Introduction
The price of conventional feed ingredients used as energy and protein sources for swine diets sharply fluctuates. Thus, the use of alternative feed ingredients such as copra meal, palm kernel expellers, and wheat distillers dried grains with solubles (DDGS) is inevitable to lower feed costs. However, alternative feed ingredients are generally high in non-starch polysaccharides (NSP) that act as anti-nutritional factors on feed energy values. Therefore, the inclusion of exogenous enzymes in swine diets is a common strategy to increase energy values by improving utilization of NSP as well as other energy- yielding nutrients in pigs.
Arabinoxylan, consisting of arabinose and xylose (Saulnier et al., 2012), is the most common NSP found in cereal grains such as corn, wheat, and barley (Masey O’Neill et al., 2014). Supplemental xylanase has been reported to break down NSP, particularly arabinoxylan, in monogastric animals (Kiarie et al., 2013). Recently, a xylanase product (Nutrase Xyla®, Nutrex, Lille, Belgium) containing endo-1,4-β xylanase (9,000 U/g) originating from Bacillus subtilis has been reported to increase nutrient digestibility of a corn-soybean meal-based diet, and subsequently, to improve growth performance of growing pigs (Lee et al., 2018). The effect of an enzyme is largely dependent on the substrate, and thus, the ingredient composition is one of the most important factors for the inclusion of supplemental enzymes in swine diets. However, the effects of exogenous xylanase from Bacillus subtilis on various feed ingredients have rarely been compared. In vitro assays have been used to evaluate the effect of supplemental enzymes on nutrient digestibility because these assays are less expensive and laborious compared with in vivo assays (Kong et al., 2015; Ha et al., 2020).
Therefore, the objective of this study was to evaluate the effects of supplemental xylanase on in vitro ileal disappearance (IVID) and in vitro total tract disappearance (IVTTD) of dry matter (DM) in various feed ingredients.
Materials and methods
Enzyme and sample preparation
A xylanase product (Nutrase Xyla®, Nutrex, Lille, Belgium) used in the present work originated from Bacillus subtilis and contained endo-1,4-β xylanase (9,000 U/g). The xylanase product was provided by Morning Bio Inc. (Cheonan, Republic of Korea). Nine feed ingredients were used to measure IVID and IVTTD of DM with or without supplemental xylanase. The ingredients used were corn, wheat, barley, wheat flour, copra meal, palm kernel expellers, corn DDGS, wheat DDGS, and wheat bran. Each ingredient was ground to pass a 1-mm screen (Cyclotech 1093; Foss Tecator AB, Höganäs, Sweden) and divided into two groups. Each group was supplemented with either cornstarch or xylanase at 1.0% result- ing in 90 U of xylanase/g of each ingredient. As the current study was a pilot study for an in vivo study, the inclusion rate of xylanase was set to be high to determine whether DM disappearance was increased or not by supplemental xylanase, and subsequently, to screen potential feed ingre dients for xylanase.
In vitro procedure
The IVID procedure was a two-step enzymatic degradation that simulates digestion in the stom- ach and small intestine of the pig (Boisen and Fernández, 1995). In the first step, 1 g of ingredi- ent sample was transferred into 100 mL conical flask and then 25 mL of sodium phosphate buffer solution (0.1 M, pH 6.0), and 10 mL of HCl (0.2 M, pH 0.7) were added. To simulate digestion conditions in the stomach, the pH was adjusted to 2.0 using 1 M HCl or NaOH solution, and 1 mL of freshly prepared pepsin solution (10 mg/mL; ≥250 U/mg solid, P7000, Pepsin from porcine gastric mucosa, Sigma-Aldrich, St. Louis, MO, USA) was added to the flask. To avoid bacterial fermentation, 0.5 mL of chloramphenicol (C0378, Chloramphenicol, Sigma-Aldrich, St. Louis, MO, USA) solution (5 g/L of ethanol) was also added. The flasks were closed with a silicon stopper and incubated in a shaking incubator at 39 °C for 6 h. After incubation, the second step simulated the digestion in the small intestine of pigs. Firstly, 10 mL of phosphate buffer solution (0.2 M, pH 6.8), and 5 mL of 0.6 M NaOH solution were added to the flasks. Then the pH was adjusted to 6.8 using 1 M HCl or NaOH solution, and 1 mL of fresh- ly prepared pancreatin solution (50 mg/mL; 4 × USP, P1750, Pancreatin from porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA) was added. Then, the flasks were incubated in a shaking in- cubator at 39 °C for 18 h. After incubation, 5 mL of 20% sulfosalicylic acid solution was added and samples were left for 30 min at room temper- ature to precipitate the indigestible protein. After 30 min of precipitation, undigested samples were filtered through pre-dried and weighed glass fil- ter crucibles (Filter Crucibles CFE Por. 2, Robu, Hattert, Germany) containing 400 mg of celite as filter aid using the Fibertec System (Fibertec System 1021 Cold Extractor, Tecator, Hӧganӓs, Sweden). The flasks were rinsed twice with 1% sulfosalicylic acid solution, and 10 mL of 95% ethanol and 99.5% acetone were added twice to the glass filter crucibles. Glass filter crucibles with undigested samples were dried at 80 °C for 24 h. After 1 h of cooling in a desiccator, glass filter crucibles were weighed.
The IVTTD procedure used was a three-step enzymatic degradation that simulates the diges- tion in the stomach, the small intestine, and the large intestine of pigs (Boisen and Fernández, 1997). The first and second steps were similar to the IVID procedure except the weight of the sample, concentration of the enzymes, and in- cubation time. For IVTTD, 0.5 g of sample was used, and the concentrations of pepsin and pan- creatic solutions were increased to 25 and 100 mg/mL, respectively, while the incubation times were reduced to 2 and 4 h, respectively. In the third step, 10 mL of 0.2 M EDTA solution was added to the flasks. The pH was then adjusted to 4.8 by adding 30% acetic acid or 1 M NaOH. Samples were supplemented with 0.5 mL of multi-enzyme (V2010, Viscozyme® L, Sigma- Aldrich, St. Louis, MO, USA) as a substitute for microbial enzymes, and incubated in a shaking incubator for 18 h at 39 °C. After incubation, the undigested residues were collected and glass filter crucibles with undigested residues were dried at 130 °C for 6 h. The number of replications per each treatment was 3 (one replication per batch).
Chemical analysis
Ingredients were analyzed for DM (Ahn et al., 2014), crude protein (method 990.03; AOAC, 2005), ether extract (method 2003.05; AOAC, 2005), neutral detergent fiber (method 2002.04; AOAC, 2005), acid detergent fiber (method 973.18; AOAC, 2005), and ash (method 942.05; AOAC, 2005).
Calculation and statistical analysis
The IVID and IVTTD of DM were calculated with the following equations, respectively (Ha et al., 2020): IVID or IVTTD of DM (%) = [(DMTI - DMUR) ÷ DMTI] × 100, where DMTI and DMUR are the weight of DM concentration in the test ingredient and undigest ed residues, respectively.
Data were analyzed by the GLM procedure of SAS, version 9.3 (SAS Inst. Inc., Cary, NC, USA; 2012). The model included xylanase ad- dition as a fixed variable (Seo et al., 2018). The experimental unit was the test flask. Statistical significance of treatment effects was declared at p<0.05.
Results
Neutral detergent fiber of test ingredients ranged from 10.4 to 62.0% (Table 1).
Xylanase addition increased the IVID of DM in wheat, barley, wheat flour, and wheat bran compared with the non-supplemental xylanase group (p<0.05; Table 2).
The IVTTD of DM in barley and wheat bran increased by xylanase addition (p<0.05; Table 3). In contrast, supplemental xylanase did not affect IVID and IVTTD of DM in the other ingredients.
Table 2
In vitro ileal disappearance of dry matter in feed ingredients with or without xylanase addition1.
Discussion
In the present study, chemical composition of the test ingredients was within the range of values reported in the literature (Stein et al., 2016; Son et al., 2019). The in vitro DM disappearance procedure has been used to estimate the in vivo digestibility of energy and nutrients in feed ingredients (Park et al., 2012) and to determine the efficacy of exogenous enzyme complexes (Park et al., 2016; Ha et al., 2020). The IVID and IVTTD of DM in test ingredients in the present work were within the range of reported values (Boisen and Fernández, 1995, 1997; Kong et al., 2015; Park et al., 2016). In previous studies, supplemental xylanase improved ileal and total tract digestibility of energy in pigs fed diets containing 30% wheat millrun or 30% wheat screening (Nortey et al., 2007), but not in pigs fed diets mainly composed of wheat and wheat DDGS (Widyaratne et al., 2009). The increased energy digestibility by xylanase was likely due to the hydrolysis of wheat arabinoxylans in the wheat millrun and wheat screening. As NSP are concentrated during the distillation process, corn DDGS and wheat DDGS contain relatively large amounts of NSP. Thus, dramatic effects of xylanase on digestibility of distillers dried grains were anticipated. In the present work, however, xylanase did not improve IVID or IVTTD of DM in corn DDGS and wheat DDGS. This result is likely due to changes or degradation of NSP in corn and wheat during the fermentation and drying processes that may result in xylanase being ineffective (Widyaratne et al., 2009). Similarly, no effect of xylanase was found in IVID and IVTTD of copra meal and palm kernel expellers, and this may be partially explained by the low concentration of arabinoxylan (approximately 3%) in these ingredients, which is a main substrate for xylanase (Alshelmani et al., 2014).
In the present study, IVTTD was greater than IVID in test ingredients regardless of xylanase addition. The greater IVTTD value is likely to be attributed to additional digestion by multi-enzymes in the step 3, which mimics digestion in the large intestine. While IVID of DM in wheat and wheat flour was increased by supplemental xylanase, IVTTD of DM in these ingredients was not affected by xylanase. This may be explained by masking effects of Viscozyme® containing arabanase, beta-glucanase, hemicellulase, and xylanase during the third step of the IVTTD procedure (Kong et al., 2015). On the other hand, both IVID and IVTTD of DM were increased by supplemental xylanase in barley and wheat bran. The significant effect of xylanase on DM disappearance in wheat bran can be explained by the highest concentrations of arabinoxylan in wheat bran (23.2%, Bach Knudsen, 2014) among the ingredients tested in the present work. In addition, exogenous xylanase increased the energy digestibility of a diet containing mainly wheat and wheat bran (Dong et al. 2018). However, the reason for the significant effect of xylanase on DM disappearance in barley is unclear. But, the increment of ileal DM disappearance by xylanase was less (1.7 vs. 4.1% units) in barley than in wheat bran. This result may be partially attributed to the lower concentration of arabinoxylan in barley (8.4%) than that in wheat bran (23.2%; Bach Knudsen, 2014).
In conclusion, exogenous xylanase could increase IVID of DM in wheat, barley, wheat flour, and wheat bran, but no effect on IVTTD in wheat and wheat flour was found. Further research is warranted to determine the effects of supplemental xylanase on other feed ingredients and confirm the effect of xylanase in experiments with animals.