Combination of Xylanase and Debranching ... - ACS Publications

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Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers Zhao Lei, Yuxin Shao, Xiaonan Yin, Dafei Yin, Yuming Guo, and Jianmin Yuan* State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, PR China S Supporting Information *

ABSTRACT: Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers. KEYWORDS: xylanase, debranching enzymes, synergistic effect, xylan oligosaccharides, broilers, growth performance, gut health

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arabinofuranose residues,9 and FAE catalyzes the phenolic acids cross-linked to arabinose residues of the arabinoxylan.10 The synergistic action of ABF and xylanase has been verified in studies of WE-AX11 and WU-AX.12 Therefore, similar activity could be utilized for the production of AXOS and XOS.13 The cleavage of the ferulic acid ester bond by FAE has been shown to improve the biodegradation of corn stover, and the synergistic effect with endoxylanase has also been confirmed in studies of WU-AX.14 The hydrolyzates of AX are XOS and AXOS, which can be used as prebiotics. Their prebiotic effects have been proven with in vitro15 and in vivo experiments,16 which show selectively enhanced probiotic bacterial proliferation of Lactobacillus and Bifidobacterium, respectively. Compared to FOS, XOS inhibits colonic DNA damage that is induced by protein fermentation, and it modulates the genotoxicity of the colonic environment.17 Moreover, the AXOS and XOS fermentation products were unbranched shortchain fatty acids (USCFA), especially butyrate, which plays a key role in intestinal enterocyte proliferation and gut health.18 Xylanase supplementation is thought to be an effective way to alleviate poor gut health induced by AX, and it might improve the growth performance of broilers fed a wheat-based diet with the strict use of antibiotics in the livestock industry. The prebiotic potential of AXOS and XOS has been the focus of research in recent years. To the best of our knowledge, there is limited information on the synergistic effects of endoxylanase in combination with both ABF and FAE in the production of

INTRODUCTION Arabinoxylan (AX) is the major antinutritional factor of wheat for monogastric animals, especially broilers. AX cannot be digested in the small intestine and generates viscous chyme in the gut, which leads to gut health problems, such as the proliferation of pathogenic bacteria,1 intestinal inflammation,2 impaired intestinal barrier function, and severe intestine lesions.3 Therefore, the ingestion of AX leads to poor growth performance.4 Xylanase was thought to be a candidate for antibiotic replacement to degrade AX in commercial wheatbased diets for broilers.5 Thus, the facile approach by the addition of xylanase could dissolve the antinutritional problem for broilers. Arabinoxylan consists of a linear backbone of β-(1-4)xylopyranosyl units that are substituted with various degrees of short side chains, including L-arabinose, ferulic acid, and Oacetyl.6 For most cereals, the main substitution structure is the 7 L-arabinose monomer. Arabinoxylan can be divided into waterextractable (WE-AX) and water-unextractable (WU-AX) fractions. The predominant fraction of AX in most cereals is WU-AX, which results from covalent cross-links (ferulic acid and p-coumaric acid), and noncovalent interactions with other components.7 The complete enzymatic degradation of AX requires the cooperative action of debranching enzymes (arabinofuranosidase [ABF], feruloyl esterase [FAE], acetyl esterase [AE], and α-L-glucuronidase) and backbone-degrading enzymes (endo1,4-L-xylanase and β-xylosidase) because of its complex substitution structure.8 Endo-1,4-L-xylanases randomly attack the β-1,4-glycosidic bonds within the backbone and generate AXOS or XOS, whereas β-xylosidases attack the nonreducing ends of XOS to release xylose.8 ABF targets the α-linked L© XXXX American Chemical Society

Received: March 21, 2016 Revised: June 2, 2016 Accepted: June 2, 2016

A

DOI: 10.1021/acs.jafc.6b01272 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Synergistic Interaction among Xylanase, ABF, and FAE. The synergistic activity among xylanase, ABF, and FAE was determined by comparing the simultaneous reactions of enzyme combinations and the sequential reactions of individual debranching enzymes, or their combinations, to those of xylanase alone, as described by Raweesri et al.9 The hydrolysis reaction lasted 36 h. Analysis of Hydrolyzed Arabinoxylan Products and Scanning Electron Microscopy. The enzymatic hydrolysis products of the supernatants were analyzed using an ICS-3000 high-performance anion exchange chromatography system (Dionex Cooperation, Sunnyvale, CA) with pulsed amperometric detection (HPAEC-PAD) using a Carbo-Pac PA200 column (3 × 250 mm as described by Zheng et al.20 Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were used as external standards. The hydrolyzates of destarched wheat bran from different treatments were air-dried and placed on stubs mounted with silver tape and sputter coated with gold. The surface morphology was examined to confirm the enzymatic degradation effect on destarched wheat bran using a scanning electron microscope (SEM) (JSM 7500F, JEOL). Images were taken at a magnification of 500× and at a voltage of 5 kV. Bacterial Strains and Fermentation Experiments. The bacterial strains used to test the fermentability potential of hydrolyzed arabinoxylan were B. subtilis (GIMI.372) and L. brevis (GIMI.730), which were purchased from GIMCC (Microbial Culture Collection Center of the Guangdong Institute of Microbiology, Guangdong, China). Both strains were precultivated twice using MRS broth (in which the carbon source is glucose). The experimental group used enzymatic hydrolyzed WE-AX and WU-AX as carbon sources, in contrast to the control group, which used glucose. Before cultivation, the sterile hydrolyzed products were added to the medium, prepared without a carbon source, at a concentration of 10 mg/mL on the basis of total sugar or hydrolyzed products as described by Falck et al.15 The inoculum was 1% (v/v) for both strains. Anaerobic and aerobic cultivation of L. brevis and B. subtilis were done at 37 °C for 48 and 24 h, respectively. Following fermentation, samples were collected for analysis of growth, pH, short-chain fatty acid (SCFA), and enzyme activities. Cell density was used as a measure of bacterial growth and was determined by measuring the optical density (OD) at 600 nm. Chyme from the cecum was collected under anaerobic conditions from six 21 day old Arbor Acre (AA) male broilers fed a corn-soybean-based diet free of antibiotics to investigate the fermentation properties of AX and XOS in vitro. The chyme was immediately placed in a prewarmed CO2-filled container for transfer to the laboratory, followed by a 1:5 (w/v) dilution with prewarmed sterile saline under anaerobic conditions. The samples were then homogenized using magnetic stirrers for 60 s and strained through a double layer of cheesecloth. The fluid was continually flushed with CO2 and stirred. The inoculum was then dispensed into a prewarmed bottle containing the reaction substrate and medium as described by Hughes et al.23 Each bottle consisted of 0.5 g of substrate (WE-AX, WU-AX, and XOS hydrolyzates), 5 mL of inoculum, and 45 mL of media. All additions were made under a stream of CO2, and the bottles were capped with an aluminum-sealed rubber stopper and kept in a water bath at 39 °C for 48 h. Blank bottles were filled with 5 mL of inoculum and 45 mL of media. After fermentation, the broth was

XOS, and data associated with the effects of their application in the feed industry are particularly scarce. This study investigated the synergistic effects of ABF and FAE, in combination with xylanase, on the production of XOS as a carbon source for probiotics in vitro. Furthermore, their effectiveness in vivo as feed enzymes used to improve the gut health and growth performance of broilers fed a wheat-based diet was also examined. These results may provide new approaches for XOS production, which could improve the use of wheat for broilers in the livestock industry via the utilization of novel complex feeding enzymes.

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MATERIALS AND METHODS Ethics Statement. The animal experimental procedures used were approved by the China Agriculture University Animal Care and Use Committee in Beijing, China. Materials. Xylose (X1500, 99% purity, Mw = 150.13), arabinose (E003256, 99% purity, Mw = 150.13), p-nitrophenylα-L-arabinofuranoside, xylan from beech wood (X4252, 90% purity), acetic acid, propionic acid, isobutyric acid, butyrate, and isovaleric acid were purchased from Sigma-Aldrich (Shanghai, China). Methyl ferulate was from Alfa Aesar (Beijing, China). Xylobiose (90% purity, Mw = 282.2), xylotriose (95% purity, Mw = 414.4), xylotetraose (95% purity, Mw = 546.5), xylopentaose (95% purity, Mw = 678.6), xylohexaose (95% purity, Mw = 810.7), high-viscosity water-soluble wheat arabinoxylan (P-WAXYH, 95% purity, avDAS = 0.58, Mw = 370 Kd), water-insoluble wheat arabinoxylan (P-WAXYI, 80% purity, avDAS = 0.71), and feruloyl esterase (EC 3.1.1.73, Clostridium thermocellum) were purchased from Megazyme (Bray, County Wicklow, Ireland). Endo-Xylanase (EC 3.2.1.8, Neocallimastix patriciarum) was purchased from Asiapac (Dongguan, China), and arabinofuranosidase (EC 3.2.1.55, Bacillus pumilus) was provided by Professor W. Shao of the Research Center for Biotechnology and Biomass Energy (Nanjing, China). Destarched wheat bran was prepared according to the method described by Mukherjee et al.19 All other chemicals and solvents used in this study were of analytical grade. Enzyme-Activity Assays. Endoxylanase activity was assayed in the reaction mixtures containing 1 mL of 0.1 M phosphate sodium citrate buffer (pH 5.5), 100 μL of appropriately diluted enzyme, and 0.4 mL of 0.5% (w/v) beech-wood xylan.20 The reaction mixtures were incubated for 10 min at 40 °C, and the reducing sugar was determined by the dinitrosalicylic acid (DNS) method. Arabinofuranosidase activity was assayed by using p-nitrophenyl α-L-arabinofuranosidase as a substrate, as described by Shi et al.21 The reaction mixtures contained 250 μL of 2 mM substrate, 150 μL of 1 mL 0.1 M phosphate sodium citrate buffer (pH 5.5), and 100 μL of appropriately diluted enzyme. The reaction mixtures were incubated for 10 min at 40 °C and terminated by adding 1.5 mL of 1 M sodium carbonate. Feruloyl esterase activity was measured by using methyl ferulate (MFA) as a substrate, as described by Yue et al.22 The reaction mixtures contained 2 mL of diluted enzyme solution and 4 mL of 100 μM MFA in 0.1 M 3-(N-morpholino) propane sulfonic buffer (pH 6.0) and were incubated at 40 °C for 15 min. A single unit of xylanase, α-Larabinofuranosidase, or feruloyl esterase was defined as the amount of enzyme released (1 μmol of xylose, p-nitrophenyl, or ferulic acid per min under standard conditions, respectively). Each reaction was performed in triplicate. B


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of BW), and jugular exsanguination. The ileal section between the terminal ileum and Meckel’s diverticulum was dissected and removed. Digesta from the jejunum was collected for analysis of viscosity. Chyme from the distal part of the ileum and cecum were collected immediately for pH measurement. On day 36, 16 birds per treatment were examined for intestinal lesion scores according to the method described by Liu et al.25 Viscosity of the jejunal digesta was determined using an Obarma’s viscosimeter. The digesta were centrifuged at 12000g for 5 min, the supernatant was withdrawn, and the viscosity of a 5 mL aliquot was measured. Distilled water was used as a control. Statistical Analysis. Individual treatment means were compared using Duncan’s multiple comparison when a significant difference was observed between treatments using SPSS Version 18.0. A value of P < 0.05 was considered statistically significant. Results are given as the mean ± standard error of the mean.

centrifuged, and enzyme activity and SCFA content in the supernatant were analyzed. Chemical Analyses of SCFA. SCFA were analyzed by gas chromatography GC-17A (Shimadzu, Kyoto, Japan) with a flame ionization detector (FID) fitted with a DB-FFAP column (30 m × 0.53 mm) (J&W Scientific). Procedures were described by Williams et al.24 Acetic, propionic, isobutyric, butyric, and isovaleric acids were used as standards. Feeding-Experiment Design and Bird Management. A total of 480 1 day old AA male broilers were assigned to five treatments with eight replicates of 12 birds each. The control group (CT) was fed an entirely wheat-based diet without any addition of enzymes. The experimental groups are as follows: Treatment X (CT plus xylanase at 2500 u·kg−1); Treatment X + A (X plus ABF at 800 u·kg−1); Treatment X + F (X plus FAE at 60 u·kg−1); and Treatment X + A + F (X plus ABF and FAE). The feeding period lasted 36 d. The chickens had free access to water and feed formulated to meet or exceed the established Nutrient Requirements of Poultry (NRC, 1994) requirement without the addition of antibiotics. The composition of the diet and nutritional levels are presented in Table 1. Feed consumption and body weight

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RESULTS Synergistic Actions among Xylanase, ABF, and FAE in Xylan Hydrolysis. A moderate degree of synergy (DS) was observed for both simultaneous reactions and sequential reactions on WE-AX, WU-AX, and xylan from beech wood (Table 2). In both cases, the DS was smaller in simultaneous reactions than in sequential reactions for ABF and xylanase. A lower DS was found between FAE and xylanase on WE-AX in either the simultaneous and sequential reactions. A higher DS was found with the combination of ABF and FAE as the first enzyme and xylanase as the second enzyme (after the heat inactivation of first enzyme) on WE-AX, beech-wood xylan, and WU-AX. This was especially true for the latter case, in which the highest DS was observed (DS = 1.72). Analysis of AX Hydrolyzates. In accordance with the practical use of feeding enzymes, which are usually added directly to the feed, only the hydrolyzates from the simultaneous reactions were measured by HPAEC-PAD. Xylobiose and xylotriose were the main hydrolysis products in all treatments (Table 3). The hydrolysis action of xylanase on WU-AX was weaker than that observed on WE-AX. The addition of either ABF or FAE, in combination with xylanase, accelerated hydrolysis of WE-AX and WU-AX, and this increased the production of XOS (xylobiose, xylotriose, and xylotetraose) as compared to the use of xylanase alone. The highest production of oligosaccharides was obtained when ABF and AFE acted simultaneously on WE-AX and WU-AX. Scanning Electron Microscopy of Wheat Bran from Different Treatments. The SEM images of native and hydrolyzed destarched wheat bran are shown in Figure 1a. Free destarched wheat bran showed a smooth flat surface, and a faveolate structure was observed after xylanase treatment. A more obvious and remarkable honeycomb form was observed with further supplementation of ABF, FAE, or both in combination with xylanase as compared to the sole use of xylanase. Bacterial Growth on AX and Hydrolyzates in Vitro. Results from the in vitro fermentation experiments showed that untreated WE-AX (Table 4) and WU-AX (Table 5) could not be fermented by either of the two strains. AX hydrolyzates, but not hydrolyzates from WU-AX on B. subtilis, could be efficiently used by B. subtilis and L. brevis, as shown by an increase in the OD, and an increase or decrease in pH, respectively. Hydrolyzates from WE-AX increased the growth of both

Table 1. Composition and Nutrient Levels of Diets ingredient (%)

1−21 d

21−35 d

wheat soybean meal soybean oil dicalcium phosphate limestone sodium chloride choline chloride (50%) L-Lys HCl trace minerala DL-met ethoxyquin vitamin premixb total calculated composition ME, Mcal/kg CP4, % available P, % Ca, % Lys, % Met + Cys, % Thr, %

64.53 28.52 2.89 1.97 0.93 0.35 0.25 0.17 0.20 0.14 0.03 0.03 100.00

68.50 23.72 4.00 1.63 0.96 0.35 0.25 0.24 0.20 0.11 0.03 0.03 100.00

2.90 21.50 0.45 1.00 1.10 0.93 0.82

2.99 19.95 0.40 0.90 1.00 0.79 0.74

a

The trace minerals supplied the following per kilogram of complete feed: copper, 8 mg; zinc, 75 mg; iron, 80 mg; manganese, 100 mg; selenium, 0.15 mg; and iodine, 0.35 mg. bThe vitamin premix supplied the following per kilogram of complete feed: vitamin A, 12 500 IU; vitamin D3, 2500 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; vitamin B12, 0.025 mg; vitamin E, 30 IU; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; and niacin, 50 mg.

of each replicate were measured on d 21 and 36. Average daily gain (ADG), average daily feed intake (ADFI), and feedconversion ratio (FCR) were calculated during d 1−21 and d 1−36. The diet composition is shown in Table 1. Intestinal Lesion Scores, Chyme pH, and Viscosity Measurements. On days 21 and 36, two birds per replicate (16 birds per treatment) were randomly selected and killed by intracardiac administration of sodium pentobarbital (30 mg/kg C


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Journal of Agricultural and Food Chemistry Table 2. Synergistic Action among Xylanase, ABF, and FAE on Xylans WE-AX

WU-AX

reducing sugara (μg/ mL)

treatment first reaction X

control reaction 1 simultaneous reaction 2 3 4 sequential reaction 5 6 7 8 9 10

second reaction −

DSb

2001.44 ± 3.01

1

beech-wood xylan

reducing sugar (μg/ mL)

DS

781.55 ± 4.86

X+A X+F X + A + Fc

− − −

2281.72 ± 12.45 2035 ± 23.69 2361.04 ± 13.91

1.14 1.02 1.18

830.53 ± 7.06 917.55 ± 14.86 1038.16 ± 28.52

X A X F X A+F

A X F X A+F X

2301 ± 13.89 2327.95 ± 13.87 2061.48 ± 14.09 2072.48 ± 15.42 2486.88 ± 6.23 2497.28 ± 15.65

1.15 1.16 1.03 1.04 1.24 1.25

950.58 1070.53 900.28 860.15 1266.03 1342.20

± ± ± ± ± ±

reducing sugar (μg/ mL)

34.66 38.60 34.86 11.33 42.76 27.21

DS

1

1353.81 ± 10.40

1

1.06 1.17 1.33

1457.55 ± 17.87 1557.81 ± 17.60 1590.48 ± 69.87

1.08 1.15 1.18

1.22 1.37 1.15 1.10 1.62 1.72

1549.81 1715.41 1671.41 1769.28 1666.88 1840.75

± ± ± ± ± ±

24.00 21.60 17.60 21.60 63.20 84.54

1.14 1.27 1.24 1.31 1.23 1.36

a This value was calculated by taking the amount of liberated reducing sugar in supernatant. bThe degree of synergy values were calculated as the activity divided by the sum of the activities of the control reactions. cX, xylanase; A, ABF; and F, FAE.

Table 3. Hydrolyzatesa of WE-AX and WU-AX Measured by HPAEC-PAD WE-AX treatment X X+A X+F X + A + Fb a

xylose 5.42 4.98 6.60 11.18

± ± ± ±

0.05 0.03 0.01 0.09

xylobiose 49.28 88.61 98.05 96.10

± ± ± ±

3.20 5.45 6.55 5.89

WU-AX xylotriose

38.88 53.66 36.03 75.11

± ± ± ±

3.89 4.02 3.01 4.56

xylotetraose 1.80 9.53 8.26 11.71

± ± ± ±

0.08 0.28 0.33 0.43

xylose 2.12 3.24 5.44 8.42

± ± ± ±

0.03 0.13 0.26 0.09

xylobiose 45.12 74.76 64.90 94.27

± ± ± ±

3.67 4.78 5.02 6.09

xylotriose 30.97 36.73 51.54 53.85

± ± ± ±

2.88 2.98 3.64 4.04

xylotetraose 2.66 8.44 5.85 6.05

± ± ± ±

0.03 0.21 0.32 0.06

μg/mL. bX, xylanase; A, ABF; and F, FAE.

Consistent with the properties of the AX hydrolyzates fermented by B. subtilis, acetate and propionate were the dominant USCFA fermented by L. brevis on AX hydrolyzates, FOS, and glucose (Figure 3). Unlike the fermentation capacity of B. subtilis, which showed limited activity on WU-AX hydrolyzates, L. brevis could efficiently use both WE-AX (Figure 3A) and WU-AX hydrolyzates (Figure 3B). For either WE-AX or WU-AX, the hydrolysis products produced by the simultaneous action of xylanase, ABF, and FAE show the highest USCFA production compared with other treatments (P < 0.05). Unlike the fermentation properties of WE-AX and WU-AX that were fermented by a single strain, both WE-AX and WUAX could be adequately fermented by broiler cecum content (Figure 4). There was no significant difference in USCFA concentrations between all treatments for either WE-AX or WU-AX, with the exception of butyrate. The highest butyrate concentration was produced in the (X + F) and (X + A + F) treatments on WE-AX and WU-AX, respectively. Enzyme Activities during Fermentation. Xylanase activities in FOS treatment were lower than those observed in other treatments (P < 0.05), and the highest xylanase activity was obtained with (X + A + F) treatment in of B. subtilis fermentation (Figure 5A). Arabinofuranosidase activities in treatments (X + A) and (X + A + F) were higher compared with other treatments (P < 0.05). However, the highest feruloyl esterase activity was found in the glucose treatment. Owing to the weak proliferation of B. subtilis on XOS produced from WU-AX, the activities of all three enzymes were lower than was observed for XOS originating from WE-AX (Figure 5B). Xylanase activity in all XOS hydrolyzates treatments was lower than glucose (P < 0.05) but not for

strains, which achieved the highest growth with hydrolyzates from (X + A + F) treatment. This result was especially true for B. subtilis in comparison with glucose and FOS. However, hydrolyzates from WU-AX showed a selective effect on L. brevis but not for B. subtilis. Similar to WE-AX, WU-AX treated with X + A + F exerted the best proliferative effect on L. brevis as compared to that of glucose and FOS. Short-Chain Fatty-Acids Production during Fermentation. The concentration of the SCFA, including unbranched SCFA (mainly acetate, propionate, and butyrate) and branched SCFA (isobutyrate and isovalerate), are presented in Figure 2 after B. subtilis fermentation on different substrates. After 24 h of fermentation for all substrates, acetate was found to be the dominant component in total USCFA. Notably, the concentrations of USCFA and total USCFA were significantly lower when WU-AX hydrolyzates (Figure 2B) were used as carbon source for B. subtilis than WE-AX hydrolyzates (Figure 2A), which could be efficiently fermented by B. subtilis for the production of USCFA. The highest concentration of acetate (P < 0.05) and total USCFA (P < 0.05) were obtained when WEAX hydrolyzates from (X + A + F) were used as a carbon source. There were no significant differences in total USCFA concentration when hydrolyzates from (X + A) and (X + A + F) were fermented compared with FOS and glucose fermentation. Furthermore, the fermentation of FOS and glucose generated much more branched SCFA in the presence of isobutyrate and isovalerate, which was thought to be an indicator of protein fermentation, than did AX hydrolyzate fermentation. Thus, the use of AX hydrolyzates, especially WEAX hydrolyzates, could efficiently increase the growth of B. subtilis, which produced USCFA and inhibited protein fermentation. D


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Figure 1. Scanning electron microscopy of free and hydrolyzing destarched wheat bran from different treatments at 500×. (a) Free-wheat-bran treatment; (b) wheat bran treated by xylanase; (c) wheat bran treated by xylanase and ABF; (d) wheat bran treated with xylanase and FAE; and (e) wheat bran treated with xylanase, ABF, and FAE.

Table 4. Relative Growth Compared with Glucose; pH of B. Subtilis and L. brevis with Glucose, FOSb, and Hydrolyzates of WE-AX after a Period of 24 and 48 h of Fermentation B. subtilis substrate Glu WE-AX WE-AX; WE-AX; WE-AX; WE-AX; +F FOS

a, X b, X+A c, X+F d. X+A

growth (24 h)

Table 5. Relative Growth Compared with Glucose and pH of B. Subtilis and L. brevis with Glucose, FOSb, and Hydrolyzates of WU-AX after a Period of 24 and 48 h of Fermentation

L. brevis

± ± ± ± ± ±

B. subtilis

growth (48 h)

pH

* − + ++++ ++ +++++

6.36 6.65 7.01 7.53 7.60 7.41

0.10 0.22 0.28 0.18 0.12 0.28

* − ++ ++ ++ ++

++

6.86 ± 0.23

++

+ + + +

+ + + +

+ + + +

pH 4.23 6.01 4.02 4.01 3.95 3.80

± ± ± ± ± ±

substrate

0.10 0.12 0.20 0.11 0.10 0.23

Glu WU-AX WU-AX; WU-AX; WU-AX; WU-AX; +F FOS

4.25 ± 0.22

a

a, X b, X+A c, X+F d, X+A

growth (24 h)

L. brevis growth (48 h)

pH

* − − − − −

6.36 6.61 6.68 6.68 6.70 6.66

± ± ± ± ± ±

0.21 0.20 0.19 0.13 0.20 0.17

* − ++ ++ ++ ++

++

6.86 ± 0.18

++

+ ++ ++ +++

pH 4.30 6.01 4.50 4.31 4.20 3.83

± ± ± ± ± ±

0.10 0.11 0.20 0.19 0.18 0.20

4.25 ± 0.10

X, xylanase; A, ABF; and F, FAE. a, b, c, d: WE-AX hydrolyzed by X alone and simultaneous action of X + A, X + F, and X + A + F, respectively. bFructo-oligosaccharides. *, Growth relative to growth on glucose (OD600); −, no growth; + , 1%−10% growth; + +, 11%−20% growth; + + +, 21%−30% growth; + + + +, 31%−40% growth; and + + + + +, 41%−60% growth. Growth experiments were conducted in duplicate.

X, xylanase; A, ABF; F, FAE. a, b, c, d: WE-AX hydrolyzed by X alone and simultaneous action of X + A, X + F, and X + A + F, respectively. b Fructo-oligosaccharides.*, growth relative to growth on glucose (OD600); −, no growth; + , 1%−10% growth; + +, 11%−20% growth; + + +, 21%−30% growth; + + + +, 31%−40% growth; and + + + + +, 41%−60% growth. Growth experiments were conducted in duplicate.

FOS. Arabinofuranosidase activity in treatments X and (X + A) was higher than other treatments (P < 0.05). Consistent with WE-AX hydrolyzates fermentation, the highest feruloyl esterase activity was found in the glucose treatment (P < 0.05).

In the in vitro fermentation by L. brevis, xylanase activity in all WE-AX hydrolyzates was significantly lower than glucose and FOS (P < 0.05) (Figure 6A). There was no difference in arabinofuranosidase activity for all treatments. Feruloyl esterase

a

E


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Figure 3. Short-chain fatty-acid production fermented by L. brevis on WE-AX, WU-AX, and corresponding hydrolyzates from different treatments. Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). (A) WE-AX and (B) WU-AX treated by X, X + A, X + F, and X + A + F. X, xylanase; A, ABF; F, FAE; FOS, fructo-oligosaccharides; and Glu, glucose.

Figure 2. Short-chain fatty-acid production fermented by B. subtilis on WE-AX, WU-AX, and corresponding hydrolyzates from different treatments. Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). (A) WE-AX and (B) WU-AX treated by X, X + A, X + F, and X + A + F. X, xylanase; A, ABF; F, FAE; FOS, fructo-oligosaccharides; and Glu, glucose.

activity in glucose was higher than other treatments (P < 0.05). Both of these enzyme activities were lower than those obtained in B. subtilis fermentation. In contrast, WU-AX hydrolyzates fermented by L. brevis behaved differently (Figure 6B). Xylanase activity in all WU-AX hydrolyzates was significantly higher than in the glucose and FOS group (P < 0.05). Arabinofuranosidase and feruloyl esterase activities in glucose treatment were lower and higher, respectively, but this was not observed in the FOS and X treatments. The highest xylanase activity was found in the control and (X + A + F) treatment when WE-AX and hydrolyzates were used as substrates fermented by broiler cecum content (Figure 7). Arabinofuranosidase activity in the X treatment was higher than in other treatments (P < 0.05). There was no significant difference between all of the treatments in feruloyl esterase activity. In contrast to WE-AX and corresponding hydrolyzate fermentation, xylanase activity in the control group was lower than that in other groups when WU-AX and hydrolyzates were used. Arabinofuranosidase activity was higher in treatment (X + A + F) than in other treatments (P < 0.05). The highest feruloyl esterase activity was obtained in the control treatment (P < 0.05). Growth Performance of Broilers. Supplementation of xylanase increased ADG (P > 0.05) and decreased FCR (P < 0.05; Table 6) than the control group from day 1 to 21. The combination of xylanase and ABF further improved the growth

Figure 4. Short-chain fatty-acid production fermented by broiler cecum content on WE-AX, WU-AX, and corresponding hydrolyzates from different treatments in vitro. Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). (A) WE-AX and (B) WU-AX hydrolyzed by X, X + A, X + F, and X + A + F. X, xylanase; A, ABF; F, FAE; and CT, control.

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Figure 5. Enzyme activities in the fermentation broth of B. subtilis on hydrolyzates of WE-AX and WU-AX from different treatments. Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). (A) WE-AX and (B) WU-AX hydrolyzed by X, X + A, X + F, and X + A + F; X, xylanase; A, ABF; F, FAE; FOS, fructo-oligosaccharides; and Glu, glucose.

Figure 6. Enzyme activities in the fermentation broth of L. brevis on hydrolyzates of WE-AX and WU-AX from different treatments. Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). (A) WE-AX and (B) WU-AX hydrolyzed by X, X + A, X + F, and X + A + F; X, xylanase; A, ABF; F, FAE; FOS, fructo-oligosaccharides; and Glu, glucose.

performance of broilers compared to xylanase alone. The addition of xylanase and FAE significantly increased ADG and decreased FCR as compared to the control (P < 0.05). Moreover, the combination of xylanase, ABF, and FAE (treatment E) produced even higher ADG than xylanase treatment alone (P < 0.05), and it showed the best growth performance of all treatments. Consistent with the growth performance results observed in broilers from day 1 to 21, supplementation of xylanase increased ADG (P > 0.05) and decreased FCR of broilers from day 1 to 36 as compared to the control group (P < 0.05; Table 6). The combination of xylanase, with either ABF or FAE, further improved the growth performance of broilers compared with the control group. The highest ADG and lowest FCR was obtained in treatment X + A + F as compared to other treatments (P < 0.05). Viscosity and pH of Digesta in the Gut. Digesta viscosities, measured in the jejunum, are shown on Table 7. The addition of xylanase significantly decreased the digesta viscosity compared to the control (P < 0.05). Additional supplementation of ABF or FAE further reduced the digesta viscosity, and this was highlighted in the ABF treatment, which produced a lower digesta viscosity than did the supplementa-

tion of xylanase alone (P < 0.05). Compared to the other treatments, the combination of xylanase, ABF, and FAE showed the best synergistic action regarding decreased digesta viscosity. The supplementation of xylanase led to lower pH of both ileum and cecum digesta than was observed for the control group (P > 0.05; Table 7). The further addition of ABF and FAE significantly decreased the pH of cecum digesta relative to the control group (P < 0.05). Regarding the lowering of pH values of ileum and cecum digesta, the combination of xylanase, ABF, and FAE were the most effective, especially for cecum digesta. Intestinal Lesion Scores of Broilers. The addition of xylanase reduced intestinal lesions induced by an exclusive wheat-based diet (Table 8). The extra supplementation of either ABF or FAE further reduced duodenum, jejunum, and ileum lesion scores. The combined use of xylanase, ABF, and FAE resulted in the lowest lesion scores of duodenum, jejunum, and ileum compared to that of the control (P < 0.05).

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DISCUSSION Synergistic Effects among Xylanase, ABF, and FAE. AX is considered the main antinutritional factor in wheat, resulting in highly viscous digesta in the gut of livestock, which further

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Table 7. Effects of Enzymes Supplementation on pH and Viscosity of the Digesta in the Gut of Broilers on d 36a treatmentb

viscosityc

pH

CT X X+A X+F X+A+F

ileum

cecum

jejunum

± ± ± ± ±

5.83 ± 0.09, a 5.67 ± 0.19, ab 5.41 ± 0.10, bc 5.40 ± 0.15, bc 5.21 ± 0.13, c

1.49 ± 0.05, a 1.29 ± 0.08, b 1.10 ± 0.06, c 1.30 ± 0.07, b 1.03 ± 0.01, c

6.81 6.64 6.66 6.63 6.51

0.07 0.08 0.10 0.06 0.09

a

Values with the same letter within a column are not significantly different at P < 0.05. bCT: control; X: xylanase; A: ABF; and F: FAE. Data are means for eight replicates of 16 chickens per group. cRelative viscosity on distilled water.

Table 8. Effects of Enzyme Supplementation on Intestinal Lesions Scores of Broilers on d 36a treatmentb CT X X+A X+F X+A+F

duodenum 0.86 0.56 0.39 0.54 0.33

± ± ± ± ±

0.20, 0.16, 0.17, 0.14, 0.13,

jejunum a ab ab ab b

0.76 0.41 0.40 0.33 0.22

± ± ± ± ±

ileum

0.22, 0.14, 0.11, 0.13, 0.07,

a bc ab bc c

0.46 0.38 0.53 0.43 0.03

± ± ± ± ±

0.19, 0.13, 0.14, 0.21, 0.01,

a ab ab ab b

a Values with the same letter within a column are not significantly different at P < 0.05. bCT: control; X: xylanase; A: ABF; and F: FAE. Data are means for eight replicates of 16 chickens per group.

AX, which indicates that the degradation of WU-AX is more difficult than that of WE-AX.12 Extra supplementation of ABF or FAE is required, which leads to a more pronounced DS in WU-AX. In this study, xylose release from WU-AX by xylanase alone accounted for about 39% of that from WE-AX, which was consistent with the results obtained by Sorensen et al. (a reported percentage of approximately 42%).12 Moreover, the addition of either ABF or FAE increased xylose production. The SEM of destarched wheat bran from different enzymatic treatments, shown in Figure 1, further supported this result. The synergism between xylanases and ABF28 or FAE14 on AX and cereal byproducts has been demonstrated. However, the synergistic effect of the combined use of xylanase, ABF, and FAE has not been extensively studied. The DS is largely dependent on the structure of AX20 and the order in which enzymes are added.9 Moreover, the prior removal of side chains in AX via debranching enzymes in the first reaction is beneficial in the degradation of AX.9,12 Our data clearly demonstrated that xylanase had synergistic effects with both ABF and FAE, especially sequential reactions, in the hydrolysis of WE-AX, WU-AX, and beech-wood xylan, which suggests that removing the side chains (L-arabinose and ferulic acids) from the

Figure 7. Enzyme activities in the fermentation broth of broiler cecum content on hydrolyzates of WE-AX and WU-AX from different treatments of 48 h fermentation in vitro. Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). (A) WE-AX and (B) WU-AX hydrolyzed by X, X + A, X + F, and X + A + F; X, xylanase; A, ABF; F, FAE; and CT, control.

leads to poor production performance. AX cannot be totally degraded by endoxylanase alone owing to its complex side chains and heterogeneous structure.12 Thus, debranching enzymes are required to cooperate with xylanase and degrade AX. The arabinose and ferulic acid side chains impede the action of xylanase, owing to the steric hindrance effect, which retards the enzymatic degradation of AX.26 ABF and FAE particularly hydrolyze the α-linked L-arabinofuranose residues27 and ferulic acid esterase linkages,14 respectively, which enables xylanase to access the AX backbone.26 The average degree of polymerization (avDP) and average degree of arabinose substitution (avDAS) of WU-AX is higher than that of WE-

Table 6. Effects of Enzymes Supplementation on Growth Performance of Birdsa 1−21 d treatmentb CT X X+A X+F X+A+F

21 d body weight (kg) 0.69 0.72 0.73 0.74 0.76

± ± ± ± ±

0.02, 0.01, 0.01, 0.01, 0.01,

a ab ab bc c

ADGc (g/bird per d) 30.99 32.43 32.54 33.09 34.45

± ± ± ± ±

0.71, 0.63, 0.56, 0.57, 0.52,

a ab ab bc c

1−36 d ADFI (g/bird per d) 46.94 47.51 48.12 47.13 49.18

± ± ± ± ±

1.31 0.56 1.25 0.71 0.84

FCR (g/g) 1.52 1.47 1.46 1.43 1.43

± ± ± ± ±

0.01, 0.02, 0.01, 0.01, 0.01,

a bc bc c c

36 d body weight (kg) 1.85 1.94 1.82 1.92 2.08

± ± ± ± ±

0.03, 0.02, 0.04, 0.03, 0.05,

a a a a b

ADG (g/bird per d) 48.97 51.24 48.32 50.75 55.04

± ± ± ± ±

0.85, 0.65, 0.98, 0.79, 1.34,

a a a a b

ADFI (g/bird per d) 96.46 96.26 90.90 93.48 97.39

± ± ± ± ±

1.83 1.05 2.32 1.86 4.14

FCR (g/g) 1.97 1.88 1.88 1.84 1.77

± ± ± ± ±

0.02, 0.02, 0.02, 0.03, 0.05,

a b b bc c

a

Values with the same letter within a column are not significantly different at P < 0.05. bCT: control; X: xylanase; A: ABF; and F: FAE. cAverage daily gain; average daily feed intake; and feed-conversion ratio. Data are means for eight replicates of 16 chickens per group. H


Article


However, the ABC mechanism involved in the uptake of XOS for L. brevis is still unclear. Growth Performance and Intestinal Health of Broilers. AX results in highly viscous chyme in the gut of broilers, and it leads to poor growth performance4 and impaired intestinal health.38 Exogenous supplementation of xylanase for a wheatbased feed has become a common approach in the animal-feed industry. Xylanase increases growth performance4 and inhibits pathogenic colonization in the gut of broilers.3,38 WU-AX was the major AX in wheat,39 and it could be transformed into WEAX by degradation via xylanase.6 However, WU-AX could not be totally degraded by xylanase alone owing to its complex structure, and the process would be even weaker in vivo due to limitations like digesta transit time.40 Further supplementation of side-chain enzymes accelerated the degradation effect, as was confirmed by Mathlouthi et al.41 This study demonstrated that the combination of side-chain enzymes and xylanase reduced the viscosity of feedstuffs more efficiently compared to xylanase alone in vitro. Nevertheless, the effect has not been proven in vivo to date. Our data clearly show that xylanase significantly reduced the viscosity of chyme in the jejunum of broilers, and the extra addition of ABF, but not FAE, further improved the effect. The result may stem from the fact that FAE specifically targets the ferulic acid ester bond that is a key structure for WU-AX,6 which did not affect the viscosity of chyme in the gut. A study by Bedford et al.42 reported that the addition of xylanase in a wheat-based diet resulted in an increase in the concentration of short-chain xylo-oligomers in the cecum of broilers. In this study, XOS obtained from hydrolysis reactions could be adequately fermented by bacteria from the cecum of broilers in vitro, and the combination of xylanase with ABF or FAE both decreased pH of ileum and cecum in vivo. This was especially true for the latter, in which the major fermentation took place. These results may be linked to the XOS released in the hindgut, which was further fermented to produce USCFA, and it also lowed the pH of the gut. A lower pH helps to inhibit the proliferation of pathogenic bacteria (e.g., Enterobacteriaceae) by dissipating the proton motive force across the bacterial cell membrane while promoting the growth of probiotics and improving gut health.43 Intestinal lesion scores are a direct and vital indicator of intestinal health, especially for pathogenic bacterial infections.25 In this study, xylanase alleviated the intestinal lesions to some extent compared to the control group, and this was consistent with the results obtained by Liu et al.3 Both ABF and FAE further improved the effect, and the combination of xylanase, ABF, and FAE significantly reduced the intestine lesion score compared to that of CT group, which indicated that the gut health has been improved. The Ussing chamber of the ileum further demonstrated that xylanase combined with ABF and FAE decreased the intestine permeability and improved intestinal barrier function (data not shown). Xylanase significantly decreased the FCR for all feeding trials, and the addition of ABF or FAE further improved the growth performance. Moreover, the combination of xylanase, ABF, and FAE exhibited the best production performance of all treatments. As previously stated, USCFA (especially for butyrate) plays an important role in the energy requirements and gut health of livestock.18,43 Our data showed that the combination of xylanase, ABF, and FAE increased the production of XOS, and it generated more USCFA when further fermented by bacteria in the cecum. This was especially true for butyrate in comparison to the control group and xylanase used alone in vitro. These findings may support the

backbone of xylan before hydrolysis by xylanase could accelerate the hydrolysis reaction. Growth and Fermentation of Bacteria on XOS Hydrolyzates. L. brevis and B. subtilis are two kinds of probiotic bacteria used as feed additives in the livestock industry. Results from our data show that XOS hydrolyzates, but not polymeric xylan, could be fermented by L. brevis and B. subtilis, which was in accordance with previous studies.15 However, there are limited studies on the probiotic effect on XOS hydrolyzates from WE-AX and WU-AX. In this study, XOS hydrolyzates from WE-AX improved the growth of both B. subtilis and L. brevis compared with glucose and FOS. This indicates that the XOS hydrolyzates from WE-AX were preferred by these two strains over FOS. XOS hydrolyzates from WE-AX treated by (X + A + F) also improved the growth of B. subtilis more than other treatments, which could be explained by a higher concentration of XOS during fermentation. Nevertheless, XOS hydrolyzates from WU-AX did not affect the growth of B. subtilis. The reason may be that the XOS hydrolyzates from WU-AX had a higher and more complex substitution structure, especially ferulic acid substitution, which was shown to hinder fermentation.29 Compared to B. subtilis, L. brevis showed a greater capacity to utilize XOS, which may be due to the difference in the ATPblinding cassette transporter (ABC)-type sugar-transport system.15 In contrast to single-strain fermentations, both WEAX and WU-AX could be adequately fermented by cecum chyme from broilers in vitro. AX could be degraded by endoxylanase-related enzymes encoded by polysaccharide utilization loci (PULs) of the bacteria.30 These loci are found mainly in Roseburia and Bacteroide,31 especially the latter, which is the predominant bacterial genus in the gut of broilers.32 SCFA concentrations during fermentation were consistent with growth results but not with enzyme activity. XOS fermentation only generated only USCFA,33 and branched SCFA (isobutyrate and isovalerate) was an indicator of protein fermentation,34 which is harmful to intestinal health.35 The fermentation of B. subtilis generated the highest amount of branched SCFA when glucose was used as the carbon source. Replacing glucose with XOS hydrolyzates inhibited the production of branched SCFA, which suggested that protein fermentation was suppressed.6 Unlike B. subtilis, the fermentation of L. brevis on XOS, FOS, and glucose did not give rise to any branched USCFA. Higher USCFA amounts, especially for butyrate, were obtained during the fermentation of XOS by the broiler cecum than with complete AX, which indicated that the low molecular mass and reduced substitution of the XOS structure were preferred.36 Because of the PULs, bacterial fermentation on AXOS, XOS, or AX produced enzymes associate with AX degradation, including endoxylanase, ABF, and FAE. For B. subtilis, higher enzyme activity (xylanase, ABF, and FAE) was obtained during the fermentation of AXOS hydrolyzed from WE-AX than from WU-AX. However, the fermentation of XOS hydrolyzed from WU-AX by L. brevis showed a higher enzyme activity of xylanase, ABF, and FAE. Notably, all enzyme activity was lower in L. brevis than that in B. subtilis fermentation. These results indicated that the expression of these enzymes is influenced by both bacterial proliferation and substrate-induced effects.37 Moreover, the higher tendencies of the L. brevis ABC for XOS hydrolyzed from WU-AX may also result in XOS utilization, which might reduce the secretion of extracellular enzymes. I


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(7) Izydorczyk, M. S.; Biliaderis, C. G. Cereal arabinoxylans: Advances in structure and physicochemical properties. Carbohydr. Polym. 1995, 28, 33−48. (8) Biely, P. Diversity of microbial endo-beta-1,4-xylanases. Applications of Enzymes to Lignocellulosics. 2003, 855, 361−380. (9) Raweesri, P.; Riangrungrojana, P.; Pinphanichakarn, P. alpha-LArabinofuranosidase from Streptomyces sp PC22: Purification, characterization and its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural residues. Bioresour. Technol. 2008, 99, 8981−8986. (10) Uraji, M.; Kimura, M.; Inoue, Y.; Kawakami, K.; Kumagai, Y.; Harazono, K.; Hatanaka, T. Enzymatic Production of Ferulic Acid from Defatted Rice Bran by Using a Combination of Bacterial Enzymes. Appl. Biochem. Biotechnol. 2013, 171, 1085−1093. (11) Sorensen, H. R.; Pedersen, S.; Vikso-Nielsen, A.; Meyer, A. S. Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue. Enzyme Microb. Technol. 2005, 36, 773−784. (12) Sorensen, H. R.; Pedersen, S.; Meyer, A. S. Synergistic enzyme mechanisms and effects of sequential enzyme additions on degradation of water insoluble wheat arabinoxylan. Enzyme Microb. Technol. 2007, 40, 908−918. (13) Goncalves, T. A.; Damasio, A. R. L.; Segato, F.; Alvarez, T. M.; Bragatto, J.; Brenelli, L. B.; Citadini, A. P. S.; Murakami, M. T.; Ruller, R.; Paes Leme, A. F. P.; Prade, R. A.; Squina, F. M. Functional characterization and synergic action of fungal xylanase and arabinofuranosidase for production of xylooligosaccharides. Bioresour. Technol. 2012, 119, 293−299. (14) Wong, D. W. S.; Chan, V. J.; Liao, H.; Zidwick, M. J. Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases. J. Ind. Microbiol. Biotechnol. 2013, 40, 287−295. (15) Falck, P.; Precha-Atsawanan, S.; Grey, C.; Immerzeel, P.; Stålbrand, H.; Adlercreutz, P.; Nordberg Karlsson, E. Xylooligosaccharides from hardwood and cereal xylans produced by a thermostable xylanase as carbon sources for Lactobacillus brevis and Bifidobacterium adolescentis. J. Agric. Food Chem. 2013, 61, 7333−7340. (16) Lecerf, J. M.; Depeint, F.; Clerc, E.; Dugenet, Y.; Niamba, C. N.; Rhazi, L.; Cayzeele, A.; Abdelnour, G.; Jaruga, A.; Younes, H.; Jacobs, H.; Lambrey, G.; Abdelnour, A. M.; Pouillart, P. R. Xylooligosaccharide (XOS) in combination with inulin modulates both the intestinal environment and immune status in healthy subjects, while XOS alone only shows prebiotic properties. Br. J. Nutr. 2012, 108, 1847−1858. (17) Belobrajdic, D. P.; Bird, A. R.; Conlon, M. A.; Williams, B. A.; Kang, S.; McSweeney, C. S.; Zhang, D.; Bryden, W. L.; Gidley, M. J.; Topping, D. L. An arabinoxylan-rich fraction from wheat enhances caecal fermentation and protects colonocyte DNA against diet-induced damage in pigs. Br. J. Nutr. 2012, 107, 1274−1282. (18) Fukunaga, T.; Sasaki, M.; Araki, Y.; Okamoto, T.; Yasuoka, T.; Tsujikawa, T.; Fujiyama, Y.; Bamba, T. Effects of the soluble fibre pectin on intestinal cell proliferation, fecal short chain fatty acid production and microbial population. Digestion 2003, 67, 42−49. (19) Mukherjee, G.; Singh, R. K.; Mitra, A.; Sen, S. K. Ferulic acid esterase production by Streptomyces sp. Bioresour. Technol. 2007, 98, 211−213. (20) Zheng, F.; Huang, J.; Yin, Y.; Ding, S. A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase. J. Ind. Microbiol. Biotechnol. 2013, 40, 1083−1093. (21) Shi, P. J.; Chen, X. Y.; Meng, K.; Huang, H. Q.; Bai, Y. G.; Luo, H. Y.; Yang, P. L.; Yao, B. Distinct Actions by Paenibacillus sp Strain E18 alpha-L-Arabinofuranosidases and Xylanase in Xylan Degradation. Appl. Environ. Microb. 2013, 79, 1990−1995. (22) Yue, Q.; Yang, H. J.; Li, D. H.; Wang, J. Q. A comparison of HPLC and spectrophotometrical methods to determine the activity of ferulic acid esterase in commercial enzyme products and rumen contents of steers. Anim. Feed Sci. Technol. 2009, 153, 169−177.

results obtained in vivo. In addition, our research also suggested the potential applications of ABF and FAE for the improvement of the growth performance and gut health of broilers that are fed a wheat-based diet, and it may provide a novel enzyme strategy for use in the broiler-feeding industry.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01272. A table showing the Ussing chamber result of ileum of broilers on d 36 expressed in fluorescein isothiocyanate concentration. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 010 62732337; fax: +86 010 62732712; e-mail: [email protected]. Funding

This research was supported by the System for Poultry Production Technology, Beijing Innovation Research Team of Modern Agriculture (CARS-PSTP), the National Natural Science Foundation of China (no. 31572424). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor W. Shao for providing the arabinofuranosidase used in this study. All authors read and approved the final version of the manuscript.

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ABBREVIATIONS USED AX, arabinoxylan; AXOS, arabinoxylan oligosaccharides; ABF, arabinofuranosidase; FAE, feruloyl esterase; WE-AX, watersoluble arabinoxylan; WU-AX, insoluble arabinoxylan; ADG, average daily growth; ADFI, average daily feed intake; FCR, feed-conversion ratio

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REFERENCES

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