Prebiotic Xylooligosaccharides Research Articles

Hyperthermophilic xylanase and thermophilicity analysis by molecular dynamic simulation with quantum mechanics

Thermophilic xylanases catalyzing the cleavage of β-1,4-glycosidic bonds in xylan have applications in food, feed, biorefinery, and pulp industries. In this study, a hyperthermophilic endo-xylanase was obtained by further enhancement of thermal tolerance of a thermophilic GH11 xylanase originated from metagenome of bagasse pile based on rational design. Introducing N13F and Q34L to the previously reported X11P enzyme shifted the optimal working temperature to 85 °C and led to 20.7-fold improvement in thermostability at 90 °C along with a marked increase in Tm to 93.3 °C. X11PNQ enzyme converted xylan to prebiotic xylooligosaccharides with high specificity on xylobiose to xylohexaose and high operational stability at 85 °C, resulting in 10.3-folds yield improvement compared to the parental enzyme. Molecular dynamic simulation and quantum mechanical analysis revealed improved H-bonding networks within GH11 xylanase principal domains and greater dynamic cross-correlations. A novel thermostabilization mechanism by π-amide interaction with slightly lower interaction energy than the native H-bond, but compensated by increased occurrence frequency was firstly demonstrated for thermophilic enzymes. The enzyme represents one of the most thermostable xylanases ever reported with biotechnological potential.Key points• Hyperthemophilic xylanase X11PNQ was obtained by rational design engineering.• X11PNQ showed specificity to prebiotic xylooligosaccharides (XOS) at 85 °C with improved t1/2 at 90 °C.• Novel thermostabilization by π-amide interaction was demonstrated by MD/QM.

Advanced technological approaches and market status analysis of xylose bioconversion and utilization: Xylooligosacharides and xylonic acid as emerging products.

The efficient conversion of xylose is a short board of cask effect to lignocellulosic biorefining, by markedly affecting the total economic and environmental benefits. Based on a comprehensive analysis of the current commercial status of traditional xylose utilization and industrial technology development, this review outlines new technological avenues for the efficient utilization of xylose from lignocellulosic biomass, focusing on super prebiotic xylo-oligosaccharides and multifunctional platform compound xylonic acid. Firstly, the traditional products that can be derived from lignocellulosic xylose, including xylitol (447.88 billion USD in 2022), furfural (662 million USD in 2023), and bioethanol (46.18 billion USD in 2022), are introduced along with the current market status and latest production technologies. Then, the discussion covers the industrial development and production methods of xylo-oligosaccharides, and highlights the potential of xylonic acid, focusing on innovative whole-cell catalysis in a sealed oxygen supply-bioreactor system. Finally, other directions for efficient and high-value utilization of lignocellulosic xylose are summarized, including lactic acid, succinic acid, and 2,3-butanediol. This review aims to provide new perspectives on the utilization and valorization of xylose by summarizing main traditional industrial products and emerging products, thereby promoting the development of the entire lignocellulosic biomass field.

Modulation of the Cardiovascular Risk in Type 1 Diabetic Rats by Endurance Training in Combination with the Prebiotic Xylooligosaccharide.

Diabetic cardiomyopathy is a major etiological factor in heart failure in diabetic patients, characterized by mitochondrial oxidative metabolism dysfunction, myocardial fibrosis, and marked glycogen elevation. The aim of the present study is to evaluate the effect of endurance training and prebiotic xylooligosaccharide (XOS) on the activity of key oxidative enzymes, myocardial collagen, and glycogen distribution as well as some serum biochemical risk markers in streptozotocin-induced type 1 diabetic rats. Male Wistar rats (n = 36) were divided into four diabetic groups (n = 9): sedentary diabetic rats on a normal diet (SDN), trained diabetic rats on a normal diet (TDN), trained diabetic rats on a normal diet with an XOS supplement (TD-XOS), and sedentary diabetic rats with an XOS supplement (SD-XOS). The results show that aerobic training managed to increase the enzyme activity of respiratory Complex I and II and the lactate dehydrogenase in the cardiomyocytes of the diabetic rats. Furthermore, the combination of exercise and XOS significantly decreased the collagen and glycogen content. No significant effects on blood pressure, heart rate or markers of inflammation were detected. These results demonstrate the beneficial effects of exercise, alone or in combination with XOS, on the cardiac mitochondrial enzymology and histopathology of diabetic rats.

Youth-derived Lactobacillus rhamnosus with prebiotic xylo-oligosaccharide exhibits anti-hyperlipidemic effects as a novel synbiotic

Changes in dietary patterns and living habits have led to an increasing number of individuals with elevated cholesterol levels. Excessive consumption of high-cholesterol foods can disrupt the body’s lipid metabolism. Numerous studies have firmly established the cholesterol-lowering effects of probiotics and prebiotics, with evidence showing that the synergistic use of synbiotics is functionally more potent than using probiotics or prebiotics alone. Currently, the screening strategy involves screening prebiotics for synbiotic development with probiotics as the core. However, in comparison to probiotics, there are fewer types of prebiotics available, leading to limited resources. Consequently, the combinations of synbiotics obtained are restricted, and probiotics and prebiotics are only relatively suitable. Therefore, in this study, a novel synbiotic screening strategy with prebiotics as the core was developed. The synbiotic combination of Lactobacillus rhamnosus S_82 and xylo-oligosaccharides was screened from the intestinal tract of young people through five generations of xylo-oligosaccharides. Subsequently, the cholesterol-lowering ability of the medium was simulated, and the two carbon sources of glucose and xylo-oligosaccharides were screened out. The results showed that synbiotics may participate in cholesterol-lowering regulation by down-regulating the expression of NPC1L1 gene, down-regulating ACAT2 and increasing the expression of ABCG8 gene in vitro through cell adsorption and cell absorption in vitro, and regulating the intestinal microbiota. Synbiotics hold promise as potential candidates for the prevention of hypercholesterolemia in humans and animals, and this study providing a theoretical foundation for the development of new synbiotic products.

The impact of probiotic Lactiplantibacillus plantarum S61 and prebiotic Xylooligosaccharide on the functional properties of fermented orange juice

The impact of probiotic Lactiplantibacillus plantarum S61 and prebiotic Xylooligosaccharide on the functional properties of fermented orange juice

A sequential bioconversion-purification process to produce high purity xylo-oligosaccharides (XOS) from Nothophagus pumilio sawdust wastes

Xylo-oligosaccharide (XOS) production from lignocellulosic biomass (LCB) is an ongoing technology in which the best raw material, catalyst, and reaction conditions remain unclear. This study proposed a three-step sequential operation to produce prebiotic Xylo-oligosaccharide (XOS) from lignocellulosic biomass (LCB) Nothophagus pumillio sawdust. First, a commercial (Enzeco Xylanase S) and recombinant (GtXyl10A) xylanase were evaluated in XOS production from pre-extracted xylan. Then, the effect of auxiliary enzyme addition (α-glucuronidase) was evaluated in xylan hydrolysis, and finally, XOS were purified by three steps of membrane filtrations: an ultrafiltration (UF) followed by two nanofiltrations (NF). From the above, XOS production yield reach 333 [mgxos∙gxylan−1] of highly purified XOS (95 %) when the process was carried out without auxiliary enzyme addition and 473 [mgxos∙gxylan−1] of 96 % purified XOS when auxiliary enzyme was added, resulting in an improvement of the XOS production yield in 42 %.This shows the feasibility to produce high purity prebiotics from sawdust wastes of the forestry industry.

A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-β-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

A prebiotic diet modulates the oral microbiome composition and results in the attenuation of oropharyngeal candidiasis in mice.

Oral bacteria can influence the ability of Candida albicans to cause oropharyngeal candidiasis (OPC). We recently reported that a Lactobacillus johnsonii-enriched oral microbiota reduced C. albicans virulence in an immunosuppressed OPC mouse model. As a follow-up, in this work, we aimed to enrich the resident oral Lactobacillus communities with a prebiotic diet to further assess their effect on the severity of OPC. We tested the effect of a prebiotic xylo-oligosaccharides (XOS)-enriched diet in the oral global bacterial composition and severity of OPC. We assessed changes in the oral microbiome composition via 16S-rRNA gene high-throughput sequencing, validated by qPCR. The impact of the prebiotic diet on Candida infection was assessed by quantifying changes in oral fungal and bacterial biomass and scoring tongue lesions. Contrary to expectations, oral Lactobacillus communities were not enriched by the XOS-supplemented diet. Yet, XOS modulated the oral microbiome composition, increasing Bifidobacterium abundance and reducing enterococci and staphylococci. In the OPC model, the XOS diet attenuated Candida virulence and bacterial dysbiosis, increasing lactobacilli and reducing enterococci on the oral mucosa. We conclude that XOS attenuates Candida virulence by promoting a bacterial microbiome structure more resilient to Candida infection. IMPORTANCE This is the first study on the effects of a prebiotic diet on the oral mucosal bacterial microbiome and an oropharyngeal candidiasis (OPC) mouse model. We found that xylo-oligosaccharides change the oral bacterial community composition and attenuate OPC. Our results contribute to the understanding of the impact of the oral bacterial communities on Candida virulence.

Expression and characterization of α-L-arabinofuranosidase derived from Aspergillus awamori and its enzymatic degradation of corn byproducts with xylanase

In this study, α-L-arabinofuranosidase (AF) from Aspergillus awamori was heterologously expressed in Pichia pastoris X33, with a 1-fold increase in AF activity after codon and vector optimization. AF remained stable at 60–65 °C and displayed a broad pH stability range of 2.5–8.0. It also demonstrated considerable resistance to pepsin and trypsin. Furthermore, compared with xylanase alone, AF with xylanase exhibited a marked synergistic effect in the degradation of expanded corn bran, corn bran, and corn distillers’ dried grains with solubles, reducing sugars by 3.6-fold, 1.4-fold, and 6.5-fold, respectively, with the degree of synergy increasing to 4.61, 2.44, and 5.4, respectively, while in vitro dry matter digestibility values were 17.6%, 5.2%, and 8.8%, respectively. After enzymatic saccharification, corn byproducts were converted to prebiotic xylo-oligosaccharides and arabinoses, thereby demonstrating the favorable properties of AF in the degradation of corn biomass and its byproducts.

The production of stirred yogurt fortified with prebiotic xylooligosaccharide, probiotic and synbiotic microcapsules

Xylooligosaccharides are considered safe emergent food ingredients with various promising biological activities. Herein, xylooligosaccharides were produced through enzymatic hydrolysis of beech wood xylan and their prebiotic activity was evaluated. The reported results indicated that the produced xylooligosaccharides own confident influence for stimulating the growth of different useful bacterial strains with a significant resistance to simulated intestinal juice digestion but decomposed by more than 50% in simulated gastric juice. Therefore, encapsulation of the produced xylooligosaccharides was assessed to enhance its gastric-digestion resistance. The results indicated that the encapsulation matrix composed of sodium alginate and whey protein concentrate led to an entrapment efficiency of 85.26% for xylooligosaccharides with 100% protective efficiency in simulated gastric juice. Moreover, the incorporation of the produced xylooligosaccharides in the preparation of synbiotic microcapsules including the probiotic Lactobacillus acidophilus was evaluated. The results indicated that the entrapment efficiency for XOS in the prepared synbiotic microcapsule was 93.89%. Finally, the applicability of the produced xylooligosaccharides in the manufacture of stirred yogurt fortified with the probiotic Lactobacillus acidophilus was evaluated. Fortification with the produced xylooligosaccharides maintained the viability of the probiotic strain along the storage period with significant viability propagation (10.45 log cfu/mL after 20 days storage period) by the use of synbiotic microcapsules.

Recovery of xylan from Acacia mearnsii using ultrasound‐assisted alkaline extraction

AbstractNovel and effective biorefinery methods are necessary to fractionate lignocellulosic biomass into separate components for the valorization of these components into value‐added products. For example, the xylan fraction can be used as an adhesive, a thickener, an additive to plastics, an emulsifier, a drug carrier, and as a precursor of prebiotic xylooligosaccharides. Unfortunately, conventional xylan extraction procedures require lengthy extraction periods and are characterized by poor product purity and yields. In this study, a non‐conventional ultrasound‐assisted alkaline extraction method was developed for the effective extraction of xylan from Acacia mearnsii, an industrially relevant hardwood in South Africa. The effect of the additional ultrasonication step on the conventional alkaline extraction of xylan was evaluated. After this, basic structural analysis was conducted on both acacia xylan extracts and commercially available beech‐derived xylan. Composition analysis data showed the acacia‐derived glucuronoxylan to have an Ara:GluA:Xyl ratio of 0.5:1:5, while that from beechwood had a 0:1:4 ratio. Ultrasound‐assisted alkaline extraction also gave a better yield (24%) than conventional alkaline extraction (21%) and had a quicker extraction period of 1 h versus 4 h. Finally, the acacia‐derived xylan exhibited antioxidant potential, with an EC50 value of ≈1.5 mg/mL against 2,2‐diphenyl‐1‐picryl‐hydrazyl‐hydrate (DPPH). In conclusion, ultrasound‐assisted alkaline extraction was successfully employed to extract high‐purity, bioactive xylan from acacia. The purified acacia xylan is a suitable replacement for birchwood glucuronoxylan, which is no longer commercially available, or for expensive beechwood substitute for numerous applications. © 2023 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.

Debranching enzymes decomposed corn arabinoxylan into xylooligosaccharides and achieved prebiotic regulation of gut microbiota in broiler chickens

BackgroundCorn arabinoxylan (AX) is a complicated and multibranched antinutritional factor, thereby proving the use of endo-xylanase (EX) to be marginally valid. This study focused on specific types of AX-degrading enzymes (ADEs) to exert the synergy of debranching enzymes and track the prebiotic potential of enzymatic hydrolysates. This study investigated the effects of ADEs on the growth performance, intestinal histomorphology, absorption functions, changes in polysaccharide components, fermentation, and gut microbiota of broiler chickens. Five hundred seventy-six five-day-old Arbor Acres male broiler chickens were randomly allocated into eight treatments with six replicates each. Corn basal diets supplemented with or without enzymes were fed for a 21-day period, specifically including EX, its compatible use with arabinofuranosidase (EXA) or ferulic acid esterase (EXF), and compound groups with the above three enzymes (XAF).ResultsSpecific ADEs stimulated the jejunal villus height and goblet cell number and evidently decreased the crypt depth (P < 0.05), while the ratio of ileal villus height to crypt depth was significantly increased in EXF (P < 0.05). Maltase activities of ileal mucosa in XAF groups were extremely enhanced (P < 0.01), and EX boosted the activity of Na+-K+ ATPase in the small intestine (P < 0.01). The insoluble AX concentrations comparatively lessened, thereby notably raising the sundry xylooligosaccharide (XOS) yield in the ileal chyme (P < 0.05), which was dominant in xylobiose and xylotriose. Improvements in the abundance and diversity of ileal microbial communities within the EXA, EXF, and XAF treatments were observed (P < 0.05). Positive correlations between microbiota and XOS were revealed, with xylobiose and xylotriose being critical for ten beneficial bacteria (P < 0.05). EXF increased the BWG and FCR of broiler chickens in this phase (P < 0.05), which was attributed to the thriving networks modified by Lactobacillus. The intracecal contents of acetic acid, butyric acid, and propionic acid were greatly enhanced in most ADE groups, such as EXF (P < 0.05).ConclusionsDebranching enzymes appreciably targeted corn AX to release prebiotic XOS in the posterior ileum and facilitated intracaecal fermentation. It was beneficial for improving gut development, digestion and absorption and modulating the microflora to promote the early performance of broiler chickens.Graphical

Green production of prebiotic xylooligosaccharides via enzymatic hydrolysis from xylan black liquor of oil palm frond

The black liquor generated from the alkaline pretreatment of lignocellulosic biomass is usually disposed of into wastewater, which could lead to environmental pollution. Alkaline black liquor (ALBL) contains a large amount of xylan with a small fraction of lignin, making it a promising raw material for the production of xylooligosaccharides (XOS). In this study, xylan was extracted from the ALBL generated upon treating the oil palm frond (OPF) with sodium hydroxide via two-stage precipitation for the separation of lignin and recovery of xylan. As a result, approximately 84.0% of xylan retrieved from the ALBL was recovered. Subsequently, enzymatic hydrolysis was optimized to recover the maximum amount of XOS from xylan. The results showed that enzymatic hydrolysis produced the highest XOS (62.5%) under optimal conditions of 50 °C, 4 U/mL xylanase, and 3% xylan loading for 48 h. The study provides insight for maximizing utilization of ALBL of OPF for future biorefinery economy.

Dose-dependent effects of xylooligosaccharides on glycemic regulation with L. rhamnosus CCFM1060 in diabetic mice.

Dietary intervention with the probiotic Lactobacillus rhamnosus CCFM1060 has been proved to be effective on glycemic regulation in diabetic mice. Therefore characterization of the potential symbiotic effect of prebiotic xylooligosaccharides (XOS) with L. rhamnosus CCFM1060 would be desirable. In this study, we evaluated any dose-dependent relationship between XOS and L. rhamnosus CCFM1060, and the potential impact on glycemic regulation. Diabetic mice were randomly assigned to receive 5 × 109 CFU mL-1L. rhamnosus CCFM1060, 5 × 109 CFU mL-1L. rhamnosus CCFM1060 with 250 mg kg-1 XOS (L-LXOS), or 5 × 109 CFU mL-1L. rhamnosus CCFM1060 with 500 mg kg-1 XOS (L-HXOS) for 7 weeks. In addition to characterization of the host metabolism, the intestinal microbiota were analyzed using 16S rRNA gene sequencing. The results showed that L. rhamnosus alone and L-LXOS intervention significantly alleviated diabetes symptoms and increased the populations of short-chain fatty acid (SCFA)-producing bacteria. The intake of L-HXOS had an adverse effect on glucose metabolism, causing increased insulin resistance and inflammation. Although a significant increase in the relative abundance of Bifidobacterium was observed in the L-HXOS group, the abundance of SCFA-producing bacteria, such as Romboutsia and Clostrudium sensu stricto 1, decreased. KEGG pathway analysis revealed that the adverse effects of L-HXOS intervention might be attributed to the metabolic pathways involved in amino acid, cofactor, and vitamin metabolism. This study revealed that L. rhamnosus CCFM1060 combined with different doses of XOS exerted dose-dependent effects on glucose metabolism. Therefore, the type and dose of prebiotics should be carefully evaluated when developing individualized symbiotic formula.

Engineering mesophilic GH11 xylanase from Cellulomonas flavigena by rational design of N-terminus substitution.

Xylanase, a glycoside hydrolase, is widely used in the food, papermaking, and textile industries; however, most xylanases are inactive at high temperatures. In this study, a xylanase gene, CFXyl3, was cloned from Cellulomonas flavigena and expressed in Escherichia coli BL21 (DE3). To improve the thermostability of xylanase, four hybrid xylanases with enhanced thermostability (designated EcsXyl1-4) were engineered from CFXyl3, guided by primary and 3D structure analyses. The optimal temperature of CFXyl3 was improved by replacing its N-terminus with the corresponding area of SyXyn11P, a xylanase that belongs to the hyperthermostable GH11 family. The optimal temperatures of the hybrid xylanases EcsXyl1-4 were 60, 60, 65, and 85°C, respectively. The optimal temperature of EcsXyl4 was 30C higher than that of CFXyl3 (55°C) and its melting temperature was 34.5°C higher than that of CFXyl3. After the hydrolysis of beechwood xylan, the main hydrolysates were xylotetraose, xylotriose, and xylobiose; thus, these hybrid xylanases could be applied to prebiotic xylooligosaccharide manufacturing.

Xylooligosaccharides and aerobic training regulate metabolism and behavior in rats with streptozotocin-induced type 1 diabetes.

Type 1 diabetes mellitus is characterized with decreased microbial diversity. Gut microbiota is essential for the normal physiological functioning of many organs, especially the brain. Prebiotics are selectively fermentable oligosaccharides [xylooligosaccharides (XOS), galactooligosaccharides, etc.] that promote the growth and activity of gut microbes and influence the gut-brain axis. Aerobic exercise is a non-pharmacological approach for the control of diabetes and could improve cognitive functions. The potential beneficial effect of XOS and/or aerobic training on cognition, the lipid profile and oxidative stress markers of experimental rats were evaluated in this study. Male Wistar rats were randomly divided into three streptozotocin-induced diabetic groups and a control group. Some of the rats, either on a XOS treatment or a standard diet, underwent aerobic training. The results showed that the aerobic training independently lowered the total cholesterol levels compared to the sedentary diabetic rats (p = 0.032), while XOS lowers the malondialdehyde levels in the trained diabetic rats (p = 0.034). What is more the exercise, independently or in combination with XOS beneficially affected all parameters of the behavioral tests. We conclude that aerobic exercises alone or in a combination with the prebiotic XOS could ameliorate the dyslipidemia, oxidative stress, and cognitive abilities in experimental type 1 diabetic animals.

Glutamic acid assisted hydrolysis strategy for preparing prebiotic xylooligosaccharides.

Since the immune-boosting properties as well as the benefit of promoting the growth of gut bacteria, xylooligosaccharides as prebiotics have attracted considerable interest as functional feed additives around the world. A growing number of studies suggest that acidic hydrolysis is the most cost-effective method for treating xylan materials to prepare xylooligosaccharides, and organic acids were proved to be more preferable. Therefore, in this study, glutamic acid, as an edible and nutritive organic acid, was employed as a catalyst for hydrolyzing xylan materials to prepare xylooligosaccharides. Further, xylooligosaccharide yields were optimized using the response surface methodology with central composite designs. Through the response surface methodology, 28.2 g/L xylooligosaccharides with the desirable degree of polymerization (2-4) at a yield of 40.5 % could be achieved using 4.5% glutamic acid at 163°C for 41 min. Overall, the application of glutamic acid as a catalyst could be a potentially cost-effective method for producing xylooligosaccharides.

Production of Xylooligosaccharide from Cassava Pulp’s Waste by Endo-β-1,4-D-Xylanase and Characterization of Its Prebiotic Effect by Fermentation of Lactobacillus acidophilus

This study explores the production of prebiotic xylooligosaccharide (XOS) from cassava pulp waste and its effectiveness for the growth of Lactobacillus acidophilus (L. acidophilus). We successfully produced and characterized XOS from cassava pulp xylan using a Bacillus sp. endo-β-1,4-D-xylanase. The XOS was added to modify the MRS medium (MRSm) in various concentrations (0, 1, 3 and 5%) in which the L. acidophilus was inoculated. The growth of L. acidophilus was observed every 12 h for 2 days, and the fermentation products were analyzed for pH, sugar content, and short-chain fatty acids (SCFA) in terms of types and amount. The study showed that L. acidophilus grew well in MRSm. The optimum XOS concentration in MRSm was 5%, indicated by the highest growth of L. acidophilus (8.61 log CFU mL−1). The profile of SCFA products is 14.42 mM acetic acid, 0.25 mM propionic acid, 0.13 mM isobutyric acid, 0.41 mM n-butyric acid, 0.02 mM n-valeric acid, 0.25 mM isovaleric acid, and 25.08 mM lactic acid.

Expression of an extremophilic xylanase in Nicotiana benthamiana and its use for the production of prebiotic xylooligosaccharides

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.