In Vitro Fermentation of Xylooligosaccharides Produced from

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In Vitro Fermentation of Xylooligosaccharides Produced from Miscanthus x giganteus by Human Fecal Microbiota Ming-Hsu Chen, Kelly S. Swanson, George C. Fahey, Bruce Dien, Alison N Beloshapka, Laura L. Bauer, Kent Rausch, Mike Tumbleson, and Vijay Singh J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04618 • Publication Date (Web): 09 Dec 2015 Downloaded from http://pubs.acs.org on December 15, 2015

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Journal of Agricultural and Food Chemistry

TITLE PAGE In Vitro Fermentation of Xylooligosaccharides Produced from Miscanthus x giganteus by Human Fecal Microbiota

Ming-Hsu Chen1, Kelly S. Swanson2, George C. Fahey Jr.2, Bruce S. Dien3, Alison N. Beloshapka2, Laura L. Bauer2, Kent D. Rausch1, M. E. Tumbleson1, and Vijay Singh1*

1. Department of Agricultural and Biological Engineering, University of Illinois at UrbanaChampaign, 1304 W. Pennsylvania Avenue, Urbana, IL 61801, USA 2. Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, IL 61801, USA 3. Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, 1815 North University Street, Peoria, IL 61604, USA

*Corresponding author. Tel: 217-333-9510, Fax: 217-244-0323 E-mail address: [email protected] (Dr. Vijay Singh)

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ABSTRACT Purified xylooligosaccharides from Miscanthus x giganteus (MxG XOS) were used in an

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in vitro fermentation experiment inoculated with human fecal microbiota. A commercial XOS

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product and pectin were used as controls. Decreases in pH by 2.3, 2.4, and 2.0 units and

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production of short-chain fatty acids (SCFA; acetic acid: 7764.2, 6664.1, 6387.9 µmol/g;

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propionic acid: 1006.7, 1089.5, 661.5 µmol/g; and butyric acid: 955.5, 1252.9, 917.7 µmol/g)

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were observed in MxG XOS, commercial XOS, and pectin medium after 12 h of fermentation,

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respectively. Titers of Bifidobacterium spp., Lactobacillus spp., and Escherichia coli increased

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when fed all three substrates as monitored by qPCR. There was no significant trend for

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Clostridium perfringens. During fermentation, MxG XOS was statistically equivalent in

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performance to the commercial XOS sample as measured by culture acidification and growth of

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health-promoting bacteria and resulted in the highest SCFA production among the three

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substrates.

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Key words: Miscanthus x giganteus, Xylooligosaccharides, Prebiotics, In Vitro Fermentation,

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Fecal Microbiota, Co-products, Biorefinery.


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Introduction Plant fiber-derived nondigestible oligosaccharides (NDO), such as xylooligosaccharides

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(XOS), are an emerging category of prebiotics. These low molecular weight dietary fibers

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represent an opportunity to realize further value from lignocellulosic material with generation of

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coproducts for the agricultural food industry.1 Some NDOs possess nutraceutical properties and

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are used widely in manufacturing of health foods. They offer multiple physiological benefits to

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consumers, including healthier gut microecology, reduced concentrations of blood cholesterol,

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reduction of stomach ulcer lesions, regulation of the immune response, and reduced incidence of

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colorectal cancer.2,3 NDOs pass through the small intestine and upon entering the large intestine

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promote the growth of beneficial bacteria such as Bifidobacterium spp. and Lactobacillus spp.3

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Short-chain fatty acids (SCFA), including acetic, propionic, and butyric acids, produced during

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NDO fermentation acidify the colon; SCFA can be absorbed, utilized as an energy source for

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colonocytes, participate in gluconeogenesis, and act as signaling molecules to activate free fatty

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acid receptors.4

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Xylooligosaccharides (XOS) are sugar oligomers composed of xylose in β-1,4 glycosidic

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bonds with other sugars and uronic and acetic acids substituted at the C2 or C3 positions. XOS

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can be produced either through the enzymatic hydrolysis of xylan-containing material or through

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the partial hydrolysis of biomass by steam, hydrothermal, and dilute acid treatment. Compared

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with other NDOs, XOS appear to elevate bifidobacteria numbers in the human gut at a lower

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dosage (2 g) than galactooligosaccharides (2.5 g), fructooligosaccharides (8 g), or

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isomaltooligosaccharides (10 g).3 Major commercial XOS are produced from corn cobs using

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enzymatic methods; however, recent investigations have focused on producing XOS from other

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sources such as wheat bran, rice husk, and dedicated bioenergy crops.5-7 Variation in structure 3 ACS Paragon Plus Environment


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and degree of polymerization (DP) of XOS leads to distinct prebiotic properties, which

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necessitates assaying each new product for bioactivity.8,9

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Miscanthus x giganteus (MxG) is an energy crop that has been well studied in terms of

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cultivation and harvesting. High carbohydrate content of the MxG cell wall makes it a suitable

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source of biomass for fuel and chemical conversion.10 There have been several reports describing

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pretreatment and enzymatic hydrolysis of MxG for ethanol production.11-13 Development of

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novel co-products will promote continued commercialization of the cellulosic ethanol industry.

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XOS from MxG hemicelluloses is a promising co-product provided it can be shown that the

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biological activity of XOS derived from MxG is beneficial.

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Previously, we produced XOS from MxG using autohydrolysis and purification via

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activated carbon adsorption coupled with ion exchange resin treatments.7,14 Purified XOS was

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cultured with two pure Bifidobacterium spp. to elucidate bacterial growth and substrate

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utilization. The pure culture study provided preliminary evidence that XOS would support

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microbial growth associated with the human gut.14 However, using XOS in the diet is more

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complicated because of the complex microbial ecology present in the gut, where microbes

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compete for energy-containing substrates with other taxa with similar enzymatic machinery and

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cross-feed with others that possesses complementary enzymatic and/or metabolic capacities.

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The objective of this study was to determine the potential utilization of MxG XOS as a

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prebiotic for humans and provide data regarding its effects on the gut ecosystem. The results

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could be used to evaluate whether XOS are worth recovering as a coproduct of the cellulosic

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ethanol process. The purified MxG XOS powder with 45.4% (w/w) XOS present as short

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oligomers (DP 2 to 6) was subjected to in vitro fermentation using human fecal inoculum.

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Commercial Wako XOS and pectin were used for comparison and as a positive control,


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respectively. The change in pH of the medium, SCFA concentrations, and abundance of

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Bifidobacterium spp, Lactobacillus spp., Escherichia coli and Clostridium perfringens were

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measured for each culture.

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Materials and methods

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Raw materials and chemicals

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Harvested MxG was obtained from the research farm at the University of Illinois at

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Urbana-Champaign in 2012 (planted at 40.06°N, 88.19°W). The whole plant was dried in a static

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oven at 49 °C and comminuted using a hammer mill equipped with 250 µm openings screen

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(model MHM4, Glen Mills, Clifton, NJ). The MxG sample contained (w/w, dry matter basis)

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39.4% glucan, 22.3% xylan, 17.0% acid insoluble lignin, 0.7% acid soluble lignin, and 8.0%

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extractives following NREL standard procedures (TP 510 42618; TP 510 42619). The milled

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powder was used for XOS sample preparation. Commercial XOS (Wako XOS) was purchased

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from Wako Chemicals USA (Richmond, VA) for comparison to an existing XOS product. The

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Wako XOS contained 77.8% (w/w) XOS (xylobiose and xylotriose) and 93.4% total substituted

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oligosaccharides (TSOS; TSOS represented the total amount of arabinose, glucose, and xylose

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oligomers and substituted acetyl groups in XOS sample), 0.03% ash, and 4.9% moisture. Pectin

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(pectin HM rapid, Tic Gums, Belcamp, MD) was used as a positive control for in vitro

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fermentation. The pectin powder had 66.3% total dietary fiber and 2.1% crude protein.15 All

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chemicals and reaction reagents, unless stated otherwise, were of analytical quality and were

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supplied by Fisher Scientific (Springfield, NJ).

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MxG XOS preparation 5 ACS Paragon Plus Environment


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XOS were produced by autohydrolysis through partial hydrolysis of MxG hemicelluloses.

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Autohydrolysis was performed in 500 mL stainless pipe reactors with a solid: water ratio at 1:7

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(12.5% w/w solids). Reactors were heated to 200 °C and incubated for 20 min in a fluidized sand

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bath.7 Pretreated liquid was separated from solids by filtration through glass fiber filters

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(Whatman GF/D, Fisher Scientific, Springfield, NJ) to acquire an XOS-enriched solution.

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Activated carbon powder 10% (w/v) (Darco G60, Sigma Aldrich, St. Louis, MO) was

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added to the XOS-enriched solution to adsorb XOS. The carbon slurry was incubated at 30 °C

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and agitated at 80 rpm for 60 min. Added carbon was separated from the slurry by vacuum

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filtration in the form of carbon cake. The adsorbed XOS were washed from the carbon cake by

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sequential elution with 5, 30, 50, 70, and 95% (v/v) ethanol solutions. The collected 30, 50, and

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70% ethanol fractions were combined; ethanol was evaporated and adjusted to an XOS

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concentration of 2.74 g/100 mL. To further remove impurities, the recovered XOS solution was

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treated with strong cation exchange resin (Amberlite IR120Na), weak anion exchange resin

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(Amberlite IR96), and polishing resin (Amberlite FPA90Cl) at the mass ratio of resin:liquid

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(1:10) in sequence. The polished solution was dried and milled to obtain MxG XOS.14 The

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overall MxG XOS preparation procedure is shown in Figure 1.

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The composition of XOS was analyzed following standard procedure NREL TP 510

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42623 (Laboratory Analytical Procedure, National Renewable Energy Laboratory, U.S.

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Department of Energy, available online at http://www.nrel.gov/docs/gen/fy08/42623.pdf).

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Briefly, XOS were hydrolyzed in 4% H2SO4 at 121 °C for 60 min. An increased concentration of

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monosaccharides (arabinose, xylose, and glucose) and acetic acid was used to calculate oligomer

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composition. XOS of each degree of polymerization (DP) were quantified by high performance

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anion exchange chromatography with a pulsed amperometric detection system, HPAEC-PAD 6 ACS Paragon Plus Environment

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(Dionex ICS 3000, Sunnyvale, CA). The HPAEC-PAD was equipped with a Dionex PA-100

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column (4 x 250 mm, Dionex) and operated at a flow rate of 1 mL/min running a linear gradient

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from 100% 100 mM NaOH to 12% 100 mM NaOH containing 1 M sodium acetate in 20 min.

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Commercially available xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaoase

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were used as standards.16

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Purified MxG XOS comprised 74.9% xylose oligomers, 3.0% arabinose oligomers, 4.5%

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glucose oligomers, and 6.7% bound acetyl groups. The total purity was 89.1% (w/w, TSOS) with

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additional 1.7% sugar monomers. Through oligomer analysis by HPAEC-PAD, the MxG XOS

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had 8.9% xylobiose, 11.3% xylotriose, 8.8% xylotetraose, 9.0% xylopentose, and 5.0%

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xylohexaose.

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Donors and microbiota solution

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Three human fecal samples were collected and homogenized to provide the microbial

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inoculum. All donors were adult males, free of gastrointestinal disease, and had no antibiotic

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treatment in the three months before initiation. The experimental protocol was approved by the

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University of Illinois at Urbana-Champaign Institutional Review Board. All subjects signed an

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informed consent form before the start of the experiment. Fresh fecal samples were delivered in a

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Commode Specimen Collection System (Sage Products, Crystal Lake, IL) in the early morning.

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Samples were mixed with anaerobic dilution solution at a ratio of 1:10 and blended for 15 sec in

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a Waring blender with CO2 injection. The diluted solution was filtered through four layers of

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cheesecloth into 125 mL serum bottles under stable and continuous CO2 injection. The solution

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was used immediately for inoculation.

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In vitro fermentation cultures 7 ACS Paragon Plus Environment


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Culture medium was prepared one day before inoculation.17 All components

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(Supplementary data 1), except vitamin and hemin solutions, were mixed and autoclaved at 121

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°C for 20 min. Vitamin and hemin solutions were filter-sterilized using a 0.22 µm membrane and

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added before dispensing medium.

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Anaerobic cultures were conducted using 1% (w/v) substrate in 50 mL culture tubes

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capped with butyl rubber stoppers. Twenty-six milliliters of the medium were added to culture

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tubes with either 300 mg of MxG XOS, Wako XOS, or pectin; blanks were prepared without

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substrate. Tubes were stored at 4 °C for 12 h to improve substrate hydration and incubated in a

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37 °C water bath 30 min prior to inoculation.

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Each culture tube was inoculated with 4 mL microbial inoculum and incubated at 37 °C

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for 12 h. Every 4 h, tubes were removed from the incubator for periodic mixing and immediately

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sampled. The pH of the medium was measured using a standard pH meter (Denver Instrument

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Co., Arvada, CO); 2 mL of medium was collected for SCFA analysis and 1 mL of medium was

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collected for bacterial population analysis.

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Short-chain fatty acid quantification

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A volume of 0.5 mL 25% (w/v) metaphosphoric acid was added to the 2 mL aliquot. The

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solution was incubated for 30 min at room temperature and centrifuged at 20,000 x g for 20 min

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and the supernatant recovered. Short-chain fatty acids (acetic, propionic, and butyric) present in

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the supernatants were analyzed by a Hewlett Packard 5890A series II gas chromatography (Palo

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Alto, CA) with a glass column packed with 10% SP-1200/1% H3PO4 on 80/100+ mesh

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Chromosorb-WAW (Supelco Inc., Bellefonte, PA). Gas chromatographic settings were:

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injection temperature, 175 °C; oven temperature, 125 °C; FID temperature, 180 °C; nitrogen 8 ACS Paragon Plus Environment

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flow , 50 mL/min; head pressure, 18 psi; threshold, 0, and running time 20 min. The

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concentrations of SCFA in each sample were calculated by comparing the peak area with the

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standards. The SCFA concentrations then were corrected with blanks, and converted to µmol/g

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for each tested substrate.

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Quantitative polymerase chain reaction (qPCR)

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The microbial groups of interest were quantified by qPCR using bacterial DNA as a

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template. DNA was extracted from sampled medium using the PowerLyzerTM PowerSoil®

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DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) and quantified by a Qubit® 2.0

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Fluorometer (Life TechnologiesTM, InvitrogenTM, Grand Island, NY).

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qPCR was performed to quantify the abundance of Bifidobacterium spp., Lactobacillus

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spp., Escherichia coli, and Clostridium perfringens as described previously.18 DNA

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amplification was performed following DePlancke et al. (2002) with slight modifications and

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specific primers for each bacteria were used.19 The 10 µL reaction mixture had 5 µL of 2x SYBR

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Green PCR Master Mix (Applied Biosystems, Foster City, CA), 15 pmol of forward and reverse

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primers, and 10 ng of extracted bacterial DNA. The standard curve for each bacterium was

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prepared by single strain culture sampled in exponential growth phase. Bacterial cells were

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collected by centrifugation and serial diluted. DNA was extracted from each pure strain dilution

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and used for qPCR. The number of colony forming units (CFU) of each standard curve serial

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dilution was determined by plating the E. coli grown on Luria-Bertani Medium (10 g/L tryptose,

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5 g/L yeast extract, and 5 g/L NaCl [pH 7]), the Lactobacillus genus grown on Difco Lactobacilli

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MRS broth (Becton, Dickinson, and Company, Sparks, MD), and the C. perfringens and

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Bifidobacterium genera grown on Difco Reinforced Clostridial Medium (Becton, Dickinson, and



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Company). Standard curves were plotted as log10CFU per mL vs. cycle threshold (Ct) values

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from qPCR.

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Statistical analysis

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The in vitro fermentation was performed in triplicate. The pH, SCFA, and bacterial

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population values were analyzed as a completely randomized design model using the Proc glm

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procedure of SAS (version 9.4, SAS Institute, Inc., Cary, NC). The means were compared using

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Tukey's Studentized Range (HSD) test; statistical significance was accepted at a probability of P

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< 0.05.

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Results and discussion

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pH change during fermentation

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The pH values for all the cultures dropped during anaerobic fermentation as expected

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from the production of organic acids. The pectin-fed control culture began at a pH of 6.98 and

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experienced a rapid pH drop during the first 4 h of fermentation. The pH decreased less for MxG

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XOS and Wako XOS than pectin for the first 4 h, but had greater drops from 4 to 8 h compared

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to pectin. At the end of fermentation (t = 12 h), pH values for pectin, Wako XOS, and MxG XOS

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were 5.26, 5.01 and 5.04, respectively. The two XOS treatments had statistically similar drops in

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pH during fermentation (Figure 2; Supplementary data 2).

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Increased acidity during fermentation is an indicator of substrate utilization and organic

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acid production. When Flickinger et al. (2000) used in vitro fermentation to evaluate

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fructooligosaccharides (FOS) and glucooligosaccharides (GOS) with fecal microbiota, the pH of


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FOS tubes decreased more rapidly than those with GOS, which was taken as evidence that FOS

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was utilized faster.20 Hernot et al. (2009) tested several commercial fructans,

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galactooligosaccharides, and polydextrose with human fecal microbiota; during 12 h of

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incubation, the pH decrease for fructans was greater and ranged from 0.9 to 1.32.17 Compared

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with our results, the pH drops were 1.72 for pectin, 2.11 for Wako XOS, and 2.05 for MxG XOS.

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The significant drops in pH demonstrated that all of the substrates were readily utilized by fecal

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microbiota. The similar pH curves observed for fermentation of MxG XOS and Wako XOS

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indicate that both XOS samples would be expected to have a similar pattern of utilization by

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microbiota present in the colon (Figure 2).

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Short-chain fatty acid (SCFA) production

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Organic acid production profiles for the three substrates are listed in Table 1. MxG- and

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Wako XOS-fed cultures yielded much more of each organic acid than did pectin. MxG XOS

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cultures produced 16.5% more acetic acid, 8.2% less propionic acid, and 31.2% less butyric acid

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than Wako XOS cultures. In terms of total SCFA (12 h), the Wako XOS and pectin cultures

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produced 7.4% and 18.5% less than the MxG XOS cultures.

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In the colon, SCFAs are produced as a result of the microbial fermentation of

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nondigestible carbohydrates. The pentose substrate (present in both xylan and pectin) is

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metabolized through the pentose phosphate pathway to pyruvate. Pyruvate can be converted to

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acetyl-CoA and then to acetic and butyric acids, or converted to succinic and then to propionic

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acids. The ratio of acetic, propionic, and butyric acids produced varies with substrate.21 Rycroft

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et al. (2001) evaluated commercial XOS (Suntory, Osaka, Japan) using human fecal microflora

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and found that 2670 µmol/g of acetic, 721 µmol/g of propionic, and 175 µmol/g of butyric acids

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were produced.22 Hughes et al. (2007) fermented wheat arabinoxylan using human fecal 11 ACS Paragon Plus Environment


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microflora and found that 2870 µmol/g acetic, 650 µmol/g propionic, and 1470 µmol/g butyric

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acids were produced from 66 kDa arabinoxylan.5 Pastell et al. (2009) tested Wako XOS using

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human fecal microflora and found that 3340 µmol/g acetic, 860 µmol/g propionic, and 800

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µmol/g butyric acids were produced.23 From our data, Wako XOS resulted in higher acetic acid

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production compared with Pastell et al. (2009), which may have been due to variance in mixed

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strain fermentation. The higher acetic acid production from MxG XOS can be explained by 6.7%

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(w/w) bound acetyl groups on the XOS, which are enzymatically released by acetyl esterase (EC

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3.1.1.6).

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Bacterial population changes during fermentation Bacterial taxa of interest were measured using qPCR on extracted microbiota DNA

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(Table 2). Beneficial bacteria genera increased during the fermentation of all three substrates.

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For Bifidobacterium spp., the total cell number started with an average of 10.12 (log10CFU/tube)

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and then increased to 10.97 (log10CFU/tube) for pectin, to 11.62 (log10CFU/tube) for Wako XOS,

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and to 11.54 (log10CFU/tube) for MxG XOS. The two XOS substrates resulted in statistically

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similar Bifidobacterium spp. growth, which was statistically higher than for growth on pectin.

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For Lactobacillus spp., the total cell number started with an average of 11.18 (log10CFU/tube)

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and increased to 12.17 (log10CFU/tube) for pectin, to 12.08 (log10CFU/tube) for Wako XOS, and

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to 12.12 (log10CFU/tube) for MxG XOS. There was no substrate effect observed, and the growth

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of Lactobacillus spp. was statistically similar at the end of fermentation for the three substrates.

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An increase in the E. coli population was observed from 0 to 4 h, but all three treatments had

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lower populations than did the control blank at the end of the fermentation. The trend for

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Clostridium perfringens cell counts was unaffected over time by substrate. 12 ACS Paragon Plus Environment

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Rycroft et al. (2001) observed a 6.5 fold increase in Bifidobacterium spp. and a 2.5 fold

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increase in E. coli populations in 1% (w/v) XOS-containing tubes after inoculum for five hours.22

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Hughes et al. (2007) reported a 6.5 fold increase in Bifidobacterium, 2.6 fold increase in

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Clostridium, and 9.3 fold increase in Lactobacillus spp. populations using 66 kDa arabinoxylan

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as a substrate.5 Moura et al. (2008) tested XOS from Eucalyptus (EUC), corn cobs (CC), and

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brewer’s spent grain (BSG); Bifidobacterium spp. populations were increased with the

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fermentation of EUC and CC, but not BSG.24 In our study, the qPCR results validated that XOS

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were able to improve beneficial bacterial population growth. Compared to 0 h data,

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Bifidobacterium spp., Lactobacillus spp., and E. coli increased 22.9-, 8.3-, and 4.4-fold,

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respectively, for MxG XOS after 12 h of fermentation. Compared to health-promoting bacteria,

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the increase in potential pathogens was relatively small and may have been due to the expanding

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of total cell numbers in the medium. The prebiotic function of MxG XOS was comparable to that

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of Wako XOS, based on the increased cell number of these beneficial bacteria.

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Production of XOS during autohydrolysis affords an opportunity to realize a valuable co-

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product in conjunction with pretreatment during the cellulosic ethanol process. Purification of

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XOS from crude hydrolysate is challenging; the liquid phase of autohydrolysis contains a variety

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of undesirable compounds derived from lignin, protein, pigment, and sugars.2,8. In this study,

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highly purified XOS were directly produced from MxG hydrolysate. MxG XOS was determined

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to be equally as effective as a commercial XOS product and more so than pectin as judged by the

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responses observed using in this experiment.

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Acknowledgement



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We thank Dr. Michael Bowman and Loren Iten from the Bioenergy Research Unit,

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Agricultural Research Service, United States Department of Agriculture for their help on the

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oligosaccharides analysis and autohydrolysis of Miscanthus x giganteus. We also thank Drs.

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Michael Cotta and Terence Whitehead from the Bioenergy Research Unit, Agricultural Research

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Service, United States Department of Agriculture, for their technical advice.

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Supporting information

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Composition of medium used for in vitro fermentations (Supplementary data 1)

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pH change in media during in vitro fermentation of pectin and XOS (Supplementary data 2)

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References

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1) Ho, A. L.; Carvalheiro, F.; Duarte, L. C.; Roseiro, L. B.; Charalampopoulos, D.; Rastall, R.

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A. Production and purification of xylooligosaccharides from oil palm empty fruit bunch fibre

280

by a non-isothermal process. Bioresour. Technol. 2014, 152, 526-529.

281

2) Moure, A.; Gullón, P.; Domı́nguez, H.; Parajó, J. C. Advances in the manufacture,

282

purification and applications of xylooligosaccharides as food additives and nutraceuticals.

283

Process Biochem. 2006, 41, 1913-1923.

284 285 286

3) Mussatto, S. I.; Mancilha, I. M. Non-digestible oligosaccharides. Carbohyd. Polym. 2007, 68, 587-597. 4) Brüssow, H.; Parkinson, S. J. You are what you eat. Nat. Biotechnol. 2014, 32, 243-245.


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5) Hughes, S. A.; Shewry, P. R.; Li, L.; Gibson, G. R.; Sanz, M. L.; Rastall, R. A. In vitro

288

fermentation by human fecal microflora of wheat arabinoxylans. J. Agric. Food Chem. 2007,

289

55, 4589-4595.

290

6) Gullón, P.; González-Múñoz, M. J.; van Gool, M. P.; Schols, H. A.; Hirsch, J.; Ebringerová,

291

A.; Parajó, J. C. Production, refining, structural characterization and fermentability of rice

292

husk xylooligosaccharides. J. Agric. Food Chem. 2010, 58, 3632-3641.

293

7) Chen, M.-H.; Bowman, M. J.; Dien, B. S.; Rausch, K. D.; Tumbleson, M. E.; Singh, V.

294

Autohydrolysis of Miscanthus x giganteus for the production of xylooligosaccharides (XOS):

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kinetics, characterization and recovery. Bioresour. Technol. 2014, 155, 359-365.

296 297 298 299 300 301 302

8) Vázquez, M. J.; Alonso, J. L.; Domı́nguez, H.; Parajó, J. C. Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol. 2000, 11, 387-393. 9) Rastall, R. A. Functional oligosaccharides: application and manufacture. Annu. Rev. Food Sci. Technol. 2010, 1, 305-339. 10) Brosse, N.; Dufour, A.; Meng, X.; Sun, Q.; Ragauskas, A. Miscanthus: a fast-growing crop for biofuels and chemicals production. Biofuels, Bioprod. Bioref. 2012, 6, 580-598. 11) Murnen, H. K.; Balan, V.; Chundawat, S. P. S.; Bals, B.; Sousa, L. D. C.; Dale, B. E.

303

Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of

304

Miscanthus x giganteus to fermentable sugars. Biotechnol. Prog. 2007, 23, 846-850.

305

12) Brosse, N.; Sannigrahi, P.; Ragauskas, A. Pretreatment of Miscanthus x giganteus using the

306

ethanol organosolv process for ethanol production. Ind. Eng. Chem. Res. 2009, 48, 8328-

307

8334.

308 309

13) Khullar, E.; Dien, B. S.; Rausch, K. D.; Tumbleson, M. E.; Singh, V. Effect of particle size on enzymatic hydrolysis of pretreated Miscanthus. Ind. Crop Prod. 2013, 44, 11-17.



310

Page 16 of 23

14) Chen, M.-H.; Bowman, M. J.; Cotta, M. A., Dien, B. S.; Iten, L. B.; Whitehead, T. R.;

311

Rausch, K. D.; Tumbleson, M. E.; Singh, V. Evaluation of xylooligosaccharide purification

312

and fermentation from Miscanthus x giganteus. Carbohyd. Polym.(accepted, pending

313

revision)

314

15) Sunvold, G. D.; Hussein, H. S.; Fahey Jr., G. C.; Merchen, N. R.; Reinhart, G. A. In vitro

315

fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from

316

cats, dogs, horses, humans, and pigs and ruminal fluid from cattle. J. Anim. Sci. 1995, 73,

317

3639-3648.

318

16) Bowman, M. J.; Dien, B. S.; O’Bryan, P. J.; Sarath, G.; Cotta, M. A. Selective chemical

319

oxidation and depolymerization of switchgrass (Panicum virgatum L.) xylan with

320

oligosaccharide product analysis by mass spectrometry. Rapid Commun. Mass Spectrom.

321

2011, 25, 941-950.

322

17) Hernot, D. C.; Boileau, T. W.; Bauer, L. L.; Middelbos, I. S.; Murphy, M. R.; Swanson, K. S.;

323

Fahey Jr., G. C. In vitro fermentation profiles, gas production rates, and microbiota

324

modulation as affetcted by certain fructans, galactooligosaccharides, and polydextrose. J.

325

Agric. Food Chem. 2009, 57, 1354-1361.

326

18) Middelbos, I. S.; Godoy, M. R.; Fastinger, N. D.; Fahey, G. C. A dose-response evaluation of

327

spray-dried yeast cell wall supplementation of diets fed to adult dogs: Effects on nutrient

328

digestibility, immune indices, and fecal microbial populations. J. Anim. Sci. 2007, 85, 3022–

329

3032.

330

19) DePlancke, B.; Vidal, O.; Ganessunker, D.; Donovan, S. M.; Mackie, R. I.; Gaskins, H. R.

331

Selective growth of mucolytic bacteria including Clostridium perfringens in a neonatal piglet

332

model of total parenteral nutrition. Am. J. Clin. Nutr. 2002, 76, 1117–1125. 16 ACS Paragon Plus Environment

Page 17 of 23

333


20) Flickinger, E. A.; Wolf, B. W.; Garleb, K. A.; Chow, J.; Leyer, G. J.; Johns, P. W.; Fahey Jr.,

334

G.C. Glucose-based oligosaccharides exhibit different in vitro fermentation patterns and

335

affect in vivo apparent nutrient digestibility and microbial populations in dogs. J. Nutr. 2000,

336

130, 1267-1273.

337 338

21) Hiljova, E.; Chmelarova, A. Short chain fatty acids and colonic health. Bratisl. Lek. Listy 2007, 108, 354-358.

339

22) Rycroft, C. E.; Jones, M. R.; Gibson, G. R.; Rastall, R. A. A comparative in vitro evaluation

340

of the fermentation properties of prebiotic oligosaccharides. J. Appl. Microbiol. 2001, 91,

341

878-887.

342

23) Pastell, H.; Westermann, P.; Meyer, A. S.; Tuomainen, P.; Tenkanen, M. In vitro

343

fermentation of arabinoxylan-derived carbohydrates by bifidobacterium and mixed fecal

344

microbiota. J. Agri. Food Chem. 2009, 57, 8598-8606.

345

24) Moura, P.; Cabanas, S.; Lourenco, P.; Girio, F.; Loureiro-Dias, M.; Esteves, M. P. In vitro

346

fermentation of selected xylo-oligosaccharides by piglet intestinal microbiota. LWT-Food Sci.

347

Technol. 2008, 41, 1952-1961.

348

349

350

351

352

353



354

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Figure Captions: Figure 1.

Schematic diagram of XOS produced and purified from MxG. The procedure was adapted from Chen et al., submitted.14

Figure 2.

pH change after 4, 8, and 12 h of in vitro fermentation of MxG XOS, Wako XOS, and pectin. Error bars represent ± one standard deviation.


Page 19 of 23


Table 1. SCFA Production (µmol/g) During In Vitro Fermentation of Pectin and XOS Samples Substrate Time 0 4 8 12

Acetic acid 52.0 (5.2)b 116.2 (3.4) 139.0 (11.4) 156.9 (5.0)

SCFA Propionic acid 14.4 (1.8) 35.4 (1.1) 39.3 (2.2) 49.6 (2.1)

Pectinc

4 8 12

3208.2 (180.8)E 5638.9 (131.3)D 6387.9 (146.5)BC

274.7 (12.1)F 620.3 (31.4)D 661.5 (12.8)D

267.3 (32.5)F 633.5 (27.6)E 917.6 (3.8)BC

3750.2 (221.9)FG 6892.7 (129.0)E 7967.0 (139.8)CD

Wako XOSc

4 8 12

2569.4 (115.6)F 5975.9 (26.8)CD 6664.1 (329.3)B

319.7 (6.0)EF 824.2 (38.0)C 1089.5 (18.4)A

278.1 (15.9)F 872.4 (19.2)C 1252.9 (37.2)A

3167.2 (132.7)G 7672.5 (15.0)D 9006.5 (360.5)B

Blanka

Butyric acid 12.5 (0.4) 31.1 (0.9) 34.7 (1.4) 36.7 (1.1)

Total SCFA 78.9 (7.1) 182.7 (5.2) 213.0 (14.8) 243.2 (7.3)

4 3524.0 (29.5)E 342.1 (21.6)E 255.0 (15.6)F 4121.1 (46.6)F B C D 8 6979.0 (196.9) 858.2 (10.9) 708.9 (39.9) 8546.1 (166.3)BC 12 7764.2 (428.0)A 1006.7 (19.7)B 955.5 (7.2)B 9726.3 (424.1)A a b Blank (no treatment) values were expressed as µmol of fatty acids produced during fermentation. Mean of three replicates; Standard deviation was given in parentheses. cValues were expressed as µmol/g of dry substrate. The results of the Tukey's Studentized Range (HSD) test at a = 0.05 were shown in superscript symbols. Columns with the same superscript letters indicate that there were not significant differences in fatty acid production. MxG XOSc



Page 20 of 23

Table 2. Microbiota Concentrations (log10CFU/tube) in Batch Culture Fermentations with Pectin and XOS Substratesa (log10CFU/tube)

Bifidobacterium spp.de b

0h

4h C

8h C

Lactobacillus spp.e 12 h

C

0h C

4h C

Blank

9.25

9.98

9.84

10.20

10.78

Pectin

10.04C

10.90C

10.97C

10.97BC

Wako XOS

10.15C 11.18BC

11.13C

MxG XOS

10.18C 11.15BC

11.50AB

Pooled SEMc

0.30

8h C

12 h C

11.09

Escherichia colie 0h C

4h D

8h

BCD

AB

Clostridium perfringens 12 h

0h A

4h

8h

12 h

11.03

11.29

8.76

9.54

9.89

10.10

7.66 7.63 7.55 7.82

11.11C 11.92BC

12.07AB

12.17AB

8.98D

9.86AB

9.93AB

9.89AB

7.89 8.17 8.28 8.34

11.62A

11.25C 11.98BC

12.28A

12.08AB

9.25CD

9.83BC

9.80BC

9.83BC

7.79 8.25 8.20 7.98

11.54A

11.20C 11.95BC

12.18AB

12.12AB

9.01D

9.84B

9.83BC

9.65BCD 8.02 8.13 8.40 7.81

0.22

0.24

0.18

a

Values with the same superscript letters indicate that there were not significant differences in growth. Colony-forming unit. c Standard error of the means. d Substrate effect (P < 0.05). e Time effect (P < 0.05). b


Page 21 of 23


Figure 1 21 ACS Paragon Plus Environment


Page 22 of 23

0.0 MxG XOS Wako XOS Pectin

pH change

-0.5

-1.0

-1.5

-2.0

-2.5 0

2

4

6

8

10

12

Time (h)

Figure 2


Page 23 of 23


(TOC Graphic)


In Vitro Fermentation of Xylooligosaccharides Produced from

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