Effects of xylanase supplementation to wheat-based diets on growth performance, nutrient digestibility and gut microbes in weanling pigs

Objective This study was designed to investigate the effects of an Aspergillus sulphureus xylanase expressed in Pichia pastoris on the growth performance, nutrient digestibility and gut microbes in weanling pigs. Methods A total of 180 weanling pigs (initial body weights were 8.47±1.40 kg) were assigned randomly to 5 dietary treatments. Each treatment had 6 replicates with 6 pigs per replicate. The experimental diets were wheat based with supplementation of 0, 500, 1,000, 2,000, and 4,000 U xylanase/kg. The experiment lasted 28 days (early phase, d 0 to 14; late phase, d 15 to 28). Results In the early phase, compared to the control, average daily gain (ADG) was higher for pigs fed diets supplemented with xylanase and there was a quadratic response in ADG (p<0.05). In the entire phase, ADG was higher for the pigs fed 1,000 or 2,000 U/kg xylanase compared to the control (p<0.05). The gain to feed ratio was higher for pigs fed diets supplemented with 1,000 or 2,000 U/kg xylanase compared to the control (p<0.05). Increasing the amount of xylanase improved the apparent total tract digestibility of dry matter, crude protein, neutral detergent fiber, calcium, and phosphorus during both periods (p<0.05). Xylanase supplementation (2,000 U/kg) decreased the proportion of Lachnospiraceae (by 50%) in Firmicutes, but increased Prevotellaceae (by 175%) in Bacteroidetes and almost diminished Enterobacteriaceae (Escherichia-Shigella) in Proteobacteria. Conclusion Xylanase supplementation increased growth performance and nutrient digestibility up to 2,000 U/kg. Supplementation of xylanase (2,000 U/kg) decreased the richness of gut bacteria but diminished the growth of harmful pathogenic bacteria, such as Escherichia-Shigella, in the colon.


INTRODUCTION
Wheat has long been used as a major feedstuff for monogastric animals. In 2012, the amount of wheat and its byproducts used in feed was 59.8 million tons (China Feed Industry Annual 20122013) in China. However, wheat contains nonstarch polysaccharides, including xylan, glucan, cellulose, and mannan, that reduce feed efficiency and nutrient digestibility. Of these, xylan makes up dominant proportions averaging 5.4% to 8.0% in Australian wheat [1], 5.5% to 6.5% in North American wheat [2] and 6.6% to 8.2% in Chinese wheat [3]. Xylan, the most abundant polysaccharide in plant cell walls in nature, is therefore considered to be primarily responsible for the antinutritional effects of wheat. Mammals cannot digest xylan because they lack endogenous xylanase. Xylanase (EC 3.2.1.8) can degrade xylan by randomly hydrolyzing the β1,4glycosidic bonds producing different length of xylooligosaccha rides. Thus supplementation of xylanase in feed has been widely applied for chicks and pigs to promote growth performance [4]. It is believed that xylanase, as other nonstarch polysaccharide (NSP) degrad ing enzymes, has several actions: partial hydrolysis of non starch polysaccharides, decreasing the viscosity of digesta and rupturing plant cell walls to release cellular nutrients for digestion [5,6]. Moreover, the supplementation of xylanase in wheat based diets may produce more short chain oligo saccharides and these products will act as the substrates for gastrointestinal ecology [7]. In this study, we evaluated the ef fect of an acidic xylanase, cloned from Aspergillus sulphureus and constitutively expressed in Pichia pastoris in our labora tory, in weanling pig diets. Its beneficial effects on hind gut bacterial community was also investigated.

Preparation of xylanase
A xylanase was prepared by fermentation in our lab as previ ously described [8]. The actual xylanase product used in this study was obtained by mixing 66% of the liquid fermentation broth produced above with 34% soybean meal and then air dried for 24 h. This resulted in a xylanase preparation contain ing approximately 400,000 units (U) of xylanase per kg. One unit of xylanase is defined as the amount of enzyme which liberates 1 μmol of total reducing sugar (xylose) per min at the optimal enzymatic reaction conditions of pH 2.4 and 50°C. The enzyme preparation was tested for contaminating levels of other enzymes using the method of dinitroalicylic acid [9]. Briefly, 5 g of enzyme powder was dissolved in 50 mL Na 2 HPO 4 citric acid buffer (pH 2.0) and incubated at room temperature for 30 min. At the same time, the substrate solu tion was prepared by dissolving 0.16 g of pure mannan, xylan, αgalactose and βglucan (Sigma, St Louis, MO, USA) in 20 mL of Na 2 HPO 4 citric acid buffer (pH 2.4). Equal amounts (400 μL) of the enzyme and substrate solutions were mixed and incubated at 65°C for 20 min before stopping the reaction with 1 mL of 3,5dinitrosalicylic acid solution. The optical density of the solution was assayed on a spectrophotometer (Beijing PuxiGeneral TU1901, Beijing, China) at 540 nm. No mannanase, βglucanase or αgalactosidase activity was detected in the enzyme preparation.

Animal and facilities
All animal procedures and animal care were approved by the Institutional Animal Care and Use Committee of China Agri cultural University (Beijing, China). The experiments were conducted in the Pig Research Facility at the Swine Nutrition Research Centre of National Feed Engineering Technology Research Centre (Chengde, Hebei, China). One nursery barn was used in the study. The barn was a closed facility with me chanical ventilation equipment. The barn was equipped with 36 pens, each pen contained 6 pigs (three barrows and three gilts), resulting in 0.45 m 2 per pig ([1.8 m×1.5 m]/6). The floor was onehalf slatted concrete. Each pen was equipped with 1 nipple waterer and 1 feeder. A total of 180 crossbred pigs (Duroc×Landrace×Yorkshire) with an average initial body weight (BW) of 8.47±1.40 kg (average weaning age was 28 d) were blocked according to gender, ancestry and BW. Pigs were allotted to one of five dietary treatments (0, 500, 1,000, 2,000, and 4,000 U/kg xylanase in wheat based diets) with six repli cates (pen) in each treatment. The diets were formulated to contain 3,400 kcal/kg of digestible energy, 3,265 kcal/kg of metabolisable energy (ME), 18.76% of crude protein (CP) and 1.14% of total lysine (Table 1) and in meal form. As an indigestible marker, 0.3% chromic oxide (Cr 2 O 3 ) was added to each diet to calculate apparent total tract digestibility (ATTD) ( Table 1). We formulated the diets in a reduced nutrient level (ME: 4% lower than Nutrient Research Council [10] and 5% lower of standard ileum digestibility [SID] lysine). The aim was to monitor the significant effects of supplemented xyla nase on improvement of the diet digestibility assuming xylanase released more oligosaccharides from wheat based diets. The experiment lasted 28 days divided into 2 stages (early phase, d 0 to 14; late phase, d 15 to 28). Pigs had ad libitum access to feed and water. On d 28 of the experiment, one pig from each pen (total three barrows and three gilts for each treatment) was selected to be slaughtered. Colon digesta were collected aseptically and immediately immersed in liquid nitrogen and stored at -80°C for analysis of bacterial community.
Each piglet was weighed on d 0, 14, and 28 of the experi ment. Feed consumption was recorded daily by weighing out any residual feed from the previous day prior to adding new feed, which was average daily feed intake (ADFI). Average daily gain (ADG) was calculated by dividing total weight gain of pigs with days of experiment. Gain to feed ratio (G:F) was calculated by dividing ADG with ADFI.
Feed samples for each treatment were collected from every batch of feed produced, pooled and mixed within treatment. Fresh fecal samples were taken from each pen on d 13 and 14 of the experiment (phase 1) as well as on d 27 and 28 of the experiment (phase 2) and frozen for later analysis. Fecal sam ples were collected at least six times a day from the floor of each pen. The fecal samples were pooled within pen and dried in a forcedair drying oven at 65°C for 72 h, ground through a 1mm screen and thoroughly mixed.
The digestibility of various chemical constituents was de termined using the reported method [11].

Chemical analyses
Feed and fecal samples were analyzed according to the me thods of the Association of Official Analytical Chemists (AOAC [12]). Analyses were conducted for moisture (AOAC method 930.15), CP (AOAC method 984.13), calcium (AOAC method 968.08) and phosphorus (AOAC method 965.17). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using fiber bags and fiber analyzer equipment (Fiber Analyzer, Ankom Technology, Macedon, NY, USA) [13]. Gross energy was measured via an adiabatic oxygen bomb calorimeter (Parr Instruments, Moline, IL, USA). The chromium concentrations of diets and fecal samples were determined after nitric acidperchloric acid wet ash sample preparation using a Polarized Zeeman Atomic Absorption Spectrometer (Hitachi Z2000, Tokyo, Japan). All analyses were performed in duplicate and repeated when the results differed by more than 5%.

Statistical analysis
Data were analyzed using oneway analysis of variance (ANOVA) in accordance with the general linear model pro cedures of SAS 9.2 (SAS Institute Inc., Cary, NC, USA) utilizing a randomized complete block design by weight, including the terms for treatments and blocks. Each pen was deemed as one experimental unit for growth performance, while an individual pig was considered as the experimental unit for other indices. Interactive matrix algebra procedure (IML) of SAS was adopted to generate the coefficients of unequally spaced contrasts. Subsequently, the linear and quadratic re sponses of xylanase level were assessed by the orthogonal polynomial contrast. Significance level was set at p<0.05. Processing of sequencing data: Raw fastq files were demul tiplexed, qualityfiltered using QIIME (version 1.17) with the following criteria: i) The 300 bp reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window, discarding the truncated reads that were shorter than 50 bp. ii) exact barcode matching, 2 nucleotide mismatch in primer matching, and reads containing ambiguous characters were removed. iii) only sequences that overlap longer than 10 bp were assembled according to their overlap sequence. Reads which could not be assembled were discarded. Opera tional taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE (version 7.1 http://drive5. com/uparse/) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier (http://rdp. cme.msu.edu/) against the silva (SSU115)16S rRNA database using confidence threshold of 70% [14].

Xylanase Supplementation improved growth performance, and ATTD
The growth performance of the weanling pigs is presented in Table 2. In the early phase (d 0 to 14 of the experiment), com pared to pigs fed the control diet, ADG were higher for pigs fed diets supplemented groups, and there was a quadratic re sponse in ADG (p<0.05). G:F of supplemented groups showed a quadratic response compared to the control in the early phase (p<0.05). In the entire phase (d 0 to d 28 of the experi ment), ADG was higher for the pigs fed 1,000 or 2,000 U/kg xylanase compared to pigs fed the control diet (p<0.05). The G:F was higher for pigs fed diets supplemented with 1,000 to 2,000 U/kg xylanase compared to the control (p<0.05). In creasing the amount of xylanase in the diet improved (p<0.05) the ATTD of dry matter (DM), CP, NDF, ADF, calcium, and phosphorus during both periods (Table 3).

Diversity of bacterial community in pig colonic digesta
To further investigate the mechanism of dietary xylanase on improved growth performance, the colon digesta bacterial richness and diversity in the group of 2,000 U/kg xylanase were determined. After size filtering, quality control and chimera removal, a total of 41,408 and 44,581 valid sequences in co lonic digesta of unsupplemented (Control) and supplemented pigs (Xylanase) were obtained, respectively. The OTU numbers of bacterial community were classified from valid sequence with 97% similarity. The indices of sobs (OTUs), Ace and Chao represent the richness of bacterial community, while Shannon, Simpson, and Coverage values represent the diver sity (Table 4). Venn analysis (data not shown) showed that the colonic digesta of control and xylanase treatment shared 250 OTUs, which accounted for 77.88% of the total OTUs of the control group and 88.65% of the xylanase group. Firmicutes, Bacteroidetes, and Proteobacteria were dominant phyla in weanling pig colonic digesta, representing over 99% propor tion of total bacterial community compared to the control ( Figure 1A). However, the distribution of individual propor tions of these three phyla were not equivalent. In Xylanase group (2,000 U/kg), Firmicutes represented 43% of the total proportion (vs 60% in control); Bacteroidetes was taken 54% (vs 21% in control). Proteobacteria was not detectable in Xy lanase group (vs 17% in control). When further dissected to bacterial compositions at the family level ( Figure 1B), xylanase supplementation decreased the proportion of Lachnospiraceae (by 50%) in Firmicutes and increased Prevotellaceae (by 175%) in Bacteroidetes. Xylanase supplementation almost diminished Enterobacteriaceae in Proteobacteria. At the Genus level (Figure 2), we found that the majority of the reduction in Lachnospiraceae was related to the genus Eubacterium_rectale_group (by 55%). And the diminished Enterobacteriaceae abundance (Figure 3) was related to Escherichia-Shigella genus, a serious pathogenic bac terial strain in gut. The increased Prevotellaceae was related to a series of Prevotella genera (Provotella_9, Prevotellaceae_ NK3B31_group, Prevotella_1 etc), and their variation profile was similar to that of the family of Prevotella.

DISCUSSION
Weaning is the most challenging event for young piglets as they are forced to encounter nutritional stress changing from  liquid sow milk to a less digestible dry feed, immature immune system, and social stress caused by separation from mothers and commingling with other nonlittermate piglets. These disruptions usually cause damage to intestinal epithelia increas ing the likelihood for infection by pathogenic microorganisms. Additionally, the immature gastrointestinal tract cannot secret sufficient amount of digestive enzymes for proper digestion and absorption, which worsened the growth of postweaning pigs. NSP can impact the gastrointestinal tract in two aspects. One is that NSP functions as a substrate to increase the vis cosity [15]. This biochemical characteristic is accompanied by some consequent effects such as increased digesta transit times, rate of mucosa cell turnover, mucin secretion and un digested contents. These effects increase microbial size (colony forming unites) [16] and composition [17]. On the other hand, NSPase, such as xylanase, can reduce digesta viscosity by hy drolysis of NSP and NSPcontaining cell walls to decapsulate nutrients for digestion. Xylanase produces numerous short chain xylooligomers [18], which improve small intestinal absorption and limits substrates for large intestinal microbial fermentation.
In this study, we investigated the effect of an acidic β1,4 xylanase, cloned from Aspergillus sulphureus and constitutively expressed in Pichi pastoris, in wheatbased diets on the growth of weanling pigs. NRC recommends nutrient requirements for an optimal growth for pigs. In this study, we formulated the diets with a reduced nutrient level (ME: 4% lower than NRC and 5% lower of SID lysine). The aim was to monitor the significant effects of supplemented xylanase on improve ment of the diet digestibility assuming xylanase released more oligosaccharides from wheat based diets. If the nutrient level had already met the growth requirement, the additional effect by supplemented xylanase could be saturated. Indeed, we observed that at this nutrient level, ADG and G:F were sig nificantly improved. At this dietary condition, the weanling pigs encountered pathogenic infection which was reflected by colonic Enterobacteriaceae (Escherichia-Shigella). Xylanase supplementation at 2,000 U/mg significantly diminished pathogenic Enterobacteriaceae (Escherichia-Shigella) in the colon. We found increased ADG and G:F in the early phase (d 0 to d 14 of the experiment) and the entire phase (d 0 to d 28 of the experiment). The ATTD of DM, CP, NDF, calcium, and phosphorus were all improved indicating that, besides digested plant cell walls, the supplemented xylanase also sys temically improved action of other digestive enzymes on the wheatbased diet. It enhanced an overall absorption of dietary nutrients including crude protein, crude fat, crude fiber, or ganic matter etc. These results were consistent with similar reports in weanling pigs [19,20]. We used soybean meal as the carrier for xylanase preparation, and conducted the sub stitution of extruded soybean. As the proportion of xylanase preparation increased, the extruded soybean amount decreased. Soybean meal contains slightly lower fat than extruded soy bean, which might be the reason that at the highest dose of xylanase (4,000 IU), ADG and G:F showed quadratic respons es. On the other hand, the created gut microenvironment favored the growth of beneficial bacteria and inhibited harm ful bacterial strains. Escherichia_Shigella can cause severe diarrhea in animals. In this study, the pathogenic Esche-richia_Shigella were almost diminished by dietary xylanase supple mentation. The xylanase supplementation decreased the overall diversity of bacterial community which agrees with a previous report that fermented swine feces in the presence of starch reduced the bacterial diversity [21].
It is interesting that xylanase supplementation (2,000 U/kg) decreased abundance of the phylum Firmicutes which were mainly contributed by f Eubacterium_rectale_group (21.5% to 13.1% in Firmicutes in genus level), an acetateconverting butyrate producer in human colon bacteria [22]. Selenomonas_ bovi spp (3.4% to 11.5% in Firmicutes in genus level) and Megasphaera elsdenii spp (2.9% to 7.5% in Firmicutes in ge nus level) were increased. Selenomonas_bovi spp can produce acetate, propionate and lactate [23] and Megasphaera elsdenii spp are lactateutilizing bacteria to produce lactic acid [24]. This indicates that xylanase supplementation (2,000 U/kg) modulated the content of short chain fatty acids in the intes tine which may act to counteract complications during post weaning period.
The pylum Bacteroidetes were increased from 22.7% to 54.5%, which were mainly represented by Prevotella_9, Pre-tellaceae_NK3B31_group and Prevotella_1 etc. Prevotella are most abundant in the rumen and hind gut of cattle and sheep to help the breakdown of carbohydrates. The abundance of Prevotella is considered a discriminative taxon in agrarian residence [25]. In this study, the increased proportion of Prevotella increased with xylanase supplementation. It indicates that digestible carbohydrate availability along the length of the intestinal tract allowed enrich the growth of Prevotella.
In conclusion, with modulating the balance of ecophysiol ogy of the total bacterial communities in the intestine including diminished the growth of Escherichia_Shigella and increased Prevotella and series of beneficial bacteria strains, dietary xyl anase supplementation enhanced growth and feed utilization of weanling piglets up to 2,000 U/kg.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.

ACKNOWLEDGMENTS
This work was supported by the Scientific and Technical Sup porting Programmes (2013BAD10B01) and Beijing Science and Technology Project (D161100006116001).