Abstract Variation in trypsin inhibitor activity (TIA) and protein solubility within commercial soybean meals (SBM) is believed to affect animal performance. The objective of this research was to investigate the proteolytic effect of purified pancreatic trypsin/chymotrypsin and a purified protease from Nocardiopsis prasina on 9 commercial SBM samples varying in chemical composition, TIA and protein solubility characteristics. SBM was incubated with or without protease (pH 7, 3 hours, 40°C) and the supernatants were analyzed by SDS-page and for level of free soluble amino ends using the o-phthal-dialdehyde method. SDS-page analysis showed differences in the amount and composition of the soluble protein fraction of the SBM. The proteolytic efficiency of the lower dosages of pancreatic protease varied extensively between SBM batches whereas at the highest dose the efficiency was comparable for all SBM. The difference could not be explained by differences in protein solubility and TIA values as isolated variables, but the data strongly suggest that the hydrolysis of soya protein by pancreatic proteases depends on the commercial batch of SBM used. Incubation with N. prasina protease showed similar response for the different SBM, the main difference being a somewhat lower effect at high protease dose for one of the SBM batches, which might be in part explained by its distinct lower protein solubility.

Variations in soybean meal and efficiency of protein hydrolysis by pancreatic protease and microbial protease

K. Pontoppidan1, G. G. Mateos2, R. Angel3 and A. Smith4

1 Feed Applications, Novozymes A/S, Bagsvaerd, Denmark; 2 Animal Science Department,

Universidad Politécnoca de Madrid, 28040 Madrid, Spain; 3 Department of Animal and Avian Sciences, University of Maryland, maryland, United States; 4 DSM Nutritional Products, Heanor,

Derbyshire, United Kingdom

Corresponding author: kpon@novozymes.com

SBM batch, origin #1

USA #2

USA #3

USA #4

Arg #5

Arg #6

Arg #7

Bra #8

Bra #9

Bra

DM, % 87.9 88.7 89.0 88.6 89.8 88.4 87.7 87.1 88.5

CP, % 53.8 52.4 52.9 53.6 52.9 52.8 53.7 54.2 53.2

PDI, % 25.6 24.8 9.7 10.4 11.7 10.1 7.0 15.2 12.5

KOH, % 80.2 79.7 78.6 75.4 77.3 71.4 67.9 77.2 81.4

Urease act., mg N/g DM 0.02 0.00 0.02 0.01 0.00 0.00 0.05 0.05 0.01

TIA, mg/g DM 3.75 3.49 3.15 2.71 2.67 2.49 2.39 2.76 2.71

Heat Damage Index 5 2 8 11 12 15 25 14 16

Table 1. Analyzed values for dry matter (DM, %), crude protein (CP, %), protein dispersibility index (PDI, %), protein solubility in KOH (KOH, %), urease activity (mg N/g DM), TI activity (TIA, mg/g DM) and Heat Damage Index (Aminored-Evonik)

Figure 2. Protease effect measured as release of soluble free amino groups after incubation of the different SBMs at pH 7, 3 h, 40°C with different proteases. Top: Pancreatic trypsin/chymotrypsin, bottom: Protease from N. prasina.

Results SDS-page analysis of non-protease treated SBM supernatants (data not shown) showed that samples had different amounts of solubilized protein. Protein intensities were generally higher for SBM batches with higher protein solubility (KOH, PDI) and low Heat Damage Index. Pancreatic trypsin/chymotrypsin was more efficient in protein hydrolysis at low dose on SBMs 2, 4, 5 and 6 compared to the other batches. However, with sufficiently high inclusion level of protease, all SBMs were hydrolyzed to approximately the same extent. The difference in response to pancreatic protease could not be explained by differences in protein solubility and TI activity as isolated parameters. The efficacy of the protease from N. prasina was very similar on all SBMs except for SBM 7, which was hydrolyzed to a lower degree. The lower efficiency on SBM 7 might be explained in part by its lower protein solubility, meaning that less protein was readily available for hydrolysis. Conclusions The results clearly showed that the efficiency of pancreatic trypsin/chymotrypsin varied among SBMs. Incubation with N. prasina protease resulted in effective and very similar responses for the different SBMs and did not appear to be affected by TI activity. Hence, N. prasina protease seems to represent a promising tool to ensure a sufficient amount of active protease in the intestinal tract of animals fed diets with moderately elevated levels of TI. Literature DE COCA-SINOVA et al. (2008) Poult. Sci. 87, 2613 HAMMERSTRAND et al. (1981) Cereal Chem. 15, 215-218 KAKADE et al. (1974) Cereal Chem. 51, 376-382 NIELSEN et al. (2001) J. Food. Sci. 66, 642-646 PERILLA et al. (1997) Br. Poult. Sci. 38, 412-416 WELHAM & DOMONEY (2000) Plant. Sci. 159, 289-299

Introduction The crude protein fraction of SBM consists roughly of 80% storage proteins, 5% anti nutritional proteins including trypsin inhibitors (TI) and 15% other proteins. De Coco-Sinova et al. (2008) demonstrated that commercial SBMs varied in chemical composition and nutritional value. TI inhibits the activity of the pancreatic digestive enzyme trypsin and high levels of TI in SBM have been associated with impaired animal performance (e.g. Perilla et al., 1997). Commercial availability of microbial proteases for inclusion in diets offers an opportunity to complement the animals own digestive proteases. Methods SBMs were obtained at the port and analysis conducted at the University of Madrid (Table 1). TI activity was assayed according to Hammerstrand et al., (1981). Proteases used were a preparation of trypsin/chymotrypsin from porcine pancreas and a preparation of S1 protease from N. prasina (Novozymes A/S, DK). SBMs were incubated as shown in Figure 1. Protein composition of the supernatants from non-protease treated samples were analyzed by SDS-page. Protease effect was evaluated by analyzing the level of soluble free amino groups in supernatants using the o-phthal-dialdehyde (OPA) method according to Nielsen et al., (2001) (Figure 2).

SBM (0.06 g DM/mL), pH 7, +/- protease

Add stop reagent (2:1 v:v)

Supernatant ready for analysis

Incubate 3h, 40°C, 350 rpm

Centrifuge 10 min, 4000 rpm, 5°C

Figure 1. Procedure for incubation of SBM with protease.