APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53 QUALITY CONTROL OF FULL-FAT SOYBEAN USING UREASE ACTIVITY: CRITICAL ASSESSMENT OF THE METHOD Dragan V. Palić, Jovanka D. Lević, Slavica A. Sredanović and Olivera M. Đuragić A critical assessment of the method for determination of urease activity, as an indicator of the degree of full-fat soybean (FFSB) processing, has been undertaken. FFSB was processed by dry extrusion at five temperatures, ranging from 5 0 C to 65 0 C, and analysed for urease activity by two laboratories and by two analysts at each laboratory, using the ph-difference procedure. The in vivo trial with chickens fed the processed FFSB has also been conducted. While the results of two analysts at each laboratory did not differ significantly (P>0.05), there was a significant difference (P<0.05) in urease activity results between the two laboratories. The overall conclusion of this study is that the urease activity determined by ph-difference method can not be recommended as a reliable indicator for FFSB quality control. KEYWORDS: Full-fat soybean, dry extrusion, degree of processing, quality control, urease activity INTRODUCTION The soybean is by far the most important oilseed crop in the world. Crude protein content of about 38%, with high content of essential amino acids, oil content of minimum 8% and low content of fibre, make soybean a superior feedstuff. As an animal feed, it is used as a protein source (). If the oil is not extracted from the beans, it is referred to as full-fat soybean (FFSB). Raw FFSB contains anti-nutritional factors () which make it unfit for feeding of monogastric and immature ruminant animals. Processing of the raw FFSB by means of heat and mechanical treatment destroys the anti-nutrients. Only an optimum level of heat treatment will produce maximal availability of the amino acids to the animal, thus securing the best animal performance. Insufficient heating, i.e. underprocessing of the FFSB limits amino acid availability due to only partial destruction of the anti-nutritional factors (ANFs). On the other hand excessive heating (over-proces- Dr. Dragan V. Palić, Sen. Res., Agricultural Research Council, Animal Production Institute, Private Bag X, Irene 006, South Africa, palic@arc.agric.za; Dr. Jovanka D. Lević, Sen. Res., Slavica A. Sredanovic, B. Sc., Olivera M. Đuragić, M.Sc., University of Novi Sad, Institute for Food Technology of Novi Sad, Cara Lazara, 000 Novi Sad, Serbia 47
APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53 sing) decreases amino acid availability as a result of the Maillard reaction that occurs between the aldehyde groups of sugar and free amino groups (3). The main objective of heat processing of FFSB is to achieve an optimum balance between degradation of ANFs and maintenance of amino acid availability. The aim of processed FFSB quality control is to establish if that balance has been achieved. There are a number of analytical procedures for processed FFSB quality control (4). Official methods are Urease Activity (5), Protein Dispersibility Index (6) and Nitrogen Solubility Index (7). Commonly used unofficial methods are those for determining Trypsin Inhibitor Activity (8), Available Lysine (9) and Protein Solubility in Potassium Hydroxide (0). It has been clearly illustrated (, ) that there are problems associated with the application of these methods in practice, such as that the use of the same method by different laboratories gives different results of analyses of the same FFSM sample, which makes confusion among the FFSB processors, feed manufacturers and end-users. The AOCS Official Method Ba. 9-58 (5) for the determination of urease activity is based on measurement of ph change and, due to its simplicity, is probably the most widely used method for the FFSB quality control in laboratories. The aim of this study was to assess this widely used method and make appropriate recommendations. MATERIAL AND METHODS Full-fat soybean Full-fat soybean was processed by dry extrusion at five different temperatures: 5C 0, 5C 0, 35C 0, 45C 0, and 65C 0. Urease activity determination Urease activity was determined according to the AOCS official procedure (5). 00 mg of FFSB sample was incubated in 0.0 ml of phosphate buffered urea solution at 30 0 C for 30 minutes, after which the increase in ph units ( ph) from ph 7.00 was recorded. Five processed FFSB samples were analysed by two laboratories (Lab A and Lab B) and by two analysts in each laboratory. Seven replicates of each sample were done by each analyst. In vivo trial A total of 5 diets, containing the FFSB processed at different temperatures, and a control diet without FFSB, were used. Ross 788 as hatched chickens were housed in floor pens, with 0 birds per pen and 6 replicates per diet, thus a total of 70 chickens were used. The birds received the diets from the hatching to the 4 th day, when they were weighed along with the reminding feed. The total body weight gain (BWG) and feed conversion ratio (FCR) were recorded. Statistical analysis The SAS statistical package (3) has been used to analyse the data. 48
APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53 RESULTS AND DISCUSSION Results of the in vivo trial are shown in Table. and Fig.. Table. Average body weight gain (BWG) and feed conversion ratio (FCR) of chickens fed FFSB processed at different temperatures in the period from 0 to 4 days of age Temperature ( 0 C) BWG (g) FCR (kg/kg) 5 0 C 9. bc.953 bc 5 0 C 05. b.735 c 35 0 C 35.5 a.350 a 45 0 C 38.6 a.335 a 65 0 C 85.3 c.899 c SEM CV% 8.9 7.5 0.07 3. a,b,c Means in the same column with common superscript do not differ significantly (P>0.0) SEM = Standard error of means CV% = Coefficient of variation 50.5 40 Body weight gain - BWG (g) 30 0 0 00 90 80 70 Well-processed FFSB range.5 0.5 Feed conversion ratio - FCR (kg/kg) 60 0 0 5 0 5 30 35 40 45 50 55 60 65 70 Temperature (ºC) BWG (g) FCR (kg/kg) Figure. Average body weight gain (BWG) and feed conversion ration (FCR) of chickens fed FFSB processed at different temperatures in the period from 0 to 4 days of age Statistical analysis of the results showed that the best performance was achieved by chickens that were fed the FFSB processed at 35 0 C and 45 0 C and that there was no significant difference between them (P>0.05). However, the difference between the groups that received the FFSB processed at 5 0 C and 35 0 C, as well as 45 0 C and 65 0 C, were significant (P<0.05). Based on these parameters, the relation between the temperature of extruding and the in vivo assessment of the degree of FFSB processing has been derived, and is shown in Table. 49
APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53 Table. Relation between the temperature of extruding and the in vivo assessment of the degree of FFSB processing Degree of FFSB processing Temperature of extrusion ( 0 C) Under-processed < 35 Adequately processed 35 45 Over-processed > 45 Results of urease activity determined by two analysts at each of the two laboratories (Lab A and Lab B) are shown in Table 3. Table 3. Urease activity (expressed as ph) in FFSB processed at different temperatures obtained by two analysts in Lab A and Lab B T 0 C 5 0 C 5 0 C 35 0 C 45 0 C 65 0 C LAB A x.63 a.4 a 0.436 b 0.430 b 0.073 c 0.087 c 0.030 d 0.03 d 0.08 d 0.09 d SD 0.035 0.060 0.03 0.03 0.008 0.005 0.008 0.008 0.005 0.0 LAB B x.886 a.866 a 0.3 b 0.47 b 0.07 c 0.066 c 0.044 d 0.043 d 0.039 d 0.03 d SD 0.030 0.09 0.03 0.09 0.00 0.00 0.005 0.008 0.0 0.004 a,b,c,d Means in the same raw with common superscript do not differ significantly (P>0.0); x = Means of seven replicates; SD = Standard deviation The application of the Student s t test on urease activity results obtained by the two analysts in Lab A and Lab B, confirmed that the two analysts results, at each laboratory and for all five temperatures, did not differ significantly (P>0.05). However, the results of the determination of urease activity were significantly different (P < 0.05) between Lab A and Lab B for the samples processed at 5 0 C and 5 0 C (Table 4). Table 4. Urease activity in FFSB processed at different temperatures obtained by two laboratories Urease Activity ( ph) Temperature ( 0 C) Lab Lab 5 0 C.89 a.876 b 5 0 C 0.433 a 0.39 b 35 0 C 0.080 c 0.069 c 45 0 C 0.06 c 0.044 c 65 0 C 0.08 c 0.035 c a,b,c Means without common superscript in the same row differ significantly (P<0.0) Mean of values obtained by two analysts in a laboratory 50
APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53 The relation between the urease activity determined by the two laboratories and the body weight gain of chickens in in vivo trial is shown in Fig. 3..5 60 Urease activity (ph-difference).0.5.0 0.5 Well-processed FFSB range 40 0 00 80 60 40 0 Body weight gain - BWG (g) 0.0 0 0 5 0 5 30 35 40 45 50 55 60 65 70 Temperature (ºC) Lab Lab BWG (g) Figure 3. Relation between the urease activity (expressed as ph) determined by two laboratories and the body weight gain (BWG) of chickens in in vivo trial Globally accepted values for urease activity in describing the degree of FFSB processsing (4) are shown in Table 6. Table 6. Globally accepted relation between the degree of FFSB processing and urease activity Degree of FFSB processing Urease Activity ( ph) Under-processed > 0.0 Adequately processed 0.05-0.0 Over-processed < 0.05 It has to be noted that a value of ph=0 does not necessarily indicate that the FFSB was over-processed, as it has been shown in trials with chickens (5). Based on the results of this study, the following conclusions can be derived:. Urease activity, as an indicator of the degree of FFSB processing, can be used to identify only under-processed FFSB (Fig. 3) and its use in FFSB quality control is therefore limited. 5
APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53. Whereas there was no significant difference (P>0.05) between results obtained by analysts in each laboratory for all five samples, there was a significant difference (P<0.05) in results of urease activity obtained by the two laboratories for the samples processed at 5 0 C and 5 0 C, which is the ground for questioning the reliability of this method. 3. The urease activity values fell below ph=0., thus indicating adequately-processed FFSB (Table 6) much before the temperature reached 35 0 C, an actual entry point to well-processed FFSB temperature range (Fig. 3). 4. In addition, it has to be noted that when the increase in ph is used as an indicator of the degree of FFSB processing, data may be influenced by whether or not the FFSB had been treated with organic acids, preservatives or sterilizing agents prior to the processing. CONCLUSION The overall results of this study show that, if determined by the ph-difference procedure, the value of urease activity in monitoring the FSSB processing is highly questionable and it therefore can not be recommended as a reliable indicator for the FFSB quality control. REFERENCES. Wiseman, J.: Recent Advances in Animal Nutrition. Butterworths, London (986), p... Maclachlan, R.G.: Soybeans: Antinutrients. Symposium: Processing of full fat soya. Irene, South Africa, March 998, Proceedings p. 0. 3. Palic, D. Laboratory methods for determining the degree of soya processing: South African reality. AFMA Simposium: Use of soya in animal feeds, Pretoria, South Africa, 3 August 004, Proceedings p. 77 4. Monary, S.: Fullfat Soya Handbook, American Soybean Association, Brussels (989), p. 6. 5. AOCS Official Methods (997). Sampling and Analysis of Oilseed By-products, method Ba 9-58. 6. AOCS Official Methods (999). Sampling and Analysis of Oilseed By-products, method Ba 0-65. 7. AOCS Official Methods, (997). Sampling and Analysis of Oilseed By-products, method Ba -65. 8. Kakade, M.L., J.J. Rackis, J.E. Mc Gee and G. Puski: Determination of trypsin inhibitor activity of soy products: A collaborative analysis of an improved procedure. Cereal Chem. 5 (974) 376. 9. Carpenter, K.J.: The Estimation of the Available Lysine in Animal-Protein Foods. Biochem. J. 77 (960), 604. 5
APTEFF, 39, - (008) UDC: 633.34:66.0.39:543.645 DOI: 0.98/APT0839047P BIBLID: 450-788 (008) 39, 47-53 0. Araba, M., N.M. Dale: Evaluation of protein solubility as an indicator of under-processing of soybean meal. Poultry Sci. 69 (990) 749.. Davies, H.: Manufacturing of full-fat soybeans. Symposium: Processing of full fat soya. Irene, South Africa, March 998, Proceedings p... Palic, D., A.A. Grove: Validation of Methods for Determining the Degree of Soya Processing: Preliminary study. nd Joint Congress of Grassland Society of Southern Africa and South African Society of Animal Science, Gaudini Spa, South Africa, 8 June July 004, Book of Abstracts, p. 8. 3. SAS/STAT User's Guide, Version 8, SAS Institute Inc., Cary (999), NC:SAS Institute. 4. Palic, D.: Quality control of processed full-fat soybeans: Choice of method. XI International Feed Technology Symposium, Vrnjačka Banja, 30 March 3 June 005, Proceedings p. 96. 5. Araba, M., N.N. Dale: Evaluation of protein solubility as an indicator of over processing of soybean meal. Poultry Sci. 69 (990) 76. АКТИВНОСТ УРЕАЗЕ КАО ИНДИКАТОР КВАЛИТЕТА ПУНОМАСНЕ ПРЕРАЂЕНЕ СОЈЕ: КРИТИЧКА ОЦЕНА МЕТОДЕ Драган В. Палић, Јованка Д. Левић, Славица А. Средановић и Оливера М. Ђурагић У овом раду је дата критичка оцена методе базиране на одређивању активности уреазе, која се широко примењује у лабораторијама за праћење квалитета пуномасне прерађене соје. Сирова соја је прерађена сувим екструдирањем на пет различитих температура у опсегу од 5 0 C до 65 0 C. Узорци прерађене соје су анализирани у две лабораторије, од стране два аналитичара у свакој лабораторијa, методом за одређивање активности уреазе која се заснива на мерењу пораста ph вредности. Док се резултати два аналитичара, у свакој од лабораторија, нису међусобно значајно разликовали (P>0.05), разлике у вредностима активности уреазе у узорцима соје прерађене на 5 0 C и 5 0 C између две лабораторије су биле значајне (P<0.05). На основу приказаних резултата, закључено је да се активност уреазе, одређена методом заснованом на мерењу ph разлике, не може препоручити као поуздан индикатор за праћење квалитета пуномасне прерађене соје. Received 0 November 007 Accepted 8 January 008 53