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

Effect of protease supplementation on apparent ileal crude protein and amino acid digestibility... 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. Keywords: Amino acids, Apparent ileal digestibility, Broilers, Exogenous protease * Correspondence: sergio.salazarvillanea@ucr.ac.cr Escuela de Zootecnia, Universidad de Costa Rica, San José, Costa Rica Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 2 of 8 Background autoclaved for 30 or 60 min at 120 °C) and protease sup- The nutritional value of feed ingredients can be nega- plementation (not supplemented and supplemented; tively affected by hydrothermal processing. Protein de- RONOZYME ProAct, Novozymes, Krogshoejvej, naturation during processing could lead to changes of Denmark) as factors. This design resulted in 6 experi- their structural conformation, resulting in the formation mental diets: unautoclaved SBM without and with en- of protein aggregates, which render the hydrolytic sites zyme supplementation (SBM and SBM + E, respectively), inaccessible to enzymes [1, 2]. In addition, processing 30 min autoclaved SBM without and with enzyme sup- can induce chemical modifications to the structure of plementation (SBM30 and SBM30 + E, respectively) and amino acids (AA) resulting in the formation of Maillard 60 min autoclaved SBM without and with enzyme sup- reaction products [3–5]. The chemically modified AA plementation (SBM60 and SBM60 + E, respectively). cause a stearic hindrance effect, which also limits en- zyme accessibility for hydrolysis [6]. The main protein- Experimental diets bound AA affected, lysine and arginine, are also the tar- Commercial SBM was purchased (Concentrados Gastón get AA of trypsin, one of the main proteolytic endogen- Fernández, Cartago, Costa Rica) and divided in 3 ous enzymes, which likely reduces the efficiency of batches: one was left unautoclaved, whilst the other two hydrolysis [2]. The overall result is a reduction in the were autoclaved at 120 °C for 30 and 60 min, respect- rate of protein hydrolysis [7, 8], which results in a de- ively. Temperatures during autoclaving were measured crease of protein digestibility [8]. Fermentation of un- with internal thermocouples and ranged from 118 to digested proteins can result in undesired putrefaction 123 °C. products in the gut (e.g. biogenic amines, phenolic and Experimental diets were formulated according to the indolic compounds) and increase the proliferation of recommendations from Ravindran et al. [15] for the pathogenic bacteria [9]. Moreover, undigested proteins evaluation of amino acid digestibility of raw materials in contribute to N emissions to the environment and can broilers (Table 1) and fed as mash. Chromium oxide therefore be considered as a pollutant [10]. (Cr O ) was included in the experimental diets as a 2 3 Exogenous enzymes, such as proteases, are a tool in marker. The exogenous protease was supplemented at 3 state-of-the-art animal nutrition to increase the nutri- tional value of feed ingredients and improve animal per- Table 1 Ingredient and nutritional composition of the formance at a lower cost [11]. The proteolytic experimental diets, g/kg as is mechanism of exogenous proteases is complementary to Ingredients Basal diet Basal diet + enzyme that of endogenous proteases [12]. For example, trypsin, Dextrose 507 506.4 one of the main endogenous digestive enzymes, is highly Soybean meal 416 416 specific for Lys and Arg; whilst, the exogenous Soybean oil 40 40 subtilisin-like proteases have high affinities towards large Dicalcium phosphate 19 19 hydrophobic amino acids, such as Phe and Tyr [13]. Calcium carbonate 10 10 Therefore, an increase in the rate of hydrolysis and amino acid digestibility could be expected when exogen- Cr O 33 2 3 ous proteases are supplemented in the diets [14]. Sodium bicarbonate 2 2 Our objective was to determine the effect of exogen- Salt 2 2 ous protease supplementation on the productive per- Vitamins and minerals premix 11 formance and apparent ileal digestibility (AID) of crude Enzyme – 0.6 protein (CP) and AA of soybean meals (SBM) with dif- Nutritional composition (calculated) ferent degrees of hydrothermal processing. We hypothe- sized that exogenous protease supplementation would Metabolizable energy, kcal/kg 3110 3107 increase the rate of hydrolysis of thermally damaged pro- Crude protein 196.10 196.10 teins, thereby increasing AA digestibility at the end of Available phosphorus 5.02 5.02 the small intestine. Calcium 8.49 8.49 Fufeng Group, China Methods Commercial soybean meal used was unautoclaved, autoclaved 30 min at Experimental setup 120 °C or autoclaved 60 min at 120 °C Vitamins and minerals supplied per kg of diet: Cu (sulfate), 12 mg; Fe (sulfate), Animal experimental procedures were approved by the 40 mg; I (iodide), 1 mg; Se (selenate), 0.30 mg; Mn (sulfate and oxide), 80 mg; Animal Care and Use Committee, University of Costa Zn (sulfate and oxide), 80 mg; retinol, 10,000 UI; cholecalciferol, 3000 UI; tocopheryl acetate, 35 UI; menadione, 2.50 mg; thiamine, 1.75 mg; riboflavin, Rica, under the authorization number CICUA-110-17. 6.50 mg; niacin, 55 mg; pantothenate 10.70 mg; pyridoxine, 3.60 mg; folate, The experiment involved a 3 × 2 factorial arrangement, 1.50 mg; cyanocobalamin, 15 μg; biotin, 110 μg with SBM autoclaving time (commercial SBM or RONOZYME ProAct, Novozymes, Krogshoejvej, Denmark Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 3 of 8 times the commercial recommendation (commercial rec- Each pen was considered as an experimental unit. Birds ommendation is 15,000 PROT/kg), in order to assure had ad libitum access to water and a commercial pre- that the enzyme was not a limiting factor for hydrolysis. starter (1–7 d) and starter diets (8–14 d). From d 15–21, One PROT unit is defined as the amount of serine pro- the birds received ad libitum access to the experimental tease that liberates 1 μmol para-nitroaniline (pNA) from diets. Average daily gain (ADG) of each bird was calcu- 1 mmol/L Suc-Ala-Ala-Pro-PhepNA (C H N O ) lated using the average weight at the start and the end of 30 36 6 9 (Sigma-Aldrich, St. Louis, MO, USA) substrate per mi- the experimental period (14 and 21 d, respectively), di- nute at pH = 9.0 and at 37 °C. The analyzed nutritional vided by the number of birds in each experimental unit. composition of the experimental diets is depicted in Average daily feed intake (ADFI) was determined as the Table 2. difference between the feed offered during the 14–21 d period and feed refusal at 21 d and calculated per bird. Animals and housing Feed conversion ratio (FCR), corrected for mortality, was A total of 504 one-day-old Cobb 500 broilers were allo- calculated as the ratio between ADFI and ADG. cated at a density of 12 birds/m in an open environ- ment facility. Temperatures during the experiment Sample collection and chemical analysis averaged 22.84 ± 2.61 °C and a light regimen of 12 h light On d 21, 6 birds were randomly selected per pen and and 12 h dark was used. There were 7 repetitions per euthanized by cervical dislocation. Digestive content treatment for the experimental diets, for a total of 42 ex- from the last 20 cm of the ileum (anterior to the ileo- perimental units, which were allotted to a total of 6 ex- cecal valve) of each bird was collected by gentle strip- perimental blocks, where each experimental diet was ping and pooled per experimental unit. Samples were represented at least once in every experimental block. immediately frozen and kept at − 70 °C, followed by Table 2 Analyzed nutrient composition of the soybean meals and experimental diets, g/kg as is Nutrient SBM – autoclaving time Experimental diets 0 min 30 min 60 min SBM SBM + E SBM30 SBM30 + E SBM60 SBM60 + E DM 868.0 856.4 847.5 880.9 877.5 882.5 875.8 874.7 869.9 CP 441.6 437.8 437.9 192.3 192.5 192.9 205.4 209.8 215.8 Cr –– – 0.040 0.030 0.070 0.043 0.075 0.079 Protease (PROT/kg) –– – nd 56,470 LOQ 48,680 nd 53,900 Essential amino acids Arg 27.9 26.4 24.8 12.9 10.9 11.6 16.5 10.3 12.7 His 13.7 10.2 12.7 6.0 5.3 5.9 8.0 5.6 6.5 Ile 18.0 17.6 17.7 8.6 7.6 8.5 11.2 8.2 9.3 Leu 30.4 29.3 28.8 13.7 12.5 13.6 18.5 13.4 15.1 Lys 24.8 22.4 20.6 10.1 8.4 8.8 12.5 7.9 8.8 Met 6.1 6.2 5.9 2.9 2.8 3.1 3.5 2.8 2.9 Phe 23.6 19.7 22.1 10.5 9.6 9.5 14.4 9.9 11.7 Thr 15.3 15.1 15.0 7.2 6.5 7.2 9.6 7.1 8.0 Trp 7.6 6.7 6.5 2.9 2.2 3.9 2.2 2.5 1.9 Val 18.4 17.8 17.8 8.6 7.8 8.3 11.2 8.2 9.2 Non-essential amino acids Ala 16.2 15.3 15.6 7.4 6.8 7.6 10.0 7.3 7.9 Asx 45.8 44.1 44.6 22.3 19.5 21.9 29.1 21.1 24.1 Cys 6.1 5.5 4.9 2.8 2.6 3.1 3.3 2.5 2.5 Glx 71.2 70.8 66.5 32.7 29.5 32.2 43.6 31.8 35.0 Gly 14.2 14.2 14.1 6.8 6.1 6.7 9.0 7.1 7.4 Pro 20.9 18.9 20.0 9.0 8.7 8.5 12.6 8.8 9.8 Ser 18.4 18.5 17.5 8.1 7.6 8.2 11.2 8.3 9.3 Tyr 12.0 13.0 12.5 5.2 4.8 5.4 7.8 4.5 5.9 Abbreviations: SBM soybean meal, E enzyme, DM dry matter, CP crude protein, Cr chromium content, nd not detected, LOQ below limit of quantification Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 4 of 8 freeze-drying and grinding to pass a 1-mm sieve. Chro- Results mium content was determined by inductively The interaction between the effects of SBM processing coupled plasma-mass spectrometry (iCAP™ RQ ICP- and protease supplementation was significant for the MS, Thermo Fisher Scientific, Waltham, MA) follow- AID of CP (P = 0.01), Trp (P = 0.01), Gly (P = 0.03) and ing microwave digestion of the samples with nitric Pro (P = 0.03). The interaction effect on CP, Gly and Pro acid [16, 17]. Nitrogen content of the diets and the responds to a numerical increase in the AID of SBM and freeze-dried digesta was determined by combustion SBM30 diets with protease supplementation, though a using a nitrogen analyzer (Rapid N Exceed, Elemen- reduction in the SBM60 diets. In addition, other amino tar, Langenselbold, Germany) and the CP content acids (Ile, Leu, Lys, Met, Phe, Val, Ala, Cys, and Glu) ex- was calculated using a 6.25 conversion factor. Amino hibited a similar response, which was shown as a ten- acid contents were determined by ion exchange dency for a significant effect of the interaction between chromatography using post-column derivatization SBM processing and protease supplementation. One ex- with ninhydrin in an amino acid analyzer (Hitachi L- ception to this trend was the AID of Trp, which for the 8900, Tokyo, Japan), after in vacuo hydrolysis with 6 SBM30 diet already showed a decrease of −6.34%, whilst mol/L HCl and 1% phenol at 110 °C for 24 h [18, the decrease for the SBM60 diet reached −27.37%. 19], using norleucine as an internal standard. For Increasing the processing time of SBM decreased (P < the analysis of Met and Cys, the samples were oxi- 0.0001) the AID of all amino acids (Table 3). The de- dized overnight at 2 °C using performic acid before crease in CP digestibility reached −13.4% and −37.3% for hydrolysis [20]. Tryptophan content was determined the SBM30 and SBM60 diets, respectively, compared to after in vacuo alkaline hydrolysis (4.2 mol/L NaOH) the SBM diets. The average AID of essential amino acids at 110 °C for 24 h [21]. Serine protease activity in decreased from 88% in the SBM diets to 82% and 60% in the experimental diets was determined using a col- the SBM30 and SBM60 diets, respectively. The largest orimetric method described by Yasar [22], where the decrease in digestibility for the essential amino acids amount of yellow complex released by serine prote- after the hydrothermal treatment was present for Lys, ase enzyme from the substrate “Suc-Ala-Ala-Pro- which was reduced by −11.9% and −46.96% comparing Phe-pNA (C H N O )” at pH = 9.0 and at 37 °C is the SBM30 and SBM60 diets with the SBM diet, respect- 30 36 6 9 related to the enzymatic activity measured at 405 ively. The average AID of the non-essential amino acids nm, using a standard curve of a certified Ronozyme decreased from 85% in the SBM diets to 75% and 46% in ProAct™ serine protease standard. The limit of quan- the SBM30 and SBM60 diets, respectively. For the non- tification of the method was 1000 PROT/kg. essential amino acids, the largest decrease after the hydrothermal treatment was present for Cys, which was Calculations and statistical analysis reduced by −14.1% and −64.3% comparing the SBM30 Apparent ileal digestibility of CP and AA was calculated and SBM60 diets with the SBM diet, respectively. according to the following equation (Eq. 1): The effect of protease supplementation was only sig- nificant for the AID of Phe (P = 0.02) and Tyr (P = 0.01). Protease supplementation increased the AID of Phe and AIDðÞ % ¼ 1- X =X  Cr =Cr  100 ð1Þ d f f d Tyr in +3.2% and +5.2%, respectively, whilst for Trp there was a decrease of −9.18%. where X is the concentration of CP or amino acid in Productive performance of the birds during the 14–21 the ileal digesta (g/kg of DM), X is the concentration of d period is presented in Table 4. Overall mortality dur- CP or amino acid in the experimental diet (g/kg of DM), ing the experiment was 1%. The effect of protease sup- Cr is the concentration of chromium in the experimen- plementation was not significant for any of the tal diet (g/kg of DM) and Cr is the concentration of productive parameters measured (P > 0.05). The ADFI of chromium in the ileal digesta (g/kg of DM). the birds consuming the SBM diets (76.96 g/d) was The AID of CP and AA were statistically analyzed higher (P = 0.02) than that of the birds consuming the using a two-way ANOVA procedure of SAS software, SBM60 diets (73.43 g/d), whilst the birds consuming the Version 9.4 m6 (SAS Institute Inc., Cary, NC). The SBM30 diets had an intermediate ADFI (76.36 g/d). The model included the effects of the experimental block, interaction between SBM processing and protease sup- SBM processing, protease supplementation and the plementation was significant for the ADG (P = 0.01) and interaction between SBM processing and protease sup- the FCR (P = 0.04). Similar to the interaction for the plementation. Significance was considered at P-values < AID of CP and some amino acids, the interaction be- 0.05 and tendencies were declared at P-values between tween SBM processing and protease supplementation 0.05–0.10. Post-hoc comparisons were performed using for ADG and FCR originates in a numerical improve- the Tukey-Kramer adjustment. ment after protease supplementation in the SBM30 Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 5 of 8 Table 3 Apparent ileal digestibility of crude protein (CP) and amino acids, % Nutrient SBM processing Protease Experimental diets P-value SBM SBM30 SBM60 No Yes SBM SBM + E SBM30 SBM30 + E SBM60 SBM60 + E SEM P E P × E ab a bc ab cd d CP 85.47 72.05 48.19 68.21 68.93 82.36 88.59 67.23 76.87 55.05 41.33 10.62 < 0.0001 0.83 0.01 Essential amino acids x x y Arg 93.25 89.06 75.74 84.67 87.36 91.54 94.95 86.51 91.61 75.96 75.52 5.07 < 0.0001 0.10 0.35 x y z His 90.24 82.61 61.88 76.71 79.78 87.98 92.50 78.53 86.69 63.63 60.14 7.39 < 0.0001 0.19 0.12 x x y Ile 87.55 82.67 64.84 76.67 80.03 84.51 90.59 78.81 86.53 66.70 62.97 7.17 < 0.0001 0.14 0.09 x x y Leu 87.68 84.26 68.95 78.53 82.07 84.47 90.90 80.63 87.89 70.48 67.41 6.59 < 0.0001 0.10 0.09 x y z Lys 89.76 77.86 42.80 68.68 71.59 87.42 92.11 72.19 83.52 46.44 39.15 10.71 < 0.0001 0.39 0.09 x y z Met 90.96 84.29 66.32 80.20 80.85 88.61 93.31 82.12 86.47 69.87 62.78 7.00 < 0.0001 0.77 0.06 x x y Phe 91.53 89.46 80.36 85.53 88.71 89.03 94.03 86.57 92.36 81.00 79.73 4.27 < 0.0001 0.02 0.08 x x y Thr 81.23 71.04 42.22 62.22 67.44 77.16 85.31 64.67 77.41 44.83 39.60 12.05 < 0.0001 0.17 0.14 a a a ab b c Trp 82.41 74.25 39.15 69.86 60.68 79.33 85.49 77.42 71.08 52.83 25.46 13.72 < 0.0001 0.04 0.01 x x y Val 86.84 80.88 61.23 74.56 78.07 83.58 90.10 76.56 85.19 63.55 58.91 7.92 < 0.0001 0.17 0.08 Non-essential amino acids x x y Ala 86.28 79.36 55.59 72.13 75.36 82.96 89.60 74.82 83.91 58.60 52.58 9.14 < 0.0001 0.26 0.08 x y z Asp 86.73 69.56 30.80 60.45 64.28 83.92 89.54 62.95 76.16 34.47 27.13 13.09 < 0.0001 0.35 0.13 x x y Cys 77.66 63.55 13.36 50.30 52.75 70.78 84.54 58.79 68.30 21.31 5.40 16.67 < 0.0001 0.64 0.06 x y z Glu 90.81 82.44 60.54 76.99 78.87 88.85 92.78 78.38 86.51 63.74 57.33 7.63 < 0.0001 0.43 0.05 ab a bc ab cd d Gly 82.22 69.09 37.73 61.11 64.92 77.96 86.49 61.92 76.26 43.44 32.01 12.60 < 0.0001 0.34 0.03 ab a b ab c c Pro 86.72 77.04 50.25 68.80 73.87 82.82 90.61 70.08 84.00 53.49 47.00 13.56 < 0.0001 0.10 0.03 x x y Ser 84.26 75.34 51.45 67.80 72.90 80.47 88.06 69.12 81.57 53.82 49.08 10.32 < 0.0001 0.12 0.10 x x y Tyr 89.02 85.46 66.78 77.83 83.01 85.02 93.02 81.98 88.94 66.50 67.06 6.13 < 0.0001 0.01 0.24 Abbreviations: SEM standard error of the mean, P soybean meal processing, E enzyme supplementation a,b,c,d Means with different superscript letters within a row are significantly different (P < 0.05) x,y,z Means with different superscript letters within a row are significantly different (P < 0.05) diets, almost reaching the levels for the SBM diets, can be considered as low and closely resembles the whilst a decrease after supplementation to the SBM60 lower values reported by Ibáñez et al. [24] and Ravin- diets. The effects on FCR are mainly originating from dran et al. [25] in meals from different countries of ori- differences in ADG. gin, which could be related to harsh processing conditions during the production of the meal. Recent Discussion studies performed in Australia [26], reported SBM with There is variability in the nutritional quality of SBM, ori- lysine contents averaging 4.82 g/100 g CP. Similar con- ginating in the wide range of processing conditions used tents can only be found in the SBM that was autoclaved in the production plants [23–25]. Lysine contents from for 60 min in the present study (4.70 g/100 g CP). different SBM production plants in United States ranged The effects of hydrothermal processing are progres- from 6.35 to 6.43 g/100 g CP [23]. In the current study, sively severe on protein solubility and lysine content lysine content in the SBM was 5.62 g/100 g CP, which [27], which reflect on slower rates of protein hydrolysis Table 4 Productive performance from 14 to 21 days of the broilers fed with the experimental diets Parameter SBM processing Protease Experimental diets SEM P-value SBM SBM30 SBM60 No Yes SBM SBM + E SBM30 SBM30 + E SBM60 SBM60 + E P E P × E 1 x xy y ADFI , g/d 76.96 76.36 73.43 75.95 75.22 77.01 76.91 75.96 76.76 74.86 71.99 3.34 0.02 0.49 0.34 a ab bc ab cd d ADG, g/d 47.78 44.17 38.83 43.67 43.52 48.53 47.01 42.27 46.08 40.21 37.45 2.79 < 0.0001 0.86 0.01 d cd abc bcd ab a FCR 1.61 1.73 1.90 1.75 1.75 1.59 1.64 1.80 1.66 1.86 1.95 0.12 < 0.0001 0.99 0.04 Abbreviations: ADFI average daily feed intake, ADG average daily gain, FCR feed conversion ratio, SEM standard error of the mean, P soybean meal processing, E enzyme supplementation a,b,c,d Means with different superscript letters within a row are significantly different (P < 0.05) x,y Means with different superscript letters within a row are significantly different (P < 0.05) Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 6 of 8 [8, 28] and decreased CP and amino acid digestibility [8, enzymes increased the activity of pancreatic trypsin by 29]. Lysine contents in the present study were linearly 27.41%, whilst the inclusion of the protease at levels of reduced by 0.015 g/100 g CP for every minute of hydro- 80 and 160 mg/kg (also in combination with the non- thermal treatment of the SBM (r = 0.997). The decrease starch polysaccharide hydrolytic enzymes) decreased the of Lys content after hydrothermal processing was prob- activity of pancreatic trypsin by 10.75% and 25.88%, re- ably due to the formation of advanced Maillard reaction spectively. Endogenous enzymatic activity might have products, which cannot be reversed by acid-hydrolysis been reduced in the present study, as the exogenous during the amino acid analysis, in contrast to Amadori protease was included at three times the recommended products, that can be partly reversed to Lys [30]. Lysine dose. A reduction of the secretion of endogenous en- digestibility was the most affected essential amino acid zymes due to an excess of exogenous enzymes supple- by the hydrothermal treatment in the present study. For mentation is likely to affect the digestibility of the every 1 g/kg loss in Lys content of the soybean meals, substrate, especially when the substrate has suffered there was a decrease of −18.6% of CP digestibility and extreme physical and chemical modifications due to −23.5% of Lys digestibility (r = 0.97 and 0.93, respect- hydrothermal over-processing. Additionally, Maillard ively). This is not surprising, as Lys is one of the target reactions could increase the formation of organic amino acids of the pancreatic proteolytic enzymes acids [35, 36], which could have reduced the intes- (mainly trypsin). In addition, the deleterious effect of tinal pH below the optimum for the activity of the autoclaving the SBM for 60 min was especially notorious exogenous protease. The combination of these three for the AID of Cys. Harsh conditions during hydrother- factors: a reduction in endogenous proteolytic activity, mal processing cause an increase in the formation of di- a reduction of the optimum pH for the exogenous sulfide bonds, especially in the insoluble protein fraction, protease and the profound structural and chemical formed between Cys residues in proteins [31, 32]. The modifications of the proteins due to hydrothermal formation of these covalent bonds likely reduced overall processing, are likely to explain the reduction in CP accessibility of enzymes for proteolysis and the digest- and amino acid digestibility with the inclusion of ex- ibility of this particular amino acid. ogenous enzymes in the SBM60 diet. The AID of Phe and Tyr also decreased with increas- The negative effects of hydrothermal damage on CP ing autoclaving time of the SBM (P < 0.0001). However, and amino acid digestibility were also noticeable on the in contrast to the rest of the AA, the AID of Phe and ADG of the birds. However, the influence of the digest- Tyr increased by exogenous protease inclusion in the di- ibility on ADG does not appear to be linear. For ex- ets (P = 0.02 and P = 0.01, respectively). Subtilisin-like ample, whilst a reduction of approximately 13% in the proteases, such as the one used in the present study, AID of Lys in the SBM30 compared to the SBM diets have a high affinity towards large hydrophobic AA, such caused a reduction of 7.5% in the ADG of the birds, a re- as Phe and Tyr [13], which explains that these were the duction of approximately 50% in the AID of Lys in the only AA affected by inclusion of the exogenous protease SBM60 compared to the SBM diets only caused a reduc- across all diets. tion of less than 20% of the ADG of the birds. Although The numerical increase in CP digestibility and amino it was not determined in the present study, it is possible acid digestibility with protease supplementation in the that catabolism of unbalanced amino acids in the diets SBM and SBM30 diets contrasts with the observed de- with higher degrees of processing resulted in a change of crease in the SBM60 diets. There seems to be a thresh- the body composition of the birds, which increased the old where protease supplementation cannot recover the deposition of body fat. Changes in body composition negative effects of thermal protein damage. Furthermore, due to catabolism of unbalanced amino acids have been contrary to what we expected, inclusion of the exogen- reported in studies with broilers on the effects of differ- ous protease in the SBM60 diets decreased the digestibil- ent dietary digestible Lys levels in the diets [37] and ity of CP and most AA. Although most studies [33] have studies that tested diets with low-protein contents [38]. reported an increase in CP and amino acid digestibility It is worth mentioning that the diets in the present study with the inclusion of exogenous proteases, there is lim- were not formulated to maximize the performance of ited information regarding the effects on thermally dam- the birds, but to test the digestibility of SBM as an ingre- aged ingredients. Moreover, the effects of the inclusion dient. Therefore, the AA content in the diets were likely of exogenous proteases seem to be dependent on the limiting for growth. Furthermore, the significant inter- dose of the enzyme used and the duration of the period action between the effects of SBM processing and prote- of supplementation, as described by Yuan et al. [34]. ase supplementation on ADG and FCR, which shows a These authors reported that, after 42 days of supplemen- positive effect of protease supplementation on the tation, the inclusion of 40 mg/kg of an acid protease in SBM30 diets and a negative effect on the SBM60 diets, combination with non-starch polysaccharide hydrolytic is likely to be caused by the AID of CP and most amino Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 7 of 8 acids, which also exhibit a similar pattern for the inter- Received: 3 January 2022 Accepted: 8 May 2022 action. Amino acid digestibility directly influences the productive performance of broilers [39]. References 1. Carbonaro M, Maselli P, Nucara A. Relationship between digestibility and Conclusions secondary structure of raw and thermally treated legume proteins: a Fourier In conclusion, exogenous protease supplementation at transform infrared (FT-IR) spectroscopic study. Amino Acids. 2012;43(2):911– 21. https://doi.org/10.1007/s00726-011-1151-4. three times the commercial dose does not seem to offset 2. Salazar-Villanea S, Hendriks WH, Bruininx EMAM, Gruppen H, van der Poel the negative effects of hydrothermal processing of SBM AFB. Protein structural changes during processing of vegetable feed on the AID of CP and amino acids or performance of ingredients used in swine diets: implications for nutritional value. Nutr Res Rev. 2016;29(1):126–41. https://doi.org/10.1017/S0954422416000056. broilers. Whilst positive numerical improvements of di- 3. Friedman M. Food browning and its prevention: an overview. J Agric Food gestibility and performance (ADG and FCR) were no- Chem. 1996;44(3):631–53. https://doi.org/10.1021/jf950394r. ticed with protease supplementation at relatively mild 4. van Rooijen C, Bosch G, van der Poel AFB, Wierenga PA, Alexander L, Hendriks WH. 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Protein fermentation in SS-V conceived and designed the experiment; SS-V and AM-M processed the the gut; implications for intestinal dysfunction in humans, pigs, and poultry. materials for the experimental diets.; SS-V, MA-U, AM-M, JIH-M and CS-D col- Am J Physiol Gastrointest Liver Physiol. 2018;315(2):G159–70. https://doi. lected the experimental samples and data; SS-V performed the crude protein org/10.1152/ajpgi.00319.2017. analysis; SS-V and MA-U performed the calculations and statistical analysis; 10. Meda B, Hassouna M, Aubert C, Robin P, Dourmad JY. Influence of rearing SS-V wrote the original draft of the manuscript; MA-U, AM-M, JIH-M and CS- conditions and manure management practices on ammonia and D revised and edited the original draft of the manuscript; SS-V and CS-D su- greenhouse gas emissions from poultry houses. Worlds Poult Sci J. 2011; pervised the execution of the experiment; SS-V acquired the funding and ad- 67(3):441–56. https://doi.org/10.1017/S0043933911000493. ministered the project. All authors read and approved the final manuscript. 11. Cowieson AJ, Roos FF. Toward optimal value creation through the application of exogenous mono-component protease in the diets of non- ruminants. Anim Feed Sci. 2016;221:331–40. https://doi.org/10.1016/j.a Funding nifeedsci.2016.04.015. This research was funded by Consejo Nacional para Investigaciones 12. Murugesan GR, Romero LF, Persia ME. Effects of protease, phytase and a Científicas y Tecnológicas (CONICIT), Costa Rica, grant number RE-006-17. Bacillus sp. direct-fed microbial on nutrient and energy digestibility, ileal brush border digestive enzyme activity and cecal short-chain fatty acid Availability of data and materials concentration in broiler chickens. PLoS One. 2014;9:e101888. https://doi. 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Effect of protease supplementation on apparent ileal crude protein and amino acid digestibility of over-processed soybean meals in broilers

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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. Keywords: Amino acids, Apparent ileal digestibility, Broilers, Exogenous protease * Correspondence: sergio.salazarvillanea@ucr.ac.cr Escuela de Zootecnia, Universidad de Costa Rica, San José, Costa Rica Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 2 of 8 Background autoclaved for 30 or 60 min at 120 °C) and protease sup- The nutritional value of feed ingredients can be nega- plementation (not supplemented and supplemented; tively affected by hydrothermal processing. Protein de- RONOZYME ProAct, Novozymes, Krogshoejvej, naturation during processing could lead to changes of Denmark) as factors. This design resulted in 6 experi- their structural conformation, resulting in the formation mental diets: unautoclaved SBM without and with en- of protein aggregates, which render the hydrolytic sites zyme supplementation (SBM and SBM + E, respectively), inaccessible to enzymes [1, 2]. In addition, processing 30 min autoclaved SBM without and with enzyme sup- can induce chemical modifications to the structure of plementation (SBM30 and SBM30 + E, respectively) and amino acids (AA) resulting in the formation of Maillard 60 min autoclaved SBM without and with enzyme sup- reaction products [3–5]. The chemically modified AA plementation (SBM60 and SBM60 + E, respectively). cause a stearic hindrance effect, which also limits en- zyme accessibility for hydrolysis [6]. The main protein- Experimental diets bound AA affected, lysine and arginine, are also the tar- Commercial SBM was purchased (Concentrados Gastón get AA of trypsin, one of the main proteolytic endogen- Fernández, Cartago, Costa Rica) and divided in 3 ous enzymes, which likely reduces the efficiency of batches: one was left unautoclaved, whilst the other two hydrolysis [2]. The overall result is a reduction in the were autoclaved at 120 °C for 30 and 60 min, respect- rate of protein hydrolysis [7, 8], which results in a de- ively. Temperatures during autoclaving were measured crease of protein digestibility [8]. Fermentation of un- with internal thermocouples and ranged from 118 to digested proteins can result in undesired putrefaction 123 °C. products in the gut (e.g. biogenic amines, phenolic and Experimental diets were formulated according to the indolic compounds) and increase the proliferation of recommendations from Ravindran et al. [15] for the pathogenic bacteria [9]. Moreover, undigested proteins evaluation of amino acid digestibility of raw materials in contribute to N emissions to the environment and can broilers (Table 1) and fed as mash. Chromium oxide therefore be considered as a pollutant [10]. (Cr O ) was included in the experimental diets as a 2 3 Exogenous enzymes, such as proteases, are a tool in marker. The exogenous protease was supplemented at 3 state-of-the-art animal nutrition to increase the nutri- tional value of feed ingredients and improve animal per- Table 1 Ingredient and nutritional composition of the formance at a lower cost [11]. The proteolytic experimental diets, g/kg as is mechanism of exogenous proteases is complementary to Ingredients Basal diet Basal diet + enzyme that of endogenous proteases [12]. For example, trypsin, Dextrose 507 506.4 one of the main endogenous digestive enzymes, is highly Soybean meal 416 416 specific for Lys and Arg; whilst, the exogenous Soybean oil 40 40 subtilisin-like proteases have high affinities towards large Dicalcium phosphate 19 19 hydrophobic amino acids, such as Phe and Tyr [13]. Calcium carbonate 10 10 Therefore, an increase in the rate of hydrolysis and amino acid digestibility could be expected when exogen- Cr O 33 2 3 ous proteases are supplemented in the diets [14]. Sodium bicarbonate 2 2 Our objective was to determine the effect of exogen- Salt 2 2 ous protease supplementation on the productive per- Vitamins and minerals premix 11 formance and apparent ileal digestibility (AID) of crude Enzyme – 0.6 protein (CP) and AA of soybean meals (SBM) with dif- Nutritional composition (calculated) ferent degrees of hydrothermal processing. We hypothe- sized that exogenous protease supplementation would Metabolizable energy, kcal/kg 3110 3107 increase the rate of hydrolysis of thermally damaged pro- Crude protein 196.10 196.10 teins, thereby increasing AA digestibility at the end of Available phosphorus 5.02 5.02 the small intestine. Calcium 8.49 8.49 Fufeng Group, China Methods Commercial soybean meal used was unautoclaved, autoclaved 30 min at Experimental setup 120 °C or autoclaved 60 min at 120 °C Vitamins and minerals supplied per kg of diet: Cu (sulfate), 12 mg; Fe (sulfate), Animal experimental procedures were approved by the 40 mg; I (iodide), 1 mg; Se (selenate), 0.30 mg; Mn (sulfate and oxide), 80 mg; Animal Care and Use Committee, University of Costa Zn (sulfate and oxide), 80 mg; retinol, 10,000 UI; cholecalciferol, 3000 UI; tocopheryl acetate, 35 UI; menadione, 2.50 mg; thiamine, 1.75 mg; riboflavin, Rica, under the authorization number CICUA-110-17. 6.50 mg; niacin, 55 mg; pantothenate 10.70 mg; pyridoxine, 3.60 mg; folate, The experiment involved a 3 × 2 factorial arrangement, 1.50 mg; cyanocobalamin, 15 μg; biotin, 110 μg with SBM autoclaving time (commercial SBM or RONOZYME ProAct, Novozymes, Krogshoejvej, Denmark Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 3 of 8 times the commercial recommendation (commercial rec- Each pen was considered as an experimental unit. Birds ommendation is 15,000 PROT/kg), in order to assure had ad libitum access to water and a commercial pre- that the enzyme was not a limiting factor for hydrolysis. starter (1–7 d) and starter diets (8–14 d). From d 15–21, One PROT unit is defined as the amount of serine pro- the birds received ad libitum access to the experimental tease that liberates 1 μmol para-nitroaniline (pNA) from diets. Average daily gain (ADG) of each bird was calcu- 1 mmol/L Suc-Ala-Ala-Pro-PhepNA (C H N O ) lated using the average weight at the start and the end of 30 36 6 9 (Sigma-Aldrich, St. Louis, MO, USA) substrate per mi- the experimental period (14 and 21 d, respectively), di- nute at pH = 9.0 and at 37 °C. The analyzed nutritional vided by the number of birds in each experimental unit. composition of the experimental diets is depicted in Average daily feed intake (ADFI) was determined as the Table 2. difference between the feed offered during the 14–21 d period and feed refusal at 21 d and calculated per bird. Animals and housing Feed conversion ratio (FCR), corrected for mortality, was A total of 504 one-day-old Cobb 500 broilers were allo- calculated as the ratio between ADFI and ADG. cated at a density of 12 birds/m in an open environ- ment facility. Temperatures during the experiment Sample collection and chemical analysis averaged 22.84 ± 2.61 °C and a light regimen of 12 h light On d 21, 6 birds were randomly selected per pen and and 12 h dark was used. There were 7 repetitions per euthanized by cervical dislocation. Digestive content treatment for the experimental diets, for a total of 42 ex- from the last 20 cm of the ileum (anterior to the ileo- perimental units, which were allotted to a total of 6 ex- cecal valve) of each bird was collected by gentle strip- perimental blocks, where each experimental diet was ping and pooled per experimental unit. Samples were represented at least once in every experimental block. immediately frozen and kept at − 70 °C, followed by Table 2 Analyzed nutrient composition of the soybean meals and experimental diets, g/kg as is Nutrient SBM – autoclaving time Experimental diets 0 min 30 min 60 min SBM SBM + E SBM30 SBM30 + E SBM60 SBM60 + E DM 868.0 856.4 847.5 880.9 877.5 882.5 875.8 874.7 869.9 CP 441.6 437.8 437.9 192.3 192.5 192.9 205.4 209.8 215.8 Cr –– – 0.040 0.030 0.070 0.043 0.075 0.079 Protease (PROT/kg) –– – nd 56,470 LOQ 48,680 nd 53,900 Essential amino acids Arg 27.9 26.4 24.8 12.9 10.9 11.6 16.5 10.3 12.7 His 13.7 10.2 12.7 6.0 5.3 5.9 8.0 5.6 6.5 Ile 18.0 17.6 17.7 8.6 7.6 8.5 11.2 8.2 9.3 Leu 30.4 29.3 28.8 13.7 12.5 13.6 18.5 13.4 15.1 Lys 24.8 22.4 20.6 10.1 8.4 8.8 12.5 7.9 8.8 Met 6.1 6.2 5.9 2.9 2.8 3.1 3.5 2.8 2.9 Phe 23.6 19.7 22.1 10.5 9.6 9.5 14.4 9.9 11.7 Thr 15.3 15.1 15.0 7.2 6.5 7.2 9.6 7.1 8.0 Trp 7.6 6.7 6.5 2.9 2.2 3.9 2.2 2.5 1.9 Val 18.4 17.8 17.8 8.6 7.8 8.3 11.2 8.2 9.2 Non-essential amino acids Ala 16.2 15.3 15.6 7.4 6.8 7.6 10.0 7.3 7.9 Asx 45.8 44.1 44.6 22.3 19.5 21.9 29.1 21.1 24.1 Cys 6.1 5.5 4.9 2.8 2.6 3.1 3.3 2.5 2.5 Glx 71.2 70.8 66.5 32.7 29.5 32.2 43.6 31.8 35.0 Gly 14.2 14.2 14.1 6.8 6.1 6.7 9.0 7.1 7.4 Pro 20.9 18.9 20.0 9.0 8.7 8.5 12.6 8.8 9.8 Ser 18.4 18.5 17.5 8.1 7.6 8.2 11.2 8.3 9.3 Tyr 12.0 13.0 12.5 5.2 4.8 5.4 7.8 4.5 5.9 Abbreviations: SBM soybean meal, E enzyme, DM dry matter, CP crude protein, Cr chromium content, nd not detected, LOQ below limit of quantification Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 4 of 8 freeze-drying and grinding to pass a 1-mm sieve. Chro- Results mium content was determined by inductively The interaction between the effects of SBM processing coupled plasma-mass spectrometry (iCAP™ RQ ICP- and protease supplementation was significant for the MS, Thermo Fisher Scientific, Waltham, MA) follow- AID of CP (P = 0.01), Trp (P = 0.01), Gly (P = 0.03) and ing microwave digestion of the samples with nitric Pro (P = 0.03). The interaction effect on CP, Gly and Pro acid [16, 17]. Nitrogen content of the diets and the responds to a numerical increase in the AID of SBM and freeze-dried digesta was determined by combustion SBM30 diets with protease supplementation, though a using a nitrogen analyzer (Rapid N Exceed, Elemen- reduction in the SBM60 diets. In addition, other amino tar, Langenselbold, Germany) and the CP content acids (Ile, Leu, Lys, Met, Phe, Val, Ala, Cys, and Glu) ex- was calculated using a 6.25 conversion factor. Amino hibited a similar response, which was shown as a ten- acid contents were determined by ion exchange dency for a significant effect of the interaction between chromatography using post-column derivatization SBM processing and protease supplementation. One ex- with ninhydrin in an amino acid analyzer (Hitachi L- ception to this trend was the AID of Trp, which for the 8900, Tokyo, Japan), after in vacuo hydrolysis with 6 SBM30 diet already showed a decrease of −6.34%, whilst mol/L HCl and 1% phenol at 110 °C for 24 h [18, the decrease for the SBM60 diet reached −27.37%. 19], using norleucine as an internal standard. For Increasing the processing time of SBM decreased (P < the analysis of Met and Cys, the samples were oxi- 0.0001) the AID of all amino acids (Table 3). The de- dized overnight at 2 °C using performic acid before crease in CP digestibility reached −13.4% and −37.3% for hydrolysis [20]. Tryptophan content was determined the SBM30 and SBM60 diets, respectively, compared to after in vacuo alkaline hydrolysis (4.2 mol/L NaOH) the SBM diets. The average AID of essential amino acids at 110 °C for 24 h [21]. Serine protease activity in decreased from 88% in the SBM diets to 82% and 60% in the experimental diets was determined using a col- the SBM30 and SBM60 diets, respectively. The largest orimetric method described by Yasar [22], where the decrease in digestibility for the essential amino acids amount of yellow complex released by serine prote- after the hydrothermal treatment was present for Lys, ase enzyme from the substrate “Suc-Ala-Ala-Pro- which was reduced by −11.9% and −46.96% comparing Phe-pNA (C H N O )” at pH = 9.0 and at 37 °C is the SBM30 and SBM60 diets with the SBM diet, respect- 30 36 6 9 related to the enzymatic activity measured at 405 ively. The average AID of the non-essential amino acids nm, using a standard curve of a certified Ronozyme decreased from 85% in the SBM diets to 75% and 46% in ProAct™ serine protease standard. The limit of quan- the SBM30 and SBM60 diets, respectively. For the non- tification of the method was 1000 PROT/kg. essential amino acids, the largest decrease after the hydrothermal treatment was present for Cys, which was Calculations and statistical analysis reduced by −14.1% and −64.3% comparing the SBM30 Apparent ileal digestibility of CP and AA was calculated and SBM60 diets with the SBM diet, respectively. according to the following equation (Eq. 1): The effect of protease supplementation was only sig- nificant for the AID of Phe (P = 0.02) and Tyr (P = 0.01). Protease supplementation increased the AID of Phe and AIDðÞ % ¼ 1- X =X  Cr =Cr  100 ð1Þ d f f d Tyr in +3.2% and +5.2%, respectively, whilst for Trp there was a decrease of −9.18%. where X is the concentration of CP or amino acid in Productive performance of the birds during the 14–21 the ileal digesta (g/kg of DM), X is the concentration of d period is presented in Table 4. Overall mortality dur- CP or amino acid in the experimental diet (g/kg of DM), ing the experiment was 1%. The effect of protease sup- Cr is the concentration of chromium in the experimen- plementation was not significant for any of the tal diet (g/kg of DM) and Cr is the concentration of productive parameters measured (P > 0.05). The ADFI of chromium in the ileal digesta (g/kg of DM). the birds consuming the SBM diets (76.96 g/d) was The AID of CP and AA were statistically analyzed higher (P = 0.02) than that of the birds consuming the using a two-way ANOVA procedure of SAS software, SBM60 diets (73.43 g/d), whilst the birds consuming the Version 9.4 m6 (SAS Institute Inc., Cary, NC). The SBM30 diets had an intermediate ADFI (76.36 g/d). The model included the effects of the experimental block, interaction between SBM processing and protease sup- SBM processing, protease supplementation and the plementation was significant for the ADG (P = 0.01) and interaction between SBM processing and protease sup- the FCR (P = 0.04). Similar to the interaction for the plementation. Significance was considered at P-values < AID of CP and some amino acids, the interaction be- 0.05 and tendencies were declared at P-values between tween SBM processing and protease supplementation 0.05–0.10. Post-hoc comparisons were performed using for ADG and FCR originates in a numerical improve- the Tukey-Kramer adjustment. ment after protease supplementation in the SBM30 Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 5 of 8 Table 3 Apparent ileal digestibility of crude protein (CP) and amino acids, % Nutrient SBM processing Protease Experimental diets P-value SBM SBM30 SBM60 No Yes SBM SBM + E SBM30 SBM30 + E SBM60 SBM60 + E SEM P E P × E ab a bc ab cd d CP 85.47 72.05 48.19 68.21 68.93 82.36 88.59 67.23 76.87 55.05 41.33 10.62 < 0.0001 0.83 0.01 Essential amino acids x x y Arg 93.25 89.06 75.74 84.67 87.36 91.54 94.95 86.51 91.61 75.96 75.52 5.07 < 0.0001 0.10 0.35 x y z His 90.24 82.61 61.88 76.71 79.78 87.98 92.50 78.53 86.69 63.63 60.14 7.39 < 0.0001 0.19 0.12 x x y Ile 87.55 82.67 64.84 76.67 80.03 84.51 90.59 78.81 86.53 66.70 62.97 7.17 < 0.0001 0.14 0.09 x x y Leu 87.68 84.26 68.95 78.53 82.07 84.47 90.90 80.63 87.89 70.48 67.41 6.59 < 0.0001 0.10 0.09 x y z Lys 89.76 77.86 42.80 68.68 71.59 87.42 92.11 72.19 83.52 46.44 39.15 10.71 < 0.0001 0.39 0.09 x y z Met 90.96 84.29 66.32 80.20 80.85 88.61 93.31 82.12 86.47 69.87 62.78 7.00 < 0.0001 0.77 0.06 x x y Phe 91.53 89.46 80.36 85.53 88.71 89.03 94.03 86.57 92.36 81.00 79.73 4.27 < 0.0001 0.02 0.08 x x y Thr 81.23 71.04 42.22 62.22 67.44 77.16 85.31 64.67 77.41 44.83 39.60 12.05 < 0.0001 0.17 0.14 a a a ab b c Trp 82.41 74.25 39.15 69.86 60.68 79.33 85.49 77.42 71.08 52.83 25.46 13.72 < 0.0001 0.04 0.01 x x y Val 86.84 80.88 61.23 74.56 78.07 83.58 90.10 76.56 85.19 63.55 58.91 7.92 < 0.0001 0.17 0.08 Non-essential amino acids x x y Ala 86.28 79.36 55.59 72.13 75.36 82.96 89.60 74.82 83.91 58.60 52.58 9.14 < 0.0001 0.26 0.08 x y z Asp 86.73 69.56 30.80 60.45 64.28 83.92 89.54 62.95 76.16 34.47 27.13 13.09 < 0.0001 0.35 0.13 x x y Cys 77.66 63.55 13.36 50.30 52.75 70.78 84.54 58.79 68.30 21.31 5.40 16.67 < 0.0001 0.64 0.06 x y z Glu 90.81 82.44 60.54 76.99 78.87 88.85 92.78 78.38 86.51 63.74 57.33 7.63 < 0.0001 0.43 0.05 ab a bc ab cd d Gly 82.22 69.09 37.73 61.11 64.92 77.96 86.49 61.92 76.26 43.44 32.01 12.60 < 0.0001 0.34 0.03 ab a b ab c c Pro 86.72 77.04 50.25 68.80 73.87 82.82 90.61 70.08 84.00 53.49 47.00 13.56 < 0.0001 0.10 0.03 x x y Ser 84.26 75.34 51.45 67.80 72.90 80.47 88.06 69.12 81.57 53.82 49.08 10.32 < 0.0001 0.12 0.10 x x y Tyr 89.02 85.46 66.78 77.83 83.01 85.02 93.02 81.98 88.94 66.50 67.06 6.13 < 0.0001 0.01 0.24 Abbreviations: SEM standard error of the mean, P soybean meal processing, E enzyme supplementation a,b,c,d Means with different superscript letters within a row are significantly different (P < 0.05) x,y,z Means with different superscript letters within a row are significantly different (P < 0.05) diets, almost reaching the levels for the SBM diets, can be considered as low and closely resembles the whilst a decrease after supplementation to the SBM60 lower values reported by Ibáñez et al. [24] and Ravin- diets. The effects on FCR are mainly originating from dran et al. [25] in meals from different countries of ori- differences in ADG. gin, which could be related to harsh processing conditions during the production of the meal. Recent Discussion studies performed in Australia [26], reported SBM with There is variability in the nutritional quality of SBM, ori- lysine contents averaging 4.82 g/100 g CP. Similar con- ginating in the wide range of processing conditions used tents can only be found in the SBM that was autoclaved in the production plants [23–25]. Lysine contents from for 60 min in the present study (4.70 g/100 g CP). different SBM production plants in United States ranged The effects of hydrothermal processing are progres- from 6.35 to 6.43 g/100 g CP [23]. In the current study, sively severe on protein solubility and lysine content lysine content in the SBM was 5.62 g/100 g CP, which [27], which reflect on slower rates of protein hydrolysis Table 4 Productive performance from 14 to 21 days of the broilers fed with the experimental diets Parameter SBM processing Protease Experimental diets SEM P-value SBM SBM30 SBM60 No Yes SBM SBM + E SBM30 SBM30 + E SBM60 SBM60 + E P E P × E 1 x xy y ADFI , g/d 76.96 76.36 73.43 75.95 75.22 77.01 76.91 75.96 76.76 74.86 71.99 3.34 0.02 0.49 0.34 a ab bc ab cd d ADG, g/d 47.78 44.17 38.83 43.67 43.52 48.53 47.01 42.27 46.08 40.21 37.45 2.79 < 0.0001 0.86 0.01 d cd abc bcd ab a FCR 1.61 1.73 1.90 1.75 1.75 1.59 1.64 1.80 1.66 1.86 1.95 0.12 < 0.0001 0.99 0.04 Abbreviations: ADFI average daily feed intake, ADG average daily gain, FCR feed conversion ratio, SEM standard error of the mean, P soybean meal processing, E enzyme supplementation a,b,c,d Means with different superscript letters within a row are significantly different (P < 0.05) x,y Means with different superscript letters within a row are significantly different (P < 0.05) Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 6 of 8 [8, 28] and decreased CP and amino acid digestibility [8, enzymes increased the activity of pancreatic trypsin by 29]. Lysine contents in the present study were linearly 27.41%, whilst the inclusion of the protease at levels of reduced by 0.015 g/100 g CP for every minute of hydro- 80 and 160 mg/kg (also in combination with the non- thermal treatment of the SBM (r = 0.997). The decrease starch polysaccharide hydrolytic enzymes) decreased the of Lys content after hydrothermal processing was prob- activity of pancreatic trypsin by 10.75% and 25.88%, re- ably due to the formation of advanced Maillard reaction spectively. Endogenous enzymatic activity might have products, which cannot be reversed by acid-hydrolysis been reduced in the present study, as the exogenous during the amino acid analysis, in contrast to Amadori protease was included at three times the recommended products, that can be partly reversed to Lys [30]. Lysine dose. A reduction of the secretion of endogenous en- digestibility was the most affected essential amino acid zymes due to an excess of exogenous enzymes supple- by the hydrothermal treatment in the present study. For mentation is likely to affect the digestibility of the every 1 g/kg loss in Lys content of the soybean meals, substrate, especially when the substrate has suffered there was a decrease of −18.6% of CP digestibility and extreme physical and chemical modifications due to −23.5% of Lys digestibility (r = 0.97 and 0.93, respect- hydrothermal over-processing. Additionally, Maillard ively). This is not surprising, as Lys is one of the target reactions could increase the formation of organic amino acids of the pancreatic proteolytic enzymes acids [35, 36], which could have reduced the intes- (mainly trypsin). In addition, the deleterious effect of tinal pH below the optimum for the activity of the autoclaving the SBM for 60 min was especially notorious exogenous protease. The combination of these three for the AID of Cys. Harsh conditions during hydrother- factors: a reduction in endogenous proteolytic activity, mal processing cause an increase in the formation of di- a reduction of the optimum pH for the exogenous sulfide bonds, especially in the insoluble protein fraction, protease and the profound structural and chemical formed between Cys residues in proteins [31, 32]. The modifications of the proteins due to hydrothermal formation of these covalent bonds likely reduced overall processing, are likely to explain the reduction in CP accessibility of enzymes for proteolysis and the digest- and amino acid digestibility with the inclusion of ex- ibility of this particular amino acid. ogenous enzymes in the SBM60 diet. The AID of Phe and Tyr also decreased with increas- The negative effects of hydrothermal damage on CP ing autoclaving time of the SBM (P < 0.0001). However, and amino acid digestibility were also noticeable on the in contrast to the rest of the AA, the AID of Phe and ADG of the birds. However, the influence of the digest- Tyr increased by exogenous protease inclusion in the di- ibility on ADG does not appear to be linear. For ex- ets (P = 0.02 and P = 0.01, respectively). Subtilisin-like ample, whilst a reduction of approximately 13% in the proteases, such as the one used in the present study, AID of Lys in the SBM30 compared to the SBM diets have a high affinity towards large hydrophobic AA, such caused a reduction of 7.5% in the ADG of the birds, a re- as Phe and Tyr [13], which explains that these were the duction of approximately 50% in the AID of Lys in the only AA affected by inclusion of the exogenous protease SBM60 compared to the SBM diets only caused a reduc- across all diets. tion of less than 20% of the ADG of the birds. Although The numerical increase in CP digestibility and amino it was not determined in the present study, it is possible acid digestibility with protease supplementation in the that catabolism of unbalanced amino acids in the diets SBM and SBM30 diets contrasts with the observed de- with higher degrees of processing resulted in a change of crease in the SBM60 diets. There seems to be a thresh- the body composition of the birds, which increased the old where protease supplementation cannot recover the deposition of body fat. Changes in body composition negative effects of thermal protein damage. Furthermore, due to catabolism of unbalanced amino acids have been contrary to what we expected, inclusion of the exogen- reported in studies with broilers on the effects of differ- ous protease in the SBM60 diets decreased the digestibil- ent dietary digestible Lys levels in the diets [37] and ity of CP and most AA. Although most studies [33] have studies that tested diets with low-protein contents [38]. reported an increase in CP and amino acid digestibility It is worth mentioning that the diets in the present study with the inclusion of exogenous proteases, there is lim- were not formulated to maximize the performance of ited information regarding the effects on thermally dam- the birds, but to test the digestibility of SBM as an ingre- aged ingredients. Moreover, the effects of the inclusion dient. Therefore, the AA content in the diets were likely of exogenous proteases seem to be dependent on the limiting for growth. Furthermore, the significant inter- dose of the enzyme used and the duration of the period action between the effects of SBM processing and prote- of supplementation, as described by Yuan et al. [34]. ase supplementation on ADG and FCR, which shows a These authors reported that, after 42 days of supplemen- positive effect of protease supplementation on the tation, the inclusion of 40 mg/kg of an acid protease in SBM30 diets and a negative effect on the SBM60 diets, combination with non-starch polysaccharide hydrolytic is likely to be caused by the AID of CP and most amino Salazar-Villanea et al. Journal of Animal Science and Biotechnology (2022) 13:74 Page 7 of 8 acids, which also exhibit a similar pattern for the inter- Received: 3 January 2022 Accepted: 8 May 2022 action. Amino acid digestibility directly influences the productive performance of broilers [39]. References 1. Carbonaro M, Maselli P, Nucara A. Relationship between digestibility and Conclusions secondary structure of raw and thermally treated legume proteins: a Fourier In conclusion, exogenous protease supplementation at transform infrared (FT-IR) spectroscopic study. 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Journal

Journal of Animal Science and Biotechnology – Springer Journals

Published: Jul 11, 2022

Keywords: Amino acids; Apparent ileal digestibility; Broilers; Exogenous protease

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