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Department of Animal Sciences, University of Illinois, Urbana 61801
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Fermentation • In Vitro • Oligosaccharides • Pigs
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Intestinal gases (H2, CO2, and CH4) originate from colonic fermentation of the nondigestible oligosaccharides, raffinose and stachyose (Delzenne and Roberfroid, 1994). Yet oligosaccharide fermentation yields products that are beneficial to the host, namely the short-chain fatty acids (SCFA) acetate, propionate, and butyrate (Buddington, 2001). Short-chain fatty acids have been implicated in a number of important physiological events. These include utilization of butyrate by rat colonic epithelial cells as the preferred energy substrate (Roediger, 1982) and prevention of colon cancer in humans (Young and Gibson, 1991). Additionally, growing swine can obtain as much as 12% of their ME requirement from SCFA (Imoto and Namioka, 1978; Kass et al., 1980).
Lactobacilli and bifidobacteria ferment carbohydrates through a pathway mediated by the glycolytic enzymes they produce in which the main end products are SCFA (Zani et al., 1974). These bacteria, in addition to other bacterial species, are capable of hydrolyzing glycosidic linkages that mammalian enzymes are incapable of hydrolyzing. Yazawa et al. (1978) reported that all species of bifidobacteria of human origin, except B. bifidum, utilize raffinose and stachyose. These galactooligosaccharides have a higher specificity for utilization by bifidobacteria than other intestinal strains, such as E. coli (Kawamura et al., 1968). Therefore, these bacteria, which inhabit the large intestine of swine, should efficiently utilize galactooligosaccharides. The objective of this study was to evaluate the fermentation characteristics resulting from the fermentation of galactooligosaccharides in comparison with other oligosaccharides used in animal nutrition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Central Soya, Gibson City, IL), granular transgalactooligosaccharides (Borculo Domo Ingredients, Borculo, The Netherlands), liquid transgalactooligosaccharides (Borculo Domo Ingredients); and 3) glucooligosaccharides (Bioecolia; Solabia, Pantin Cedex, France), mannanoligosaccharides (Alltech, Nicholasville, KY), and xylooligosaccharides (Xyloligo 95; Suntory, Tokyo, Japan). Three healthy pigs (approximately 25 kg) served as sources of feces from which the inoculum was prepared. The donors consumed a standard corn–soybean meal-based diet appropriate for their stage of growth for 2 wk prior to fecal collection. The pigs originated from an antibiotic-free herd and were housed in a temperature-controlled room with no exposure to other pigs or antibiotics for the duration of this study. The pigs selected from the antibiotic-free herd had no exposure to in-feed antibiotics for the duration of their life cycle; in addition, these three pigs never received injected antibiotics at any time prior to or during the study. The University of Illinois Institutional Animal Care and Use Committee approved all experimental procedures prior to experiment initiation (protocol No. 00199).
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Fermentation Procedures
The composition of the semi-defined medium used for the fermentations is presented in Table 2. All components except for the vitamin solutions were mixed before autoclave sterilization of the medium. Filter-sterilized vitamin solutions were added just before dispensing the medium, which was maintained under anaerobic conditions at all times after preparation. Aliquots (10 mL) of medium were aseptically transferred into Balch tubes, capped with butyl rubber stoppers, and sealed with aluminum caps. All tubes were stored at 4°C for approximately 12 h to enable hydration of the substrates before initiating fermentations. Tubes were placed in a 37°C water bath approximately 30 min before inoculation.
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) by blending for 15 s in a Waring blender under a stream of CO2. Blended, diluted feces were filtered through four layers of cheesecloth and sealed in 125-mL serum bottles under CO2.
Appropriate sample and blank tubes were aseptically inoculated with 1.5 mL of diluted feces. Tubes were incubated at 37°C with periodic mixing for the respective fermentation times. At the appropriate time, tubes were removed from the 37°C incubator and processed immediately for analyses. First, gas production was determined by fluid displacement (water with 5% HCl and resazurin) at equal pressure using a manometer (Campbell and Fahey, 1997). Corrections were made for temperature, pressure, and headspace contained in the Balch tube prior to initiation of fermentation. Gas production (mL) was calculated as gas production from the substrate minus gas production from the blank divided by original sample weight expressed on an OM basis. The pH of tube contents was measured with a standard pH meter (Denver Instrument Co., Arvada, CO) at 0, 2, 4, 8, and 12 h. Finally, a 2-mL subsample was taken from each tube for SCFA analyses.
Chemical Analyses
Samples to be analyzed for SCFA were mixed with 0.5 mL of 250 g/L m-phosphoric acid, precipitated at room temperature for 30 min, and then centrifuged at 25,900 x g for 20 min. The supernatant was decanted and frozen at -20°C in microfuge tubes. After freezing, the supernatant was thawed and centrifuged in microfuge tubes at 13,000 x g for 10 min. Concentrations of SCFA were determined via gas–liquid chromatography. Briefly, concentrations of acetate, propionate, and butyrate were determined in the supernatant of the tubes using a Hewlett-Packard 5890A Series II gas chromatograph (Palo Alto, CA) and a glass column (180 cm x 4 mm i.d.) packed with 10% SP-1200/1% H3PO4 on 80/100 mesh Chromosorb WAW (Supelco Inc., Bellefonte, PA). Nitrogen was the carrier gas with a flow rate of 75 mL/min. Oven temperature, detector temperature, and injector temperature were 125, 175, and 180°C, respectively. Short-chain fatty acid concentrations were corrected for the quantities of SCFA produced in the blank tubes. Lactate concentration was determined using a method adapted from Barker and Summerson (1941) and a Dionex Technical Bulletin (Dionex, Sunnyvale, CA).
The composition of soy solubles was analyzed by the University of Illinois and the Quality Assurance Laboratory of Central Soya; results are presented in Table 1
. The methods for these analyses are as described by Smiricky et al. (2002).
Calculations and Statistical Analyses
Data were analyzed as a randomized complete block design with fecal donor serving as block. Treatments, which were factorially arranged, included substrate and length of fermentation. Therefore, donor, substrate, time, and substrate x time were used in the statistical model. All ANOVA were performed according to the GLM procedures of SAS (SAS Inst., Inc., Cary, NC). Least squares means were reported along with the pooled SEM for all response criteria. When treatment differences were detected (P < 0.05), means were compared using least significant difference (Milliken and Johnson, 1984).
The data for gas and SCFA production were fitted to a logistic model equation to determine the rate of production and the time to attain maximal rate of production for each substrate. This function is frequently used to model biological, and especially microbial, growth (Koops and Grossman, 1993). It is a sigmoidal curve that can describe accelerating and, after passing through an inflection point, decelerating phases of growth (Koops and Grossman, 1991). The time at which maximal rate of gas or SCFA production occurred was calculated according to the following equation:
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where Y = gas or SCFA production, A = asymptote, or maximal gas or SCFA production, t = incubation time in hours, C = time in hours at which the rate of gas or SCFA production is maximum (the inflection point), and B = a measure of the duration of gas or SCFA production expressed in milliliters and millimoles per gram of OM, respectively.
Variables (A, B, and C) were estimated for each donor and substrate combination using nonlinear regression (NLREG; Sherrod, 2002). The model explained 95% or more of the variation in gas and SCFA production in all cases.
Additionally, maximal rates of gas and SCFA production were estimated using the derivative of the logistic function according to the following equation:
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where A = asymptote, or maximal gas or SCFA production; t = time in hours; C = time in hours at which maximal rate of gas or SCFA production occurs; and B = measure of the duration of gas or SCFA production expressed as milliliters and millimoles per gram of OM, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. The fructans produced similar amounts of gas at similar rates when compared to each other at each time point. Additionally, the fructans reached maximal rate of gas production at similar times (average 5.7 h).
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The other substrates tested, glucooligosaccharides, mannanoligosaccharides, and xylooligosaccharides, produced similar (P > 0.05) amounts of gas when compared with each other at 2 and 4 h. However, at 8 and 12 h of fermentation, mannanoligosaccharides produced less (P < 0.05) gas when compared to xylooligosaccharides. Furthermore, time to attain maximal rate of gas production for these substrates did not differ (P > 0.05). Rate of gas production was similar (P > 0.05) between glucooligosaccharides and mannanoligosaccharides, but was higher (P < 0.05) for xylooligosaccharides.
Gas production was highest (P < 0.05), on average, for the pure soy oligosaccharides (i.e., raffinose, stachyose, and raffinose + stachyose) at all time points. Gas production by the other galactooligosaccharides was not different (P > 0.05) from the fructans or glucooligosaccharides, mannanoligosaccharides, or xylooligosaccharides at 2 and 4 h. However at 8 and 12 h, gas production by mannanoligosaccharides was lower (P < 0.05) than that for the galactooligosaccharides and the fructans. Rate of gas production was similar (P > 0.05) for the fructans, soy solubles, both transgalactooligosaccharide forms, and xylooligosaccharides. Glucooligosaccharides and mannanoligosaccharides had the lowest (P < 0.05), and raffinose + stachyose the highest (P < 0.05), rate of gas production. Time to attain maximal rate of gas production did not differ (P > 0.05) among substrates.
Changes in pH
Generally, pH decreased as time of fermentation increased (Table 4). All substrates had similar pH values (6.5) at 0 h. No differences (P > 0.05) in pH were noted among fructans at any time point evaluated.
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Fermentations of glucooligosaccharides, mannanoligosaccharides, and xylooligosaccharides resulted in similar (P > 0.05) pH values at both 2 and 4 h. However, at 8 and 12 h, xylooligosaccharide fermentation resulted in the lowest (P < 0.05) pH, followed by glucooligosaccharides, with mannanoligosaccharides having the highest (P < 0.05) pH.
Overall, pH values for the fructans, granular transgalactooligosaccharides, and xylooligosaccharides at 12 h were similar (P < 0.05). The pH values for fermentations of glucooligosaccharides, soy solubles, and liquid transgalactooligosaccharides at 12 h were similar (P > 0.05) but lower (P < 0.05) than pH values for fermentations of mannanoligosaccharides and the pure galactooligosaccharides.
SCFA Production
Short-chain fatty acid production data are reported in Table 5. Whereas lactate concentration was measured for all substrates tested, there was no measurable lactate present as a result of oligosaccharide fermentation. The fructans resulted in similar (P > 0.05) concentrations of acetate, propionate, butyrate, and total SCFA when compared to each other at each time point.
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Fermentation of glucooligosaccharides, mannanoligosaccharides, and xylooligosaccharides resulted in similar (P > 0.05) acetate, propionate, and butyrate concentrations when compared to each other at 2 and 4 h. However, at 8 and 12 h, mannanoligosaccharide fermentation produced the least (P < 0.05) acetate and propionate, and xylooligosaccharides produced the most (P < 0.05) acetate. Propionate production was similar (P > 0.05) between xylooligosaccharides and glucooligosaccharides at 8 and 12 h. Butyrate production was similar (P > 0.05) for each substrate at all time points. Total SCFA production was similar (P > 0.05) for glucooligosaccharides, mannanoligosaccharides, and xylooligosaccharides at 2 and 4 h. However, at 8 and 12 h, mannanoligosaccharide fermentation resulted in the lowest (P < 0.05), and xylooligosaccharide fermentation the highest (P < 0.05), total SCFA production.
When comparing all oligosaccharides tested at the 12 h fermentation time, mannanoligosaccharide fermentation produced the least amounts (P < 0.05) of acetate and propionate when compared to all other substrates. Fermentation of the fructans, soy solubles, granular transgalactooligosaccharides, liquid transgalactooligosaccharides, and glucooligosaccharides resulted in similar (P > 0.05) acetate and propionate productions. Fermentation of pure galactooligosaccharides and xylooligosaccharides resulted in the highest (P < 0.05) acetate production. Glucooligosaccharide, mannanoligosaccharide, and xylooligosaccharide fermentation resulted in the lowest (P < 0.05) butyrate concentration, whereas fermentation of all other substrates resulted in similar (P > 0.05) butyrate concentrations. Total SCFA production was similar (P > 0.05) for the fructans, soy solubles, both transgalactooligosaccharides forms, and glucooligosaccharides. Fermentation of raffinose and mannanoligosaccharides resulted in the lowest (P < 0.05) total SCFA production. Stachyose, raffinose + stachyose, and xylooligosaccharide fermentation resulted in the most (P < 0.05) total SCFA production at 12 h.
Rate of SCFA production and time to attain maximal rate of SCFA production data are presented in Table 6. Rates of acetate, propionate, and butyrate production and time to attain maximal rate of production were similar (P > 0.05) among the fructans (average 0.4, 0.4, and 0.2 mmol•g OM in sample-1 •h-1 and 5.5, 6.4, and 6.0 h, respectively).
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Fermentation of mannanoligosaccharides resulted in the lowest (P < 0.05), and xylooligosaccharides the highest (P < 0.05), rates of acetate and propionate production. Rate of butyrate production was similar (P > 0.05) for glucooligosaccharides, mannanoligosaccharides, and xylooligosaccharides.
Overall, rates of acetate production were similar and lowest (P < 0.05) for glucooligosaccharides, mannanoligosaccharides, medium-chain fructooligosaccharides, and long-chain fructooligosaccharides. Fermentation of the galactooligosaccharides (except granular transgalactooligosaccharides) resulted in the highest (P < 0.05) rates of acetate and propionate production when compared to other substrates. The rates of propionate production were similar and lowest (P < 0.05) for glucooligosaccharides and mannanoligosaccharides. Fermentation of the fructans and xylooligosaccharides resulted in moderate and similar (P > 0.05) rates of propionate production. Rates of butyrate production did not differ (P > 0.05) among substrates.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Gas production was similar among all substrates tested except for mannanoligosaccharides and the pure galactooligosaccharides. Fermentation of soy solubles resulted in lower amounts of gas production when compared with pure forms of galactooligosaccharides found in soy solubles. Additionally, the rate at which pure raffinose + stachyose produced gas greatly exceeded that of the soy solubles. These data indicate that raffinose and stachyose present in the soy solubles matrix behave much differently than they do in the pure form. Hence, the increase in gas production observed in fermentations of pure galactooligosaccharides may provide indirect evidence that genera other than bifidobacteria and lactobacilli were stimulated since they do not produce gas during homolactic fermentation (Lengeler et al., 1999). Homolactic-fermenting organisms, such as bifidobacteria and lactobacilli, produce exclusively D- or L-lactate from hexoses (Lengeler et al., 1999). Whether growth of other genera is a direct result of fermentation processes or from cross feeding on bifidobacterial metabolites is unclear. Soy solubles contain 2.4% N and a significant amount of sucrose. Therefore, fermentation of the oligosaccharides in soy solubles may have been delayed due to the availability of more fermentable components. Additionally, they may be complexed and less available for fermentation when present in the whole plant matrix. Carbohydrates are the preferred substrates for fermentation and, thus, the sucrose in soy solubles would have been quickly transported via a phosphotransferase carrier system into the cell wall of bacteria and converted to fructose 1, 6 bisphosphate (Lengeler et al., 1999). This compound can then enter the Embden-Meyerhof pathway and be converted to 2 pyruvate + 2 ATP + 2 NADH (Lengeler et al., 1999). Pyruvate can be converted to SCFA, resulting in release of 1 mol of CO2. The minimal amount of gas released as a result of sucrose fermentation may be the reason that less gas is produced by soy solubles fermentation when compared to fermentation of the pure galactooligosaccharide components of soy solubles. Additionally, gas production as a result of fermentation of raffinose was higher than for stachyose or raffinose + stachyose. Since raffinose fermentation did not follow a logistical model, and rate of production and time to attain maximal rate of production could not be calculated, the difference between raffinose and stachyose is perhaps due to a slower rate of stachyose fermentation. Stachyose contains an extra 
1-6 linked galactose unit. This additional bond that must be broken by bacterial -galactosidase may have slowed fermentation and decreased the rate of fermentation and, thus, gas production when compared with raffinose fermentation. The potential reason that raffinose + stachyose fermentation did not result in gas production rates intermediate to raffinose and stachyose fermentation is the slower rate of fermentation as affected by the presence of stachyose and, thus, gas production did not rebound to the production level of pure raffinose.
Mannanoligosaccharide fermentation resulted in the lowest quantity of gas production, the lowest rate of gas production, and the longest time to attain maximal rate of gas production. This is coupled with high pH and low SCFA production. Unlike most of the oligosaccharides tested in this experiment, mannanoligosaccharide is a crude extract from yeast and contains 6% N, 41% total dietary fiber, 8% fat, and 44% total monosaccharides on a dry matter basis. Therefore, ingredients that do not ferment as rapidly, such as N and fat, could potentially inhibit fermentation. Additionally, the fiber component of mannanoligosaccharides could slow its fermentability.
The change in pH as a result of fructan fermentation was similar among all fructan forms. Flickinger et al. (2000) reported a 17% decrease in pH values when short-chain fructooligosaccharides were fermented for 11 h with canine fecal microflora. Similarly, in the present study, the pH value for short-chain fructooligosaccharides decreased 17% over a 12 h fermentation period.
At every time point, soy solubles fermentation resulted in a lower pH than did the pure galactooligosaccharide substrates. A potential explanation could be that fermentation of raffinose + stachyose was rapid and, once complete, proteolytic bacteria present in the fermentation vessel began consuming spent bacteria as substrates for growth, yielding end products that were of a higher pH. Fermentations of soy solubles and liquid transgalactooligosaccharides generally resulted in similar pH values, indicating potential similarity in fermentative characteristics.
Fermentation of xylooligosaccharides and the fructans resulted in the lowest pH values of all oligosaccharides tested. These data indicate that bacteria present in the large intestine of swine may more extensively ferment these substrates compared with the other oligosaccharides tested.
Short-chain fatty acid production among fructan sources was not different in this study. Flickinger et al. (2000) reported a fivefold increase in acetate and a ninefold increase in propionate and butyrate production after 11 h of short-chain fructooligosaccharides fermentation. Our data indicate greater increases in acetate (10-fold) and butyrate (11-fold) production. This may be due to the different sources of fecal inoculum (canine vs. swine). The SCFA production data presented by Flickinger et al. (2000) are consistent with data for other in vitro fermentations of short-chain fructooligosaccharides using canine fecal microflora (Sunvold et al., 1995; Vickers et al., 2001). In agreement with our data, Vickers et al. (2001) reported similar SCFA production as a result of fermentation of short-chain fructooligosaccharides and long-chain fructooligosaccharides (0.97 vs. 1.1 mmol of total SCFA/g of OM fermented, respectively) with canine fecal microflora.
Stachyose and raffinose + stachyose fermentations produced more SCFA, had higher rates of SCFA production, and attained a maximal rate of SCFA production earlier when compared with other oligosaccharides. Galactooligosaccharides in the soy solubles matrix are fermented more slowly than pure raffinose and stachoyse present in the same concentrations as in soy solubles. Therefore, in the ingredient matrix, galactooligosaccharides may be more efficacious as fermentative substrates in the large intestine when compared with their pure counterparts because they are more slowly fermented, and thus, may escape degradation by terminal small intestinal microflora and be available to serve as fermentative substrates in the large intestine of swine. Additionally, at 8 and 12 h, fermentation of raffinose resulted in no propionate formation, indicating that bacteria contributing to fermentation were not generating propionate or the propionate in the fermentation vessels was metabolized.
Fermentation of mannanoligosaccharides resulted in less production of SCFA when compared with all other substrates except raffinose. Also, rate of SCFA production was lowest for mannanoligosaccharides, indicating a lower fermentative capacity. In agreement with our data, Vickers et al. (2001) reported substantially lower acetate (0.89 vs. 2.3 mmol/g of OM sample), propionate (0.3 vs. 0.9 mmol/g of OM), and butyrate (0.2 vs. 0.3 mmol/g of OM) values when mannanoligosaccharide was fermented for 12 h when compared to short-chain fructooligosaccharides.
Flickinger et al. (2000) reported lower amounts of SCFA production when glucooligosaccharides were fermented by canine microflora over an 11-h period in comparison to our data. They reported a fourfold increase in acetate, an eightfold increase in propionate, and a fourfold increase in butyrate production when glucooligosaccharides was fermented. Our results show an eightfold increase in acetate, a 10-fold increase in propionate, and a fivefold increase in butyrate production when glucooligosaccharides was fermented with swine fecal microflora over a 12-h period.
Short-chain fatty acids play several important roles in animal metabolism. For example, Roediger (1982) reported that butyrate was the preferential energy source of colonocytes in rats. Also, nonruminant hindgut fermentors utilize acetate largely as a fuel source for peripheral tissues (Cummings, 1991). Propionate has been suggested to spare amino acids that would be used in gluconeogensis in the postabsorptive state (Demigne and Remesy, 1991). Additionally, SCFA can contribute up to 28% of the total maintenance energy requirements of pigs (Imoto and Namioka, 1978). Therefore, substrates that produce high amounts of SCFA in vitro, namely the fructans, xylooligosaccharides, and the pure galactooligosaccharides, may do the same in vivo. However, dietary inclusion of pure galactooligosaccharides may result in problems, namely high amounts of gas production and fermentation rates that are too rapid. Substrates resulting in lower amounts of gas production and moderate to high quantities of SCFA, such as soy solubles and the fructans, may be better substrates for fermentation in vivo.
The differences in amounts of SCFA produced by oligosaccharides tested in our study when compared with previous studies may be a result of the source of fecal inoculum. Flickinger et al. (2000) reported data utilizing canine fecal donors, whereas our study utilized swine fecal donors. Different potential concentrations of bacteria known to ferment oligosaccharides may have been a factor in the increased SCFA production noted in our study.
In conclusion, fermentation of most of the oligosaccharides in this study resulted in a rapid decline in pH and an increase in organic acid production. This indicates that most of these oligosaccharides are rapidly fermentable. Higher amounts of gas and SCFA production were synonymous with faster rates of production and earlier times to attain maximal rate of production. Lower amounts of gas and SCFA production were indicative of slower rates of production and longer times to attain maximal rates of production. The greater rate of fermentation of the pure galactooligosaccharides indicates that these oligosaccharides may serve as fermentable substrates in the terminal small intestine or proximal large intestine. Fermentation of mannanoligosaccharides resulted in the lowest SCFA production, indicating that this substrate is fermentable but not to the same extent as the other oligosaccharides tested. Fermentation of xylooligosaccharides resulted in the highest SCFA production by 12 h when compared with the pure galactooligosaccharides and soy solubles; however, time to attain maximal rate of SCFA production, when compared with that of the pure galactooligosaccharides and soy solubles, was longer. The slower rate of fermentation of xylooligosaccharides when compared with galactooligosaccharides and soy solubles may indicate that it is a better source of fermentable substrate in the distal portion of the gastrointestinal tract, such as in the transverse and descending regions of the large intestine.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence: 132 Animal Sciences Laboratory 1207 W. Gregory Dr. (phone: 217-333-2361; fax: 217-244-3169; E-mail: gcfahey{at}uiuc.edu).
Received for publication June 25, 2002. Accepted for publication June 6, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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