Production of Xylo-oligosaccharides from Corncob Using High Efficiency Xylanase from Trichoderma harzianum 4FR8

doi:10.21203/rs.3.rs-4647331/v1

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Production of Xylo-oligosaccharides from Corncob Using High Efficiency Xylanase from Trichoderma harzianum 4FR8

https://doi.org/10.21203/rs.3.rs-4647331/v1

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Xylo-oligosaccharides (XOS) are oligosaccharides components derived from hemicelluloses. Classified as non-digestible sugars, XOS can promote the growth of beneficial bacteria within the gastrointestinal tract, thereby contributing to human health; XOS have been widely used in industry as a prebiotic. Therefore, this research investigated optimizing the conditions for xylanase and XOS production using corncob and wheat bran. The parameters investigated included pH, moisture content, and incubation time. Purified xylanase with ammonium sulfate used an optimum fraction of 40–60%, dialysis, and column chromatography (DEAE-cellulose). The molecular mass of the purified enzyme was 18 kDa. The purified enzyme remained at over 80% relative activity at 40 min at the optimal temperature. The K_m and V_max values using beechwood xylan were 6.57 mg.mL^− 1 and 18,298.26 U.mg^− 1 respectively. XOS produced through xylanase hydrolysis of corncob exhibited the ability to promote the growth of probiotic strains Lactobacillus lactis TISTR1464 and Bifidobacterium bifidum TISTR2129, suggesting its potential application in prebiotic development. These findings highlight the feasibility of utilizing sustainable agricultural residues like corncob for the production of prebiotic XOS via an enzymatic approach. This enzymatically derived product demonstrates potential for various commercial applications, with particular viability for the food and nutraceutical industries.

xylanase

agricultural waste

corncob

purification

xylo-oligosaccharides

solid state fermentation

Xylo-oligosaccharides (XOS) are sugar oligomers produced during xylan hydrolysis from lignocellulosic materials, mostly agricultural waste. XOS includes a degree of polymerization (DP) in the range of 2 to 6 xylose units linked through β-(1,4)-xylosidic bonds [1, 2]. XOS can be produced during xylan hydrolysis from lignocellulosic materials, mostly agricultural waste. Furthermore, XOS are classified as non-digestible food ingredients because they aren’t hydrolyzed and absorbed in the human gastrointestinal tract, which has beneficial host effects [3]. The importance of XOS as a valuable food ingredient is enhanced currently as it has several health-beneficial effects [4]. XOS exhibits enhanced resistance to digestion, leading to a greater ability to stimulate the growth of Bifidobacteria [5, 6]. XOS have been implicated in exerting beneficial effects on gastrointestinal health. These effects are postulated to be mediated by an increase in the Bifidobacteria population and the production of short-chain fatty acids (SCFAs) [7]. XOS promoted the growth of lactic acid bacteria (LAB) such as Bifidobacterium spp. and Lactobacillus spp. used as probiotics [3]. Consequently, XOS have been advantageous and utilized in various applications, including food additives, animal feed, nutraceuticals, healthcare products, and agricultural products [8].

Generally, XOS are obtained through the enzymatic hydrolysis of xylan, as xylan is a main component of hemicellulose, which contains a xylan-lignin complex. Corncob is an agricultural waste, which contains 38% cellulose, 35% hemicellulose, and 14% lignin [9]. A cheap material with readily available biomass, it has a high xylan content, offers great potential, and is frequently used as a substrate for production of XOS. Moreover, corncob has both high fiber content and antioxidant activity [10]. Xylan can be used in various industries, including bioethanol production and the manufacturing of XOS [11, 12]. Lignocellulosic material constitutes a significant component of wastes generated across diverse industries, including agriculture, forestry, and municipalities. This biomass is composed of three distinct polymeric constituents: cellulose, hemicellulose, and lignin. Notably, these polymers are intricately linked and exhibit resistance to enzymatic degradation [12].

The production of XOS involves the fractionation of lignocellulosic material to acquire soluble xylan, followed by enzymatic hydrolysis utilizing xylanolytic enzymes [13], such as xylanase (endo-1,4-β-D-xylanohydrolase; EC 3.2.1.8) is a hemicellulolytic enzyme that randomly catalyzes β-1,4-xylosidic bonds of xylan backbone into small oligomers, such as xylo-oligosaccharides (XOS). Xylanase can be found in a wide range of microorganisms. The production of high-potential xylanase obtains high-quality products that can be further processed into XOS for other applications, particularly in health supplements, sweeteners, nutritional enhancers, and food additives. Fungi can produce xylanases more than other species [14] and they are also more desirable for industrial applications [15]. Enzymes produced by microorganism are an eco-friendly alternative due to low environmental impact [16]. Xylanase is crucial for depolymerization of the xylan backbone [17, 18]; converting xylan is crucial for the lignocellulose-production process to be economically feasible, as it can be developed in various ways to produce biofuels and chemicals [19]. Consequently, the pretreatment process for agricultural waste is significant in enhancing enzymatic hydrolysis and enabling fungi to have easier access to biomass [20, 21]. SSF technique encourages microbial growth and metabolism in still water, which is comparable to fermentation in a natural environment; the SSF has a low water content, and the microorganisms are almost in contact with gaseous oxygen and substrate [22]. Furthermore, the advantages of SSF use include high productivity, decreased operating costs, and lower energy consumption [23].

This study explored a sustainable approach to XOS production by agricultural waste. Corncob, a readily available agricultural residue, was chosen as the substrate for xylanase enzyme production optimization. Following purification and characterization of the xylanase, its efficacy in generating XOS was investigated. Finally, the ability of the resulting XOS to promote the growth of beneficial probiotic strains Lactobacillus lactis TISTR1464 and Bifidobacterium bifidum TISTR2129 was evaluated, paving the way for potential applications in gut health modulation.

2.1 Microorganism

Trichoderma harzianum strain 4FR8, isolated from a fruit sample collected within the area of the Plant Genetic Conservation Project under The Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn, located at Chulabhorn Dam, Konsan district, Chaiyaphum province, northeastern Thailand, was employed in this study. The strain was obtained from the Biohydrogen and Microbial Enzymes Research Group at Khon Kaen University, Thailand. For inoculum preparation, T. harzianum 4FR8 was incubated on potato dextrose agar (PDA) at 30°C for 5 days.

2.2 The optimal conditions for xylanase production

T. harzianum 4FR8 under solid-state fermentation (SSF) was determined by varying the initial pH (4, 5, and 6), moisture content (60, 65, and 70%), and incubation time (3, 5, and 7 days). Six formulations (Table 1) consisting of corncob and wheat bran were performed by polypropylene bag (12 × 18 inches). A plastic tube (2 × 4.5 inches) was covered in a bag with cotton wool [24] and sterilized at 121°C for 60 min. The concentration of spore suspension was 1 × 10⁸ spores.mL^− 1 and incubated at 30°C.

Table 1

Formula of the cultivation materials for xylanase production.
Formula	Corncob (g)	Wheat bran (g)	C/N ratio
A	10	10	33:1
B	8	12	28:1
C	12	8	37:1
D	16	4	45:1
E	18	2	49:1
F	20	-	53:1

2.3 Enzyme extraction

The crude enzyme was extracted from SSF with 0.05 M citrate phosphate buffer, pH 5 (30 mL), incubated at 4°C for 24 h, then filtered through muslin cloth and centrifugated at 6,950 ×g for 10 min at 4°C.

2.4 Xylanase activity assay

The enzyme activity was investigated with 3,5-dinitrosalicylic acid [25]. The enzyme (0.5 mL) and 1% (w/v) beechwood xylan (0.5 mL) (Sigma, Germany) were incubated at 45°C for 30 min and the reaction stopped with 1 mL of 3,5-dinitrosalicylic acid (DNS) [26]. The released reducing sugars were measured at an absorbance of 540 nm, using xylose as a standard. One unit of xylanase activity (IU) was defined as the amount of enzyme-hydrolyzed xylan to 1 µmol of xylose per minute under experimental conditions [27].

2.5 Protein determination

The amount of protein was determined by Lowry method [28], using bovine serum albumin (BSA) as the standard. Protein presence and quantification of the protein were measured at 280 nm.

2.6 Purification of xylanase

The purification of the enzyme began with the salting-out procedure, performed with ammonium sulfate added to saturation of 20, 40, 60, 80, and 100%, respectively. The precipitated enzyme solution was subjected to centrifugation at 6,950 ×g for 10 min at 4 ºC and dissolved in 0.05 M citrate phosphate buffer (pH 5). Dialysis was performed in a dialysis bag against 0.05 M citrate phosphate buffer (pH 5) at 4 ºC. The dialysis was finished when the dialysate conductivity was constant. The dialyzed enzyme solution was further purified with column chromatography. The enzyme solution with dialysis was purified by DEAE-cellulose chromatography. Consequently, 0.05 M citrate phosphate buffer (pH 5) was used for equilibrating and washing the column. Additionally, 1 M sodium chloride was eluted at a flow rate of 0.5 mL.min^− 1 [29].

2.7 Analysis of electrophoresis (SDS-PAGE) and zymogram

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was retained to evaluate protein profiles. The gels were composed of 12.5% (w/v) acrylamide and stained with Coomassie Brilliant Blue R-250 for protein visualization. Subsequent de-staining was performed using a solution of methanol, acetic acid, and double-distilled water in a ratio of 4:1:5 (v/v/v) [19]. The standard proteins (Rainbow™ Marker, RPN800E, Sigma, USA) were used for calculating the mass of molecules from the relative mobility of the proteins. For zymogram analysis, adapted from [30], 0.1% (w/v) beechwood xylan (Sigma, Germany) was employed to visualize xylanase activity within the SDS-PAGE. Briefly, 0.1% (w/v) beechwood xylan (Sigma, Germany) was incorporated into the separating gel during preparation. Following electrophoresis, the gel was washed twice with 0.05 M citrate phosphate buffer (pH 5) to remove the unbound substrate. Subsequently, the gel was incubated in the same buffer at 45°C to allow the enzyme within the gel matrix to hydrolyze the embedded beechwood xylan substrate. The gel was stained with Congo red solution. Finally, the gel was de-stained using a 1 M sodium chloride (NaCl) solution. Xylanase activity zones appeared as clear bands against a red background due to the selective binding of Congo red to the unhydrolyzed beechwood xylan.

2.8 Effect of temperature on xylanase activity

The reaction was incubated at 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 ºC on xylanase assay conditions using beechwood xylan (Sigma, Germany) as a substrate for 30 min. Thermal stability was incubated at 30–80 ºC for 1 h, then xylanase activity measured [31].

2.9 Effect of pH on xylanase activity

The optimum pH was resolved using different 0.05 M buffers such as citrate phosphate buffer pH (3–5), sodium phosphate buffer (pH 5–7), Tris-HCl (pH 7–9), and sodium glycine buffer (pH 9–11) at 30°C for 30 min [32]. The stability of pH was incubated in buffer ranges of pH 3–11 at 30°C for 1 h and measured xylanase activity [33].

2.10 Half-life of xylanase activity

The pure xylanase was incubated at 45°C with increasing intervals of time (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min) and xylanase activity measured.

2.11 Kinetics

The enzyme was incubated with 1% (w/v) beechwood xylan (Sigma, Germany) with concentrations varying from 1, 2, 5, 10, and 20 mg.mL^− 1. The Michaelis-Menten constant (K_m, mg.mL⁻¹) and the maximum velocity (V_max, U.mg⁻¹) were determined using the Lineweaver-Burk linearization method [34]. This approach involves plotting the reciprocal of the substrate concentration (1/[S]) against the reciprocal of the reaction velocity (1/v). The K_m and V_max values can then be obtained from the slope and y-intercept [35].

2.12 Analysis of hydrolysis products

Purification of the enzyme (5 mL) and 0.05 g of corncob (0.044 mm.) was then carried out at 40°C. After 0, 12, 24, 36, and 48 h of incubation. Subsequently, the supernatant was collected to determine hydrolysate composition [36]. The purity of XOS was analyzed by thin layer chromatography (TLC). The hydrolytic product (10 µL) was spotted on the TLC silica gel plate (60 F254, Merck). The mobile phase consists of butanol, acetic acid, and deionized water (5:3:2, v/v/v). After running for two ascents, the TLC plate was detected by spraying with ethanol containing 5% (v/v) sulfuric acid (H₂SO₄). Subsequently, TLC plates were dried at 150°C for 5 min until brown spots appeared on the plate. The mixture containing xylose (Sigma, USA), xylobiose (Tokyo Chemical Industry, Japan), xylotetraose (Megazyme, Ireland), and xylohexaose (Megazyme, Ireland) (1 mg.mL^− 1) were used as standard sugars [37]. The data from TLC were confirmed quantitatively by HPLC (Shimadzu, Tokyo, Japan) with a Fortis Amino FNH column. Xylose, xylobiose (X2), xylotriose (X3), and xylotetraose (X4) were used as standards for the quantification of sugars.

2.13 Application of XOS by corncob

The potential effects of XOS produced from corncob through an enzymatic reaction investigated two probiotic strains of lactic acid bacteria (LAB) including Lactobacillus lactis TISTR1464 and Bifidobacterium bifidum TISTR2129. These strains were cultured in their respective optimized medium: MRS medium was utilized for L. lactis TISTR1464, while MRSL medium was utilized for B. bifidum TISTR2129. Notably, XOS served as the primary carbon source for both strains throughout the experiment.

Growth and potential XOS utilization were monitored over a 72-h incubation period at 37°C under static conditions. The hydrolysates were collected for analysis at each period (0, 12, 24, 48, and 72 h). Firstly, pH measurements provided insights into metabolic activity and possible acidification associated with lactic acid production. Secondly, viable cell counts were determined using the established drop-plate technique [38] under anaerobic conditions. The viable cells were enumerated as colony-forming units (CFU) per mL^− 1.

2.14 Statistical analysis

Statistic analysis was performed using IBM SPSS statistics 28.0.0.0. Multiple comparisons of the ANOVA procedure determined the significance of enzyme production with the least significant difference (LSD), while p < 0.05 was used to determine any significant differences, that were identified between treatments.

3.1 optimization of xylanase production

The effects of SSF were analyzed independently in static cultures at different parameters. All treatments significantly affect the activity of enzyme (p < 0.01). The highest xylanase production occurred at formula B (8 g. of corncob and 12 g. of wheat bran), pH 6 with 70% moisture content after 3 days of cultivation (178,623 IU.mL^− 1 or 267,934.5 IU.gds^− 1) by T. harzianum 4FR8 (Fig. 1). Therefore, this condition was selected for use in subsequent experiments. Other reports are available on the production of xylanase in SSF by Trichoderma sp. and the enzyme yield was lower than this research. Similar observation was reported form T. harzianum PPDDN10 NFCCI-2925 using wheat bran incubation with initial pH 6, 1:3 of moisture ratio at 34°C for 4 days (1,208.6 ± 54.3 IU.gds^− 1) [39]. T. harzianum was reported xylanase from wheat bran (4.4 IU.mL^− 1) and corncob (3.03 IU.mL^− 1) [40]. The highest activity by T. harzianum strain HZN11 under SSF utilized wheat bran (1,673 ± 0.25 IU.mL^− 1) and corncob (1,472 ± 0.40 IU.mL^− 1) for 7 days [41]. Maximum xylanase yield was discovered using corncob at 96 h from T. harzianum (2,968.69 IU.mL^− 1) [42]. The xylanase production by T. harzianum kj831197.1 used wheat bran at 30°C, pH 5 (510 ± 0.32 IU.mL^− 1) [43].

3.2 Biochemical characterization of the purified xylanases

The crude xylanase was subjected to a multi-step purification process. Firstly, ammonium sulfate precipitation was 40–60% saturation. Following this, dialysis was performed to remove residual ammonium sulfate. Finally, ion-exchange chromatography using DEAE-cellulose was utilized for further purification (data not shown).

3.2.1 Analysis of electrophoresis (SDS-PAGE) and zymogram

Analysis by SDS-PAGE revealed the presence of a single protein band, indicating the purified xylanase exists as a monomer composed of a single polypeptide chain [35]. A purified enzyme in 12.5% polyacrylamide gels for detecting a homogeneous single band with a predictable molecular mass and the zymogram of the active band of xylanase demonstrated the presence of a clear zone of hydrolysis in lane 1, with a molecular mass of 18 kDa (Fig. 2). The molecular mass of xylanases depends on the fungal species and the purification method. According to the literature, the xylanase molecular mass of Trichoderma harzianum was reported to vary between 22, 23, 32, and 72 kDa, respectively [44, 45, 43, 46].

3.2.2 Optimal temperature and stability for xylanase activity

The purified xylanase activity increased at the range of 35 to 45°C, representing the highest activity at

45°C, where it retained 100% of relative activity. The xylanase was found to be highly stable, retaining 100% relative activity over temperatures between 30 to 45°C. At 50°C, xylanase retained 99% of its relative activity, while at 55 and 60°C, it retained 98% and 60% of relative activity, respectively. However, at 65°C and above, the enzyme activity and thermostability declined rapidly within 1 h (Fig. 3). The optimal temperature was reported between 40–70°C [43, 44, 39, 46]. The stability of temperatures was reported at 55–60°C [39].

T. harzianum 4FR8 under solid state fermentation (SSF) for xylanase production. The experiments were replicated three times and error bars are included in the figure. Significant difference according to four-way ANOVA by LSD (p < 0.01).

Lane 1: zymogram.

3.2.3 Optimal pH and stability for xylanase activity

The maximum purified xylanase activity was obtained with 0.05 M citrate phosphate buffer, pH 5 at 30°C for 1 h (Fig. 4). The pH ranged from 3 to 7, and the xylanases retained exceeded 50% relative activity. However, at sodium phosphate buffer, pH 5 the enzyme activity declined. Therefore, xylanase activity slightly declined when the pH was above pH 5. For pH stability, the enzyme revealed 100% of maximum activity in citrate phosphate buffer, pH 5 while in the range of pH 4 to 7, the enzyme revealed up to 80%. Currently, most xylanases of fungi origin show higher activity at a pH range between 3 to 5, and stability over a wide pH range (3 to 10) [47, 48].

3.4 Half-life

The stable of purified xylanase was found at 45°C for 0 to 50 minutes of incubation (Fig. 5). This means the enzyme retained its full activity for up to 50 minutes when incubated at 45°C. This is a significant finding, as it indicates the xylanase enzyme is relatively thermostable. This could make it a useful enzyme for industrial applications where high temperatures are required. The estimated half-life of xylanase was reported at 50°C after 70 min [44], further substantiating its potential for industrial use.

3.5 Kinetics of xylanase activity

The kinetics parameters of xylanase enzymes are important for understanding how enzymes work and for optimizing their use in industrial applications. The Michaelis constant (K_m) and maximum velocity (V_max) were estimated to the corresponding Lineweaver-Burk plot and found to be 6.57 mg.mL^− 1 and 18,298.26 U.mg^− 1, respectively (Fig. 6). These results indicate that the xylanase from T. harzianum 4FR8 exhibits both high affinity (low K_m) and high catalytic efficiency (high V_max) for beechwood xylan. A comparable value of K_m and V_max for pure xylanase was greater than other researchers. The kinetics of xylanase from T. harzianum revealed K_m 5.56 mg.mL^− 1 and V_max 1,052.63 µmol.min.mg^− 1, respectively [46]. The xylanase from T. harzianum 4FR8 possesses a significantly higher V_max compared to both strains highlighted, while maintaining a comparable K_m value. The ratio of V_max to K_m, also known as the catalytic efficiency (CE), provides a valuable metric for comparing the overall efficiency of enzyme activity. Furthermore, the T. harzianum 4FR8 xylanase exhibited a higher V_max/K_m ratio compared to the xylanases from both T. harzianum and T. harzianum T4. The higher CE value indicates the T. harzianum 4FR8 enzyme exhibits significantly greater catalytic efficiency in hydrolyzing beechwood xylan, signifying its potential for more efficient applications compared to the other evaluated strains.

3.6 Hydrolysis of corncob by xylanase

Xylo-oligosaccharides (XOS) hydrolyzed from corncob by pure xylanase from T. harzianum 4FR8 as shown in Fig. 7. The impact of reaction time was studied at 40°C for 0, 12, 24, 36, and 48 h. The results showed that the highest XOS yield was xylobiose (X2). Meanwhile, other XOS, such as xylose, xylotriose (X3), and xylotetraose (X4) were also detected. Table 2 shows that the xylose and XOS with the highest yield of XOS produced was xylobiose (X2) after 48 h. This suggests the xylanase from T. harzianum 4FR8 can hydrolyze xylan into a variety of XOS, which is advantageous for XOS production. Furthermore, different XOS have differing nutritional properties. This study also found that the amount of XOS produced increased along with hydrolysis time. This suggests the xylanase from T. harzianum 4FR8 can continuously hydrolyze xylan from agricultural waste materials, such as corncob. XOS production from maize straws was xylose (18.42% ± 0.59), xylobiose (58.37% ± 1.63), and xylotriose (17.57% ± 0.32) [49].

Table 2

Analysis of xylo-oligosaccharides and xylose using HPLC.
Sugar	Concentration (mg.mL^− 1)
Xylose (X1)	0.57 ± 0.01
Xylobiose (X2)	2.51 ± 0.03
Xylotriose (X3)	0.12 ± 0.00
Xylotetraose (X4)	0.08 ± 0.00

3.7 Application of XOS

This study assessed the potential of XOS derived from corncob via enzymatic hydrolysis (XOS_corncob) by evaluating their utilization by lactic acid bacteria (LAB) strains e.g. Lactobacillus lactis TISTR1464 and Bifidobacterium bifidum TISTR2129. Table 3 summarizes the growth and acidification profiles of these bacteria when XOS_corncob was provided as the primary carbon source for the growth promotion of these beneficial microbes.

The results confirmed the ability of L. lactis TISTR1464 to effectively utilize XOS_corncob for growth. Viable cell counts progressively increased over the incubation period, reaching a maximum of 1.14 ± 0.1 log CFU mL^− 1 at 48 h (Table 3). Similarly, B. bifidum TISTR2129 exhibited growth on XOS_corncob media, with viable cell counts ranging from 0.94 ± 0.04 log CFU mL^− 1 at 12 h to 1.17 ± 0.03 log CFU mL^− 1 at 72 h (Table 3). Notably, acid production by both strains displayed a positive correlation with their growth, suggesting XOS_corncob fermentation.

These findings align with previous reports indicating the preferential utilization of shorter chain XOS by LAB [50]. These bacterial genera are recognized for their potential health benefits in humans [51], as evidenced by Moura et al [52]. In addition, Bifidobacterium strains are known for their efficient fermentation of xylose-based oligo- and polysaccharides, aligning with their potential utilization of corncob-derived XOS. Additionally, prebiotic compounds are often employed to stimulate growth of the beneficial Lactobacillus genus within the gut microbiota [53].

Table 3

The growth parameters of probiotics on MRS/MRSL with XOS prepared by agricultural residue xylan from corncob (XOS _corncob).
Probiotic	Time	XOS_corncob		Probiotic	Time	XOS_corncob
Probiotic	Time	pH	Cell viability (log CFU.mL^− 1)	Probiotic	Time	pH	Cell viability (log CFU.mL^− 1)
L. lactis TISTR1464	0 h	5.47	0.85 ± 0.12	B. bifidum TISTR2129	0 h	5.54	0.6 ± 0.26
	12 h	5.38	0.94 ± 0.2		12 h	5.41	0.94 ± 0.04
	24 h	5.11	0.78 ± 0.13		24 h	5.14	0.82 ± 0.14
	48 h	5.11	1.14 ± 0.1		48 h	5.14	0.97 ± 0.17
	72 h	5.13	1.04 ± 0.16		72 h	5.17	1.17 ± 0.03

While previous studies, such as corncob-derived XOS to promote Bifidobacterium and Lactobacillus growth, further insights into specific growth capacities are valuable. Building upon this prior research: the fermentation of xylo-oligosaccharides from banana pseudostem xylan by Lactobacillus plantarum (ATCC 8014) and Lactobacillus fermentum (ATCC 9338). After 72 h, the growth of the two strains were 1 × 10¹¹ CFU.mL^− 1 and 8 × 10¹⁰ CFU.mL^− 1, respectively [53].

This study explored the prebiotic potential of XOS derived from corncob using xylanase produced by Trichoderma harzianum 4FR8. The results demonstrated that T. harzianum 4FR8 is a potent xylanase producer, capable of generating high enzyme yields from sustainable agricultural waste materials like corncob and wheat bran. This finding highlights its potential as a cost-effective and environmentally friendly approach for industrial XOS production. Optimal enzyme activity was observed at 45°C and pH 5. Furthermore, the enzyme exhibited stability for 40 min while retaining activity. XOS produced from corncob fermentation demonstrated the ability to promote the growth of L. lactis TISTR1464 and B. bifidum TISTR2129. These promising findings pave the way for further research to comprehensively evaluate the prebiotic potential of XOS derived from corncob. Future studies should include a detailed analysis of XOS composition and its impact on gut microbiota. Additionally, a rigorous evaluation of XOS efficacy within in vivo models and a comprehensive assessment of its commercial viability for diverse food and nutraceutical applications are warranted.

Acknowledgements

This research was co-supported by the research capability enhancement program through graduate student scholarship, Faculty of Science, Khon Kaen University; and the framework of Plant Genetic Project under The Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn, Khon Kaen University which was supported by the Fundamental Fund of Khon Kaen University and the National Science, Research and Innovation Fund (NSRF), Thailand.

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Production of Xylo-oligosaccharides from Corncob Using High Efficiency Xylanase from Trichoderma harzianum 4FR8

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Microorganism

2.2 The optimal conditions for xylanase production

2.3 Enzyme extraction

2.4 Xylanase activity assay

2.5 Protein determination

2.6 Purification of xylanase

2.7 Analysis of electrophoresis (SDS-PAGE) and zymogram

2.8 Effect of temperature on xylanase activity

2.9 Effect of pH on xylanase activity

2.10 Half-life of xylanase activity

2.11 Kinetics

2.12 Analysis of hydrolysis products

2.13 Application of XOS by corncob

2.14 Statistical analysis

3. Results and discussion

3.1 optimization of xylanase production

3.2 Biochemical characterization of the purified xylanases

3.2.1 Analysis of electrophoresis (SDS-PAGE) and zymogram

3.2.2 Optimal temperature and stability for xylanase activity

3.2.3 Optimal pH and stability for xylanase activity

3.4 Half-life

3.5 Kinetics of xylanase activity

3.6 Hydrolysis of corncob by xylanase

3.7 Application of XOS

4. Conclusions

Declarations

Acknowledgements

References

Status:

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