The production of value-added bioproducts from renewable carbon sources has gained increasing attention in recent years. Bacillus subtilis MR42 was used to produce specific xylooligosaccharides (XOS) from corn stalks. This strain, through a genetic modification targeting the xynC gene, can convert arabinoxylan into specific XOS without generating xylose as a byproduct. Under optimal conditions, a high yield of XOS (703.4 mg/g xylan) was obtained. The purified XOS were further characterized using various techniques including TLC, HPLC, ESI-MS and 1HNMR spectroscopy. The analysis revealed that the XOS primarily consisted of xylotriose (289 ± 0.03 mg/g), xylotetraose (237 ± 0.05 mg/g) and xylopentaose (267 ± 0.03 mg/g), accounting for approximately 80% of the total XOS. The prebiotic effect of XOS was evaluated by examining its impact on the growth of Lactobacillus, the production of short-chain fatty acids, and its antioxidant activity. B. subtilis MR42 showed a promising ability to produce short DP of XOS without xylose.
Research Article
Harnessing the Power of Bacillus subtilis MR42: Direct Production of Defined Xylo-Oligosaccharides (XOS) from Corn Stalks with Enhanced Prebiotic Properties
https://doi.org/10.21203/rs.3.rs-3769836/v1
This work is licensed under a CC BY 4.0 License
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Xylooligosaccharides
Bacillus subtilis
Corn stalks
Lactobacillus
Xylo-oligosaccharides (XOS) are oligosaccharides produced synthetically, composed of 2–7 xylose units linked by β-1,4-glycosidic bonds. These valuable compounds have a wide range of applications in diverse industries (Karlsson et al., 2018; Aachary et al., 2011). XOS with a degree of polymerization (DP) less than 4 have been shown to promote the proliferation of beneficial bacteria such as bifidobacteria in the human intestinal tract. The worldwide XOS market is projected to reach a valuation of $130 million (USD) by 2025, exhibiting a Compound Annual Growth Rate (CAGR) of 4.1% throughout the forecast period (Patel and Shah, 2021).
Lignocellulosic biomass, renowned for its high hemicellulose content (approximately 30%), presents a promising feedstock for XOS production (Ostadjoo et al., 2019). XOS are primarily obtained though the extraction and hydrolysis of xylan from corncobs, which contain approximately 40% xylan (Yang et al., 2005). However, corncobs have limited availability and relatively higher cost, resulting in increased production costs for XOS. As the most representative type of lignocellulosic biomass in China, corn stalks (CS) accounts for approximately 60% of total straw production and offers a cost-effective and readily available alternative. Unfortunately, CS is either burned or discarded after harvesting, resulting in resource wastage and environmental pollution (Chapla et al., 2012). The hemicellulose in corn stalks consists mainly of L-arabino-β-(1→4)-D-glucuronoxylan units in the extracted form. Efficient extraction of hemicellulose is a prerequisite for its utilization. However, the production and purification processes for XOS from hemicellulose are complex and costly, hindering their widespread use (Chen et al., 2016; Rathamat et al., 2021). Therefore, there is significant value in developing a technology that can efficiently extract hemicellulose from biomass and produce specific XOS.
Oligosaccharides are commonly produced by enzymatic hydrolysis of lignocellulosic biomass or agricultural residues, requiring the addition of relatively expensive enzymes for polysaccharide degradation (Jian et al., 2018; Karboune et al., 2022). To overcome these limitations, a potential strategy is to develop bacterial biocatalysts capable of secreting highly efficient enzymes for polysaccharide breakdown into oligosaccharides. Various microbial strains, such as Bacillus subtilis (Rhee et al., 2014), Aspergillus niger (Tan et al., 2020), Trichoderma reesei (Marjamaa et al., 2022), Thermobifida fusca (Shi et al., 2020), and Trichoderma viride (Precup et al., 2022), have been extensively studied for their capacity for their enzymatic secretion capacity and proficient assimilation of oligosaccharides. Our research team has investigated the metabolic capabilities of Bacillus subtilis strains, specifically their ability to enzymatically degrade hemicellulose extracted from agricultural biomass to produce XOS, acidic XOS, and other valuable products (Wei et al., 2016; Rhee, et al., 2016; Wei et al., 2018). Additionally, Wan et al. investigated the potential of B. amyloliquefaciens WX-1 as biocatalyst for the direct conversion of polysaccharides from Amorphophallus konjac C. Koch into functional prebiotics (Wan et al., 2021). Reque et al. identified that the probiotic Bacillus subtilis FTC01 has the capability to bioprocess wheat middlings and produce prebiotics such as XOS (Reque et al., 2019). These studies highlight the efficient metabolic systems of various bacterial species, enabling more effective bioconversion and minimizing the requirement for complex multi-step procedures to obtain targeted bio-based products.
Bacillus subtilis 168 has been shown to produce GH11 (Xyn11A) and GH30 (Xyn30C) endoxylanases (St. John et al, 2006; Tjalsma et al, 2004). This strain is capable of enzymatically processing hardwood methylglucuronoxylans (MeGXn) and monocots methylglucuronoarabinoxylans (MeGAXn), resulting in the accumulation of methylglucuronoxylotriose (MeGX3) in the culture, while xylobiose (X2), xylotriose (X3), and a small fraction of xylose are released and uptaked for cell growth (Rhee et al., 2014). Our previous studies have also demonstrated the ability of Bacillus subtilis MR44, which secretes GH30 (Xyn30C) endoxylanases and GH43 arabinoxylan 𝛼-1,3-arabinofuranohydrolase (Axh43), to efficiently degrade xylan extracted from biomass to accumulate acidic-XOS in abundant yields and purity (Wei et al., 2015). Considering the generally regarded as safe (GRAS) status of several B. subtilis strains, B. subtilis MR42, which is a mutant strain of B. subtilis 168 with the xynC gene disrupted, was developed. This disruption is achieved by introducing a kanamycin resistance gene fragment to prevent xylose release. B. subtilis MR42 secretes only the endo-1,4-β-xylanase Xyn11A, which can randomly catalyze the hydrolysis of 1,4-β-xylose linkages and converts MeGXn to predominantly xylobiose, xylotriose and MeGX4 (Rhee et al., 2014). In addition to Xyn11A, B. subtilis MR42 also produces an Axh43 enzyme from the GH43 family of arabinoxylan 𝛼-1,3-arabinofuranohydrolases to effectively cleave the arabinose side chain attached to main chain (Sawhney et al, 2015; Wei et al, 2016; Wei et al, 2018). Hence, the selective secretion of Xyn11A and Axh43 by MR42 suggests its potential for XOS production from certain types of hemicellulose, such as arabinoxylan.
With the rising demand for XOS in the market, the objective of this study was to explore the utilization of B. subtilis MR42 for the direct production of targeted XOS from corn stalks within the context of a corn stalks biorefinery. To the best of our knowledge, there have been limited reports investigating on the direct production of defined XOS by engineered strains. The entire production chain, including pretreatment with sodium chlorite and sodium hydroxide (lignin removal and xylan extraction), bacterial hydrolysis, purification, structural characterization, and the assessment of the final product’s nutritional properties (prebiotic potential and antioxidant activity), was investigated. Qualitative and quantitative analysis of XOS was performed using TLC, HPLC, ESI-MS and NMR spectroscopy. Furthermore, the prebiotic effects of XOS on three different probiotic Lactobacillus strains, as well as their antioxidant activities, were evaluated.
2.1 Materials and chemicals
Corn stalks were obtained from the Guanzhong area of Shaanxi Province, China, and then oven-dried at 60°C for 24 h. The dried stalks were ground to a particle size of ≤ 1 mm and stored in a sealed plastic bag.
Bacillus subtilis MR42, which was described in reference (Rhee et al., 2014), was obtained from the Department of Microbiology and Cell Science at University of Florida. Lactobacillus plantarum CGMCC 1.9087 and Lactobacillus casei CGMCC 1.575 were purchased from China General Microbiological Culture Collection Center, while Lactobacillus brevis YM 1301 was provided by the Yunnan Institute of Microbiology.
Monosaccharides, including xylose, xylobiose, xylotriose, xylotetraose, mannose, rhamnose, arabinose, were purchased from Sigma-Aldrich Corporation (Shanghai, China). Additional chemicals and reagents were purchased from various suppliers such as Jingbo Biotechnology Co., Ltd (Shanxi, China), Merck (Germany), and Jinan manalab Co., Ltd. (Shandong, China).
2.2 Methods
The technical flowchart for this paper is shown in Fig. 1.
2.3 Pretreatment and delignification of corn stalks
The raw material was pretreated with deionized water and ethanol according to Zhang’s method with slight modifications (Zhang et al., 2018). Water pre-treatment was performed with a solid to liquid ratio of 1:20 (g/mL) at 90°C for 2 h, and the ethanol pre-treatment was performed with a solid to liquid ratio of 1:5 (g/mL) at room temperature for 2 h. The pretreated CS was delignified with 8% sodium chlorite at 90°C for 2 h (Wang et al, 2021) until the CS powder turned white. The delignified CS was thoroughly washed to neutrality and dried in an oven at 45°C overnight.
2.4 Extraction of xylan from delignified corn stalks
20 g of corn stalks was treated with 10% NaOH (w/w) and incubated in an oven for 2 h at a temperature of 80°C. The supernatant fraction was collected by centrifugation at 7500 rpm for 20 min and subsequently neutralized to pH 6–7 with hydrochloric acid. After alkaline extraction, alcohol precipitation was applied to prepare the xylan.
2.5 Analysis of main components of the extracted xylan from corn stalks
The NREL method (National Renewable Energy Laboratory [NREL], 2013) was used to determine the xylan content of corn stalks. 0.3 g of the extracted powder was transferred to a 10 mL centrifuge tube and treated with 3 mL of 72% H2SO4 in a water bath at 35°C for 1 h. The resulting sample was carefully transferred to a 100 mL flask. 84 mL of ultrapure water was added to the flask and the combined solution was weighed. The resulting mixture was then sealed before being subjected to sterilization at a temperature of 121°C for a duration of 1 h. The sterilized mixture was then subjected to filtration using a Buchner funnel. The pH of the filtrate was adjusted to a range of 1–3 using hydrochloric acid. The concentrations of glucose, xylose, and arabinose in the filtrate were determined using high-performance liquid chromatography (Shimadzu, LC2050). A Shimadu C18 column (250 mm×4.60 mm, 3 µm) equipped with a PDA detector set at a wavelength of 254 nm was employed for the analysis. The column temperature was maintained at a constant 30°C throughout the experiment. The mobile phase was acetonitrile-ammonium acetate solution (18.5:81.5, V/V) at a flow rate of 0.8 mL/min. A 10 µL injection volume was applied, and the total analysis process lasted for 46 minutes.
To characterize the xylan extracted from the delignified corn stalks, Fourier transform infrared (FTIR) spectroscopy was performed using the Bruker Tensor 27 instrument. Dried KBr powder and the xylan sample were thoroughly ground and mixed in a ratio of 100:1 before being pressed into tablets. Detection was performed after correction for potassium bromide background, and the spectra was scanned over the range of 4000-400cm− 1.
2.6 B. subtilis strain and growth medium
The MR42 strain (168, ΔxynC-Km) was described in ref (Rhee et al., 2014). The MR42 strain was cultured in LB broth with 100 µg/mL of kanamycin (Lennox L broth) for 24 h at 37°C. For cultivation on xylan extracted from corn stalks, Spizizen's medium (Spizizen, 1958) containing tryptophan at 25 ug/mL was used.
2.7 Optimization of XOS production
The effects of xylan concentration, culture temperature and culture time on XOS production were investigated in single factor experiments. Xylan concentration was in the range of 10 g/L-100 g/L, culture temperature was in the range of 37°C -57°C, and culture time was in the range of 0 h-20 h. One variable was examined in each experiment while the other factors remained constant. The hydrolysis rate of xylan by MR42 was calculated using the following formula.
2.8 TLC analysis of MR42 hydrolysis products
TLC analysis of MR42 cultures was carried out on 20 × 20 cm silica gel glass plates with a developer solution of ethyl acetate : n-butanol : glacial acetic acid : water at a ratio of 4:3:2:2 (v: v: v: v) (Song et al., 2018). The colorant was prepared by dissolving 0.41 g of lichenol and 250 mL of methanol in 41 mL of sulfuric acid. The spots were showed by spraying the colorants onto TLC plates, followed by heating at 80°C for 10 min. Related monosaccharides and polysaccharides at a concentration of 5 g/L were employed as standard references. Cultures grown for 0 h, 2 h, 4h, 6 h, 8 h, 10 h, 15 h and 20 h with extracted arabinoxylan by the strain MR42 were used as samples.
2.9 Separation and purification of Xylooligosacchrides
The activated carbon adsorption method is widely applied to separate and purify the XOS from mixture. After the MR42 culture was centrifuged at 6000 rpm for 15 min to separate the cells, the pH of the resulting supernatant was adjusted to 2 with hydrochloric acid, and an acidic precipitation reaction was performed at 60°C for 1 h. The resulting supernatant was collected by centrifugation, and 0.04 g/mL of activated carbon was added for an adsorption reaction. This mixture was then incubated at 50°C for 40 min. Following the adsorption reaction, diatomaceous earth (2%, w/v) was added, and the activated carbon filter cake was obtained through filtration. To elute the activated carbon cake, 30% ethanol was used as eluent, and the eluates were further concentrated and lyophilized to obtain pure XOS (Zhang et al., 2021).
2.10 HPLC analysis of PMP derivatives of XOS
The PMP derivatization method for XOS analysis followed the procedure described by Zhang et al. (2003). Analysis of the PMP derivatives of XOS was performed using an HPLC system equipped with a Shimadu C18 column (250 mm × 4.60 mm, 3 µm) and a mobile phase consisting of sodium phosphate buffer (40 mM, pH 8.0) and acetonitrile (79:21, v/v). The HPLC system operated at a flow rate of 0.5 mL/min. Detection of the PMP derivatives of XOS was conducted at 245 nm.
2.11 Electrospray ionization mass-spectrometry (ESI-MS) analysis of purified XOS
The possible structure of purified XOS was analyzed using an Agilent 1290 UPLC & 6540B Q-TOF-MS equipped with an electrospray ion (ESI) source system by performing positive ion mode continuum mode operations. The parameters utilized were as follows: an interface temperature of 250°C, a nebulizer gas (N2) flow of 90 L h− 1, an ionization voltage of 4.0 kV, and a detector voltage of 1.6 kV. The scan rate was set to normal for a mass range of m/z 25-20000.
2.12 1HNMR analysis of purified XOS
A Mercury 600 MHz spectrometer, equipped with a 5 mm pulsed-field gradient broadband probe, was utilized for the 1HNMR spectroscopic studies. Purified XOS produced by MR42 were dialyzed three times using dialysis tubing (12 KD, Merck Co.) against 2 L of water. The resulting samples, with a total carbohydrate concentration of 2.5 g/L, were freeze-dried under vacuum, dissolved in D2O (99.9 atom % D, Sigma-Aldrich) and lyophilized three times. Subsequently, the lyophilized samples were dissolved in 0.55 mL of D2O containing 0.05% of 3-(trimethylsilyl) propionic-2,2,3,3-d4-acid (98 atom % D, Sigma-Aldrich) as a chemical shift reference (0.0 ppm). The exchanged samples were then injected to Wilmad 505-PS NMR tubes and detected using the 1HNMR spectrometer. MestReNova 14.0 was utilized to process the data and spectra obtained from the 1HNMR spectrometer ( Mikkelson, Maaheimo, & Hakala, 2013; Wan et al., 2021).
2.13 In vitro fermentation of purified XOS and SCFAs detection
The growth-promoting potential of XOS was assessed with Lactobacillus plantarum CGMCC 1.9087, Lactobacillus casei CGMCC 1.575, and Lactobacillus brevis YM 1301 strains. These strains are lactic acid bacteria, which have health-promoting properties. (Qin et al., 2023; Wan et al., 2021; Wei et al., 2023).
All three strains were incubated overnight at 37°C in MRS medium. Fermentation was carried out in a minimal MRS broth medium, supplemented separately with no carbohydarate, 5 g/L of extracted xylan, and 5 g/L of XOS as the sole carbon source. The fermentation medium was sterilized at 121°C for 20 min, and the cultures of the three strains were added to 10 mL of autoclaved medium. The mixtures were then incubated at 37°C for 12 h. A UV spectrophotometer was used to measure the optical density of microbial growth at 600 nm every two hours for 12 h, and the pH of the samples before and after fermentation was recorded using a digital pH meter (Wang et al., 2022).
The content of SCFAs was detected by GC-MS (QP2010 Ultra, Shimadzu Corporation, Japan), using the following conditions: an HPINNOWAX column with dimensions of 25 m × 0.20 mm × 0.40 µm, N2 used as the carrier gas with a flow rate of 1.0 mL/min, and an inlet temperature of 240°C. The oven temperature program started at 100°C and was held for 5 min, followed by an increase to 150°C at a rate of 5°C /min. Subsequently, the temperature was further increased to 240°C at a rate of 30°C /min and held constant for 30 minutes. For the mass spectrometry conditions, the transfer-line temperature was set at 250°C and ion source temperature was set at 200°C. The electron ionization mode using an ionization voltage of 70 eV was utilized.
2.14 Antioxidant activity of purified xylooligosaccharides
The antioxidant activity of purified XOS was evaluated by measuring its ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. To determine the DPPH free radical scavenging activity, different XOS concentrations of 0.3 g/L, 0.6 g/L, 0.9 g/L, 1.2 g/L, 1.5 g/L, 2.1 g/L, and 4.5 g/L were prepared with freshly prepared 80 mg/L DPPH solution in methanol. The mixture was then incubated at room temperature for 30 min. After centrifugation at 3,000 rpm for 2 min, the absorbance of the sample was measured spectrophotometrically at 517 nm (Veenashri & Muralikrishna, 2011). The following equation was used to express the radical scavenging activity of the samples:
3.1 Comprehensive analysis of chemical composition and structural characteristics of hemicellulose from delignified corn stalks
The delignification process resulted in a yield of 85.71% w/w delignified corn stalk. After alkaline extraction, the extraction rate of hemicelluloses was 28.35% w/w. The HPLC analysis revealed the chemical composition of the extracted hemicelluloses sample (Fig. 2A). Xylose, arabinose and glucose were readily observed, with xylose accounts for the majority. The primary macromolecular components was 91.07% of xylan and 5.58% of arabinan, which supported the structure of arabinoxylan-type hemicelluloses. Components of galactose, glucose and arabinose could be efficiently absorbed and utilized by B. subtilis MR42 for its growth. Several reports have found that the xylose content of various agricultural residues such as sugarcane straw, corn stalks, sunflower stalks, tobacco stalks, cotton stalks, and wheat straw xylans varies from 85 to 95%, which is similar to the results of this study (Carvalho et al., 2013; Carvalho et al., 2017; Yuan et al., 2019; Puițel et al., 2022 ). The considerable quantity of xylan obtained in this study may be ascribed to the extraction of delignified corn stalks, rather than the raw lignocellulosic biomass.
The physicochemical characterization of the extracted hemicellulose was performed using FTIR. The FTIR spectrum (Fig. 2B) displayed characteristic absorption peaks in the range of 1600 to 800 cm− 1, corresponding to the glycosyl units in the hemicellulose (Zhang et al. 2021; Liu et al. 2021). The presence of an absorption peak at 899 cm− 1 indicated the C-O-C stretching of β-glucosidic bonds, confirming the presence of β-glycosidic linkages in the sugar units of the hemicellulose main chain (Sarker et al., 2020). Additional absorption peaks at 980, 1043, and 3350 cm− 1 were attributed to vibrational C3-H bending, C-O-C stretching and O-H stretching of xylosyl groups in the sugar units, respectively. A prominent and broad band in the range of 1170 to 1000 cm− 1 was observed, indicating the typical characteristic absorption of xylan. In addition, a weak absorption peak at 1165 cm− 1 was associated with the arabinose side chain (Zhu et al., 2022; Hussain et al., 2022; Raza et al., 2022). The FTIR spectral profile suggests that the extracted hemicellulose from corn stalks has typical signal patterns of arabinoxylan as described by other reports (Teleman et al. 2000).
Figure 2. Characterization of hemicellulose extracted from delignified corn stalks by HPLC and FTIR. (A) HPLC anlysis of extracted hemicellulose hydrolyzed by sulfuric acid. 1: mannose standard; 2: rhamnose standard; 3: glucuronic acid standard; 4: galacturonic acid standard; 5: galactose standard; 6: N-acetylgalactose standard; 7: glucose standard; 8: xylose standard; 9: arabinose standard; 10: fructose standard. (B) FTIR spectra of extracted hemicellulose from corn stalks.
3.2 Optimization of XOS production by B. subtilis MR42 from extracted xylan
In order to enhance the XOS content in the hydrolysate of B. subtilis MR42, a number of single-factor conditions were optimized, as depicted in Fig. 3.
In Fig. 3A, when the xylan concentration was 10g/L and the culture time was 15 h, the optimum growth temperature for MR42 was found to be 42°C, resulting in a xylan hydrolysis rate of 36.48%. As the temperature was changed, the growth curve first increased and then decreased. Figure 3B showed that when the growth temperature was 42°C and the culture time was 15 h, the hydrolysis rate at a xylan concentration of 10 g/L and 25 g/L was 36.12% and 35.67%, respectively. As the xylan concentration in medium increased, the hydrolysis rate decreased. Considering the final XOS yield, a xylan concentration of 25 g/L was determined to used in this study. Figure 3C showed that with a growth temperature of 42°C and a xylan concentration of 25 g/L, the hydrolysis rates of xylan varied during different culture times ranging from 0 to 20 h. It was observed that as the incubation time increased, the hydrolysis rate of xylan exhibited a rapid increase within the time range of 0 to 10 h. However, beyond 10 h, the hydrolysis rate gradually declined with further increase in culture time. Based on these findings, the optimal conditions for XOS production by MR42 were set, including a CS xylan concentration of 25 g/L, a culture temperature of 42°C, and a culture time of 10 h. Under these optimal conditions, the hydrolysis rate was measured to be 39.27% and the total yield of XOS generated reached 700.34 mg/g xylan. The reported yields of XOS can vary significantly based on the substrate and production process. Values range from approximately 100 mg/g to 800 mg/g (Liu et al., 2017; Amorim et al., 2018; da Silva Menezes et al., 2018). It is worth noting that previous studies have reported varying production times for XOS, ranging from 24 to 120 h (Liu et al., 2017; Reque et al., 2019; Kocabas & Ozben, 2014). However, this study observed a significantly shorter production time of only 10 h. These findings highlight the potential of B. subtilis MR42 as a biocatalyst for producing XOS from corn stalks due to its efficient synthesis of Xyn11A and Axh43 enzymes, resulting in a high XOS yield and shorter production time.
3.3 TLC analysis of MR42 hydrolysis products
Thin-layer chromatography (TLC) was utilized to investigate the accumulation of XOS in the culture medium of B. subtilis MR42. Figure 4 illustrated the gradual degradation of CS arabinoxylan by B. subtilis MR42 into a mixture of small oligosaccharides, with no apparent monosaccharides observed. Previous study have indicated that XOS mixtures produced from various substrates using xylanases often contain significant amounts of xylose (Zhu et al., 2006). However, the absence of xylose in XOS mixture is considered advantageous as it eliminates the need for its removal in the downstream purification process. This suggests that B. subtilis MR42 might have an advantage in XOS production compared to the use of enzymes as previously found (Rajagopalan et al., 2017). After 10 h of incubation, xylotriose (X3), xylotetraose (X4) and xylopentaose (X5) were detected by TLC, with X4 and X5 exhibiting darker colors. The absence of monosaccharides such as arabinose and xylose suggested that B. subtilis MR42 could assimilate and metabolize them for growth. In our previous study, MR42 cultivated with methylglucuronoxylans accumulated MeGX3, while X2 and X3 were gradually consumed with increasing culture time (Rhee et al, 2013). Here, B. subtilis MR42 secreting Xyn11A and Axh43 converted arabinoxylan to a mixture of small oligosaccharides with a DP of 3–5 as predominant products that were not subject to assimilation and metabolism. This finding indicates that the arabinoxylan substrate may be suitable for MR42 strain to produce XOS.
3.4 Purification of XOS in MR42 hydrolysate
Given that the presence of furfural and dissolved lignin-derived phenolic compounds in XOS can impact its prebiotic properties, and considering the fluctuating prices ranging from US $25/kg to US $50/kg for high-purity XOS (Han et al., 2020), the production of high-purity XOS is regarded as crucial. Activated carbon adsorption and ethanol elution were used to refine the xylooligosaccharides accumulated in the MR42 hydrolysate (Fig. 5A). The color value of the hydrolysate reduced from 122900 to 15400 with the removal of the remaining chromophoric group. The purity of the XOS obtained through 30% ethanol elution was determined to be 87.72%, which was consistent with previous reports, such as XOS extracted from almond shells with autohydrolysis and XOS derived from grass (Nabarlatz et al., 2007; Chen et al., 2016). The obtained XOS samples have met the standards set for commercial XOS, as per the XOS standard specifications obtained from Shandong Longli Biotechnology Co., Ltd. The purified XOS were evaluated by thin-layer chromatography, which clearly showed xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and larger oligosaccharides (presumably X6 and X7) (Fig. 5B). X3, X4 and X5 appeared to be the predominant components, which are preferred as prebiotic compounds for food and pharmaceutical applications (Reddy and Krishnan, 2016). The purified XOS were further structurally defined and quantified using HPLC, ESI-MS, and 1H NMR.
3.5 Characterization of purified XOS products from MR42 hydrolysate by HPLC, ESI-MS, and 1HNMR analysis
Purified XOS obtained from MR42 hydrolysate was analyzed using HPLC with pre-column derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP), as shown in Fig. 6. The PMP derivatives of xylotriose (X3), xylotetraose (X4), and xylopentaose (X5) were clearly separated, and their respective contents were determined to be xylotriose (289 ± 0.03 mg/g), xylotetraose (237 ± 0.05 mg/g), and xylopentaose (267 ± 0.03 mg/g), which accounted for 79.3% of the total XOS. According to previous report, XOS with a lower DP of 2–5 are considered more favorable for food-related applications due to their easy metabolism by probiotic bacteria and enhanced prebiotic activity (Mhetras et al., 2019). Therefore, it is expected that the XOS generated by MR42 using corn stalks, which mainly comprises X3-X5, may demonstrate excellent prebiotic properties.
To determine the composition and structure of the XOS accumulated in culture media, ESI-MS was used in this work. The mass spectrometer can provide an accurate molecular mass of the sample, which is an efficient technique for determining unknown chemical compounds (Xiao et al., 2018). The DPS of the purified XOS was determined using ESI-MS, by evaluating the mass-to-charge (m/z) values of the observed ions. Figure 7 confirmed that MR42 produced a range of characteristic XOS molecules with a polymerization degree ranging from 3 to 9. The peaks at m/z 437.5 and 453.5 are assigned to xyltriose with Na+ and K+ respectively.
To further evaluate the structure of XOS, 1HNMR was employed to identify the presence of substitutions. Figure 8 shows the 1HNMR spectra of purified XOS isolated from corn stalks. The main spectral signals of XOS revealed the presence of β-anomeric protons within the range of 4.0-4.7 ppm. The chemical shifts ranging from 3.2 to 5.5 ppm were indicative of β-linked xylopyranoside residues. The notable chemical shifts observed for the anomeric protons of XOS at δ 4.49 ppm to 3.22 ppm corresponded to H-1, H-2, H-3, H-4, and H-5 of the β-D-xylopyranose residue, respectively. The anomeric signal detected at 4.7 ppm was attributed to the anomeric proton of the reducing Xylp end groups, Xα and Xβ, respectively. Additionally, proton signals at δ 5.34 ppm were ascribed to the α-D-furanoarabinose end group attached to the C-3 of the main chain xylose group, indicating a slight presence of AXOS (arabino-XOS). A significant signal from the solvent (D2O) was observed at δ 4.78 ppm. The 1HNMR spectra of XOS did not exhibit any signals in the range of 5.28 to 5.32 ppm, indicating the absence of uronic acid with the 1H linked to urinate C-1.
3.6 In vitro fermentation of XOS and determination of SCFAs
To assess the utilization of XOS by Lactobacillus strains, growth curves of L. plantarum CGMCC 1.9087, L. casei CGMCC 1.575 and L. brevis YM 1301 strains were constructed using a visible ultraviolet spectrophotometer, with 1% (w/v) XOS served as the exclusive carbon source for the cultures. A minimal MRS medium containing 1% (w/v) arabinoxylan and no carbohydrate was used as a control. The SCFAs contents produced in these culture mediums were determined using GC-MS.
In Fig. 9A, it is evident that all the Lactobacillus strains exhibited strong growth, following a consistent trend, with L. plantarum demonstrating the highest growth rate, followed by L. brevis and then L. casei. All Lactobacillus strains in the medium containing no carbohydrate and arabinoxylan showed negligible growth. Previous research has revealed that XOS from sugarcane bagasse can significantly improve the growth of Lactobacillus plantarum M-13 strain, which is comparable to that of the standard MRS medium (Gupta et al., 2022). Additionally, XOS are slowly metabolized due to their oligomeric structure, providing sustained growth of probiotics for much longer periods (Gupta et al, 2022). The pH of the XOS cultures with Lactobacillus strains decreased from an initial value of 7.0 to 5.2, 5.5, and 5.9 after 18 h, indicating that Lactobacillus strains readily utilize XOS to produce short-chain fatty acids (SCFAs). Short-chain fatty acids (SCFAs), such as propionic acid and isobutyric acid, are primarily produced as metabolites during carbohydrate fermentation by specific gut microbes (Wang et al., 2023). They can inhibit the growth of harmful bacteria in the intestine, regulate lipid metabolism, and impact the immune system positively (Karakan et al., 2021).
The total amount of SCFAs is a reliable indicator of prebiotic properties. SCFAs levels in all culture mediums were analyzed and shown in Fig. 9B. The XOS-containing cultures exhibited significantly higher contents of the six SCFAs, with acetic acid being the most abundant in all culture mediums. L. plantarum CGMCC 1.9087 produced significantly higher amounts of acetic acid (625.8 µg/g), butyric acid (100.5 µg/g), and valeric acid (56.8 µg/g) compared to the other strains. L. casei CGMCC 1.575 mainly generated 556.8 µg/g of acetic acid, 29.8 µg/g of propionic acid, and 23.4 µg/g of butyric acid. L. brevis YM 1301 primarily produced 459.5 µg/g of acetic acid, 35.6 µg/g of isobutyric acid and 10.5 µg/g of propionic acid. Similar findings have been reported, where acetate acid was observed as the primary short-chain fatty acid generated as an end-product of fermentation, with concentrations ranging from 1.50 to 1.78 mg/ml (Yu et al., 2015). The high productions of SCFAs in the culture mediums containing XOS may be attributed to the presence of X3, X4 and X5, which are more easily fermentable than XOS with higher degree of polymerization. The SCFAs levels in the cultures containing xylan were significantly lower, indicating less utilization of xylan by these strains. Thus, these results suggest that XOS produced by MR42 exhibited excellent prebiotic properties, including the ability to promote the growth of Lactobacillus strains and enhance the production of SCFAs, making them a valuable dietary component for promoting optimal gut health in humans.
3.7 Antioxidant potential of produced XOS
The DPPH scavenging assay is a widely accepted method for evaluating the scavenging activity of antioxidants, and is known for its reliability, simplicity, accuracy, sensitivity, and affordability (Mishra et al., 2012). In this study, the antioxidant potential of XOS produced by MR42 was demonstrated by its ability to scavenge DPPH radicals. Various XOS concentrations of 0.3 g/L, 0.6 g/L, 0.9 g/L, 1.2 g/L, 1.5 g/L, 2.1 g/L, and 4.5 g/L were tested, and a dose-dependent increase in antioxidant activity was observed (Fig. 10). Corn stalks XOS exhibited a maximum DPPH scavenging activity of 81.3% at a concentration of 4.5 mg/mL. Similarly, 2 g/L of XOS solution prepared from sugarcane straw xylan and coffee husk xylan showed antioxidant activities of 71% and 78% respectively (Ávila et al., 2020). These findings emphasize the significant antioxidant potential of corn stalks XOS and suggest its promising applications in the food and pharmaceutical industries.
This study highlights the capacity of Bacillus subtilis MR42, which produces the enzymes Xyn11A and Axh43, to convert arabinoxylan extracted from corn stalks into a mixture of XOS composed predominantly of X3-X5, without the accumulation of xylose as a result of the xynC gene deletion. B. subtilis MR42 efficiently processed arabinoxylan from corn stalks with a yield of 703.4 mg/g XOS and a purity of 87.72% after treatment by activated carbon adsorption. The presence of XOS was confirmed by various analytical methods, including TLC, HPLC, ESI-MS, and NMR spectroscopy. Composition analysis revealed that XOS mixture contained a serious of neutral XOS with a degree of polymerization of 3–9, with X3, X4, and X5 being the predominant components, accounting for approximately 80% of the total XOS. These results demonstrate the efficient production of substantial quantities of low degree of polymerization xylooligosaccharides (XOS) from corn stalks using B. subtilis MR42. To the best of our knowledge, no reports have been published on the production of significant quantities of X3-X5 from corn stalks using engineered bacteria.
Moreover, the degree of polymerization (DP) is widely recognized as one of the crucial parameters influencing the functional properties of the molecule (Singh et al., 2015). Subsequent investigations on the produced XOS revealed their significant prebiotic potential, including enhancing the growth of all tested probiotic Lactobacillus strains, promoting the production of short-chain fatty acids (SCFAs), and exhibiting enhanced antioxidant activity. These results strongly indicate the prospective application of B. subtilis MR42 as a reliable biocatalyst for large-scale production of XOS with narrowly-distributed molecular mass derived from renewable sources like corn stalks. The resulting XOS can be employed as a potential prebiotic agent in the development of functional foods and nutraceutical products.
Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Author Contribution
Lusha Wei wrote the main manuscript text,Shuo Sun and Yuan Chen prepared figures 1-8,Xin An prepared figures 9-10, Hanning Wu interpretated the data in figure 8, Yifei Wu designed the whole work,All authors reviewed the manuscript.
Acknowledgements
This work was supported by National Natural Science Foundation of China [grant number 41802164]; Basic Research Program of Natural Science of Shaanxi Province [grant number: 2019JQ-717] ; Xi 'an Science and Technology Program [grant number: 20NYYF0020] and Fundamental Research Funds for the Central Universities [grant number GK202103102].
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No competing interests reported.