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/lp/springer-journals/physical-and-chemical-changes-of-rapeseed-meal-proteins-during-ot9IXSpF56
- Publisher
- Springer Journals
- Copyright
- Copyright © 2016 by The Author(s).
- Subject
- Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
- eISSN
- 2049-1891
- DOI
- 10.1186/s40104-016-0120-x
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Abstract
Background: Toasting during the production of rapeseed meal (RSM) decreases ileal crude protein (CP) and amino acid (AA) digestibility. The mechanisms that determine the decrease in digestibility have not been fully elucidated. A high protein quality, low-denatured, RSM was produced and toasted up to 120 min, with samples taken every 20 min. The aim of this study was to characterize secondary structure and chemical changes of proteins and glucosinolates occurring during toasting of RSM and the effects on its in vitro CP digestibility. Results: The decrease in protein solubility and the increase of intermolecular β-sheets with increasing toasting time were indications of protein aggregation. The contents of NDF and ADIN increased with increasing toasting time. Contents of arginine, lysine and O-methylisourea reactive lysine (OMIU-RL) linearly decreased with increasing toasting time, with a larger decrease of OMIU-RL than lysine. First-order reactions calculated from the measured parameters show that glucosinolates were degraded faster than lysine, OMIU-RL and arginine and that physical changes to proteins seem to occur before chemical changes during toasting. Despite the drastic physical and chemical changes noticed on the proteins, the coefficient of in vitro CP digestibility ranged from 0.776 to 0.750 and there were no effects on the extent of protein hydrolysis after 120 min. In contrast, the rate of protein hydrolysis linearly decreased with increasing toasting time, which was largely correlated to the decrease in protein solubility, lysine and OMIU-RL observed. Rate of protein hydrolysis was more than 2-fold higher for the untoasted RSM compared to the 120 min toasted material. Conclusions: Increasing the toasting time for the production of RSM causes physical and chemical changes to the proteins that decrease the rate of protein hydrolysis. The observed decrease in the rate of protein hydrolysis could impact protein digestion and utilization. Keywords: Hydrolysis rate, In vitro protein digestibility, Rapeseed meal, Reactive lysine, Secondary structure Background inactivate antinutritional factors present such as glu- Rapeseed meal (RSM) is the most important protein cosinolates [4]. Direct application of live steam is source utilized in commercial swine and poultry diets used during toasting to complete the solvent removal, after soybean meal [1–3]. The production process of which also increases the moisture content. The toast- RSM involves toasting to remove the organic solvent ing process time usually ranges from 60 to 90 min at remaining after solvent extraction of the oil and to 100–110 °C, which can increase the variation in the lysine content and ileal digestibility of most AA in RSM [4, 5]. The coefficient of variation of the appar- * Correspondence: sergio.salazarvillanea@ucr.ac.cr Wageningen Livestock Research, P.O. Box 338, 6700 AH Wageningen, The ent ileal digestibility of lysine in poultry increased Netherlands from 1.4 % in the solvent extracted meal to 5.4 % Animal Nutrition Group, Wageningen University & Research, P.O. Box 338, after toasting [5]. 6700 AH Wageningen, The Netherlands Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 2 of 11 Both physical and chemical changes of proteins due to Methods thermal processing can influence protein digestibility [6]. Materials Autoclaving at 120 °C for 20 min increased the propor- A batch of commercially available winter 00-rapeseed tion of random coil in the secondary protein structure of (Brassica napus), harvested in the southwest of France legume seeds, which is related to protein denaturation in 2013, was used. All chemicals used were of analytical and increased CP in vitro digestibility [7]. At the same grade. Pepsin (2,000 FIP U/g) was obtained from Merck time, appearance of intermolecular β-sheets, linked to (Darmstadt, Germany), whilst pancreatin (grade IV from decreased protein digestibility, was reported in the same porcine pancreas), trypsin (type IX-S, 13,000–20,000 study. The net effect on the in vitro crude protein (CP) BAEE units/mg protein), chymotrypsin (type II, >40 digestibility seems to be related to the ratio between units/mg protein), and peptidase from porcine intestinal both types of physical changes. Chemical changes can be mucosa (50–100 units/g solid) were obtained from the result of Maillard reactions or covalent crosslinking Sigma-Aldrich (St Louis, MO, USA). between amino acids (AA). Increasing the toasting time decreased the lysine and reactive lysine contents [8]. In Rapeseed meals preparation addition, the standardized ileal digestibility of CP was All processing of the rapeseed was conducted at the reduced from 66 to 60 % and that of lysine from 64 to pilot plant of CREOL (Pessac, France). The batch of 54 % when the toasting time was increased from 48 to rapeseed was dried in a warm-air dryer at 70 °C to a 93 min [8]. Chemical changes due to Maillard reactions moisture level of 5 % (w/w) (Fig. 1). After drying, the were suggested to be responsible for the decrease in pro- rapeseed was cold pressed (La Mecanique Moderne tein and amino acid digestibility. However, chemical MBU 75 type, Arras, France) at 250 kg/h. Temperatures changes of proteins due to Maillard reactions do not during pressing did not exceed 80 °C. Continuous completely explain the reduction in the digestibility of extraction of the cake by hexane was performed on a all AA, as observed in that study. This suggests that also belt extractor (B-1930, Desmet-Ballestra, Zaventem, changes in the structure of proteins (e.g. secondary and Belgium) at 160 kg/h flow of the cake and 230 L/h flow tertiary) affect the digestion process. of solvent. Temperature of the rapeseed cake during The aim of the present experiment was to characterize solvent extraction did not exceed 55 °C. The solvent the physical and chemical changes that occur to rapeseed remaining after solvent extraction of the oil was re- proteins during toasting and the influence of these changes moved using indirect heat (i.e. without use of direct live on in vitro CP digestibility. We hypothesize that increasing steam) in a desolventizer-toaster (Schumacher type, toasting times causes physical and chemical changes to Desmet-Ballestra, Zaventem, Belgium; 6 trays with a rapeseed proteins, resulting in decreased in vitro CP rotating arm and 1 m internal diameter) for 60 min. digestibility. Temperatures during desolventization were 90 ± 3 °C. Fig. 1 Schematic view of the experimental rapeseed treatment Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 3 of 11 Spot samples (5 kg) were obtained after drying, cold- For the determination of amino acid content, sam- pressing, hexane extraction, and desolventization with ples were hydrolyzed with 6 mol/L HCl at 110 °C for indirect heat. 23 h and the hydrolysates were adjusted to pH 2.2 A batch of 150 kg of the desolventized-untoasted RSM using NaOH. Amino acids were determined by post was toasted in the lower tray of the desolventizer-toaster column reaction with ninhydrin, after separation by with injection of live steam (30 kg/h), whilst indirect ion exchange chromatography. Photometric detection steam pressure was set at 3 bars and arm rotations at was performed at 570 nm and at 440 nm for proline 20 rpm. These conditions were maintained for 120 min, according to ISO 13903 [14]. Norleucine was used as with spot samples (5 kg) taken every 20 min through a an internal standard. door in the desolventizer-toaster. Monitoring of time Reactive lysine was determined using a method de- was initiated when the temperature inside the scribed by Moughan and Rutherfurd [15]. In short, re- desolventizer-toaster reached 100 °C. A second batch of active lysine was converted into homoarginine by 150 kg of the desolventized-untoasted RSM was used for incubation with O-methylisourea (OMIU) for 7 d. Sam- duplication of the toasting experiment on the next day ples were subsequently hydrolyzed with 6 mol/L HCl at (Fig. 1). The samples obtained from both toasted batches 110 °C for 23 h and the hydrolysates were adjusted using (in total 12 toasted RSM plus the untoasted RSM) were NaOH to pH 2.2. After separation by ion exchange chro- analyzed separately. Temperatures during toasting matography, the homoarginine content was determined ranged from 107 to 112 °C on the first day and from 109 by post column reaction with ninhydrin using photomet- to 112 °C on the second day. ric detection at 570 nm. The amount of OMIU-reactive lysine (OMIU-RL) was calculated from the molar Analytical methods amount of homoarginine and the molecular weight of The desolventized RSM and the toasted RSMs were lysine. ground to pass a 1 mm sieve using a centrifugal mill Glucosinolates were quantified according to ISO (ZM200, Retsch, Haan, Germany) at 8,000 rpm prior to 9167–1 [16]. Glucosinolates were extracted by a 70 % (v/ chemical analysis. Samples (5 g) were re-ground using a v) methanol in water mixture at 70 °C, using sinigrin as ball (Ø 12 mm) mill (MM2000, Retsch) at a frequency of internal standard. The glucosinolates were then linked 80 during 3 min and used for secondary structure, on an anion-exchange column, purified and on-column degree of denaturation, amino acid and reactive lysine desulphated by overnight action of sulphatase enzyme. analysis. All chemical and secondary structure analyses Desulphoderivatives were eluted with water and ana- were performed in duplicate, except for reactive lysine, lyzed using reverse phase liquid chromatography with which was performed in triplicate. gradient elution and UV detection at 229 nm. Nutrient contents Differential scanning calorimetry Dry matter (DM) content was determined by oven- Degree of denaturation of the samples was studied using drying at 103 °C to constant weight according to ISO differential scanning calorimetry (DSC12E, Mettler- 6496 [9]. Nitrogen content was analyzed by combustion Toledo, Greifensee, Switzerland). Samples (15–20 mg) according to AOAC 968.06 (Thermo Quest NA 2100 were weighed into medium pressure crucibles (ME-29990, Nitrogen and Protein Analyzer; Breda, the Netherlands) Mettler-Toledo, Greifensee, Switzerland) and approxi- [10]. Nitrogen content in the nitrogen solubility index mately 60 mg of demineralized water was added. Samples (NSI) and nitrogen linked to the acid detergent fiber were left overnight to equilibrate at 4 °C. The heating pro- (ADIN) determinations were measured using the gram ranged from 15 to 120 °C at a rate of 5 °C/min, with Kjeldahl method according to ISO 5983 [11]. A conver- an initial isothermal step of 5 min at 15 °C. A crucible sion factor of 6.25 was used for the calculation of CP filled with demineralized water was used in the reference content from nitrogen. Crude fat content was deter- cell. Enthalpy (J/g CP) of denaturation was determined mined according to ISO 734–2 [12]. The neutral deter- using the TA89E software (Version 3, Mettler-Toledo, gent fiber (NDF) and acid detergent fiber (ADF) Switzerland) for analysis of thermo-analytical data. contents were determined using a fiber analyzer equip- ment (Fiber Analyzer, Ankom Technology, Macedon, Protein solubility NY) according to a modification of the procedure of Van Protein dispersibility index (PDI) in water was mea- Soest et al. [13]. The NDF determination involved en- sured using a modification of the method of AOCS zymatic incubation with α-amylase and alcalase, but [17]. Approximately 75 mg of sample was weighed without addition of sodium sulfite. The ADIN content and mixed for 30 s with 1.5 mL of water in a vortex. was determined in the residues after hydrolysis with acid Thesamplewas then mixedfor 20 min in a rotator detergent reagents. SB2 (Stuart-Barloworld Scientific Staffordshire, UK) Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 4 of 11 with an angle of 90° and a speed of 20 rpm. Centrifu- STAT method from Pedersen and Eggum [22]. In gation was performed at 13,000 × g for 10 min at contrast to the original method, the hydrolysis was room temperature and the supernatant analyzed for extended for 120 min after the addition of the en- nitrogen content. Soluble protein in 0.2 % (w/v) zymes. The volume of 0.1 mol/L NaOH added was KOH, equivalent to nitrogen solubility index (NSI), used for the calculation of the degree of hydrolysis was measured according to ISO 14244 [18]. (DH) according to Eq. 1: DH ¼ðÞ Vb Nb =ðÞð α mp htot 1Þ Protein secondary structure Attenuated total reflectance Fourier transform infrared in which Vb is the volume of NaOH solution added, Nb spectroscopy (ATR-FTIR) (Tensor 27, Bruker, MA, is the normality of the titration solution, α is the degree USA) was used to measure the spectra ranging from 600 of dissociation of the α-NH group (i.e. 0.794 at 37 °C to 4,000/cm. The spectra were measured as absorbance and pH 8), mp is the mass of protein in grams and htot with a resolution of 4/cm and using 16 scans per spec- the total number of peptide bonds per gram of substrate tra. Spectral measurements were performed in duplicate (7.8 eq/g). The DH was used to calculate the rate of pro- and corrected for background. The OPUS software Ver- tein hydrolysis (k) based on the model described by sion 7.2 (Bruker, Billerica, MA, USA) was used for all Butré et al. [23] shown in Eq. 2: spectral transformations and calculations according to the procedure described by Hu et al. [19] with minor DH ¼ 1=b lnðÞ k ht þ 1 ð2Þ modifications. Briefly, Fourier self-deconvolution was applied to the Amide I region (1,595–1,705/cm) of the In this model b is a parameter that defines the shape original spectra, using a Lorentzian correction with a of the curve, k is the constant for the rate of protein hy- bandwidth of 25/cm and a noise reduction factor of 0.3. drolysis (/s) and ht is the hydrolysis time (s). The model The second derivative was applied to the original spectra was fitted using the MODEL procedure of SAS software and used for peak selection. Curve fitting of the selected (Version 9.3, SAS Institute Inc., Cary, NC). peaks was performed using a Gaussian approximation with the Levenberg-Marquardt algorithm as the fitting Calculations and statistical analysis method and an iteration time of 10 s. Selected peaks Degradation rate constants and half-life for the parameters were identified using existing literature [7, 20]. were calculated according to first-order reactions which were selected after fitting zero and second order reactions. In vitro digestibility Regression equations for the effect of toasting time were Two-step enzymatic digestibility generated using the GLM procedure of the statistical Dry matter and CP digestibility were determined using a software SAS. Correlations between parameters related modification of the method from Boisen and Fernández to protein changes (e.g. NSI, PDI, lysine and OMIU- [21]. Briefly, 5 g of material was mixed with 125 mL of a RL content, secondary structure) and in vitro digest- pH 6.0 disodium phosphate buffer (0.1 mol/L) and 50 mL ibility (e.g. CP digestibility, DH after 120 min, k) were of 0.2 mol/L HCl. This mixture was incubated with 5 mL determined using the CORR procedure of SAS soft- of a freshly prepared pepsin solution (0.025 g/mL) for 2 h ware. Linear or quadratic effects were considered as at 39 °C and pH 2.0. After this incubation, 50 mL of significant when the P-values were lower than 0.05 pH 6.8 sodium phosphate buffer (0.2 mol/L) and 25 mL of and as trends when P-values were between 0.05 and 0.6 mol/L NaOH were added. The pH was adjusted to 6.8, 0.10. The experimental unit was the RSM at each 5 mL of a freshly prepared pancreatin solution (0.10 g/mL) toasting time point. were added and the mixture incubated for 4 h. All buffers and solutions were preheated at 39 °C before addition, with Results the exception of the enzyme solutions. After the latter in- During the oil extraction process of the rapeseed seeds, cubation, 5 mL of a 20 % (w/v) sulfosalicylic acid solution the crude fat content was reduced from 493 g/kg DM in was added and the mixture centrifuged at 4,500 × g for the seeds to 16 g/kg DM in the untoasted meal (Table 1). 10 min at room temperature. The insoluble residue was At the same time, the NSI was decreased from 86.9 % in collected, freeze-dried and analyzed for dry matter and the seed to 79.9 % in the untoasted meal. nitrogen content. There was no effect of toasting time on the CP content of RSM, whilst there was a linear increase (P = 0.02) of pH-STAT enzymatic hydrolysis the DM content with increasing toasting time (Table 1). Enzymatic hydrolysis was performed with the addition There was a 33 % linear increase (P < 0.001) in the NDF of porcine trypsin, bovine chymotrypsin and porcine content with increasing toasting time from the untoasted intestinal peptidase using a modification of the pH- to the 120 min toasted RSM. In contrast, the ADF Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 5 of 11 Table 1 Characterization of rapeseed samples before and after toasting Material DM, CP, g/kg Crude fat, NDF, g/kg ADF, g/kg ADIN, g/kg Denaturation NSI, g/kg PDI, g/kg g/kg DM g/kg DM DM DM DM enthalpy, J/g CP CP CP Rapeseed 937 - 493 - - - 2.23 869 - Dried rapeseed 947 - 489 - - - - 861 - Rapeseed cake 923 - 135 - - - - 861 - RSM + solvent 907 - 13 - - - - 825 - Toasting time RSM 0 min 913.0 360.0 16 274.4 217.4 3.0 2.34 799.0 260.7 20 min 917.7 362.0 - 278.2 211.7 3.0 1.29 695.5 161.8 40 min 918.9 366.3 - 291.4 215.5 3.1 1.23 597.5 129.3 60 min 922.8 372.9 - 319.1 213.3 3.1 1.13 537.0 105.3 80 min 924.6 368.4 - 338.9 216.0 3.3 1.07 513.0 84.7 100 min 917.8 363.7 - 354.6 217.1 3.4 1.03 475.5 72.6 120 min 930.8 369.1 - 365.3 218.2 3.8 0.74 431.0 63.3 SEM 1.7 1.4 9.8 1.1 0.3 0.11 31 15 P-value Linear 0.02 0.19 - <0.001 0.21 <0.001 0.02 <0.001 <0.001 Quadratic 0.90 0.15 - 0.91 0.40 0.02 0.07 <0.001 <0.001 DM dry matter, CP crude protein, NDF neutral detergent fiber, ADF acid detergent fiber, ADIN nitrogen linked to the acid detergent fiber, NSI nitrogen solubility index, PDI protein dispersibility index, RSM rapeseed meal, SEM standard error of the mean content was not affected (P > 0.05) by toasting time. Glucosinolates content Linear (P < 0.001) and quadratic (P = 0.02) effects of There were linear and quadratic effects of toasting time toasting time were found on the content of ADIN. The on the content of total (P = 0.001), alkenyl (P < 0.01) and increase in the content of ADIN was more evident after indolyl plus aralkyl (P < 0.001) glucosinolates (Table 2). 60 min toasting. Toasting time had a linear effect (P = The largest decrease seems to occur after 60–80 min of 0.02) and a tendency for a quadratic effect (P = 0.07) on toasting. Not all the glucosinolate types, however, the denaturation enthalpy, which decreased with in- responded to toasting in the same manner. Whilst the creasing toasting time. There were linear (P < 0.001) and contents of epi-progoitrin, sinalbin and neoglucobrass- quadratic (P < 0.001) effects of toasting time on NSI and icin were linearly reduced (P < 0.001) with increased PDI, with more apparent effects at low toasting times. toasting times, the effect of toasting time was both linear Table 2 Content (μmol/g DM) of glucosinolates in rapeseed meal samples toasted for different times Toasting time PRO EPRO GNL GNA GBN SNB GST 4-OHGBS GBS NGBS Alk Ara + Ind Total 0 min 12.62 0.36 0.81 4.83 2.66 0.23 0.63 5.50 0.23 0.11 21.28 6.70 27.98 20 min 10.18 0.28 0.68 4.01 2.08 0.25 nd 2.69 0.18 0.09 17.21 3.20 20.41 40 min 8.15 0.25 0.56 3.22 1.67 0.18 nd 1.39 0.13 0.08 13.83 1.77 15.60 60 min 5.94 0.16 0.41 2.47 1.11 0.16 nd 0.58 0.09 0.06 10.09 0.89 10.97 80 min 3.84 0.06 0.28 1.65 0.71 nd nd 0.20 0.06 nd 6.52 0.26 6.78 100 min 2.27 nd 0.19 0.99 0.36 0.03 nd 0.06 nd nd 3.81 0.08 3.89 120 min 1.13 0.02 0.13 0.46 0.20 nd nd nd nd nd 1.94 0.00 1.94 SEM 1.05 0.04 0.06 0.40 0.23 0.03 - 0.45 0.02 0.01 1.78 0.54 2.28 P-value Linear <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 - <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Quadratic 0.001 0.19 0.006 0.04 0.003 0.73 - <0.001 0.01 0.55 0.002 <0.001 <0.001 PRO progoitrin, EPRO epi-progoitrin, GNL gluconapoleiferin, GNA gluconapin, GBN glucobrassicanapin, SNB sinalbin, GST gluconasturtin, 4-OHGBS 4- hydroxyglucobrassicin, GBS glucobrassicin, NGBS neoglucobrassicin, Alk alkenyl glucosinolates, Ara + Ind aralkyl plus indolyl glucosinolates, nd not detected, SEM standard error of the means. Alkenyl glucosinolates: PRO, EPRO, GNL, GNA, GBN; aralkyl glucosinolates: SNB, GST; indolyl glucosinolates: 4-OHGBS, GBS, NGBS Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 6 of 11 and quadratic (P < 0.05) for the other glucosinolates. proportion of α-helix decreased by half after the initial The most abundant alkenyl glucosinolates was progoi- 20 min of toasting and increased thereafter with toasting trin (Table 2) which, even after toasting for 120 min, time from 16.2 % after 20 min to 19.7 % after 120 min remained present at 9 % of its content in the toasting. Toasting time had a quadratic effect (P = 0.04) untoasted RSM. Gluconapoleiferin was the most resili- on the α-helix proportion. Linear (P =0.04) and quadratic ent alkenyl glucosinolate after toasting, as 16 % of the (P = 0.01) effects of toasting time were noticed on the T2 content of the untoasted RSM can still be found after proportion. This element increased after the first 20 min 120 min toasting. toasting, but stabilize thereafter. Linear (P <0.001) and quadratic (P = 0.004) effects of toasting time were also Amino acids content found for the proportion of A2 elements (Table 4). The The amino acid content is reported in Table 3. There increase in these elements was more apparent after the was a linear decrease (P < 0.05) in the content of alanine, first 20 min of toasting than thereafter. aspartic acid, glutamic acid and glycine with increasing toasting time. Increasing toasting time also caused a lin- Degradation rate constants ear decrease (P < 0.001) of the lysine and arginine con- Indolyl, alkenyl and total glucosinolates had the highest tent. Arginine and lysine contents were reduced by 7 degradation rate constants and the shortest half-life and 23 %, respectively, after toasting for 120 min in compared to the other parameters (Table 5). The deg- comparison with the untoasted RSM. The content of radation rate constant of indolyl glucosinolates was 2- OMIU-RL was also reduced (P < 0.001) linearly with in- fold higher than that of alkenyl glucosinolates. The creasing toasting time. After 120 min of toasting, the half-life of total glucosinolates was approximately 5.5- OMIU-RL content was 38 % lower than that in the fold lower than that of OMIU-RL and 10-fold lower untoasted sample. The reduction of the OMIU-RL con- than that of lysine. The degradation rate constant of tent after toasting was more pronounced than the reduc- OMIU-RL was almost twice that of lysine. The NSI had tion of the lysine content. This is reflected in the a degradation rate constant 2-fold as low as that of reduction of the OMIU-RL to lysine ratio from 0.98 in PDI, which is also reflected in a longer half-life. the untoasted RSM sample to 0.80 in the RSM toasted for 120 min. In vitro CP digestibility With the two-step enzymatic digestibility method, there Secondary protein structure was a tendency (P = 0.08) for a linear increase of the The proportion of intermolecular β-sheets tended (P = in vitro dry matter digestibility along with toasting time 0.06) to be affected by the quadratic effect of toasting (Table 6). In addition, there was a quadratic effect of time (Table 4). This proportion markedly increased after toasting time on the in vitro CP digestibility (P = 0.005), the initial 20 min of toasting and thereafter gradually increasing before 60 min of toasting and decreasing decreased with longer toasting times. In contrast, the thereafter. With the pH-STAT enzymatic hydrolysis Table 3 Amino acid contents (g/16 g N) and ratio between OMIU-RL and lysine in rapeseed meal samples toasted for different times Toasting Indispensable amino acids Dispensable amino acids time Arg His Ile Leu Lys OMIU-RL Phe Thr Val Ratio Ala Asp Glu Gly Ser Tyr 0 min 5.47 2.93 4.11 7.04 6.31 6.20 4.07 4.67 5.38 0.98 4.63 7.38 17.34 5.33 4.48 3.30 20 min 5.50 2.95 4.17 7.13 6.08 5.66 4.12 4.70 5.43 0.93 4.68 7.39 17.45 5.38 4.56 3.34 40 min 5.42 2.93 4.13 7.06 5.83 5.32 4.06 4.67 5.38 0.91 4.64 7.35 17.30 5.33 4.51 3.26 60 min 5.18 2.85 4.03 6.90 5.46 4.85 3.97 4.55 5.28 0.89 4.53 7.15 16.83 5.19 4.39 3.17 80 min 5.23 2.91 4.08 6.97 5.39 4.61 4.03 4.59 5.34 0.86 4.58 7.21 17.05 5.25 4.45 3.24 100 min 5.25 3.01 4.18 7.15 5.23 4.22 4.12 4.68 5.47 0.81 4.69 7.36 17.40 5.38 4.55 3.27 120 min 5.08 2.94 4.09 6.99 4.85 3.86 4.02 4.61 5.38 0.80 4.60 7.18 17.06 5.27 4.47 3.23 SEM 0.04 0.01 0.02 0.03 0.13 0.21 0.02 0.02 0.02 0.02 0.02 0.03 0.07 0.02 0.02 0.02 P-value Linear <0.001 0.09 0.09 0.15 <0.001 <0.001 0.37 0.15 0.07 <0.001 0.05 0.02 0.03 0.04 0.06 0.35 Quadratic 0.72 0.24 0.23 0.24 0.74 0.32 0.29 0.17 0.17 0.92 0.14 0.30 0.16 0.16 0.12 0.16 Arg arginine, His histidine, Ile isoleucine, Leu leucine, Lys lysine, OMIU-RL O-methylisourea reactive lysine, Phe phenylalanine, Thr threonine, Val valine, Ratio ratio OMIU-RL to lysine, Ala alanine, Asp aspartic acid, Glu glutamic acid, Gly glycine, Ser serine, Tyr tyrosine, SEM standard error of the mean Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 7 of 11 Table 4 Proportion (%) of the secondary structures of rapeseed meals toasted for different times Toasting Intermolecular β-sheets Intramolecular β-sheets α-helix T2 A2 α-helix : β-sheet time b (1,627 – 1,630/cm) (1,634 – 1,635/cm) (1,655 – 1,656/cm) (1,674/cm) (1,692/cm) 0 min 49.0 5.5 30.5 8.9 6.1 0.62 20 min 63.1 nd 16.2 13.7 6.9 0.26 40 min 60.8 nd 17.5 14.4 7.2 0.29 60 min 60.0 nd 18.0 14.6 7.4 0.30 80 min 59.4 nd 18.0 15.0 7.5 0.30 100 min 57.5 nd 20.4 14.6 7.5 0.36 120 min 57.9 nd 19.7 14.7 7.7 0.34 SEM 1.0 - 1.1 0.5 0.1 0.03 P-value Linear 0.94 - 0.63 0.04 <0.001 0.56 Quadratic 0.06 - 0.04 0.01 0.004 0.03 T2 turns, A2 intermolecular hydrogen-bonded β-sheets, nd not detected, SEM standard error of the mean Regions of the Fourier transform infrared spectra method, there were no effects of toasting time on the Discussion DH after 120 min hydrolysis. However, the rate of pro- The small reduction in the enthalpy of denaturation and tein hydrolysis was linearly (P < 0.001) reduced with in- the NSI, along with a high ratio of OMIU-RL to total ly- creasing toasting time. sine (0.98) in the untoasted RSM can be considered indi- The in vitro CP digestibility with the two-step en- cators of a RSM with low protein denaturation and high zymatic method did not correlate with any of the pa- protein nutritional quality. A decrease in protein solubil- rameters of protein changes measured. In contrast, ity in heat-treated materials is an indication of the aggre- significant correlations were found between k and NSI gation of proteins after denaturation [24, 25]. As more (r =0.88, P < 0.001), PDI (r = 0.79, P = 0.001), lysine (r proteins become denatured and unfolded with increasing = 0.92, P < 0.001), OMIU-RL content (r = 0.91, P < toasting time, intra and intermolecular interactions 0.001), and the proportion of A2 in the secondary within and between proteins promote aggregation. structure (r = −0.74, P = 0.004). Significant correlations Both NSI and PDI have been used before as indicators were also found between the DH after 120 min hy- of the extent of thermal damage in processed protein- drolysis and the proportion of intermolecular β-sheets rich ingredients (e.g. soybean meal and RSM) [26–28]. (r = −0.66, P = 0.01), α-helices (r = 0.60, P =0.03) and Protein solubility and the standardized ileal digestibility the ratio of α-helices to β-sheets (r = 0.58, P =0.04) in of AA in cecectomized broilers were reduced with in- the secondary structure. creasing autoclaving time of a commercial RSM [28]. Pastuszewska et al. [29] suggested that rapeseed meals with a NSI in 0.5 % KOH between 55 and 60 % can be considered of a high nutritional value. These values were Table 5 Degradation rate constants and half-life (first order achieved in our experiment between 40 and 60 min reactions) of parameters measured after toasting of rapeseed meal toasting, which correspond to toasting times used during Parameter Degradation rate constant, Half-life, min commercial RSM production [29]. −3 ×10 /min The increasing NDF and ADIN contents with increas- Enthalpy of denaturation 6.1 114 ing toasting time in the present experiment was previ- NSI 4.8 144 ously described after hydrothermal treatments of canola PDI 10.6 65 and RSM [8, 29, 30]. These authors, however, also re- ported an increase in the ADF content, which was not Alkenyl glucosinolates 20.4 34 found in the present study. The difference in the results Indolyl glucosinolates 44.3 16 could be due to milder conditions used in the present Total glucosinolates 22.3 31 experiment compared to those reported previously. The Arginine 0.7 990 increase of the ADIN content was linked to a decrease Lysine 2.1 330 of the standardized ileal protein digestibility and was OMIU-reactive lysine 3.8 182 proposed as a good indicator for protein damage [30]. NSI nitrogen solubility index, PDI protein dispersibility index Although it has been suggested that heat treatment Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 8 of 11 Table 6 Coefficients of in vitro digestibility and hydrolysis of rapeseed meal samples toasted for different times Toasting time Two-step enzymatic digestibility pH-STAT enzymatic hydrolysis CDMD CCPD DH 120 min k (/s) 0 min 0.382 0.752 0.186 0.029 20 min 0.393 0.758 0.173 0.032 40 min 0.406 0.773 0.179 0.027 60 min 0.418 0.776 0.175 0.024 80 min 0.415 0.764 0.183 0.018 100 min 0.411 0.753 0.179 0.017 120 min 0.415 0.750 0.185 0.013 SEM 0.005 0.003 0.002 0.002 P-value Linear 0.08 0.32 0.23 <0.001 Quadratic 0.19 0.005 0.18 0.47 CDMD coefficient of dry matter digestibility, CCPD coefficient of crude protein digestibility, DH degree of hydrolysis, SEM standard error of the mean increases the linkage between proteins and fiber [29], it increasing toasting time, chemical changes of AA con- is possible that the increase in the content of NDF, ADF tinue to occur resulting in the formation of more early and ADIN results from the inability of the solvents used MRP and the conversion of the early into advanced to solubilize the aggregated and chemically modified MRP and melanoidins [33]. In contrast to early MRP, ad- proteins (e.g. melanoidins) [30]. vanced MRP cannot be reverted into lysine under condi- The changes observed in protein denaturation and solu- tions of 6 mol/L acid hydrolysis [15]. This was noticed bility with increasing toasting time do not parallel the by a decrease of lysine content with increasing toasting changes observed in the secondary structure of proteins. time. The decrease of the OMIU-RL to lysine ratio is Contrary to what we expected, there was an increase in probably the result of higher rate of formation of early the proportion of α-helix and a decrease of intermolecular MRP compared to advanced ones. Previous research β-sheets with increasing toasting time after the initial only showed a reduction in the ratio between lysine and 20 min of toasting. Previous research [7, 20] described a reactive lysine after 64 min of toasting [8]. This might be decrease in the proportion of α-helix and an increase in due to the low reactive lysine to lysine ratio determined that of intermolecular β-sheet structures after thermal already in the rapeseed cake (i.e. 0.81). In a recent ex- treatment, which was also expected in the present experi- periment [3], values of lysine for commercial RSM of ment with increasing toasting time. The increase in the German oil mills ranged from 5.5 to 5.3 g/100 g CP, proportion of intermolecular β-sheets was linked to a de- which correspond in our experiment to toasting times of crease in the in vitro CP digestibility [7]. It is possible that approximately 71 and 91 min, respectively. However, in with increasing denaturation, which is the rate limiting that same experiment, the OMIU-RL content ranged step, there is partial unfolding of the proteins with a sim- from 4.4 to 4.0 g/16 g N for the same RSM. This makes ultaneous increase of aggregation and (partial) refolding of the ratio of OMIU-RL to lysine much lower compared the secondary structure. Most of the literature on to those reported here. The variation in the results could thermal-induced changes to the secondary structure of be due to the shorter incubation times for the reaction proteins reports the effects after a certain period of time with OMIU used in the those studies [3, 8] compared to (e.g. autoclaving at 120 °C for 20 min) [7, 20, 31], but do the longer incubation times used in the present study not include the changes occurring during that time period. (2–2.5 vs. 7 d). It is possible that proteins with a large When considering all time points analyzed in the present extent of thermal damage and high aggregation (low study, the net results for secondary structure are still com- solubility) might need longer incubation times for the parable to the results described in literature after autoclav- OMIU reactive to penetrate within the aggregate struc- ing [7, 20, 31]. The presence of A2 bands has been related ture and bind with the free lysine. Alternatively, free ly- to aggregation of proteins due to intermolecular sine may have been formed during toasting, which hydrogen-bonded anti-parallel β-sheets [32] or to absorp- cannot be analyzed by the OMIU-RL procedure. tion of infrared light from the amino acid side chains [7]. The decrease in the ratio lysine to CP with thermal treat- The formation of Maillard reaction products (MRP) ments has been reported before [8, 30, 34–36]. According results from chemical changes to AA, for which the to these authors, lower ratios, as compared to higher ones, most susceptible ones are lysine and arginine [33]. With indicate that protein damage occurred due to the Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 9 of 11 formation of MRP. A decrease of this ratio led to a de- when the enzymes are available sufficiently long to crease of the ileal digestibility of CP and AA [8, 30, 34]. hydrolyze. However, the rate at which the enzymes access Therefore, the decrease in the lysine to CP ratio reported the substrate during hydrolysis was linearly reduced by in our experiment with increasing toasting time is indica- toasting time. A reduction in the rate of hydrolysis with tive of protein damage and could lead to a decrease of the increasing heating time has been reported before [40] for in vivo protein digestibility. The ratio lysine to CP of the glycinin from soybeans. The high correlation of the rate of 0 min toasted RSM in the present study (6.3 g lysine/100 g hydrolysis with protein solubility (in alkali and water), and CP) corresponds well to values previously reported for lysine or OMIU-RL contents could explain the decrease in non-toasted canola meal [36]. The apparent ileal digestibil- the rate of hydrolysis with increasing toasting time. It is ity of lysine of the non-toasted canola meal in broilers not possible, however, to distinguish if the formation of ag- ranged from 87 to 92 % [36]. A lower ratio of lysine to CP gregates (i.e. lower solubility) or the chemical modification (5.55 g lysine/100 g CP) was reported in that study for of the Maillard-sensitive AA is the major factor controlling solvent-extracted canola meals from 7 different production therateof hydrolysis, as bothoccur simultaneously during plants. The apparent ileal digestibility of lysine for the toasting. The decrease in the rate of protein hydrolysis with solvent-extracted canola meals was lower and more vari- increasing toasting time could explain the reduction of the able (ranging from 65.5 to 85.7 %) than the values reported ileal protein digestibility reported in other studies after for non-toasted canola meal [36]. Other authors [8, 30] toasting [5, 8]. have reported lysine to CP ratios of 5.2 g/100 g CP in com- Extensive reviews have suggested inclusion levels of mercial canola meals and 5.1 g/100 g CP in RSM toasted total glucosinolates ranging from 2 to 2.5 μmol/g diet for 48 min, indicating damage of the proteins. These values for pigs, whilst for poultry, the inclusion level ranges corresponded to standardized ileal digestibilities of lysine from 2 to 10 μmol/g diet [41, 42]. To maintain these in growing pigs of 68.2 and 64 %, respectively. In the total glucosinolates level, at the maximum rate of pro- present experiment, values of lysine to CP ratio that resem- tein hydrolysis (i.e. 20 min of toasting) in this study, the ble the ones reported by these authors were obtained after inclusion level of RSM in the diets for pigs and poultry 100 min toasting, indicating that the thermal treatments can be 9.8 and 49 %, respectively. This would also in- applied by these authors were likely more severe than the volve a loss of 4 % lysine and 9 % OMIU-RL with respect ones used herein. to the untoasted RSM. At the maximum in vitro CP di- First order reactions have been used previously to gestibility (i.e. 60 min of toasting) in this study, the in- model the decrease in glucosinolate content of red cab- clusion level in the diets for pigs can increase to 18.2 %, bage [37] and reactive (available) lysine in model systems whilst there would be no limit to the inclusion level for [38]. One of the aims of toasting during the production poultry diets. This would involve a loss of 13 % lysine of RSM is to inactivate the glucosinolates without affect- and 22 % OMIU-RL with respect to the untoasted RSM. ing the nutritional quality of proteins (e.g. lysine con- However, the inclusion level of rapeseed or canola meal tent). Glucosinolates were degraded at a faster rate than in the diets for pigs might depend not only on the con- the degradation of OMIU-RL and lysine. Furthermore, tent of total glucosinolates, but also on the type of glu- the rate constant of decrease of the solubility parameters cosinolates included [43]. Whilst the feed intake of (i.e. NSI and PDI) is higher than that of OMIU-RL and weanling pigs did not decrease after the inclusion of lysine. This could be an indication that changes in the 2.2 μmol/g diet of total glucosinolates from Brassica structure of proteins occur earlier during toasting than napus [44], the inclusion of 2.2 μmol/g diet of total glu- chemical (i.e. Maillard) changes. The higher rate con- cosinolates from Brassica juncea in diets for growing- stant of decrease of PDI could make it a better indicator finishing pigs resulted in a decrease of feed intake and of the changes in solubility after toasting of RSM than weight gain [43]. The major glucosinolate in B. juncea is NSI. Previous research in soybeans indicated that PDI gluconapin, whilst B. napus contains higher levels of reflects protein quality better than NSI, especially after progoitrin than gluconapin [45]. processing at mild conditions [39]. The range of values obtained with the two-step in vitro CP digestibility can be considered as narrow (75.0– Conclusions 77.6 %). A linear decrease of the in vitro CP digestibility Toasting of RSM for increasing time induces physical from 71 % in the 48 min toasted RSM to 62 % in the and chemical changes to the proteins and affects its nu- 93 min toasted meal was reported in a recent study [8]. tritional value. These changes are correlated to the rate This also matched the reported decrease in standardized of protein hydrolysis but not the in vitro CP digestibility ileal CP digestibility in that study. Toasting time did not or the extent of hydrolysis. Degradation of glucosinolates affect the DH after 120 min indicating that the observed occurs earlier during toasting and at higher rates than protein changes are not a restriction for protein hydrolysis that of OMIU-RL and lysine. Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2016) 7:62 Page 10 of 11 Abbreviations 13. Van Soest PJ, Robertson JB, Lewis BA. 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Geneva: International Additives BV, Ajinomoto Eurolysine s.a.s and Stichting VICTAM BV. SSV Standards Organisation; 1992. acknowledges the support from the Universidad de Costa Rica. 17. AOCS. Protein dispersibility index. Official Method Ba 10–65. Champaign: American Oil Chemists Society; 1980. 18. ISO. Oilseed meals. Determination of soluble proteins in potassium Authors’ contributions hydroxide solution. Geneva: International Standards Organisation; 2014. SSV, EMAMB, PC, AQ and AFBvdP conceived and designed the experiment. PC 19. Hu X, Kaplan D, Cebe P. Determining beta-sheet crystallinity in fibrous and AQ performed the production of the experimental samples. HG, EMAMB proteins by thermal analysis and infrared spectroscopy. Macromolecules. and AFBvdP supervised the experimental work. SSV performed all chemical 2006;39:6161–70. analyses and wrote the manuscript. EMAMB, AFBvdP, HG and WHH checked 20. Carbonaro M, Maselli P, Dore P, Nucara A. 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Journal
Journal of Animal Science and Biotechnology – Springer Journals
Published: Oct 18, 2016