Of proteins and processing: mechanisms of protein damage upon rapeseed processing and their effects on nutritional value

Hydrothermal processing is a common practice during the manufacture of protein-rich feed ingredients, such as rapeseed meal (RSM), and feeds. This processing step can induce physical and chemical changes to the proteins, thereby reducing the digestibility and utilization of crude protein (CP) and amino acids (AA). Whilst most literature has linked the chemical changes to the proteins with negative effects on protein digestibility, the effects of the physical changes of the protein conformation have not been considered simultaneously. Hence, the aim of this thesis was to provide further insight into the mechanisms of protein damage during ingredient/feed processing and their effects on protein hydrolysis/digestibility. In Chapter 2, the available literature on the physical changes that occur to vegetable proteins used in swine diets after processing was reviewed. Overall, hydrothermal processing increases the contents of intermolecular/intramolecular β-sheets and disulfide bonds, which were negatively correlated to protein digestibility. The correlations, however, were dependent on the type of protein analysed. When the physico-chemical changes in the proteins occur during processing of the ingredients, proteins usually become less responsive to further processing treatments. Rapeseed proteins were used as model, as this oilseed is hydrothermally processed during the oil extraction process and is further processed when incorporated in animal diets. Protein damage during production of rapeseed meal There is high variability in the nutritional value of commercial RSM. The variation is mainly due to the conditions used during the desolventization/toasting step. Therefore, the aim of the experiment in Chapter 3 was to characterize the secondary structure and chemical changes that occur during toasting of RSM and their effects on in vitro protein digestibility. A cold defatted RSM was toasted for 120 min with samples obtained every 20 min. Increasing the toasting time from 0 to 120 min increased protein denaturation by 3-fold and decreased protein solubility by 4-fold, lysine content by 23% and the reactive lysine content by 37%. The proportion of intermolecular β-sheets increased after the initial 20 min of toasting, but steadily decreased thereafter. The contrary was observed for the proportion of α-helices. The changes in the secondary structure of proteins were not correlated to the rest of the physical and chemical changes. Therefore, changes in the secondary structure of proteins cannot be considered good indicators of damage to proteins due to hydrothermal processing. The rate of protein hydrolysis decreased by 2-fold when toasting time was increased from 0 min to 120 min. The changes in protein solubility and lysine/reactive lysine contents were positively correlated to the rate of protein hydrolysis. Changes to the physical conformation of rapeseed proteins occur at faster rates during toasting compared to chemical changes. In Chapter 4, it was hypothesized that the decrease in the rate of protein hydrolysis with increasing toasting time was due to the reduction in protein solubility. In order to test this hypothesis, the soluble and insoluble protein fractions from each of the RSM studied in Chapter 3 were separated and hydrolysed. Hydrolysis kinetics and the molecular size distribution of the peptides resulting after hydrolysis were analysed. The extent of hydrolysis of the insoluble protein fraction was 44% higher than that of the soluble protein fraction. The rate of hydrolysis of the soluble protein fraction separated from the hydrothermally treated RSM was 3-9 fold higher than that of the insoluble protein fraction. In the insoluble fraction, formation of both disulfide bonds and Maillard reaction products (MRP) (fructosyl-lysine [FL], carboxymethyl-lysine [CML] and carboxyethyl-lysine [CEL]) was noticed, which explains the decrease in the rate of protein hydrolysis with longer toasting times. Overall, increasing the toasting time of the whole RSM and the insoluble protein fraction increased the size of the peptides resulting after enzymatic hydrolysis. A shift in the mechanism of protein hydrolysis from a more one-by-one type to a more zipper-type likely explained the correlations between the rate of hydrolysis and the molecular size distribution after hydrolysis. Protein solubility seems to be a key parameter for understanding the decrease in the rate of protein hydrolysis with increasing toasting time. The correlations between two in vitro protein digestibility methods and the standardised ileal digestibility in growing pigs of severe thermally-treated soybean and rapeseed meals were studied in Chapter 5. Soybean meal and RSM were toasted in the presence of lignosulfonate in order to induce severe thermal damage to the proteins. In vitro protein digestibility was analysed using the two-step enzymatic method (pepsin at pH 2.0 and pancreatin at pH 6.8) and the pH-STAT method. The standardised ileal digestibility values were obtained from a previous experiment, in which ileal-cannulated growing pigs were used. The degree of hydrolysis after 10 min was positively correlated (r = 0.95, P = 0.046) to the standardised ileal CP digestibility. The in vitro rate of protein hydrolysis using the pH-STAT method and CP digestibility using the two-step enzymatic method tended to be positively correlated to the standardised ileal digestibility of CP (r = 0.91, P = 0.09, for both in vitro methods). In conclusion, both in vitro methods might be used for the in vivo digestibility of severe thermally-treated ingredients. Effects of diet processing on protein digestibility of RSM with different extents of damage The processed ingredients (e.g. rapeseed meal) are mixed with other ingredients and processed further during the compound feed manufacturing process. The effects of the diet processing methods (e.g. pelleting and extrusion) on protein digestibility could depend on the extent of the damage of the ingredients used. The aim of Chapter 6 was to test the effects of toasting time of rapeseed meal, diet processing method and the interaction between both on protein digestion along the gastrointestinal tract and apparent/standardised ileal digestibility of CP and AA. Mash, pelleted and extruded diets were manufactured using either 0, 60 or 120 min toasted RSM as the only protein source, for a total of 9 different experimental diets. Whilst increasing the toasting time decreased the contents of lysine and reactive lysine in the diets, no effects were noticed after pelleting or extrusion of the diets compared to the mash. The mean particle size of the diets was reduced from 479 μm in the mash diets to 309 and 211 μm after pelleting and extrusion, respectively. A total of 81 growing boars were individually fed with one of the experimental diets. Following euthanasia, the small intestine was divided in 3 sections of equal length and the contents of the final 1.5 m of each small intestine section were sampled. The apparent CP digestibility for each section of the gastrointestinal tract was used to calculate the rate of CP digestion based on a second order equation. The rate of digestion was higher in the diets containing 0 min toasted RSM compared to the diets that contained 60 or 120 min toasted RSM. The diet processing method tended to affect the rate of protein digestion, with higher rates for the extruded > pelleted > mash diets. Significant effects of the interaction between toasting time and diet processing method were found on the apparent ileal CP digestibility. Whilst a lower apparent ileal CP digestibility was found in the 120 min toasted RSM mash diet compared to the 0 and 60 min toasted RSM mash diets, no differences were observed between the different toasting times in the pelleted and extruded diets. Similar significant interactions were noticed for the apparent ileal digestibility of some dispensable and indispensable AA (e.g. arginine, isoleucine, leucine, methionine, threonine, alanine, glycine, proline, serine). Pelleting of the 60 and 120 min toasted RSM diets increased the standardized ileal digestible CP content by 6 and 15%, respectively, compared to the 60 and 120 min toasted mash diets. Extrusion of the 0, 60 and 120 min toasted RSM diets increased the standardized ileal digestible CP content by 5, 9 and 12%, respectively, compared to the 0, 60 and 120 min toasted mash diets. Similar positive effects of pelleting and extrusion were obtained for the apparent ileal digestible contents of lysine and reactive lysine, especially for the diets that contained RSM toasted for longer times. In conclusion, the severe effects of protein damage during the production of RSM on protein digestibility can be (partially) ameliorated by processing of the diets. Processing of ingredients and diets can lead to the formation of early (e.g. FL) and advanced (e.g. CML and CEL) MRP. These MRP have been associated with common metabolic disorders, for example atherosclerosis. Absorption of dietary MRP has been previously estimated based on indirect measurements, such as concentrations in blood, urine and faeces, which could be biased by endogenous formation of MRP and deposition in tissues. Hence, the aim of Chapter 7 was to measure the apparent ileal digestibility of early (FL, determined as furosine after acid hydrolysis) and advanced (CML and CEL) MRP. The same diets and ileal digesta samples as in Chapter 6 were used in this study. The 0 min toasted RSM diets (mash, pelleted and extruded) were excluded from this study. Sucrose was added to these diets immediately before feeding in order to increase their feed intake, which could confound the determination of the apparent ileal digestibility of the MRP for these treatments. The content of FL was higher in the 120 min compared to the 60 min toasted RSM diets, whereas it was lower in the extruded diets compared to the pelleted and mash diets. The decrease in FL content after extrusion of the diets can probably be related to conversion of the early into advanced MRP, as the content of CML was higher in the extruded diets compared to the pelleted and mash diets. The contents of FL, CML and CEL were positively correlated to the contents of these compounds in the ileal chyme. The apparent ileal digestibility of FL, CML and CEL for the different diets ranged from -8.5 to 19.1%, -0.2 to 18.3% and 3.6 to 30%, respectively. In conclusion, the apparent ileal digestibility of the early and advanced MRP from thermally-treated RSM diets in growing pigs were overall low and did not seem to be related to the contents of these compounds in the diets. The results of this thesis indicate that the changes to the physical conformation of proteins during toasting of RSM occur at a faster rate than chemical changes. Both types of changes affect protein solubility, which is important in determining the rate of protein hydrolysis. It is suggested in this thesis that the rate of protein hydrolysis of hydrothermally-processed ingredients is probably the main contributing factor for the in vivo protein digestibility. This was demonstrated, as increasing the toasting time of RSM decreased the rate of CP digestion in growing pigs. The negative effects of longer toasting times on CP and AA digestibility, however, could be ameliorated by pelleting and extrusion of the diets. The positive effects of diet processing methods (pelleting and extrusion) on the digestibility of damaged proteins from thermally-treated ingredients should be taken into account in feed evaluation studies and formulation of practical diets.</p

Activación del sistema inmune en cerdos y su requerimiento de metionina, treonina y triptófano

Las altas densidades de animales en granjas porcinas son un desafío para el adecuado estado de salud de los animales, favoreciendo la presencia de diversas enfermedades. Estas enfermedades provocan que el organismo del animal realice ajustes costosos desde el punto de vista metabólico, incrementando el gasto energético y provocando una desviación de los nutrientes utilizados para cubrir los requerimientos de mantenimiento, crecimiento y reparación tejidos, hacia el funcionamiento del sistema inmune. Por lo tanto, el objetivo de esta revisión literaria es analizar los efectos de la activación del sistema inmune en cerdos desafiados sanitariamente sobre el requerimiento de algunos aminoácidos, con el fin de valorar el componente inmune como una parte clave para una nutrición más efectiva. Ante la activación del sistema inmune se producen citoquinas proinflamatorias, las cuales a su vez estimulan la síntesis de proteínas de fase aguda. Esta síntesis incrementa el requerimiento de ciertos aminoácidos, de tal forma que si la necesidad de aminoácidos sobrepasa la disponibilidad a nivel sérico, se presentará una inducción al catabolismo de proteína muscular; además, estos aminoácidos también son utilizados como fuente de energía y precursores antioxidantes. La metionina, treonina y triptófano son los principales aminoácidos que influyen en la respuesta del sistema inmune a enfermedades clínicas y subclínicas. En conclusión, una dieta que supla, ante un reto inmunológico, únicamente los requerimientos de mantenimiento y crecimiento de los cerdos, sin considerar los requerimientos del sistema inmune, puede producir una deficiencia nutricional y menor rendimiento productivo del animal. Por lo tanto, se debe valorar la necesidad de suplementar estos aminoácidos por encima de la cantidad recomendada en un perfil de proteína ideal, especialmente bajo condiciones de estrés sanitario.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro de Investigación en Nutrición Animal (CINA

Effect of protease supplementation on apparent ileal crude protein and amino acid digestibility of over-processed soybean meals in broilers

Abstract Background: Nutritional value of proteins in feed ingredients can be negatively affected by hydrothermal processing, which causes large variation in the bioavailability of amino acids (AA) and negatively affects animal productive performance. Supplementation of exogenous proteases could increase the rate of digestion of damaged proteins, thereby increasing overall AA digestibility and bioavailability. The aim was to determine the effect of exogenous protease supplementation on the apparent ileal digestibility (AID) of crude protein (CP) and AA of soybean meals (SBM) with different degrees of hydrothermal processing in broilers. Methods: The experiment involved a 3 × 2 factorial arrangement, with SBM processing time (commercial SBM or autoclaved for 30 or 60 min at 120 °C) and protease supplementation (not supplemented and supplemented) as factors. Protease was included at three times the recommended dose (0.06%) and the experimental diets were fed from 15 to 21 d. Results: The interaction between the effects of SBM processing and protease supplementation was significant for the AID of CP (P = 0.01), Trp (P = 0.01), Gly (P = 0.03) and Pro (P = 0.03), and also for the average daily gain (P = 0.01) and feed conversion ratio (P = 0.04). Increasing the processing time of SBM decreased (P < 0.0001) the AID of all amino acids, whilst the effect of protease supplementation was only significant for the AID of Phe (P = 0.02) and Tyr (P = 0.01). Conclusions: Exogenous protease supplementation at three times the commercial dose does not seem to offset the negative effects of hydrothermal processing of SBM on the apparent ileal digestibility of CP and amino acids or performance of broilers. Whilst positive numerical improvements of digestibility and performance (ADG and FCR) were noticed with protease supplementation at relatively mild processing levels, negative results were obtained with the harsh-processed meals.Consejo Nacional para Investigaciones Científicas y Tecnológicas/[RE-006-17]/CONICIT/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro de Investigación en Nutrición Animal (CINA)UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni

Suplementación de extracto de Cynara scolymus L. y betaína en cerdas durante la lactancia

Introducción. La cerda moderna tiene la capacidad genética de producir un mayor número de lechones por camada y una mayor producción de leche, lo que conlleva que el animal tenga mayores requerimientos de nutrientes. Objetivo. Evaluar el desempeño productivo de las cerdas y su progenie a la suplementación de un producto comercial a base de extracto seco de alcuacil (Cynara scolymus) e hidrocloruro de betaína durante el período de lactancia. Materiales y métodos. El estudio se llevó a cabo durante el primer semestre del año 2020, en la provincia de Limón, Costa Rica. Se utilizaron un total de 42 cerdas en etapa de lactancia que fueron distribuidas en tres tratamientos: dieta control (T1), dieta control + 0,25 kg t-1 de producto comercial (T2) y dieta control + 0,50 kg t-1 de producto comercial (T3), todos los tratamientos fueron isoproteicos e isoenergéticos. Resultados. La inclusión del aditivo mostró una tendencia a la mejora (p=0,09) de los parámetros evaluados en la cerda. La pérdida de condición corporal al final de la lactancia fue menor (p=0,03) en T2, mientras que la suplementación con 0,50 kg t-1 de producto comercial presentó resultados intermedios. Los parámetros evaluados en la camada (peso al destete, ganancia de peso y cantidad de lechones destetados por cerda) no presentaron diferencias significativas (p>0,05) entre tratamientos. Conclusión. El uso del aditivo mostró una tendencia a mejorar el consumo de alimento en la cerda durante la etapa de lactancia, lo que disminuyó las pérdidas de peso y condición corporal durante este periodo. La inclusión del aditivo no mejoró el desempeño productivo de los lechones

Processing temperature and sugar type affect the rate and the extent of proteolysis of a model soy protein isolate system

Processing of ingredients and diets using hydrothermal treatments can lead to negative effects on the nutritional value of proteins, depending on the processing conditions and the matrix of the ingredient or diet. The aim of this study was to evaluate the effects of processing temperature and sugar type on the extent and rate of protein hydrolysis using soy protein isolate as a model system. The experiment utilized a 3☓3 factorial design, with three repetitions per treatment, using autoclaving temperature (not autoclaved, 100 °C, 120 °C) and type of sugar (not added, glucose, xylose) as main factors, also evaluating the interaction between both. Limited formation of Maillard reaction products occurred in the absence of sugars, whilst the addition of xylose increased the formation of MRPs at all processing temperatures. Crosslinks between amino acids (lysinoalanine, lanthionine) occurred with increasing temperatures, with sugar addition being inhibiting. There was a significant interaction (P < 0.01) between the effects of processing temperature and type of sugar for nitrogen solubility, the extent and rate of proteolysis. The effect of autoclaving at 100°C on the rate of protein hydrolysis of the samples without sugars added was as high as the additional effect of autoclaving after the addition of sugars. In contrast, the effect of autoclaving at 120°C on the rate of protein hydrolysis was higher for the samples that contained sugars, compared to those that did not had sugars added. In conclusion, the effect of sugar addition on the extent and rate of proteolysis seems to be dependent on processing temperature, which might indicate different protein damage mechanisms limiting enzymatic protein hydrolysis.</p

Effects of Toasting Time on Digestive Hydrolysis of Soluble and Insoluble 00-Rapeseed Meal Proteins

Thermal damage to proteins can reduce their nutritional value. The effects of toasting time on the kinetics of hydrolysis, the resulting molecular weight distribution of 00-rapeseed meal (RSM) and the soluble and insoluble protein fractions separated from the RSM were studied. Hydrolysis was performed with pancreatic proteases to represent in vitro protein digestibility. Increasing the toasting time of RSM linearly decreased the rate of protein hydrolysis of RSM and the insoluble protein fractions. The extent of hydrolysis was, on average, 44% higher for the insoluble compared with the soluble protein fraction. In contrast, the rate of protein hydrolysis of the soluble protein fraction was 3–9-fold higher than that of the insoluble protein fraction. The rate of hydrolysis of the insoluble protein fraction linearly decreased by more than 60% when comparing the untoasted to the 120 min toasted RSM. Increasing the toasting time elicited the formation of Maillard reaction products (furosine, Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine) and disulfide bonds in the insoluble protein fraction, which is proposed to explain the reduction in the hydrolysis rate of this fraction. Overall, longer toasting times increased the size of the peptides resulting after hydrolysis of the RSM and the insoluble protein fraction. The hydrolysis kinetics of the soluble and insoluble protein fractions and the proportion of soluble:insoluble proteins in the RSM explain the reduction in the rate of protein hydrolysis observed in the RSM with increasing toasting time

Apparent ileal digestibility of Maillard reaction products in growing pigs

The absorption of Maillard reaction products (MRP) from dietary origin has been linked to the occurrence of chronic diseases. The aim of the present study was to determine the effects of toasting time of rapeseed meal (RSM) and the processing method of the diets (pelleting and extrusion) that included RSM on the apparent ileal digestibility (AID) of total lysine, fructosyl-lysine (FL), carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), lanthionine (LAN) and lysinoalanine (LAL) in growing pigs. The study consisted of a 2×3 factorial design with toasting time of RSM (60, 120 min) and diet processing method (mash, pelleted, extruded) as factors. Fifty growing pigs were individually fed one of the experimental diets for 4.5 consecutive days. Following euthanasia, samples of digesta were collected from the terminal 1.5 m of the small intestine. Increasing the toasting time of RSM increased the contents of FL, CML and CEL, whereas the additional effects of the diet processing methods were relatively small. Lysinoalanine and lanthionine were not detected in the diets; therefore, digestibility of these compounds could not be determined. The contents of FL, CML and CEL in the ileal chyme were positively correlated to their contents in the diets. The AID of the MRP from thermally-treated RSM were overall low and were not related to their contents in the diets. The AID of FL ranged between -8.5 and 19.1%, whilst AID of CML and CEL ranged from -0.2 to 18.3 and 3.6 to 30%, respectively. In conclusion, thermal treatments have clear effects on the contents of MRP in the diets. These compounds have relatively low digestibility in growing pigs.</p

Physical and chemical changes of rapeseed meal proteins during toasting and their effects on in vitro digestibility

La Universidad de Costa Rica proporcionó una beca para el programa de doctorado de Sergio Salazar VillaneaBackground: 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.UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Zootecni