Dragan V. Palić a *, Kedibone Y. Modika b, Andre Oelofse c and Marijana B. Sakač a

Transcription

1 MODIFICATION OF THE METHOD FOR DETERMINING PROTEIN SOLUBILITY OF HEAT TREATED FULL-FAT SOYBEANS USING EXTRACTION IN POTASSIUM HYDROXIDE: INTER-LABORATORY STUDY Dragan V. Palić a *, Kedibone Y. Modika b, Andre Oelofse c and Marijana B. Sakač a a University of Novi Sad, Institute of Food Technology, Bulevar Cara Lazara 1, Novi Sad, Serbia b Agricultural Research Council, Private Bag X2, Irene, 0062, South Africa c University of Pretoria, Faculty of Natural Sciences, Lynnwood Road, Hatfield, South Africa Among a number of laboratory methods used to estimate the adequacy of heat treatment applied to full-fat soybean (FFSB) protein solubility in potassium hydroxide (PSKOH) is an unofficial method, but it is used by many laboratories due to its simplicity. It has been reported that the results of PSKOH analysis of the same sample of FFSB on the degree of heat treatment vary widely between laboratories. In this study, the original PSKOH method has been modified and subjected to an inter-laboratory test, with participation of 9 laboratories. Eight FFBS samples were processed by dry extrusion at temperatures ranging from 110 to 164 C and analysed on PSKOH. Processed FFSB samples were also assessed in a growth trial with broilers. Analysis of FFSB by the PSKOH method generated for adequately-processed FFSB values between 67.1 and 76.5%. The values above 76.5% corresponded to under-processed and below 67.1% to over-processed FFSB. The PSKOH method generated good precision, i.e. the repeatability and reproducibility limits of 3.48 and %, respectively. The modified PSKOH method can be recommended as a reliable indicator for quality control of heat processed FFSB for the use in routine laboratory practice. This study could pave the way for establishing PSKOH as an official method for determining the nutritional value of FFSB following heat treatment. KEY WORDS: broilers, protein solubility in potassium hydroxide, inter-laboratory analysis INTRODUCTION The soybean is by far the most important oilseed crop in the world. Soybeans contain highly valuable proteins and oils (crude protein ranging from 39-41% and oil from 18-21%), which make them good feed alternatives to animal proteins and oils. As an animal feed, it is used as a high-protein source (1). Soybean prior to oil extraction is referred to * Correspoding author: Dragan V. Palić, University of Novi Sad, Institute of Food Technology, Bulevar cara Lazara 1, Novi Sad, Serbia, dragan.palic@fins.uns.ac.rs 69

2 as full-fat soybean (FFSB). Raw FFSB contains anti-nutritional factors (ANFs) (2), which limits its use in diets for monogastric animals. The ANFs contained in soybeans may cause unfavourable physiological effects (3, 4), and may decrease weight gain in animals (5). It has been shown (6, 7) that ANFs can be destroyed by moderate heating, which leads to denaturation of tertiary and quaternary protein structures, allowing more effecttive penetration of digestion enzymes and increasing digestibility of soybean protein in non-ruminants. 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. Under-processing of the FFSB limits amino acid availability due to only partial destruction of the ANFs. Over-processing, on the other hand, decreases amino acid availability as a result of the Maillard reaction that occurs between the aldehyde groups of sugars and free amino groups of amino acids (mostly epsilon-group of lysine) (8). The main objective of heat processing of FFSB is to achieve an optimum balance between degradation of ANFs and maintenance of amino acid availability. There are a number of laboratory methods that can be used to estimate the adequacy of FFSB heat treatment. Commonly used methods for assessing the processed FFSB quality are those for the determination of urease activity index (UAI), trypsin inhibitor activity (TIA), nitrogen solubility index (NSI), protein dispensability index (PDI), and protein solubility in potassium hydroxide (PSKOH) (9). It has been demonstrated (10, 11) that the results of analysis of the same sample of FFSB on the degree of heat treatment obtained by currently available analytical techniques vary widely between laboratories, causing uncertainty among soya processors, feed manufacturers and end-users. The provision of amino acids, either free or as protein, contributes a substantial amount to the cost of animal feedstuffs. The objective of any nutritionist is therefore to formulate diets that will provide the correct amounts of nutrients required by the animal at the lowest possible cost (12). This implies that dietary formulations need to be done on an available amino acid basis in order to optimise the dietary amino acid levels, thereby minimizing the cost. As a result of the uncertainties regarding the effect of processing on the amino acid availability of full-fat soybeans (9), some nutritionists using FFSB in diets have been forced to compensate for the possibility of reduced amino acid availability by over-formulating diets on a total amino acid level. It is therefore recognised that, if the full utilisation of FFSB used in diets is to be realised, reliable analytical procedures need to be available for quality control of processed FFSB, which would enable feed manufacturers to determine the exact degree of processing of the soybeans (10). The results of this would in turn provide an estimate of the availability of the amino acids contained in the FFSB for the use in feed formulations and enable nutritionists to lower the cost of feed formulations. There are reports (13, 14) that protein solubility might be the most reliable indicator for FFSB quality control and that therefore NSI, PDI and PSKOH would be the preferred methods. Although being an unofficial method, PSKOH has been widely used by many 70

3 laboratories, due to its simplicity. The preference in this study has been therefore given to PSKOH method. The aim of this study was to standardise, through an inter-laboratory study, the PSKOH method, to establish its ranges for describing under-, adequate- and over-processed FFSB and its precision, i.e. the values for its repeatability and reproducibility limits. EXPERIMENTAL Raw soybeans, with moisture of 10-11%, were processed by dry extrusion, using industrial Insta-Pro 2000R single screw extruder at 7 temperatures: 110, 120, 127, 136, 140, 145, 151 and 164 C. In vivo trial. A total of 336 male Ross broilers were randomly allocated to 42 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 heat treated FFSB, with six replicates per treatment. The average body weight gain (ADWG) in the period from day 0 to day 14 and feed conversion ratio (FCR) on day 14 were monitored as production parameters. The protocol was approved by the Animal Ethics Committee no. APIEC 04/01 (ARC-Irene, South Africa) to ensure that it complies with South African and international standards for the care and use of animals for experimental purposes. Laboratory procedure. The procedure of the original PSKOH method of Araba and Dale (15) was as follows. An amount of 1.5 g of a FFSB sample, ground in a mill so it would pass through a 0.5 mm screen, was mixed with 75 ml of 0.2 % (0.036 N, ph 12.5) potassium hydroxide, and then stirred for 20 min on a magnetic stir plate. The mixture was centrifuged at 2700 rpm for 15 min. The supernatant was decanted, and filtered through glass wool. Fifteen ml, in duplicate from a single filtrate, were transferred to digestion tubes. Total nitrogen was determined by the Kjeldahl method and the protein content was calculated. For the original FFSB samples, the crude-protein content was also determined. Protein solubility was expressed as a percentage of the total protein soluble in a 0.2 % solution of potassium hydroxide. A review of this original PSKOH method revealed the following possible sources of differences in results of analysis of the same FFBS sample obtained by different laboratories. 1. Milling through a 0.5 mm screen. (It was very difficult to obtain a representative sample of FFSB when this is milled through a 0.5 mm screen. This has been shown to be especially true for samples processed above 130 C). 2. Mixing of FFSB sample with 75 ml of 0.2 % KOH. (The volume and shape of the beaker, which both affect the protein solubility, was not specified). 71

4 3. Stirring of the sample on a magnetic stirring plate. (The speed of stirring and the size of magnetic stir bar, have an effect on protein solubility, were not specified). 4. Centrifugation of mixture at 2700 rpm. (Specification of rpm value means little in practice, where different types of centrifuges are used). The modification of the original PSKOH method was then made, which resulted in the following procedure. 1. The FFSB sample was milled using a laboratory hammer or cyclone mill equipped with a 1.0 mm screen. 2. Milled sample was sieved using mm and mm sieves. The fraction that passed through a mm sieve and was retained on a mm sieve was taken for analysis. Thus, improved sample homogeneity was obtained g (± g) of the sieved sample was placed in a 250 ml Erlenmeyer flask (bottom diameter = 85 mm and height = 145 mm) ml of 0.2 % KOH solution was added to the sample using a calibrated pipette and the flask was sealed with a stopper. 5. The flask was placed on a horizontal shaker immediately after addition of the KOH solution and was shaken at 100 cycles per minute for exactly 20 minutes at room temperature. 6. The flask was left for exactly 2 minutes for its content to settle. 7. Without delay, the content of the flask was filtered through a wide-pore filter paper (Whatman no. 1) ml of the supernatant was placed into a 250 ml flask and total nitrogen was determined by Kjeldahl method. Crude protein content (% nitrogen x 6.25), i.e. soluble protein of the sample, was calculated. The crude protein content of the original FFSB sample was also determined. 9. Protein solubility was expressed as a percentage of the total protein soluble in a 0.2 % solution of potassium hydroxide. Eight samples of FFSB used in the in vivo trial with chickens were analysed by 9 laboratories in duplicate using the improved PSKOH method. The method has therefore been subjected to an inter-laboratory study, conducted according to the AOCS Official Methods (16). Data were analysed using the statistical programme SAS/STAT (17). 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. Precision of the improved PSKOH method was determined according to the AOCS Official Methods (18). 72

5 RESULTS AND DISCUSSION In vivo trial The results of broiler trial are shown in Table 1. Table 1. Mean average daily weight gain (ADWG) during the period from day 0 to day 14 and feed conversion ratio (FCR) on day 14 of broiler chickens fed FFSB processed by dry extrusion at different temperatures Treatment ( C) ADWG (g) FCR bc 96.0 bc bc a a a 97.2 b 79.8 c d cd c a a a c cd a,b,c,d Values in the same column with different superscript differ significantly (P<0.05). The results showed that the best performance was achieved by chickens that were fed the FFSB processed at 136, 140 and 145 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 127 and 136 C, as well as at 145 and 151 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 ( C) Under-processed < 136 Adequately-processed Over-processed > 145 It was concluded that the temperatures between 136 and 145 C define the range for adequately-processed FFSB. Inter-laboratory results Average values of PSKOH in FFSB samples analysed by 9 laboratories in duplicate are shown in Table 3. 73

6 Table 3. Results of the determination of protein solubility in potassium hydroxide (PSKOH) in FFSB samples processed by dry extrusion at different temperatures 1 PSKOH (%) Lab No 110ºC 120ºC 127ºC 136ºC 140ºC 145ºC 151ºC 164ºC Average SD Average of two replicates; SD = Standard deviation The influence of temperature of processing on PSKOH values is shown in Fig. 1. A high correlation (R 2 = 0.94) was established between PSKOH values and treatment temperature. 100 PSKOH (%) y = x R² = RSD = Temperature ( 0 C) Figure 1. The influence of processing temperature on PSKOH values The FFBS samples processed in this study at temperatures between 136 and 145 C represented adequately-processed FFSB (Table 1). The PSKOH values for these samples obtained in the inter-laboratory study, shown in Table 3, were 76.5 and 67.1, respectively. Consequently, the PSKOH values for describing the degree of FFSB processing have been established and are shown in Table 4. 74

7 Table 4. PSKOH values for describing the degree of FFSB processing Degree of FFSB processing PSKOH (%) Under-processed > 76.5 Adequately-processed Over-processed < 67.1 The PSKOH range for adequately-processed FFSB was between 67.1 and 76.5%. PSKOH values above 76.5 % corresponded to under-processed, whereas below 67.1% corresponded to over-processed FFSB. Precision parameters of the PSKOH method are shown in Table 5. The results obtained under the conditions of this study showed that the repeatability limit (r), i.e. absolute difference between two single results of analysis of the same sample obtained in one laboratory, should not exceed 3.48 %. It was found that the reproducibility limit (R) of the PSKOH method, i.e. absolute difference between single results of analysis of the same sample obtained in different laboratories, should not exceed %. Table 5. Precision parameters of the PSKOH method Parameter Number of laboratories Number of laboratories retained after eliminating outliers PSKOH values (%), average of 9 laboratories Repeatability standard deviation (s r ), % Repeatability relative standard deviation (RSD r ), % Repeatability limit (r) [r = 2,8 x s r ], % Reproducibility standard deviation (s R ), % Reproducibility relative standard deviation (RSD R ), % Reproducibility limit (R) [R = 2,8 x s R ], % Sample 110ºC 120ºC 127ºC 136ºC 140ºC 145ºC 151ºC 164ºC Average values According to the in vivo trial with broilers (Table 1), the samples which represent adequately-processed FFSB were those processed between 136 and 145 C. The PSKOH values for those temperatures were between 67.1 and 76.5%. These results are lower than those established by Arabe and Dale (15). According to their results, a range of protein solubility between 80 and 85 % appeared to be consistent with optimum soybean processing for broiler chickens. Values below 75 % indicated excessive heating. Globally accepted PSKOH values for adequately processed soybean are between 75 and 85 % (9). It is to be noted that the above-mentioned authors obtained their values in single laboratory 75

8 studies, contrary to the present study, which might be the explanation for the differences in the results. There is a good correlation (R 2 =0.94) between the PSKOH values and extrusion temperatures. A reduction in the protein solubility in 0.2 % KOH was found to be associated with an increased heat treatment in soybeans and a decreased growth performance in chickens (19). The PSKOH changed consistently as soybeans were heated at higher temperatures. The only slightly inconsistent response of PSKOH was observed at 151 C, where the value was 66.62%, thus being on the border of the range of adequately- and over-processed FFSB. The same FFSB sample was assessed as over-processed in the in vivo trial. Previous studies by Parsons et al. (20), Anderson-Haferman et al. (21) and Batal et al. (22) reported that PSKOH is a good indicator of FFSB over-processing. Regarding results of the present study on precision of the PSKOH method, two single determinations performed in one laboratory should not differ by more than 3.48 %, whereas two single determinations performed in different laboratories should not differ by more than %. Attempts to find in accessible literature the results on precision parameters for the PSKOH method obtained elsewhere were unsuccessful. The optimum temperature for dry extrusion of full-fat soybean for use in poultry feeding obtained in this study (between 136 and 145 C) is in line with the results of Ruiz et al. (6) who established, in a trial with broilers fed extruded FFSB, that body weight gain and feed conversion ratio were best at treatment temperatures between 126 and 140 C. Nelson et al. (23) stated that temperatures most commonly used commercially for extruding raw soybeans were between 135 and 140 C. 76 CONCLUSION Inter-laboratory analysis of FFSB by the improved PSKOH method generated for adequately-processed FFSB values between 67.1 and 76.5 %. Values above 76.5 % corresponded to under-processed and below 67.1 % to over-processed FFSB. Improved PSKOH method generated good precision, i.e. repeatability and reproducibility limits of 3.48 and %, respectively. Based on the results of this inter-laboratory study, the PSKOH method can be recommended as a reliable indicator for quality control of heat processed FFSB, for the use in routine laboratory practice. This study could pave the way for establishing PSKOH as an official method for determining the nutritional value of FFSB following heat treatment. Acknowledgement This study was supported by the Protein Research Foundation of South Africa. REFERENCES 1. Wiseman, J.: Recent Advances in Animal Nutrition, Butterworths, London (1986) p. 2.

9 2. Maclachlan, R.G.: Soybeans: Antinutrients - What? Why? Workshop on processing of full fat soya, 12 March 1998, Irene, South Africa, Proceedings pp Buttle, L.G., Burrells, A.C., Good, J.E., Williams, P.D., Southgate, P.J. and Burrells, C.: The binding of soybean agglutinin (SBA) to the intestinal epithelium of Atlantic salmon, Salmo salar and Rainbow trout, Oncorhynchus mykiss, fed high levels of soybean meal. J. Veter. Immun. Immunopath. 80 (2001) Vasconcelos, I.M., Maia, A.A.B., Siebra, E.A., Oliveira, J.T.A., Carvalho, A.F.F.U., Melo, V.M.M., Carlini, C.R. and Castelar, L.I.M.: Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of anti-nutritional and toxic proteins. J. Nutr. Biochem. 12 (2001) Palacios, M.F., Easter, R.A., Soltwedel, K.T., Parsons, C.M., Douglas, T., Hymowitz, M.W. and Pettigrew, J.E.: Effect of soybean variety and processing on growth performance of young chicks and pigs. J. Anim. Sci. 82 (2004) Ruiz, N., De Belalcazar, F., Diaz, G.J.: 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 Wiseman, J.: Influence of processing of full fat soya beans included in diets for piglets. I. Performance. Anim. Feed. Sci. Tech. 118 (2005) Vohra, F. and Kratzer, F.K.: Evaluation of soybean meal determines adequacy of heat treatment. Feedstuffs 63 (1991) Monary, S., Fullfat Soya Handbook, 2 nd ed., American Soybean Assosiation, Brussels (1996) pp Davies, H.: Manufacturing of full-fat soybeans. Workshop: Processing of full-fat soya, 12 March 1998, Irene, South Africa, Proceedings, pp Palić, D., Morey, L., Modika, K.Y., Kokić, B., Djuragić, O., and Spasevski, N.: Precision of laboratory methods based on protein solubility in quality control of heat treated feedstuffs. Chem. Industry 66, 1 (2012) Palic, D., Moloto, K., Coetzee, S.E. and Djuragic, O.: Critical assessment of laboratory methods for full -fat soybean quality control. 1 st International Congress on Food Technology, Quality and Safety, November 2007, Novi Sad, Serbia, Proceedings, pp Peisker, M. and Dersjan-Li, Y.: Best use of soy proteins. Feed-mix 12 (2004) Caprita, R., Caprita, A. and Cretescu, I.: Protein solubility as quality index for processed soybean. Anim. Sci. Biotech. 43 (2010) Araba, M. and Dale, N. M.: Evaluation of protein solubility as an indicator of over processing of soybean meal. Poultry Sci. 69 (1990) AOCS Official Methods: Collaborative Study Procedures, AOCS Procedure M 4-86, SAS/STAT. SAS Institute Inc.: User's Guide, Version 6, 4th Edition, Volume 2, Cary, NC:SAS Institute, AOCS Official Methods: Determination of Precision of Analytical Methods, AOCS procedure M 1-92, Wiriyaumpaiwong, S., Soponronnarit, S. and Prachayawarakorn, S.: Comparative study of heating processes for full-fat soybeans. J. Food Engineer. 65 (2004)

10 20. Parsons, C.M., Hashimoto, K., Wedekind, K.J. and Baker, D.H.: Soybean protein solubility in potassium hydroxide: An in-vitro test of in-vivo protein quality. J. Anim. Sci. 69 (1991) Anderson-Haferman, J.C., Ahang, Y., Parsons, C.M. and Hymowitz, T.: Effect of heating on the nutritional quality of Kunitz-trypsin-inhibitor-free and conventional soybeans for chicks. Poultry Sci. 71, 10 (1992) Batal, A.B., Douglas, M.W., Engram, A.E. and Parsons, C.M.: Protein dispersibility index as an indicator of adequately processed soybean meal. Poultry Sci. 79 (2000) Nelson, A.I., Wijeratne, W.B., Weh, S.W., Wei, T.M. and Wei, L.S.: Dry extrusion as an aid to mechanical expelling of oil from soybeans. J. Am. Oil Chem. Soc. 64 (1987) МОДИФИКАЦИЈА МЕТОДЕ ЗА ОДРЕЂИВАЊЕ РАСТВОРЉИВОСТИ ПРОТЕИНА У ТЕРМИЧКИ ТРЕТИРАНОЈ ПУНОМАСНОЈ СОЈИ КОРИШЋЕЊЕМ ЕКСТРАКЦИЈЕ У КАЛИЈУМ ХИДРОКСИДУ: МЕЂУ- ЛАБОРАТОРИЈСКО ИСПИТИВАЊЕ Драган В. Палић a, Кедибоне Модика б, Андре Оелофсе в и Маријана Б. Сакач a a Универзитет у Новом Саду, Институт за прехрамбене технологије, Булевар цара Лазара 1, Нови Сад, Србија б Agricultural Research Council, Јужно-афричка република в Универзитет у Преторији, Јужно-афричка република Међу методама за процену адекватности термичког третмана пуномасне соје је и растворљивост у калијум хидроксиду (PSKOH), која је неофицијелна метода, али је многе лабораторије користе због њене једноставности. У овом раду PSKOH метода је модификована и примењена у 9 лабораторија. Осам узорака пуномасне соје је екструдирано на температурама од 110 до 164 C и анализирано модификованом PSKOH методом. Исти узорци су испитани и у in vivo огледима на пилићима. Анализом пуномасне соје модификованом PSKOH методом су добијене вредности од 67,1 до 76,5% за адекватно третирану соју. Вредности растворљивости изнад 76,5% одговарају недовољно термички третираној соји, а испод 67,1% сувише третираној. Модификована PSKOH метода је генерисала добру прецизност: репетабилност од 3,48% и репродуктивност од 10,86%. Модификована PSKOH метода се може препоручити као поуздан индикатор за контролу квалитета термички третиране соје. Кључне речи: бројлери, растворљивост протеина у калијум хидроксиду, интерлабораторијска анализа Received: 25 July 2012 Accepted: 28 September