Impact of Steam Treatment on Protein Quality Indicators of Full Fat Soybeans from Different Origins

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Impact of steam treatment on protein quality indicators of full fat soybeans from different origins

Pieter Bos

20-10-2019

Wageningen University

ASG - Animal Nutrition Group

Impact of steam treatment on protein quality indicators of full fat soybeans from different origins

Author : Bos, P.

Registration nr. : 940221104030

Code : ANU-80436

Supervisor(s) : A.F.B. van der Poel, G. Bosch

Wageningen, Oktober 2018

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No part of this publication may be reproduced or published in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the head of the Animal Nutrition Group of Wageningen University, The Netherlands.

Summary The production of soybeans in the EU-28 in 2016 was 2.4 million tons, which is only 0.7% of the global production. From a social perspective, there is a stimulus in the Netherlands to a protein transition in which regional proteins are used for livestock farming. Heat-treated full-fat soybeans (FFSB) can be an important protein source. Due to the gap in knowledge about European soybeans, more research can provide clarity about protein quality of European FFSB. In this study, raw GMO- free, unprocessed soybeans from European zone (France, FFSBFR; Netherlands, FFSBNL ) and common used beans (Ukraine, FFSBUKR; Brazil, FFSBBR) were steam-toasted for 9 different time- temperature combinations, and analysed on different in-vitro protein quality indicators: Trypsin inhibitor activity (TIA), total and reactive lysine (rLys, tLys), crude protein (CP), pH-stat digestibility at 10 minutes (DH10) and 120 minutes (DH120), and protein dispersibility index (PDI). For Lysine, the degree of heat damage was determined by calculating the rLys:CP ratio. TIA sharply decreased for all origins when temperature increased, with remaining TIA levels at 130 °C of 0.51 mg/g DM or lower. At 130 °C, the rLys:CP ratio decreases between 4.5 – 6.8%. FFSBBR processed at 115 °C had the highest overall rate of hydrolysis (11.82 %) for DH10. FFSBFR seems to have a better result under higher temperatures, whereas for both DH10 and DH120 the observed values at 130 °C was higher (respectively 10.37% and 18.73%) compared to lower temperatures. For FFSBUKR, the DH10 and DH120 decreased slightly for the 130 °C compared to the 115 °C. For FFSBNL, results between DH10 and DH120 were in contradiction with eachother, and therefore based on pH-stat it is confusing which process temperature gives the best protein quality. Unprocessed, raw soybeans have an initial PDI value between 84.2 – 87.4. After steam-toasting of the raw beans for different temperatures (100 – 130 °C) for different durations (2.5 – 30 minutes) the PDI decreases and becomes stable at PDI levels of approximately 10-12 %. Based on processing temperature at a toasting time of 10 minutes, 115 °C seems to be the most favourable temperature when looking at TIA and rLys:CP ratio. This, because of the sufficient inactivation of ANF below the threshold of 4mg/g on the one hand (underprocessing), and maintaining the lysine availability which can be reduced by Maillard reaction on the other hand (overprocessing). Based on pH-stat, degree of hydrolysis of both DH10 and DH120 was the highest for common soybeans (FFSBBR, FFSBUKR). This is in conflict with the rLys:CP ratio, which was the highest in FFSBNL. Based on PDI and DH120 by pH-stat, European beans seems to act better after more intensive processing conditions. Because of a decrease in rLys:CP ratio at 130 °C, rLys:CP is in conflict with the optimum degree of hydrolysis at DH120 for FFSBFR and FFSBNL, which indicates best protein quality at processing temperature of 130 °C. For this reason, it is assumed that other processes must have influenced an overall higher degree of hydrolysis in commonly used soybeans and the preference of European beans for a more intensive processing. In conclusion, it is assumed that, based on TIA and rLys:CP ratio, European FFSB could have an competitive value compared to common used FFSB. However, because the overall protein hydrolysis was higher in common beans, it seems that other processes play a part in determining the digestibility of protein.

Contents Summary ...... 4 Introduction ...... 6 Production and origin ...... 6 Composition of full-fat soybeans from different origins ...... 7 Processing of full-fat soybeans ...... 7 Indicators for protein quality ...... 8 Objectives ...... 9 Material and Methods ...... 10 Materials ...... 10 Steam treatments ...... 10 Chemical and physical analyses ...... 11 Results ...... 12 Discussion ...... 17 Data analyses ...... 17 Protein quality indicators ...... 17 Evaluating protein quality indicators ...... 19 Conclusion ...... 20 Recommendations ...... 21 References ...... 22 Appendix I ...... 24

Introduction The feed and food industry is worldwide becoming increasingly dependent upon vegetable protein sources (Henchion et al., 2017). In 2001, the EU banned the use of meat and bone meal (MBM) as an ingredient in animal feed in order to halt the spread of mad cow disease. In the year following the ban, some 16 million tonnes of MBM in animal feed was substituted by 23 million tonnes of soybean meal (De Ridder, 2015). FAO (2009) stated that the expected increase per capita income will increase the demand for food products that are responsive to higher incomes, such as livestock and dairy products. To meet the demand for animal protein, plant-based proteins are needed to produce such products (FAO, 2009). As the major vegetable protein commodity, soybean are an important dietary raw material. Mainly due to the high protein content, full-fat soybeans (FFSB) can be used for increasing plant-based protein demand, but is in addition, an even more excellent source of energy and fatty acids (Willis, 2003). For the feed industry, most of the FFSB fed were first oil-extracted by an desolventizing-toasting (D-T) step, whereas the oil is being used for human purposes (Mustakas et al., 1981). Because the remaining protein rich soybean meal is the main source of protein for the feed industry worldwide, it has become an ingredient that is strategically traded around the globe every day of the year. The global production of soybeans by farmers has increased from 108 million tons in 1990 to 335 million tons in 2016, mainly due to the high amount of protein and an excellent amino acid pattern for animals and human food in soybeans in combination with the growing demand for human edible protein (Zarkadas et al., 2007; FAO, 2018)

Production and origin In 2016, the five largest producing countries were contributing for 89% of the 335 millions tons of global soybean production, which is represented in Figure 1 (FAO, 2018). The import- export- and production for the most important continents or countries is represented as a flow chart in Appendix 1. It can be concluded that EU-28 and China have an high import of soybeans and soybean meal (respectively 33 Mton and 97 Mton). This is mainly imported from countries as Brazil, Argentina and USA.

The production of soybeans in the EU-28 in 2016 was 2.4 million tons, which is only 0,7% of the global production (FAO, 2018). However, it is suggested that the demand for European soybeans will increase. From a social perspective, there is a stimulus to a protein transition in which regional proteins are used for livestock farming. The European Parlement (2018) recognizes that soy production in South America plays an important role in the change in land use, and causes many environmental problems, such as pollution of groundwater with pesticides, soil erosion, declining water supplies and deforestation, resulting in a huge loss of biodiversity. To counter this, for the Netherlands in 2011, the “Verbond van Den Bosch”, a covenant between retail, feed industry, government and NGO’s set the goal of using at least 50% of the protein-rich animal feed out of Europe by 2020 (Doorn, 2011). In 2018, the Dutch agricultural and horticultural organization and the Dutch dairy organization came up with a future policy in which the goal is to get 65% of the protein for dairy cattle feed (included roughages, compound feed and wet co-products) from the region by 2025 (Loman, 2017).

SOYBEANS PRODUCTION (MTONS)

1990 2000 2016

335

161

117

108 96

20 20

11 11 5 3

W O R L D UNITED STATES BRAZIL ARGENTINA INDIA CHINA PRODUCTION Figure 1: Global production of soybeans and the countries that make a major contribution to this.

Composition of full-fat soybeans from different origins Based on biological function in plants, seed proteins are of two types: metabolic proteins and storage proteins (Clarke, 1998). Metabolic proteins include enzymatic and structural proteins and are involved in normal cellular activities, including synthesis of storage proteins. Glycinin and β- conglycinin are the most important storage proteins in soybeans, which function as a protein source for the growing seedling. The metabolic proteins have to be inactivated by heating to eliminate unwanted and anti-nutritional effects when they are used in human foods or animal feeds (Renkema et al., 2001). In comparison with other legumes, according to CVB (2016), FFSB has a high protein content (40.4 %) and a relatively high oil content (21.9%) on a dry weight basis.

From a research perspective, the nutritional characteristics of commonly used soybeans from countries as Brazil, USA and Argentina are usually well-known and well described. On the other hand, little is known about the nutritional characteristics of other origins like European soybeans. There are several associations worldwide (e.g. USSEC, ASA, CSIA) who measure and control the quality of soybeans dependent on growing region. Unfortunately, there is no European association for soybeans. Based on literature, there is a scarcity of information concerning protein quality of soybean grown in EU-28 region. The quality (e.g. standardized ileal digestibility of protein, composition of amino acids, trypsin inhibitor activity) between origin can possible differ due to difference in climate, soil, breed and cultivation. Kaewtapee et al. (2018) compared different defatted soybean products from Germany and Austria with traditional soybean products including GMO-free Brazilian soybean meal. European soybeans showed similar or sometimes higher SID of CP (standardized ileal digestibility of crude protein) compared to other origins. He concluded that European soya beans can be a suitable alternative to imported soya. However, critical studies about characteristics of soybeans from other European zones are not obtained. Due to the gap in knowledge about these origins, more research can provide clarity. In the end, getting more insight in the quality (which is explained later in the report) of European soybeans compared to more common soybeans is a necessary part to judge if soybeans have added value for cultivation in Europe.

Processing of full-fat soybeans For an optimal performance of livestock animals, digestible amino acids are essential nutrients. For this reason, striving for an optimal treatment of FFSB is important to make amino acids available for digestion in non-ruminants.

For non-ruminants, the nutritional potential of FFSB is limited by the presence of antinutritional factors, mainly trypsin inhibitors, which interfere with digestion, absorption, and metabolism of nutrients (Liener and Kakade, 1980). According to the A.S.A (American Soybean Association), antinutritional factors (ANF) can be divided into the following categories (Table 1):

Table 1: Types of antinutritional factors with their specific substances (Clarke and Wiseman, 1998).

ANF group Heat stable Specific substances Trypsin inhibitor No Kunitz inhibitor

Bowman Birk inhibitor Lectins No Agglutinin

Antigenic and No β-conglycinin antigen

toxic proteins Glycinin antigen

Carbohydrates Yes Oligosaccharides

Raffinose

Stachyose Phytic acid Yes

Trypsin inhibitors form complexes with pancreatic proteases, thus reducing their acitivity in the small intestine. Trypsin inhibitors are more heat stable compared to other ANF’s like lectins (Qin et al.,

1996). Therefore, sufficient inactivation of trypsin inhibitors is crucial for optimal protein digestibility. In ruminants, the rumen microbial population can degrade these compounds (Susmel et al., 1995).

The steam toasting technology, if adequately controlled, can be used to reduce the contents or the activities of the proteinaceous ANFs in soybean (Melcion and van der Poel, 1993). Houdijk (1992) made a comparison on the processing cost between different processing technologies. It was found that the cost per unit of processed product with pressurized steaming (toasting) was much lower than that processed with extrusion. In terms of economy, pressurized steaming, could therefore, be a better method than extrusion. Based on the exposure of proteins to several conditions, like heat, pressure, shear and pH, reactions will occur. These reactions can be considered positive (e.g. denaturation and inactivation of ANF’s) or negative (e.g. aggregation and Maillard reaction). Insufficient ANF inactivation or protein denaturation, indicates underprocessing, caused by a too mild treatment. Conversely, in case of too extreme treatment, the Maillard reaction or protein aggregation will occur, indicators of overprocessing. The final result of these changes on digestibility seems to be a counterbalance of the occurrence of each phenomenon (Salazar-Villanea et al., 2016a). To meet the optimal protein digestibility of full-fat soybeans (FFSB), the influences of both underprocessing and overprocessing of the beans must be taken into account. The Maillard reaction, mainly a reaction between a free Ɛ-amino group of lysine and a reducing sugar which causes a reaction, can be divided in three phases: early, advanced, and final. Mauron (1990) described the influences of the toasting process on protein as the change in digestibility of amino acids (AA) by modifying the tertiary and secondary structure (denaturation), changing AA side-chains (hindering peptide bonds from enzymatic hydrolysis), forming cross-links within or between molecules and the conversion of L-AA to their D-form (racemization).

Indicators for protein quality There are many parameters to evaluate the protein quality of FFSB after processing. In the end, most parameters are used to assess the true digestibility of amino acids of FFSB. Examples of these parameters are Protein Dispersibility Index (PDI), Nitrogen Solubility Index (NSI), Protein Solubility (PSKOH), which are mainly focussed on the solubility of the FFSB in water (PDI and NSI) or potassium hydroxide (PSKOH). Palic et al. (2007) concluded that protein solubility was the most reliable indicator for FFSB quality control and therefore, that NSI, PDI and PSKOH would be the preferred methods. Batal et al. (2000) reported that the PDI displayed the most constant response to the heating of FFSB, while Dudley-Cash (2001) stated that the PDI may indicate soybean quality better than other indices. Therefore, the preference in this study has been given to the PDI method.

Urease Index (UI) is used as an indicator of trypsin inhibitor activity (TIA). The urease enzyme is much easier and cheap to measure than is trypsin inhibitor and both molecules show similar characteristics of heat sensitivity (Dourado, 2011). On the other hand, Qin et al. (1996) concluded that urease activity was more sensitive to high temperature than TIA. Qin et al. (1996) also found that the nitrogen digestibility was associated with the inactivation of antinutritional factors. This was confirmed by Frikha et al. (2012), who found a high correlation (P<0.01) between TIA concentration in soybean meal and SID of CP for broilers in an in-vivo trial. The lower the ANF’s, the higher the digestibility. For this reason, it is assumed that TIA is an good protein quality indicator for processed FFSB.

In contrast with in-vivo measurements like measuring the SID of CP (standardized ileal digestibility of crude protein), in-vitro measurements are developed for rapid assessment of nutritional quality. More recently, in-vitro digestibility was measured by the pH-stat method, described by Boisen and Fernández (1995). Salazar-Villanea et al. (2016b) found correlations between SID of CP and in-vitro pH-stat results after 10 minutes incubation with an enzyme substrate for soybean meal and rapeseed meal. Therefore, it is assumed by Salazar-Villanea et al. (2016b) that the degree of hydrolysis after 10 minutes (DH10) by pH-stat method is an accurate method to predict the protein quality of soybean meal after toasting on several conditions.

González-Vega et al. (2011) stated that the ratio total lysine to crude protein (tLys:CP) is a relatively quick method to estimate if a given source of soybean meal is heat damaged, and this procedure may potentially be used to evaluate the quality of soybean meal.

Fontaine et al. (2007) also concluded this for tLys:CP ratio, but concluded a higher sensitivity for the ratio reactive lysine to crude protein (rLys:CP) compared to tLys:CP regarded to heat damage in 8

soybean products. Frikha et al. (2012) stated that both the CP and rLys content in soybean meal were significant correlated to the SID of CP in broilers.

In the end, an in-vivo trial with animals is the most precise method to access the true digestibility of amino acids of processed soybeans. Unfortunately, in-vivo research is time-consuming, expensive and an ethical issue. Therefore, in-vitro or physical parameters like NSI, PDI, PSKOH, UA, TIA, tLys rLys and pH-stat are relatively easy and cheap indicators to assess the amino acid digestibility of FFSB.

In the present study, trypsin inhibitor activity (TIA), OMIU-(reactive) lysine (rLys and tLys), crude protein (CP) degree of hydrolysis by pH-stat method (pH-stat) after 10 minutes (DH10) and 120 minutes (DH120) and protein dispersibility index (PDI) were determined for raw and steam toasted Dutch (FFSBNL), French (FFSBFR), Ukrainian (FFSBUKR) and Brazilian (FFSBBR) soybeans, to evaluate the effect of different steam-heating temperatures on protein quality of the four origins. For PDI, besides different temperatures, also differences in durations were evaluated.

Objectives The objective of this research is to provide a descriptive insight into the current situation of knowledge about the effect of toasting of European FFSB’s on protein quality compared to commonly used FFSB’s. To reach these objectives, the following sub-questions were formulated:

1. What is the effect of different toasting conditions on the indicator protein dispersibility index (PDI)? 2. What is the effect of toasting temperature on FFSB protein quality indicators? 3. Is there an effect of origin between European FFSB and common used FFSB based on different protein quality indicators?

Material and Methods Materials Raw, unprocessed non-GMO soybeans were gained from different commercial suppliers. European (FFSBFR, FFSBNL ) beans were gained from Cargill (Velddriel, the Netherlands). Commonly used beans (FFSBUKR, FFSBBR) were gained from Bunge Netherlands (Amsterdam, the Netherlands). All soybeans were transported to the Netherlands for the production of animal feed. For FFSBNL, FFSBFR and FFSBUKR the year of harvest was 2017. FFSBBR were harvested in 2018. FFSBUKR were loaded at the harbour of Nikolaev (Ukrain), and FFSBBR were loaded at the harbour of Paranaqua (Brazil). Furthermore specific infomation about growing region or genotypes was not available. Only one sample of about 15 kg hwas collected per origin. The raw, unprocessed beans were first passed through a 4mm Retsch sieve to remove contaminants.

Steam treatments Batches of 500 g of different soybeans were steam-toasted for 9 time-temperature combinations, which were described in Table 2. Besides the 9 treatments per origin, for each origin an untreated sample was prepared. Because the untreated sample has been exposed to a temperature for a certain period of time during storage, the untreated sample is shown with a treatment temperature of 25 degrees and a treatment duration of 0 minutes.

Table 2: The different time-temperature conditions on which the beans were toasted.

Temperature (°C) / time (min) 2.5 5 10 20 30 100 x X x 115 x x X 130 x x x

The batches were processed at Carus experimental facilities (Wageningen, Netherlands) using a laboratory-scale pressurized steam toaster (Figure 2). This toaster is developed by Wageningen University as described by van der Poel et al. (1990). The batches were processed sequentially in order of temperature. Before toasting a batch, the toaster was set to the correct settings, with the temperature being dependent on the steam pressure. For the temperatures of 100, 115 and 130 degrees, an inside steam pressure of respectively 0, 0.8 and 1.7 bar was applied. After toasting each batch, the processed soybeans were cooled to room temperature immediately after toasting on a drying plate (example, Figure 3), and air-dried in a Thermo Scientific forced-draught oven at 40°C for 24 h. Sequentially, all batches were divided by a sample splitter, and half of the sample (about 250 g) was used for analyses. Then, samples were milled by a Retsch SM2000 hammer mill to pass through a 3 mm screen. After that, the milled samples were furthermore milled by a Retsch ZM200 centrifugal mill to pass through a 1 mm screen for additional analyses. The milled batches were again divided by an sample splitter, and for each batch two identical samples of about 125 g each were used for internal and external analyses.

Figure 2: The steam-toaster at Carus experimental facilities. 10

Chemical and physical analyses For every batch, the processed beans were analysed on dry matter (DM) and PDI. The untreated batches in combination with the batches processed at 10 minutes for different temperatures were additionally analysed on TIA, tLys, rLys and pH-stat. For DM, PDI and pH-stat, analyses were executed at the laboratory of the Animal Nutrition Group (Wageningen, Netherlands). For TIA, rLys, tLys and CP (nitrogen analysis), external laboratories were used. All results will be represented as 100% dry matter.

Dry matter The DM content of all the batches after drying in the forced-draught ovenwere measured in duplicate by drying for 4 h at 103˚C and weight. Samples were again dried for 16 hours at 70˚C followed by 4 hours at 103˚C. The dry matter content was calculated as dried mass divided by original massdrying process..

Crude protein The crude protein content was calculated based on the nitrogen content., Samples were shipped to NutriControl in Veghel (Netherlands). The analyses were executed following the Kjeldahl principle which is described in NEN-ISO-8968-1. For each origin, the untreated sample was analysed in simplo. It was assumed that for the other samples after toasting, the crude protein level (on dry- matter level) was unchanged.

Trypsin inhibitor activity For the analysis of the TIA, samples were shipped to MasterLab in Putten (Netherlands). The TIA was measured with the help of benzoyl-I-arginine-p-nitro-anilide (L-BAPA) as substrate. The amount of p-nitro-anilide existing, is measured spectrophotometricaly. The analyses were executed following the principle which is described in NEN-EN-ISO 14902. The TIA was measured in simplo for the untreated samples and the samples treated at 10 minutes. For TIA, a regression model was applied. The model was calculated using lineair regression of IBM SPSS Statistics (Version 23, IBM Corp.)

Protein hydrolysis using the pH-stat method The degree of hydrolysis (DH) was determined in simplo for the untreated samples and for the samples treated at 10 minutes using a method of Salazar-Villanea et al. (2017), which was a modified edition of Pedersen and Eggum (1983),. Enzymatic incubation was extended to 120 min using 1.61 mg porcine trypsin (Sigma, 13,000 to 20,000 BAEE units/mg protein) and 3.96 mg bovine chymotrypsin (Sigma, > 40 units/mg protein) per milliliter of water. The discribed addition of porcine intestial peptidase by Salazar-Villanea et al. (2017) was not used in this study, because this enzyme was not available. Results of unpublished data made it clear that this had no effect on the results of this method. The volume of NaOH added during the titration was used to calculate DH. Initial pH of ingredient solutions and DH after 10 (DH10) and 120 min (DH120) hydrolysis were selected. The DH curve was used to calculate the rate of protein hydrolysis (k) based on the model described by Butré et al. (2012).The model was fitted using non-lineair cruvefit of GraphPad Prism (Version 4, GraphPad Software, Inc., San Diego, CA).

OMIU-(reactive) lysine For the analyses of tLys and rLys, samples were shipped to NutriControl in Veghel (Netherlands). The analyses were executed following an modified method of (Moughan and Rutherfurd, 1996), to determine the reactive lysine by using O-methylisourea (OMIU). The total and reactive lysine was measured singular for the untreated samples and the samples treated at 10 minutes.

Proten dispersibility index The PDI was determined duplicate for the untreated samples and the samples treated at 10 minutes. The other samples were treated singular. The PDI was calculated using the method of AOCS (1997).

Results

The results of PDI are shown in table 3. Results are showing an decrease in dispersibility of protein when duration and temperature of processing increases. An increase of both time and temperature decreases the PDI till it becomes approximately stable at 115 °C / 20 min and beyond. The obtained results of analysed protein quality indicators for the untreated and 10 minutes-toasted samples are represented in table 4. CP levels were the highest in FFSBFR (434 g/kg DM), whereas other origins had about the same CP level. Results from the table are described in this chapter. The removed contamination by sieving before toasting was 3.8% for FFSBBR, 1.0% for FFSBFR, 3.3% for FFSBUKR and 1.5% for FFSBNL. Figure 3 shows the differences in colour of toasted beans after drying between three different temperatures and Figure 4 shows FFSBNL inside the toaster.

Figure 3: Difference in colour between toasting temperatures of 100 Figure 4: Dutch beans inside (middle), 115 (right) and 130 degrees (left) at a toasting time of 10 the steam-toaster. minutes.

Table 3: Protein Dispersibility Index (PDI) for different origins after processing on different conditions.

Origin of bean Toasting Toasting time Brazil France Ukraine Netherlands temperature 25* 0* 84.2 87.4 85 85.8 10 59.3 68.8 60 69.3 100 20 42.2 65.9 51.7 59 30 38.6 60.8 41.7 53 5 21.3 39.4 18 38.1 115 10 14.9 27.3 15.3 24.4 20 10.7 12.3 10.2 13.8 2.5 11.6 13.8 11 17 130 5 10.7 10.5 10.1 12.3 10 11.1 10.3 10.1 12.2

*untreated sample

130 395 25.4 23.8 93.8 6.43 6.03 9.08 0.72 6.84 12.2 <0.4 17.94 10 115 395 2.64 26.1 24.7 94.5 6.61 6.25 9.48 0.81 6.97 24.4 17.93 95 1.3 6.3 100 395 7.33 26.1 24.8 6.61 6.28 69.3 Netherlands 10.23 16.64 0 25 25 395 26.1 95.7 6.61 6.33 1.83 9.78 0.13 6.51 85.8 39.74 6.8 130 394 24.9 23.1 92.9 6.32 5.86 9.73 0.77 10.1 <0.4 18.55 24 94 115 394 2.32 25.5 6.47 6.09 0.89 6.83 15.3 10 10.45 18.71 Ukraine 60 100 394 10.5 25.2 23.7 94.3 6.40 6.02 8.37 0.51 6.65

18.83

. 0 25 85 394 25.5 24.2 95.1 6.47 6.14 3.32 6.31 2.27 6.39 34.94 Origin of bean of Origin 130 434 0.51 27.2 25.3 93.1 6.27 5.83 0.87 6.85 10.3 10.37 18.73 10 94 115 434 2.83 27.6 25.9 6.36 5.97 0.93 6.89 27.3 10.34 18.33 France 100 434 27.8 26.2 94.1 6.41 6.04 9.55 1.01 6.44 68.8 10.92 16.96 0 25 28 434 26.5 94.8 6.45 6.11 1.48 9.28 0.08 7.94 87.4 33.84 25 23 0.8 130 403 0.46 92.1 6.20 5.71 9.96 6.76 11.1 18.65 10 2.3 115 403 26.5 24.8 93.8 6.58 6.15 0.96 6.79 14.9 11.82 20.05 Brazil 26 95 8.2 100 403 10.8 24.7 6.45 6.13 0.69 6.55 59.3 16.97 0 25 26 1.3 403 24.6 94.8 6.45 6.10 7.42 0.14 7.08 84.2 35.21 ¯¹) )(s -4 pH-initial DH10 (%) DH10 DH120 (%) DH120 k (*10

tLys (g/kg DM) rLys (g/kg DM)

: Different protein quality indicators for soybeans of different origins treated on conditions origins different treated different of soybeans for quality indicators protein : Different 4 tLys : CP (%)ratio rLys : CP (%)ratio

rLys : tLys (%)ratio Table Toasting time Toasting (min) (C°) temperature Toasting CP (g/kg DM) TIA (mg/g DM) Lysine pH-STAT PDI (%) trypsin CP= TIA= activity;protein; inhibitor crude tLys= total lysine; lysine;rLys= reactive rLys : tLys lysine ratio= to reactive total lysine ratio; tLys : CP ratio= total lysine hydrolysis ratio; torLys min of protein 10 incubation; crude :lysineafter degree CP ratio; ratio= toDH10= protein reactive crude hyrdolysis incubation; k=of hydrolysis;120min incubation protein after of rate degree the initialpH-initial= sample DH120= of before pH 13

Trypsin inhibitor activity (TIA) The initial level of trypsin inhibitor activity (TIA) was the highest for FFSBNL with 39.74 mg/kg (Figure 5). After the steam treatment, TIA sharply decreased for all the origins with approximately equal remaining TIA levels of 0.51 mg/kg or lower. For FFSBNL treated at 100 °C / 10 min, the residual TIA value decreased to 18,5% of the activity of that in the untreated sample (25 °C / 0 min). For all the origins, the inactivation pattern of TIA by an increase of temperature were highly correlated to each other. This is represented in Table 5, were a regression model is applied. FFSBNL being somewhat more extreme in initial value and decrease after an increase in processing temperature.

Figure 5: Trypsin inhibitor activity (mg/g DM) of raw FFSB and FFSB treated at a duration of 10 minutes for different origins on different temperatures.

Table 5: Regression model for the change of trypsin inhibitor activity (TIA) measured as untreated sample and for three different temperatures (100, 115, 130 C) at 10 minutes treatment.

Model R for origin Brazil France Ukraine Netherlands TIA=44.55-0.35t 0.990 0.992 0.992 0.989 TIA=trypsin inhibitor activity in mg/g dm t=temperature in °C

OMIU-(Reactive) lysine For an overview of the amino acid stability during processing, lysine was determined as total lysine (tLys), reactive lysine (rLys) and the ratio between each other was calculated (rLys:tLys). Also the tLys:CP and rLys:CP ratio was calculated. tLys was measured as highest in FFSBFR (28 g/kg). For the other beans, initial total lysine was approximately equal. The lysine availability after processing (tLys: CP and rLys:CP) is showed in Figure 6 and 7, and shows a decrease during an increase in processing temperature, especially for the samples treated at 130 °C / 10 min. The difference in rLys:CP ratio between the highest (FFSBNL; 25 °C / 0 min) and lowest sample (FFSBBR; 130 °C / 10 min) measured is 9.8%. FFSBNL seems to have the highest lysine availability ratio before and after processing for all temperatures. The figure shows on average a decrease of both ratios tLys:CP and rLys:CP when temperature is increasing, whereas the initial untreated samples have the highest values.

Figure 6: tLys : CP ratio of raw FFSB (25 C°) and FFSB Figure 7: rLys : CP ratio of raw FFSB (25 C°) and FFSB treated at a duration of 10 minutes for different origins at treated at a duration of 10 minutes for different origins at different temperatures. different temperatures. pH-STAT method The initial pH before incubating the enzyme substrate was different for the origins, with pH-levels ranging from 6.3 (FFSBNL ; 100 °C / 10 min) till 7.94 (FFSBFR ; 25 °C / 0 min). There seems to be a pattern in the initial pH value among the origins. Except for the FFSBUKR untreated sample, all the samples of the origins are following the pattern: a decrease in pH between the untreated sample and processing at 100 °C / 10 min followed by an increase when temperatures of 115 and 130 °C are being reached. Compared to the untreated samples, the processed samples by the steam-toaster had an higher rate of hydrolysis (k), except for the FFSBUKR untreated sample. For all origins, k decreased by a temperature of 130 °C, compared to 100 and 115 °C. The observed results for degree of hydrolysis after 10 minutes (DH10; Figure 8) and 120 minutes (DH120; Figure 9) showed similar patterns, as DH120 had an overall higher degree of hydrolysis. The steam-treatment for different temperatures increases the degree of hydrolysis for all origins compared to the untreated samples. The highest degree of hydrolysis was observed in FFSBBR (DH120; 20.05%) processed at 115 °C for 10 minutes.

Figure 8: degree of hydrolysis after 10 min (DH10) of raw Figure 9: degree of hydrolysis after 120 min (DH120) of raw FFSB (25 C°) and FFSB treated at a duration of 10 FFSB (25 C°) and FFSB treated at a duration of 10 minutes minutes for different origins at different temperatures. for different origins at different temperatures.

PDI The PDI was measured at 10 different time-temperature combinations for every origin. The decrease of the PDI is represented in Figure 10. European soybeans processed at 115 °C/5 min (FFSBFR and FFSBNL) which have a PDI of respectively 39.4 and 38.1, seems to differ in PDI value from the non- European origins (FFSBBR and FFSBUKR), which generates a PDI of respectively 21.3 and 18.0 for the same conditions. This difference then decreases until it is approximately equal at 115 °C/20 min and beyond.

Brasil France Ukraine Netherlands

100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 25 ° C / 0 100 100 100 115 115 115 130 130 130 MIN ° C / 1 0 ° C / 2 0 ° C / 3 0 ° C / 5 ° C / 1 0 ° C / 2 0 ° C / 2 . 5 ° C / 5 ° C / 1 0 MIN MIN MIN MIN MIN MIN MIN MIN MIN

Figure 10: Protein Dispersibility Index (PDI) of FFSB for different origins after processing at conditions between 100 - 130 °C temperature and 2.5 - 30 minutes duration.

Discussion

Data analyses Due to the design of the research, most of the analyses were carried out in simplo. Due tot the fact that only one sample per origin was collected, statistical analyses were not possible. For this reason, the results are open for discussion. The advantage of this was that a wide spectrum of origins and process conditions could be treated and measured on different protein quality parameters. On the other hand, the power of expression about the effects that have been investigated is less.

The batches of soy beans obtained were supplied by a specific supplier per origin. This is not representative for the average characteristic properties of soybean of a certain origin. Unfortunately, due to lack of time, more detailed research was not possible. Also furthermore specific information about growing region, genotype and cultivation of the beans was not available, and could improve the quality of the research.

Protein quality indicators Trypsin inhibitor activity For all the origins, the trypsin inhibitor acitivy (TIA), decreased rapidly by an increasing temperature. It was assumed that the TIA is an good parameter to estimate the overall ANF activity in the soybeans. In this study, a correlation between TIA-level and temperature was found which can be applied for all the origins. This is well-established for common soybeans, but results in this study are showing a high regression for FFSBBR, FFSBFR, FFSBUKR and FFSBNL when a model is applied. Therefore, it is assumed that the all the origins have a similar tendency in response to the treatment in which the untreated sample and the 10min-treatment for different temperatures (100, 115, 130 °C) were used.

For indicating well-processed FFSB, Chang (1987) suggested that the TIA level should be below 4 mg/g on product base. Mian and Garlich (1995) concluded that a dietary TIA of 4.4 mg/g or greater decreased digestibility in turkey’s. When the standard of 4 mg/g product is applied, sufficient inactivation of TIA is determined in beans steam-toasted for 10 minutes at 115 °C and 130 °C. This applies for all origins, whereas the untreated samples and samples processed at 100 °C are for all origins above the threshold of 4mg/g, and thus underprocessed. On the other hand, based on TIA levels, it is not clear to what extent these beans are overprocessed.

Reactive lysine Due to the Maillard reaction, the reactive and total lysine content will decrease when toasting conditions become more extreme. As expected, the results are showing a trend towards a decrease of total lysine, reactive lysine and the ratio between them. This decrease is clearly visible after toasting temperature of 130 °C at 10 minutes. For the other conditions, it is difficult to link results, due to the small differences. For the untreated, raw soybeans, besides FFSBFR, the other origins seems to have the same initial total lysine levels. The higher amount of total lysine for France beans (28 g/kg) can be explained by an higher crude protein level (García-Rebollar et al., 2016). Therefore, crude protein is an important parameter when (reactive) lysine levels are compared. Fontaine et al.( 2007) stated that for rLys:CP, it is highly probable that heat damage is the reason for the obtained variability. Therefore, rLys:CP could be an parameter to assess protein quality of FFSB after processing. Table 6 shows the percentage of losses in reactive lysine after processing for different conditions compared tot the rLys:CP ratio of the untreated samples (25 °C / 0 min).

It is assumed to use rLys rather than total lysine content as measure for available lysine, because rLys being a more sensitive indicator to measure heat damage (Fontaine et al., 2007). This comes due to the fact that rLys is taking into account the amount of non-reactive residual lysine, as tLys is the sum of rLys and residual lysine. The degree of heat damage is reflected in the ratios of tLys:CP or rLys:CP (Fontaine et al., 2007), because the intensity of heat treatment has a major impact on lysine but not on CP content (Stein, Connot, & Pedersen, 2009). In this study, it was made clear that, according to the rLys:CP ratio, only at high temperatures (130 C for 10 minutes) the available lysine for all origins decreased, whereas FFSBBR has the highest losses in rLys:CP ratio, as a consequence of heat damage due to Maillard reaction. For all processing temperatures, the rLys:CP

ratio of FFSBNL was the highest. According to rLys:CP ratio, the protein quality of all the beans decreases due to heat damage at 130 °C compared to 100 and 115 °C.

Table 6: Changes in rLysine:CP ratio in percentage of rLysine:CP ratio in the untreated sample.

Origin of soybean / 100 115 130 processing temperature Brazil +0.4 +0.8 -6.5 France -1.1 -2.3 -4.5 Ukraine -2.1 -0.8 -4.5 Netherlands -0.8 -1.2 -4.8 pH-stat method Salazar-Villanea et al. (2016b) obtained a correlation between DH10 and SID of CP in growing pigs. Pedersen and Eggum (1983) found the highest correlation between degree of hydrolysis after 10 minutes (DH10) by pH-stat method and faecal digestibility in rats. In this study, the differences of results from the pH-stat method after 10 minutes of hydrolysis (DH10) method were small for each treatment. FFSBBR processed at 115 °C had the highest rate of hydrolysis (11.82 %) for DH10. FFSBFR seems to have a better result under higher temperatures, whereas for both DH10 and DH120 the observed values at 130 °C was higher (respectively 10.37% and 18.73%) compared to lower temperatures. For FFSBUKR, the DH10 and DH120 decreased slightly for the 130 °C compared to the 115 °C. FFSBNL tends to react the same on toasting for different temperatures. The differences between 100, 115 and 130 °C are small for both DH10 and DH120. For FFSBNL, DH10 shows an optimum of hydrolysis at 100 °C, but DH120 shows an optimum at 130 °C. For this reason, it is difficult to conclude someting based on the results for FFSBNL.

Based on pH-stat, FFSBBR and FFSBUKR seems to act most favorable at temperature of 115 °C. For FFSBFR, it seems to act, despite small differences, most favorable at 130 °C. For Dutch beans, because of small differences between 100, 115 and 130 °C treatment in combination in with a contradiction in results between DH10 and DH120, it was not clear based on pH-stat which process temperature gives the best protein quality.

Salazar-Villanea et al. (2016b) also assumed that initial pH after processing could provide a rapid indication of protein damage. This is not in line with the results from this study. Except for the FFSBUKR untreated sample, all the samples of the origins are following the pattern: a decrease in pH between the untreated sample and processing at 100 °C / 10 min followed by an increase when temperatures of 115 and 130 °C are being reached. According to the rLys:CP ratio, heat damage is only occuring at high temperatures (Table 5). The drop in pH between the untreated samples and the samples treated at 100 °C can not be assigned to heat damage, and therefore, other processes must have influenced this drop in pH.

PDI For adequate processed soybeans, the National Soybean Processors Association recommend a range of 15 to 30% PDI (Balloun, 1980). In an in-vivo trial, Batal et al. (2000) generally indicated that soybean meal containing a PDI of 45% or lower is adequately heat processed. On the other hand, Palic (2007) stated that adequately-processed FFSB values between 8.5% and 10.3%. Qin et al. (1996) compared the PDI of steam-toasted FFSB for different conditions with the apparant ileal digestibility of nitrogen in pigs. Soybeans treated at 102 °C / 40 min, 120 °C / 7.5 min and 134 °C / 1.5 min showed similar and suficient results in terms of nitrogen digestibility. Corresponding PDI values were respectively 20.4, 12.4 and 10.4. Optimal PDI values for protein digestibility are very different upon different studies. The experimental design of this study is near to the design of Qin et al. (1996), mainly due to similar processing technique (steam-toasting of FFSB). Therefore, the PDI values (10.4 – 20.4) corresponding to sufficient nitrogen digestibility from Qin et al. (1996) are prefered to use as optimum for adequate processing of FFSB. Based on this, FFSBNL and FFSBFR seems to prefer a more intensive treatment compared to FFSBBR and FFSBUKR. At 115 °C / 10 min, PDI values for FFSBFR and FFSBNL are respectively 27.3 and 24.4. This PDI value indicates that FFSBFR and FFSBNL are underprocessed. In contrast, FFSBBR and FFSBUKR were well-processed for the same conditions, whereas the PDI values were respectively 14.9 and 15.3. At 130 °C / 10 min, PDI values for FFSBFR and FFSBUKR, are below 10.4, which could indicate overprocessing of FFSB. 18

Evaluating protein quality indicators As discussed before, different studies in the past have shown an relationship between SID of CP and protein indicators. CP, rLys and TIA concentration in soybean meal were correlating with SID of CP in an in-vivo trial with broilers by (Frikha, 2012), Qin et al. (1996) stated that TIA and PDI in steam-toasted FFSB had high correlation coefficients with nitrogen digestibility. Kaewtapee et al. (2018) reported a correlation between both TIA concentrations and tLys:CP ratio in European soy products and the SID of CP in growing pigs. For DH10 (pH-stat) Salazar-Villanea et al. (2017) reported a correlation between SID of CP and DH10 for soybean meal and rapeseed meal. However, only two batches of soybean meal were analyzed in this studie. Gebhardt (2015) obtained a correlation between SID of Lysine and growth performance for growing pigs. A higher concentration of SID Lysine improved daily gain and feed conversion ratio. Therefore, it is assumed that all protein quality indicators described in this studie are correlated to the SID of CP, and thus, indirect related to animal performance.

Effect of different toasting conditions on the Protein dispersibility Index (PDI) Results from this study are showing an decrease in dispersibility of protein when duration and temperature of processing increases. Unprocessed, raw soybeans have an initial PDI value between 84.2 – 87.4. After steam-toasting of the raw beans for different temperatures (100 – 130 °C) for different durations (2.5 – 30 minutes), the PDI decreases rapidly and becomes stable at PDI levels of approximately 10-12 %. This happens under conditions of 115 °C / 20 min and for the durations 2.5 min, 5 min and 10 min at 130 °C.

Effect of toasting temperature on FFSB protein quality indicators TIA sharply decreases when temperature is increasing, with remaining TIA levels at 130 °C of 0.51 mg/g DM or lower. When threshold levels of 4mg/g are applied for well-processed FFSB, a minimum toasting temperature of 115 °C is necessary for toasting at 10 minutes. Mainly due to Maillard reaction, available lysine decreases at high temperature (130 °C). The degree of heat damage can be determined by calculating rLys:CP ratio. At 130 °C, the rLys:CP ratio decreases between 4.5 – 6.8%, which indicates overprocessing. Based on this ratio, toasting temperatures of 100 and 115 °C are preferred for optimal lysine availability. For pH-stat, degree of hydrolysis after 10 minutes (DH10) differs widely among the temperatures. For FFSBBR and FFSBUKR, processing at 115 °C gave the highest DH10 value. This was 100 °C for FFSBNL, and 130 °C for FFSBFR. Due to small differences in degree of hydrolysis, it is hard to conclude the optimal temperature for each origin. For initial pH, it was not clear what influences the drop in pH between the untreated samples and samples processed at 100 °C / 10 minutes.

Effect of origin between European FFSB and common used FFSB on different protein quality indicators A high regression for TIA dependent on temperature for all origins was determined. The characteristics of TIA were about the same for all the origins, and thus, no obvious differences were observed in TIA behaviour between European and common used FFSB. Reactive lysine, mainly measured as rLys:CP, was the highest in FFSBNL. The behaviour of rLys:CP is about the same between European and common used FFSB, whereas the European varieties seems to have on average an higher rLys:CP ratio. The degree of hydrolysis after 10 minutes (DH10) was the highest in the common used beans (FFSBBR and FFSBUKR) with a degree of hydrolysis of respectively 11.82% and 10.45% at 115 °C / 10 minutes treatment. The FFSBFR, seems to act, despite small differences, most favorable at 130 °C. For FFSBNL, the DH10 was the highest at 100 °C treatment. But, because of small differences between 100, 115 and 130 °C treatment in combination in with a contradiction in results between DH10 and DH120, it was confusing based on pH-stat which process temperature gives the best protein quality in FFSBNL. Based on PDI, the European beans seems to need a more intensive treatment, to achieve the optimal PDI of about 10.4 - 20.4. For commonly used beans, this PDI is reached at 115 °C/ 5 min (FFSBUKR) or 115 °C / 10 min (FFSBBR) and beyond. European beans need at least a treatment of 115 °C / 20 min to obtain a PDI value between 10.4 - 20.4. This is confirmed by DH120 from pH stat, where the highest degree of hydrolysis is obtained at 115 °C for common used beans, and 130 °C for European beans.

Conclusions Based on processing temperature at a toasting time of 10 minutes, 115 °C seems to be the most favourable temperature. This, because of the sufficient inactivation of ANF below the threshold of 4mg/g on the one hand (underprocessing), and maintaining the lysine availability which can be reduced by Maillard reaction on the other hand (overprocessing). This applies for all the origins.

Despite these agreements, the characteristics of the indicators PDI and pH-stat towards the statement above are sometimes contradictory: the degree of hydrolysis was the highest for the commonly used soybeans, compared to the European beans. However this result, the rLys:CP ratio was the highest in FFSBNL, which indicates a higher protein quality. Based on PDI, European beans seems to act better after more intensive processing conditions. The need for an intensive processing for European beans was confirmed by DH120 from pH-stat, whereas the highest degrees of hydrolysis were measured at 130 °C, compared to 115 °C for commonly used beans.

After comparing FFSB grown in European zone (Netherlands, France) to FFSB from common growing zone (Brazil, Ukraine), based on protein quality indicators TIA and rLys:CP ratio in our study, it is assumed that European soybeans could have a competitive value for non-ruminants. However, because the overall protein hydrolysis was higher in commonly used beans, it seems that other processes play a part in determining the digestibility of protein.

Recommendations

In present study, the objective of this research was to provide a descriptive insight into the current situation of knowledge about the effect of toasting of European FFSB’s on protein quality compared to common used FFSB’s. Results were not statistical proven. Further research can provide more detailed information about the protein quality of European soybeans. In most studies, the standardized ileal digestibility of crude protein (SID of CP) is determined by an in-vivo trial. By determining the SID of CP of different independent sources of soybeans from different European countries with their characteristics (e.g. growing region, breed, cultivation) in duplicate, statistical proven data can give more clarification about this subject. This information can be compared with data of common used beans, whereas this information is widely available.

In this study, it is assumed that European beans seems to act better after more intensive processing conditions, and common beans (FFSBUKR and FFSBBR) have an higher overall degree of in-vitro protein hydrolysis. Based on results from this study, it seems that this is not related to TIA inactivation or rLysine availability (Maillard reaction), and other processes might will influence on this. Furthermore research can give clarity about this phenome. More specific research into amino acid composition, protein composition (e.g. structure and proportion of metabolic- and storage proteins) and behavior of other ANF’s can possible explain this.

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Appendix I