- Email: [email protected]
Effects of Inulin Chain Length on Fermentation by Equine Fecal Bacteria and Streptococcus bovis
Accepted Manuscript Effects of inulin chain length on fermentation by equine fecal bacteria and Streptococcus bovis Brittany E. Harlow, Isabelle A. Kagan, Laurie M. Lawrence, Michael D. Flythe PII:
S0737-0806(15)30018-6
DOI:
10.1016/j.jevs.2015.11.010
Reference:
YJEVS 1987
To appear in:
Journal of Equine Veterinary Science
Received Date: 10 September 2015 Revised Date:
29 November 2015
Accepted Date: 30 November 2015
Please cite this article as: Harlow BE, Kagan IA, Lawrence LM, Flythe MD, Effects of inulin chain length on fermentation by equine fecal bacteria and Streptococcus bovis, Journal of Equine Veterinary Science (2016), doi: 10.1016/j.jevs.2015.11.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title:
Effects of inulin chain length on fermentation by equine fecal
Authors:
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bacteria and Streptococcus bovis
Brittany E. Harlowa, Isabelle A. Kagana,b, Laurie M. Lawrencea,
a
University of Kentucky, Department of Animal and Food
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Affiliations:
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Michael D. Flythea,b
Sciences; Lexington, Kentucky 40546
b
USDA,
Agricultural
Research
Service,
Forage-Animal
“Proprietary or brand names are necessary to report factually on
available data; however, the USDA neither guarantees nor
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Disclaimer:
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Mandatory
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Production Research Unit; Lexington, Kentucky 40546
warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, nor exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.”
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Key Words:
fructan, inulin, oligofructose, streptococci, hindgut acidosis
Correspondence:
Michael D. Flythe USDA, ARS
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N-220 Ag. Science North University of Kentucky
Phone: (859) 421-5699 Fax: (859) 257-3334
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Lexington, KY 40546
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Email: [email protected]
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Abstract
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Grass fructans can be fermented by Gram-positive bacteria (e.g., Streptococcus bovis) in
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the equine hindgut, increasing production of lactic acid and decreasing pH. The degree of
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polymerization (DP) of fructans has been suggested to influence fermentation rates. The
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objective of the current study was to determine how DP impacts fermentation by equine
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fecal bacteria and a model S. bovis. Fecal microbes from 3 mares were harvested by
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differential centrifugation, washed, and re-suspended in anaerobic media containing
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short-chain (SC; DP ≤ 10) or long-chain (LC) inulin (DP ≥ 23) from 0-2% w/v. After 24
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h of incubation (37 ºC) samples were collected for pH determination. Data were
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analyzed using the GLM procedure testing for the effect of treatment, concentration, and
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treatment × concentration (SAS v. 9.3). At all concentrations, the pH was lower in SC
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fermentations than in LC (P < 0.0001, in all cases). To determine the effect of DP on S.
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bovis, cultures were grown (39 ºC, 9 h) with 0.1, 0.5, or 1.3% SC or LC inulin. Optical
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density (OD600) was determined by spectrophotometry. Maximum specific growth rates
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(µ) were determined by linear regression (2-5 h). Data were analyzed using the one-way
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ANOVA procedure (SAS v. 9.3). The final OD600, µ and yield were higher with SC than
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with LC fermentation (P < 0.05). These results indicate that SC inulin may be more
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available for fermentation than LC inulin by equine fecal bacteria and S. bovis,
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specifically.
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Highlights
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Inulin DP influences equine fecal bacteria and S. bovis fermentations in vitro.
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Short-chain inulin is more available for fermentation than long-chain inulin.
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S. bovis JB1 grows faster on low DP inulin than on high DP inulin.
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1. Introduction Pasture associated laminitis (PAL) is a multifactorial disorder, and risk factors
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ranging from diet to reduced insulin sensitivity have been implicated [1]. One of the risk
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factors is grazing on pastures rich in water-soluble carbohydrates (WSC, mono- and
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disaccharides and fructans), which can lead to gastrointestinal disturbances [2]. Cool
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season grasses produce fructans, fructose polymers that usually have a terminal sucrose
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[3] and that may accumulate at certain times of the year [4,5,6]. Fructans are not
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completely digested in the foregut and are fermented in the hindgut by Gram-positive
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bacteria (e.g., Streptococcus spp.) that produce lactic acid, decreasing pH [7,8,9].
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Decreasing hindgut pH can lead to increases in intestinal permeability, enabling bacteria
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or bacterial metabolites to enter the blood stream [10]. Several bacterial metabolites
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have been implicated in the pathogenesis of PAL including endotoxins [11], amines [12]
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and proteinases [13]. Streptococcus bovis is commonly implicated as the primary
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etiological agent in PAL because it has the capacity to produce amines and proteinases,
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besides being abundant in carbohydrate-excess conditions [12,14].
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Inulins, the β-2,1 linked fructans found in a few dicotyledonous and
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monocotyledonous families in addition to the Poaceae [15], have been commonly used as
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a model fructan to study the pathogenesis of PAL both in vitro [12,16,17] and in vivo
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[7,18,19]. However, grass fructans can have β-2,6 linkages or a mixture of β-2,1 and β-
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2,6 linkages [20], sometimes within the same molecule [21,22]. Fructans with β-2,6
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linkages are sometimes called levans [3] or phleins [23], and those with both linkage
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types may be referred to as graminans [23]. Due to the structural diversity of grass
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fructans, it is uncertain if they would be fermented in the same manner as inulin. One
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parameter contributing to this uncertainty, in terms of both inulins and grass fructans, is
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the effect of the number of monomers, referred to as chain length or degrees of
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polymerization (DP). Several factors can influence the composition and chain length of
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grass fructan, including time of year, climate, and grass species [24]. It has been
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suggested that short-chain (SC) fructans are more rapidly fermented than long-chain (LC)
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fructans in the equine hindgut [9]. However, to the best of our knowledge, the
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relationship between fructan chain length and fermentation by equine hindgut bacteria
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has not been experimentally determined. Therefore, the objective of the current study
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was to determine how chain length impacts fermentation by equine fecal bacteria, using
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inulins as model fructans. The advantage of using inulins is that the effects of chain
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length can be tested without other differences in structure. The hypothesis was that
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equine fecal bacteria and a model S. bovis would more rapidly ferment an inulin with a
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shorter chain length.
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2. Material and methods
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2.1. Media composition
The cell suspension medium was weakly buffered to allow pH to decrease in
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response to acid production, and contained (per liter): 240 mg KH2PO4, 240 mg K2HPO4,
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480 mg (NH4)2SO4, 480 mg NaCl, 64 mg CaCl2 · 2H2O, 100 mg MgSO4 · 7H2O, 600 mg
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cysteine hydrochloride; initial pH 6.7; autoclaved to remove O2 and cooled under N2.
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The growth medium, based on Mantovani and Russell [25], was heavily buffered
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to maximize bacterial growth. This medium contained (per liter): 240 mg KH2PO4, 240
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mg K2HPO4, 480 mg (NH4)2SO4, 480 mg NaCl, 64 mg CaCl2 · 2H2O, 100 mg MgSO4 ·
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7H2O, 600 mg cysteine hydrochloride, 1,000 mg Trypticase, 500 mg yeast extract; initial
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pH 6.7; autoclaved to remove O2 and cooled under O2 –free CO2. The buffer (4,000 mg
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Na2CO3 per liter) was added to the medium before dispensing and autoclaving for
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sterility.
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Short chain (SC; chain length: 1 to 13, mean DP documented as ≤ 10; Orafti®
OPS; BENEO Inc., Morris Plaines, NJ, USA) or long chain (LC; chain length: 13 to 60,
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mean DP documented as ≥ 23; Orafti® HP; BENEO Inc.) inulin from chicory (Fig. 1)
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was added to either media type when indicated. A preliminary experiment indicated that
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autoclaving (121 °C, 1 bar) did not hydrolyze fructans at pH 6 to 7. The chain length
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thought to be represented by each peak (indicated by numbers above selected peaks) was
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based on the retention time determined for a standard of 1-kestose (a trisaccharide), and
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the fact that each successive inulin peak after 1-kestose generally represents an increase
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of one DP [21]. The SC inulin was not entirely free from non-inulin compounds (glucose,
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fructose, and sucrose, indicated on Figure 1A), but is referred to as SC inulin instead of
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SC inulin-enriched fraction for brevity.
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2.2. Animals and fecal collection
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All animal care, handling and procedures were approved by the University of
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Kentucky Institutional Animal Care and Use Committee. Horses were selected from the
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University of Kentucky, Department of Animal and Food Sciences herd at Maine Chance
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Farm, Lexington KY. The feces donors were three mature Thoroughbred mares
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maintained on hay with limited access to pasture. Replicate experiments (n = 3) were
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performed on separate days using the feces from one horse on each day.
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When feces were needed for an experiment, horses were observed for defecation and feces were collected by catch sampling without ground contact. Each sample was
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thoroughly mixed by hand and placed in a plastic bag. The bag was then purged of air
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with CO2 and transported to the laboratory (within 0.5 h of collection) in a pre-warmed
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container (37 °C) for processing.
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2.3. Fecal Cell Suspensions
Upon arrival at the laboratory fecal cell suspensions were prepared as previously
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described [17]. In short, feces (450 g) were placed in a blender, under N2, and mixed (3
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min or until homogenous) with 750 mL of cell suspension medium (see above). The
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mixture was then squeezed through cheesecloth to remove large plant particles and
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underwent low-speed centrifugation (341.6 × g, 5 min) to remove protists and remaining
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plant fibers. The supernatants were collected and then subjected to high-speed
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centrifugation (25654.3 × g, 5 min) to collect bacteria. Supernatants were then discarded
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and bacterial pellets were washed via re-suspension in anaerobic cell suspension medium.
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Bacterial cells were then harvested by a second high-speed centrifugation (25654.3 × g,
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10 min). Again the supernatants were discarded, and the bacterial pellets were re-
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suspended and pooled in a N2-sparged glass vessel. The optical densities (600 nm; OD600)
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of the fecal cell suspensions were adjusted to ~15 with anaerobic cell suspension
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medium. Microscopic analysis of fecal cell suspensions revealed bacteria sized cells with
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no plant particles or protists.
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An initial experiment was conducted to determine the effect of inulin
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concentration and chain length on pH of the cell suspension medium. SC or LC inulin (0
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– 2.0% w/v, in 0.1% increments) was added to a fecal cell suspension that was aliquoted
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into duplicate anaerobic Hungate tubes (N2 atmosphere). The tubes were then incubated
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in a shaking water bath (37 °C, 160 rpm) for 24 h. Samples were collected via tuberculin
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syringes for pH measurement and later product analysis. The pH was measured
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immediately with a pH meter. Supernatants for product analysis were clarified by
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centrifugation (21,000 × g, 2 min), and frozen (-20 °C) until analyzed, as described
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below. The experiment was replicated three times with each replicate using feces from a
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different horse.
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2.4. Streptococcus bovis JB1 experiments
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A pure culture of S. bovis JB1 was originally obtained from the culture collection
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of James B. Russell; United States Department of Agriculture, Agricultural Research
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Service at Cornell University, Ithaca, NY, USA. S. bovis was routinely transferred in
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growth medium with glucose (0.4% w/v), but it was transferred at least five times with
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inulin from chicory (0.4% w/v; chain length: 10 to 50, mean DP documented as 35,
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Sigma Aldrich, St Louis, MO, USA) as the sole carbon source prior to the described
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experiments.
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Of the twenty inulin concentrations used to check effects on pH, three (0.1%,
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0.5% and 1.3% w/v) were selected to determine the effect of inulin concentration and
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chain length on S. bovis JB1 growth and metabolism. SC or LC inulin was added to
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bottles of growth medium at the three concentrations listed above. Stationary phase (16 h)
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S. bovis JB1 cultures were used to inoculate bottles (0.1% w/v inoculum), and they were
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incubated in a shaking water bath (37 °C, 160 rpm). Growth (OD600) was monitored in a
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spectrophotometer every 30 min for 9 h. After 9 h of incubation, samples were collected
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with tuberculin syringes for pH measurement and later product analysis (see section 2.3.).
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2.5. Chemical Analyses
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2.5.1. Inulin quantification
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Clarified supernatants of S. bovis incubated 9 h with 0.1%, 0.5%, or 1.3% inulin (SC or LC) were vacuum-filtered through Extract-Clean Prevail C18 solid-phase
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extraction columns (Grace Davison Discovery Sciences, Deerfield, IL, USA). This step
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removed lipophilic compounds. Because the concentration of medium affected the
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detector response (data not shown), filtrates were diluted so that all injections contained
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10% medium, and 10% medium was injected as a blank. The 0.1% (1 mg/mL) inulin
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incubations were diluted tenfold in water to 0.1 mg/mL in 10% medium, and the 0.5% (5
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mg/mL) and 1.3% (13 mg/mL) inulin incubations were diluted with water and medium to
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0.25 mg/mL in 10% medium. Dilutions (20 µL) were separated on a Dionex (Westmont,
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IL, USA) CarboPac PA200 anion-exchange column (guard column dimensions: 3 mm
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i.d. × 50 mm length, analytical column dimensions: 3 mm i.d. × 250 mm length).
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Detection was by pulsed amperometry with a quadruple potential waveform [26]. A
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sodium acetate in sodium hydroxide gradient separation [6] was used. Chromatographic
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profiles were compared to those of standards of glucose, fructose, sucrose, and solutions
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of SC or LC inulin prepared in 10% medium. Glucose, fructose, and sucrose were
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quantified with calibration curves, also prepared in 10% medium, and summed to give
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the majority of inulin hydrolysates. Additional small peaks in samples were not
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quantified because the majority were present in the media blanks as well.
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Acid hydrolysis was performed on unfermented inulin solutions to determine the
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total amount of mono- and disaccharides available to S. bovis. Solutions of SC or LC
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inulin (0.375 mg/mL in 15% medium) were acidified to pH 1 by adding 1 M HCl to a
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final concentration of 0.1 M. An equal volume of water was added to controls. All
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samples were incubated 1 h in a 50 °C sonicating water bath (Branson Ultrasonics,
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Danbury, CT, USA). The pH of acid-treated samples was raised by adding 1 M NaOH to
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a final concentration of 0.1 M (final pH 7 to 9 when checked with pH paper), and an
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equal volume of water was added to controls. Solutions (0.31 mg/mL in 12.3% medium
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after base/water addition) were diluted so that the final medium concentration was 10%.
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Water-treated controls were diluted to 0.25 mg/mL in 10% media, and SC and LC inulin
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acid hydrolysates were diluted to 0.1 and 0.075 mg/mL in 10% medium, respectively, to
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keep fructose concentrations within the range bracketed by the fructose standard curve.
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Concentrations of glucose, fructose, and sucrose were summed to give total inulin
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hydrolysates, and the total mono- and disaccharides remaining in the S. bovis
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fermentations after 9 h incubation was subtracted from the total in the hydrolyzed inulin
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solutions to determine the amounts of mono- and disaccharides utilized by S. bovis.
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2.5.2. Fermentation end product quantification
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Supernatant samples were thawed and clarified in a micro-centrifuge (21,000 × g, 2 min) for short chain fatty acid (SCFA) analyses. SCFA were quantified on a Summit
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HPLC (Dionex; Sunnyville, CA, USA). Extracts (0.1 mL) were injected onto an anion
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exchange column (Aminex HP-87H; Bio-Rad, Hercules, CA, USA) at 50 °C, separated
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isocratically with 5 mM sulfuric acid (0.4 mL/min flow rate), and detected by refractive
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index, as well as by UV absorbance at 210 nm.
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2.5.3. Other analyses
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The relationship between S. bovis JB1 OD600 and cell protein was previously determined [27] using the Folin-phenol method of Lowry et al. [28]. Ammonia
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concentration was determined in clarified culture supernatants with the colorimetric
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method of Chaney and Marbach [29], using 6-fold concentrated reagents.
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2.6. Statistical analyses The pH data from the initial concentration experiment were analyzed using the
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GLM procedure of SAS v. 9.3 (SAS Institute, Cary, NC, USA). The model included
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treatment, concentration and the interaction between these variables (treatment by
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concentration). Horse was included as a random effect. Means were separated using
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Fisher’s protected LSD test. Maximum specific growth rates of S. bovis were calculated
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using linear regression of OD600 values during the exponential phase of growth (2 – 5h).
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All other data (products, growth rates, OD600, pH of S. bovis incubations) were analyzed
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using the one-way ANOVA procedure of SAS v. 9.3. For all analyses, statistical
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significance was set at P < 0.05.
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3. Results
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3.1. Effects of inulin chain length and concentration on pH and SCFA production by equine fecal cell suspensions
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When SC or LC inulins, added to a final concentration of 0% to 2% w/v inulin in
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0.1% increments, were fermented by equine fecal cell suspensions, pH declined (Fig. 2).
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The average starting pH of the fecal cell suspensions was 6.8, and the extent of pH
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decline over the 24 h incubation period was dependent on both inulin chain length and
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concentration (P < 0.0001). The lowest concentration (0.1% w/v inulin) resulted in
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average pH values of 5.74 and 5.27 for LC and SC inulin, respectively. The greatest
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differences in pH between SC and LC inulin were observed at 0.5% w/v inulin (0.32 pH
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units). Furthermore, the 1.3% w/v inulin concentration was the lowest concentration
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tested that elicited maximal pH effects for both inulin chain lengths after 24 h of
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incubation (pH 3.95 and 3.86 for LC and SC inulin, respectively). When SC or LC inulins were added at 0.5% w/v inulin, SC inulin fermentations
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accumulated more lactate and total SCFA than LC fermentations over the 24 h incubation
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(P = 0.0220 and P = 0.0006, respectively; Table 1). Furthermore, the lactate to
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propionate ratio and lactate + propionate (representing total lactate produced)
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concentration was higher in SC than LC fermentations at 24 h (P = 0.0251 and P =
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0.0189, respectively). However, no differences in acetate, propionate or butyrate
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concentrations were observed after 24 h of incubation (P > 0.05, in all cases).
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3.2. Effect of inulin chain length on S. bovis JB1
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3.2.1. S. bovis JB1 inulin utilization and growth
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utilized by Streptococcus bovis JB1 incubated with 0.1% inulin (Table S1). At these
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lower concentrations, utilization was almost complete, and the peak areas of the
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quantified mono- and disaccharides were very small. Consequently, a substantial amount
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of error was associated with the quantification of most of the mono- and disaccharides
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remaining after 9 h incubation in 0.1% and 0.5% SC and LC inulin (Table S1.
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Furthermore, the residual glucose concentrations are so low that the lower utilization of
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glucose in the 0.1% inulin incubations is not biologically relevant. The total mono- and
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disaccharide concentrations present after 9 h incubation indicated that S. bovis JB1 was
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able to utilize > 98% of the available inulin hydrolysates at 0.1% and 0.5% w/v inulin,
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regardless of chain length, indicating that substrate was limiting in these fermentations.
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At 1.3% w/v inulin, no inulin polymers were detected (data not shown). However,
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glucose, fructose, and sucrose, summed to give total inulin hydrolysates, were present
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after 9 h (Fig. 3). A greater percentage of these mono- and disaccharides disappeared
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from 0 to 9 h in SC than in LC inulin incubations (Fig. 3; P < 0.05). Thus, S. bovis JB1
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was able to utilize more SC inulin than LC inulin in 9 h (P = 0.0216).
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Concomitantly, S. bovis JB1 grew rapidly when inulin was provided as the
primary substrate (Fig. 4), and reached stationary phase in < 6 h (data not shown). S.
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bovis had greater maximum specific growth rates when fermenting SC inulin than LC
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inulin (P = 0.0001, P = 0.0004, and P = 0.0178 for 0.1, 0.5 and 1.3% w/v inulin
240
concentrations, respectively; Fig. 4a). For example, at 0.5% w/v inulin the maximum
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specific growth rate of S. bovis was twice as fast with SC as with LC inulin fermentation.
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Similarly, S. bovis JB1 growth yield at 9 h was greater in SC inulin fermentations than in
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LC inulin fermentations (P < 0.0001, P = 0.0011, and P < 0.0001 for 0.1, 0.5 and 1.3%
244
w/v inulin concentrations, respectively; Fig. 4b). The greatest difference in OD600 at 9 h
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was observed at 1.3% w/v inulin, in which LC and SC inulin fermentations had average
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OD600 of 4.8 and 7.8, respectively. The total bacterial protein yield per unit of inulin
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substrate was higher in SC than in LC inulin fermentations (P < 0.0001 for all inulin
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concentrations; Fig. 4c).
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3.2.2. S. bovis JB1 pH and SCFA
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At stationary phase (9 h), no remaining fructose was detected in 0.1% or 0.5%
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w/v SC or LC inulin fermentations (0.1%, data not shown; 0.5%, Table 2), which
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indicates that the substrate was limiting. However, fructose was detected in the media by
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HPLC with refractive index and UV absorbance detection after 9 h fermentation when
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1.3% w/v inulin was included, and the LC inulin fermentations had more fructose than
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SC (P < 0.0001). This latter result agrees with the percent disappearance of hydrolyzed
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inulin determined by HPLC with pulsed amperometric detection (Fig. 3). Other
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metabolic products included formic, acetic and lactic acids, and ethanol, which were
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similar in LC and SC inulin fermentations when the substrate concentrations were 0.1 and
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0.5% (Table 2). However, when 1.3% w/v inulin was used, LC inulin fermentations had
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greater formate and ethanol concentrations compared to SC inulin fermentations at 9 h (P
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< 0.0001 and P = 0.001, respectively). Concurrently, SC inulin fermentations had a
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greater concentration of acetate at 9 h than LC fermentations (P = 0.0213), and lactate
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concentrations were similar between treatments (P = 0.0742). At stationary phase (9 h),
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S. bovis fermentations of SC inulin elicited a greater pH decline than fermentation of LC
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inulin (P = 0.0038, P = 0.0049, and P = 0.0034 for 0.1, 0.5 and 1.3% w/v inulin
266
concentrations, respectively, Fig. 5). The greatest difference in pH was observed at the
267
1.3% w/v inulin concentration in which LC and SC fermentations had an average pH of
268
5.73 and 4.99, respectively. S. bovis assimilated more ammonia in 9 h when SC inulin
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was the substrate (data not shown).
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4. Discussion
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Subsequent to the original assertion that the accumulation of fructan in pasture grasses
272
could cause PAL [2], researchers have concluded that PAL is a complex, multifactorial
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condition that involves the metabolic state of the horse [1]. However, laminitis can be
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induced in horses under laboratory conditions by administration of bolus doses of inulins
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or other oligofructose [1,7,18,19]. Furthermore, others have suggested that short-chain
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fructans are more rapidly fermented than long-chain fructans in the equine hindgut [9],
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and in vitro experiments to study factors contributing to dysbiosis have also employed
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fructans [9, 12,16,17]. When microbiological analyses have been performed,
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Streptococcus bovis, a well-known inulin-catabolizing organism, has been either isolated
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or detected, [12,16,17]. These reasons justify examining the ability of S. bovis to
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metabolize fructans with different chemical characteristics, the simplest of which is the
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length of the polymer.
Previous studies have suggested that equine hindgut bacteria may more rapidly
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ferment SC fructans than LC fructans [9]. A difference in effects of fructan chain length
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has been demonstrated in the fermentation of long- and short-chain inulin by the human
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isolate Bifidobacterium longum subspecies infantis [30], and in the fermentation of inulin
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of DP 31 and 57 by swine gut bacteria [31]. However, this relationship has not been
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previously demonstrated with equine gut bacteria, and some bacterial species were
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reported to preferentially utilize fructans of longer chain length [32]. Therefore, the
290
purpose of the current study was to determine if equine fecal bacteria and a model S.
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bovis would equally ferment SC and LC inulins. Lower pH values and greater lactic acid
292
concentrations indicated that the SC inulin was fermented to a greater degree by equine
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fecal bacteria. Streptococcus bovis JB1 readily fermented all of either inulin when inulin
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was limiting. However, at all concentrations tested, the maximum specific growth rate,
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the yield of cell protein per unit substrate, and the final optical density were greater when
296
SC inulin was used. These results indicate that SC inulin was a more easily utilized
297
substrate for equine fecal bacteria and this model S. bovis.
298 299
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It is important to note that S. bovis JB1 made copious amounts of lactic acid from either SC or LC inulin. However, the observed pH decline was greater in the SC inulin S.
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bovis fermentations. Although lactate production in both treatments was similar, acetate,
301
formate, and ethanol concentrations were different. Furthermore, more ammonia (a
302
strong base) was assimilated in the SC inulin treatment, which is consistent with the cell
303
protein yields calculated in the growth experiments. Therefore, the pH differences
304
observed could be attributed to these differences in acid production and ammonia
305
utilization.
In the S. bovis fermentations with the highest inulin concentration, significant
SC
306
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quantities of free fructose were detected after 9 h with either treatment, indicating that
308
substrate was in excess. However, 42% more residual fructose was detected in LC than in
309
SC inulin fermentations. This latter result is consistent with the observation that the
310
growth yield was greater on SC inulin (i.e. there were more cells to utilize the sugar), and
311
supports the hypothesis that SC inulin is utilized more rapidly than LC inulin by S. bovis
312
JB1. Residual fructose in early stationary phase indicates that the catabolic capacity of
313
extracellular fructan hydrolases, which are widespread among streptococci [33],
314
exceeded the ability of the S. bovis cells to transport and metabolize the fructose. This
315
latter result is an informative laboratory artifact, but it is unlikely that sugars would
316
accumulate in vivo because other microorganisms in the equine hindgut can utilize them
317
The concept of crossfeeding between microorganisms is well established in the rumen
318
[34]. For example, cellulose, like fructan, is depolymerized outside of the cells.
319
Cellulolytic bacteria transport some of the glucose, but much is also utilized by non-
320
cellulolytic, saccharolytic bacteria. Another well-known example is interspecies
321
hydrogen transfer, in which methanogens utilize H2 and CO2 produced by bacteria. It is
322
almost certain that crossfeeding also occurs in the equine hindgut. In this way, other
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bacteria could convert fructose to acids and contribute to acidosis after fructan catalysis
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by S. bovis or similar bacteria.
325 5. Conclusions
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The congruence of results between the experiments that employed uncultured fecal
328
cell suspensions and S. bovis JB1 support the hypothesis that bacteria of the S. bovis/S.
329
equinus group or similar bacteria are responsible for fructan fermentation in the equine
330
hindgut. These results indicate that inulin chain length influences fermentation by equine
331
fecal bacteria and a model S. bovis. Specifically, inulin of shorter chain length was more
332
available for fermentation in vitro suggesting that SC fructan could also present a greater
333
risk for PAL than LC fructans in vivo. However, an additional consideration is that chain
334
length could be reduced by catalysis prior to the large intestine [9]. In this way, hindgut
335
fermentation could be increased in vivo. It is also important to note that the inulin utilized
336
in the current study was extracted from Jerusalem artichoke (Helianthus tuberosus). We
337
acknowledge that inulins are structurally simpler (i.e. unbranched, a single type of
338
linkage) than many grass fructans. However, the advantage of using structurally simple
339
inulins is that the difference in chain length was tested without other differences in
340
structure. Even greater differences in fermentation might be obtained utilizing native
341
grass fructans that can vary dramatically in chain length depending on species and time of
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year, and that include phleins and graminans [24].
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Acknowledgements
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The information reported in this paper (#15-07-096) is part of a project of the Kentucky
346
Agricultural Experiment Station and is published with the approval of the Director. MF
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and IK were supported by USDA-ARS, National Program 215 – Pasture, Forage &
348
Rangeland Systems. The authors thank Beneo, Inc. for donating the inulins used in the
349
study, and Taylor Donley and Gloria Gellin for technical assistance.
350
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cheatgrass (Bromus tectorum L.). New Phytologist 1993; 124: 389-396.
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[26] Rocklin RD, Clarke AP, Weitzhandler M. Improved long-term reproducibility for
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pulsed amperometric detection of carbohydrates via a new quadruple-potential waveform.
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[27] Russell J, Robinson P. Compositions and characteristics of strains of Streptococcus
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bovis. J Dairy Sci 1984; 67: 1525-1531.
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[28] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the
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Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
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[29] Chaney AL, Marbach EP. Modified reagents for determination of urea and
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[30] Perrin S, Fougnies C, Grill J, Jacobs H, Schneider F. Fermentation of chicory
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fructo-oligosaccharides in mixtures of different degrees of polymerization by three strains
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of bifidobacteria. Can J Microbiol 2002; 48: 759-763.
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[31] Paßlack N, Al-Samman M, Vahjen W, Männer K, Zentek J. Chain length of inulin
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affects its degradation and the microbiota in the gastrointestinal tract of weaned piglets
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after a short-term dietary application. Livestock Sci 2012; 149: 128-136.
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[32] Tsujikawa Y, Nomoto R, Osawa R. Difference in degradation patterns on inulin-type
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fructans among strains of Lactobacillus delbrueckii and Lactobacillus paracasei. Biosci
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Microbiota, Food Health 2013; 32: 157.
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[33] Kersters K, Vancanneyt M. Bergey’s Manual of Systemic Bacteriology. Williams
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and Wilkins Co., Baltimore, MD, USA 2005; 9th ed.: 552 – 558.
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[34] Russell JB. Rumen Microbiology and Its Role in Ruminant Nutrition. Ithaca, NY
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2002
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Figure Legends
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Fig 1.
468
Chromatographic profiles of (A) SC inulin and (B) lLC inulin. Numbers above peaks
469
represent chain length.The SC inulin is actually a SC inulin-rich fraction that also
470
contains some contaminating glucose (G), fructose (F), and sucrose (S). Scales on
471
chromatograms A and B differ: chromatogram B has been enlarged for better viewing of
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peaks. Both inulin samples were injected at 0.25 mg/mL, but electrochemical response
473
decreases with increasing chain length [21].
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Fig. 2.
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Relationship between pH and inulin concentration after 24 h fermentation by suspensions
476
of uncultivated, washed equine fecal microorganisms. The suspensions (n = 3, horses)
477
had initial pH values of 6.8 (dashed line). Inulin substrates included short chain inulin
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(squares) and long chain inulin (circles) at concentrations ranging from 0 to 2 % w/v, in
479
0.2% increments. Asterisks indicate that means are different between treatments within a
480
given concentration (P < 0.05).
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SC
481 Fig. 3.
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Mono- and disaccharide concentration as the sum of glucose, fructose, and sucrose (left
484
y-axis to solid vertical line), or percentage of mono- and disaccharides fermented (solid
485
vertical line to right y-axis). Amount present is shown at 0 h in acid-hydrolyzed inulin
486
(LC, solid bars; SC, open bars), and in inulin solutions incubated (39 °C) for 9 h with S.
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bovis. Percentage fermented by 9 h in the S. bovis incubations is shown on the right-hand
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side of the graph. The experiment was performed in triplicate. Asterisks indicate that
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means are different between treatments within timepoint (P < 0.05); 0h, trt: P = 0.2288,
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SEM: 0.2194; 9h, trt: P = 0.0313, SEM: 0.0100; % disappearance, trt: P = 0.0216, SEM:
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0.1710.
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Fig. 4.
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Effect of inulin chain length on the growth of inulin-fermenting Streptococcus bovis JB1.
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The culture was inoculated into media with 0.1, 0.5 or 1.3% long chain (solid bars) or
496
short chain (open bars) inulin (w/v) and incubated (37 °C) for 9 h. The maximum specific
497
growth rates are shown on panel (a), the final growth values (OD600) are shown on panel
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(b), and the cell yield (mg bacterial protein/mg inulin) at 9 h are shown on panel (c). The
499
experiment was performed in triplicate. Asterisks indicate that means are different
500
between treatments within concentration (P < 0.05); (a) 0.1%, trt: P = 0.0001, SEM: 0;
501
0.5%, trt: P = 0.0004, SEM: 0.0002; 1.3%, trt: P = 0.0178, SEM: 0.0001; (b) 0.1%, trt: P
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< 0.0001, SEM: 0; 0.5%, trt: P = 0.0011, SEM: 0.0001; 1.3%, trt: P < 0.0001, SEM:
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0.0003; (c) 0.1%, trt: P < 0.0001, SEM: 0.345; 0.5%, trt: P = 0.0001, SEM: 1.0897;
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1.3%, trt: P < 0.0001, SEM: 0.2484.
SC
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505 Fig. 5.
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Effect of inulin degree of polymerization on the pH of inulin-fermenting Streptococcus
508
bovis JB1 suspensions at stationary phase (9 h). The culture was inoculated into media
509
with 0.1, 0.5 or 1.3% long chain (solid bars) or short chain (open bars) inulin (w/v) and
510
incubated (37 °C) for 9 h. The experiment was performed in triplicate. Asterisks indicate
511
that means are different between treatments within concentration (P < 0.05); 0.1%, trt: P
512
= 0.0038, SEM: 0.0004; 0.5%, trt: P = 0.0049, SEM: 0.0010; 1.3%, trt: P = 0.0034,
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SEM: 0.0028.
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Table 1. Effect of inulin degrees of polymerization after 24 h of fermentation (0.5% inulin) on short chain fatty acid (SCFA) concentrations in equine fecal cell suspensions.
Lactate Acetate Propionate Butyrate Lactate:Propionate Lactate + Propionate Total SCFA
Long Chain Inulin 6.0 1.6 1.2 1.1 5.1 7.2 10.0
P – value
SEM
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Short Chain Inulin 7.8 1.3 Trace 1.2 7.8 8.8 11.3
P = 0.0220 P = 0.1296 P = 0.0742 P = 0.4226 P = 0.0251 P = 0.0189 P = 0.0006
SC
SCFA (mM)
0.107 0.027 0.005 0.015 0.292 0.268 0.002
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Statistically different concentrations between treatments are shown in bold. Each value is a mean of three replicates. Quantities < 1.0 mM were considered below the limit of quantitation (trace) and included as 1 mM in the statistical analysis.
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Long Chain Inulin 0 11 3.0 3.1 2.0 24.7 23.7 1.9 1.7 1.0
P – value P = 1.0000 P = 1.0000 P = 0.4479 P = 0.2380 P = 0.1625 P < 0.0001 P = 0.0742 P < 0.0001 P = 0.0213 P = 0.001
SEM 0 0 0.349 0.442 0.471 0 0.707 0 0.248 0.041
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Short Chain Inulin 0 11 2.8 3.7 1.1 14.3 21.7 1.4 3.0 0
SC
SCFA (mM) Fructose Lactate Formate Acetate Ethanol Fructose Lactate Formate Acetate Ethanol
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1.3% w/v
0.5% w/v
Table 2. Effect of inulin degrees of polymerization after 24 h of fermentation (0.5% and 1.3% w/v inulin) on metabolite concentrations in Streptococcus bovis JB1 cultures.
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1
Highlights •
Inulin DP influences equine fecal bacteria and S. bovis fermentations in vitro.
3
•
Short-chain inulin is more available for fermentation than long-chain inulin.
4
•
S. bovis JB1 grows faster on low DP inulin than on high DP inulin.
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Table S1. Residual inulin hydrolysates after fermentation (9 h) by Streptococcus bovis.
SC, 0.5%
LC, 0.5%
SC, 1.3%
LC, 1.3%
mg/mL remaining post incubation
glucose fructose
0.056 ± 0.004 0.59 ± 0.03
0.007 ± 0.004 0.008 ± 0.006
sucrose
0.071 ± 0.008
total glucose
0.72 ± 0.04 0.041 ± 0.004
0.016 ± 0.002 0.013 ± 0.014
fructose
0.76 ± 0.08
0.006 ± 0.005
sucrose total
0.010 ± 0.0005 0.82 ± 0.00
0.019 ± 0.015
glucose
0.28 ± 0.02
0.002 ± 0.003
fructose sucrose
2.96 ± 0.14 0.35 ± 0.04
0.056 ± 0.005 0.001 ± 0.000
total
3.59 ± 0.18
glucose fructose
% utilized
0
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87 99
100
98 69 99
0
100 98
SC
LC, 0.1%
mg/mL initial, based on inulin hydrolysis
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mono- or disaccharide
99
98 99.6
0.059 ± 0.009
98
0.21 ± 0.020 3.82 ± 0.42
-0.001 ± 0.004 0.027 ± 0.009
100 99
sucrose
0.051 ± 0.003
0.001 ± 0.0001
99
total glucose
4.08 ± 0.44 0.72 ± 0.05
0.027 ± 0.013 0.28 ± 0.05
99 61
fructose
7.69 ± 0.36
2.08 ±0.19
73
sucrose total
0.92 ± 0.10 9.34 ± 0.47
0.46 ± 0.07 2.82 ± 0.29
50 70
0.54 ± 0.05
0.33 ± 0.06
38
9.94 ± 1.09 0.13 ± 0.01
4.08 ± 0.90 0.18 ± 0.01
59 -36
10.6 ± 1.1
4.59 ± 0.97
57
glucose fructose sucrose
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S0737-0806(15)30018-6
DOI:
10.1016/j.jevs.2015.11.010
Reference:
YJEVS 1987
To appear in:
Journal of Equine Veterinary Science
Received Date: 10 September 2015 Revised Date:
29 November 2015
Accepted Date: 30 November 2015
Please cite this article as: Harlow BE, Kagan IA, Lawrence LM, Flythe MD, Effects of inulin chain length on fermentation by equine fecal bacteria and Streptococcus bovis, Journal of Equine Veterinary Science (2016), doi: 10.1016/j.jevs.2015.11.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title:
Effects of inulin chain length on fermentation by equine fecal
Authors:
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bacteria and Streptococcus bovis
Brittany E. Harlowa, Isabelle A. Kagana,b, Laurie M. Lawrencea,
a
University of Kentucky, Department of Animal and Food
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Affiliations:
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Michael D. Flythea,b
Sciences; Lexington, Kentucky 40546
b
USDA,
Agricultural
Research
Service,
Forage-Animal
“Proprietary or brand names are necessary to report factually on
available data; however, the USDA neither guarantees nor
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Disclaimer:
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Mandatory
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Production Research Unit; Lexington, Kentucky 40546
warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, nor exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.”
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Key Words:
fructan, inulin, oligofructose, streptococci, hindgut acidosis
Correspondence:
Michael D. Flythe USDA, ARS
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N-220 Ag. Science North University of Kentucky
Phone: (859) 421-5699 Fax: (859) 257-3334
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Lexington, KY 40546
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Email: [email protected]
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Abstract
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Grass fructans can be fermented by Gram-positive bacteria (e.g., Streptococcus bovis) in
3
the equine hindgut, increasing production of lactic acid and decreasing pH. The degree of
4
polymerization (DP) of fructans has been suggested to influence fermentation rates. The
5
objective of the current study was to determine how DP impacts fermentation by equine
6
fecal bacteria and a model S. bovis. Fecal microbes from 3 mares were harvested by
7
differential centrifugation, washed, and re-suspended in anaerobic media containing
8
short-chain (SC; DP ≤ 10) or long-chain (LC) inulin (DP ≥ 23) from 0-2% w/v. After 24
9
h of incubation (37 ºC) samples were collected for pH determination. Data were
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analyzed using the GLM procedure testing for the effect of treatment, concentration, and
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treatment × concentration (SAS v. 9.3). At all concentrations, the pH was lower in SC
12
fermentations than in LC (P < 0.0001, in all cases). To determine the effect of DP on S.
13
bovis, cultures were grown (39 ºC, 9 h) with 0.1, 0.5, or 1.3% SC or LC inulin. Optical
14
density (OD600) was determined by spectrophotometry. Maximum specific growth rates
15
(µ) were determined by linear regression (2-5 h). Data were analyzed using the one-way
16
ANOVA procedure (SAS v. 9.3). The final OD600, µ and yield were higher with SC than
17
with LC fermentation (P < 0.05). These results indicate that SC inulin may be more
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available for fermentation than LC inulin by equine fecal bacteria and S. bovis,
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specifically.
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Highlights
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•
Inulin DP influences equine fecal bacteria and S. bovis fermentations in vitro.
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•
Short-chain inulin is more available for fermentation than long-chain inulin.
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•
S. bovis JB1 grows faster on low DP inulin than on high DP inulin.
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1. Introduction Pasture associated laminitis (PAL) is a multifactorial disorder, and risk factors
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ranging from diet to reduced insulin sensitivity have been implicated [1]. One of the risk
28
factors is grazing on pastures rich in water-soluble carbohydrates (WSC, mono- and
29
disaccharides and fructans), which can lead to gastrointestinal disturbances [2]. Cool
30
season grasses produce fructans, fructose polymers that usually have a terminal sucrose
31
[3] and that may accumulate at certain times of the year [4,5,6]. Fructans are not
32
completely digested in the foregut and are fermented in the hindgut by Gram-positive
33
bacteria (e.g., Streptococcus spp.) that produce lactic acid, decreasing pH [7,8,9].
34
Decreasing hindgut pH can lead to increases in intestinal permeability, enabling bacteria
35
or bacterial metabolites to enter the blood stream [10]. Several bacterial metabolites
36
have been implicated in the pathogenesis of PAL including endotoxins [11], amines [12]
37
and proteinases [13]. Streptococcus bovis is commonly implicated as the primary
38
etiological agent in PAL because it has the capacity to produce amines and proteinases,
39
besides being abundant in carbohydrate-excess conditions [12,14].
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Inulins, the β-2,1 linked fructans found in a few dicotyledonous and
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SC
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monocotyledonous families in addition to the Poaceae [15], have been commonly used as
42
a model fructan to study the pathogenesis of PAL both in vitro [12,16,17] and in vivo
43
[7,18,19]. However, grass fructans can have β-2,6 linkages or a mixture of β-2,1 and β-
44
2,6 linkages [20], sometimes within the same molecule [21,22]. Fructans with β-2,6
45
linkages are sometimes called levans [3] or phleins [23], and those with both linkage
46
types may be referred to as graminans [23]. Due to the structural diversity of grass
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fructans, it is uncertain if they would be fermented in the same manner as inulin. One
48
parameter contributing to this uncertainty, in terms of both inulins and grass fructans, is
49
the effect of the number of monomers, referred to as chain length or degrees of
50
polymerization (DP). Several factors can influence the composition and chain length of
51
grass fructan, including time of year, climate, and grass species [24]. It has been
52
suggested that short-chain (SC) fructans are more rapidly fermented than long-chain (LC)
53
fructans in the equine hindgut [9]. However, to the best of our knowledge, the
54
relationship between fructan chain length and fermentation by equine hindgut bacteria
55
has not been experimentally determined. Therefore, the objective of the current study
56
was to determine how chain length impacts fermentation by equine fecal bacteria, using
57
inulins as model fructans. The advantage of using inulins is that the effects of chain
58
length can be tested without other differences in structure. The hypothesis was that
59
equine fecal bacteria and a model S. bovis would more rapidly ferment an inulin with a
60
shorter chain length.
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2. Material and methods
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2.1. Media composition
The cell suspension medium was weakly buffered to allow pH to decrease in
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response to acid production, and contained (per liter): 240 mg KH2PO4, 240 mg K2HPO4,
66
480 mg (NH4)2SO4, 480 mg NaCl, 64 mg CaCl2 · 2H2O, 100 mg MgSO4 · 7H2O, 600 mg
67
cysteine hydrochloride; initial pH 6.7; autoclaved to remove O2 and cooled under N2.
68
The growth medium, based on Mantovani and Russell [25], was heavily buffered
69
to maximize bacterial growth. This medium contained (per liter): 240 mg KH2PO4, 240
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mg K2HPO4, 480 mg (NH4)2SO4, 480 mg NaCl, 64 mg CaCl2 · 2H2O, 100 mg MgSO4 ·
71
7H2O, 600 mg cysteine hydrochloride, 1,000 mg Trypticase, 500 mg yeast extract; initial
72
pH 6.7; autoclaved to remove O2 and cooled under O2 –free CO2. The buffer (4,000 mg
73
Na2CO3 per liter) was added to the medium before dispensing and autoclaving for
74
sterility.
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Short chain (SC; chain length: 1 to 13, mean DP documented as ≤ 10; Orafti®
OPS; BENEO Inc., Morris Plaines, NJ, USA) or long chain (LC; chain length: 13 to 60,
77
mean DP documented as ≥ 23; Orafti® HP; BENEO Inc.) inulin from chicory (Fig. 1)
78
was added to either media type when indicated. A preliminary experiment indicated that
79
autoclaving (121 °C, 1 bar) did not hydrolyze fructans at pH 6 to 7. The chain length
80
thought to be represented by each peak (indicated by numbers above selected peaks) was
81
based on the retention time determined for a standard of 1-kestose (a trisaccharide), and
82
the fact that each successive inulin peak after 1-kestose generally represents an increase
83
of one DP [21]. The SC inulin was not entirely free from non-inulin compounds (glucose,
84
fructose, and sucrose, indicated on Figure 1A), but is referred to as SC inulin instead of
85
SC inulin-enriched fraction for brevity.
86
2.2. Animals and fecal collection
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All animal care, handling and procedures were approved by the University of
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Kentucky Institutional Animal Care and Use Committee. Horses were selected from the
89
University of Kentucky, Department of Animal and Food Sciences herd at Maine Chance
90
Farm, Lexington KY. The feces donors were three mature Thoroughbred mares
91
maintained on hay with limited access to pasture. Replicate experiments (n = 3) were
92
performed on separate days using the feces from one horse on each day.
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When feces were needed for an experiment, horses were observed for defecation and feces were collected by catch sampling without ground contact. Each sample was
95
thoroughly mixed by hand and placed in a plastic bag. The bag was then purged of air
96
with CO2 and transported to the laboratory (within 0.5 h of collection) in a pre-warmed
97
container (37 °C) for processing.
98
2.3. Fecal Cell Suspensions
Upon arrival at the laboratory fecal cell suspensions were prepared as previously
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described [17]. In short, feces (450 g) were placed in a blender, under N2, and mixed (3
101
min or until homogenous) with 750 mL of cell suspension medium (see above). The
102
mixture was then squeezed through cheesecloth to remove large plant particles and
103
underwent low-speed centrifugation (341.6 × g, 5 min) to remove protists and remaining
104
plant fibers. The supernatants were collected and then subjected to high-speed
105
centrifugation (25654.3 × g, 5 min) to collect bacteria. Supernatants were then discarded
106
and bacterial pellets were washed via re-suspension in anaerobic cell suspension medium.
107
Bacterial cells were then harvested by a second high-speed centrifugation (25654.3 × g,
108
10 min). Again the supernatants were discarded, and the bacterial pellets were re-
109
suspended and pooled in a N2-sparged glass vessel. The optical densities (600 nm; OD600)
110
of the fecal cell suspensions were adjusted to ~15 with anaerobic cell suspension
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medium. Microscopic analysis of fecal cell suspensions revealed bacteria sized cells with
112
no plant particles or protists.
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An initial experiment was conducted to determine the effect of inulin
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concentration and chain length on pH of the cell suspension medium. SC or LC inulin (0
115
– 2.0% w/v, in 0.1% increments) was added to a fecal cell suspension that was aliquoted
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into duplicate anaerobic Hungate tubes (N2 atmosphere). The tubes were then incubated
117
in a shaking water bath (37 °C, 160 rpm) for 24 h. Samples were collected via tuberculin
118
syringes for pH measurement and later product analysis. The pH was measured
119
immediately with a pH meter. Supernatants for product analysis were clarified by
120
centrifugation (21,000 × g, 2 min), and frozen (-20 °C) until analyzed, as described
121
below. The experiment was replicated three times with each replicate using feces from a
122
different horse.
123
2.4. Streptococcus bovis JB1 experiments
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A pure culture of S. bovis JB1 was originally obtained from the culture collection
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of James B. Russell; United States Department of Agriculture, Agricultural Research
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Service at Cornell University, Ithaca, NY, USA. S. bovis was routinely transferred in
127
growth medium with glucose (0.4% w/v), but it was transferred at least five times with
128
inulin from chicory (0.4% w/v; chain length: 10 to 50, mean DP documented as 35,
129
Sigma Aldrich, St Louis, MO, USA) as the sole carbon source prior to the described
130
experiments.
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Of the twenty inulin concentrations used to check effects on pH, three (0.1%,
132
0.5% and 1.3% w/v) were selected to determine the effect of inulin concentration and
133
chain length on S. bovis JB1 growth and metabolism. SC or LC inulin was added to
134
bottles of growth medium at the three concentrations listed above. Stationary phase (16 h)
135
S. bovis JB1 cultures were used to inoculate bottles (0.1% w/v inoculum), and they were
136
incubated in a shaking water bath (37 °C, 160 rpm). Growth (OD600) was monitored in a
137
spectrophotometer every 30 min for 9 h. After 9 h of incubation, samples were collected
138
with tuberculin syringes for pH measurement and later product analysis (see section 2.3.).
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2.5. Chemical Analyses
140
2.5.1. Inulin quantification
141
Clarified supernatants of S. bovis incubated 9 h with 0.1%, 0.5%, or 1.3% inulin (SC or LC) were vacuum-filtered through Extract-Clean Prevail C18 solid-phase
143
extraction columns (Grace Davison Discovery Sciences, Deerfield, IL, USA). This step
144
removed lipophilic compounds. Because the concentration of medium affected the
145
detector response (data not shown), filtrates were diluted so that all injections contained
146
10% medium, and 10% medium was injected as a blank. The 0.1% (1 mg/mL) inulin
147
incubations were diluted tenfold in water to 0.1 mg/mL in 10% medium, and the 0.5% (5
148
mg/mL) and 1.3% (13 mg/mL) inulin incubations were diluted with water and medium to
149
0.25 mg/mL in 10% medium. Dilutions (20 µL) were separated on a Dionex (Westmont,
150
IL, USA) CarboPac PA200 anion-exchange column (guard column dimensions: 3 mm
151
i.d. × 50 mm length, analytical column dimensions: 3 mm i.d. × 250 mm length).
152
Detection was by pulsed amperometry with a quadruple potential waveform [26]. A
153
sodium acetate in sodium hydroxide gradient separation [6] was used. Chromatographic
154
profiles were compared to those of standards of glucose, fructose, sucrose, and solutions
155
of SC or LC inulin prepared in 10% medium. Glucose, fructose, and sucrose were
156
quantified with calibration curves, also prepared in 10% medium, and summed to give
157
the majority of inulin hydrolysates. Additional small peaks in samples were not
158
quantified because the majority were present in the media blanks as well.
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Acid hydrolysis was performed on unfermented inulin solutions to determine the
160
total amount of mono- and disaccharides available to S. bovis. Solutions of SC or LC
161
inulin (0.375 mg/mL in 15% medium) were acidified to pH 1 by adding 1 M HCl to a
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final concentration of 0.1 M. An equal volume of water was added to controls. All
163
samples were incubated 1 h in a 50 °C sonicating water bath (Branson Ultrasonics,
164
Danbury, CT, USA). The pH of acid-treated samples was raised by adding 1 M NaOH to
165
a final concentration of 0.1 M (final pH 7 to 9 when checked with pH paper), and an
166
equal volume of water was added to controls. Solutions (0.31 mg/mL in 12.3% medium
167
after base/water addition) were diluted so that the final medium concentration was 10%.
168
Water-treated controls were diluted to 0.25 mg/mL in 10% media, and SC and LC inulin
169
acid hydrolysates were diluted to 0.1 and 0.075 mg/mL in 10% medium, respectively, to
170
keep fructose concentrations within the range bracketed by the fructose standard curve.
171
Concentrations of glucose, fructose, and sucrose were summed to give total inulin
172
hydrolysates, and the total mono- and disaccharides remaining in the S. bovis
173
fermentations after 9 h incubation was subtracted from the total in the hydrolyzed inulin
174
solutions to determine the amounts of mono- and disaccharides utilized by S. bovis.
175
2.5.2. Fermentation end product quantification
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Supernatant samples were thawed and clarified in a micro-centrifuge (21,000 × g, 2 min) for short chain fatty acid (SCFA) analyses. SCFA were quantified on a Summit
178
HPLC (Dionex; Sunnyville, CA, USA). Extracts (0.1 mL) were injected onto an anion
179
exchange column (Aminex HP-87H; Bio-Rad, Hercules, CA, USA) at 50 °C, separated
180
isocratically with 5 mM sulfuric acid (0.4 mL/min flow rate), and detected by refractive
181
index, as well as by UV absorbance at 210 nm.
182
2.5.3. Other analyses
183 184
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The relationship between S. bovis JB1 OD600 and cell protein was previously determined [27] using the Folin-phenol method of Lowry et al. [28]. Ammonia
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concentration was determined in clarified culture supernatants with the colorimetric
186
method of Chaney and Marbach [29], using 6-fold concentrated reagents.
187
2.6. Statistical analyses The pH data from the initial concentration experiment were analyzed using the
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GLM procedure of SAS v. 9.3 (SAS Institute, Cary, NC, USA). The model included
190
treatment, concentration and the interaction between these variables (treatment by
191
concentration). Horse was included as a random effect. Means were separated using
192
Fisher’s protected LSD test. Maximum specific growth rates of S. bovis were calculated
193
using linear regression of OD600 values during the exponential phase of growth (2 – 5h).
194
All other data (products, growth rates, OD600, pH of S. bovis incubations) were analyzed
195
using the one-way ANOVA procedure of SAS v. 9.3. For all analyses, statistical
196
significance was set at P < 0.05.
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3. Results
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3.1. Effects of inulin chain length and concentration on pH and SCFA production by equine fecal cell suspensions
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When SC or LC inulins, added to a final concentration of 0% to 2% w/v inulin in
202
0.1% increments, were fermented by equine fecal cell suspensions, pH declined (Fig. 2).
203
The average starting pH of the fecal cell suspensions was 6.8, and the extent of pH
204
decline over the 24 h incubation period was dependent on both inulin chain length and
205
concentration (P < 0.0001). The lowest concentration (0.1% w/v inulin) resulted in
206
average pH values of 5.74 and 5.27 for LC and SC inulin, respectively. The greatest
207
differences in pH between SC and LC inulin were observed at 0.5% w/v inulin (0.32 pH
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208
units). Furthermore, the 1.3% w/v inulin concentration was the lowest concentration
209
tested that elicited maximal pH effects for both inulin chain lengths after 24 h of
210
incubation (pH 3.95 and 3.86 for LC and SC inulin, respectively). When SC or LC inulins were added at 0.5% w/v inulin, SC inulin fermentations
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accumulated more lactate and total SCFA than LC fermentations over the 24 h incubation
213
(P = 0.0220 and P = 0.0006, respectively; Table 1). Furthermore, the lactate to
214
propionate ratio and lactate + propionate (representing total lactate produced)
215
concentration was higher in SC than LC fermentations at 24 h (P = 0.0251 and P =
216
0.0189, respectively). However, no differences in acetate, propionate or butyrate
217
concentrations were observed after 24 h of incubation (P > 0.05, in all cases).
218
3.2. Effect of inulin chain length on S. bovis JB1
219
3.2.1. S. bovis JB1 inulin utilization and growth
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utilized by Streptococcus bovis JB1 incubated with 0.1% inulin (Table S1). At these
222
lower concentrations, utilization was almost complete, and the peak areas of the
223
quantified mono- and disaccharides were very small. Consequently, a substantial amount
224
of error was associated with the quantification of most of the mono- and disaccharides
225
remaining after 9 h incubation in 0.1% and 0.5% SC and LC inulin (Table S1.
226
Furthermore, the residual glucose concentrations are so low that the lower utilization of
227
glucose in the 0.1% inulin incubations is not biologically relevant. The total mono- and
228
disaccharide concentrations present after 9 h incubation indicated that S. bovis JB1 was
229
able to utilize > 98% of the available inulin hydrolysates at 0.1% and 0.5% w/v inulin,
230
regardless of chain length, indicating that substrate was limiting in these fermentations.
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At 1.3% w/v inulin, no inulin polymers were detected (data not shown). However,
232
glucose, fructose, and sucrose, summed to give total inulin hydrolysates, were present
233
after 9 h (Fig. 3). A greater percentage of these mono- and disaccharides disappeared
234
from 0 to 9 h in SC than in LC inulin incubations (Fig. 3; P < 0.05). Thus, S. bovis JB1
235
was able to utilize more SC inulin than LC inulin in 9 h (P = 0.0216).
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Concomitantly, S. bovis JB1 grew rapidly when inulin was provided as the
primary substrate (Fig. 4), and reached stationary phase in < 6 h (data not shown). S.
238
bovis had greater maximum specific growth rates when fermenting SC inulin than LC
239
inulin (P = 0.0001, P = 0.0004, and P = 0.0178 for 0.1, 0.5 and 1.3% w/v inulin
240
concentrations, respectively; Fig. 4a). For example, at 0.5% w/v inulin the maximum
241
specific growth rate of S. bovis was twice as fast with SC as with LC inulin fermentation.
242
Similarly, S. bovis JB1 growth yield at 9 h was greater in SC inulin fermentations than in
243
LC inulin fermentations (P < 0.0001, P = 0.0011, and P < 0.0001 for 0.1, 0.5 and 1.3%
244
w/v inulin concentrations, respectively; Fig. 4b). The greatest difference in OD600 at 9 h
245
was observed at 1.3% w/v inulin, in which LC and SC inulin fermentations had average
246
OD600 of 4.8 and 7.8, respectively. The total bacterial protein yield per unit of inulin
247
substrate was higher in SC than in LC inulin fermentations (P < 0.0001 for all inulin
248
concentrations; Fig. 4c).
249
3.2.2. S. bovis JB1 pH and SCFA
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At stationary phase (9 h), no remaining fructose was detected in 0.1% or 0.5%
251
w/v SC or LC inulin fermentations (0.1%, data not shown; 0.5%, Table 2), which
252
indicates that the substrate was limiting. However, fructose was detected in the media by
253
HPLC with refractive index and UV absorbance detection after 9 h fermentation when
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1.3% w/v inulin was included, and the LC inulin fermentations had more fructose than
255
SC (P < 0.0001). This latter result agrees with the percent disappearance of hydrolyzed
256
inulin determined by HPLC with pulsed amperometric detection (Fig. 3). Other
257
metabolic products included formic, acetic and lactic acids, and ethanol, which were
258
similar in LC and SC inulin fermentations when the substrate concentrations were 0.1 and
259
0.5% (Table 2). However, when 1.3% w/v inulin was used, LC inulin fermentations had
260
greater formate and ethanol concentrations compared to SC inulin fermentations at 9 h (P
261
< 0.0001 and P = 0.001, respectively). Concurrently, SC inulin fermentations had a
262
greater concentration of acetate at 9 h than LC fermentations (P = 0.0213), and lactate
263
concentrations were similar between treatments (P = 0.0742). At stationary phase (9 h),
264
S. bovis fermentations of SC inulin elicited a greater pH decline than fermentation of LC
265
inulin (P = 0.0038, P = 0.0049, and P = 0.0034 for 0.1, 0.5 and 1.3% w/v inulin
266
concentrations, respectively, Fig. 5). The greatest difference in pH was observed at the
267
1.3% w/v inulin concentration in which LC and SC fermentations had an average pH of
268
5.73 and 4.99, respectively. S. bovis assimilated more ammonia in 9 h when SC inulin
269
was the substrate (data not shown).
270
4. Discussion
271
Subsequent to the original assertion that the accumulation of fructan in pasture grasses
272
could cause PAL [2], researchers have concluded that PAL is a complex, multifactorial
273
condition that involves the metabolic state of the horse [1]. However, laminitis can be
274
induced in horses under laboratory conditions by administration of bolus doses of inulins
275
or other oligofructose [1,7,18,19]. Furthermore, others have suggested that short-chain
276
fructans are more rapidly fermented than long-chain fructans in the equine hindgut [9],
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and in vitro experiments to study factors contributing to dysbiosis have also employed
278
fructans [9, 12,16,17]. When microbiological analyses have been performed,
279
Streptococcus bovis, a well-known inulin-catabolizing organism, has been either isolated
280
or detected, [12,16,17]. These reasons justify examining the ability of S. bovis to
281
metabolize fructans with different chemical characteristics, the simplest of which is the
282
length of the polymer.
Previous studies have suggested that equine hindgut bacteria may more rapidly
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ferment SC fructans than LC fructans [9]. A difference in effects of fructan chain length
285
has been demonstrated in the fermentation of long- and short-chain inulin by the human
286
isolate Bifidobacterium longum subspecies infantis [30], and in the fermentation of inulin
287
of DP 31 and 57 by swine gut bacteria [31]. However, this relationship has not been
288
previously demonstrated with equine gut bacteria, and some bacterial species were
289
reported to preferentially utilize fructans of longer chain length [32]. Therefore, the
290
purpose of the current study was to determine if equine fecal bacteria and a model S.
291
bovis would equally ferment SC and LC inulins. Lower pH values and greater lactic acid
292
concentrations indicated that the SC inulin was fermented to a greater degree by equine
293
fecal bacteria. Streptococcus bovis JB1 readily fermented all of either inulin when inulin
294
was limiting. However, at all concentrations tested, the maximum specific growth rate,
295
the yield of cell protein per unit substrate, and the final optical density were greater when
296
SC inulin was used. These results indicate that SC inulin was a more easily utilized
297
substrate for equine fecal bacteria and this model S. bovis.
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It is important to note that S. bovis JB1 made copious amounts of lactic acid from either SC or LC inulin. However, the observed pH decline was greater in the SC inulin S.
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bovis fermentations. Although lactate production in both treatments was similar, acetate,
301
formate, and ethanol concentrations were different. Furthermore, more ammonia (a
302
strong base) was assimilated in the SC inulin treatment, which is consistent with the cell
303
protein yields calculated in the growth experiments. Therefore, the pH differences
304
observed could be attributed to these differences in acid production and ammonia
305
utilization.
In the S. bovis fermentations with the highest inulin concentration, significant
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quantities of free fructose were detected after 9 h with either treatment, indicating that
308
substrate was in excess. However, 42% more residual fructose was detected in LC than in
309
SC inulin fermentations. This latter result is consistent with the observation that the
310
growth yield was greater on SC inulin (i.e. there were more cells to utilize the sugar), and
311
supports the hypothesis that SC inulin is utilized more rapidly than LC inulin by S. bovis
312
JB1. Residual fructose in early stationary phase indicates that the catabolic capacity of
313
extracellular fructan hydrolases, which are widespread among streptococci [33],
314
exceeded the ability of the S. bovis cells to transport and metabolize the fructose. This
315
latter result is an informative laboratory artifact, but it is unlikely that sugars would
316
accumulate in vivo because other microorganisms in the equine hindgut can utilize them
317
The concept of crossfeeding between microorganisms is well established in the rumen
318
[34]. For example, cellulose, like fructan, is depolymerized outside of the cells.
319
Cellulolytic bacteria transport some of the glucose, but much is also utilized by non-
320
cellulolytic, saccharolytic bacteria. Another well-known example is interspecies
321
hydrogen transfer, in which methanogens utilize H2 and CO2 produced by bacteria. It is
322
almost certain that crossfeeding also occurs in the equine hindgut. In this way, other
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bacteria could convert fructose to acids and contribute to acidosis after fructan catalysis
324
by S. bovis or similar bacteria.
325 5. Conclusions
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The congruence of results between the experiments that employed uncultured fecal
328
cell suspensions and S. bovis JB1 support the hypothesis that bacteria of the S. bovis/S.
329
equinus group or similar bacteria are responsible for fructan fermentation in the equine
330
hindgut. These results indicate that inulin chain length influences fermentation by equine
331
fecal bacteria and a model S. bovis. Specifically, inulin of shorter chain length was more
332
available for fermentation in vitro suggesting that SC fructan could also present a greater
333
risk for PAL than LC fructans in vivo. However, an additional consideration is that chain
334
length could be reduced by catalysis prior to the large intestine [9]. In this way, hindgut
335
fermentation could be increased in vivo. It is also important to note that the inulin utilized
336
in the current study was extracted from Jerusalem artichoke (Helianthus tuberosus). We
337
acknowledge that inulins are structurally simpler (i.e. unbranched, a single type of
338
linkage) than many grass fructans. However, the advantage of using structurally simple
339
inulins is that the difference in chain length was tested without other differences in
340
structure. Even greater differences in fermentation might be obtained utilizing native
341
grass fructans that can vary dramatically in chain length depending on species and time of
342
year, and that include phleins and graminans [24].
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Acknowledgements
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The information reported in this paper (#15-07-096) is part of a project of the Kentucky
346
Agricultural Experiment Station and is published with the approval of the Director. MF
347
and IK were supported by USDA-ARS, National Program 215 – Pasture, Forage &
348
Rangeland Systems. The authors thank Beneo, Inc. for donating the inulins used in the
349
study, and Taylor Donley and Gloria Gellin for technical assistance.
350
References
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[8] Milinovich GJ, Burrell PC, Pollitt CC, Klieve AV, Blackall LL, Ouwerkerk D,
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bacteria producing amines in an in vitro model of carbohydrate overload. Appl Environ
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[14] Milinovich GJ. Trott DJ, Burrell PC, Croser EL, Al Jassim RAM, Morton JM, van
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Eps AW, Pollitt CC. Fluorescence in situ hybridization analysis of hindgut bacteria
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associated with the development of equine laminitis. Environ Microbiol 2007; 9: 2090-
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[15] Nelson CJ, Spollen WG. Fructans. Physiologia Plantarum 1987; 71:512-516.
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[16] Bailey S, Rycroft A, Elliott J. Production of amines in equine cecal contents in an in
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vitro model of carbohydrate overload. J Anim Sci 2002; 80: 2656-2662.
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[17] Harlow BE, Lawrence LM, Kagan IA, Flythe MD. Inhibition of fructan‐fermenting
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equine faecal bacteria and Streptococcus bovis by hops (Humulus lupulus L.) β‐acid. J
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[18] Crawford C, Sepulveda M, Elliott J, Harris P, Bailey S. Dietary fructan carbohydrate
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increases amine production in the equine large intestine: implications for pasture-
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associated laminitis. J Anim Sci 2007; 85: 2949-2958.
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[19] Kalck KA, Frank N, Elliott SB, Boston RC. Effects of low-dose oligofructose
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treatment administered via nasogastric intubation on induction of laminitis and associated
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alterations in glucose and insulin dynamics in horses. Am J Vet Res 2009; 70: 624-632.
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[20] Chatterton N, Harrison P. Fructan oligomers in Poa ampla. New Phytologist 1997;
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cheatgrass (Bromus tectorum L.). New Phytologist 1993; 124: 389-396.
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[22] Chatterton NJ, Harrison PA. Fructans in crested wheatgrass leaves. J Plant Physiol
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in transgenic tobacco and chicory plants expressing barley sucrose: fructan 6-
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fructosyltransferase. FEBS Lett 1997; 400: 355-358.
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Microbiol 2001; 67: 808-813.
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[26] Rocklin RD, Clarke AP, Weitzhandler M. Improved long-term reproducibility for
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pulsed amperometric detection of carbohydrates via a new quadruple-potential waveform.
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Anal Chem 1998; 70: 1496-1501.
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[27] Russell J, Robinson P. Compositions and characteristics of strains of Streptococcus
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bovis. J Dairy Sci 1984; 67: 1525-1531.
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[28] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the
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Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
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[29] Chaney AL, Marbach EP. Modified reagents for determination of urea and
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ammonia. Clin Chem 1962; 8: 130-132.
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[30] Perrin S, Fougnies C, Grill J, Jacobs H, Schneider F. Fermentation of chicory
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fructo-oligosaccharides in mixtures of different degrees of polymerization by three strains
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of bifidobacteria. Can J Microbiol 2002; 48: 759-763.
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[31] Paßlack N, Al-Samman M, Vahjen W, Männer K, Zentek J. Chain length of inulin
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affects its degradation and the microbiota in the gastrointestinal tract of weaned piglets
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after a short-term dietary application. Livestock Sci 2012; 149: 128-136.
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[32] Tsujikawa Y, Nomoto R, Osawa R. Difference in degradation patterns on inulin-type
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fructans among strains of Lactobacillus delbrueckii and Lactobacillus paracasei. Biosci
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Microbiota, Food Health 2013; 32: 157.
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and Wilkins Co., Baltimore, MD, USA 2005; 9th ed.: 552 – 558.
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[34] Russell JB. Rumen Microbiology and Its Role in Ruminant Nutrition. Ithaca, NY
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2002
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Figure Legends
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Fig 1.
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Chromatographic profiles of (A) SC inulin and (B) lLC inulin. Numbers above peaks
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represent chain length.The SC inulin is actually a SC inulin-rich fraction that also
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contains some contaminating glucose (G), fructose (F), and sucrose (S). Scales on
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chromatograms A and B differ: chromatogram B has been enlarged for better viewing of
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peaks. Both inulin samples were injected at 0.25 mg/mL, but electrochemical response
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decreases with increasing chain length [21].
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Fig. 2.
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Relationship between pH and inulin concentration after 24 h fermentation by suspensions
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of uncultivated, washed equine fecal microorganisms. The suspensions (n = 3, horses)
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had initial pH values of 6.8 (dashed line). Inulin substrates included short chain inulin
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(squares) and long chain inulin (circles) at concentrations ranging from 0 to 2 % w/v, in
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0.2% increments. Asterisks indicate that means are different between treatments within a
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given concentration (P < 0.05).
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SC
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Mono- and disaccharide concentration as the sum of glucose, fructose, and sucrose (left
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y-axis to solid vertical line), or percentage of mono- and disaccharides fermented (solid
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vertical line to right y-axis). Amount present is shown at 0 h in acid-hydrolyzed inulin
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(LC, solid bars; SC, open bars), and in inulin solutions incubated (39 °C) for 9 h with S.
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bovis. Percentage fermented by 9 h in the S. bovis incubations is shown on the right-hand
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side of the graph. The experiment was performed in triplicate. Asterisks indicate that
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means are different between treatments within timepoint (P < 0.05); 0h, trt: P = 0.2288,
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SEM: 0.2194; 9h, trt: P = 0.0313, SEM: 0.0100; % disappearance, trt: P = 0.0216, SEM:
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0.1710.
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Fig. 4.
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Effect of inulin chain length on the growth of inulin-fermenting Streptococcus bovis JB1.
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The culture was inoculated into media with 0.1, 0.5 or 1.3% long chain (solid bars) or
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short chain (open bars) inulin (w/v) and incubated (37 °C) for 9 h. The maximum specific
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growth rates are shown on panel (a), the final growth values (OD600) are shown on panel
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(b), and the cell yield (mg bacterial protein/mg inulin) at 9 h are shown on panel (c). The
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experiment was performed in triplicate. Asterisks indicate that means are different
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between treatments within concentration (P < 0.05); (a) 0.1%, trt: P = 0.0001, SEM: 0;
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0.5%, trt: P = 0.0004, SEM: 0.0002; 1.3%, trt: P = 0.0178, SEM: 0.0001; (b) 0.1%, trt: P
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< 0.0001, SEM: 0; 0.5%, trt: P = 0.0011, SEM: 0.0001; 1.3%, trt: P < 0.0001, SEM:
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0.0003; (c) 0.1%, trt: P < 0.0001, SEM: 0.345; 0.5%, trt: P = 0.0001, SEM: 1.0897;
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1.3%, trt: P < 0.0001, SEM: 0.2484.
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Effect of inulin degree of polymerization on the pH of inulin-fermenting Streptococcus
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bovis JB1 suspensions at stationary phase (9 h). The culture was inoculated into media
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with 0.1, 0.5 or 1.3% long chain (solid bars) or short chain (open bars) inulin (w/v) and
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incubated (37 °C) for 9 h. The experiment was performed in triplicate. Asterisks indicate
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that means are different between treatments within concentration (P < 0.05); 0.1%, trt: P
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= 0.0038, SEM: 0.0004; 0.5%, trt: P = 0.0049, SEM: 0.0010; 1.3%, trt: P = 0.0034,
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SEM: 0.0028.
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Table 1. Effect of inulin degrees of polymerization after 24 h of fermentation (0.5% inulin) on short chain fatty acid (SCFA) concentrations in equine fecal cell suspensions.
Lactate Acetate Propionate Butyrate Lactate:Propionate Lactate + Propionate Total SCFA
Long Chain Inulin 6.0 1.6 1.2 1.1 5.1 7.2 10.0
P – value
SEM
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Short Chain Inulin 7.8 1.3 Trace 1.2 7.8 8.8 11.3
P = 0.0220 P = 0.1296 P = 0.0742 P = 0.4226 P = 0.0251 P = 0.0189 P = 0.0006
SC
SCFA (mM)
0.107 0.027 0.005 0.015 0.292 0.268 0.002
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Long Chain Inulin 0 11 3.0 3.1 2.0 24.7 23.7 1.9 1.7 1.0
P – value P = 1.0000 P = 1.0000 P = 0.4479 P = 0.2380 P = 0.1625 P < 0.0001 P = 0.0742 P < 0.0001 P = 0.0213 P = 0.001
SEM 0 0 0.349 0.442 0.471 0 0.707 0 0.248 0.041
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Short Chain Inulin 0 11 2.8 3.7 1.1 14.3 21.7 1.4 3.0 0
SC
SCFA (mM) Fructose Lactate Formate Acetate Ethanol Fructose Lactate Formate Acetate Ethanol
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Table 2. Effect of inulin degrees of polymerization after 24 h of fermentation (0.5% and 1.3% w/v inulin) on metabolite concentrations in Streptococcus bovis JB1 cultures.
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Highlights •
Inulin DP influences equine fecal bacteria and S. bovis fermentations in vitro.
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•
Short-chain inulin is more available for fermentation than long-chain inulin.
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S. bovis JB1 grows faster on low DP inulin than on high DP inulin.
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Table S1. Residual inulin hydrolysates after fermentation (9 h) by Streptococcus bovis.
SC, 0.5%
LC, 0.5%
SC, 1.3%
LC, 1.3%
mg/mL remaining post incubation
glucose fructose
0.056 ± 0.004 0.59 ± 0.03
0.007 ± 0.004 0.008 ± 0.006
sucrose
0.071 ± 0.008
total glucose
0.72 ± 0.04 0.041 ± 0.004
0.016 ± 0.002 0.013 ± 0.014
fructose
0.76 ± 0.08
0.006 ± 0.005
sucrose total
0.010 ± 0.0005 0.82 ± 0.00
0.019 ± 0.015
glucose
0.28 ± 0.02
0.002 ± 0.003
fructose sucrose
2.96 ± 0.14 0.35 ± 0.04
0.056 ± 0.005 0.001 ± 0.000
total
3.59 ± 0.18
glucose fructose
% utilized
0
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87 99
100
98 69 99
0
100 98
SC
LC, 0.1%
mg/mL initial, based on inulin hydrolysis
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mono- or disaccharide
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98 99.6
0.059 ± 0.009
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0.21 ± 0.020 3.82 ± 0.42
-0.001 ± 0.004 0.027 ± 0.009
100 99
sucrose
0.051 ± 0.003
0.001 ± 0.0001
99
total glucose
4.08 ± 0.44 0.72 ± 0.05
0.027 ± 0.013 0.28 ± 0.05
99 61
fructose
7.69 ± 0.36
2.08 ±0.19
73
sucrose total
0.92 ± 0.10 9.34 ± 0.47
0.46 ± 0.07 2.82 ± 0.29
50 70
0.54 ± 0.05
0.33 ± 0.06
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9.94 ± 1.09 0.13 ± 0.01
4.08 ± 0.90 0.18 ± 0.01
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10.6 ± 1.1
4.59 ± 0.97
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glucose fructose sucrose
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