INTRODUCTION
Despite the usage of cereal bran as a feedstuff, the existence of high level of arabinoxylan (Abx) may limit its application in animal diets [
1], because Abx is resistant to digestion by endogenous digestive enzymes. More importantly, Abx can cause viscous digesta with subsequent intestinal disorders such as reduced digestibility of nutrients, increased pathogen load, gut leakage, and inflammation [
2,
3]. Thereby, Abx is considered as a critical anti-nutritional factor for monogastric animals especially for those at young age (e.g., piglets) [
2]. These raise a necessity to explore strategies to efficiently degrade Abx in cereal bran that may benefit its application in animal diets.
It is known that Abx represents a typical member of non-starch polysaccharides in multiple plant ingredients, comprising a linear backbone of β-D-(1,4)-xylopyranosyl units onto which L-arabinose units are linked as the side chains [
4]. Generally, arabinose residues that are attached to the C-2, C-3, or both positions of Abx backbone can be further substituted in the O-5 position by ferulic acid ester groups [
4]. Because of the complex molecular structure, the efficient degradation of dietary Abx may demand a combination of depolymerizing enzymes such as endo-xylanase (Xyn) with debranching enzymes such as arabinofuranosidase (Afd), and feruloyl esterase (FE) [
5,
6]. Thereinto, Xyn stochastically cleaves the β-1,4-glycosidic bonds within Abx backbone and produces smaller polysaccharides or oligosaccharides [
5], which can be used as prebiotics to improve intestinal health by targeting host gut microbiota [
2]. However, the presence of side chains enhances the degradation resistance of Abx backbone via impeding recognition of cleavage sites in the backbone by Xyn [
6,
7]. This effect may be overwhelmed by the action of debranching enzymes, among which Afd removes the arabinose residues to expose more cleavage sites and provide a convenience for Xyn action [
6,
7], while FE breaks ferulic acid ester bonds cross-linked to arabinose residues to liberate ferulic acid [
8]. These actions aid in simplifying the molecular structure of Abx and enhancing Xyn accessibility to produce reducing sugars [
8–
10]. Accordingly, there can be an efficient degradation of Abx in plant-sourced ingredients under the synergy among Xyn, Afd, and FE, which may in turn diminish the viscosity of intestinal content, thus promoting a sufficient contact between digesta and digestive enzymes with a resultant benefit on nutrient digestion [
2,
3]. In addition, combination of Xyn, Afd, and FE may prompt plant cell wall fragmentation by efficiently degrading Abx and cleaving the ferulic acid ester bonds linking feruloyl residues in Abx with other cell wall components (e.g., cellulose), presumptively favoring the access of digestive enzymes to the cell wall or intracellular components [
8–
10]. Thereby, combination of Xyn, Afd, and FE might be superior to Xyn alone for enhancing digestion of cereal bran with subsequent promotions of growth and health of animals ingesting a bran-containing diet. Although there were numerous studies revealing the positive effects of Xyn and its combination with Afd on chicken growth and health performance [
11,
12], there were few studies regarding the combined application of Xyn, Afd, and FE in pig diets.
Comprehensively, we raised the hypothesis that combination of Xyn, Afd, and FE was more favorable to promote degradation of Abx with a destruction of cell wall of cereal bran, possibly ameliorating the application effect of cereal bran. To test this hypothesis, the current study was conducted to investigate the effects of a combination of Xyn, Afd, and FE on degradation of Abx-riched cereal bran as well as growth and health of piglets received bran-containing diet.
DISCUSSION
A study on the enzymological characteristics of Xyn, Afd, and FE can favor the understanding of their application potentials in animal diet. A previous study by Wang et al [
13] indicated that over 70% of Xyn activity was kept between pH 3.0 and 10.0, while FE retained more than 80% of the activity at the pH range of 2.0 to 12.0, besides, both Xyn and FE were stable from 4°C to 40°C and drastically reduced its activities at temperature higher than 50°C. Similarly, this study revealed that both Xyn, Afd, and FE could maintain relatively high activities (more than 50% relative activity) within wide ranges of pH value (from 3.0 to 10.0) and temperature (from 30°C to 85°C). Moreover, Xyn, Afd, and FE particularly the first two had a relatively high thermal endurance and acid resistance, which could enable a feasibility of these enzymes applied in animal diets to withstand the hostile conditions during diet processing and gastrointestinal digestion.
The present result showed that Xyn treatment alone sharply increased the RRS from destarched wheat bran, hinting that Xyn exerted a leading role among combination of xylanolytic enzymes in degradation of Abx that subsequently released reducing sugars [
5]. However, the existence of arabinose groups attached to Abx backbone could reduce the depolymerizing effect of Xyn on Abx, by hampering the recognition of cleavage sites within the backbone by Xyn [
6,
7]. But this steric hindrance may be alleviated by the usage of Afd that removes arabinose groups from side chains, thus enabling further action of Xyn on Abx [
5–
7]. In support of this view, we noted that combination of Xyn and Afd at various ratios (from 3,000:1 to 100:1) resulted in a more pronounced RRS from destarched wheat bran relative to Xyn acting alone, implying a cooperation between Xyn and Afd on degradation of Abx in destarched wheat bran [
21]. Intriguingly, there was almost no elevation in the RRS with the increased proportion of Afd dose. Based on the overall consideration of efficacy and cost, the combination of Xyn and Afd at a ratio of 3,000:1 (3 U/g Xyn + 0.001 U/g Afd) seemed to be suitable. Thereby, this combination scheme of Xyn and Afd was used for further analysis.
The α-L-arabinofuranosyl residues linked in Abx backbone can further be substituted by ferulic acid ester [
4,
5], which acts as another essential side chain group of Abx to impede the depolymerization of Xyn, therefore retarding Abx degradation [
6,
10]. But this steric hindrance effect can be attenuated by the action of FE capable of hydrolyzing ferulic acid ester bonds attached to Abx side chains [
7–
9]. It has been found that the RRS from Abx-riched crop products under the synergy between Xyn and FE was higher than that under Xyn acting alone [
13]. Similarly, this study demonstrated an advantage of combination of Xyn and FE at varying ratios (from 3,000:1 to 100:1) over Xyn alone in promoting the RRS from destarched wheat bran, which might emphasize a cooperation between Xyn and FE on degradation of Abx. Due to generation of the numerically highest RRS from destarched wheat bran, the combination of Xyn (3 U/g) and FE (0.006 U/g) at a ratio of 500:1 was selected for further analysis.
To validate the synergy among Xyn, Afd, and FE at selected doses (3, 0.001, and 0.006 U/g, respectively), SF and IF of wheat bran and oat bran were employed as different Abx sources. Remarkably, the present study revealed that combining Xyn with Afd had a superiority over Xyn alone to increase the RRS from all bran fibers, while combining Xyn with FE increased the RRS from SF and IF of oat bran instead of wheat bran. The difference between the degrading actions on oat bran fiber and wheat bran fiber might be related to their different level of ferulic acid ester that can be targeted by FE [
22]. Although Abx contains a linear backbone substituted with various degrees of side chains including arabinose and ferulic acid [
4,
5], arabinose seems to be the main group linked as the side chain of Abx in most cereal brans [
4]. It was thus deduced that Afd elicited a more important role than FE in removing the side chains of Abx, thus being more beneficial for the depolymerizing action of Xyn on Abx in brans. This might account for the current findings: i) combining Xyn with Afd caused a higher RRS from Abx sources (especially wheat bran fiber) relative to combination of Xyn and FE; ii) the synergy degree between Xyn and Afd was obviously greater than that between Xyn and FE. Strikingly, integration of Xyn, Afd, and FE increased the RRS from all bran fibers relative to combining Xyn with Afd or FE, which corresponded to the observed higher synergy degree among these three enzymes than that between Xyn and Afd or between Xyn and FE, highlighting that combining Xyn, Afd, and FE had an advantage over combining Xyn with Afd or FE in accelerating degradation of Abx in brans. This was most likely due to that cooperation of debranching enzymes (Afd and FE) was more efficient than their single action in eliminating the spatial obstacles (branch points) of Abx [
5–
7], thereby favoring the access of Xyn to Abx backbone and its depolymerization.
Treatment with Xyn has been confirmed to be effective in promoting digestion of dietary fiber in animals [
23,
24], nevertheless, few studies are available concerning the influence of Xyn in combination with debranching enzymes on nutrient digestibility of piglet diet. In the current study, combination of Xyn, Afd, and FE or Xyn alone tended to improve the
in vitro digestibility of dry matter, crude protein, crude ash, and gross energy of piglet diet containing bran, with numerically higher digestibility of these nutrients induced by combined enzyme treatment relative to Xyn treatment alone. It seems that the efficient degradation of Abx under the observed synergy among Xyn, Afd, and FE potentially translated into a benefit for cell wall destruction of bran, which facilitated the access of digestive enzymes to the cell wall, intracellular or both constituents of bran [
8–
10], thus assisting with nutrient digestion of diet. Alternatively, the degradation of Abx in bran-containing diet under the action of combination of Xyn, Afd, and FE or of Xyn alone profited the reduction of digesta viscosity, allowing a sufficient contact between digesta and digestive enzymes [
2,
3]. This might be also partially responsible for the observed increase in
in vitro nutrients = of diet exposed to enzyme treatments.
Previous studies evidenced a variable efficacy of Xyn treatment alone in enhancing pig growth performance [
23,
24]. We herein observed that combination of Xyn, Afd, and FE was more beneficial than Xyn alone to induce increasing trends of FBW, ADG, and G/F of piglets fed bran-containing diet. This was likely related to the observed increasing trend of nutrient digestibility of bran-containing diet treated with enzymes especially combination of Xyn, Afd, and FE. Alternatively, the potential production of xylooligosaccharides induced by Xyn or its combination with Afd and FE modulated gut microbiota [
2], which possibly contributed to the improvements of piglet performance. Regarding the diarrhea rate of piglets, it was found to be reduced by the treatment with either Xyn alone or combination of Xyn, Afd, and FE, with a lower diarrhea rate found in combined treatment group versus Xyn alone group. It was probable that the elevated nutrient digestibility of diet induced by Xyn alone or combination of Xyn, Afd, and FE could reduce the nutrient residuals utilized by certain pathogens in hindgut, thereby decreasing diarrhea prevalence of piglets [
25,
26]. Besides, the more efficient degradation of dietary Abx under the synergy among Xyn, Afd, and FE might be more favorable to lower the viscosity of chyme, which was hypothesized to avoid overgrowth of intestinal harmful bacteria and subsequently reduce diarrhea rate of piglets [
2,
27].
Bacteria-produced VFA via fermentation of dietary fibers were characterized as a typical improver of gut health of pigs [
20,
28]. It was suggested that dietary Xyn treatment modulated fermentation pattern of gut microbiota and increased the concentrations of certain VFA in gut of pigs [
23,
24]. The present study indicated that combination of Xyn, Afd, and FE or Xyn alone could increase the concentrations of acetic acid and total VFA in both cecum and colon coupled with cecal butyric acid concentration of piglets fed bran-containing diet, with higher concentrations of these parameters observed in combined treatment group versus Xyn treatment alone. Moreover, an elevation was recorded in cecal and colonic propionic acid concentration of piglets due to the treatment with combination of Xyn, Afd, and FE. These results revealed a distinct advantage of combination of Xyn, Afd, and FE over Xyn alone in improving intestinal VFA profile, which might be due to that the efficient degradation of dietary Abx into oligoscaccharides under the synergy among Xyn, Afd, and FE could improve gut fermentation by several beneficial bacteria (namely the prebiotic effect) [
2,
24]. The elevated concentrations of certain VFA in gut following combined treatment with Xyn, Afd, and FE were postulated to translate into a lower pH value of gut. Herein, although treatment with Xyn alone had little influence on intestinal pH value, we indeed detected a reduction of cecal pH value together with a decreasing trend of colonic pH value in piglets treated with combination of Xyn, Afd, and FE. These results basically coincided to the simultaneous increases in cecal and colonic concentrations of VFA of piglets treated with combination of Xyn, Afd, and FE. It could be speculated that the increased concentrations of VFA coupled with the resulting decrease of pH value in gut due to combined treatment with Xyn, Afd, and FE were at least partially responsible for the observed reduction of diarrhea rate of piglets, since acid microenvironment in gut could resist pathogen invasion as well as boost gut health and growth performance of piglets [
29,
30].