Quinoa Stalks Glucuronoarabinoxylan : Biorefinery,xylooligosaccharides production and potential applications

Quinoa stalks were used as a source of hemicellulose for XOs production and further as prebiotic source for probiotic bacteria. Three methods for extraction of hemicellulose were used, of which alkaline extraction (NaOH [0.5 M]) was the optimum method. This methodology allowed the establishment of a sequential extraction platform, including saponins obtention via PHWE, hemicellulose via alkaline extraction and cellulose via acid purification. Maximum yields of 15.4, 120 and 296 mg/g raw material of saponins, hemicellulose and cellulose yield were obtained, respectively. Saponins and hemicellulose extractions were dependent on PHWE conditions used, while cellulose extraction was not dependent on conditions in previous steps and resulted in purest fraction. The purified hemicellulose fraction consisted of glucuronoarabinoxylan (GAX) (HPAEC-PAD, FT-IR) with an estimated Mw of 1758 ± 31 kDa (SEC). The xylopyranosyl backbone was linked via β-(1,4) bonds, and substituted with 4-O-Methyl glucuronosyl and arabinofuranosyl residues. The later was linked as oligomers via α-(1-5) or as monomers α-(1-3) or α-(1-2) to the xylose skeleton (NMR). The monosaccharides composition of the GAX included xylose, glucuronic acid, arabinose and galactose in a molar ratio of 114:23:5:1, respectively. The GAX purified from quinoa stalks was treated by two methodologies to produce XOs: an enzymatic method (using xylanases) and a dilute acid method using H2SO4 [0.25 M]. In the enzymatic method, in house produced thermostable endoxylanases from GH10 (from R. marinus and B. halodurans) and the commercial GH11 PENTOPAN® , were used. The maximum yield of linear XOs obtained from the hydrolysis of GAX was 1.217 g XOs/100 g of GAX. Dilute acid treatment resulted in a maximum yield of 0.564 g XOs/100 g GAX. However, the DP in dilute acid treatment was more widely distributed, ranging from xylobiose to xylohexaose compared to the enzymatic method. B. adolescentis ATCC15703 and Weissella sp. strain 92 were cultivated using XOs from quinoa stalks GAX as carbon source. After 48 hours of cultivation, B. adolescentis grew to a maximum OD600 of 0.326, producing the followig amounts of SCFA and lactate (g/L): Acetate (1.243); lactate (1.013); propionate (0.812); formate (0.179) and; butyrate (0.124). Weissella sp. 92 grew to an OD600 of 0.626, producing Acetate (0.379 g/L) and lactate (0.259 g/L). The consumption of XOs by Weissella sp. 92 was mainly consuming monosaccharides (xylose and arabinose), and XOs: xylobiose, xylotriose and xylotetraose, while B. adolescentis was able to consume all linear XOs at different ratios, and was, moreover, able to consume substituted XOs (according to HPAEC-PAD). A draft genome sequencing of Clostridium boliviense strain E-1 was made, and a number of genes encoding hemicellulose-active enzymes were identified. Two enzymes were fully characterized; one two domain GH43-like endo-β-xylanase (CbE1Xyn43, MW 52.9 kDa) and one bifunctional acetyl esterase/endo-β-xylanase (CbE1Est1XynX, MW 44.2 kDa), also consisting of two domains (one carbohydrate esterase and one potential glycoside hydrolase). Both enzymes were thermostable and most active at neutral pH. Both enzymes also were active on birchwood glucuronoxylan, wheat bran arabinoxylan and quinoa stalks GAX. No xylosidase activity was determined. CbE1Xyn43 kinetic parameters was determined to Kcat (1.587 min-1) and Km (0.126 mM) using p-nitrophenyl xylobioside (pNPX2) as substrate. For CbE1Est1XynX the Kcat (6.645 min-1) and Km (0.233mM) was determined using pNPX2 and, using p-nitrophenyl acetate (pNPAc) Kcat was 243.363 min-1 and Km 2.25 mM. Despite the sequence similarity of CbE1Xyn43 to enzymes in GH43, the conserved catalytic residues of GH43 could not be identified, making classification of the enzyme difficult. In conclusion, the potential of quinoa stalks as a raw material for valorization has been demonstrated (separating saponins, GAX and cellulose) for further biorefinery applications. GAX in particular, resulted in successfully XOs production by enzymatic and acid methodologies for prebiotic usage. Also additional xylanases were explored, and were demonstrated as potential tools to diversify and increase linear and substituted XOs production from different material

Glucuronosylated and linear xylooligosaccharides from Quinoa stalks xylan as potential prebiotic source for growth of Bifidobacterium adolescentis and Weissella cibaria

Quinoa stalks glucuronoarabinoxylan (QSGAX) has been extracted by alkali and further utilized for production of two-types of xylooligosaccharides (XOs): i) enzymatically produced glucuronosylated-XOs (GXOs), and ii) a dilute acid produced mixture of non-substituted/substituted XOs of different degree of polymerization (DP). The respective mixtures were then separately evaluated as prebiotics by analysis of their consumption by two phylogenetically different potential probiotic bacterial strains (Bifidobacterium adolescentis ATCC15703 and Weissella cibaria strain 92), from which release of short chain fatty acids (SCFA) was also monitored. The GXOs mixture was produced using a glucuronosyl-requiring family 30 glycoside hydrolase (GH) Bacteroides ovatus (BoXyn30A), while the XOs mixture was produced by a 30 min acid treatment of QSGAX with H2SO4 [24.5 g/L] at 90 °C. B. adolescentis consumed both GXOs and XOs (DP 2–6), in both cases releasing acetate, lactate, propionate, formate and butyrate as metabolic products. W. cibaria only consumed XOs (DP 2–4), releasing acetate, lactate and minor amounts of butyrate. This is the first study reporting the ability of GXOs consumption by B. adolescentis and shows the potential of GXOs to selectively stimulate B. adolescentis, while XOs stimulated both types of potential probiotics (B. adolescentis ATCC15703 and W. cibaria strain 92)

Extraction of Glucuronoarabinoxylan from Quinoa Stalks (Chenopodium quinoa Willd.) and Evaluation of Xylooligosaccharides Produced by GH10 and GH11 Xylanases

Byproducts from quinoa are not yet well explored sources of hemicellulose or products thereof. In this work, xylan from milled quinoa stalks was retrieved to 66% recovery by akaline extraction using 0.5 M NaOH at 80 °C, followed by ethanol precipitation. The isolated polymer eluted as a single peak in size-exclusion chromatography with a molecular weight of >700 kDa. Analysis by Fourier transform infrared spectroscopy and nuclear magnetic resonance (NMR) combined with acid hydrolysis to monomers showed that the polymer was built of a backbone of β(1 → 4)-linked xylose residues that were substituted by 4-O-methylglucuronic acids, arabinose, and galactose in an approximate molar ratio of 114:23:5:1. NMR analysis also indicated the presence of α(1 → 5)-linked arabinose substituents in dimeric or oligomeric forms. The main xylooligosaccharides (XOs) produced after hydrolysis of the extracted glucuronoarabinoxylan polymer by thermostable glycoside hydrolases (GHs) from families 10 and 11 were xylobiose and xylotriose, followed by peaks of putative substituted XOs. Quantification of the unsubstituted XOs using standards showed that the highest yield from the soluble glucuronoarabinoxylan fraction was 1.26 g/100 g of xylan fraction, only slightly higher than the yield (1.00 g/100 g of xylan fraction) from the insoluble fraction (p 0.05). This study shows that quinoa stalks represent a novel source of glucuronoarabinoxylan, with a substituent structure that allowed for limited production of XOs by GH10 or GH11 enzymes

Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)

World quinoa production is increasing due its high nutritional value. As a consequence, large quantities of stalks accumulate as unused byproducts. Here, we verify the presence of saponins in the stalks and present a biorefinery approach with quinoa stalks as feedstock, using an integrated processing scheme to separate saponins, xylan and cellulose. Saponins were extracted using pressurized hot water extraction (PHWE), optimized by a central composite experimental design (rotatable 22) with temperature and extraction time as factors. Xylan was extracted from the residual solid material after PHWE by an alkaline method using 0.5 M NaOH at 80 °C. Cellulose was purified from the remaining residuals using acetic and nitric acid at 120 °C, which resulted in recovery of white cotton-like cellulose, showing no need of further bleaching. The saponin yield was significantly increased at temperatures exceeding 110 °C, with highest amounts obtained at 195 °C (15.4 mg/g raw material). The yield in the following xylan extraction (maximum 120 mg/g raw material) was however significantly reduced when preceded by PHWE above 110 °C, indicating degradation of the polymer. Cellulose recovery (maximum 296 mg/g raw material) was less affected by variations in temperature and time in the preceding PHWE. The results obtained shows that tuning between saponin and xylan extraction is critical. This approach is foreseen to be applicable to the valorisation of residual fiber-rich biomass from various types of crops, besides quinoa

Data on saponins, xylan and cellulose yield obtained from quinoa stalks after pressurized hot water extraction

The data we present below are linked to our research paper “Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)” (Gil-Ramírez et al., 2018) [1]. The objective is to provide supplementary information in order to facilitate the comprehension of the central composite experimental design (rotatable 22) used in the integrated process of extractions. Two factors, temperature and time of extraction are considered in the design. The responses are the yield of saponin, xylan and cellulose. First, the desirable linear regression obtained by the observed vs. predicted yields plot for each variable response confirm the validation of the model (Fig. 1). Second, the data presented here through Standardized Pareto Charts (Fig. 2), provides information about the effect of the time and temperature, as well as their interactions, in the yield of saponins, xylan and cellulose obtained in an integrated sequential extraction