PROTOCOL FOR USING PROTEIN SOLUBILITY AS AN INDICATOR OF FULL-FAT SOYBEAN HEAT TREATMENT. Dragan V. Palić and Sophia E. Coetzee

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1 PROTOCOL FOR USING PROTEIN SOLUBILITY AS AN INDICATOR OF FULL-FAT SOYBEAN HEAT TREATMENT Dragan V. Palić and Sophia E. Coetzee When the degree of full-fat soybean (FFSB) processing is determined using protein solubility as an indicator of heat treatment extent, a problem represents the lack of a standard with known value of protein solubility, against which the protein solubility of heat treated FFSB would be determined. Also, a special practical problem imposes the fact that universal ranges of units for describing the degree of FFSB processing are used globally, without taking into consideration specific regional differences. In this paper, a protocol was proposed for establishing unit ranges for defining under-, adequately- and over-processed FFSB when protein solubility is used as an indicator of the extent of heat treatment. KEY WORDS: Full-fat soybeans, degree of processing, extent of heat treatment, protein solubility INTRODUCTION The use of full-fat soybeans (FFSB) in animal feeds has been limited because of the uncertainty of the exact availability of the amino acids. This arises due to both the presence of biologically active substances with an anti-nutrient action, which are contained in raw soybean, as well as the effect that processing has on the availability of the amino acids contained therein. Processing of the raw FFSB by means of heat destroys the anti-nutrients, thus making them fit for use in monogastric diets. The problem relating to the availability of the amino acids in the heat-treated soybeans arises due to the fact that only an optimum level of heat treatment will produce maximal availability of the amino acids to the animal. Both under- and over-processing result in decreased availability of amino acids (4). Amongst other authors, Holmes (1), Ruiz et al. (2) and Zarkadas et al. (3) showed that moderate heating is necessary to increase the digestibility of soybean protein for nonruminants. A comparatively mild heating leads to partial protein degradation (denaturation of tertiary and quaternary structures), allowing more effective penetration by digestion enzymes. One of the major concerns is: what happens when FFSB is under- or over-processed? Is the one more detrimental than the other? To define under- and over-processing is easy Dr. Dragan Palić, dragan.palic@fins.uns.ac.rs, Institute for Food Technology, Bulevar Cara Lazara 1, Novi Sad, Serbia; Sophia E. Coetzee, M. Sc., Agricultural Research Council, ARC-Animal Production Institute, Private Bag X2, Irene 0062, South Africa 71

2 in theoretical terms, but is it easy to define it in practice? The following mechanisms are involved in under- and over-processing: Under-processing: Residual trypsin inhibitor mediates its effects via the digestive processes, affecting both endogenous and exogenous amino acid losses. It also binds and inactivates the pancreatic enzyme trypsin (4). The result is that protein digestibility is reduced and swelling of the pancreas occurs, caused by the production of additional trypsin and chymotrypsin. Over-processing: In this process, proteins are more than partially denaturated and amino acid availability is reduced. This is because the Maillard reaction takes place, i.e. reducing sugars react with the epsilon-amino group of lysine (4). As a consequence, the objective of heating processes for full-fat soybeans, intended for inclusion in diets for poultry and pigs, is to maintain optimum balance between degradation of anti-nutrients on the one hand and maintenance of protein digestibility on the other. Commonly used methods for assessing the processed FFSB quality are those for the determination of: Urease Activity Index (UAI) 2. Trypsin Inhibitor Activity (TIA) 3. Protein Solubility in KOH (PSKOH) 4. Nitrogen Solubility Index (NSI) 5. Protein Dispersibility Index (PDI) 6. Lysine availability In a critical assessments of methods, Palic et al. (5, 6), established that some of commonly used methods, e.g. UAI, have limitations, as they can be used only to determine the under-processed FFSB, that some of the methods are very complicated to perform, such as TIA and Lysine availability, and concluded that protein solubility is the best indicator for FFSB quality control and that therefore PSKOH, NSI and PDI methods would be the prefferd choice. A problem represents the fact that there is a lack of a standard with known protein solubility, against which the protein solubility of processed FFSB would be determined. Special practical problem imposes the fact that universal ranges of units for PSKOH, PDI and NSI methods, for describing the under-, adequately- and over-processed FFSB, are used globally, without taking into consideration specific regional differences (7). The aim of this study was to develop a protocol for establishing unit ranges for defining under-, well- and over processed FFSB, when protein solubility is used as an indicator of the extent of heat treatment. EXPERIMENTAL In the absence of a standard with known value of protein solubility, the solution is to use an indirect standard, which can be obtained through in vivo trial with animals. Therefore, the proposed protocol for establishing the degree of FFSB processing, when protein solubility is used as an indicator of heat treatment extent, consists of the following steps.

3 Step 1. Raw soybeans processing A number, but not less than five, samples of FFSB processed at different temperatures, i.e. exposed to different extents of heat, is produced. In this study, raw soybeans, with moisture of 10-11%, were processed by dry extrusion, using industrial Insta-Pro 2000R single screw extruder at 8 temperatures: 110, 120, 127, 136, 140, 145, 151 and 165 C, with the processing time ranging between 30 and 40 seconds. Step 2. In vivo trial Samples of FFSB produced in Step 1 are fed to animals and their performance is monitored. In the work presented, a total of 384 male Ross broilers were randomly allocated to 48 pens, each containing 8 birds. On arrival, all broilers were sorted into equal weight groups, and assigned at random to the different treatment pens, such that initial average weight and weight distribution were similar for all pens. They were allocated to one of eight dietary treatments containing the processed FFSB. The average body weight gain (ADWG), in the period from 0 to 14 days of age, and feed conversion ratio (FCR), on day 14, were monitored as production parameters. Step 3. Choice of laboratory method A laboratory method for determining protein solubility is chosen. In this study, as a model-method for determining protein solubility as an indicator of the extent of soybean processing, the Protein solubility in potassium hydroxide (PSKOH), as described by Palic (8), has been chosen. Eight samples of FFSB processed in Step 1 were analysed for PSKOH in duplicates by five laboratories. Step 4. Establishing ranges of units for chosen method for describing the degree of FFSB processing The ranges of units for chosen method, for describing under-, adequately- and overprocessed FFSB, are established. An illustration of the relation between in vivo animal performance, measured by average daily weight gain (g), and the PSKOH values (in %) is shown in Figure 1. Assuming that the FFSB samples at 135 o C and 145 o C are adequately processed, the values X and Y of PSKOH (%) for those two samples, represent the border-points of the range of adequately-processed FFSB. Consequently, unknown FFSB sample, whose PSKOH protein solubility value falls between X and Y points, would be assessed as adequately-processed. 73

4 Average daily weight gain (g) KOH (%) X Y Temperatures ( C) Fig 1. Illustration of the relation between in vivo animal performance, measured by average daily weight gain (g), and the KOH protein solubility (%) for FFSB samples processed at different temperatures Statistical analysis. Data were analyzed using the statistical program SAS/STAT (9). The experiment was designed as a randomized complete block with five replicates per treatment. Analysis of variance (ANOVA) was used to test for differences between treatments. Treatment means were separated using Fishers' protected t-test least significant difference (LSD) at the 5 % level of significance. RESULTS AND DISCUSSION The results of the in vivo trial are shown in Table 1 and Figure 2. Table 1. Average body weight gain (ABWG) and feed conversion ratio (FCR) for chickens fed FFSB processed at different temperatures in the period from 0 to 14 days of age AWG Temperature ( o C) ABWG (g) FCR (kg/kg) 110 C 120 C 127 C 136 C 140 C 145 C 151 C 164 C 87.8 bc 96.0 bc bc a a a 97.2 b 79.8 c d cd c a a a c cd SEM 1 LSD 2 CV% a,b,c,d Values in the same column with different superscript differ significantly (P<0.05); 1 SEM = Standard error of the means; 2 LSD = Least significant difference; 3 CV% = Coefficient of variation KOH 40

5 Body weight gain (g) Well prosessed FFSB range Feed conversion ratio (kg/kg) Temperature (ºC) In-vivo body weight gain In-vivo FCR Fig. 2. Average daily body weight gain in the period from 0 to 14 days of age, and feed conversion ratio on day 14, of broiler chickens fed FFSB processed at different temperatures Statistical analysis of the results showed that the best performance was achieved by chickens that were fed the FFSB processed at 136 o C, 140 o C and 145 o 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 C and 136 o C, as well as at 145 o C and 151 o C, were significant (P<0.05). Based on these parameters, a 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 2. Table 2. Relation between the temperature of extruding and the in vivo assessment of the degree of FFSB processing Degree of FFSB processing Temperature of extrusion ( o C) Under-processed < 136 Adequately - processed Over-processed > 145 The results of the protein solubility in potassium hydroxide (PSKOH) are shown in Table 3. 75

6 Table 3. Results of the determination of protein solubility in potassium hydroxide (PSKOH) in FFSB samples processed by dry extrusion at different temperatures 76 Temperature ( o C) PSKOH (%) C C C C C C C C Mean values of the results obtained in five laboratories FFBS samples processed at temperatures between 136 o C and 145 o C, represented adequately-processed FFSB (Table 1). The mean values for PSKOH for these two samples, obtained at five laboratories (Table 3), were % and 67.14%, or for the practical application, 77 % and 67% respectively. Therefore, the following ranges, shown in Table 4, for describing the degree of FFSB processing when PSKOH method is used, have been established. Table 4. Ranges for describing the degree of FFSB processing using PSKOH method Degree of FFSB processing PSKOH (%) Under-processed >77% Adequately- processed 67% - 77% Over-processed <67% CONCLUSION Using the protocol described in this study, the ranges for any laboratory method which uses protein solubility as an indicator of the extent of FFSB heat treatment, can be established. The numerical value of the units for the method(s) established by using the proposed protocol, take into consideration regional differences such as soybean quality and may be safely applied for FFSB quality control, regardless of what the globally accepted unit ranges for the specific method(s) are. ACKNOWLEGMENT This study has been supported by the Protein Research Foundation of South Africa. REFERENCES 1. Holmes, B: Quality control of raw materials and final products in fullfat soybean production. Proc. of Fullfat Soybean Regional Conference, Milan, April 1987, Book of Abstracts, p. 246.

7 2. Ruiz, N., F. de Belalcazar and G. J. Diaz: Quality Control Parameters for Commercial Full-Fat Soybeans Processed by Two Different Methods and Fed to Broilers. J. Appl. Poult. Res. 13 (2004) Zarkadas, L. N. and J. Wiseman: Influence of processing of full fat soya beans included in diet for piglets. 1. Performance. Animal Feed Science and Technology 118 (2005) Monary, S.: Fullfat Soya Handbook, American Soybean Association, Brussels (1989) p Palic, D., K. Moloto, E. S. Coetzee and O. Djuragic, O: Critical assessment of laboratory methods for full-fat soybean quality control. 1 st International Congress on Food Technology, Quality and Safety, Novi Sad, November 2007, Proceedings p Palic, D., J. Levic, S. Sredanovic, O. Djuragic: Quality control of full-fat soybeans using urease activity: critical assessment of the method. Acta Periodica Technologica, 39 (2008) Palic, D: Quality control of processed full-fat soybeans: Choice of method. XI International Feed Technology Symposium, Vrnjacka Banja, 30 March 3 June 2005, Proceedings p Palic, D: Quality control of processed full-fat soybeans using protein solubility in KOH: Critical review and modification of the method. 11 th International Feed Technology Symposium, Vrnjacka Banja, 30 March 3 June 2005, Proceedings p SAS/STAT User's Guide, Version 8, SAS Institute Inc., Cary, NC:SAS Institute (1999). ПОСТУПАК ЗА КОРИШЋЕЊЕ РАСТВОРЉИВОСТИ ПРОТЕИНА КАО ИНДИКАТОРА ТЕРМИЧКОГ ТРЕТМАНА ПУНОМАСНЕ СОЈЕ Драган В. Палић и Sophia E. Coetzee Када се растворљивост протеина користи као индикатор степена термичке обраде пуномасне соје, проблем представља недостатак стандарда у односу на који би се одређивала растворљивост протеина обрађеног сојиног зрна. Посебан практичан проблем представља чињеница да се за изражавање различитих степена термичког третмана, примењује опсег јединица које су глобално прихваћене, не водећи при томе рачуна о регионалним разликама у квалитету сирове соје. У овом раду је предложен поступак за утврђивање опсега јединица за дефинисање недовољно, адекватно и сувише термички обрађене пуномасне соје, када се растворљивост протеина користи као индикатор степена термичког третмана. Received 17 June 2009 Accepted 28 September