubility. Body weight gains were significantly (P<.5) reduced by feeding SBM autoclaved for 2 or min, but the feed-to-gain ratios did not differ significantly (Table 5). Supplementing diets containing the meals autoclaved for, 2, and min with a mixture of.2% lysine,.2% arginine, and.1% methionine TABLE 3. Effect of autoclaving of soybean meal on chick performance (1 to 21 days) and on protein solubility, urease activity, and orange G-binding capacity. Experiment 2 Treatment (min) Weight gain' Feed:gain ratio Protein solubility Urease activity (ph units of change).2.1 'No significant differences were observed at the.5 level of probability within each column. Orange G bound (mg/g of meal)

5 8 ARABA AND DALE TABLE 4. Effect of autoclaving of soybean meal on chick performance (1 to 21 days) and on protein solubility, urease activity, and orange G-binding capacity. Experiment 3 Treatment Weight gain Feed:gain ratio Protein solubility Urease activity Orange G bound (min) ab 452 a 444 a 5 b 254 c 2.26 b 2.12 b 2.24 b 2.36 b 2.6 a (ph units of change) (mg/g of meal) c Means within each column with no common superscripts are significantly different (P<.5). Treatment (min) significantly (P<.5) improved BW gains. The greatest improvement in weight gain was achieved by adding amino acid to the diet with meal that had been autoclaved for min. For the diet with SBM autoclaved for 2 min, the relative improvement in weight gain from amino-acid supplementation was intermediate between that observed at and min. Also, feed conversions were consistendy improved when diets were supplemented with amino acids. Experiment 5. The urease activity of the SBM used in this experiment was units of ph change before any autoclaving heat was applied (Table 6). Heating for min decreased the meal's orange G-binding capacity from 78.6 to 75.1 mg/g of meal. Protein solubility was reduced from 8.3 to 48.2%. Feeding the meal autoclaved for min significantly reduced weight gain and increased the feed-to-gain ratio. Adding.2% arginine or.1% methionine, either individually or in combination, produced weight gains that did not differ from those for groups fed overprocessed, unsupplemented meal. However, overprocessed SBM supplemented with.2% lysine, either singly or in combination with.2% arginine or.1% methionine, or both, resulted in a significantly improved weight gain compared to that of chicks fed the unsupplemented, overprocessed meal or the unsupplemented, nonautoclaved meal. Thus, weight gains were significandy (P<.5) improved in treatments employing lysine. No further improvement resulted from the addition of either methionine or arginine. DISCUSSION Previous experiments at the authors' laboratory indicated that commercial SBM samples TABLE 5. Effect of supplementing lysine, methionine, and arginine in overprocessed soybean meal on chick performance (1 to 21 days). Experiment 4 Weight gain 453 c 548 a 41 d 531 a 324 e 493 b Improvement Feed:gain ratio 2 ab 1.77 bc 2.8" 1.68 c 2.1" 1.86 abc Protein solubility Urease activity (ph units of change) Orange G bound 2 (mg/g of meal) a_e Means within each column with no common superscript are significantly different (P<.5). 'Protein solubility in a dilute solution of potassium hydroxide. 2range G-binding capacity of the meal. 3 Diet supplemented with.2% L-lysine (98.5% lysine-hcl),.2% L-arginine (98% arginine-hcl) and.1% DL-methionine in addition to supplementation of the basal diet.

6 SOYBEAN OIL MEAL OVERPROCESSING AND PROTEIN SOLUBILITY 81 TABLE 6. Effect of lysine, arginine, or methionine supplementation, or all three, on chick performance, Experiment 5 Results, 21 days Amino-acid supplement 2 Weight Feed Treatment 1 Lysine Arginine Methionine gain ratio (nun) with urease activity values of units of ph change did not necessarily result in reduced nutritive value for broiler chicks (Dale et al., 1986). This observation was supported by the results of Experiment 2. The weight gains of chicks fed diets containing SBM with urease levels of.2 and.1 units of ph change did not differ from those for others fed SBM with a zero urease activity. As demonstrated by McNaughton and Reece (198), urease destruction during heat treatment precedes lysine degradation. In Experiments 1 and 2, the urease-activity values were quickly decreased by autoclaving and reached zero. Thus, these values could not differentiate between the effects of further heating on the nutritive value of SBM as reflected by chick growth. The urease test is the traditional assay used to evaluate SBM for overprocessing; however, as has been demonstrated repeatedly, the test is not applicable to the detection of over-heated meals (Caskey and Knapp, 1944; Bird et al., 1947; Balloun et al, 1953). McNaughton et al. (1981) provided further evidence that urease activity does not adequately reflect overprocessing. The results of the present study provide additional evidence that the urease-activity test is not a reliable indicator of either overprocessing or the degree of overprocessing for SBM. In all five experiments, protein solubility in a dilute solution of potassium hydroxide b a 52" 529 a 53 l a 2.7" " 2.25 bc 2.33 ab 2.36 ab 2 d 2.7 cd 1.95 d 1.87 d Means within each column with no common superscripts are significantly different (P<.5). ^otein solubility, urease activity (units of ph change), and orange G-binding capacity of the meal (mg/g of meal) autoclaved for and min were 8.3 and 48.2, and, and 78.6 and 75.1, respectively. 2 Lysine (98.5% L-lysine HC1), arginine (98% L-arginine HC1), and DL-methionine replaced an equivalent amount of com in the basal diet. Methionine added in addition to that in the basal diet. decreased markedly and steadily with each increase in processing time. In contrast to urease activity, the solubility test reflected the effect of greater heating throughout the total range of times used in these studies. Even with severe overheating (8 min, Experiment 1), the protein solubility values did not approach zero. Thus, the protein-solubility assay overcomes a basic disadvantage of urease activity in that the former is able to distinguish between degrees of overprocessing. This marked decrease in protein solubility in an alkaline solution supports the findings of del Cueto et al. (196), who reported a decrease in the solubility of chick-pea flour protein in aqueous sodium hydroxide after autoclaving the flour; and of Lyman et al. (1953), who reported a relationship between the solubility of cottonseed-meal protein in a solution of.2 N of sodium hydroxide and the nutritional value of the meal as evaluated by chick growth. In Experiments 1 and 3 of the present series, significantly depressed weight gains were associated with SBM that had reduced protein solubility. Neither study, however, showed urease activity of the overheated meals as a useful indicator of impaired nutritional value. This observation was most notable in Experiment 3, in which the urease activity of the control feed was zero. In Experiment 2, solubility decreased with each increase in heating time; but no significant (P<.5) differ-

7 82 ARABA AND DALE ences in body weight gain were observed. The reduced capacity to bind orange G associated with increased autoclaving times agrees with the findings of Moran et al. (1963). Orange G, under acidic conditions, binds specifically either to free amino-group, imidazole-group, or guanidyl-group (Fraenkel- Conrat and Cooper, 1944). On that basis, and since lysine was shown to be damaged most by heat (Experiment 5), the present results agree with Moran et al. (1963). As the epsilon amino-group of lysine is increasingly bound to reducing sugars by an increase in heat treatment (Mauron, 1981), the low capacity of the meal to bind orange G after autoclaving suggests that lysine has been made unavailable. In the present studies, binding capacity was more useful than urease activity in reflecting the degree of heat treatment. However, the difference in dye-binding values for meals heated over markedly different time periods was quite narrow. In Experiment 1, for example, identical dye-binding values were obtained when the SBM samples were heated for 2 and for min. By contrast, protein solubility was reduced from 65.4 to 48.1% in the same samples. Thus, the narrow scale of the orange G-binding values corresponding to gradually rising heat treatments indicates that this test may not be able to differentiate well between overprocessed SBM. Under the conditions of the present series of experiments, protein solubility more closely reflected the overprocessing of SBM and the corresponding chick response than did urease activity or the orange G-binding assays. The positive response of broiler chicks to the addition in the mixture of methionine, arginine, and lysine to nonautoclaved SBM was expected since the diets were formulated to be deficient in protein, especially lysine. The greater improvement in growth rate observed when overprocessed SBM was supplemented with the three amino acids suggests that at least one of them was partially destroyed or rendered unavailable by heat (Experiment 4). This observation was confirmed in Experiment 5. Additions of lysine singly or in combination with methionine or arginine, or both, totally overcame the effects of overheating the meal. Similar results were obtained by Block et al. (1946), Fritz et al. (1947), Balloun et al. (1953), Moran et al. (1963), Hill and Olsen (1967), Wamick and Anderson (1968), and others. The performance of chicks fed overprocessed SBM supplemented with arginine or methionine, or both, did not differ from those fed unsupplemented meal, indicating that arginine and methionine did not become limiting under the conditions of the present study. The protein-solubility assay described here seems to be preferable to urease activity and the orange G-binding tests for detecting overprocessed SBM. Solubility values closely reflected the degree of heating and the impaired chick performance associated with overprocessed SBM. Ford and Shorrock (1971) stated that free amino-groups (mainly the epsilon amino-group of lysine) react with other groups to form enzyme-resistant, inter- and intra-molecular bonds, thereby reducing the solubility and digestibility of the protein. These effects seem to explain the decrease in protein solubility wkhin potassium hydroxide produced by longer autoclaving time, making that decrease specifically related to the nutritional value of SBM. Results obtained in these experiments indicate that protein solubility in a solution of.2% potassium hydroxide detects overprocessed SBM well and differentiates effectively between degrees of overprocessing for SBM. Meals with protein solubility values below 7% probably are of impaired nutritive value for the chick. Values of less than 65% almost certainly indicate overprocessing. ACKNOWLEDGMENT The encouragement and able assistance of Egerton Whittle is gratefully acknowledged. REFERENCES Abraham, J., J. Adrian, C. Calet, G. Charlet-Lery, J. Delort- Laval, J. Guillaume, M. Gutton, J. Lougnon, A Rerat, and S. Z. Zelter, Heat treatment and quality of soybean protein. VIII. Sensitivity of three animal species (rat, pig and chicken) and of various biochemical tests to the intensity of the heat treatment of the oil meal. Ann. Zootech. (Paris) 2: Balloun, S. L., E. L. Johnson, and L. K. Arnold, Laboratory estimation of the nutritive value of soybean oil meals. Poultry Sci. 32: Bird, H. R., R. F. Boucher, C. D. Caskey, J. W. Hayward, and J. F. Hunter, Urease activity and other chemical criteria as indicators of inadequate heating of soybean oil meal. J. Assoc. Off. Agric. Chem. 3: Block, R. J., P. R. Cannon, R. W. Wissler, C. H. Steffee, Jr., R. R. Straube, and R. L. Woodridge, The effect of baking and toasting on the nutritional value of

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