Debranching enzymes decomposed corn arabinoxylan into xylooligosaccharides and achieved a prebiotic regulation of gut microbiota in broilers

doi:10.21203/rs.3.rs-1991906/v1

Download PDF

Research Article

Debranching enzymes decomposed corn arabinoxylan into xylooligosaccharides and achieved a prebiotic regulation of gut microbiota in broilers

https://doi.org/10.21203/rs.3.rs-1991906/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 09 Mar, 2023

Read the published version in Journal of Animal Science and Biotechnology →

You are reading this latest preprint version

BACKGROUND

Corn arabinoxylan (AX) is a complicated and multi-branched antinutritional factor, thereby proving the use of endo-xylanase (EX) to be marginally valid. This study focused on specific types of AX-degrading enzymes (ADE) to exert the synergy of debranching enzymes and track prebiotic potential of enzymatic hydrolysates. It aimed to investigate the effects of ADE on growth performance, intestinal histomorphology, absorption functions, changes of polysaccharide components, fermentation, and gut microbiota of broilers. To this purpose, 576 five-day-old Arbor Acres male broilers were randomly allocated into eight treatments with six replicates each. Corn basal diets supplemented with or without enzymes were fed for a 21-day period, specifically including EX, its compatible use with arabinofuranosidase (EXA) or ferulic acid esterase (EXF), and compound groups with the above three enzymes (XAF).

RESULTS

Specific ADE stimulated the jejunal villus height and goblet cell number, and evidently decreased the crypt depth (P < 0.05), while the ratio of ileal villus height to crypt depth was significantly increased in EXF (P < 0.05). Maltase activities of ileal mucosa in XAF groups were extremely enhanced (P < 0.01), and EX boosted the activity of Na⁺-K⁺ ATPase in the small intestine (P < 0.01). The insoluble AX concentrations comparatively lessened, thereby notably raising the sundry xylooligosaccharide (XOS) yield in the ileal chyme (P < 0.05), which was dominant in xylobiose and xylotriose. Improvements in the abundance and diversity of ileal microbial communities within EXA, EXF, XAF treatments were observed (P < 0.05). Positive correlations between microbiota and XOS were revealed, with xylobiose and xylotriose being critical for ten beneficial bacteria (P < 0.05). EXF escalated the BWG and FCR of broilers in this phase (P < 0.05), attributing to the thriving networks modified by Lactobacillus. The intracecal contents of acetic acid, butyric acid, and propionic acid were greatly enhanced in most ADE groups such as EXF (P < 0.05).

CONCLUSIONS

Debranching enzymes appreciably targeted corn AX to release prebiotic XOS in the posterior ileum and facilitated intracaecal fermentation. It was beneficial for improving gut development, digestion and absorption, and modulating the microflora to promote early performance of broilers.

Corn

Arabinoxylan

Specific xylanase

Xylooligosaccharide

Prebiotic

The relatively complex gut microecosystem plays an indispensable role in maintaining intestinal health, and the diversifications in microbiota are inseparable from dietary factors. The common non-starch polysaccharides (NSP) in the diet, especially the grain-derived arabinoxylan (AX), are inclined to increase the viscosity of chyme in the intestinal lumen and disturb the normal microflora. This can result in reductions in the nutrient utilization efficiency and growth performance of the host. Although exogenous endo-xylanase (EX) eliminates the negative effects of AX to a certain extent, its efficacy can be abridged by factors such as the source of enzyme-producing strains, the expressed enzyme activities, the inclusion levels, and the experimental animals. Furthermore, due to the specificity of enzymes, the structure of the degraded substrate is of paramount importance. A higher frequency of branched attachments and substituent groups hinders enzymatic cleavage of the AX backbone [1]. Constructed as such, it might require a battery of enzymes projected not to differ so much in the core EX but rather in conjunction with debranching enzymes to attack side chains or indestructible linkages [2].

An abundance of data are focused on wheat-based grains, where efficacy for both in vitro and in vivo settings has been varied [3, 4]. Additionally, solitary EX has proven to have limited validity. Conversely, our previous study systematically demonstrated that specific arabinoxylan-degrading enzyme (ADE) collaborations among EX, arabinofuranosidase (EA), and ferulic acid esterase (EF) increased AX degradation to more oligomers, which improved the growth performance and gut health of broilers fed wheat diets [5–7]. However, corn, a nearly universal ingredient in poultry feeds, retains structural complexity and varies considerably in AX solubility compared with wheat. There is a lack of insight reporting the potential for specific ADE synergism on corn typical diets. Relevant reports have emphasized the in vitro solubilization or fermentation of corn AX by these carbohydrases [8, 9], as opposed to in-depth tracking and monitoring of their potential in vivo effects, especially involving gut microbiota or microecosystems. Based on the synergistic mechanism of the debranching enzymes and the main enzyme EX, we hypothesize that it may require diverse combinations or even multiple doses of specific ADE preparations for corn type basal diets. Moreover, greater attention needs to be paid to whether there are certain differences in the enzymatic hydrolysis products of corn AX, and the product components are beneficial to maintain intestinal health.

The formation of xylooligosaccharides (XOS) is recognized as a merit of AX degradation, in which xylobiose and xylotriose are the more potent functional components. Scholars compared the prebiotic effects and fermentation abilities of five commonly used fibers through in vitro system measurements [10], and found that XOS significantly increased the abundance of Bifidobacterium after 24 hours and promoted the formation of short-chain fatty acids (SCFAs). Lactobacillus plantarum S2 had higher cell density and faster growth rates when cultured on the corncob-derived XOS, and acetate was the predominant SCFAs in the final fermentation products. Much attention has also been devoted to their prebiotic roles of mediating intestinal homeostasis and maintaining the mutually beneficial relationship among microbes [11–13]. The in situ production of corn XOS is of great significance to the regulation of intestinal microflora. However, there is a lack of understanding about the AX conversion, as well as the different sizes and quantities of XOS fractions (xylobiose, xylotriose, xylotetraose, xylopentose, etc.) manufactured in the gastrointestinal tract as a result of feeding birds basal diets supplemented with specific ADE. The scientific doubt needs to be answered in combination with the analysis of intestinal content products and microbiome technology.

Collectively, this study aimed to evaluate the effects of different specific ADE combinations or inclusion levels on the growth performance, intestinal morphology, digestive enzyme activities, enzymatic hydrolysis products composition, fermentation metabolism, and gut microbiota of broilers via a feeding trial. Allowing for the possible clarification of the differential regulation of the enzymatic hydrolysis products of corn-type AX on the endogenous intestinal probiotics of broilers, and provide a solid theoretical foundation for the improvement of intestinal health and application promotion of prebiotic ADE.

2.1 Ethical statements

Animal experimental design and procedures were approved and conducted under the supervision of the China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethical Committee (No. AW80011202-1-3) (Beijing, China).

2.2 Enzyme preparations in the animal trial

The Aspergillus niger-derived endo-xylanase (EX), arabinofuranosidase (EA), and ferulic acid esterase (EF) (90,000, 10,000, and 1,000 U/g, respectively) were provided by Bestzyme Bio-products, Co., Ltd. (Jinan, China).

2.3 Experimental design, diets, and management

In total, 576 5-day-old Arbor Acres (AA) male broilers were weighed and randomly allocated to eight treatments with six replicates each so that their initial body weights were similar across all the groups. The birds were fed with basal diet (control, CTL), whose composition and nutritional levels were shown in Table 1, and the corresponding diets supplemented with specific arabinoxylan-degrading enzymes (ADE) (Table 2). Briefly, including single xylanase (EX), its compatible addition with EA (EXA) or EF (EXF), and compound groups with the above three enzymes (XAF-1,2,3,4). Supplemental enzymes levels were based on the in vitro screening results. The growth trial lasted for 21 days. All chicks were reared in cages with nipple drinkers, and the house was environmentally controlled. The lighting schedule was 20 h light and 4 h dark throughout the period. Feed and freshwater were available ad libitum.

2.4 Growth performance

Bodyweight and feed consumption were recorded for each replicate at 21 d. Thus, body weight gain (BWG), along with feed intake (FI), and the feed to gain ratio (F/G) during the starter period were calculated. Moreover, the mortality (MRT) and European comprehensive production index (EPI), based on the death and elimination of broilers in this stage, were calculated.

2.5 Slaughter method and sample collection

At 21 d of age, six healthy chicks from each treatment were bled from the carotid artery after being anesthetized by an injection of sodium pentobarbital (50 mg/kg body weight) in the wing vein. Then, mid-segments of the jejunum and ileum at necropsy were immediately removed and fixed in 4% paraformaldehyde solution for intestinal morphology measurements. Digesta and mucosa from the remaining intestinal portions and ceca were separately harvested and stored at -80 ℃ for further analysis.

2.5.1 Intestinal histomorphology and absorption function

5-μm consecutive sections of jejunal and ileal segments were prepared for morphological observations after staining with hematoxylin-eosin (HE). Representative and well-oriented villi were used to determine the villus height (VH), crypt depth (CD), as well as the villus height to crypt depth ratio (VCR). The number of goblet cells (GC), between each set of per 100 micrometers intestinal epithelial columnar cells, were counted. Activities of mucosal sucrase, maltase, alkaline phosphatase, and Na⁺-K⁺ ATPase were determined colorimetrically via commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to instruction protocols. These indices were normalized by the total protein contents of the intestinal mucosa, which were quantified using BCA protein assay kits (CWBiotech Co. Ltd, Beijing, China).

2.5.2 Analysis of NSP in distal ileum chyme of broiler chickens

The soluble and insoluble NSP or xylan contents were measured as newly illustrated with minor modifications [14].Ileal chyme samples were pre-treated with fat extraction and enzymatic hydrolysis of starch. Subsequently, the supernatant and residue were subjected to different complicated steps such as hydrolysis, washing, centrifugation, and drying. The glycan degradation products were then analyzed for individual sugar concentration by high-performance liquid chromatography (HPLC); the quantity of arabinose and xylose determined AX content and the total sugars represented the total NSP content. Monosaccharide standards consist of galactose (Gal), glucose (Glu), mannose (Man), arabinose (Ara), xylose (Xyl), fucose (Fuc), rhamnose (Rha), galacturonic acid (Glc), and glucuronic acid (GlcA) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA), which were subjected to the same procedures as the samples.

2.5.3 Analysis of XOS in distal ileum and cecal chyme of broiler chickens

The XOS contents were evaluated by high-performance anion-exchange chromatography (HPAEC). Samples were dissolved in ultrapure water and oscillated by ultrasound, then centrifuged at 6000 × g for 15 min and 1 mL of the supernatant was collected. Standards of xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) were purchased from Megazyme (Wicklow, Ireland, UK). The following steps were conducted referring to the method [14].Analysis of the standards and filtered samples was carried out on a Dionex ICS3000 system equipped with a pump and an amperometric detector. The chameleon chromatography management system (Dionex, Sunnyvale, CA) was used for sugar identification and quantification. An analytical CarboPac PA10 pellicular anion-exchange resin column (250 × 4 mm) was used for sugar separation. The monoses were eluted with 250 mM NaOH at a flow rate of 1.0 mL/min.

2.5.4 Bacterial 16S rDNA sequencing of ileal microbiota

The concentration and purity of total genomic DNA, which was extracted from ileal digesta following the CTAB method, was monitored on 1% agarose gel electrophoresis. 16S rDNA sequences spanning the distinct regions V3-V4 were amplified with the primer sets of 515 F and 806 R with the barcode. All procedures were conducted by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China) as described previously [15]. Beta diversity was visualized by multivariate statistical methods such as principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS), etc. Visual heat maps were acquired according to the Spearman correlation and its significance between microbial species and intestinal apparent index. Redundancy analysis (RDA) and variance partial analysis (VPA) were adopted to quantify interpretations of the distribution of microbial communities by certain factors. Graphviz was used to draw the microbial interaction network for each treatment considering the species abundance and correlation between each genus.

2.5.5 Determination of SCFAs concentrations in the caeca

Referring to the detailed method and process described by Wu et al. [16],the composition and concentration of SCFAs, such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid, were used as standards and were analyzed by gas chromatography SCION-456-GC with a flame ionization detector.

2.5.6 Statistical analysis

One-way ANOVA and Duncan’s test for multiple comparisons were employed to identify differences in this trial (IBM SPSS statistics software version 21). A probability of P < 0.05 was considered statistically significant, P < 0.01 was described as extremely significant, and 0.05 < P < 0.10 was defined as a tendency towards significance.

3.1 Specific ADE improved the morphology and absorption of the small intestine in broilers

As shown in Fig. 1A, specific ADE combinations, except EX, EXA, and XAF-4, significantly increased the VH of the jejunum at 21 d (P < 0.05). All enzyme treatments were demonstrated to significantly stimulate (P < 0.01) the jejunal VCR and goblet cell counts of broiler chickens, and reduce the corresponding CD (Fig. 1A, C). Specifically, EX, EXF, and XAF-1 equally resulted in higher VCR (P < 0.05) and more GC quantities (P < 0.01) in the ileum (Fig. 2A, C). Broilers fed the XAF-2/3 diets had greater (P < 0.01) maltase activities in the ileal mucosa (Fig. 2B), while EX and EXF extremely upregulated (P < 0.01) the Na⁺-K⁺ ATPase activities in the small intestinal mucosa at 21 d of age (Fig. 1B, 2B).

3.2 Specific ADE promoted the partial enzymatic hydrolysis of corn AX to XOS in the ileum of broilers

There were extremely significant hoists (P < 0.01) for soluble arabinoxylan (SAX) concentration in the chyme among the treatments except for the EX group (Fig. 3A). Broilers in the EXF and XAF-2 groups exhibited higher (P < 0.05) concentrations of soluble NSP (SNSP) (Fig. 3A). The amount of insoluble arabinoxylan (IAX) and INSP consistently comparatively lessened (P > 0.05) in birds fed with ADE than those in the CTL group (Fig. 3A). Moreover, the supplementation of XAF-2/3 significantly enhanced (P < 0.01) the concentration of X2 (Fig. 3B), whereas much higher concentrations of X3 and X5 (P < 0.01) were detected in enzyme treatments apart from EX (Fig. 3B). The EXA, EXF, and XAF-1/3 groups notably increased (P < 0.05) the total xylooligosaccharide (T-XOS) content of the ileal chyme at 21 d (Fig. 3A). Intuitively, the complex of single xylanase and specific debranching enzyme (EXA) dominated in X4, while the three-enzyme complex treatments (XAF) concentrated on X2 and X3 (Fig. S1). On the contrary, all the components and proportions of XOS in the EX group were close to those of the CTL group (Fig. S1).

3.3 Specific ADE modulated gut microbiota of broilers through the prebiotic effects of its enzymatic hydrolyzate XOS

Overall, alpha diversity parameters reflecting the microbial richness and evenness sharply increased after supplementation with EXA, EXF, XAF-1, and XAF-2 (P < 0.01) (Fig. 4A); specifically, the EXF group had the most unique OTUs (Fig. 4A). According to the composite analysis of UPGMA, PCoA, and NMDS, the community structures from different specific ADE occupied distinct positions, except for EX which was clustered with the CTL group (Fig. 4B, C). Likewise, it was consistent with the higher coefficients of dissimilarity between the other specific ADE and the control group (Fig. 4B). The dominant bacteria at the genus level (Top 35) for specific ADE treatments were Lactobacillus, Subdoligranulum, Butyricicoccus, Lachnospiraceae, Bacteroides, and Candidatus Arthromitus, while EX and CTL groups trended higher in relative abundance for Streptococcus and Enterococcus (Fig. S2). The ileal microbial diversity index was remarkably (P < 0.05) positively correlated with soluble xylose, SAX, X2, X3, X5, and T-XOS contents by the clustering heatmaps (Fig. 4D). These mentioned soluble sugars and XOS components, were analogously critical for ten families of bacteria (P < 0.05), including Ruminococcaceae, Lachnospiraceae, Butyricicoccaceae, Bacillacea, Christensenellacea, and Erysipelotrichaceae (Fig. 5A). Soluble sugars and oligosaccharides distinguished the communities along the first coordinate axis (34.48%), which was specifically embodied as higher contributions of SAX, X3, X5, and T-XOS to the microbial diversity (Fig. 5B). Moreover, glycan variables (SAX, SNSP, IAX, and INSP) explained 46.48% of the variance based on the VPA analysis. However, the XOS produced by specific enzymes treatments explained 43.28% of the community changes, of which 35.39% could be explained by the induction of glycan dependence (Fig. 5C).

3.4 Specific ADE triggered the production of major SCFAs in the caecal site of broilers

Unlike the specific dominant relationship of XOS in the posterior ileum, the contents of X3 in the ceca were decreased in all enzymatic treatments (Fig. 6B), and the corresponding changes in XAF were significant (P < 0.01). The contents of X2 in EXF and XAF groups were increased significantly (P < 0.05) (Fig. 6A). Compared with the CTL group, the contents of X5 in the ceca of all ADE groups were increased (Fig. 6C), and the promotion effects of EX, EXA, and EXF were the most significant (P < 0.01). This was consistent with the changes in the total amount of XOS in the ceca (P < 0.05) (Fig. 6D), while X4 and X6 components were not detected in chyme samples. As shown in Fig. 7, the production of acetic acid, butyric acid, and propionic acid were dominant in the ceca of 21-day-old broilers, while the contents of formic acid and valeric acid were extremely low. Specifically, the concentrations of acetic acid were significantly raised in EX, EXA, and EXF groups (Fig. 7A), and the contents of butyric acid and propionic acid were significantly advanced in most ADE groups (P < 0.05) (Fig. 7B, C). According to Spearman’s correlation test (Fig. S3), formic acid and butyric acid were significantly positively correlated with X5, X6, and TXOS in ileum, and acetic acid was significantly positively correlated with BWG of 21-day-old broilers (P < 0.05). However, isobutyric acid was significantly negatively correlated with X2 and the goblet cells counts (P < 0.05). The branched clustering tree on the left side of the Fig. S3 intuitively reflects the close relationships between sugar composition, gut development, and growth performance regulated by SCFAs.

3.5 Beneficial microbes ameliorated ileal development and promoted the growth performance of broilers

Dietary EXF treatment had significant effects (P < 0.05) on BWG of broilers from d 14 to 21, and it tended to increase (P < 0.10) the feed conversion efficiency and EPI of broilers at 5-21 days (Table 3). As shown in Fig. 8B, the VH and CD were significantly positively correlated with the diversity and abundance of ileal microbes (P < 0.05). Besides, the longer intestinal villi were closely related to Lachnospiraceae, Ruminococcaceae, Anaeroplasmataceae, and Christensenellaceae (P < 0.05). Lactobacillaceae effectively (P < 0.01) stimulated the secretory functions of goblet cells (Fig. 8D). The F/G of broilers had strikingly (P < 0.05) negative correlations with Enterococcaceae, Bacteroidaceae, and Sphingomonadaceae (Fig. 8C). Microbes with noteworthy differences (P < 0.05) were filtered to construct co-occurrence networks (Fig. S4). It demonstrated that community interactions were richer and more complex, and the multiplication of species was modified by Lactobacillus after the EXF supplementation, which was critical to elevating the growth of broilers (Table 3).

Intestinal villi are the main sites of nutrient absorption, and their structural integrity and functional manifestations greatly affect the growth performance and overall health of animals. Long villi and shallow crypts are generally interrelated to support wider contact surface areas for higher absorption capacities and healthy development of the intestine [17, 18],but they also support greater tissue turnover accounting for the optimal status of the gut. The current study showed that most specific ADE combinations significantly increased the jejunal VH, VCR, and reduced the corresponding CD at 21 d, which was similar to the previous report by Wang et al [19]. Moreover, microscopic analysis and statistical quantification of the villi also indicated that ADE supplements resulted in more goblet cells in both the jejunum and ileum of broiler chickens, thereby synthesizing and secreting lubricant mucus to protect the small intestine [20].It has been demonstrated that digestive and absorptive functions of the small intestine were closely related to morphological changes in VH and CD [18, 19].In this study, as expected, dietary ADE treatments had significant effects on the maltase activities in the ileal mucosa and upregulated Na⁺-K⁺ ATPase activities in the small intestine mucosa at 21 d in broilers. Thus, the enhanced intestinal growth might improve absorption abilities or nutrient utilization, and further benefit the subsequent growth performance.

AX in corn comprises the majority of the NSP in this grain tissue. It is frequently distinguished with reference to the water solubility, an important benchmark for enzymatic hydrolysis, while corn contains less-soluble AX [21].On the other hand, the abundance of arabinose residues, along with the attachments decorated with various linkages, is key to its multifarious and complex character. Of the phenolic cross-links in corn AX, ferulic acid makes up 90% or more of the total and comprises 5% of the cell wall [22].These covalent linkages offer strength and rigidity to the structure with lower solubility, which may trigger another way to impede access to the primary xylan-backbone. It is reasonable to think that debranching enzymes, such as EA and EF, could work in collaboration with main chain enzymes to increase enzymatic accessibility and to improve the efficiency of core EX. Positive impacts of ADE supplements could be explained by their ability to generate prebiotic XOS from dietary AX [23].In this study, chromatographic technology was used to track the ileal chyme of broilers fed corn-type diets containing specific ADE, to highlight the conversion characteristics of corn AX and its product components. The data showed that specific ADE were responsible for the increased solubilization of IAX or INSP in the ileum of enzyme-fed broilers. Recently, the term “kick-starter” has been proposed to suggest that XOS could form a signaling agent for microbiota to boost the development of their fiber-degrading capacity in the young broiler [24].This provides evidence for our findings that higher levels of IAX solubilization in the ileum were also reported even though these oligosaccharides were generated in a small quantity. There were dissimilarities in the types of XOS products in the terminal ileum corresponding to the different specific ADE combinations. The results indicated that synergistic and sequential removal of substituents allowed for more rapid degradation of corn AX. Hence, it proves once more that the pattern of released XOS reaffirms a chronological process - the removal of branched components is followed by xylan backbone degradation [5, 25].

XOS, sugar oligomers produced by xylan enzymatic hydrolysis, are considered as emerging or candidate prebiotics. The beneficial effects of XOS on performance characteristics and the overall host health status have been well documented in the last decade, which was well-known for their prebiotic potential to broilers [26, 27].Among the explicit functions of XOS, the selective stimulation of beneficial bacterial population is the most vital demonstration and manifestation. In the present study, it was demonstrated that the microbial richness and diversity produced by specific ADE groups (EXA, EXF, XAF-1, and XAF-2) significantly increased, suggesting that their effects on the composition of the ileal microbial community were more prominent. The results were consistent with the findings of Wang et al, who reported that xylanase supplementation mixture modified the overall structure of the fecal bacterial community in terms of β-diversity, with observably distinctive OTUs appearing [19]. Evidently positive correlations between ileal microbial diversity indexes and XOS also indicated that the oligosaccharides products were essential in early nutrition to stimulate and establish gut microbiota in the broilers' intestine [28, 29]. The dominant bacteria at the genus level for specific ADE treatments were Lactobacillus, Butyricicoccus, Lachnospiraceae, and Bacteroides, and this observation was in line with many fresh reports [20, 30, 31].These probiotics could lead to the competitive exclusion of pathogenic bacteria and benefit the gut health of broilers [32].However, the differential flora enriched in the ileum was dissimilar for each specific ADE treatment. The variable results may be attributed to explanations that the effects of multienzyme complexes containing debranching enzymes on XOS production are inseparable from their associated cleavage properties and the order of action when bounding to EX [25].In brief, different ADE combination types yielded specific XOS components in the ileum of broilers, thereby promoting the selective proliferation of beneficial bacteria. SAX, X2, X3, X5, and T-XOS, for instance, were critical for ten families of bacteria, such as Ruminococcaceae, Lachnospiraceae, Christensenellacea, and Butyricicoccaceae. Collectively, the conversion of corn AX to assorted XOS under the ADE treatments benefited to sculpt microbial homeostasis.

The formation of XOS in the lower intestinal tract is considered as a benefit of AX degradation, acting as prebiotics for anaerobic bacteria in the ceca for energy and other purposes [12].The common microbes in the hindgut, including Bifidobacterium and Lactobacillus, are conducive to ferment AX hydrolysis products to SCFAs (mainly acetate, propionate and butyrate) [12].A wealth of data indicate that SCFAs are the superior energy sources for intestinal epithelial cells and for their role as signaling molecules to modulate gut integrity and immune response [33].Besides, these microbial metabolites fermented from complex carbohydrates play a major role in microbiota-host interactions. Thus, the composition of XOS and the contents of SCFAs in the ceca were also monitored to clearly assess the dynamic changes in the posterior intestinal segment of broilers. Herein, the markedly increased concentrations of X2, X5, and T-XOS in the cecal chyme after most specific ADE treatments were associated with subsequent SCFAs production. It was also demonstrated that the concentrations of acetic acid, butyric acid, and propionic acid were significantly advanced in most ADE groups, suggesting that supplemental specific ADE combinations effectively promoted the production of XOS in the hindgut segment of broilers and the fermentation to output SCFAs.

Although there is no shortage of studies indicating that the exogenous xylanase escalated the nutrient digestibility and growth performance of broilers fed corn basal diets, few specific enzyme mixtures included debranching enzymes or their transformative effects in vivo were not clear [20, 31, 34, 35].In the current study, improvements of growth performance in supplementation with EXF might be ascribed to the beneficial bioactivities of feruloylated XOS via prebiotic, immunomodulatory, and/or antioxidant effects [36].This discrepancy could be explained by the complex community interactions, with the multiplication of species modified by Lactobacillus after the EXF supplementation. Additionally, in order to further understand how specific ADE-induced changes of the gut microbiota modulate host health, spearman association analysis was used to expound potential impacts on the growth and development of broilers. The higher VH was significantly positively correlated with the diversity and abundance of ileal microbes (Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae), revealing the internal factors of intestinal morphology and microflora. Similarly, a previous study concluded that Lactobacillus enrichment was positively correlated with ileal VH and lactate concentration, Enterobacter, Staphylococcus, and Pseudomonas were negatively associated with intestinal development [20].Accordingly, it was speculated that the specific EXF might effectively connect corn AX, product XOS, microbial community, and physical health to playing prebiotic roles.

In summary, specific xylanase supplementations to corn basal diets were capable of improving the broilers' growth performance. Xylanase mixtures potentially advanced the conversion output of corn AX to XOS. Such improvements were necessarily associated with the positive shifts in the microbial community, intestinal morphology, ileal absorptive capacity, and intracecal fermentation. Therefore, it is recommended that debranching enzymes be used in future studies of fabricating prebiotic XOS in broiler basal diets.

NSP, nonstarch polysaccharides; AX, arabinoxylan; DDGS, corn distillers dried grains with solubles; EX, endo-xylanases; EA, arabinofuranosidase; EF, ferulic acid esterase; ADE, arabinoxylan-degrading enzymes; XOS, xylooligosaccharides; SCFAs, short-chain fatty acids; OTU, operational taxonomic units; PCoA, principal coordinates analysis; RDA, redundancy analysis; SEM, standard error of the mean.

Ethics approval and consent to participate

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was financially supported by the System for Poultry Production Technology, Beijing Agriculture Innovation Consortium (Project Number: BAIC04-2020). The funder had no roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions

WW: Data curation, Investigation, Project administration, Writing - original draft; HZ: Investigation, Resources, Software; YC: Investigation; YG: Conceptualization, Supervision; JY: Conceptualization, Methodology, Project administration, Supervision, Validation, Writing - review & editing. All authors read and approved the final manuscript.

Acknowledgments

The authors gratefully thank Fengliang Chen, Xintang Jing, and Fei Zheng (Bestzyme Bio-products, Co., Ltd.) for their diligent technical assistance. Furthermore, all authors are particularly grateful to Xiangli Sun, Hong Ma, and Chaoyi Chi colleagues (Ministry of Agriculture Feed Industry Center, Beijing) for their numerous guidance and suggestions in the application of chromatographic analysis.

Supporting Information/Additional Files

Fig. S1. Diagram of the proportion of specific ADE treatments in respective XOS components (DOCX).

Fig. S2. Ileal microbial community distribution heatmap at the genus level (top 35) (DOCX).

Fig. S3. Correlation of SCFAs components in hindgut of broiler chickens with multiple indicators (DOCX).

Fig. S4. Co-occurrence networks analysis of ileal microbiota at the genus level for EXF supplementation (DOCX).

Nordberg KE, Schmitz E, Linares-Pastén JA, Adlercreutz P. Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties. Appl Microbiol Biotechnol. 2018;102(21): 9081-8. https://doi.org/10.1007/s00253-018-9343-4.
Ward NE. Debranching enzymes in corn/soybean meal-based poultry feeds: A review. Poult Sci. 2021;100(2): 765-75. https://doi.org/10.1016/j.psj.2020.10.074.
Bautil A, Buyse J, Goos P, Bedford MR, Courtin CM. Feed endoxylanase type and dose affect arabinoxylan hydrolysis and fermentation in ageing broilers. Anim Nutr. 2021;7(3): 787-800. https://doi.org/10.1016/j.aninu.2020.11.015.
González-Ortiz G, Olukosi O, Bedford MR. Evaluation of the effect of different wheats and xylanase supplementation on performance, nutrient and energy utilisation in broiler chicks. Anim Nutr. 2016;2(3): 173-9. https://doi.org/10.1016/j.aninu.2016.06.005.
Lei Z, Shao Y, Yin X, Yin D, Guo Y, Yuan J. Combination of xylanase and debranching enzymes specific to wheat arabinoxylan improve the growth performance and gut health of broilers. J Agric Food Chem. 2016;64(24):4932-42. https://doi.org/10.1021/acs.jafc.6b01272.
Lei Z, Wu Y, Nie W, Yin D, Yin X, Guo Y, et al. Transcriptomic analysis of xylan oligosaccharide utilization systems in Pediococcus acidilactici strain BCC-1. J Agric Food Chem. 2018;66(18): 4725-33. https://doi.org/10.1021/acs.jafc.8b00210.
Wu Y, Lei Z, Wang Y, Yin D, Aggrey SE, Guo Y, et al. Metabolome and microbiota analysis reveals the conducive effect of Pediococcus acidilactici BCC-1 and xylan oligosaccharides on broiler chickens. Front Microbiol. 2021;12. https://doi.org/10.3389/fmicb.2021.683905.
Ravn JL, Glitsø V, Pettersson D, Ducatelle R, Van Immerseel F, Pedersen NR. Combined endo-β-1,4-xylanase and α-l-arabinofuranosidase increases butyrate concentration during broiler cecal fermentation of maize glucurono-arabinoxylan. Anim Feed Sci Technol. 2018;236:159-69. https://doi.org/10.1016/j.anifeedsci.2017.12.012.
Tu T, Li X, Meng K, Bai Y, Wang Y, Wang Z, et al. A GH51 α-l-arabinofuranosidase from Talaromyces leycettanus strain JCM12802 that selectively drives synergistic lignocellulose hydrolysis. Microb Cell Fact. 2019;18(1). https://doi.org/10.1186/s12934-019-1192-z.
Carlson J, Erickson J, Hess J, Gould T, Slavin J. Prebiotic dietary fiber and gut health: Comparing the in vitro fermentations of beta-glucan, inulin and xylooligosaccharide. Nutrients. 2017;9(12): 1361. https://doi.org/10.3390/nu9121361.
Holscher HD. Dietary fiber and prebiotics and the gastrointestinal microbiota. Gut Microbes. 2017;8(2): 172-84. https://doi.org/10.1080/19490976.2017.1290756.
Payling L, Fraser K, Loveday SM, Sims I, Roy N, McNabb W. The effects of carbohydrate structure on the composition and functionality of the human gut microbiota. Trends Food Sci Technol. 2020;97:233-48. https://doi.org/10.1016/j.tifs.2020.01.009.
Zhang Y, Jiang D, Jin Y, Jia H, Yang Y, Kim IH, et al. Glycine attenuates citrobacter rodentium-induced colitis by regulating ATF6-mediated endoplasmic reticulum stress in mice. Mol Nutr Food Res. 2021;65(15): e2001065. https://doi.org/10.1002/mnfr.202001065.
Morgan NK, Wallace A, Bedford MR, Hawking KL, Rodrigues I, Hilliar M, et al. In vitro versus in situ evaluation of xylan hydrolysis into xylo-oligosaccharides in broiler chicken gastrointestinal tract. Carbohydr Polym. 2020;230:115645. https://doi.org/10.1016/j.carbpol.2019.115645.
Wang W, Li Z, Lv Z, Zhang B, Lv H, Guo Y. Effects of Kluyveromyces marxianus supplementation on immune responses, intestinal structure and microbiota in broiler chickens. PLoS One. 2017;12(7): e180884. https://doi.org/10.1371/journal.pone.0180884.
Wu W, Xiao Z, An W, Dong Y, Zhang B. Dietary sodium butyrate improves intestinal development and function by modulating the microbial community in broilers. PLoS One. 2018;13(5): e197762. https://doi.org/10.1371/journal.pone.0197762.
Li C, Wang J, Zhang H, Wu S, Hui Q, Yang C, et al. Intestinal morphologic and microbiota responses to dietary Bacillus spp. in a broiler chicken model. Front Physiol. 2019;9. https://doi.org/10.3389/fphys.2018.01968.
Wang Y, Wu Y, Chen J, Guo X, Yan L, Guo Y, et al. The duration of food withdrawal affects the intestinal structure, nutrients absorption, and utilization in broiler chicken. FASEB J. 2021;35(1): e21178. https://doi.org/10.1096/fj.202001773R.
Wang Y, Heng C, Zhou X, Cao G, Jiang L, Wang J, et al. Supplemental Bacillus subtilis DSM 29784 and enzymes, alone or in combination, as alternatives for antibiotics to improve growth performance, digestive enzyme activity, anti-oxidative status, immune response and the intestinal barrier of broiler chickens. Br J Nutr. 2021;125(5): 494-507. https://doi.org/10.1017/S0007114520002755.
Wang J, Liu S, Ma J, Piao X. Changes in growth performance and ileal microbiota composition by xylanase supplementation in broilers fed wheat-based diets. Front Microbiol. 2021;12:706396. https://doi.org/10.3389/fmicb.2021.706396.
Choct M. Feed non-starch polysaccharides for monogastric animals: Classification and function. Anim Prod Sci. 2015;55(12): 1360. https://doi.org/10.1071/AN15276.
Agger J, Viksø-Nielsen A, Meyer AS. Enzymatic xylose release from pretreated corn bran arabinoxylan: Differential effects of deacetylation and deferuloylation on insoluble and soluble substrate fractions. J Agric Food Chem. 2010;58(10): 6141-8. https://doi.org/10.1021/jf100633f.
Lee SA, Apajalahti J, Vienola K, González-Ortiz G, Fontes CMGA, Bedford MR. Age and dietary xylanase supplementation affects ileal sugar residues and short chain fatty acid concentration in the ileum and caecum of broiler chickens. Anim Feed Sci Technol. 2017;234:29-42. https://doi.org/10.1016/j.anifeedsci.2017.07.017.
Bautil A, Verspreet J, Buyse J, Goos P, Bedford MR, Courtin CM. Arabinoxylan-oligosaccharides kick-start arabinoxylan digestion in the aging broiler. Poult Sci. 2020;99(5): 2555-65. https://doi.org/10.1016/j.psj.2019.12.041.
Santibáñez L, Henríquez C, Corro-Tejeda R, Bernal S, Armijo B, Salazar O. Xylooligosaccharides from lignocellulosic biomass: A comprehensive review. Carbohydr Polym. 2021;251:117118. https://doi.org/10.1016/j.carbpol.2020.117118.
Ribeiro T, Cardoso V, Ferreira LMA, Lordelo MMS, Coelho E, Moreira ASP, et al. Xylo-oligosaccharides display a prebiotic activity when used to supplement wheat or corn-based diets for broilers. Poult Sci. 2018;97(12): 4330-41. https://doi.org/10.3382/ps/pey336.
Singh AK, Mishra B, Bedford MR, Jha R. Effects of supplemental xylanase and xylooligosaccharides on production performance and gut health variables of broiler chickens. J Anim Sci Biotechnol. 2021;12(1). https://doi.org/10.1186/s40104-021-00617-8.
Jha R, Fouhse JM, Tiwari UP, Li L, Willing BP. Dietary fiber and intestinal health of monogastric animals. Front Vet Sci. 2019;6:48. https://doi.org/10.3389/fvets.2019.00048.
Singh AK, Kim WK. Effects of dietary fiber on nutrients utilization and gut health of poultry: A review of challenges and opportunities. Animals. 2021;11(1): 181. https://doi.org/10.3390/ani11010181.
Shin N, Whon TW, Bae J. Proteobacteria: Microbial signature of dysbiosis in gut microbiota. Trends Biotechnol. 2015;33(9): 496-503. https://doi.org/10.1016/j.tibtech.2015.06.011.
Singh AK, Mandal RK, Bedford MR, Jha R. Xylanase improves growth performance, enhances cecal short-chain fatty acids production, and increases the relative abundance of fiber fermenting cecal microbiota in broilers. Anim Feed Sci Technol. 2021;277:114956. https://doi.org/10.1016/j.anifeedsci.2021.114956.
Gao P, Ma C, Sun Z, Wang L, Huang S, Su X, et al. Feed-additive probiotics accelerate yet antibiotics delay intestinal microbiota maturation in broiler chicken. Microbiome. 2017;5(1). https://doi.org/10.1186/s40168-017-0315-1.
Adebowale TO, Yao K, Oso AO. Major cereal carbohydrates in relation to intestinal health of monogastric animals: A review. Anim Nutr. 2019;5(4): 331-9. https://doi.org/10.1016/j.aninu.2019.09.001.
González-Ortiz G, Dos Santos TT, Bedford MR. Evaluation of xylanase and a fermentable xylo-oligosaccharide on performance and ileal digestibility of broiler chickens fed energy and amino acid deficient diets. Anim Nutr. 2021;7(2): 488-95. https://doi.org/10.1016/j.aninu.2020.07.008.
Rabello CBV, Costa MJ, Nogueira WCL, Barbosa JG, Rios-Alva JC, Wyatt CL, et al. Effects of graded levels of exogenous xylanase in corn-soy diets with two amino acid density and fat levels postpellet in broiler chickens: Live performance, energy utilization, digestibility, and carcass characteristics. Poult Sci. 2021;100(2): 820-34. https://doi.org/10.1016/j.psj.2020.10.046.
Lin S, Agger JW, Wilkens C, Meyer AS. Feruloylated arabinoxylan and oligosaccharides: Chemistry, nutritional functions, and options for enzymatic modification. Annu Rev Food Sci Technol. 2021;12(1): 331-54. https://doi.org/10.1146/annurev-food-032818-121443.

Table 1. Composition and nutrient levels of corn basal diet (as-fed basis)

items, % unless noted	d 1 to 21
ingredients
corn (7.8%, crude protein)	51.50
soybean meal (46%, crude protein)	30.58
DDGS (28%, crude protein)	5.00
corn protein flour (63.5%, crude protein)	4.00
wheat flour	2.00
soybean oil	2.40
limestone	1.27
sodium chloride	0.35
dicalcium phosphate	1.94
choline chloride (50%)	0.20
DL-methionine (98%)	0.24
L-lysine·HCl (99%)	0.26
antioxidant	0.02
multivitamin ^a	0.03
multimineral ^b	0.20
phytase 10,000	0.01
total	100.00
calculated nutrient levels
metabolic energy, Mcal/kg	2.95
crude protein	22.5
available phosphorus	0.45
calcium	1.00
lysine	1.30
methionine	0.59
threonine	0.85
tryptophan	0.25

^aSupplied per kilogram of diet: retinyl acetate, 9500 IU; cholecalciferol, 2500 IU; α-tocopherol acetate, 30 IU; menadione, 2.65 mg; thiamin, 2 mg; riboflavin, 6 mg; cyanocobalamin, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; and niacin, 50 mg.

^bSupplied per kilogram of diet: copper, 8 mg; zinc, 75 mg; iron, 80 mg; manganese, 100 mg; selenium, 0.15 mg; and iodine, 0.35 mg.

Table 2. Treatments with specific arabinoxylan-degrading enzymes and relevant inclusion levels in animal trial ^a

treatments	CTL	EX	EXA	EXF	XAF-1	XAF-2	XAF-3	XAF-4
EX	0	54 (600)	54 (600)	54 (600)	54 (600)	63 (700)	63 (700)	54 (600)
EA	0	0	5 (500)	0	5 (500)	6 (600)	4 (400)	6.7 (668)
EF	0	0	0	0.4 (400)	0.4 (400)	0.5 (500)	0.5 (500)	0.4 (400)

^aTreatments: CTL, a basal diet (control); EX, the basal diet supplemented with core xylanase; EXA, a basal diet supplemented with xylanase accompanied by EA; EXF, a basal diet supplemented with xylanase accompanied by EF; XAF-i (i=1-4), compound groups with the above three enzymes. The same as below, also involving all Figures. Bold values in parentheses were additive dosages (mg/kg) in this animal experiment, while the values outside brackets were the appropriate inclusion levels (U/g) obtained from the in vitro screening process.

Table 3. Effects of specific arabinoxylan-degrading enzymes on growth performance of broilers ^a

items	BWG, g	FI, g	F/G, g/g	EPI	MRT, %
phase 1 (d 14 to 21)
CTL	466.53^b	632.27	1.355	929.70	0.00
EX	469.45^ab	633.11	1.371	932.16	1.39
EXA	456.82^b	637.35	1.374	907.06	0.00
EXF	486.15^a	644.08	1.327	988.55	0.00
XAF-1	462.57^b	630.08	1.353	931.74	2.78
XAF-2	467.67^b	631.73	1.326	940.83	1.39
XAF-3	465.08^b	629.63	1.354	915.86	1.39
XAF-4	473.08^ab	627.56	1.327	928.83	2.78
SEM	2.192	3.539	0.006	6.920	0.554
P-value	0.044	0.971	0.137	0.136	0.824
phase 2 (d 5 to 21)
CTL	789.08	1045.78	1.325	391.36	0.00
EX	799.65	1065.37	1.332	389.20	1.39
EXA	796.05	1057.27	1.328	387.52	1.39
EXF	831.80	1076.85	1.305	420.42	0.00
XAF-1	795.80	1058.14	1.339	387.21	4.17
XAF-2	795.35	1049.67	1.323	396.60	1.39
XAF-3	804.93	1063.23	1.323	400.86	1.39
XAF-4	796.18	1054.51	1.325	400.50	2.78
SEM	4.753	6.150	0.003	2.857	0.590
P-value	0.478	0.958	0.065	0.055	0.717

^a Values were given as the means based on six birds for each treatment (n = 6). The abbreviations of groups were consistent with those in Table 2. BWG, body weight gain (g); FI, feed intake (g); F/G, feed to gain ratio (g/g); EPI, European comprehensive production index; MRT, mortality (%). SEM, standard error of the mean. ^a,b Means in a column with superscripts without a common letter differ, P < 0.05.

Download PDF

Journal Publication

published 09 Mar, 2023

Read the published version in Journal of Animal Science and Biotechnology →

Editorial decision: Major revision
21 Sep, 2022
Reviewers agreed at journal
31 Aug, 2022
Reviewers invited by journal
29 Aug, 2022
Editor assigned by journal
24 Aug, 2022
First submitted to journal
23 Aug, 2022

You are reading this latest preprint version

Debranching enzymes decomposed corn arabinoxylan into xylooligosaccharides and achieved a prebiotic regulation of gut microbiota in broilers

Status:

Journal Publication

Version 1

Abstract

BACKGROUND

RESULTS

CONCLUSIONS

Figures

1. Introduction

2. Materials And Methods

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1