Optimization of Xylooligosaccharides Production by Native and Recombinant Xylanase Hydrolysis of Chicken Feed Substrates

Abstract

Poultry production faces several challenges, with feed efficiency being the main factor that can be influenced through the use of different nutritional strategies. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their excellent ability to modulate the composition of the gut microbiota. The aim of the study was to apply crude and purified fungal xylanases, from Trichoderma harzianum, as well as a recombinant glycoside hydrolase family 10 xylanase, derived from Geobacillus stearothermophilus T6, as additives to locally produced chicken feeds. A Box-Behnken Design (BBD) was used to optimize the reducing sugar yield. Response surface methodology (RSM) revealed that reducing sugars were higher (8.05 mg/mL, 2.81 mg/mL and 2.98 mg/mL) for the starter feed treated with each of the three enzymes compared to the treatment with grower feed (3.11 mg/mL, 2.41 mg/mL and 2.62 mg/mL). The hydrolysis products were analysed by thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) analysis and showed that the enzymes hydrolysed the chicken feeds, producing a range of monosaccharides (arabinose, mannose, glucose, and galactose) and XOS, with xylobiose being the predominant XOS. These results show promising data for future applications as additives to poultry feeds.

Keywords: chicken feed; lignocellulosic biomass; response surface methodology; xylanase; xylooligosaccharides.

Figure 1

Pareto chart of standardized effects for the BBD for enzyme dosage (X1), feed loading (X2), incubation time (X3), incubation temperature (X4), and pH (X5) for the hydrolysis of (a) starter feed and (b) grower feed with crude T. harzianum xylanase. The orange line represents p = 0.05.

Figure 2

Pareto chart of standardised effects for the BBD for enzyme dosage (X1), feed loading (X2), incubation time (X3), incubation temperature (X4), and pH (X5) for the hydrolysis of (a) starter feed and (b) grower feed with purified T. harzianum xylanase. The orange line represents p = 0.05.

Figure 3

Pareto chart of standardised effects for the BBD for enzyme dosage (X1), feed loading (X2), incubation time (X3), incubation temperature (X4), and pH (X5) for the hydrolysis of (a) starter feed and (b) grower feed with purified recombinant XT6 xylanase. The orange line represents p = 0.05.

Figure 4

3D-response surface plots and contour plots of the combined effects of feed loading and incubation time (a) and incubation time and temperature (b) on the yield of reducing sugars from the hydrolysis of starter chicken feed by the crude T. harzianum xylanase.

Figure 5

3D-response surface plots and contour plots of the combined effects of enzyme dose and incubation time (a) and incubation time and temperature (b) on the yield of reducing sugars from starter chicken feed hydrolysed by the purified T. harzianum xylanase.

Figure 6

3D-response surface plots and contour plots of the combined effects of enzyme dose and feed loading (a) and incubation time and temperature (b) on the yield of reducing sugars from grower chicken feed hydrolysed by the purified T. harzianum xylanase.

Figure 7

3D-response surface plots and contour plots of the combined effects of dose and feed loading (a) and incubation time and temperature (b) on the yield of reducing sugars from the starter chicken feed hydrolysed by the purified recombinant XT6.

Figure 8

3D-response surface plot and contour plot of the combined effects of incubation time and temperature on the yield of reducing sugars from the grower chicken feed hydrolysed by the purified recombinant XT6.

Figure 9

Thin Layer Chromatography (TLC) profile of XOS produced from chicken feed hydrolysis by the crude (a) and purified (b) T. harzianum xylanases and the recombinant XT6 xylanase (c). (d) shows the TLC profile of the monosaccharides resulting from chicken feed hydrolysis by the three enzyme preparations. STD—Xylooligosaccharides standards, SC (S)—substrate control (no xylanase) starter feed, SC (G)–Substrate control (no xylanase) grower feed, T.h S1—starter feed with crude T. harzianum xylanase (sample 1), T.h S2—starter feed with crude T. harzianum xylanase (sample 2), T.h G—grower feed with crude T. harzianum xylanase (sample 1) and T.h G2—grower feed with crude T. harzianum xylanase (sample 2). T.hP S1—starter feed with purified T. harzianum xylanase (sample 1), T.hP S2—starter feed with purified T. harzianum xylanase (sample 2), T.hP G1–grower feed with purified T. harzianum xylanase (sample 1) and T.hP G2—grower feed with purified T. harzianum xylanase (sample 2). X2—Xylobiose; X3—Xylotriose; X4—Xylotetraose; X5—Xylopentaose; X6—Xylohexaose. Monosaccharide standards; Xyl—xylose, Ara–arabinose, Man—mannose, Glu—glucose, and Gal—galactose. T.h SF—starter feed with crude T. harzianum xylanase, T.h GF—grower feed with crude T. harzianum xylanase, T.hP SF—starter feed with purified T. harzianum xylanase, T.hP GF—grower feed with purified T. harzianum xylanase, XT6 SF—recombinant XT6 xylanase with starter feed and XT6 GF–recombinant XT6 xylanase with grower feed.

Figure 9

Thin Layer Chromatography (TLC) profile of XOS produced from chicken feed hydrolysis by the crude (a) and purified (b) T. harzianum xylanases and the recombinant XT6 xylanase (c). (d) shows the TLC profile of the monosaccharides resulting from chicken feed hydrolysis by the three enzyme preparations. STD—Xylooligosaccharides standards, SC (S)—substrate control (no xylanase) starter feed, SC (G)–Substrate control (no xylanase) grower feed, T.h S1—starter feed with crude T. harzianum xylanase (sample 1), T.h S2—starter feed with crude T. harzianum xylanase (sample 2), T.h G—grower feed with crude T. harzianum xylanase (sample 1) and T.h G2—grower feed with crude T. harzianum xylanase (sample 2). T.hP S1—starter feed with purified T. harzianum xylanase (sample 1), T.hP S2—starter feed with purified T. harzianum xylanase (sample 2), T.hP G1–grower feed with purified T. harzianum xylanase (sample 1) and T.hP G2—grower feed with purified T. harzianum xylanase (sample 2). X2—Xylobiose; X3—Xylotriose; X4—Xylotetraose; X5—Xylopentaose; X6—Xylohexaose. Monosaccharide standards; Xyl—xylose, Ara–arabinose, Man—mannose, Glu—glucose, and Gal—galactose. T.h SF—starter feed with crude T. harzianum xylanase, T.h GF—grower feed with crude T. harzianum xylanase, T.hP SF—starter feed with purified T. harzianum xylanase, T.hP GF—grower feed with purified T. harzianum xylanase, XT6 SF—recombinant XT6 xylanase with starter feed and XT6 GF–recombinant XT6 xylanase with grower feed.

Figure 10

The High-Performance Liquid Chromatography (HPLC) profile of monosaccharides and xylooligosaccharides resulting from chicken feed hydrolysis. E1 + SF Crude T. harzianum xylanase + starter chicken feed, E1 + G—crude T. harzianum xylanase + grower chicken feed, E2 + S—purified T. harzianum xylanase + starter chicken feed, E2 + G—purified T. harzianum xylanase + grower chicken feed, E3 + S—purified recombinant XT6 xylanase + starter chicken feed and E3 + G—purified recombinant XT6 xylanase + grower chicken feed. Data points represent the mean values ± SD (n = 3).