Production of xylooligosaccharides from hardwood xylan by using immobilized endoxylanase of Clostridium strain BOH3

Abstract

Prebiotic xylooligosaccharides (XOS) are indispensable components in food preparations. Thus, developing a cost-effective process for XOS production is important to meet increasing market demands. In this study, an endoxylanase excreted by a solventogenic Clostridium strain BOH3 is exploited to hydrolyze xylan and produce XOS with a high yield. Furthermore, a technique is developed to immobilize the endoxylanase in calcium alginate gel beads to produce XOS from hardwood xylan. In addition to calcium alginate, the gel beads also contain 3.5% w/v of silica gel, which increases the hardness by 1.6 times and significantly reduces leaching of xylanase from the beads. As a result, the immobilized xylanase can be reused for 7 cycles while retaining >62% of its initial activity. Moreover, when beechwood and teakwood xylans are used as substrates, the immobilized xylanase liberates only xylobiose and xylotriose. This phenomenon is distinctly different from free xylanase which releases a wide range of XOS. Overall, the immobilized xylanase can produce 0.37–0.48 mg of XOS (per mg of xylan). The XOS produced by immobilized xylanase shows prebiotic effects on Bifidobacterium animalis, and Lactobacillus acidophilus.

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Introduction

Endoxylanases (EC 3.2.18, endo-β-1,4-D-xylanase) excreted by high-butanol-producing clostridial strains are not reported very often. To date, there are only three strains including Clostridium acetobutylicum ATCC824, Clostridium beijerinckii G117 and Clostridium strain BOH3 known to excrete endoxylanases when they were cultured in xylan-containing media.^1–3 Interestingly, these endoxylanases can release xylooligosaccharides (XOS) as main products during hydrolysis of xylan. Because XOS show prebiotic effects, they are used by food and pharmaceutical industries in large quantities.^4,5 Among xylanases from bacterial sources, xylanase from strain BOH3 is very unique and has several advantages. (1) It can be produced by using low-cost substrates such as sugarcane bagasse. (2) It shows high-level substrate specificity for xylan (195–212 U mg⁻¹ protein) and no activity for cellulose, cellobiose, starch and XOS. (3) It catalyzes the hydrolysis of xylan and releases XOS as main products (>95%) with very little xylose (<1%). (4) It is highly resistant to common enzyme inhibitors including p-aminobenzoic acid, Tween 80, EDTA, urea and β-mercaptoethanol.³ These characteristics are usually only observed in recombinant xylanases (BlxA and XynB) from engineered bacteria.⁴ Therefore, xylanase excreted by strain BOH3 can be considered as a potential candidate for the production of XOS.

Even though xylanases are effective biocatalysts, their high cost remains a problem for mass production of XOS.⁶ To recycle xylanases, the enzymes should be immobilized in solid carriers, which can be collected and reused many times after hydrolysis. Purification of XOS will also be simpler because the immobilized enzymes can be easily separated from the reaction mixture. In addition, immobilization can enhance thermal stability and pH tolerance of xylanases. Therefore, it is a key step for developing a cost-effective bioprocess for mass production of XOS. To immobilize xylanases, several immobilization techniques such as covalent linkage and physical adsorption have been reported as shown in Table 1. In most techniques, immobilized xylanases still show activities similar to free enzymes (18–117% after covalent immobilization or 42–126% after physical adsorption). Notably, these immobilized xylanases could be reused for 3–17 cycles while producing 1.92–15.5 mg ml⁻¹ of XOS in batch processes (Table 1). Because XOS are often used in food and pharmaceutical preparations, solid carriers for enzyme immobilization also need to be selected carefully.⁵ Obviously, they should be biocompatible and chemically inert while toxic chemicals such as glutaraldehyde cannot be used as a cross-linker.^5,7

Table 1 Production of xylooligosaccharides by various immobilized xylanases reported in literatures^a

Xylanase sources	Matrix used for immobilization (type)	Immobilization yield/xylanase activity yield	Reusability (% of activity retaining)	Substrate (%) used	XOS (DP2-6) concentration
a ND – not detected.
^8Commercial recombinant xylanase	Eudragit L-100 (adsorption)	95%/87–126%	3 cycles (100%)	Birchwood xylan (1%)	8.0 mg ml^−1
^9Streptomyces halstedii xylanase	Glyoxyl-agarose (covalent)	95%/65%	10 cycles (80%)	Beechwood xylan (1%)	5.6 mg ml^−1
^10Aspergillus versicolor xylanase	Glyoxyl-agarose (covalent)	90%/84%	ND	Birchwood xylan (1.8%)	3.1 mg ml^−1
^11Streptomyces olivaceoviridis E–86 xylanase	Eudragit S-100 (adsorption)	90%/92%	4 cycles (81%)	Pretreated corncob powder (4%)	15.5 mg ml^−1
^12Aspergillus niger A25 xylanase	Dialdehyde starch-chitosan (covalent)	71%/61%	ND	Birchwood xylan (2%)	ND
^13Talaromyces thermophilus xylanase	Gelatin-glutaraldehyde (covalent)	99%/99%	13 cycles (94%)	Birchwood xylan (1%)	2.0 mg ml^−1
^14Recombinant Penicillium occitanis xylanase 3	Chitosan-glutaraldehyde (covalent)	99%/95%	5 cycles (50%)	Corncob extracted xylan (1%)	5.3 mg ml^−1
^15Recombinant Penicillium occitanis xylanase 2	Nickel-chelate Eupergit C (covalent)	93%/65%	5 cycles (50%)	Corncob extracted xylan (2%)	15.3 mg ml^−1
^16Armillaria gemina xylanase	Functionalised silicon oxide nanoparticle (covalent)	117%/ND	17 cycles (92%)	Birchwood xylan (1%)	7.5 mg ml^−1
^17Aspergillus niger xylanase	Fe3O4-coated chitosan magnetic nanoparticles (covalent)	ND/57%	7 cycles (85.5%)	Birchwood xylan (1%)	3.8 mg ml^−1
				Wheat bran xylan (1%)	1.2 mg ml^−1
^18Bacillus pumilus xylanase	HP-20 beads (covalent)	18%/42%	7 cycles (70%)	Birchwood xylan (1%)	9.8 mg ml^−1
	Ca-alginate (entrapment)	70%/30%	ND	ND	ND
Clostridium strain BOH3 xylanase^This work	Alginate-silica gel (entrapment)	100%/>70%	7 cycles (>62%)	Beechwood xylan (5%)	23.7 mg ml^−1

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Because of the limitations mentioned above, we propose that entrapment of xylanase in calcium alginate beads is a suitable technique to immobilize xylanase for producing food-grade XOS. Alginate is a nontoxic, natural polysaccharide (from brown seaweed) containing 1,4-linked β-D-mannuronic and α-L-guluronic acid. In the past, it has been widely used as a gelling and thickening agent in food and pharmaceutical preparations. Moreover, calcium alginate provides a suitable solid matrix for immobilization of various enzymes.^19,20 For example, enzymes such as xylanase from B. pumilus MK001 was successfully immobilized in calcium alginate beads. However, the immobilization yield was only 70% and the immobilized xylanase only retained 30% of its original activity.¹⁸ The low activity was caused by leaching of xylanase from the alginate beads and poor operational stability. To address these issues, additives such as silica gel, chitosan and pectin were mixed with the sodium alginate solution before gelation to improve mechanical strength and operation stability.^21–24

In this study, strategies for immobilization of xylanase in calcium alginate beads were investigated and optimized.

Subsequently, the beads were used for the production of food-grade XOS with good prebiotic effects. This is the first study of using endoxylanase produced by wild-type, obligate anaerobic, high-butanol-producing Clostridium as a biocatalyst to produce XOS.

Experimental

Materials

Beechwood xylan, sodium alginate, calcium chloride, xylose, mannose, galactose, rhamnose, arabinose, glucose, luria broth (Miller), lactobacillus MRS broth, M9 minimal medium, buffering salts, other medium components and minerals were purchased from Sigma (USA). Silica gel (150 Å) was obtained from Merck (Germany). Xylobiose (X2), xylotriose (X3), xylotetarose (X4) and xylopentaose (X5) were procured from Megazyme (Ireland). Probiotic microbes including Bifidobacterium animalis (ATCC®27674™) and Lactobacillus acidophilus (ATCC®4357™) were purchased from Bio-Rev (Singapore). Escherichia coli BL21 was a strain in our laboratory. Membrane ultrafiltration (UF) and nanofiltration (NF) were procured from Sterilitech (USA).

Methods

Production and purification of xylanase. Clostridium strain BOH3 was cultured in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate. After 48 h of fermentation, extracted xylanase from the fermented broth was purified by using FPLC with an anionic exchange Q-column. Detailed methodology for fermentation, purification of xylanase and assay can be found elsewhere.³

Enzyme immobilization. Final volume of 20 ml sodium alginate solution (2–5% w/v) containing 100 U of xylanase (by adding 1.34 ml from the stock concentration of 74.52 ± 1.25 U ml⁻¹) was prepared. The solution was injected into 200 ml of calcium chloride solution (5% w/v) through a needle (ID 0.8 mm) by using a syringe pump (0.5 ml min⁻¹) to immobilize xylanase. The solution was stirred constantly at room temperature to promote gelling. In some cases, silica gel (1–5% w/v), chitosan (1% w/v) and pectin (1% w/v) were added to the slurry before gelation. After 1 h, alginate gel beads were collected by filtration through a Buchner funnel. Subsequently, they were washed twice with MilliQ water to remove residual calcium chloride solution and air-dried for 10–15 min. They were stored at 4 °C before use. The immobilization yield was defined as:

Immoblization yield (%) = (A − B)/A × 100 (1)

where A is the total enzyme activity, B is the enzyme activity in the calcium chloride solution after the immobilization. The activity was determined after calcium chloride was removed by using a PD-10 column (GE Singapore). Xylanase activity yield of the immobilized beads is defined as:

Activity yield = (C/A) × 100 (2)

where C is activity of the immobilized xylanase in the beads.

Extraction of xylan from teakwood sawdust. Teakwood sawdust with a particle size smaller than 50 μm was used for extracting xylan. Initially, lignin present in the sawdust was removed by using sodium chlorite–acetic acid method as described by Kumar et al.²⁵ Later, the holocellulose obtained from the sawdust was soaked in a NaOH solution (15% w/v) for 24 h to solubilize xylan. Afterwards, the solution containing soluble xylan was filtered through muslin cloth to remove residual sawdust. The soluble xylan was neutralized with concentrated sulphuric acid (3 M) to precipitate xylan. The precipitate was collected after centrifugation at 10 000 rpm for 30 min and then dialyzed against DI water to remove NaOH and salts. Pure xylan was dried at 80 °C and weighted. Total sugar content in the extracted xylan was analyzed by using phenol–sulphuric acid method²⁶ whereas monosaccharides present in extracted xylan was analyzed by using HPLC with a Aminex-HPX-87H column (Bio-Rad, USA) as specified by National Renewable Energy Laboratory (USA).²⁷ All reactions were conducted in triplicates.

Production of XOS. A reaction mixture consisted of 5% (w/v) beechwood xylan (or teakwood xylan) and 20 U of xylanase in 20 ml of acetate buffer (10 mM, pH 5.5). For the case of immobilized xylanase, beads containing 20 U of xylanase were used instead. The reaction mixture was incubated at 50 °C for 24 h with mild agitation (50 rpm) in a temperature-controlled water bath. To monitor hydrolysis of xylan and formation of XOS, samples were drawn at regular intervals for analysis. They were centrifuged at 12 000 rpm for 20 min at 4 °C. Subsequently, reducing sugar concentration in the supernatant was measured by using 3,5-dinitrosalicylic acid method.²⁸ In parallel, XOS formed in the reaction were analyzed and estimated by using HPLC (Agilent, USA) with a Zorbax carbohydrate column (Agilent, USA) and a refractive index detector (maintained at 30 °C).³

Estimation of xylanase in solutions and alginate beads. After hydrolysis of xylan for 24 h, the immobilized beads were removed from the reaction mixture. Subsequently, the reaction mixture was centrifuged at 8000 rpm for 20 min at 4 °C to remove all suspended solids. Ammonium sulphate was added to the supernatant until 70% saturation (at 25 °C) under mild agitation (50 rpm). This solution was incubated at room temperature for 30 min, and then centrifuged at 12 000 rpm for 30 min to precipitate proteins. Afterwards, the proteins were re-suspended in phosphate buffer (pH 6.5, 50 mM) and passed through a PD-10 column to remove excess ammonium sulphate in the sample. In parallel, alginate beads at the end of the reaction were washed thoroughly with Milli-Q water and then suspended in 10 mM of ethylenediaminetetraacetic acid (EDTA) solution for 30 min under mild agitation (50 rpm). Afterwards, undissolved beads were gently grounded with a mortar and pestle. The solution containing dissolved alginate beads was filtered through filter paper (Whatman-1). The filtrate was loaded into a snakeskin dialysis tubing (MWCO: 10k, Thermo Fisher Scientific, USA) and dialyzed against phosphate buffer (pH 6.5, 50 mM) for 24 h at 10 °C. The dialyzing buffer was replaced with fresh buffer every 4 h for the next 24 h. Later, centrifugal concentrators (Vivaspin-20, USA) were employed to concentrate the dialyzed xylanase at 4 °C to a desired volume. Eventually, the proteins extracted from reaction mixture as well as alginate beads were assayed for xylanase activity at 50 °C using beechwood xylan (1% w/v) as a substrate. One unit of xylanase activity is defined as the amount of enzyme that liberates 1 μmole of reducing sugar (xylose equivalent) per minute.³

Hardness measurement. Hardness of calcium alginate beads with (or without) silica gel were measured by using a sophisticated texture analyzer (TA-X2Ti Stable Microsystem, UK) with a probe length of 10 cm and a diameter of 5 mm. The target mode was distance, and the test mode was compression with a test speed of 0.2 mm s⁻¹ along with pre- and post-test speeds of 0.1 mm s⁻¹. The trigger type was auto (force) and response was drawn at 200 points per second. For each type of alginate bead, a minimum of 25 beads were tested and their average value was reported.

Prebiotic effects of XOS on probiotic microbes. Effects of XOS on probiotic microbes including B. animalis, and L. acidophilus were investigated as specified by Chapla et al.²⁹ with some modifications: glucose (20 g l⁻¹) was used as positive control and XOS produced in house were supplemented to the fermentation media. Initial pH of the medium was adjusted to pH 7.0 ± 0.1 using 0.05 N of NaOH/HCl. An inoculum size of 10% v/v for both B. animalis, and L. acidophilus was used, and the fermentation was conducted at 37 °C under rotation (120 rpm) for 3–5 days. Meanwhile, E. coli was cultured in LB medium for 12–16 h at 37 °C. Later, 5% v/v of inoculum was transferred to M9 minimal medium supplemented with 20 g l⁻¹ of glucose or XOS. E. coli was allowed to grow at 37 °C under 120 rpm for 1–3 days. In all cases, samples were drawn at regular intervals (12–24 h) to estimate cell proliferation based on dry cell weight (mg ml⁻¹) and pH change in the media.

Results and discussion

Immobilization of xylanase in alginate–silica beads

To immobilize xylanase in alginate beads, purified xylanase produced in house were dissolved in different sodium alginate solutions with concentrations between 2–6% (w/v). Fig. 1 shows immobilization yields and xylanase activity yields when different concentrations of sodium alginate (2–6% w/v) were used. These results reveal a close relationship between alginate concentrations and immobilization yields. For example, the immobilization yield gradually increased from 76.5 to 100% when the alginate concentration was increased from 2 to 4% w/v (Fig. 1A). This result suggests that a higher sodium alginate concentration led to formation of a denser matrix such that entrapment of xylanase was more effective. On the other hand, a denser matrix also means smaller pores which can significantly reduce diffusion of substrates into the matrix.^19,21 As shown in Fig. 1A, the immobilized xylanase activity yield decreased from 86.3 to 45.2% when the sodium alginate concentration was increased from 2% to 6%. Next, we used alginate beads as biocatalysts to hydrolyze xylan and determine reducing sugar concentrations. Because the activity of immobilized xylanase was lower than free xylanase, the hydrolysis reaction time was extended to 24 h. Fig. 1B shows the reducing sugar concentrations produced by free xylanase and immobilized xylanase. When the free xylanase was used, the reaction was completed within 6 h. However, when the immobilized xylanase was used, a reaction time between 18–22 h was needed to complete the hydrolysis. The longer reaction time can be attributed to slow diffusion of large substrates such as xylan into the alginate beads.¹⁹ Enzyme immobilization is important when enzymes need to be reused for multiple reactions. To evaluate reusability of immobilized enzymes, alginate beads containing immobilized xylanase were reused for 5 cycles.

Fig. 1 Effect of sodium alginate concentrations on the immobilization of xylanase. (A) Immobilization yields and xylanase activity yields at different sodium alginate concentrations (2–6% w/v). (B) Comparison of free and immobilized xylanase in alginate beads for hydrolysis of xylan. Amounts of reducing sugars reflected xylan activities.

Table 2 shows that alginate beads prepared from lower concentrations of alginate (2–3% w/v) lost their xylanase activity significantly (75–100%) in 1–2 cycles. Additionally, these beads were fragile in nature such that approximately 20% of the beads disintegrated in each cycle. In contrast, beads prepared from higher concentrations of alginate (4–6% w/v) retained more than 50% of their xylanase activity in subsequent cycles, and only ∼5% of the beads disintegrated in each cycle. Based on the production of XOS from 5 repeated cycles (Table 2), sodium alginate concentration was fixed at 4% w/v in the following experiments. However, there are two critical problems need to be addressed. The first one is rapid loss of xylanase activity in the alginate beads. This problem was likely due to leaching of enzyme as shown in the literature.^19,20 The second one is poor stability of the beads due to suboptimal hardness. To investigate leaching of xylanase from the alginate beads, xylanase activity in the reaction mixture was measured after each cycle as shown in Fig. 2A. On the basis of xylanase activity, it can be estimated that >65% of the immobilized xylanase in the beads leached out after 3 cycles of reactions. To reduce leaching of xylanase and improve hardness of alginate beads, different additives including silica gel, chitosan and pectin were tested. They were mixed with 4% sodium alginate solution before gelation. Among them, silica gel was the best additive. Not only did it improve the hardness of the beads, but it also preserved the xylanase activity.

Table 2 Effect of alginate concentration in multiple uses of immobilized beads^c

Alginate (% w/v)	Hardness estimated^a (kgf)	Reducing sugars produced (mg) in repeated use (cycle)^b					Total (mg)
		1	2	3	4	5
a For each type of alginate bead, a minimum of 25 beads were tested and their average value was reported.b Reducing sugars produced from 20 ml of reaction volume; all reactions were conducted in triplicates. To further minimize the error, values estimated from three independent batches of reactions were pooled together and average values were presented.c ND – not detected.
2	0.719	623.70	ND	ND	ND	ND	623.70
3	0.752	561.21	188.17	31.04	ND	ND	780.42
4	0.855	514.55	275.12	92.81	21.4	ND	903.88
5	0.879	406.08	237.37	94.05	24.4	ND	761.90
6	0.926	324.20	210.40	102.25	57.18	21.35	715.38

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Fig. 2 Investigation of xylanase leach-out from the immobilized beads. (A) Xylanase leach-out estimated from beads prepared from alginate (4% w/v) and (B) alginate (4% w/v) with silica gel (3.5% w/v), respectively.

Table 3 shows the hardness of alginate beads with different concentration of silica gel (1–5% w/v).

Table 3 Effect of silica gel concentration on the catalytic performance of immobilized xylanase in alginate beads

Silica gel (% w/v)	Hardness estimated^a (kgf)	Reducing sugars produced (mg) in each cycle^b					Total (mg)
		1	2	3	4	5
a For each type of alginate bead, a minimum of 25 beads were tested and their average value was reported.b Reducing sugars produced from 20 ml of reaction volume; all reactions were conducted in triplicates. To further minimize the error, values estimated from three independent batches of reactions were pooled together and average values were presented.
1	0.980	501.32	370.20	294.20	231.73	164.40	1561.85
2	1.144	497.07	428.80	361.40	279.18	213.36	1779.81
3	1.278	483.60	462.35	435.20	399.06	363.40	2143.66
4	1.466	453.57	421.40	380.17	336.63	300.56	1892.33
5	1.556	416.20	391.00	361.40	331.12	292.80	1792.52

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Addition of silica gel (1% w/v) improved the hardness of the alginate beads from 0.855 to 0.980 kgf. When the silica gel was increased to 5% w/v, the hardness also increased to 1.556 kgf. Consequently, the alginate beads with silica gel (1–5% w/v) were used in 5 consecutive cycles of reactions (Table 3). It can be observed that addition of silica gel (1–5% w/v) slightly reduced the xylanase activity (2.5–9.7%), but the beads remained stable for 5 cycles of reactions, and a large amount of reducing sugars were also produced.

Presumably, addition of silica gel enhanced the hardness of alginate beads and reduced the leaching of xylanase as a result. To test this hypothesis, xylanase leached out to the reaction mixture and remaining xylanase inside the beads were evaluated separately. Fig. 2B shows that after 5 cycles of reaction, only 11.25% of xylanase leached out from the beads. The result shows that silica gel plays a dual role for improving hardness and reducing xylanase leach-out from the alginate beads.

Eventually, the optimized concentrations of sodium alginate (4% w/v) and silica gel (3.5% w/v) were used to prepare alginate beads with immobilized xylanase. These beads had an average diameter of 2.3 ± 0.1 mm. When they were used as biocatalysts for hydrolysis of xylan, they can be used for 7 cycles with >62% of remaining xylanase activity at the end of the 7^th cycle (Fig. 3A).

Fig. 3 Studies on (A) operational efficiency and (B) stability of alginate–silica gel immobilized beads.

Moreover, the immobilized xylanase in the alginate beads were stable at 25 °C in phosphate buffer (pH 6.5), and retained >90% of their activity even after 35 days. In contrast, free xylanase lost ∼50% of their activity in 7 days (Fig. 3B). In literatures, xylanase immobilized in calcium alginate beads had a lower immobilization yield (<70%) and activity yield (<30%). Both issues also led to poor operational stability (5 cycles with 50% of remaining activity).^18,19 Conversely, the immobilization technique reported herein has a very high immobilization yield (∼100%) as well as activity yield (>70%) with good operational stability (7 cycles with >62% of its initial activity).

Production of XOS by free and immobilized xylanase

Next, free xylanase and alginate–silica gel beads containing immobilized xylanase were used to hydrolyze beechwood xylan. Fig. 4A shows the formation of XOS after hydrolysis of beechwood xylan as a function of time. Interestingly, when the immobilized xylanase was used as a catalyst, only xylobiose (51.79 ± 1.50%), xylotriose (46.12 ± 0.65%) and a small amount of xylose (<1%) were generated. No other larger XOS (DP > 3) was detected in the hydrolysate. After 24 h of hydrolysis, the total XOS concentration was 23.74 ± 0.85 mg ml⁻¹ in the hydrolysate. This is the highest concentration ever reported for the production of XOS in a batch process by using immobilized xylanase (Table 1). Moreover, when immobilized xylanases were used to produce XOS in the past, xylose (>10%), a potential contaminant was often produced.^10,13,14 Its presence lowered the value of XOS. In contrast, the immobilized xylanase reported herein naturally liberates XOS (>97%) and very little xylose (<1%) from beechwood xylan (Fig. 4A-inset).

Fig. 4 Production of XOS from xylan hydrolysis by using (A) immobilized (B) free xylanase. XOS produced include xylobiose (hollow square), xylotriose (hollow triangle), xylotetarose (filled square), xylopentaose (filled triangle), xylohexaose (hallow circle), xyloheptaose (filled circle) and xylose (hollow diamond) production by immobilized xylanase. Inset in (A) shows the formation of only xylobiose (X2) and xylotriose (X3) with negligible amount of xylose (X1).

In the case of immobilized Aspergillus niger xylanase, the XOS production and product distribution in the hydrolysate were dependent on the sources of xylan.¹⁷ To investigate this effect with the immobilized xylanase, xylan extracted from teakwood sawdust was used to produce XOS. Teakwood xylan from sawdust was extracted with a yield of 124.8 g kg⁻¹. It contained monosaccharides such as xylose (83.5 ± 4.5%), arabinose (7.3 ± 1.5%), glucose (6.0 ± 1.2%) and galactose (3.2 ± 0.5%). Among them, xylose was the predominant monosaccharide. These values agree with literature values³⁰. When teakwood xylan was hydrolyzed by using the immobilized xylanase, a pattern similar to the case of beechwood xylan was found. The XOS product contained xylobiose (57.50 ± 0.55%), xylotriose (41.25 ± 1.05%), and a small amount of xylose (<0.5%). After 24 h of reaction, the total XOS concentration was 19.75 ± 1.18 mg ml⁻¹. These results show that the immobilized xylanase can act on various sources of xylan, and produce a significant amount of XOS.

In the case of beechwood xylan, hydrolysis reaction catalyzed by free xylanase led to a wide range of XOS product from xylobiose to xyloheptaose (DP = 7) after 30 min (Fig. 4B). Among them, the predominant species were xylobiose (3.45 mg ml⁻¹) and xylotriose (1.85 mg ml⁻¹). Notably, when the reaction progressed, the concentrations of xylohexaose and xyloheptaose gradually increased with time during the first hour. Then, the xylohexaose and xyloheptaose concentration slowly dwindled and completely disappeared after 5 h (Fig. 4B).

Thereafter, no change in XOS concentration was observed over 24 h. The final concentration of XOS was 33.87 ± 1.20 mg ml⁻¹. The XOS produced contained xylobiose (33.72 ± 3.50%), xylotriose (32.10 ± 0.75%), xylotetraose (22.57 ± 0.34%) and xylopentaose (4.39 ± 0.15%) without xylose (<0.5%). Both free and immobilized xylanase released xylobiose and xylotirose as predominant products when xylan was hydrolyzed. However, the immobilized xylanase only liberated xylobiose and xylotriose from xylan; this phenomenon is very unique and has not been reported before. One possible explanation is limited availability of xylan in the alginate beads caused the unique product distribution. Moreover, immobilization may change the protein conformation and catalytic behaviors to a large extend.^8,19,31 For instance, immobilized xylanases of Armillaria gemina produced more xylopentaose (78%) than its free enzyme counterpart (61%).¹⁶ Similarly immobilized xylanase of Pholiota adioposa produced more XOS (10.3 mg ml⁻¹) than free enzyme (5.7 mg ml⁻¹).³² However, to understand this unique phenomenon fully, a molecular-level analysis is necessary. Even though the immobilized xylanases in alginate–silica gel beads only retained 70.1% activity (based on the amount of XOS produced) compared to free xylanase, they were more stable during multiple cycles of hydrolysis. After 10 successful repeated cycles, the immobilized xylanase produced 3342.65 mg of XOS while free xylanase yielded only 677.40 mg of XOS. Thus, immobilization of xylanase in alginate–silica gel matrix can enhance the XOS production by nearly 5 folds compared to free xylanase.

Even though many xylanase immobilization techniques are available, the proposed immobilized technique of using alginate–silica gel is simpler and less expensive (Table 1). Notably, the amount of xylanase required in the current study was only 20 U g⁻¹ xylan, which is significantly less than other cases (50 and 200 U g⁻¹ xylan) reported in the literature.^14,16 Importantly, unlike other XOS-forming xylanase, xylanase produced by strain BOH3 generated a lot of XOS (>95%) and little xylose (<1%) in the hydrolysate. Because of the high XOS concentration, it can be easily purified from the hydrolystate. These advantages make this technique suitable for the mass production of food-grade XOS.

In vitro fermentation of XOS

Because many XOS species show prebiotic effects, the XOS produced from beechwood xylan (BX) and teakwood xylan (TX) were further fermented by various probiotic microbes. For B. animalis and L. acidophilus cultured in BX supplemented medium, cell weight plateaued at 5.35 ± 0.10 mg ml⁻¹ and 2.15 ± 0.06 mg ml⁻¹, respectively, after 48 h of fermentation. In contrast, for the same strains cultured in a glucose-containing medium, the cell weight reached 3.42 ± 0.06 mg ml⁻¹ and 1.82 ± 0.07 mg ml⁻¹, respectively, after 36 h. The result suggests that BX can significantly promote the growth of B. animalis (1.56 times) and L. acidophilus (1.18 times). Similarly, TX can also enhance the growth of B. animalis (1.40 times) and L. acidophilus (1.13 times) compared to glucose. Additionally, when both strains were cultured in BX and TX supplemented medium, the pH dropped from neutral to acidic ranges for both B. animalis (pH 6.25–6.31) and L. acidophilus (pH 6.50–6.58). The decrease in pH can be attributed to excretion of short chain fatty acids (e.g. acetic acid and butyric acid) by probiotic microbes.³³ Notably, E.coli cultured in BX and TX supplemented medium did not grow. In contrast, when a glucose-based medium was used, the cell weight of E. coli increased to 1.55 ± 0.12 mg ml⁻¹ after 24 h of culturing. These results are comparable to other prebiotic XOS reported in the literature.^33,34

Conclusions

Xylanase can be immobilized in alginate (4% w/v) beads through entrapments with a high immobilization yield near 100%. Addition of silica gel (3.5% w/v) to the sodium alginate solution before gelation can enhance the hardness by 1.6 times and gives stronger and more robust beads. The alginate–silica gel beads can be reused for 7 cycles while retaining >62% of the initial activity. They can be used as a catalyst to hydrolyze xylan from various sources and produce 19–24 mg ml⁻¹ of XOS as a final product. This is the highest concentration ever reported for XOS production by using immobilized xylanase. Additionally, the XOS produced by the immobilized xylanase shows significant prebiotic effects on probiotic microbes.

Acknowledgements

This work was supported by the Singapore-MIT Alliance for Research and Technology (SMART) Innovation grant – ING137063-BIO (IGN).

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